3aS^E=iF=i^F^F=^E^F=?SSI=i^B3 1 I Marine Biological Laboratory Library E E Woods Hole, Mass II I I I I I I I J . . lll&m R, Amberson J Universi " Maryland E B July 1, 1955 ID I EE »/^v?«^N* Presented ty JEaSB^^^^B^B^^^^ESE The Microtomist's Formulary and Guide The Microtomist's Formulary and Guide by Peter Gray, Ph.D., D.I.C., F.R.M.S. Head, Department of Biological Sciences University of Pittsburgh THE BLAKISTON COMPANY, INC. New York Toronto Copyright 1954, by The Blakiston Company, Inc. This book is fully protected by copyright, and no part of it, with the exception of short quotations for review, may be reproduced without the written permission of the publisher Library of Congress Catalog Card Number: 53-11567 PRINTED IN THE UNITED STATES OF AMERICA BY THE MAPLE PRESS COMPANY, YORK, PA. To Freda ^/<^IC/^ ^i^CAl Preface The preface of a book affords the author an opportunity of speaking to his reader in a comparatively direct and personal manner, and of acquaiiiting the prospective user of the book with the considerations which impelled the author to write it. (From The Bookman's Glossary. 3d ed. New York, Bowker [c. 1951]. Reprinted by permission of the R. R. Bowker Co.) A few generations ago the English periodical Punch offered to its readers a "letter of advice to those about to be married": the applicants received the single word "Don't." The advice is pertinent for those about to write a source reference work. You may well, in reading this book, become incensed at what you beheve to be its inaccuracies, errors, and faulty arrangements. This is exactly how I felt, twenty years ago, when I struggled with the reference books on micro- technique which I was then using. You may decide, as I did, to try to write a better book. You will find it a wearisome and disillusioning task. The research will, of course, be wholly dehghtful, but it will be followed by a period of brutal hard labor. Not only will you have to write, but then, if you are to produce a pub- Hshable book, it will have to be condensed and rewritten. Add to this the fact that the finished work has then to be reread four separate times as it goes through press, and you will join me in hoping that your activities do not too strongly resemble those of the dog mentioned in the Book of Proverbs. This book would never have been completed without the help of the librarians of the University of Edinburgh, the Wood's Hole Marine Biological Labora- tory, the University of Rochester, the Carnegie Library of Pittsburgh, and the University of Pittsburgh. I am especially indebted to Miss Lorena Garloch, and her assistants in the Reference Department of the University of Pitts- burgh Library, for their extraordinary skill in tracking down obscure journals and securing them for me on inter-library loan. The illustrations for this work, as for my Handbook of Basic Microtechnique, were prepared from my phot()grai)hs and sketches by Mrs. Gloria Green Hirsch. 1 am glad thai r('\ icwcrs of the published book share my enthusiasm for her work. The number of those, including the author and his wife, who have had a hand in typing this book is legion. It should be recorded, however, that Mrs. Mary Roman single-handed produced the first complete (1500 page) typescript vii Vlll PREFACE and that Mrs. Dolores Johnson, and Miss Kristine Pallesen, have stood by the author durmg the distressing hurly-burly known as "getting the book into press." Dr. James Lackey, then scientific editor of the Blakiston Company, encour- aged me over long periods to persevere in producing a publishable book. His successor, Mr. WiUiam Keller, approved what had been done and, with Mr. Willard Shoener converted my efforts to their present form. My debt to these gentlemen, and to their editorial assistants, is immense. Acknowledgement is made with thanks to the American Optical Company for figures 56, 57, and 84, to the Fisher Scientific Company for figures 34 and 38, and to the Carbide and Chemical Corporation for some of the data in Chapter 25. Permission to reproduce copyright material of the R. R. Bowker Company and the Oxford University Press is specifically acknowledged at the places where these reproductions occur. Peter Gray Edinburgh 1933 Pittsburgh 1953 List of Contents Preface vii Introduction , . 1 PART I— THE ART OF MAKING MICROSCOPE SLIDES Foreword to Part 1 7 1. Dry Wholemounts 10 Slides for dry mounts — coverslips — cells — background — cell cements — • coverslip cements for dry mounts — typical preparation: Strewn slide of foraminifera or radiolaria. 2. Fluid Wholemounts — Aqueous Type 21 Cells for aqueous mounts — cell cements — preservation media — coverslip cements — sealing the coverslip — typical preparations: Wholemount of Microcystis, Wholemount of a rotifer. 3. Fluid Wholemounts — Non-aqueous Type 32 Choice of mounting media — dichromate-gelatin seals — hot resin seals — typical preparations: Nematode in glycerine, Diatom in monobromnaphtha- lene. 4. Wholemounts in Gum Media 42 Choice of mounting medium — types of object to be mounted — finishing slides — typical preparation: Mite in Berlese's medium. 5. Wholemounts in Jelly Media 46 Process of mounting — finishing jelly mounts — typical preparation: Wholemount of a small crustacean. 6. Wholemounts in Resinous Media 51 Narcotizing and fixation — choice of stains — dehydration — clearing — mounting in balsam — finishing balsam mounts — typical preparations: Carmine stained wholemount of Pectinatella, Skeleton of an insect, Double stained alga in venice turpentine, Minute fresh-water organisms. 7. Smear Preparations from Fluid Material 69 Preparation of smears — fixing smears — drying smears — typical prepa- ration : Monocystis from the seminal vesicle of the earthworm. ix O^t^t^^ x list of contents 8. Smeae Preparations from Cut Surfaces 74 Preparation of smears — typical preparation : Diagnostic smear of Negri bodies. 9. Squash Preparations from Solid Bodies 76 The process of maceration — staining and mounting squash prepara- tions— typical preparations: Macrosporocytes of Crocus, Dissociated Hydra. 10. Ground Sections 80 Preparation of the crude section — grinding and polishing agents — typical preparations: Transverse section of bone, Section of coral with polyp in situ. 11. Sections of Free Material 88 Nature of sections — microtomes for free sections — methods of holding material — hardening and fixing material — staining and mounting sec- tions— typical preparations: Transverse section of leaf of Ligustrum, Section of wood. 12. Paraffin Sections 94 Selection of a fixative — dehydrating agents — clearing agents — embed- ding media — technique of dehydrating, clearing and embedding — micro- tomes— knives and knife sharpening — block mounting — cutting paraffin ribbons — staining and mounting sections — cleaning and labelling slides — typical preparations: Transverse section of frog's intestine, Section of amphibian embryo. Sagittal section of whole mouse. 13. Nitrocellulose Sections 142 Nitrocellulose — preparation of nitrocellulose solutions — infiltration — casting celloidin blocks — cutting sections — staining and mounting — typical preparation : Transverse section of lily bud. 14. Sections from Double Embedded Material 153 Explanation of process — typical preparation : Sections, intended for recon- struction, of pluteus larva. 15. Frozen Sections 157 Choice of a supporting medium — refrigerants — cutting frozen sections — staining and mounting — typical preparation: Section of fatty tissue. 16. Injections 162 Selection of injection mass — methods of injection — typical preparations: India ink injection of chicken embryo. Lead chromate injection of kidney glom- eruli, Carmine-gelatin injection of intestinal capillaries. PART II— METHODS AND FORMULAS USED IN MAKING MICROSCOPE SLIDES Foreword to Part II 173 LIST OF CONTENTS ■ XI 17. Preservatives (Referenced AS P) 175 P 00 General observations — P 10 Preservatives miscible with water — 11 inorganic reagents, 12 organic reagents, 13 other preservatives — P 20 Preservatives not miscible with water. 18. Fixatives (Referenced as F) 182 General observations — Standard fixative solutions — Formulas arranged by classes — ^1 osmic, 2 platinic, 3 mercuric, 4 cupric, 5 picric, 6 chromic, 7 dichromate, S other inorganic salts, 9 other organic reagents: alone, combined together, or with 0.1 formaldehj-de, 0.2 acetaldehyde, 0.3 acetone, 0.4 other mod'i&er : with or icithout 0.001 acetic, 0.002 trichloracetic, 0.003 formic, 0.004 nitric, 0.005 sulphuric, 0.006 hydrochloric, 0.007 oxahc, 0.008 other inorganic acids, 0.009 other organic acids — Basal fixative solutions — Formulas arranged alphabetically. 19. Accessory Fixative Formulas (Referenced as AF) 254 AF 00 General observations — AF 10 fixative removers — AF 20 De- calcifying AGENTS and AGENTS FOR SOFTENING CHITIN AF 30 BLEACHING AGENTS — AF 40 Macerating agents — AF 50 Narcotizing agents. 20. Formulas and Techniques for Dye Stains of General Applica- tions (Referenced as DS) 267 DS 00 General observations — DS 10 Dye staining techniques of GENERAL APPLICATION — DS 11 NucLEAR STAINS, 11.1 hematoxylin (typical preparations: Kat testis with iron-hematoxylin, Chicken embryo wholemount with alum hematoxylin, Chicken embryo sections with acid-alum hematoxylin), 11.2 carmine (typical preparations: Liver fluke with carmalum, Medusa with alcoholic borax-carmine, Chromosomes with iron aceto-carmine) , 11.3 other natural dyes, 11.4 synthetic dyes (typical prepsirations: Pollen grains with safranin, chromosomes with magenta) — DS 12 Plasma stains, 12.1 single con- trast formulas, 12.2 double contrasts from one solution (typical prepara- tions : Squalus embryo with 'picro-indigocarmine, Rat tongue with celestin blue — picro-acid fuchsin) , 12.3 complex contrast formulas (typical preparations : Section of earthworm with hematoxylin-acid fuchsin-anilin blue, Section of mouse head with hematox^jlin- ponceau 2R-light green) — DS 13 Complex TECHNIQUES INVOLVING BOTH NUCLEAR AND PLASMA STAINING, 13.1 thiazin eosinates (typical preparation: Blood smear with methylene blue-azur A- methylene violet-eosin Y), 13.2 thiazin eosinates with other dyes, 13.3 methyl green techniques (typical preparation: Suprarenal body with methyl green- acid fuchsin-orange G), 13.4 acid fuchsin techniques (typical preparation: Section of Amphioxus with acid fuchsin-anilin blue-orange G), 13.5 safranin techniques, 13.6 hematoxylin techniques, 13.7 other complex techniques. 21. Formulas and Techniques for Dye Stains of Special Applica- tions (Referenced as DS) 374 DS 20 Dye staining techniques op special application — DS 21 Sk- LECTivE stains FOR HISTOLOGICAL ELEMENTS, 21.1 skeletal tissues (typical preparations: Bones in wholemount of small salamander mth alizarin, Carti- lage in embryo with methylene blue. Root skeleton with acid fuchsin-iodine XU LIST OF CONTENTS green), 21,2 nervous tissues (t3T3ical preparations: Section of brain with methylene blue, Section of spinal cord with hematoxylin. Neuroglia with crystal violet), 21.3 blood, 21.4 other histological elements — DS 22 Stains foe CYTOLOGiCAL ELEMENTS, 22.1 nuclei (typical preparation: Mitosis with rose bengal-orange G-ioluidin blue), 22.2 mitochondria and Golgi (typical prepa- ration: Mitochondria in pancreas with acid fuchsin-toluidin blue-aurantia) , 22.3 Nissl granules, 22.4 yolk and fat granules, 22.5 plastids, 22.6 starch, glycogen and amyloid granules, 22.7 mucin, 22.8 other cell inclusions and extrusions — DS 23 Selective stains for specific organisms, 23.1 virus, Rickettsiae and Negri bodies (typical preparations : Rickettsiae in guinea pig scrotum with magentathionin, Negri bodies in guinea pig brain with ethyl eosin-methylene blue), 23.2 bacteria (typical preparations: Bacterial smear with crystal violet, Demonstration of Gram positive bacteria. Demonstration of tubercle bacilli, Flagella of Proteus vulgaris, Diploccocci in liver of rabbit), 23.3 other parasites and commensals (typical preparations •.Pencillium mycelia in orange rind with thionin-light green-orange G-erythrosin, Fungi in tissue scrapings), 23.4 other animals, 23.5 other plants — DS 24 Miscellaneous TECHNIQUES. 22. Accessory Dye Staining Formulas (Referenced as ADS) .... 514 ADS 10 Mordants and tissue revivers, 11 miscellaneous formulas, 12 mordants — ADS 20 Differentiating solutions, 21 for hematoxylin, 22 for other stains. 23. Formulas and Techniques for ]\Ietal Stains (Referenced as MS) 522 MS 00 General observations — MS 10 Osmic acid, 11.0 typical prepa- rations: (Golgi network in earthworm ovary), 11.1 staining solutions, 11.2 neurological techniques, 11.3 histological techniques, 11.4 techniques for cell inclusions — MS 20 Gold — MS 21 Gold used alone, 21.0 typical preparations: {Nerve termination in muscle), 21.1 staining solutions, 21.2 techniques — MS 22 Gold in combination with mercury, 22.0 typical preparation (Protoplasmic neuroglia in the cerebral cortex), 22.1 staining solutions, 22.2 neurological techniques — MS 23 Gold in other combina- tions, 23.0 typical preparation (Spinal cord with ammonium dichromate — gold chloride), 23.1 staining solutions, 23.2 neurological techniques, 23.3 cytological techniques, 23.4 other techniques — MS 30 Silver — MS 31 Silver nitrate, 31.0 typical preparations (Nervous elements of retina. Neuroblasts and axons in chicken embryo, Spirochaetes in sections), 31.1 staining solutions, 31.2 neurological methods, 31.3 cytological methods, 31.4 histological methods, 31.5 bacteriological methods — MS 32 Protein silver, 32.0 typical preparation (Sciatic nerve of cat to show axis cylinders), 32.1 neurological methods, other methods — MS 33 Silver diammine, 33.0 typical preparations (Nerve endings in taste buds, Oligigodendria and mi- croglia, microglia), 33.1 staining solutions, 33.2 neurological methods, 33.3 cytological methods, 33.4 histological methods, 33.5 bacteriological methods, 33.6 other silver diammine methods — MS 34 Silver in combination with other metals, 34.0 typical preparations (Purkinje cells in the cerebellar cortex. Structure of superior cervical ganglion. Neurons and dendrites in brain LIST OF CONTENTS Xlll of rabbit embryo), 34.1 staining solutions, 34.2 neurological methods, 34.3 histological methods, 34.4 cytological methods, 34.5 bacteriological methods — MS 35 Other Silver methods — MS 40 Other metals, 41.1 staining solutions, 41.2 neurological methods, 41.3 histological methods. 24. Accessory Metal Staining Formulas (Referenced as AMS) . . 612 AMS 10 Accelerators and mordants, 11 formaldehyde mixtures, 12 alcohol mixtures, 13 other mixtures — ADS 20 Solutions used after stain- ing, 21 developers, 22 toners, 23 differentiators, 24 fixers. 25. Solvents and Oils (Referenced as S) 622 S 10 Dehydrating agents — S 20 Clearing agents, 21 essential oils, 22 synthetic clearing agents — S 30 "Universal" solvents — S 40 Mix- tures. 26. Mounting Media (Referenced as M) 630 M 10 Mountants miscible with water, 11 gum arable media, 12 gelatin media, 13 other media — M 20 Mountants miscible with alcohol, 21 mastic media, 22 Venice turpentine media, 23 sandarac media — M 30 Mountants not miscible with water or alcohol, 31 canada balsam media, 32 damar media, 33 other natural resins, 34 synthetic resins. 27. Embedding Media (Referenced as E) 642 E 10 Media miscible with water — E 20 Media not miscible with water, 21 wax media, 22 nitrocellulose media, 23 resinous media, 24 other media. 28. Various Formulas (Referenced as V) 650 V 10 Cements, lutes and varnishes, 11 fluid, 12 solid, 13 other mix- tures— ^V 20 Adhesives, 21 for attaching sections to slides, 22 for attaching whole objects to slides, 23 for other purposes — ^V 30 Injection media — V 40 Cleaning formulas — V 50 Miscellaneous formulas. List of Abbreviations Used 669 List of Books and Journals Cited 670 Books — Journals not listed in "World List" — Journals hsted in "World List." Index 681 Introduction jOiC/^^ Scope of the Book This work consists of two parts. Part I (Chapters 1-16) is a treatise on the art of making microscope sUdes from biological specimens. Part II (Chapters 17-28) is a classified list of the formulas and tech- niques used in this art. Arrangement of Part I Each chapter deals with a specific type of microscope shde and is divided into two parts. The first part discusses problems in- volved in the preparation of such a sUde and the general methods by which these problems have been overcome. The second part is devoted to one or more specific ex- amples which describe in detail the appli- cation of the general methods to the production of an actual slide. The few literature references in Part I are confined to places where the author is describing a method of which he lacks personal experi- ence, or where he is giving opinions at variance with his own. Arrangement of Part II The chapters in Part II are devoted to specific types of formulas and give, where necessary, the techniques by which these formulas are used. Each chapter is sub- divided decimally in accordance with a scheme given in full at the beginning of the chapter and explained in the first para- graph of the chapter. Every formula or technique is thus identified by a number which is used, together with two or three letters identifying the chapter, in all cross references. Thus: DS 11.122 Mayer 1891 identifies a specific alum-hematoxylin of Mayer (he published five other alum- hematoxylin formulas) in any of the fifty places that reference is made to it. The formula is given only once and then in as- sociation with all the other alum-hema- toxylin (DS 11.122) formulas in the book. These decimal reference numbers are added to the page numbers all through, thus making it easy to run down a given type of formula or technique. Pet Names Some biologists have a pernicious habit of omitting literature references and using what Conn 1938 (20540b, 13:121) in a well-organized attack on them, calls "pet names." In cases where these pet names — such as paracarmine or B 15 — have be- come embedded in the folklore of micro- technique, the present author puts them in italics, immediately after the decimal reference, thus: DS 11.22 Mayer 1892 paracarmine — compl. script. The appended conipl. script, indicates that the word has occurred in a "great many writings." When only the originator of the technique appears to have used the pet name it is referred to with auct. Pet names should never be used in scientific writing but such sloppy scholarship as is inherent in a reference to "Bouin's Fluid" is almost worse. Bouin is the originator of many fixatives of which one happens to be popular at the moment; quite another was popular twenty years ago, when a casual reference to Bouin's Fluid meant a mer- curic formaldehyde mixture. Method of Giving Literature References The author indicates, after every for- mula or technique, the source from which he is quoting. The form used varies accord- ing to the type of source; and an example of each will be given. INTRODUCTION Direct Quotations from Books. The author's name, date, and page only are used, thus: DS 11.123 Anderson Anderson 1929, 129 At the end of Part II there is a list of books cited where "Anderson 1929" is ex- panded to a full bibliographic reference. Indirect Quotations from Books. It too frequently happens that a book quotes a formula by name, either without giving a reference at all or with an incorrect reference. When the present author has been unable to find the original he uses the abbreviation test. Thus: DS 11.123. Conklin test. 1930 Guyer Guyer 1930, 232. This indicates that the volume in ques- tion contains, on page 232, a formula for Conklin's picro-hematoxylin but offers no information as to where the original can be checked. Where the author of a book cited is quoting at second hand, the abbreviation cit. is used. Thus DS 11.24 Vignal test. 1907 Bohm and Oppel cit. Henneguy Bohm and Oppel 1907, 118 indicates that, on page 118 of the volume in question, there is a statement to the effect that Henneguy proposed, following the method of Vignal, to prepare a picro- carmine by this particular method. Where the author of a book cites him- self, or the authors cite themselves, with- out reference, the abbreviation used is test. ips. (standing for teste ipso or testihus ipsis). Thus: MS 31.22 Cajal 1925 test. 1933 ips. Cajal and de Castro 1933, 262 The present author would plead in self defense that he has tracked more than a thousand such references to the originals and that these test, and cit. references are used only where he has failed to find the original or where the original is incorrectly quoted. Direct Quotations from Journals. The author has used, in place of the name of the journal, the number assigned to that journal in the World List of Scientific Periodicals (Oxford, The University Press, 1927). Thus: DS 11.122 Carazzi 1911 23632, 28:273 indicates that Carazzi's formula is given on page 273 of volume 28 of the Zeitschrift far wissenschdftUche Mikroskopie und fiir mikroskopische Technik. The full titles of the two-hundred-odd journals cited will be found immediately preceding the index. The use of this number not only saves space, an important consideration in a volume of this magnitude, but also permits exact identification of the journal. The author decided to use these numbers quite shortly after he started checking refer- ences in the /. Anat. (either of two jour- nals) and the /. Bot. (any one of three journals). Indirect Quotations from Journals. The abbreviations test., test. cit. and test, ips. are used with journal references ex- actly as described for book references. Unpublished Information. The ab- breviations in verb, and in litt. indicate that the author has received unpubhshed information either verbally or in a letter. Thus: V 12.2 Fant 1932 in verb. indicates that Mr. Fant told the author the unpubhshed composition of his seahng medium for glycerol mounts in 1932. .Slavonic Names Where the author has cited Slavonic names from a Cyrillic alphabet original, he has transliterated according to the rules of the Library of Congress (Beetle, Clara, ed. A. L. A. Cataloging Rules for Author and Title Entries. Chicago, Ameri- can Library Association, 1949, p. 246) without regard for the writer's preference as indicated in, say, a German summary of his paper. Thus Yasvoyn, not Jasswoin, is cited from the original. Slavonic names cited from a Latin alphabet original are transcribed directly even though this in- volves referring to the same individual by several names. Slavonic names cited at second hand are also transcribed directly, no matter how obviously they may have been mistransliterated. INTRODUCTION Citing Latin alphabet names from Cyrillic alphabet originals has involved even more uncertainty. Russian writers not only omit references but also follow varying rules of transliteration, some tak- ing a phonetic and others a Hteral ap- proach. The name Huygens, for example, can be transliterated into a Cyrillic form which can then be phonetically trans- literated in German as Geugantz. Thus Roskin 1946 attributes to an individual whose name may be transliterated Shteve, Sleeve, Stieve, or Stive a fixative which re- sembles, but is not identical with, the formula attributed, also without refer- ence, to Stieve by Romeis 1948. The author has given the one as Stieve test. 1946 Roskin — and the other as Stieve test. 1948 Romeis. Faulty scholarship can certainly increase the confusion originated by the architects of the Tower of Babel. Names of Dyes The author has, with one exception, changed the names of all dyes to accord with the synonym preferred by Conn 1946 (Conn, H. J. Biological Stains 5th ed. Geneva, N. Y., Biotech, 1946). The author prefers, however, to use the name magenta, rather than basic fuchsin, to describe the mixture of magenta 0, magenta I* ma- genta II now sold as basic fuchsin. There is no discussion of the chemistry and synonymy of dyes in the present work; reference should be made to Conn (op. cit.) . The author has not given certification numbers or dye content in formulas, since they are available for so few. Names of Reagents Other than Dyes The author has in almost all cases fol- lowed the usage preferred by The Merck Index 6th ed. Rahway, N. J., Merck, 1952. The terms chromic acid, osmic acid, and picric acid, though technicallj^ incorrect, are so universal in biological literature that they have been retained. Similarly the terms alcohol and absolute alcohol (ab- breviated in the formulas to ale. and abs. ale.) have been used in place of ethanol. Chemical names used are those custom- arily found on the label of the reagent bottle and are not accompanied by chem- ical formulas, or otherwise qualified, unless the reagent is found equally commonly in several forms. Thus copper suZ/aie indicates the usual reagent CUSO4.5H2O. On the rare occasions when reference is made to the anhydrous salt, it is referred to as copper sulfate, anhydr. In any case of doubt, reference should be made to the Merck Index. Proprietary Compounds Proprietary compounds of known com- position, such as amidol and salvarsan have been referred to by the name preferred in the Merck Index. Proprietary compounds of secret composition have no place in con- temporary science and have been ignored. It is fantastic that purveyors of reagents should be permitted to sell nostrums of secret composition, thus indicating a con- tempt for technicians equal to that shown by medicine men for the yokels they gypped with snake oil. The author would make it very plain that he does not extend this attitude to "brands" of mixtures or reagents selected for technicians' use. Every maker of microscope slides is in- debted to those firms which select and blend materials specially for his use. Quantities and Measures The abbreviations ml. and Gm. have been omitted. It is to be presumed that all liquids will be measured in millihters and all sohds in grams. Formulas have been adjusted to give a rational total (usually 100) in terms of standard ingredients, no matter how the original was presented. This has been wearisome labor applied to thousands of formulas. It is doubtless con- venient to make up a solution by adding fifteen drops of a 2.5% solution of this to 30 drops of a 1.25% solution of that and then to dilute to 15 milliliters wdth 30% alcohol. As a published instruction, how- ever, it does not commend itself to writers of textbooks struggUng to avoid duplication. Index The last section of the book is a single- alphabet, fully expanded, index, alphabet- ized according to the rules of the American Library Association (Beetle, 1949, op. cit.). These terms may require explanation. INTRODUCTION A single-alphabet index is one in which all entries are placed in the same index. There are not separate indexes for authors, stains, etc. Fully expanded means that more than one entry leads to the informa- tion sought. For example, "Grenadier's alcoholic-borax-carmine" may be found whether the reader consults the word "Grenacher," "borax-carmine," alcoholic- borax-carmine," or "carmine." A con- densed index, which saves the author work and the publisher money without regard to the reader's feehngs, has "borax carmine see Grenacher," " carmine staining solutions, see author's name," etc. etc. Those who do not think it necessary to have rules of alphabetization might try indexing del Rio-Hortega, 2BD fixative, CS-IS mountant, and van't Hooft. The rules of the A.L.A. {op. cit.) may not be perfect but they are at least clearly ex- pressed and easily available. Part I The Art of Making Microscope Slides Foreword to Part I The preparation of objects for micro- scopic examination — more colloquially known as "making microscope slides" — has a twofold purpose. On the one hand it may be desired to preserve in permanent form objects too small or too dehcate to be handled by the ordinary methods of mu- seum preparation. Second, and far more important, it may be necessary to make permanent preparations of objects and tissues in such a manner that their struc- ture may be more clearly seen under the microscope. In both cases, the object is mounted on a slide, which is nowadays a standardized 3" XI" strip of thin glass. Originally microscope slides were very different and were usually made by taking a sHp of ivory, about 2" X }i", and drill- ing through it a hole of about %" in diam- eter. This hole was then enlarged from each side, about a third of the way through, to a }'i" diameter, thus leaving a ridge of ivory in the center. The depres- sion on each side of the slide was fitted ^^ith a ring of spring steel and several disks of mica of a half-inch diameter were fur- nished with each sUde. To make a mount, a piece of mica was inserted from one side and held in place by the slip ring, the ob- ject was placed on it and another disk of mica was then inserted from the other side and, in its turn, held in by a slip ring. This was the only type of shde available until about the middle of the 18th century when glass shdes first made their appearance. These glass slides were, however, of very little use, with their talc covers which re- mained the greatest bar to the progress of microtomy. Toward the close of the first half of the 19th century Messrs. Chance, Birming- ham, England discovered how to make thin glass coverslips. They were for many years (Queckett 1855, 287) the only manu- facturers, and until the discovery of oil immersion objectives microscopists were entirely dependent upon the increasing thinness with which these glasses could be supplied. Microscopists were still seeking for magnification rather than resolution, and by 1880 (Beale 1880, 351) a coversHp had been made sufficiently thin to permit the use of a ^^o-inch "high dry" objec- tive. Coverslips are now taken so much for granted that the contribution made to the development of biology through the intro- duction of thin glass is often overlooked. The earliest method of using these thin coverslips with glass shdes was by holding the cover in place with the aid of a paper label which covered all the slide except the area immediately over the object. These labels were often fancifully engraved to the design of the individual technician and an excellent and well-illustrated descrip- tion of their use is to be found in Martin 1872, pp. 46-52. Microscope mounts, as made today, consist of three types. These are, first, wholemounts, in which organisms or pieces of organisms are mounted under a cover- slip on a slide; second, smear -preparations, in which either a cut surface or a viscous fluid is smeared on a shde to form a thin layer which is subsequently preserved under a cover glass; third, sections, in which thin shces of objects are mounted under a coverslip. Where objects are cut into a series of sections, each of which is mounted in consecutive order on a slide, the preparation is known as a serial section. The simplest slide to prepare is that in which the object is mounted dry. An ex- ample of this is shown in Fig. 1 where a series of diatoms have been spread on a slide, and a covershp placed over 'them. This coversHp is held in place by a ring of cement which is prevented from running under the edges of the covershp by some 8 THE ART OF MAKING MICROSCOPE SLIDES Figs. 1 to 6. Types of microscopical preparation. 1. Dry wholemount of diatoms. 2. Freshwater hryzoan in deep cell of formaldehyde. 3. Crustacean in oval cavity in glycerol jelly. 4. Smear preparation. 5. Single section of plant stem. 6. Serial section of embryo. FOREWORD TO PART I 9 form of thin cell, which may be either of cement or paper, and which servos the additional purpose of preventing the crushing of the object by the coverslip. Most wholemounts are, however, pre- pared in a preservative medium, wliicli may be either aqueous, colloidal, or resin- ous. Many of these whole objects are rela- tively thick so that some method must be adopted of ])roviding space for them under the coversUp. Fig. 2 shows an object mounted in a deep cell of glass, while Fig. 3 shows an alternative method in which a relatively thick shde has had an oval cavity ground into it. As will be seen from the figure these mounts are heavily var- nished at the edges to prevent the evap- oration of fluid or the A\ithdrawal of water from the colloidal medium. Wholemounts prepared with | 'resinous media, w'hich harden and thus hold the coverslip in place, are frequently not varnished at the edges though some case can be made out (Gray 193G, Microsc. Rec, 38) for the ap- phcation of a ring of varnish around the edge of balsam mounts. Smear preparations (Fig. 4) are almost invarialily prepared in resinous media and equally invariably the edges of the covcr- shps are not varnished. Sections (Figs. 5 and 6), either single or serial, are univer- sally mounted in resinous media and the edges of the covershp are practically never varnished. Dry Wholemounts General Principles A dry wholemount consists essentially of an object or objects enclosed within a small, usually cylindrical, box attached to the center of a microscope slide. The floor of this box is almost invariably the surface of the slide itself while the roof is formed by the coversUp. The sides of the box are produced by the attachment of a cell, which may be a thin ring of cement, a washerlike piece of paper or plastic, or a squat cylinder of the same materials. The object may be attached directly to the glass surface of the sUde, if one desires to make a transparent mount, or the surface of the shde may be rendered opaque and the object then attached to whatever sub- stance is used to blacken the surface. There are a number of decisions to be made before preparing a dry wholemount. The considerations governing these deci- sions will be discussed in the order in which they present themselves to the technician. Selection of a Slide If the object is to be prepared as a trans- parent wholemount one has no choice but glass. None of the transparent plastics at present available have a sufficiently hard surface to be worth using. They are un- breakable and easy to handle when first made, but will become so scratched after even a few months of use as to be worth- less. The manufacturers of these slides point out that they may be repohshed at intervals, but there seems little point in preferring them to glass which does not become scratched. If the wholemount is to be prepared as an opaque object, which is the case in probably 90% of all dry wholemounts, there is little justification for using glass. It has two great disadvantages: first, it^is very easily broken; second, it is one of the most difficult materials to which to cause adhesives to stick. In the early days of microscope mounting it was customary to employ slides of well-seasoned mahogany and, though this practice is today confined to the mounters of Foraminifera, a brief description of the preparation of these shdes will be given. A piece of seasoned mahogany, 3" by 1" in section, is secured with the grain running parallel to the three-inch face. This block is set up on end in a vertical drill and a hole of the required diameter drilled as deeply into it as is possible with tools available. This hole should be about Ke of an inch smaller than the size of the coversUp which will be used; that is, if ^^-inch coversUps are customary an i He-inch drill is used to make the hole. The actual size of the covershps should be checked before drill- ing, since many coverslips which are sold as %-inch have a diameter of eighteen millimeters, or a trifle less than '*%4. When the hole has been drilled, the block of wood is transferred to a circular saw and slices about J^e of an inch in thickness cut from it. These slices are, in effect, 3" XI" microscope shdes with a hole of the required size in the center. A sheet of strong, thin card is then cut into 3" X 1" pieces, each of which is glued to the under side of one of the shdes. The best way to do this without warping the wooden strip is to use shellac as an adhesive, either em- ploying a very thick solution in alcohol and permitting it to dry under pressure, or 10 Coverslips DRY WHOLEMOUNTS 11 coating the sheet before cutting with the thick solution which is allowed to dry and then pressed under heat onto the lower surface of the slide. Sheets of photog- rapher's dry mounting tissue can be used for the same purpose, if a photographer's dry mounting press is available. This com- pletes a wooden microscope slide with a built-in, white-bottomed, cell. If a black bottom is required a disk of black paper is punched, coated with ordinary starch frame, the flanges of which are sufficiently deep to allow a thin slide to be slipped in as a cover. Selection of a Coverslip The chief difficulty in selecting a cover- slip for a dry wholemount using a deep cell is to find one which is thick enough. The only value attaching to very thin coverslips is that they permit the use of high power objectives. Most dry whole- Fig. 7. Turning a ring on a slide. paste on the underside, and pressed into position at the bottom of the hole. Numerous variations on slides of this type are possible. To mount a number of small objects on the same slide it is only necessary to drill a number of small holes at the end of the wooden slab and thus secure a slide with as many built-in cells as is required. Special shdes for Foraminifera are made where large collections are to be mounted. These consist of 3" X 1" slips of black card on the central two-thirds of which are printed 60 numbered divisions. At each end a section of thick card is glued on, so as to leave the black portion in the form of a shallow rectangular cell. The card so prepared slides into an aluminum mounts are used with low power objec- tives. The thickness commercially sold as No. 3 is the thinnest which should be con- sidered, unless high powers are certain to be used. Selection of a Cell A cell on a dry mount serves mainly to support the covershp, and the thickness required is therefore the primary con- sideration governing selection. Where the object is only a few hundredths of an inch in thickness, as in the case of diatoms or the smaller Radiolaria, a cement cell is the simplest. For a dry mount it is difficult to find a better cement than "gold size" and the preparation of a cell with this medium 12 THE ART OF MAKING MICROSCOPE SLIDES Cells will therefore be described. Cement cells are made with a turntable in the manner shown in Fig. 7. Turntables are of many- patterns but consist essentially of a rest for the hand and of a rotating circular plate bearing chps to hold the slide. These plates have the center marked, usually with a series of concentric rings engraved round it. The center of the shde must first be marked, and this is readily done by placing the slide on a sheet of paper, running a pencil around its edges, and then drawing the diagonals of the rec- tangle so formed. The shde is replaced on the rectangle and a dot made with India ink at the point immediately above the intersection of the diagonals. The shde is transferred to the circular plate of the turntable with the dot over the central point of the plate. The cement ring should be of the same diameter as the covershp and one of the circles on the plate may be used as a guide; if there are no guide hues, a ring should be marked on the underside of the slide of the same diameter as the covershp to be used. The brush is charged with the cement and the table spun quite rapidly by means of the milled ring shown in Fig. 7. It is safer to use this milled ring than to use the edge of the turntable be- cause the shde frequently projects slightly beyond the edge and may be tapped off center with the finger used for spinning. The charged brush is brought slowly down over the marked ring and held in contact with the spinning shde so that a circle of cement is drawn on the glass face. Re- member that you are not painting a thin ring of varnish on the slide; you are en- deavoring to build up a relatively thick layer of cement by allowing it to flow from the brush to the shde. The hairs of the brush should never touch the glass itself ; only the cement should touch the glass and thus be drawn off. In making a dry wholemount it is not very important how wide the ring is, but a %6-inch-wide ring for a %-inch covershp will be about cor- rect. As many shdes as are likely to be required are prepared at one time and may be left to dry indefinitely. Cells prepared with an ordinary sample of good gold size are safe to use after about 24 hrs. Building up a thick ring of cement by the applica- tion of successive coats is rarely satisfac- tory. A gold-size ring prepared in the manner described will have a thickness between one- and three-thousandths of an inch. If thicker rings are required, it is better to use cells made of paper, card- board, tin, or plastic. Paper rings are stamped from a sheet of the required thickness (a good quality bond paper runs from three- to five-thou- sandths of an inch) or from Bristol board (6- to 12-thousandths of an inch) or from cardboard (up to a thickness of about Ke of an inch). The best board to use is the dense black bookbinder's board (once known as millboard) since cheap yellow strawboards have such a rough surface that they can be made to adhere only with difficulty to a glass slide. SheUac is a good adhesive for attaching paper, or thin card, to a glass slide. A sheet of bond paper is coated on one side with com- mercial sheUac varnish and then dried. Rings of the appropriate size are stamped from this shellac-coated sheet, either by using two punches successively, or with a double punch. The outer diameter of the cell should be larger than that of the covershp, while the inner diameter should be less, so that when the cover is laid in place there wiU be an appreciable overlap of paper both inside a-nd outside. A large number of these stamped rings may be cut at one time. When required for use, one is centered on the shde and then pressed into place with a hot iron, raised to a temperature which will melt the sheUac. When the shde has cooled, it is turned upside down and observed with light reflected from it at an angle. If the cell is perfectly attached no adjustment of the angle of observation will produce mirrorhke reflections from the underside of the cell; if any considerable area of the underside of the cell shows mirrorhke re- flections, it is not properly attached and had better be rejected. Cardboard cells more than He of an inch thick are not satisfactory, for they are so porous that they admit moisture in humid weather and allow fungus growth on the specimen. The outside of the ceh may, of course, be covered with some waterproof cement, but this makes a clumsy looking mount and it Backgrounds DRY WIIOLEMOUNTS 13 is better to substitute either a plastic or a tin cell. Most plastic cells seem to be stamped out of vulcanite, though there is no reason why the numerous other plastics available today sliould not be used. It is almost im- possible to punch cells from sheets of plastic more than He of an inch thick without special machinery, so that it is better to buy them than to prepare them one's self. Excellent cells may, however, be prepared by anybody in possession of a lathe by buying extruded tubing, readily available in many types of plastic, and cutting from it lengths of the appropriate thickness. Opaque plastic should never be used to prepare cells more than i^g of an inch high, since the opaque wall interferes with the illumination of the contained object. Homemade cells are better pre- pared from tin than plastic, since an ordinary hammer and punch may be used to cut sheet tin, or sheet pewter, up to a thickness of nearly }i of an inch. When cells have been punched from sheet, rather than turned from a tube on a lathe, they will be found to have a turned-down edge where the punch came through. This edge must be removed before they are cemented by placing the cell on a sheet of fine sand- paper— sandpaper blocks sold for sharpen- ing draftsmen's pencils are excellent — and rubbing it backward and forward until a visual inspection shows that all the under- surface has come in contact with the abrasive. Cementing of cells to glass depends far more for its success on the cleanhness of the glass than on the cement selected. Gold size has the tremendous advantage that it will adhere firmly to slightly dirty glass, but it has the disadvantage that three or four days are required before it is sufficiently firm to continue mounting. If one is prepared to take the trouble to clean the glass thoroughly, almost any of the cements given in Chapter 28 under V 11.2 may be employed. To make a firm bond with liquid cements it is best to turn a ring of the cement in the appropriate place on the slide with a turntable, and to apply a thin coat of cement to the under- side of the cell. When both these coats are dry another thin coat is applied either to the slide or the cell. The two are then pressed together and dried under pressure. An easy way to apply this pressure is to take a 500-gram brass weight, which is usually to be found somewhere about the laboratory, and lay it carefully on top of the cell; or one can place another slide on top of the cell and clip the two slides to- gether with a strong spring paper clip. Whatever method, or cement, is em- ploj^ed, each slide must be inspected after it is dry to make sure that it is adhering perfectly over all, or almost all, of the base ; minute air holes will admit moisture and lead to molding of the contained speci- men. If the cell has been fixed with a cement under pressure, a small quantity of surplus cement will almost always have been extruded both outside and inside at the point of contact of the ring. That which has been extruded outside should be left in position, unless there is a great deal too much of it, but the material inside should be carefully scraped off with the edge of a scalpel. It is most unwise to endeavor to remove this cement with a solvent which is hkely to loosen the cell. Selection of a Background No background other than the glass it- self is necessary when the object is to be prepared as a transparent wholemount; but many objects prepared as a dry whole- mount are better displayed against a black or colored background. This background may either be a varnish apphed to the bottom surface of the cell, or a disk of the appropriately colored paper cemented in position. The best black paper is that used to wrap photographic plates, but colored papers of all tj^pes are available. The paper is punched into disks which are at- tached to the bottom of the cell with any adhesive. They will not be subjected to any strain, and office mucilage, or any of the formulas given in Chapter 28 under V 11.1 will be found satisfactory. If a black background is to be painted in place, the most satisfactory paint is the optical dead black, listed by some scien- tific suppHers or available occasionally from scientific instrument makers. The only one of these which may be prepared at home is the formula of Martin 1872 14 THE ART OF MAKING MICROSCOPE SLIDES Cements given in Chapter 28 under V 13.1. This is an excellent dead-black cement but, since it has a gold-size base, is very slow drjdng. The best colored backgrounds are the old wax and resin formulas, particularly those of Martin 1872, Oschatz 1842, and Mende- leef (1942); the formulas for these are in Chapter 28 under the heading V 12.2. These media have the advantage that they are thermoplastic so that they may be used both to provide a smooth back- ground and to secure the adhesion of the object to the bottom of the cell. They must be applied molten, and this is readily done if the circular stage of the turntable is heated while a small quantity of the cement is melted in a capsule. The cement is then apphed with a brush exactly as though it were a varnish and permitted to cool. These colored cement back- grounds were widely used in the old days and should receive more attention than is at present the case. Selection of a Cement to Hold the Object in a Cell This is the most difficult, as well as the most important, of the decisions which have to be made. A well-prepared dry wholemount should not have any visible cement obscuring the object, but the ob- ject must at the same time be so firmly held that it will stand the relatively rough handling to which most slides are sub- jected. If the sUde is to be mounted as a transparent wholemount, there is nothing, in the author's opinion, which can com- pare with gum tragacanth; and a simple dispersion of this gum in water, with the addition of some preservative such as thymol, is better than any of the more complex formulas. Tragacanth has the useful property of being transparent in thin films, but these thin films are not strong enough to hold objects larger than diatoms or butterfly scales. To use this gum one takes the slide on which the selected cell has already been prepared and turns, with the aid of a turntable, a very thin uniform layer on the bottom of the cell. The preparation of this thin uni- form layer requires experience and skill for which no description can substitute. The adhesive is then allowed to dry and the objects are arranged on it in the re- quired positions. As soon as all the objects have been laid on the dry film, it is placed in a moist, warm atmosphere wliich is usu- ally secured by bending open-mouthed over the shde and breathing very slowly and carefully. This makes the layer of tragacanth sticky so that the objects ad- here; it will dry again in a few moments. This was the method used to prepare those pictures, made with the scales of butterflies, or selected diatoms, which used to be a feature of the catalogs of old- time microscope preparers. There is, how- ever, no reason why the method should not be employed for scientific purposes, for it is often desirable, particularly when dealing with diatoms, to arrange them in a selected pattern. Small objects of this type may readily be placed in position if they are picked up on the end of a hair attached to a needle-holder; if they do not stick to the hair at the first trial, it is only necessary to moisten it with the hps. Gum tragacanth may also be used to attach larger objects (such as Foraminif- era and Radiolaria) to a paper background but it will not stick satisfactorily to either a resinous or wax surface. For these larger objects it is necessary to take a very small sable brush and place a drop of tragacanth on the surface of the paper background which has previously been moistened. The individual object is then picked up and pressed into the surface of the drop, which is then allowed to dry. If the appearance of the preparation is not very important a fairly thick smear of the mucilage may be placed all over the paper and the objects sprinkled on; this gives, however, a clumsy and unfinished appearance. The principal objection to the use of optical-dead-black varnish as a back- ground is that aqueous adhesives will not adhere to it. Both gum arable and gum tragacanth will stick for a certain length of time, but the author has never known a mount made with these adhesives in which the object did not loosen within a period of two or three months. Nothing is more annoying than to take the trouble to make a mount of ^elected foraminiferans and then, a few months later, to find one or two specimens rolling about inside the Cements DRY WHOLEMOUNTS 15 cell. If you are using an optical-dead-black based on gold size, clear gold size itself is th*e best cement. A series of fine drops of gold size are placed in the positions which the objects are subsequently to occupy, and the objects added one after another. This will result in perfect adhesion, but the sUde will have to be left uncovered and flat for at least two or three days, to harden the gold size before the cover is attached. When using an optical-dead- at a relatively high temperature and all too often this high temperature causes the dead-black cement to break away from the glass. When using these cements, a piece about one third of the size of the object which it is desired to attach is broken from the mass. These pieces of cement are placed on the bottom of the cell in the positions which the objects will occupy and the slide placed on a hot table to melt the cement. The author prefers Fig. 8. Using a warm table to attach cells with marine glue. black cement secured from a scientific supply house, it is necessary to secure some of the varnish medium in which the black has been suspended. The writer has seen hundreds of commercially prepared strewn sHdes of Radiolaria and Forami- nifera in which gum arable had obviously been used on black varnish backgrounds and in which from a third to a half of all the specimens were loose. As a second choice to the varnishes, there may be em- ployed one of the Canada-balsam-resin cements of the type of Fant 1932 (Chapter 28, V 12.2), or one of the Venice-turpen- tine cements of the type of Gage 1896. Un- fortunately these cements have to be used the type of table shown in Fig. 8 which consists of a strip of heavy metal, prefer- ably copper, bent back twice on itself and mounted on four legs. The top of the upper strip projects beyond the bent por- tions. A burner is placed under this pro- jection and adjusted to keep the end of the strip well above the boiUng point of water. It will be seen that the temperature steadily diminishes from shelf to shelf, that of the upper shelf being highest, that of the second shelf being lower, while the bottom shelf is scarcely warm. To deter- mine whereabouts on the shelf to place the slide it is only necessary to place a few chips of cement at about one-inch inter- 16 THE ART OF MAKING MICROSCOPE SLIDES Cements vals along the first and second shelves. After a few moments it will be apparent which point is just at the melting point of the cement in question. The slide bear- ing the small pieces in the position where the objects are to be mounted is then placed at this point and each object is individually placed in its own little pool of molten cement. It must be left at this temperature long enough for the object to reach the temperature of the cement or it will not stick. It is best not to cool these preparations suddenly, so that it is the author's practice to take them from the hot shelf on which the cement is molten, remove them to the shelf under- neath, and after they have cooled to that temperature to place them on the bench for their final cooling. For attaching opaque objects, particu- larly those of relatively large size, nothing is simpler than the wax-resin backgrounds which have been mentioned. In using these, a fairly thick coating is applied to the warmed slide and the object dropped into place. It is left until it has reached the temperature of the cement, or the cement is seen to be "creeping," and is then cooled. These media are more fre- quently employed by botanists for mount- ing dried-spore cases of mosses, and the like, but they also work admirably for zoological specimens. Selection of Cement for Attaching the Cover Glass Before discussing the selection of a cement for the attachment of a cover glass, which is the last step in the preparation of a dry wholemount, it is necessary to insert a warning that the word dry as applied to dry wholemounts must be interpreted literally. If wholemounts are being made in an American laboratory in winter at an inside temperature of 70°F. and an out- side temperature around 0°F., no diffi- culty will be encountered since the atmos- pheric humidity is practically nil. If, how- ever, the humidity is relatively high some method of drying the specimen must be used. The writer has in his possession several imperfectly sealed dry whole- mounts of ground sections of bone, made in Europe about forty years ago, which are entirely covered with fungus hyphae. The object may, it is true, be treated with some fungicide but this is rarely as effec- tive as, and usually more trouble than, making sure that the mount is dry before sealing. If the object has been attached by one of the techniques which involves heat- ing the slide and cement, it will probably have dried sufficiently, but it is desirable to make sure by leaving the uncovered mount overnight in a desiccator over some standard desiccant. When a coversUp is to be attached to a gold-size or other cement cell, it is best to use the same cement as was used in the preparation of the cell. A thin coat of this cement is applied to the top of the cell and left until it becomes tacky. A clean cover- slip is then placed on top and firmly pressed into position with a needle. It is easy to see whether adhesion is perfect and, if necessary, a small quantity of cement may be added from outside. It must be emphasized that only a very thin coat should be used because a thick layer will inevitably run in by capillary attrac- tion and thus ruin the specimens which have been mounted. It is really not important what cement is used when a coverslip is to be attached to the top of a paper, cardboard, or plastic cell. The author invariably uses gold size, largely from force of habit, but any liquid cement or varnish is adequate. A thin layer is painted on the upper surface of the cell and the coverslip pressed into place. The preparation should now be placed on one side luitil the adhesive is dry, and then finished with a coat of some black cement. Asphalt varnish [Benoit- Bazelle (1942) is an excellent formula] or Brunswick black (Beale 1880) both have the required characteristics of providing a waterproof seal while retaining a certain amount of flexibility. These formulas, and those of other suitable cements, are given under V 12.2 in Chapter 28. The old seal- ing-wax varnishes, and the modern cellu- lose-ester varnishes have the disadvantage that they tend to become brittle and break ofT after some years. Foraminifera DRY WIIOLEMOUNTS 17 Specific Example Preparation of a Strewn Slide of Foraminifera or Radiolaria Wholemoiints of the dried tests of Foraminifera, either fossil or I'cccut, are customarily referred to as strewn slides even though the individual tests may be arranged in place. Tests of Foraminifera may be obtained either from sand, from marine sludge, or from fossil deposits, and the method by which the shells are sepa- rated is in each instance different. Radio- laria are almost invariably obtained from fossil deposits. Forminiferal sands, which may be pur- chased or collected, are the best source of material. Large numbers of shells are thrown onto beaches in many parts of the world where they form whitish ridges. If such a ridge is observed it is only necessary to scoop off the surface with a spoon and to preserve it for further examination. Many scientific supply houses sell these sands. The separation of the dried shells from the sand is relatively simple. The whole may either be sprinkled onto the surface of a large vessel of cold water, in which case the majority of the shells will float, since they are filled with air; or carbon tetrachloride, the high specific gravity of which ensures that Foraminif- eral shells with only a small quantity of air enclosed will rise to the surface, may be substituted for water. If only a few shells are required they may be picked from the surface of the flotation medium with a brush and laid to dry on a disk of fine filter paper. If all the shells are re- quired, the surface layer containing the floating shells should be poured off through a fine sieve. Bolting silk is the best mate- rial from which to prepare this sieve, though fine brass screen wire may also be used. The separation of tests from marine deposits dredged from the bottom is not so easy since they are, in this case, mixed not only with particles of sand but also with considerable quantities of fine sludge which may include some organic matter. If the sludge is free from organic matter, the mass may be passed through a series of sieves under a jet of running water; but even under those circumst:iiices the tests will usually be discolored. Laporte 1946, p. 194 recommends that such tests be boiled in an alkaline solution of calcium hypochlorite {eau de Javelle) which serves the double purpose of bleaching the stains and removing any trace of organic matter which may remain. If the sludge is heavily contaminated with organic matter, the mass should be boiled for some time in a weak (2%) solution of sodium or potas- sium hydroxide before being sieved. Cush- man 1940, p. 26 suggests also that tests may be separated as the old gold miners separated gold by rotating the mass in a flattened dish. The Foraminifera, being somewhat lighter than the rest of the material present, will collect round the edges of the dish, from which they may be jerked with a circular motion. Tests of fossil Foraminifera may^be ob- tained from sandy deposits. They are also found embedded in clay deposits, or chalk. Foraminifera concreted in limestone can- not, in most cases, be removed and made into wholemounts. Foraminiferal tests ob- tained from sandy deposits may be sepa- rated by flotation in the manner already described, but these shells are usually dirty either from chalk or clay, and must be thoroughly cleaned if a satisfactory mount is to be prepared. It is best to boil them after they have been separated in a 5% solution of sodium carbonate. After they have boiled for some time the beaker containing them is removed from the flame and the Foraminifera are al- lowed to settle to the bottom. The cloudy alkahne solution is then poured off and re- placed with fresh solution and this is re- peated until the tests are sufficiently clean. If the dirt is particularly tenacious, it may often be loosened by boiling the tests in a relatively small quantity of the alkaline solution which is then poured while boiling over a mass of cracked ice. This sudden temperature change will often loosen dirt which cannot be removed by jg THE ART OF MAKING MICROSCOPE SLIDES Radiolaria any other method. When tests are cleaned blown off to diminish the pressure as in this manner it is essential that they rapidly as possible. The repetition of this should be thoroughly soaked, and prefer- process resu ts m the disintegration of ably also boiled, in a large quantity of materials which resist every other method, distilled water to remove the alkah. The separation of f oramini eral tests Methods for the separation of forami- from chalk is a relatively simple process, niferal tests from shale and clay deposits If one is only collecting the tests at ran- vary according to the degree of hardness dom, so that i does not matter if many of of the deposit The first exploratory step the more fragile forms are broken, the old should always be to boil the mass in a 5 % method of brushang under water has much solution of sodium carbonate. If the to recommend it. A piece of chalk is held solution speedily turns cloudy, it is in one hand under he surface water and a evident that the material is being dis- brush (an old tooth brush is excellent) is integrated satisfactorily and it is only scrubbed over the surface. L^^ge numbers necessary to continue boiUng long enough of tests, which fall to the bottom of the for the tests to separate. The cloudy solu- container, are removed by this method tion should be stirred up and poured off while the chalk remains m suspension and from time to time into a large cyhnder of can be poured off. Tests prepared by this distilled water. This should be allowed to method are never clean and must sub- stand for about 10 minutes, to permit all sequently be boiled in alkah to remove the foraminiferal tests to fall to the the adherent chalk. If it is desired to col- bottom and the cloudy supernatant lect the greatest possible number of shells, hauid then poured off. This may be re- chalk can often be disintegrated by boihng neated as often as experience shows to be either in 5% potassium hydroxide or m necessary to collect a mixture of forami- 5% sodium carbonate; this is, however, a niferal tests and fragments of the shale prolonged and messy business Chalk may mass at the bottom of the cyhnder. Sepa- also be disintegrated by the freezing and ration of the tests from the shale frag- thawing process, or by the autoclave proc- ments may either be by hand under a ess already mentioned. ^ ,. . . binocular microscope, or the mass may be The sihceous skeletons of radiolarians dried in an oven and sprinkled on cold are cleaned altogether differently and it water, or carbon tetrachloride, for the is difficult to improve on the method of flotation method previously described. If Roudabush 1938 {Ward's Bui 9). No the preUminary boiUng in sodium carbon- prehminary treatment is needed for ate does not result in a sufficiently rapid the easily disintegrated Barbados earths disintegration, two possible methods re- though other material may have to be dis- main The old method used to be to soak integrated by one of the methods already the mass thoroughly in water and then to described. The disintegrated pieces are reez^Tt; a household freezer giving tem- then boiled in 10% potassium hydroxide oeratures of -10° or -20°C. is excellent, for about 20 minutes. Throw the whole The frozen piece is then removed and mass into 10 times its own volume of thrown into boiUng water which almost water, stir vigorously, and allow to settle invariably breaks the mass into smaller for 10 minutes. Pour off the milky solu- Dieces This process of alternately freezing tion; refill the beaker with water, btir and boiUng is continued until the pieces vigorously, allow to settle for 15 seconds have become sufficiently smaU to enable and save the supernatant hquid. Repeat one to complete the separation of the tests the process; the two batches of decanted with boiUng alkaU. water will be found to have most of the An interesting alternative method for hberated radiolarians. , , ^, . shale has been suggested by Driver 1928 The pieces remaining at the bottom ot (J Pal 1 -253) who subjects the pieces to the beaker can be again boiled witti iu /o the action of high pressure steam in a potassium hydroxide and further batches laboratory autoclave. The pressure is run of liberated radiolarians poured off and up to about 20 lbs., maintained at this for accumulated, a few minutes, and the autoclave then The cleaned radiolarians in the ac- Foraminifera DRY WHOLEMOUNTS 19 cumulated decantations are allowed to settle for about 20 miinitos after the last batcli has been added and the suj)ernatant water poured off. Add carefully about twice as much nitric acid as there is sludge and boil for 20 minutes. Again wash with water, allow to settle antl decant. Now carefully add twice as much 10% potas- sium hydroxide as there is sludge, boil for 20 miiuites and again wash by decanta- tion. The material is now nearly clean — if it is not, repeat the nitric acid-potassium hydroxide cycle. Concentrate the nearly clean skeletons and then cover them with their own vol- ume of ammonium hydroxide. Stir at intervals for about 10 minutes, then slowlj^ add an equal volume of nitric acid. Wash thoroughly by decantation and ex- amine the skeletons; if they are not now clean, repeat the ammonium hj-droxide- nitric acid cycle. Concentrate the sludge as much as possible, wash it thoroughly with 95% alcohol, and then either allow to dry for strewn slides or store in alcohol and make balsam mounts in the manner described in Chapter 6. After the foraminiferan tests or radio- larian skeletons are accumulated in a small watch glass, they should be dry and quite free from any of the reagents used to clean them. It is also necessary to have ready on the bench a binocular dissecting micro- scope, two fine sable brushes, sHdes on which cells have already been cemented, and a container of mucilage of gum tragacanth. The author prefers to make all forami- niferal mounts in cells prepared from rings of vulcanite with bottoms of black paper. These should have been prepared the day before in the following manner. First take the required number of slides and clean them thoroughly. It is not nec- essary for the slides to be chemically clean — it is necessary only that the slide should be grease-free. A simple method of de- greasing slides is to take a commercial scouring powder, of the type used for household purposes, and make it into a paste with water. This paste is smeared Hberally on all surfaces of the required number of slides which are then dried. When the shde is dry, the scouring powder is removed by vigorous rubbing with a soft cloth. While the slides are drying, the retpiired number of vulcanite cells are laid out and a piece of fine sandpaper secured. Each cell is held on the sandpaper with the ball of the first finger and rubbed, with a circular motion, until all the lower surface has been abraded. The cell is then turned over and the process repeated. One side of the cell is then given a thin coat of gold size and placed with firm pressure on the center of a glass shde, which should then be left for three or four days. Any gold size which has been pressed out of the inner surface should be removed with the edge of a sharp pointed scalpel. A number of disks of black paper of the required size are then taken, coated on one side with any satisfactory adhesive, and pressed onto the bottom of the cell. It must be remembered that the cell should be of such a size that the coverslip, when laid on top, does not reach to the outer edge of the cell but only halfway across it. This may conveniently be done by using a %-inch cell with an 18-milhmeter covershp. The thickness of the cell selected is not of major importance but the writer usuall}^ prefers about }i2 of an inch when mount- ing Foraminifera. Let us suppose first that it is desired to prepare, from the materials at hand, an ordinary strewn shde hke those sold by biological supply houses. It is only neces- sary to moisten slightly the paper at the bottom of one of the prepared cells and to smear mucilage of tragacanth liberally over the surface. Plenty of tests are thrown onto the mucilage and the shde is placed on one side for about 10 minutes to dry. As soon as the mucilage is dry, the slide is inverted over the watch glass con- taining the tests and tapped sharply with the forefinger. This will cause all those tests which have not become attached to the mucilage to fall back into the stock, and will usually leave a continuous coat of foraminiferal tests over the black paper. It is generally, however, more satis- factory to mount selected tests in the re- quired position. In this case, the black paper on one of the prepared slides is thoroughly moistened and a fine sable brush is used to place small portions of mucilage of tragacanth in the positions which the selected tests are to occupy. 20 THE ART OF MAKING MICROSCOPE SLIDES Foraminifera Each drop of tragacanth should be slightly smaller, both in breadth and in thickness, than the test which is going to be placed on it. These drops of mucilage are most conveniently placed in the correct position with the aid of a binocular dissecting microscope. As soon as the drops have been placed the slide is pushed out of the field of the dissecting microscope and the watch glass, containing the specimens to be mounted, pushed into the field. A clean sable brush is then moistened with the lips. It should be sufficiently wet to cause the tip of the brush to come to a point but not suf- ficiently impregnated with saliva for any liquid to be showing. The tip of this brush is touched down onto the required speci- men and held in the field of the dissecting microscope with the right hand, while the left hand pushes the watch glass of speci- mens out of place and replaces the glass slide. It cannot be emphasized too much that the paper must be liberally moistened or the drops of gum tragacanth will dry in the period of time that it takes to select tests. If, when the cell is replaced under the binocular microscope, it is ob- served that the mucilage is dry, do not at- tempt to remoisten it; place another drop of mucilage on top of the dry portion. The shell, on the tip of the fine brush, is now pressed down in the selected position. If it is not exactly as required, it may be adjusted with the tip of a needle. After aU the tests required on any one slide have been placed in position, a fine sable brush is used to place a drop of water on top of each shell. This makes certain that there will be a perfect adhesion of the shell to the underlying mucilage. On a very dry day (that is, one on which the relative humidity is below 20%) the preparations may be sealed immediately; on humid days it is best to place the shdes in a desiccator overnight. In either case, the next step is simple. The top of each cell is spread with a very thin coat of gold size and a clean coverslip dropped into position. It is best to place one edge of the coverslip down first, supporting the other edge with a needle, and then to lower it by withdrawing the needle. As soon as it is in contact with the cell it is pressed down all round its circumference with a needle. It is not important that it should be in contact all over since further coats of cement will be placed on top. The initial coat of gold size is intended only to hold the coverslip in position through the next stages and it is better to have a very thin coat with an imperfect adhesion than to have a coat so thick that cement spreads onto the inner surface of the covershp. The slides, with their attached covers, are then placed on one side, preferably in a desiccator, for a period of about 24 hours to set the gold size. The shde is finished on a turntable (Fig. 7) by turning onto the upper surface of the cell a coat of any selected cement. The author prefers either asphalt varnish, or Brunswick black, though any tough and flexible cement may be used. It is quite important that the cell should be accurately centered on the turntable, and though this may be roughly done with the aid of the concentric circles engraved on the table, it is usually neces- sary to spin it once or twice and to make necessary adjustments manually. If lack of experience renders this difficult, it is suggested that a needle should be held stationary above the edge of the cell and the turntable rotated slowly. The table should be stopped when the edge of the cell (presuming it to be eccentrically placed) is at the maximum possible dis- tance from the needle. The cell is then pushed one half of this distance towards the needle, the needle replaced over the edge of the shde, turned as before, and readjusted. By this method even the most inexperienced can center a cell perfectly within a few moments. Only one coat of varnish is really necessary, though some people prefer to put on four or five, using the last coats to fill up the edge of the cell which is thus doubly protected. In the author's opinion this is not necessary and makes a clumsy mount. If, through accident, a test becomes detached in one of these slides it may be repaired easily. The coverslip should be broken by a sharp blow with the handle of a scalpel and the pieces removed with a pair of forceps. The top of the cell is then scraped clean with a scalpel and the test recemented in place. Fluid Wholemounts — Aqueous Type General Principles A fluid wholemount in an aqueous me- dium is essentially a miniature museum mount in which the glass jar has been re- placed by a cell mounted on a microscope slide. With the exception of the selection of a slide — for none other than glass is suitable — the choices confronting the technician are very much those discussed in the preparation of dry wholemounts in the last chapter, though the selection is in each instance different. It is necessary to select successively a type of cell, a cement for attaching the cell to the sUde, a cement for attaching the coverslip to the cell, and finally the mounting medium itself. Selection of a Cell The author prefers to use concave- ground glass slides instead of cells. At the present time these concave glass shdes are both difficult to obtain, and unsatis- factory when obtained, from American sources. It is, however, possible to secure in Great Britain slides into which have been ground circular concavities from 9 to 20 milhmeters in diameter, or oval con- ca\'ities in many sizes. The use of cavity sUdes avoids the difficulties of attaching a cell, and the slides are more waterproof than any cell. It is to be hoped that Ameri- can suppliers will make cavity slides avail- able to technicians who wish to make wholemounts in fluid media. If, however, cells must be used, the choice is very hmited. It is a waste of time to take paper and cardboard cells and to endeavor, by soaking them in various resins, to make them take the place of a plastic or metal cell. Cells of vulcanite anb tin are obtainable or may be pre- pared with the aid of a punch. These should, before use, be flattened on both sides in the manner described in the last chapter. Where a very deep mount is re- quired, it is better to use a glass cell which can be cut from thick-walled glass tube and ground flat on both faces. These cells are usually only obtainable in a %-inch size and care should be taken, as with other cells, to make sure that the edges of the coverslip selected will he on the sur- face of the cell. It is difficult to seal a dry wholemount, and impossible to seal an aqueous wholemount, in which the edge of the coverslip and the edge of the cell coincide. An almost perfect relationship is that of an 18-millimeter covershp to a fi-ineh cell, but unfortunately both 18 mm. and 3-^-inch appear to be used interchangeably by scientific supphers so that the measurements must be checked before mounting. When very thin objects are to be mounted, a cell can be made from gold size in the manner described in the last chapter. Selection of a Cell Cement The selection of a cement to attach the cell to the shde is of far more impor- tance in aqueous fluid mounts than in dry mounts. The cement must not only lje capable of holding the cell firmly to the glass, but must also make a waterproof seal which must remain waterproof for many years. In the author's experience no varnish is satisfactory, and one is forced to turn to the thermoplastic cements. Among these marine glue (Chapter 28, V 12.2 Beale 1880) or, if this is not obtain- 21 22 THE ART OF MAKING MICROSCOPE SLIDES Cements able, the very similar cement of Harting 1880 are the best. The marine glue here specified bears no relation to the so-called marine glue commonly sold today. The old-style marine glue, which is essentially a mixture of rubber (or gutta-percha) with shellac is one of the most water-resistant cements ever invented. This style of marine glue can still be obtained from suppliers of microscope-mounting acces- sories in Europe but does not appear at present to be on the market in the United States. If it is unobtainable, and the technician is unwilUng to make his own supply, gold size is the next best sub- stitute. This gold size must, however, be of the old-fashioned kind specified for microscope mounting and not one of the new varnishes which are placed on the market for the benefit of gilders. It should perhaps be explained at this point that gold size was the material used by the early gilders to apply sheets of gold leaf on large areas. It was partially polymer- ized and partially oxidized linseed oil mixed with small quantities of resin and diluted with turpentine. To the old gilders it had the advantage that it took a long time to harden so that it retained a tacky surface, to which the gold leaf could be applied, over a long period. The advan- tage of this material to the maker of microscope slides is that both boiled linseed oil and turpentine will selectively "wet" glass — that is, they will displace a fine film of water from the surface of the glass. They can therefore be applied to damp glass to which they will remain adherent. Modern gilder's varnishes — sometimes called gold size — have the advantage to the modern gilder that they remain tacky for any specified period; to the microscope mounter they have the disadvantage that they are made in the interests of the gilder, not of the tech- nician, and rarely contain ingredients which will adhere to moist glass surfaces. The attachment of a cell with gold size was described in Chapter 1 and need only be briefly reiterated. The slide is placed on a turntable (Fig. 7) and a ring of gold size of about the width of the cell turned on the center of the slide. The undersur- face of the cell is given a thin coat of gold size and both the slide and the cell are placed on one side until the varnished surfaces have become tacky. An additional thin coat of gold size is then applied either to the cell, or to the slide, and the two pressed together. Since, however, a water- proof seal is required, the cell must be pressed firmly against the slide until the cement has hardened, either by laying a heavy weight on top of the cell, or by placing another slide on top and clamping the two together. The attachment of a cell with marine glue is an altogether different proposition. If a solution of marine glue is used, a thick ring is turned on the sUde and a thick coat is applied to the underside of the cell. Both cell and slide are then warmed (the lowest step of the hot table shown in Fig. 8 may be employed) until all the solvent has been driven off. The slide is then laid on the upper shelf, which should be heated above the melting point of marine glue. The cell is placed on the now molten ring of cement and maintained in constant contact with it until its own coat of cement has melted and fused with the cement on the slide. The slide should next be transferred to the second or third shelf (which should be just below the melting point of the cement) and a heavy weight placed on top while the cement slowly solidifies. After a few minutes at this solidification temperature, the slide is re- moved, still with the weight or clips in position, and laid on one side to cool. It is then turned upside down and in- spected to make sure that no air bubbles have been caught in the cement. If solid marine glue is used, chips must be scraped from the block with a knife. A layer of these chips is then placed on one surface of the cell (which in this case must be of tin or some other metal) and the cell laid on the upper shelf of the hot table at a temperature which will melt the cement. A heated needle is used to remove as many air bubbles as possible from the molten cement and the glass slide is laid alongside it on the hot table. The hot slide is then pressed firmly to the molten cement on the upper surface of the ring. As soon as the ring is firmly pressed into place the slide is inverted, placed on the second Preservatives FLUID WITOLEMOUNTS — AQUEOUS TYPE 23 shelf, and a heavy weight placed on top until the cement is cooled. Whether gold size or marine glue be em- ployed, care must be taken to remove those portions which have been extruded into the interior of the cell. These cements swell up and become white in the presence of water, and even a trace of remaining cement will give an unfinished appearance to the slide. The excess cement may be removed by scraping with a scalpel, and a final cleaning may be given with a 10% solution of potassium, or sodium, hydrox- ide, which is wiped over the inside of the cell with a piece of cotton held in a pair of forceps. The cell is then thoroughly washed and laid on one side to dry. It is best to cement cells onto slides in ad- vance of requirements and thus secure an adequate reserve. Selection of a Preservative Medium The introduction of formaldehyde to microscopic technique was welcomed as the beginning of the millennium, and almost all of the older aqueous media were thrown overboard by mounters. This is to be regretted since formaldehyde is by no means a perfect medium, particu- larly for the preservation of small inverte- brates and single-celled plants, so that attention should be given to the list of aqueous preservative media in Chapter 17 (P 11.1). These media are mostly variations on the fluid of Goadby which was a mixture of sodium chloride and am- monium alum — designed to approximate an isotonic solution — containing a very small quantity of mercuric chloride as a preservative. Some of these solutions (cf. Kronecker 1907) had an alkaU added to preserve the green color of small algae. Another excellent preservative of green material is the solution of Ripart and Petit which is given in Chapter 18 (F 3000.0010 Ripart and Petit 1884) because it serves the dual purpose of fixation and preservation. A very similar formula was pubhshed by Woods in 1929 (Chapter 17, P 11.1) as a preservative for green algae. Simple solutions of various reagents may also be employed. The best of these is formaldehyde, which for purposes of mounting should never be neutraUzed since, once subjected to this treatment, it is liable to develop precipitates. The ordinary 1 to 10 dilution of 40% formalde- hyde, which is commonly employed for the preservation of gross biological speci- niens, is far too strong for microscope mounting of the type being discussed. It must be remembered that these strong solutions become greatly diluted from the water contained in the specimens placed in them, whereas in the case of a microscope mount the material will have already been impregnated with formaldehyde before being mounted. A dilution of 1 to 100 of the commercial 40% formaldehyde is adequate as a mounting fluid. Camphor water and chloroform water, which are merely saturated solutions of these rea- gents in distilled water, are also excellent preservatives for the more delicate Pro- tozoa and Algae. It must be emphasized that if glycerol is added to these media, the material will have to be handled by the special methods necessary for making glycerol mounts, which are described in the next chapter. Selection of a Coverslip Cement Though the cell is best attached to the slide with a thermoplastic cement, it must be obvious that a liquid cement must be used to attach the coversHp to a cell containing a fluid mounting medium. Numerous formulas have been developed for this purpose, and the author most warmly recommends either gold size, or the cements given under Behrens 1883 or Carany 1937 in Chapter 28 (V 12.1). These last two cements are quick drying, which is desirable, since at least three successive coats must be used properly to seal an aqueous wholemount. The first of these coats is designed to block off the water, and to provide a temporary support for a second layer of waterproof cement which would not adhere to the moist glass. This second coat of waterproof cement should always be an asphalt varnish (formulas are given in Chapter 28, V 11.2) and, if the black color is ob- jected to, any colored varnish may be coated over the asphalt to provide a more finished appearance to the mount. 21 THE ART OF MAKING MICRORCOrE SLIDES Sealing Sealing the Coverslip in Place It was pointed out in the last cliapter, and must be reiterated here, that before any sealing cement can be applied a pro- tective barrier must be erected to prevent this cement from running in by capillary attraction and mixing with the contents of the cell. More wholemounts are spoiled by this running in of cement than by any other method. The procedure, when mounting on a flat slide, or one containing a concavity, is somewhat different from that which is used in sealing a cell. The former will be described first. The process of attaching a coverslip and sealing it in place on an aqueous fluid mount in a concave shde is shown in sec- tion in Figs. 9-14. Fig. 9 shows a longi- tudinal section of the concave slide with the protective ring of cement in place. This ring should be the narrowest and the thickest which can be made with the aid of the finest sable-hair brush. It should certainly not be wider than >^4 of an inch, and if it can be built up to twice this depth, it will be all the better. This ring also assists in attaching the coverslip, so that the gold size should be given time to become tacky before the mount is made. It is a matter of convenience to run such rings on the turntable on the night before the mount is to be made. A ring made from a good specimen of gold size will remain tacky for at least 48 hours after it is turned. The diameter of this ring is quite critical. It should be at the outer edge about J-^4 of an inch less than the diameter of the coverslip. If it is smaller than this, too big a space will be left between the edge of the cell and the coverslip; if it is larger than tliis, perfect sealing is impossible. Fig. 10 shows the same slide after the object has been placed in the cavity and a sufficient quantity of the selected mount- ing medium placed on top of it. Notice that a great excess of the medium is pro- vided and permitted to rise up in a convex meniscus. After this drop has been placed in position the mount should be inspected carefully to make quite certain that the fluid is in contact with the protective ring of varnish all the way around the edge. If the slide is perfectly clean it may so happen that the meniscus does not extend to the varnish ring, leaving a small air gap which will result in a bub- ble— almost impossible to remove sub- sequently. The next step is that of placing the coverslip in position. The covershp must never be let down from one side in the manner customarily taught in making balsam mounts. It must be held between the thumb and second finger and lowered horizontally until it is in the position shown in Fig. 11. It will be seen that the object remains in the central position in which it started whereas, if the cover were lowered from the side, the object would inevitably be pulled by capillary attrac- tion to one corner whence it would be almost impossible to displace it. Fig. 12 shows that the covershp has been let down and pressed with a needle onto the surface of the tacky protective ring of gold size. The excess fluid has been pushed out and mopped up with a filter paper. Care should be taken to remove the whole of the fluid between the outer projecting edge of the covershp and the ring to which it is attached. This is one of the most critical stages in the whole procedure. The needle used to press the covershp in place should be run with a circular movement round the covershp vertically above the protective ring, and pressure should be continued until the glass is clearly in contact with the gold size at all points. The shde is then placed on the turntable, centered, and a ring of the selected second cement applied round the edge. The result of this is shown in Fig. 13, where it will be seen that the edge of the coverslip is firmly embedded in the cement which has run under as far as the pro- tective barrier. The existence of the pro- tective barrier and the overhang of the covershp insure, therefore, that there shall be a good, thick layer of this cement in position. The shde is now laid on one side until this first protective layer is thoroughly dry and then (Fig. 14) as many rings of asphalt varnish turned over the top as are required. It is an excellent thing to apply two coats of asphalt var- nish, naturally permitting the first to dry before applying the second, at the Sealing FLUID WHOLEMOUNTS — AQUEOUS TYPE 25 Cavity Gold size ring Drop of preservative Object 10 ,^ Coverslip 11 Preservative withdrawn 12 ^_ Gold size coat sealing 13 Asphalt varnish ^^^finishing coat ^^^ . 14 Figs. 9 to 14. Longitudinal section of a cavity slide showing successive stages in the preparation of an aqueous wholemount. 9. Protective ring of gold size turned. 10. Object placed in position in a large drop of preservative. 11. Coverslip held horizontally in con- tact with preservative. 12. Coverslip lowered and preservative withdrawn between protective ring and edge of coverslip. IS. Heavy sealing coat of gold size applied. I4. Finishing coat of asphalt varnish applied over gold size. 26 THE ART OF MAKING MICROSCOPE SLIDES Deep cells time of making the slide and to turn an additional coat on top every two or three years. Slides treated in this manner may be kept for as long as 20 years without any air bubbles appearing. The petroleum jelly method of Spence, which in the author's opinion is more applicable to Drop of preservative Object to the side of the cell or dissolved in the mounting medium. It is best to remove dissolved air from the medium either by boihng or by placing a small beaker of the medium under a vacuum until all the dissolved air has been removed. A pro- tective ring is turned as before (Fig. 15) Gold size ring Cell 15 Coverslip X 16 Preservative withdrawn ML 17 Gold size sealing coat 18 Asphalt varnish finishing coat 19 Figs. 15 to 19. Sections showing successive stages in the preparation of an aqueous wholemount in a deep cell. 15. The cell has been cemented to the slide, and a gold size ring turned on its inner edge, before being filled with fluid. 16. The coverslip is slid into position. 17. Coverslip pushed into place and preservative withdrawn between gold size ring and edge of cover- slip. 18. Heavy sealing coat of gold size applied. 19. Finishing coat of asphalt varnish applied over gold size. glycerol than to aqueous mounts, is given in the next chapter. For a descrip- tion of a modification of this method ai> plied to aqueous mounts, refer to Spence 1940 (il/icroscope, 4:121). The method of mounting in a relativel}' deep cell is shown in Figs. 15-19. Par- ticular care has to be taken in this case to prevent the appearance of air bubl)les which may come from air either attached but it will he seen, in this case, that this protective ring is on the inner edge of the upper surface of the cell. The cell is then filled with the preservative fluid, allow- ing an excess to rise in a concave meniscus. To make sure that no air is caught on the irregular surface of the inside of the cell one may now either place the whole under a vacuum or, more conveniently, take a clean brush and wipe the inside of Algae FLUID WHOLEMOUNTS — AQUEOUS TYPE 27 the cell with it. Particular attention should be paid to the junction of the cell with the slide, whore tnipi)ed air bubbles are often caught. The author finds it best not to lower the coverslip horizon- tally, as in the previous mount, but to shde the cover horizontally onto the cell. This is shown in Fig. 16 where the cover- slip has reached halfway across. This illustration is shghtly exaggerated since the cover may be started farther across and sUd only the last few miUimeters. Care must naturalh' be taken that the gold-size protective ring is sufficiently dry not to smear the coversUp as it is pushed. If, when the cover reaches nearly to the other side of the cell, a small air pocket is left, it may be filled with moun- tant and the coverslip pushed neatly into place. It is also possible to lower the coverslip from one side — that is, to place one edge in contact with one edge of the cell and to lower the other with a needle — provided that object is sufficiently large not to become displaced. Fig. 17 shows the covershp in place after it has been pressed in contact with the protective ring and after the mounting fluid has been wiped from the outside. This is more difficult, and must be done more carefully, than in the case of the flat mount previously discussed. A ring of the first sealing cement is then (Fig. 18) applied to fill the gap between the ovcrliip of the coverslip and the protective ring on the inner edge of the cell. It is not necessary to do this on a turntable since this cement need not come onto the top of the coverslip at all but may be applied directly from the side. After this cement has had time to dry one should then build up (Fig. 19) several layers of asphalt varnish. So many laj^ers are re- quired to fill the angle between the cell and the coversUp that it is often desirable to use some cement containing a pigment. If a pigmented cement is used it should, however, be given a coat of waterproof asphalt varnish on the top before the slide can be considered finished. The purpose of a thick layer of cement, filling the angle between the cell and the slide, is to pro- vide additional mechanical support to the cell. The most frequent cause of break- down of thick aqueous mounts is either the complete detachment of the cell from the slide, or the cracking of the ce- ment which holds the cell in place with the subsequent intrusion of small air bubbles. Specific Examples Preparation of a Wholemount of Microcystis in the Fluid of Ripart AND Petit 1884 Wholemounts of unicellular algae pre- pared in any medium except balsam are rarely seen nowadays. These balsam mounts, though they display fairly clearly the internal structure of the alga, give the student not the faintest idea of wdiat the material looks like in life. Nothing is more valuable for the laboratory instruc- tion of classes, who will subsequently study in the field, than a series of whole- mounts of phytoplankton preserved so as to resemble, as nearly as possible, the Uving material. It is the author's opinion that the solution of Ripart and Petit, used as described in this example, gives as close an approximation to the appearance of the Uving material as can be produced. Very weak solutions of formaldehyde are often used for the preservation of vials of phytoplankton concentrates for labora- tory study, but it is not, in the writer's opinion, a satisfactory medium for the preparation of a wholemount. The blue-green alga microcystis has been selected for the present example because it hajjpens to be the most common alga found in large bodies of w^ater in the district from which the author is writing. Other blue-green and green algae may just as well be prepared by the present method. It is a waste of time to endeavor to concentrate algal collections in the field. Several gallons of the greenish water containing these specimens should be collected and brought back to the laboratory for immediate processing. There are two ways of concentrating the specimens. The first is to add to the 28 THE ART OF MAKING MICROSCOPE SLIDES Algae fluid containing them considerable quanti- ties of the required preservative, to permit the algae to settle to the bottom, and then to pour off the supernatant Uquid. One of the modern plankton centrifuges will do the job twice as efficiently in half the time. These plankton centrifuges are built as miniature milk separators save for the fact that the vertical plates of the latter are missing. That is, the plankton centrifuge is merely a small cup which is rotated at high speeds while a continuous stream of the material to be concentrated is poured into the top. The plankton organisms, being the heavier, are collected round the edge while the cleared water passes out at the bottom. The material should be put through the separator twice and with its aid it is possible to con- centrate five gallons of plankton into 100 milhliters in about five minutes. These concentrates must be processed immedi- ately; within a space of ten minutes the available oxygen in the water will have been used up and the concentrate will die with a consequent degradation of its appearance. The solution of Ripart and Petit, here recommended, may be used as a fixative for animal tissues as well as a preservative for plant tissues and is accordingly given in Chapter 18 under the heading F 4000.0010. fcit is a weak solution of copper acetate and copper chloride, acid- ified with acetic acid and with a small quantity of camphor added. More modern writers (Mayer 1920, p. 232) have sug- gested the substitution of thymol for camphor, and menthol may equally well be employed. It is unwise to use a satu- rated solution of any of these compounds, for crystals are likely to form through the slight evaporation which always takes place in a mount. One therefore takes equal quantities of a saturated solution of camphor or thymol, and distilled water, and then adds to each hter of this mixture two grams each of copper acetate and copper chloride together with seven milh- liters of acetic acid. About ten times its own volume of preservative should be added to the concentrate, the bottle con- taining which is then carefully tilted backward and forward at intervals for the next twenty-four hours before the organ- isms arc allowed finally to concentrate at the bottom of the jar and the supernatant reagent poured off. This preserv^ed con- centrate may be kept indefinitely in the dark and mounts made at any time. Preparation of the actual mounts must be done on two successive days: on the first of these the cells are prepared and placed on one side to become hard; on the second the actual mount is made. Clean sHdes are absolutely necessary and may either be cleaned in the manner sug- gested in the last chapter, or may be chemically cleaned by one of the cleaning mixtures given in Chapter 28. The sKdes should be selected rather more carefully than usual for the most minute flaw in the surface of the slide will become appar- ent in an aqueous fluid mount, even though it would be in\dsible in a colloidal or resin medium of higher refractive index. Having selected and cleaned the shdes, a ring of gold size is turned on each, care being taken that the ring is smaller in diameter than is the coverslip to be em- ployed. An 18-milhmeter ring and a %-inch coverslip form an excellent com- bination. It may be pointed out that this is the reverse of what is done when mount- ing in a tin, cardboard, or plastic cell where a %-inch cell is used with an 18-milhmeter covershp. In this case the purpose of the initial ring is not only to provide support to the covershp but also to insure that the cement subsequently used for sealing shall not run in by capil- lary attraction and ruin the mount. The ring should be as narrow as can be drawn and should be about }i4 of an inch thick when in the fluid stain. With experience, and a fine sable brush, it is possible to draw these initial rings about ^i^ of an inch in width, though Ke is permissible and wiU be more hkely in the hands of the inexperienced. As many rings are prepared as mounts are to be made and placed on one side until the next day. If, through some accident, mounting cannot be con- tinued on the next day, or possibly the day after, it will be necessary to put a thin coat of fresh gold size over the dry coat and permit this to harden for 24 hours. The condition of the gold-size Rotifers FLUID WHOLEMOUNTS AQUEOUS TYPE 29 ring when mounting is critical; it must have dried to a rubbery, but not to a hard, consistency. For the final mounting one requires at hand the shdes which have been prepared, the turntable which was used to draw the original ring, some clean covershps, a pipet of the eye-dropper type, and the concentrate of algae. A slide is centered on the turntable and a twist given to the turntable to make sure that the centering is accurate. A drop of the concentrated algae is placed in the center. If the slide is clean this drop will flow outwards until it reaches the cement ring where it will be held. Under no cir- cumstances may the coverslip be placed on the preparation until the fluid touches the ring at all points. If it does not do so at once it may be brushed out with a small brush. It does not matter in the least if it flows over the ring but it will be impossible to avoid including air bubbles if it does not reach the ring. A coverslip, held between the thumb and the index finger of the right hand, is lowered horizontally until it touches the drop of algal suspen- sion. It is then dropped in such a manner that it falls onto the ring which should be centered exactly under the coverslip. If it is not centered it is still possible to ad- just it with a needle, provided it is not too far out, as long as it has been dropped and not pressed down. If it is initially pressed down, so as to make a contact with the gold size, nothing can be done and another sUde must be taken in its place. As soon as the coverslip is centered a fine needle is taken and run round immediately over the ring to press the glass into contact with the slightly tacky gold size. Any of the algal suspension which has crept onto the top surface of the coverslip is re- moved with a soft cloth, remembering to be very careful not to shift the cover- shp; any fluid which has spread out over the shde may he wiped off as far as the edge of the coverslip itself. There will still remain a small quantity of the fluid be- tween the ring, which is slightly smaller than the coverslip, and the edge of the coverslip itself. This must be removed, using the edge of a sheet of filter paper which is touched down to the fluid. The withdrawal of this superfluous fluid from between the edge of the cover- shp and the ring, is a critical part of the proceedings. The ring on which the cover- slip rests is not sufficient to prevent evaporation but is sufficient to prevent the introduction of air mechanically while this superfluous fluid is being withdrawn. If, however, the coverslip is ever so shghtly raised by the edge of the filter paper an air bubble will inevitably enter the mount which must then be thrown away. Assuming that all has gone well, and that the superfluous fluid has been with- drawn, a heavy ring of gold size is turned on and the mount placed on one side. The ring of gold size should be at least }i of an inch wide and as thick as the ma- terial can be persuaded to flow from the brush. The mount is then placed on one side and the next one taken. A single ring of gold size will hold the mount in good condition for a few months but if permanence is desired it is better to add three other rings of gold size at daily intervals, then to wait a week and to turn on top of this a coat of asphalt varnish. The degree of permanence of these mounts is variable. The writer has one in his possession which is more than 20 years old and is as good as it was the day it was made. Preparation of a Wholemount of a Rotifer by the Method of Hanley 1949 The method of Hanlev is a modification narcotic (Chapter 19, AF 50 Rousselet of the well-known method published by 1895), and the sjiibstitution of formalde- Rousselet 1895 (11479, 5:1) which has hyde for osniic^'acid in kiUing. These been quoted without alteration in the substitutions not only render the final literature for more than 50 years. Han- preparation better and more permanent ley's method involves narcotization in his l)ut also remove the difficulties both of own narcotic (Chapter 19, AF 50 Hanley working with osmic acid and of securing 1949) as a substitute for Rousselet's cocaine. This method is of great impor- 30 THE ART OF MAKING MICROSCOPE SLIDES Rotifers tance because satisfactory wholemounts of rotifers cannot be made in either resin- ous or gelatinous media, since no method of dehydration has yet been discovered which will not distort all save a very few of the toughest rotifers. The collection of rotifers is relatively simple. Planktonic forms, either marine or fresh-water, may be taken in fine plankton nets and usually occur in con- siderable quantities where they occur at all. The tube or bottle at the end of the plankton net should be emptied into a con- siderable volume of water and kept well- oxygenated unless the specimens are to be prepared immediately. The usual methods of plankton concentration are very unsatisfactory for delicate rotifers, and it is better to rely on their attraction by light, and by high concentrations of oxygen. If, on the return to the laboratory, the quart or gallon of plankton suspension be placed on a bench and one side shaded while the other is brilliantly illuminated, all the planktonic rotifers will be found to concentrate at the surface on the illumi- nated side of the bottle. They may then be picked out without difficulty with a fine pipet and transferred to a watch glass for narcotization. If there are only a few rotifers present it may be necessary to take the jar into a darkened room and to illuminate one angle of it with a small spotlight (such as the Nicholas lamp used by embryologists) which will collect all the rotifers from half a gallon of water in a few minutes. If the jar is going to be left for some time under these conditions it is desirable to use some form of heat filter between the lamp and the jar. The collection of sessile rotifers is more difficult. They will usually be found at- tached to the stems of water plants, and to the underside of water-lily leaves. It has been the author's experience that more rotifers will be found in relatively small ponds than hi large lakes, and that if one could find a body of water several feet deep but of only a few hundred square feet of surface area, and if this water is relatively choked with large water weeds but contains only a small quantity of green algae, it is likely to contain many of the rarer forms of sessile rotifers. The distribution of these forms is, however, very scattered and it is scarcely ever worth while to collect large quantities of water weeds with a drag and then to take them back to the laboratory and hunt through them. It is far more profitable to settle down and hunt the weeds as they are in the water, cutting from them short lengths of stem or small areas of leaf which bear the required forms. These are then placed in a large jar of water from the pond and brought back to the labora- tory for further treatment. The most difficult part of the prepa- ration of a mount of the rotifer is to narcotize it correctly. Hanley (Micro- scope, 7:155) has discovered that the use of alcohol in Rousselet's fixative is an- tagonistic to the cocaine in the same solu- tion and that it is, therefore, by Rous- selet's method necessary to use very large quantities of narcotic with a resultant very short interval between complete narcotization and death. With Hanley's narcotic the narcotization is relatively rapid but the interval between complete narcotization and death is relatively long. With Rousselet's fixative there is often only a period of from one to two seconds between the moment when the fixative can be applied and the moment when the rotifer dies and is then worthless. With Hanley's narcotic, this period is extended for as long as 10 to 15 seconds and only those who have mounted rotifers by Rousselet's method can appreciate how great is this advantage. For the actual process of narcotization it is necessary to have two watch glasses, one containing the rotifers swimming in their normal environment, and the other a 10% solution of formaldehyde. These two watch glasses should be sufficiently far apart that fumes from the formalde- hyde do not dissolve in the glass contain- ing the rotifers. There is also required a supply of Hanley's narcotic, a fine pipet, and a dissecting microscope having a power sufficiently high to enable the rotifers to be seen clearly. For an average watch glass containing the rotifers two drops of Hanley's narcotic are added to the water and mixed by sucking the water in and out with a rather coarse pipet. Rotifers FLUID WHOLEMOUNTS — AQUEOUS TYPE 31 It does not matter that this treatment will cause the intifei's tn contract for tlioy will have ample opportunity, at this stage, to re-expand. The watch glass is then left alone for about 20 or 30 minutes, a further drop added and vcni cautiously mixed in; after a furtiioi- five niinutes another drop is mixed in with extreme caution and the rotifers watched under a microscope. The pH of the water, as Hanley points out, very greatly affects the rai)idity of nar- cotization which may be complete in from 45 minutes to an hour and a half. No definite data are, however, available as to the adjustment of the pH in relation to the quantity of the narcotic so that one can only j)roceed by trial and error. The author differs from Hanley as to the exact momeiit at which fixation or kill- ing should take place. Hanley states that it is safe to pick out the rotifers and trans- fer them to the formaldehyde solution when they are moving sluggishly about but do not contract when they hit each other. He further saj's that it is too late to applj' the killing agent when cihary action has ceased. It has been the writer's experience that kiUing should always take place at the exact moment when the ciha cease to move. With Rousselet's narcotic this cessation of ciliary movement is fol- lowed within a second or two by death; it has been the writer's experience that with Hanley's narcotic one has at least ten seconds of leeway which permits one to flood the watch glass with a considerable quantity of 10% formaldehyde. Which- ever method is adopted, as soon as the formaldehyde has been placed in the watch glass, it is rapidly withdrawn and replaced with fresh 10% formaldehyde in which the rotifers remain until they are ready for mounting. With Rousselet's method one used to add a drop or two of 2% osmic acid to kill the rotifers and then remove them very, very rapidly from the mixture through several changes of distilled water and then in to the formalde- hyde for preservation. This method oc- casionally resulted in the destruction of the cilia, and it was also exceedingly diffi- cult to avoid retaining sufficient osmic acid to cause subsequent darkening of the mount. As Hanley's method of sealing a wet wholemount differs appreciably from the writer's, which was given in the descrip- tion of the last example, Hanley's method will 1)6 given in some detail. The following description is taken almost verbatim from the paper of Hanley cited. If cement cells are used the cell is made beforehand and allowed to dry. When mounting, rotifers are picked out with a fine pipet and placed on the floor of the cell. The slide is then placed on the microscope stage and filled to excess with 23^^% formaldehyde. The mount is examined under the micro- scope and any foreign bodies or air bubbles removed with a fine pipet — do not run a needle round inside the end of the cement ring to remove bubbles, "unless you are fond of cement scrapings in your mounts." Much of the excess fluid can be removed with the pipet, being careful not to remove the rotifers also, and a clean coversUp then placed on the mount with flat-ended forceps. The coverslip should float on the dome of fluid and then is tapped down smartly with the base of the forceps — if this is not done smartly enough the rotifers will be washed out. The surplus fluid is removed with filter paper, changing the point of application as the rotifers move, and when nearly all tlie surplus has been removed the cover can be pushed slowly into place with a bent wire. It is important to notice that no wet cement is used on the cement cell. This is quite unnecessary with cement rings. (This is Hanley's opinion not the writer's.) If the cell is properly made, the cover glass when set down adheres so firmly to the cell that it can be broken before it will move, while, when wet cement is used, the cover cannot be centered once it has been applied. The slide is then placed on a turntable and a thin ring of cement is run round and over the edge of the cover in the manner described in the last example. In the matter of sealing the writer prefers the classical method of running several rings of gold size as a seal and finishing this with a flexible black varnish. Fluid Wholemoiints in Nonaqueous Media General Principles Nature of the Process The mounting of whole objects in non- aqueous media is essentially the same process as mounting objects in aqueous media: that is, the objects are enclosed in the preservative medium in a very flat box, the floor of which is formed by the slide, the top of which is formed by the coverslip and the sides of which are formed either of cement, or by a cell. There are not, however, so many possible choices among cells and seahng media as is the case with aqueous mounts, for the choice of the medium itself dictates every subsequent step. Choice of the Medium Only three nonaqueous media are commonly used in mounting: these are glycerol, bromonaphthalene, and Uquid petrolatum. These should never be used when any aqueous substitute is available, nor should a fluid medium be used if a mountant which will harden under the coverslip (see the next three chapters) can be employed in its place. Each of these three media will be discussed in their turn. Glycerol is widely used as a mountant in those cases in which a water-miscible, high-refractive-index material is required and in which a medium of the type dis- cussed in the next chapters cannot be em- ployed. The principal reason that such media cannot be used is the difficulty of transferring delicate objects to, say, glycerol jelly without causing a collapse of their walls, while it is comparatively simple to get delicate objects into glycerol by evaporation. This technique is usually applied to nematode worms, and some- times to small arthropods or very delicate coelenterates, which should be fixed in the ordinary manner and then transferred very gradually to alcohol and from alcohol to ^ % glycerol in alcohol. The alcohol is then slowly evaporated, leaving the material in pure glycerol. It is almost impossible to seal a deep cell full of glycerol, and mounting in this material should be confined to cells built out of cement or to shdes in which a concave hollow has been ground. Sealing Glycerol Mounts with Dichro- mate Gelatin There are three ways in which a glycerol-filled cell may satisfactorily be sealed. The first is with the aid of molten gelatin, appUed from a turntable in the manner described in the last two chapters, and then varnished with any good cement; the second method involves the apphca- tion of a molten resinous medium; the third method uses petrolatum. In the case of the first method it is better to use a solution of gelatin containing potassium dichromate, which becomes insoluble on exposure to hght, than to use straight gelatin; and it is doubtful if the formula of Riiyter 1934 or 1935 (Chapter 28) can be improved. A narrow ring of material is turned on the sUde, in the manner previously de- scribed, the ring being made slightly smaller in diameter than the size of the coverslip to be used and just sufficiently thick to keep the covershp from bearing on the object. This cement shrinks on drying so that a ring must be turned 32 Sealing FLUID WHOLEMOUNTS IN NONAQUEOUS MEDIA 33 somewhat thicker than is customary with other cements. A number of slides may be prepared at the same time and left in a light place for an hour or two until the gelatin has become insolubilized. The object, together with a drop of glyc- erol, is placed in the middle of the cell and the coverslip lowered vertically as shown in Fig. 24 (Chapter 6). The covershp is then held firmly in place, either with the finger or with one of the chps shown in Fig. 25, while all traces of exuded glycerol are removed with the aid of a rag moist- ened in alcohol. The shde is then placed on the turntable and a ring of molten dichromate gelatin turned over the edges of the covershp. This ring of cement is cooled — it is not necessary to dry it — and the whole slide then thoroughly cleaned in 95% alcohol, either apphed from a rag, or by waving the slide back- ward and forward in the fingerbowl of the reagent. Great care is necessary at this stage to avoid displacing the coverslip. The purpose of the ring of gelatin, in fact, is not so much to cement the cover- slip in place as to provide a temporary seal which will hold the cover sufficiently long to permit the removal of exuded glycerol. As soon as the slide is dry, and glycerol-free, several coats of gold size are added, allowing ample time for each to dry, and then a final coat of asphalt varnish is turned on top. Slides prepared by this method have a very pleasing appearance but they require a great expenditure of time compared to the use of a thermoplastic resin cement. Sealing Glycerol Mounts with Thermo- plastic Resin Mixtures The medium most usually recom- mended for heat-sealing glycerol mounts is Noyer (Chapter 28, V 12.2 Noyer 1918), a simple mixture of rosin and lanolin. The writer prefers the formula of Fant (V 12.2 Fant 1932), containing a quantity of dried Canada balsam, which appears to make it both easier to handle and more adhesive. Whichever medium is employed, the object in glycerol is placed under the coverslip and, after crudeh' wiping away the excess fluid, a layer of molten cement is applied to the edge. For making large quantities of these preparations a most ingenious mechanism has been described by Banard (113G0, 54:29), but it is proposed here only to deal with the method of handling indi- vidual slides. This method is shown in Fig. 20 where the objects are being mounted under a square coverslip. It is the author's opinion that no satisfactory seal can be made by this method on round coverslips. The dish in the left foreground contains the objects in pure glycerol and, immedi- ately behind it in the center of the picture, there is a tin can containing the cement selected. The author always prepares the cement in such quantities as will just fill an empty boot-polish can, which is admirably adapted to the purpose. The tool being used is the same rather heavy brass tool which is shown being employed in the mounting of paraffin blocks in Fig. 65 (Chapter 12). An ordinary section lifter, sometimes recommended, is too thin and does not hold enough cement. In the illustration, it is presumed that the object has been placed under the coverslip, the coverslip lowered in place, the glycerol roughly wiped away, and the metal tool heated to about 150° to 200°C. This tool is now dipped into the can of cement, so that the edge accumu- lates molten cement along it, and then touched down on the edge of the cover- shp. It will be noticed that the edge to- ward the front of the illustrations has already been finished and that the second edge is being apphed. Before this was done, a minute drop of the cement was placed at one corner of the covershp to hold it in position. Having finished two sides in this manner, it is easy to apply cement to the third side, but the whole trick of a successful mount lies in the method in which cement is applied to the fourth side. It will be obvious that this very hot cement, when it is applied to the coverslip, will cause an instantaneous expansion of the fluid. This does not matter as long as one side remains open. The last side, however, cannot be sealed in one piece, and it is necessary to apply the cement in such a manner that about a one-milhmeter gap is left at a corner 34 THE ART OF MAKING MICROSCOPE SLIDES Sealing for the escape of the heated glycerol. The slide is then cooled, such glycerol as has been extruded from the corner is wiped away, and a small drop of very hot cement is apphed at this place. Slides sealed in this manner will last almost indefinitely and require no further finish- ing beyond a brief wash in alcohol to slip. The size of the drop is therefore critical but can onlj^ be learned by experience. The shde is now placed on a warm table, kept a few degrees above melting point of the petrolatum employed, and molten petrolatum run under the cover from a pipet. Spence prefers to take a Fig. 20. Sealing a wholemount with Fant's cement. remove excess glycerol. They are, how- ever, clumsy in appearance compared to a ringed slide made with dichromate gelatin. Sealing Glycerol Mounts with Petro- latum This method, which was developed by Spence 1940 {Microscope, 4:123) is the best yet developed, providing one is looking for chemical stability rather than mechanical strength. The object is lifted in a drop of glycerol and placed in the center of a clean shde. Three Uttle squares of petrolatum-soaked paper or card, of a thickness sufficient to prevent coverslips crushing the object, are placed round, but not in contact with, the drop. The cover- slip is now lowered vertically onto the drop which should spread out, when the coverslip is resting on the squares, until it occupies about half the area of the cover- wisp of solid petrolatum on a toothpick and to let this melt and run under the covershp. In either case, one is left with a bubble of glycerol surrounded by a thick layer of molten petrolatum. The slide is now chilled and any excess petrolatum scraped away. The mount is permanent in this form, but the petrolatum is so soft that the cover is liable to become de- tached when dust is wiped from it. A certain degree of mechanical strength can be given by turning on three or four rings of shellac, followed by three or four coats of asphalt varnish. Sealing Other Non-aqueous Liquid Mounts Mounting in liquid petrolatum is prac- tically confined to blood films, on the assumption that this inert medium pre- vents the fading of methylene blue-eosin stains. These stains are, however, best Nematodes FLUID WHOLEMOUNTS IN NONAQUEOUS MEDIA 35 kept dry, and many of the neutral niount- ing media described in Chapter 20, under the heading M 23.1, are less trouble to use, and probably just as good. Liquid petrolatum is difficult to seal, though the author has had most success with the hot- resin method described in the last para- graph. Even with this material, however, there is a slow diffusion of the brown resin through the liquid petrolatum which ultimatel}' damages the slide. Since liquid petrolatum does not evaporate, it is some- times preserved by holding a covershp in place with a drop of cement at each corner. The quantity of cement used is thus so small that diffusion through the mounting medium is negligible, while the degree of adherence is sufficiently good for all nor- mal handling. Bromonaphthalene is used only for mounting diatoms, when a medium of high refractive index is re- quired. The only satisfactory cement for sealing is a de-waxed shellac prepared by the method of Hitchcock (Chapter 28, V 11.2). Both the preparation of the cell, and the process of mounting, are special- ized procedures which are described in considerable detail in the second of the typical preparations which terminate this chapter. Specific Examples Preparation of Nematodes in Glycerol Nematodes are awkward objects from which to make v.holemounts, for their thick cuticle permits only slow diffusion of reagents, and it is almost impossible to get them into either resinous or gelati- nous media. The objection to shrinkage is not on aesthetic grounds, but on the basis that the folds and ridges of cuticle render it almost impossible to make out clearly those internal organs upon which classification depends. Nematodes are, therefore, almost invariably mounted in glycerol. No difficulty will be experienced in collecting small nematodes from the blood, or when they are free-swimming (as Anguillula). The standard method of se- curing nematodes and their eggs from feces, however, is by flotation from a strong salt solution. Fresh specimens are collected and flooded with 10 or 15 times their volume of a 20% solution of sodium chloride. This may be added directly to the cardboard containers customarily used for such samples, and the unpleasant odor may be diminished by adding small quantities of nitrobenzene both to the salt solution anfl to the feces themselves. After the sohds have settled, the top layer, on which tlie nematodes will be floating, is jerked into another dish with a quick movement of the wrist. It is almost impossible to pick the worms or eggs from the surface in a pipet so that, when a sample has been thus isolated, it should be diluted to a salt concentration of about 1 % which allows the specimens to sink to the bottom. They may then be washed with weak saline until free of fecal matter. This method of collection cannot be used with fecal specimens which have been mixed with animal charcoal as a deodor- ant, because the charcoal also floats on the surface. Worms may, however, be collected from such samples by a modified Berlese funnel (see Chapter 4, Fig. 21). In this technique a plug of glass wool is placed at the bottom of an ordinary glass funnel and the fecal material poured in. The bottom of the funnel is then lowered into a tube of 1 % salt solution until the liquid rises just to the lower edge of the fecal matter. A lamp, or some other heat source, is then placed above the feces. The worms endeavor to escape from the heat and, burrowing down through the feces, ultimately pass through the glass- wool plug and accumulate at the bottom of the tube of salt solution. The collection of small nematodes from soil samples is much more difficult than from feces. The flotation method is practically impossible because in most soil samples there are large quantities of organic matter which will also float, while the modified Berlese funnel usually permits enough clay to sift down to make it difficult to sepaiate' the worms. Pro!)- ably the best procedure is to dilute soil samples with a 1 % salt solution and then 36 THE ART OF MAKING MICROSCOPE SLIDES Nematodes survey small aliquots by strong trans- mitted light under a dissecting micro- scope. The nematodes may be recognized by their activity, picked out with a fine pipet, and transferred to fresh saline. Whatever method has been employed, one is left with a collection of nematodes in salt solution. The solution should be changed frequently, until the worms are clean; for satisfactory wholemounts cannot be made if either dirt or mucus adheres to the outside. Heat is the only fixative which will penetrate a nematode rapidly. It is conventional, therefore, to fix worms in hot 70 % alcohol, though hot water will, in point of fact, do equally well. The exact temperature is immaterial and usually 100 times as much 70% alco- hol as there is saline around the worms is warmed until bubbles appear. This is usu- ally at about 55° to 60°C. The hot alcohol is then rapidly flooded over the living worms, which are again collected by being allowed to settle to the bottom of the dish or tube. Most of the worms fixed by this method will be found to have straight- ened out, and the few which have not had better be thrown away. The worms must next, very carefully and slowly, be transferred to absolute alcohol, in which they must remain until they are completely dehydrated. This transfer is best effected through 5 % grades of alcohol; that is, from 70 to 75 to 80 to 85, etc. In the case of worms with very tough cuticles, a faster schedule may be employed. The reason the worms must be transferred to absolute alcohol before passing to glycerol is that it is almost impossible to get rid of water once it has got into the glycerol, and the high re- fractive index of the glj^cerol is lost if it is diluted. It is easy to find out how fast a schedule may be emploj'^ed by taking one of the worms from 70% and throwing it directly into, say, 95% alcohol. If, after two or three hours in this, there is no sign of tlie colla])se of the wall, the rest may follow it, but if the wall collapses one must experiment with 80% and so on until one has found the most rapid transfer which may be made. When the worms are all accumulated in absolute alcoliol, a little glycerol is added. Assuming that the worms are in 100 milliliters of absolute alcohol, it would be safe to add about 10 drops of glycerol, being very careful to shake rapidly and continuously so as to disperse the glj^cerol rapidly. The worms are left in this mixture for about 24 hours before a further 10 or 20 drops of glycerol are added and mixed. This schedule is continued until about 10 milliliters of glycerol have been added. The solution is then concentrated by evaporation, in a desiccator, at a rate which leaves the worms in concentrated glycerol at the end of about a week. It is easiest to suck air through with an aspirator, being careful that the air itself passes through a de- hydration column before entering the desiccator. This method of preparation is laborious in the extreme, but it yields a product which looks exactly like a glass model. The author knows no other method which will produce clear nema- todes without causing the collapse and wrinkUng of the cuticle. To make these cleared nematodes into permanent mounts, one now secures the necessary slides, coverslips, a metal tool of the type shown in Fig. 20, and a can either of Noyer's or Fant's cement. It is unnecessary to make a cell, or to use a concave slide, because the viscosity of the glycerol will hold the cover a reasonable distance away from the slide while the mount is being made, and the method of mounting embeds the coverslip so firmly that it does not subsequently shift. The slide is taken, cleaned by any preferred means, and the required specimen or specimens placed in the center in a drop of glycerol. Square coverslips should be employed and a little experience will soon show what amount will fill the cover- sUp to the edge. The coversHp should be lowered vertically to avoid displacing the worms, and, as soon as the glycerol has reached the edge, the heated metal tool is plunged into the cement and used to seal one edge. The success of tlie process depends on having the cement hot enough at the moment when it is applied. The opposite edge of the coverslip is then sealed, and these two seals connected by a third. The application of cement to the Diatoms FLUID WHOLEMOUNTS IN NONAQUEOUS MEDIA 37 fourth side is, however, made in such a manner that a gap of about a millimeter will be left between the cement and one of the corners of the cover. This gap is necessary to permit the heat-expanded glycerol to escape. After the slide has thus been not quite sealed it is permitted to cool and a rag moistened with 95% alcohol is used to remove excess glycerol from the httle vent which has been left. This vent is then itself sealed with a drop of very hot cement. Preparation of Diatoms in Bromonaphthalene Strewn slides of diatoms may be mounted dry in the manner described in Chapter 1. When it is necessary, however, to resolve fine structure, they should be prepared in a medium of high refractive index, and no resin has yet been found which is as satisfactory as bromonaph- thalene. No one who has ever examined diatoms mounted in bromonaphthalene will ever wish to use any other medium and, though the process is tedious, the end result justifies the trouble taken. Before mounting, diatoms must be collected and cleaned. The three great sources are fresh-water, sea-water, and fossil deposits. Diatoms occur in fresh water as part of the plankton, but are mostly found in the mud on the bottom of ponds or attached to weeds. No attempt should be made to separate diatoms from the weeds in the field; the collection should be taken back to the laboratory. The first rough separation is then carried out by cutting the plants into about ]>i- inch lengths and putting them into a flask with enough water to cover them, shaking vigorously, and then straining this water through coarse cloth into another con- tainer. More water is then added to the material, which is again shaken, and so on until after four or five washings, all the diatoms have been removed. These wash- ings may be set on one side to settle for further treatment. Diatoms may be separated from fresh mud by taking advantage of their photot- ropism. The mud, together with an ade- quate quantity of the water from which it was collected, is placed in a small saucer and a thin layer of cheesecloth is spread on the top. The mud should be sufficiently Uquid to permit diatoms to pass through readily, but sufficiently solid to prevent the cheesecloth from*sinking into it. If the dish be set in bright light for a day or two, the diatoms will migrate through tlie cheesecloth and form a d;irk greenish smear over its surface. The cloth is then removed, washed, and the diatoms ac- cumulated in a small quantity of water. The collection of diatoms from marine plants may follow the technique used for fresh-water plants, though the larger algae are better scraped with a blunt knife. These scrapings are then transferred to a jar of sea water where the diatoms and debris settle to the bottom. A rather large number of marine diatoms are, however, planktonic and can be collected from sea water with a centrifuge. A plankton-con- centrating centrifuge is described in Chap- ter- 2 and with its aid large volumes of water may be processed in a relatively short space of time. If such a planktonic centrifuge is not available, it will be neces- sary to collect the samples by towing be- hind the boat a long conical net of the finest obtainable bolting silk to the end of which is attached a small tube in which collect those specimens which have not passed through the net. Unfortunately the diatoms form a relatively small bulk, even though they may be numerous in quan- tity, of the total material collected, so that if there are many crustaceans among the plankton it is desirable to have a double net, the first layer of which will retain the crustaceans without permitting the dia- toms to pass. Whatever method of con- centration is adopted, however, one ends, as in the other processes described, by having a mixture of dirty diatoms and sludge accumulated in the bottom of the dish. Diatoms also occur in guano, and in many fossil deposits, and must be roughly separated before cleaning. If the fossil de- posit resembles guano, it is only necessary to shake it up in water and pass it through a coarse sieve to remove the sand and 38 THE ART OF MAKING MICROSCOPE SLIDES Diatoms other extraneous material. Many of the more interesting diatoms, however, are found in hard aggregates which must be broken up before the frustules can be separated. Many methods of doing this have been described, but undoubtedly one of the most useful is the technique of Swatman {Microscope, 7:132). This technique utiUzes the expansion and contraction which takes place on the sudden crystallization of a supersaturated solution of sodium acetate. The rock, or hard aggregate, containi ng the diatoms is roughly broken into j'i-inch pieces and placed at the bottom of an Erlenmeyer flask. Two or three times its own bulk of sodium acetate is then added, and thor- oughly mixed in, before adding water to the extent of about 5 % the total weight of the sodium acetate. The flask is then very carefully warmed, the flame being first ap- plied to the sides and not to the bottom, until the sodium acetate is molten. Heat- ing should then be continued until the material commences to boil and it should be maintained in a hot condition for as long as is required to cause the penetra- tion of this supersaturated solution to every part of the aggregate. The flask is then cooled slowly, care being taken to avoid jarring, and when the solution is cold a single crystal of sodium acetate is dropped into it, which causes instant crystallization. As the flask will heat up greatly during crystallization, it is then recooled in water. The mass is remelted, recooled, recrj^stallized, and so on until a sufficient disintegration of the rock has taken place. Another method of arriving at the same result is to soak the pieces of material in water, to freeze them very rapidly (either in a freezer unit or in dry ice) then to drop them into warm water, refreeze them, and so on. This process is no more effective, however, and is usually much more trouble to carry out, than the sodium acetate procedure outlined. When the mass has sufficiently disintegrated, it is strained through a coarse sieve to get rid of the lumps and the diatomacious ma- terial allowed to form a sludge at the bottom. Whatever method has been employed, one has now, from either fresh or fossil material, a sludge which should be trans- ferred to a flask. This sludge contains dia- toms together with various organic and inorganic impurities. The first thing is to get rid of any carbonates which may be present by adding hydrochloric acid cau- tiously (if there is a great deal of carbonate present effervescence may rise above the neck of the flask and cause a loss of mate- rial) until no further gas is evolved. The flask is then filled with water, the undis- solved material allowed to settle, the water poured off, and the process repeated until all the soluble chloride has been removed. If there is any appreciable amount of clay present, it will also have been removed by this process, since even the smallest dia- toms will settle relatively rapidly com- pared to the fine particles of clay. It is next necessary to remove an}^ organic matter which may be present, and many methods have been proposed for this. The conventional method, also described by Swatman (loc. cit.), is to get the diatoms into concentrated sulfuric acid which is then heated to about 120°C. This chars the organic matter which is then oxidized by dropping small crystals of potassium chlorate into the hot acid. It should per- haps be emphasized that only exceedingly small crystals should be added, and that those who do not normally wear glasses should use some form of protection against the chance of spurting acid. If there is much organic matter present, the heated acid will be from black to dark brown in color, and chlorate is added until the color is reduced to yellow. The only safe method of removing the acid is to wait until it is entirely cold and then pour it in a slow and steady stream, while constantly stirring, into a relatively large volume of water. The diatoms settle out on the bottom. Some iron may still be present, either de- rived from a fossil deposit or from the chlorophyll of the plant debris. This is best removed by suspending the diatoms in 5 % sodium hydroxide and bringing them to the boil. If there is any iron present it will ap- pear as a brownish ferric hydroxide through which the diatoms will settle readily and which may then be poured off. After repeating this process several times, the diatoms are treated with hydrochloric Diatoms FLUID WHOLEMOUNTS IN NONAQUEOUS MEDIA 39 acid to remove the last of the iron chloride and again washed by decantation. It will probably happen, howe\-er, that many of the finest markings on the dia- toms are still filled with finely divided clay which must be removed by treating the diatoms in the cold with a 10 "^t, dilution of ammonia. The frustules will be dam- aged if a hot or strong solution is used, and it is best to leave the diatoms in cold ammonia for two or three days, shaking at intervals, before washing them by de- cantation. The final stage in cleaning the diatoms is now to wash them with re- peated changes of filtered distilled water until all traces of dissolved salts have been removed. Some workers, after treating the dia- toms with ammonia, repeat the sulfuric acid-potassium chlorate treatment as a final precaution. Another variant is to precede the original treatment with sul- furic acid and potassium chlorate by treat- ment with a hot mixture of two parts of sulfuric acid with one of nitric acid. This treatment is recommended when the orig- inal collection contains very large quan- tities of vegetable matter in addition to the diatoms. Swatman (loc. cit.) points out that if diatoms are collected from mud containing coal dust this will not be satisfactorily removed by any of the pre- ceding processes and recommends that the diatoms be fused in a platinum crucible with pure potassium nitrate for removal of this contaminant. It will have been observed, either in theory or practice, that many of the proc- esses just described result in the produc- tion of noxious vapors, so that they cannot be properly carried out by anyone not ha\dng access to a chemical hood. To meet this objection Hendey 1938 (11360, 58:49) has devised a most ingenious apparatus which will permit any of the processes described to be used in a living room. All the methods so far described pre- sume that the collector has been working close to his laboratory and has, therefore, not been faced with the problem of trans- l)orting large quantities of vegetable matter. A rough method of field cleaning (Swatman 1941: 11479, 1:191) may be used to concentrate diatoms. As much water as possible is drained from the rough sludge and replaced with 10% sulfuric acid. Potassium permanganate is then added, with constant stirring, until the solution remains pink after standing for a few minutes; then enough oxalic acid is added to dissolve the brown oxide sludge. The clear solution may be poured off and the diatoms roughly washed before being transferred to a tube. By whatever method the diatoms have been cleaned, they are now presumed to be accumulated in clean distilled water. They should be roughly sorted into their kinds, since diatoms are much easier to handle under the surface of water with the aid of a fine pipet than they are when dry. The different kinds are then stored in small vials of distilled water to which a trace (about one-tenth of 1 %) of formalde- hyde is added with a view to discouraging organic growth. Larger quantities of form- aldehyde should not be used, or a fine de- posit will be found on the surface of the diatom when it is subsequently dried. To mount a strewn slide of diatoms, it is only necessary to take a drop of the dis- tilled water with the diatoms suspended in it, to let this evaporate on a coverslip, to dry the coverslip with heat, and then to mount it in the manner to be described subsequently. It may be presumed, how- ever, that the worker wishes to prepare a slide in which the diatoms are arranged in some given order on the coverslip. It must not be thought for one moment that this method of arranging diatoms on the cover- slip is of necessity confined to the produc- tion of artistic pictures. It is true that the method was developed by those who wished to build pictures, but it can also be used to line uj) in correct ranks all of the species found, for example, in one locahty. No method of arranging diatoms on, and attaching them to, a coverslip will compare with that of BelHdo 1927 (11360, 47:9). The description cited is one of very considerable complexity and goes into details not possible in the present place. It consists essentially, however, of coating a chemically clean covershp with an exceedingly thiu film of Bellido's ce- ment (Chapter 28, V 11.1 Bellido 1897) which is then dried. Bellitlo recommends 40 THE ART OF MAKING MICROSCOPE SLIDES Diatoms that the film be applied by dipping a needle into the cement and then drawing the flat of the needle sharply across the covershp. This leaves an invisible film of dry gelatin on the cover, and individual diatoms may be placed on this film to which they will not adhere until the film is slightly moistened by breathing on it. The moment this has been done the diatoms are permanently attached. Before individual diatoms may be se- lected for this technique, however, they must be dried. It is not safe to dry them on glass, to which they frequently adhere. It is better to attach a piece of mica to a slide with petrolatum and to evaporate the drop of water on this base. Individual di- atoms may then be picked up on the end of a hair under the microscope. There is usually enough grease on a normal hair to permit the diatom to adhere. Bellido de- scribes the ingenious idea of mounting a hair on the collar of a microscope objective in such a manner that the tip of the hair is in focus when the draw tube of the microscope is pulled halfway out. It fol- lows that when the draw tube is pushed home the hair will be out of focus, and also well above the plane of the object which is in focus. It is possible, therefore, to press the draw tube fuU}^ home, search one of the squares of mica for the required speci- men, pull out the draw tube until the hair is in focus, and then lower the microscope until the hair touches and picks up the object. Belhdo recommends that the hair be moistened with a little bromonaph- thalene and that the film of gelatin also be lubricated with the same reagent. He has also described, in the place quoted, a sealed chamber within which all these operations may be conducted without the risk of dust faUing on the preparation. Another device for handling individual diatoms on a mechanically operated hair has been described by Meakin 1939 {Microscope, 4:8). These mechanical de- vices are only necessary, however, for handling large quantities of rather small diatoms. A few months of practice with a hair mounted in any holder will enable the average worker to arrange diatoms di- rectly. A mechanical device is, of course, almost necessary if one is endeavoring to arrange the diatoms according to any artistic pattern. In any event, as soon as the diatoms have been arranged one breathes very gently on the coated cover- slip and pauses a moment or two. The covershp is then examined under the microscope and a few of the larger and rougher diatoms very delicately probed with a hair. If they are found to be firmly attached, it may be assumed that the smaller diatoms are also attached. If, how- ever, anything is found to be loose, one breathes again, and again probes until such a time as all the diatoms are fixed. These coverslips with the diatoms at- tached to them may now be laid on one side while the necessary cells for mounting are prepared. There will be required a turntable, shdes, a fine brush, a hot plate, and some de-waxed shellac (Chapter 28 V 11.2 Hitchcock 1884) which should be as thick as can conveniently be persuaded to flow from a brush of the size selected. A fine ring is then turned of a size shghtly smaller than the covershp. Save for the very largest diatoms a single ring of this cement will be sufficiently thick. It is ab- solutely necessary that these shellacked rings be baked if they are to become in- soluble in the bromonaphthalene used for mounting. As soon, therefore, as the alco- hol has evaporated from the shellac, the slides are placed on a hot plate, or for that matter, in an oven, and heated to just below the melting point of the shellac for at least 30 minutes. The cells, which would now be adequate for aqueous media, must be further processed for bromo- naphthalene mounting by having the top ground flat. This is done by taking some of the finest available carborundum, making it into a slurry with water, and spreading this on a sheet of fine quality plate glass. The slide, with the cell down, is then laid on this glass and, with a delicate finger placed over the center of the cell, moved gently backward and forward for a few moments. It is then picked up, washed under the tap, and examined with a strong lens to make sure that there is a flat, smooth area over the whole of the top of the cell. The slide is then washed free of Diatoms FLUID WHOLEMOUNTS IN NONAQUEOUS MEDIA 41 all traces of grit and dried in a dust-free place. As many prepared slides as are re- quired now have a small drop of bromo- iiaphthalene placed in each cell. If petro- latum was used to hold the coverslip in place while the diatoms were mounted on it, this should first be removed with ether to make quite certain that the diatom frustules are grease-free, dry, and clean. When one is satisfied as to this, the cover- slip is lowered in place on top of the bromonaphthalene and then, with some blunt instrument (Bellido recommends a toothpick) pressed on the cell until it is firmly attached. If the coversUp is flat and if the cell has been properly ground, this seal is sufficiently good to permit one to remove any exuded bromonaphthalene with a cloth before turning on an addi- tional layer of the shellac as a final seal. It is usually safer to follow this with an- other layer of some impermeable cement such as asphalt varnish. Though this process may sound labori- ous, it actually takes less time by this means to mount one each of the 200-300 species that may be found in a fossil de- posit on a single coverslip than it takes either to mount them individually on separate sHdes by any other means, or to endeavor to find them under a microscope if they are arranged at random. Wholemoiints in Gum Media General Principles Nature of the Process The preparations which have been de- scribed in the last two chapters are those in which the specimen is sealed in a pre- servative fluid. These mounts, as will be readily understood by anybody who has read the chapters, are difficult and labori- ous to prepare, so that most slides are made in a mounting as distinguished from a 'preservative medium. A mounting me- dium,, used in this sense, is one which itself hardens and holds the coverslip in place while at the same -time preserving the object contained in it. Mounting media may be divided into two large groups: first, those which are miscible with water; second, those which are not miscible with water, so that some initial treatment must be given to most objects before they are mounted. The media miscible with water are in themselves divisible into two types: first, those which are liquid at room tem- perature (dealt with in this chapter), second, those which are solid at room tem- perature and must be melted before they can be used. Water-soluble media are all colloidal dispersions of various materials, the colloids being in the sol phase for the media described in this chapter, and in the gel phase for the media described in the next. Types of Gum Media Employed Many formulas for mounting media of the sol-colloidal type are given in Chapter 27 under the heading M 11.1. They are dispersions of either natural or synthetic gums in water and must, therefore, depend for their hardening upon the evaporation of moisture from the edge of the coverslip. Were this to continue for an indefinite period, the media would naturally harden and crack; hence, most contain either glj^cerol or sorbitol to impart hygroscopic qualities. The prototype of all these media is Farrants', which is a simple dispersion of gum arable in water to which has been added a small quantity of glycerol to- gether with a preservative. All media de- rived from this follow the same pattern, differing mostly in the quantity of glycerol included and in the nature of the preserva- tive selected. The fundamental objection to gum-arabic media is that it is difficult to obtain a pure sample of the gum, and one has to go through a wearisome process of filtration to avoid having the mount filled with sand grains and pieces of stick. Another objection to this type of medium is the low index of refraction, which leaves objects mounted in it relatively opaque when they are examined by trans- mitted light. This difficulty is overcome in Berlese's medium, to which chloral hy- drate is added in considerable quantities with a view to increasing the index of re- fraction. The ordinary media of the Farrants' type have an index of refraction just over 1.3, while Berlese's medium and its modifications have indices of refraction as high as 1.47. Very few synthetic sub- stitutes for water-soluble gums are avail- able, the most promising at the present time being polyvinyl alcohol, which is used in the media of Downs, and of Gray and Wess. It is probable that the recent appearance on the market of water-soluble cellulose derivatives (for example, car- boxymethyl cellulose) may lead to the 42 Mounting GUM MOUNTS 43 ultimate suppression of gum arabic in mounting media. Types of Objects Which May Be Mounted Simple water-miscible liquid mountants are of far wider utility than is usually realized, for there has been a complete mental block on the part of most micro- scopists when faced with any mounting medium which is not a solution of a resin in a hydrocarbon. As a matter of fact, most simple objects such as the scales of fish, animal hairs, and the like, may be more readily mounted in aqueous media than in resinous ones. The actual process of mounting is so simple that it is regarded with distrust by those who have come to believe that only through complexity can good results be produced. With these media one merely takes the object which it is desired to mount, places it in the drop of mountant on the shde, and presses a coversUp onto the top. This process is not confined to relatively hard objects of the type described, but may also be apphed to many protozoa and other small inverte- brates. These do not make satisfactory permanent mounts by this method, for they ultimately reach a refractive index identical with that of the mountants and thus vanish; but a temporary mount of Paramecium, in one of these media, will show the internal structure to a class far better than will the average stained mount, and will also give a far better reaUzation of what the living object looks hke. Objects most commonly mounted however, are small arthropods of -the degree of transparency that does not re- quire that the skeleton be cleared in the manner described in Chapter 6. Gum mountants are not satisfactory in thick layers and the writer has never made a successful mount in a deep cell. There is no point in endeavoring to use shallow cells for these media, for the viscosity of the mountant is sufficiently high to pre- vent the coverslip from crushing small objects. Finishing Slides in Gum Media Exudate round the edges of the cover may be removed by washing with warm water, but it will be some time before the edges reharden. Moreover, no mounting medium containing glycerol or sorbitol can fail to absorb moisture from the air on humid days, and to lose it on dry days, so that it is usually better to finish the shde by applying a ring of varnish in the man- ner described in previous chapters. It does not matter what cement is employed, the writer's preference being for gold size, probably more from force of habit than from any other reason. Specific Example Preparation of a Wholemount op a Mite by the Method of Berlese The use of the name Berlese in the head- ing of this example is less an injunction to employ the mounting medium of that writer than a tribute to the method of col- lecting small arthropods which he intro- duced. This method is applied with the aid of the Berlese funnel which is seen in Fig. 21. This is a double-walled funnel, between the walls of which warm water may be placed and maintained at any desired temperature by applying a small flame to a projecting side arm. The temperature is not critical, so that no thermostatic mechanism is provided, but a thermom- eter may be inserted and used to read the temperature at intervals. A circle of wire gauze with a mesh of about K 6 of an inch is placed at the bottom of the inner glass funnel, and whatever material to be searched for mites is placed loosely on this gauze. The lower end of the glass funnel is then attached with modeling clay to a tube containing whatever medium is being used for the collection of the specimens. If the specimens are merely to be stored, rather than mounted at once, 95% alcohol may be placed in the tube, and it is then unnecessary to seal it to the base of the funnel. If, however, the specimens are to be mounted directly in Berlese's medium, in which better mounts can be prepared from living than from preserved material, 44 THE ART OF MAKING MICROSCOPE SLIDES Mites Fig. 21. Berlese funnel in use. the funnel must be sealed to prevent the more active forms from working their way out of the tube. After the mass has been placed in posi- tion a small lamp, certainly not of more than 15 watts, is mounted in any kind of a reflector some distance above the material. The animals in the material, therefore, find themselves surrounded by heat at the sides and plagued with hght froju above. As all of these animals are photophobic, they tend to move toward the lowest point of the mass from which they drop down the tube into the funnel. By this means it is possible in 10 or 15 minutes to collect the whole fauna from a large handful of any organic material which would, by any other means, take several hours to search. The use of the funnel is not, of course, con- fined to moss but may be used for hay, straw, shredded bark, or any other mate- rial from which small arthropods are cus- Mites GUM MOUNTS 45 tomarily collected. The only difficulty in using this equipment is in preventing the heat from getting too great. Some people use so large a lamp above, and so high a temperature around the edges, that many small arthropods are killed before they have time to fall into the trap which has been laid for them. The outer water for most uses should be at a temperature of 30° to 40°C., while the lamp above should under no circumstances raise surface tem- perature of the material above 60°C. These temperatures are for a moderately dry moss sample, and may be considerably exceeded when one is dealing with a drj^ material such as straw. Wet moss of the sphagnum type, however, requires lower temperatures. Assuming that permanent mounts are to be made, for record purposes, of all the small invertebrates which may be found in a moss sample, it is necessary to make adequate preparations to receive them while the moss is being treated. Two kinds of gum mountants are desirable: a high- refractive-index medium like Berlese, for the very heavy-walled forms such as the Oribatid mites and the Pseudoscorpion- ides; and a low-refractive-index mountant, like Gray and Wess, for the thinner-walled forms such as the Tyroglyphid and Gam- assid mites. This last medium is also suitable for Thysanura and for CoUem- boUa. Thick-walled beetles and fleas, if they are to be made into microscope slides, had better be treated as described in Chapter 6, and should be accumulated for this purpose in a tube of 95% alcohol. Sphagnum moss is also likely to yield a number of crustaceans, particularly Cla- docera and Ostracoda. These are better mounted in glycerol jelly, in the manner described in the next chapter, and should be transferred as soon as they are found to 30% alcohol where they will die with their appendages extended. They should not, however, be permitted to remain in this weak alcohol for longer than is necessary to kill them, but should then be trans- ferred to 95% alcohol. A considerable number of nematode worms are likely to turn up and should be treated as de- scribed in the last chapter, while a tube of some fixative should be provided to re- ceive any small annelids which may be found in the gathering, and which must be fixed, stained, and mounted at once. A brush will also be required, a supply of clean 3" X 1" glass slides, and a numljer of covershps. All being ready, and observation show- ing that no further forms are falling through the Berlese funnel, the collecting tube beneath it is now inspected to see roughly what one has gathered. If there are a considerable number of Gamassid mites or active insects, it is necessary gently to open a portion of the tube by pushing away the modelhng clay with the thumb and to let a minute drop of ether run down inside. When this has been done the tube is removed and the contents tipped out into a petri dish or similar con- tainer and the catch sorted. A mite, or similar form, which is to be mounted, is then picked up on the tip of a brush and transferred to a drop of the mountant. As little water as possible should be transferred with it and the mite should be pushed under the surface of the gum with the point of a needle. The mount is then inspected under the low part of the microscope and, if any large quantity of air has been carried in with the mite, the bubbles are released with the aid of a fine needle and allowed to come to the surface before the covershp is laid gently into place. The drop of gum should be of a considerable size and no endeavor should be made to press the coverslip down. If a reasonably thick layer of mountant is left almost any small arthropod will spread its legs like a textbook diagram before dying and will remain in this form indefinitely. Wholemoiints in Jelly Media General Principles Glycerol jelly is the only type of water- miscible medium known to most workers. Many objects, both plant and animal, which are usually prepared in glycerol jelly, are much better mounted by the method described in the last chapter, and the author would most warmly recom- mend to workers who have been using glycerol jelly that they try one of the methods there described. Formulas for the jelly media, the use of which is described in the present chapter, are given in section M 12.1 of Chapter 26, and it will be seen that they are all essen- tially the same. That is, they consist of a dispersion of gelatin, which has been di- luted with glycerol until the required index of refraction is obtained, and they have added to them some preservative. The older glycerol jellies, designed for use in European laboratories, do not in general contain sufficient glycerol to withstand the drying effects of an American laboratory. The author has in his possession many deep wholemouhts of fairly large crusta- ceans which remained perfect for ten years in England but which dried and cracked after only two years in the United States. There is little to choose among any of the media, apart from the consideration just given, and practice will soon permit the mounter to select the one which works best in his hands. Nature of the Process Mounts prepared in glycerol jelly may be made either on flat slides or as deep wholemounts. Glj'cerol jelly can be used for larger objects than can the gum media of the last chapter and, except for botan- ical specimens, the use of glycerol jelly is largely confined to the preparation of permanent slides of unstained crustaceans. These media are sohd at room temperature and must be melted before use. Material cannot be mounted directly from water unless the objects are soaked for a long time in molten jelly to allow some of the glycerol to penetrate. Mounts made from specimens taken directly from water are liable to have the jelly crack away from the object as it cools. Moreover, these media are not in their own right good preservatives so that material placed di- rectly from water into them is liable par- tially to decompose before the mount stabihzes. lit is conventional to transfer objects to these media from 50% alcohol, but it is better to harden the objects first in alcohol than to transfer them from alcohol to 50% glycerol and thence into the molten medium. Process of Mounting There are three separate stages in the preparation of glycerol-jelly mounts. The first is hardening the object; the second is getting the object from the hardening fluid into the 50% glycerol; and the third is the transfer from the 50% glycerol onto the slide. As these mountants cannot be used for stained specimens, there is little object in using any fixative other than alcohol. The transfer from the hardening alcohol to the glycerol, however, must be by such stages as will insure that the object does not col- lapse through osmotic pressure. This makes it impossible satisfactorily to mount the majority of nematode worms in 46 Principles JELLY MOUNTS 47 jelly, and thus necessitates the glycerol technique described in Chapter 3. The author prefers to transfer objects from 95% alcohol to 70% alcohol and then to add, by such stages as are necessary, enough glycerol to this mixture to insure that, when the alcohol has been removed by evaporation, 50% glycerol will remain. The two great difficulties in mounting material in these media are first to arrange the object on the shde before the jelly has time to solidify, and then to get the cover- in which they are left until they are thor- oughly permeated, and then placed in a stender dish on top of a water bath, seen in the left background of the picture. On this water bath, which is held about 10°C. above the melting point of the jelly, there is also placed the bottle containing the mounting medium and as many sHdes as will be required. A shde is taken, a molten drop of the medium placed on the warm shde, and the object to be mounted re- moved with a pipet and placed in this Fig. 22. Layout for jelly mounting. slip into place without disarranging the object. The writer has invented a tool for overcoming these troubles, the use of wliich will be described in some detail. The layout, when one is mounting a num- ber of objects, is shown in Fig. 22; the only object not commonly found in laboratories is the special tool shown in the left hand in the picture. This device is a short length of ^^-inch aluminum rod flattened at one end and rounded at the other. This alumi- num rod is screwed to a short length of brass tube which terminates in a wooden handle. The procedure in mounting small objects is as follows. The objects them- selves are accumulated in 50% glycerol, drop. The shde is then removed from the water bath and examined under a micro- scope while a warmed needle is used to push the object into the required position. Make sure that it is in contact with the slide and that there is a considerable amount of jelly above it. In the center of the picture will be seen three slides which have so been treated and laid on one side until the medium has hardened. As many slides as are required may be treated in this manner so that one has a series of preparations, each of which con- tains a domed drop of solid jelly with the object lying in the required position at the bottom of it. Now take a cover glass of the 48 THE ART OF MAKING MICROSCOPE SLIDES Finishing required size, moisten the underside by breathing on it, or by smearing on it a little 50% glycerol, and then lay this shde on top of the domed drop of jelly. The cylinder of aluminum is then taken, warmed in the flame until it is somewhat above the melting point of the jelly, and pressed gently on top of the center of the slide until enough jelly has been melted to permit the cover to drop down to the re- quired level. Very little practice is re- quired before one can flatten down the coverslip without in any way disturbing the arrangement of the object at the bottom, which remains throughout this whole process in a layer of solid jelly. In this way the coverslip is flattened without disturbing the object, and one avoids the constant nightmare of endeavoring to lower a coverslip onto rapidly cooling jelly without disturbing the contained object. If the specimens are so thin that pressure may be applied, a clip is attached and the slide placed for a few minutes on a hot table, to permit an equalization of the pressures between the object and the sur- rounding jelly. The method of mounting in deep cells is practically identical and is, with the de- scribed tool, just as convenient. The slide, with the cell cemented to it, is warmed on a water bath and then filled with molten glycerol jelly. Sufficient glycerol jelly should be used to leave a high domed layer protruding above the cell; air bubbles must be displaced with a hot needle. The object is then placed in the glycerol jelh', arranged in the required position within the cell, and ^then gently' laid on one side to cool. A moistened coverslip is then placed in the center of the dome and pressed down with the warmed tool until it is flattened against the top of the cell. Large cells of this nature should always be placed on a hot plate for at least two or three hours before they are finally cooled, for the commonest cause of break- down of deep jelly mounts is failure to permit the osmotic tension to equahze be- tween the object and its surrounding medium before coohng. Finishing Glycerol Jelly Mounts Most glycerol jelHes contain so much glycerol that they cannot be left unsealed. They may appear excellent for a few days but sooner or later the glycerol will spread out over the surface of the shde, even to the extent of moistening the label and causing it to fall off. The slide, however, must be cleaned before it can be sealed. Make sure that the slide is cold — a refrig- erator is very convenient — and then with a sharp razor blade or scalpel trim away all the unwanted jelly. Then remove the smears that are left with a moist cloth and use another cloth moistened with 95 % alcohol to remove any glycerol which may remain on the glass. There is always some residual gh'cerol, however, which makes it essential that the first coat of cement should be a gelatin-dichromate mixture of the type of Riiyter 1934 (Chapter 28, V 12.1). This is melted on the water bath and applied to the edges of the coverslip with a brush. The slide need not be warmed since these cements stay molten at quite low temperatures. The slide is placed on one side to dry and then given an additional coat of any waterproof ce- ment. It is a worthwhile precaution, be- fore applying the last coat of cement, to wipe off the slide with 95% alcohol to re- move any trace of glycerol. Specific Example Preparation of Small Crustaceans in Glycerol Jelly The term small crustaceans, as here used, includes all specimens up to the size of a Gammarus, as well as the numerous larvae which are found both in fresh and salt waters. This group contains a large number of fascinating forms of universal distribution. A brief word must be said on methods of collection and preservation be- fore passing to actual mounting. Free-swimming crustaceans, whether marine or fresh-water, are collected by means of a plankton net. This is a long conical net, made by the professional from bolting silk, and by the amateur from the Crustaceans JELLY MOUNTS 49 nearest woman's stocking. What distin- guishes tliese nets from all others is that the lower end of the net, instead of being tied off, is blocked by a small glass tube tied firmly in place. These nets are towed slowly through the water, a process which results in the accumulation of large quan- tities of plankton within the net. The net itself is then very slowly and with constant shaking lifted from the water with the result that all those forms which have been accumulated in the net are washed down in the small glass tube, the contents of which may then be tipped out into an- other container. Unless one is on a very long collecting trip, it is better to bring home planktonic crustaceans alive, and for this purpose the contents of the tube should be tipped into at least a gallon of well-aerated water — fresh or salt as the case may be — for transference back to the laboratory. It is almost impossible to make a satisfactory preparation from the horri- ble messes which result from endeavor- ing to preserve directly the contents of an entire tube by throwing it into formalde- hyde, a technique too often employed. When plankton samples are brought back to the laboratory they may again be con- centrated with a plankton net or other de- vice, and the still-Uving individuals are picked out with a pipet and transferred to a small bowl of clean, well-aerated water. Marine forms in particular should be washed in numerous changes of water to rid them of adherent phytoplankton which is almost impossible to remove from the fixed specimen. Though every mounter of microscope slides will go to endless lengths to narcotize many invertebrates, few ever appear to consider this necessary in the case of crustaceans. It is however much easier to identify specimens if their ap- pendages are properly spread out, and the writer always kills crustaceans either ^vith weak alcohol or with chloroform, a few drops of which are sprinkled on the sur- face of the water. As soon as they have dropped to the bottom they will be found to be flexible, and may then be picked out and placed on a sUde, the appendages ar- ranged more or less in the order required, and 95% alcohol cautiously dropped on them until they have stiffened in position. Such specimens may then he transferred directly to a tube of do% alcohol where they will retain the required shape until needed for mounting. Some small crusta- ceans are always found mixed up with weeds, both marine and fresh-water, from which they may be readily sei)arated in the laboratory. Large masses of the weeds are brought back to the laboratory, and placed in shallow dishes with just sufficient water to cover them. The lack of oxygen in the water soon forces the crustaceans to detach themselves and gather round the edges of the bowl from which they may be removed with a pipet. There are a few marine copepods {Dyspontius, for ex- ample) which are considered exceedingly rare, for the reason that nobody ever col- lects them. These forms have sucking mouth-parts which they use to extract the juices of algae. They never become de- tached from these algae in the normal course of events. They may be readily collected by taking large masses of the specific alga, placing it in weak alcohol, shaking it vigorously, and then examining the sludge which is deposited at the bot- tom of the jar. Good hauls of these forms may also often be found at marine stations by going through the sludge which collects at the bottom of the jars in which both algae and ascidians have been stored. There are also many small crustaceans which dwell in mosses, even in those which are apparently quite dry. These will not be secured if the moss is treated with a Berlese funnel in the way described in the last chapter. The only way to collect them is to soak the moss in some kind of narcotic until the crustaceans are stunned, then to rinse off the moss in a considerable volume of fluid which is allowed to settle, and to examine the sludge. The same proc- ess, apphed to marine sands between tide- marks, often discloses small forms not found by any other means. Another fruit- ful way of collecting so-called rare forms is to go netting at night, since there are many marine crustaceans (Cumacea, for example) which become planktonic only at night. Parasitic forms, particularly cope- pods, are also often overlooked, particu- larly those which inhabit invertebrates. No matter how these forms are col- 50 THE ART OF MAKING MICROSCOPE SLIDES Crustaceans lected, they should all be narcotized before killing, and should be killed in 95% alco- hol and stored in this fluid, with a trace of glycerol, until required. When it is de- cided to mount them they are removed from 90% alcohol to 70% alcohol. After they have been permeated with this, glycerol is added little by little until the total concentration of glycerol is such that, if the alcohol be evaporated, a 50% glycerol-water mixture will be left. The final evaporation is best done at a tem- perature of 30° to 40°C. and is very con- veniently conducted in a desiccator through which a current of air is drawn with any aspirator device. If the specimens are not very minute, it is desirable that the 50 % glycerol should be poured off and replaced with the molten mounting me- dium in which the specimens should re- main for an hour or two before mounting. Do not, however, transfer to the molten jelly more specimens than you are able to mount at one time, since prolonged soak- ing in the molten medium tends to soften them. The required number of slides are then warmed and a specimen, in a large drop of molten medium, placed on each. The specimen is then arranged with a needle and chilled rapidly. If there is not a large domed drop of medium over the top of the specimen, further medium is added from a pipet until there is a thick layer. When all the specimens have thus been mounted in the required position with a big dome of cooled jelly above them, a covershp thinly smeared with 50% glyc- erol is laid on each. All of the shdes may be provided with coverslips resting loosely on them before going further. The alumi- num rod shown in Fig. 22 is now warmed and pressed down on the coverslip until the latter has come to rest on the speci- men, or on the walls of the cell. A httle practice is required to be able to do this without pressing down either so hard that one crushes the specimen or so long that one melts the jelly surrounding it. If these specimens are mounted in a medium which does not contain sufficient glycerol, and which accordingly cracks and dries out when they are transferred to a drier environment, they may be re- mounted without very much difficulty by cracking the covershp, soaking the speci- men in 50% glycerol for a week or two, then removing the rest of the coversUp and remounting as though one had a fresh specimen. Wholemounts in Resinous Media General Principles Mounting whole objects by the methods described in the last five chapters involves little preparation of the specimen but a great deal of preparation of the mount. In preparing wholemounts in resinous media a great deal of attention must be paid to the preparation of material, although the actual mounting is simple. Resinous media are used for whole- mounts not only because they permit mounting stained objects but more par- ticularly because they impart to the speci- men a great degree of transparency. This transparency comes from the increase in the index of refraction when the specimen is completely impregnated with the resin. These resins are not, however, miscible with water, hence the water must first be removed {dehydration) and then the de- hydrant replaced with some material {clearing agent) with which the resin itself is miscible. Before these operations the specimen must be killed and hardened {fixed) and it is customary to stain the specimen in order to bring out those in- ternal structures which would become in- visible, were they not colored, through the increase in transparency. All of the follow- ing operations must, therefore, be con- ducted and will be discussed in turn : 1. narcotizing and fixing 2. staining 3. dehydrating 4. clearing 5. mounting Narcotizing and Fixing Specimens Hard objects such as small arthropods, hairs, and the hke may be dehydrated and mounted directly into resinous media,' but are far better prepared according to the manner described in Chapter 4. Most ob- jects which are mounted in resinous media are, however, too^soft to withstand the process of dehydration and^clearing with- out special treatment. Though hardening and fixing agents were once considered as separate, they are now usually combined into a solution known as a fixative. Before deahng with fixatives, however, it is neces- sary to point out that few small animals, on being plunged into a fixative, will re- tain their shape, so that it is necessary first to narcotize them in some solution which will render them incapable of muscular contraction. Narcotization may be caused either through the blocking of nerve impulses which cause contraction, or by some treat- ment which will inhibit the actual con- traction of the muscle. For blocking nerve impulses there are a wide range of nar- cotics available (see Chapter 19, AF 50) and making a choice between them must be a matter of experience. It is to be rec- ommended, in the absence of experience, that one of the solutions containing co- caine be first tried, since cocaine is the nearest approach to a universal narcotic known to the author. Should cocaine not be available, crystals of menthol may be sprinkled on the surface of the water con- taining the specimen. It is very important to distinguish between narcotization and kilUng, for a good wholemount cannot be made from a specimen which has been permitted to die in the narcotic. Narcotization should always proceed slowly; that is, one should add a small 51 52 THE ART OF MAKING MICROSCOPE SLIDES Narcotizing quantity of narcotic at the beginning and increase tlie quantity later, adding the fixative only after the cessation of move- ment. This is easy to judge in the case of motile forms, which may be presumed to be naiTotized shortly after they have fallen to the bottom, but in the case of sessile forms it is necessary to use a fine probe, preferably a hair, to determine the end point of narcotization. Recommended Narcotics and Fixatives for Specific Objects It must be pointed out that the primary purpose of fixing an object before making a wholemount is to retain as nearly as possible the natural shape. The fixative selected should, therefore, contain an im- mohilizing agent as well as a hardening agent. Gray 1933 (11360, 53:14), in a dis- cussion of the principles governing the selection of fixatives, came to the conclu- sion that there were only two good im- mobihzing agents. These were heat and osmic acid. It is therefore necessary, when dealing with highly contractile or imper- fectly narcotized animals either to select a fixative containing osmic acid (Chapter 18, F 1000) or to heat the fixative. Neither osmic acid nor heat are good hardening agents and should not, therefore, be used alone. The best hardening agents for ob- jects which are subsequently turned into resinous wholemounts appear to be chro- mic acid and formaldehj'de, used either singly or in combination, and these solu- tions are usuall}'' acidified with acetic acid to assist in the preservation of internal structures, particularly nuclei. Reference to the classification of fixatives at the be- ginning of Chapter IS will show a large number of solutions fulfilling these re- quirements and no specific recommenda- tion need be made here. Mercuric chloride, particularly in the solution of Gilson 1898 (Chapter 18, F 3000.0014), is another good fixative to use before wholemount- ing. The following recommendations, drawn largely from Gra}- 1935 (Microsc. Rec, 35:4), and Gray 1936 {Microsc. Rec, 37 :10), are to be taken only as suggestions representing the author's opinion and should be used as a basis for further experiment. NoNCONTRACTiLE Protozoa. These do not require nnrcotization and may be fixed directly in a weak solution of osmic acid. The writer, however, much prefers his own technique (described at the end of this chapter) for the handling of these specimens. Individual Contractile Protozoans, These are very difficult to handle. Ten per cent methanol is quite a good narcotic for Dileptus, but 1 % hydroxylamine seems better for Spirodomum. It is the writer's practice to try new forms with the follow- ing narcotics in the order given: 10% methanol, 1% hydroxylamine, 1% ure- thane, AF 51.1 Hanley 1949, AF 51.1 Rousselet 1895, and AF 51.1 Cori 1893 (Chapter 19). There are many forms, how- ever, which do not respond to these nar- cotics and of which it appears almost im- possible to make a good wholemount. Individual rhizopods, as Amoeba and Difflugia, are best fixed to a coverslip in the following manner. Take a clean cover- slip and smear on it a very slight quantity of fresh egg albumen. The solution of Mayer 1884 (Chapter 26, V 21.1), which is often recommended for the purpose, should be avoided, for the glycerol and preservative included in it inhibit the ex- pansion of the animals. Each individual protozoan is placed in the center of a covershj) and left to expand. While this is going on a flask (or kettle) is fitted with a cork. Through this cork is inserted a glass tube the outer end of which has been drawn to a fairly fine point. The water in the flask is boiled to produce a jet of steam. As soon as the protozoan is satis- factorily expanded, the coverslip is picked up very gently and the underside passed momentarily through the jet of steam. This instantly hardens the protozoans in position and at the same time cements them to the coverslip through the coagu- lation of the egg white. The coverslip should then be transferred to any standard fixative solution for a few minutes before being washed and stored in alcohol. Among the Suctoria, Acineta and Den- drocometes may be prepared by placing them in a good volume of water, sprink- ling menthol crystals on the surface, leav- ing them overnight, and then adding suffi- Fixing WHOLEMOUNTS IN RESINOUS MEDIA 53 cient 40% formaldeh3^cle to bring the total strengtli to 4%. These forms may then be transferred to alcohol for staining and preparation as resinous wholemounts or may be mounted directly in formalde- hyde as described in Chapter 2. Stalked Ciliate Protozoans. These forms are quite easy to fix provided one reahzes that double narcotization is neces- sary: once for the stalk and once for the head. The author's technique is to nar- cotize with Rousselet's solution until the snapping movements have slowed up and then very gently to add weak hydrogen peroxide. The specimens are then watched under a microscope and the selected fixative — which must contain osmic acid — is flooded onto them at the exact moment when the cilia straighten out and become stationary. This is satisfactory' with Carchesium, Zo- othamnium and Vorticella. Opercularia and Epistylis have noncontractile stalks and one need, therefore, only use hj^drogen peroxide. The writer has never made a satisfactory mount of Scalhidium or Pyxicola. CoELENTERATA. Hj'droids are usuallj' narcotized with menthol, though the writer prefers liis own mixture (Chapter 19, AF 51.1 Graj') for the purpose, and fixed in a hot mercuric-acetic mixture. A description of the narcotization of Hydra is given in Chapter 9 and a detailed ac- count of the preparation of Medusae in Chapter 20. Anthozoa, particularly the small ones likely to be prepared as whole- mounts, can be narcotized with menthol, though magnesium sulfate is better. Platyhelminthes. Some of the smaller fresh water Turbellaria (e.g. Vortex, Microstortmm) may be narcotized satis- factorily by adding small quantities of 2% chloral hydrate to the water in which they are swimming. Another good techniciue is to isolate the forms in a watch glass of water and place the watch glass under a bell jar together with a small beaker of ether. The ether vapor dissolves in the water and narcotizes these forms excel- lently. A detailed account of the method of handling the liver fluke is given in Chapter 20 and may be satisfactorily employed for other parasitic flatworms. Annelida. Small, marine, free-living Polychaetae make excellent wholemounts and do not usually need to be narcotized before killing. They should, however, be stranded on a slide and a very small quantity of the fixative dropped on them, so that they die in a flat condition which makes subsequent mounting possible. Much more reaUstic mounts are obtained by this means than if they are laboriously straightened before fixing, for they usu- ally contract into the sinuous wave which they show when swimming. There seems to be no certain method of fixing the Nereids with their jaws protruding and one has to rely on chance to obtain one in this condition. The free-swimming larvae of marine polychaetes are very difficult to fix fsatisfactorily, because the large fiota- tion chaetae usually fall out. The writer prefers for these, as for other marine invertebrate larvae, to concentrate a relatively large quantity of the plankton and then to flood over it three or four times its volume of Bouin's fixative (Chapter 18, F 5000.1010 Bouin 1897) at 70°C. The specimens are then allowed to settle, the fixative poured off, and re- placed with 70% alcohol which is replaced daily until it ceases to extract yellow from the specimens. By hunting through a large mass of plankton so fixed, one can usually obtain a considerable number of specimens in a perfectly expanded condition. Fresh water Ohgochaetes are best narcotized with chloroform, either by adding small quantities of a saturated solution of chloroform in water, or by placing them in a small quantity of water under a bell jar in which an atmosphere of chloroform vapor is maintained. Leeches are difficult to handle and the author has had most success bj' placing them in a fairly large quantity of water to which is added, from time to time, small quantities of a saturated solution of magnesium sulfate. As soon as the leeches have fallen to the bottom considerably larger quantities of magnesium sulfate can be added, which will leave the leeches, in a short time, in a perfectly relaxed, but not expanded, condition. They should then be flattened between two sUdes and 54 THE ART OF MAKING MICROSCOPE SLIDES Stains fixed in Zenker's fluid (Chapter 18, F 3700.0010 Zenker 1894). After the speci- mens have been fixed sufficiently long to hold their shape when the glass plates are removed, they are transferred for a couple of daj^s to fresh fixative and then washed in running water overnight. If the crop contains any considerable quan- tity of blood, it will be necessary to bleach this before a satisfactory stained wholemount can be made; the specimens should, therefore, be transferred to the bleach of Murdoch 1945 (Chapter 19, AF 31.1) where they should remain for a few days. Bryozoa. Marine bryozoans may be narcotized without difficulty by sprinkhng menthol on the surface of the water con- taining them. Subsequent fixation is best in some chromic-acetic mixture, for osmic acid|tends to precipitate on the test and blacken the specimen. It may be pointed out that, for taxonomic purposes, dried wholemounts of the test prepared as described in Chapter 1 are of more value than are wholemounts with the expanded animal. It is usually recommended that fresh-water bryozoans be narcotized in some cocaine solution, but the writer has found menthol just as good and much easier to use. Fresh-water bryozoans should be fixed directly in 4% formalde- hyde since they shrink badly in any other fixative. Gastrotricha. These give excellent results by the special technique for minute fresh-water animals described at the end of this chapter. Small Crustaceans. These are some- times prepared as resinous mounts, though the writer prefers to mount them in glycerol jelly in the manner described in Chapter 5. They may be narcotized in weak alcohol and fixed in almost any fixative. Other Arthropods. Wholemounts of most small arthropods are better made in gum media in the manner described in Chapter 4. A detailed description of the preparation of the skeleton of an insect for mounting in Canada balsam is among the typical preparations described at the end of this chapter. Choice of a Stain It is now to be presumed that, whatever method of narcotization and fixation has been employed, the specimens to be mounted have been washed free from fixative and accumulated either in water or 70% alcohol. The reason that so many formulas and methods for staining are given in Chapter 20, 21, and 23 is that no two people have ever agreed on the best method of staining anything. The sug- gestions which follow, therefore, are hkely to be modified by every individual reading the book; but they are included for the sake of those inexperienced in making wholemounts. Small Invertebrates and Inverte- brate Larvae, These are best stained in carmine by the indirect process : that is, by overstaining and subsequent differentia- tion in acid alcohol. For most specimens the writer prefers Grenacher's alcoholic- borax-carmine (Chapter 20, DS 11.22 Grenacher 1879). As an alternative, particularly for marine invertebrates, he has frequently used the two formulas for Mayer's paracarmine (Chapter 20, DS 11.22 Mayer 1892a and 1892b). With these stains available there are very few small invertebrates or invertebrate larvae which cannot be prepared. Larger Invertebrate Specimens, Larger specimens are better stained by the direct process: that is, exposed for a con- siderable length of time to a very weak solution of stain and subsequently not dif- ferentiated. Tills process is described in considerable detail for the fiver fluke in Chapter 20; the directions there given ap- ply equally to earthworms, leeches, or medium-sized polychaetae. Vertebrate Embryos. These seem to stain more satisfactorily in hematoxyhn than in carmine solutions, the author's preference being for the formula of Carazzi (Chapter 20, DS 11.122 Carazzi 1911). This formula is not very well known but may be used whenever the solution of Delafield is recommended. Detailed in- structions for the use of this stain on a chicken embryo are given in Chapter 20. People who wish to produce a startling, Dehydration WHOLEMOUNTS IN RESINOUS MEDIA 55 rather than a useful, mount are recom- mended to try the technique of Lynch (Chapter 20, DS 13.7 Lynch 1930). Plant Materials. Plant specimens for wholemounts often consist of only one, or at the most two, layers of cells and are easier to stain than zoological specimens. The nuclei may be stained either with saf- ranin (Chapter 20, DS 11.42) or with any of the iron hematoxyUn techniques (Chap- ter 20, DS 11.11) which in zoological procedures are rigorously confined to sec- Dehydration is carried out by soak- ing the specimen in gradually increasing strengths of alcohol, it being conventional to employ 30%, 50%, 70%, 90%, 95%, and absolute alcohol. The writer prefers to omit from this series, unless the object is very deUcate, both the 30% and the 50% steps in the process, thus starting with di- rect transfer from water to 70% alcohol. The only difficulty likely to be met in de- hydration is in the handling of small speci- mens, for if they are in specimen tubes it Fig. 23. Transferring objects between reagents with cloth-bottomed tubes. tions. A contrasting plasma stain may be used after the nuclei have been well differentiated. Dehydration It is to be presumed that the specimens, plant or animal, stained or unstained, are now accumulated either in distilled water or in 70% alcohol according to the treat- ment which they have had. It is now necessary to remove the water from them before they can be transferred .into a resinous mounting medium. Ethanol is widely used as a dehydrant and, at least in the preparation of wholemounts, only its nonavailabihty should make any sub- stitute necessary. If substitution is neces- sary, acetone or methanol, in that order of preference, may be used, but they have the disadvantage of being more volatile than ethanol and, therefore, require more care in handhng. is almost impossible to transfer them from one to the other without carrying over too much weak alcohol. The writer has long since abandoned the use of tubes in favor of the device seen in Fig. 23. This is a short length of glass tube, open at both ends, with a small piece of bolting silk or other fine cloth tied across the lower end. The specimens are placed in these httle tubes which (see illustration) are trans- ferred from one stender dish to another wdth a minimum chance of contamination. These tubes are commercially available in England but in America must either be imported or homemade. There is no means of judging when de- hydration is complete save by attempting to clear the object. It is unwise to beheve the label on an open bottle or jar if it says absolute alcohol because this reagent is hy- groscopic and rapidly absorbs water from the air. One should, therefore, keep a 56 THE ART OF MAKING MICROSCOrE SLIDES Clearing quantity of anhydrous copper sulfate at the bottom of the aljsolute alcohol bottle and cease to regard the alcohol as absolute when the salt starts turning from white to blue. More wholemounts are ruined by being imperfectly dehydrated than by any other method, and even the smallest speci- men should have at least 24 hours in abso- lute alcohol before being cleared. If the specimen is to be mounted in Canada balsam, or one of the substitutes for it, it must next be cleared, but if it is to be passed directly to Venice turpentine the reader should turn to end of the chap- ter for a detailed description of this technique. The Choice of a Clearing Agent A clearing agent must be some sub- stance which is miscible both with abso- lute alcohol and with the resinous medium which has been selected for mounting. The ideal substances for tliis purpose are essential oils for they impart just as much transparency to the specimen as does the resin used for mounting, so that one has, as it were, a preview of the finished specimen. The use of xylene or benzene, which is so widespread in the preparation of paraffin sections (see Chapter 12) has tended to spread into the preparation of wholemounts, for which purpose, in the author's opinion, they are worthless. They have a relatively low index of refraction ; hence one cannot tell whether or not the slight cloudiness of the specimen is due to imperfect dehydration until after they have been mounted in balsam. The writer's first choice is terpineol (synthetic oil of hlac) which has advan- tages possessed by no other oil. It is read- ily miscible with 90% alcohol, so that it will remove from the specimen any traces of water which may remain in it through faulty dehydration, and it has also the property of not making specimens brittle. The odor is very shght and rather pleas- ant. Oil of cloves is the most widely recommended essential oil for the prepa- ration of wholemounts and it has only two disadvantages: its violent odor and the fact that objects placed in it are rendered brittle. If a small arthropod be cleared in oil of cloves, it is almost impossible to get it into a wholemount without breaking off some appendages. Oil of cloves is, how- ever, miscible with 90% alcohol. Oil of cedar (more correctly oil of cedar wood) has been recommended in the literature and has the advantage of having a pleas- ant odor and of not rendering ol:)jects brittle. Unfortunately it is very sensitive to water so that perfect dehydration in absolute alcohol is necessary before en- deavoring to clear with it. Two clearing agents, wliich are excellent for unstained specimens, are very little known. These are turpentine and acetic acid. The acid cannot be used with stains for obvious reasons, while the turpentine is a strong oxidizing agent and cannot, therefore, be used after hematoxyhn, though it is perfectly safe with carmine. Absolute (glacial) acetic acid is miscible at all proportions both with water and with Canada balsam. If small arthropods are to be mounted in balsam, rather than in the manner described in Chapter 4, they may be dropped into acetic acid, left there until they are completely dehydrated, and then transferred directly to balsam. This little-known technique is strongly to be recommended. Mounting Specimens in Balsam Nothing is easier than to mount a speci- men in a resinous medium, provided that it has been perfectly dehydrated and cleared. A properly made wholemount should be glass-clear, but it will not be clear in balsam unless it is clear in ter- pineol or clove oil. Not more than one in a thousand wholemounts has this vitreous appearance, and the Avorker who is ac- customed to looking at rather cloudy wholemounts should take the trouble to dehydrate a specimen thoroughly, then to remove the whole of the dehydrating agent with a clearing agent, and then to mount properly in balsam. The first step, therefore, in making a mount in, say, Canada balsam is to make quite certain that the specimen in its es- sential oil is glass-clear; the second step is to make certain that one has "natural" Canada balsam and not "dried" balsam which has been dissolved in xylene. Solu- tions of dried balsam in hydrocarbons are Mounting WHOLEMOUNTS IN RESINOUS MEDIA 57 meant for mounting sections and are, for this purpose, superior to the natural bal- sam. Natural balsam is, however, just as preferable for wiiolemounts and is just as easy to obtain. If it is found to be too thick for ready use, it may be warmed gently until it reaches the desired consist- ency. A single small specimen is mounted by placing it in a drop of balsam on a slide and then lowering a covershp horizontally (Fig. 24) until the central portion touches the drop. The coverslip is then released and pressed very gently until it just touches the top of the oliject. By this means it is possible to retain the object in the center of the covershp and also, if one is using natural balsam which does not shrink much in drjing, to avoid using cells for any but the largest object. Unfortu- nately most people are accustomed to mounting sections in thin balsam by the teclmique shown in Fig. 25: that is, b}^ touching one edge of the covershp to the drop and then lowering it from one side. The objection to this is that the balsam, as is seen in the figure, immediately runs into the angle of the coverslip, taking the object with it, and it is difficult to lower the covershp in such a waj' that the object is left in the center. If one is mounting thin objects, or deep objects in a cell in which a cavity has been ground, it is desirable to hold the coverslip in place with a clip while the balsam is hardening. This proc- ess is seen in Fig. 25, the type of chp there shown being made of Phosphor bronze wire, and is far superior, in the writer's opinion, to any other type. This description presumes that one is using natural Canada balsam, which is un- questionably the best resinous medium in which to prepare wholemounts. If one is using one of the thin resinous media, many formulas for which are given in Chapter 26 under the heading M 30, a very differ- ent technique wiU have to be adopted. In the first place these media are so thin that it is almost impossible to ajiply the cover- shp as shown in Fig. 24, and one is forced to adopt the technique shown in Fig. 25. This difficulty may be avoided by placing the object on the slide, placing a drop of the medium over the object, and tlicn placing the shde in a desiccator until most of the solvent has evaporated. A second layer is then placed on toj-), and a large drop, or rather a thick coat of varnish, is thus built up over the specimen. A cover- slip is then applied and the slide warmed until the resin becomes fluid. The best use for solutions of balsam in making wholemounts is in the preparation of dehcate specimens or a large number of objects. The technique for the former is described in Chapter 20. In the latter case the objects are transferred from the clear- ing medium to a tube or dish of the solu- tion of balsam in whatever hydrocarbon has been selected, and the solvent then evaporated. When the balsam which re- mains has reached a good consistency for mounting, each specimen is taken, to- gether with a drop of balsam, and placed on a slide. A covershp is then added. By this method large numbers of shdes may be made in a short time. It is not necessary to use solutions of dried balsam, and the writer prefers, for this pur^DOse, to dilute natural balsam with benzene. Mounting large objects in a deep cell in Canada balsam is not to be recommended for the reason that the balsam becomes yellow with age and, in thick layers, tends to obscure the specimen. A wholemount of a 96-hour chicken embryo, for example, is of very doubtful value; but if it has to be made it is best first to impregnate it thoroughly with a fairly thin dilution of natural balsam. It is then placed in the cell, piling the solution up on top, and left in a desiccator. The cell is refilled as the evaporation of the solvent lowers the level. When the cell is finally completely filled with solvent-free balsam it is warmed on a hot table and the covershp applied directly. Finishing Balsam Mounts If a mount has been properly made with natural balsam, and if the size of the drop has been estimated correctly, no finishing is recjuii'cd since no balsam will overflow the edges of the coveislij). Natural balsam takes a long time to harden and, if one has a fairly thick mount the covershp of which is not supported, drying cannot be hastened by heating as this will liciuify the balsam, causing the coverslip to tip 58 THE ART OF MAKING MICROSCOPE SLIDES Finishing Fig. 24. Applying coverslip to balsam wholemount. Fig. 25 (inset). Wrong way to apply coverslip to balsam wholemount. to one side. Mounts in natural balsam are better put away for a month or two before any attempt is made to clean them. The slide should be cleaned, when it is suffi- ciently hard, first by chipping off any ex- cess balsam with a knife and secondly by wiping away the chips with a rag moist- ened in 90% alcohol. This will leave over the surface of the slide a whitish film w^hich may then be removed with a warm soap solution, and the sHde may be pol- ished before being labeled. The writer prefers to apply a ring of some cement or varnish round the edge of his balsam mounts for two reasons. In the first place such a ring diminishes the rate of oxidation so that the mounts do not start going brown around the edges. In the second place, such shdes are less Ukely to be damaged in students' hands because of the psychological effect produced by a well-finished shde. One must carefully avoid using any cement or varnish which is soluble in, or miscible with, balsam; the author prefers to use one of the numerous cellulose acetate lacquers which are avail- able on the market. The method of apply- ing such a ring has been described in Examples WHOLEMOUNTS IN RESINOUS MEDIA 59 Fig. 26. Balsam wholemount ready for drying. Chapter 1. With regard to labeUng, it may the label is to be attached should, there- be pointed out that no power on earth will fore, be cleaned more carefully than any persuade gum arable, customarily used for other. The writer prefers to moisten both attaching labels, to adhere to a greasy or sides of the label, press it firmly to the oily sKde: the portion of the sUde to which glass, and to write on it only after it is dry. Specific Examples Preparation of a Wholemount of Pectinatella Though this exposition specifically ap- pUes to preparing wholemounts of the animal named, it applies equally well to the preparation of wholemounts of any other fresh-water bryozoan or, as a matter of fact, for any small invertebrate of about the same size and consistency. Pectinatella has been picked only for the reason that it has a habit of turning up on the walls of the aquaria in the writer's laboratory. If it does not turn up in aquaria in the reader's laboratory, it will be necessary for him to collect the specimen. Fresh-water bryo- zoans are rather like gold: that is, they oc- cur where you find them. Profitable hunt- ing grounds are the underside of the leaves of large water plants and the surface of branches of trees which have fallen into the water but have not yet had time to de- cay. An old trick of European collectors was to lower a length of rope into a pond in which bryozoans were known to occur, and to leave it there for the summer. It was astonishing how frequently, when these ropes were pulled up again in the fall, they were found to be covered with colonies of bryozoans. However the bryozoans be obtained, it 60 THE ART OF MAKING MICROSCOPE SLIDES Pectinatella is necessary next that they should be nar- cotized. The material on which they are living is cut up and the pieces placed in fingerbowl or aquarium of pond water. Distilled water and tap water are lethal to these forms. There should not be so many specimens that they touch each other on the bottom of the fingerbowl, and the fingerbowl itself should be completely filled with water. Fresh-water bryozoa are a little sensitive to heat and may not re- spond well to the high temperatures found in some laboratories. In this case it is as well to put the fingerbowl containing the specimens in a refrigerator, preferably one held at about 10°C, and to leave them there overnight. They may then be brought out and narcotized before they have time to suffer from the increasing temperature. It is usually recommended that fresh- water bryozoans be narcotized with co- caine, either as a straight 2 % solution, or in one of the mixtures the formulas for which are given in Chapter 19 under the heading AF 50. The writer prefers to use menthol which is both cheaper and easier to obtain. For an ordinary fingerbowl about a gram of menthol sprinkled on the surface will be sufficient. There is no means of foreteUing how long it will take the specimens to become narcotized, therefore one must look at them at inter- vals until they are seen not to be contract- ing. This may not be due to narcotization, however, so one should take some very delicate instrument — a hair mounted in a wooden handle is excellent — and use this to push the individual polyps. If, on re- ceiving a push, they contract sharply, it is evident that little narcotization has taken place and more menthol should be sprin- kled on the surface. If, on being pushed with a hair, they contract slowly, it is evident that they are partly narcotized and one must be careful not to disturb them further for at least ten minutes, for if they contract in a narcotized condition they will not again expand. The right stage for killing has arrived when no amount of shoving with a hair will per- suade the specimens to retract, and an ex- amination under a binocular microscope shows the ciliary action on the lophophore not to have stopped. A tube is used to siphon from the fingei-bowl so much water that the remaining layer just covers the specimens. The fingerbowl is then filled with 4% formaldehyde, covered, and placed to one side. One must be careful to distinguish at this point between a killing agent such as formaldehyde, and hardening and fixing agents. In the present instance it is un- necessary, since the stain to be used con- tains in itself an adequate mordant, to use a fixative which will combine with the proteins of the specimen, but it is neces- sary that they should be hardened in order that they may withstand the treatment to which they will be subjected in staining and dehydration. Four per cent formalde- hyde hardens very slowly, and it is sug- gested that they should next be passed to alcohol for the hardening process. It is desirable to flatten the specimens before hardening into the shape that they will be required to assume after mounting. It is to be presumed that the purpose of making a microscope shde is to study the object wliich has been mounted; and the depth of focus of microscope lenses is so slight that only relatively thin objects can be studied at one time. It is extraordinary how frequently tliis simple principle is overlooked, or how frequently people en- deavor to flatten the object after it has been gotten into balsam when it is almost invariably so brittle that it will break up during the flattening process. Five min- utes' work in arranging the parts before hardening makes all the difference be- tween a first-class and a second-class mount. To arrange and flatten the objects for hardening, the 40% formaldehyde is replaced with water. The specimen is re- moved to a fingerbowl of clean distilled water where it is examined thoroughly to make sure it has no adherent dirt. The object is flattened by hardening it between two slides, but obviously, if it is just pressed between two shdes it will be squashed rather than flattened. Anything may be used to hold the two sUdes apart, though in the present instance a very thick No. 3 or two No. 2 covershps would give about the right separation. A glass shde is taken, and about an inch on each side of the center a thick No. 3 coversUp is placed and held in place by the capillary Pectinatella WHOLEMOUNTS IN RESINOUS MEDIA 61 attraction of a drop of water. The speci- men is taken from the water with a large ej^e-dropper type of pipet and placed in a considerable volume of water on the slide. It is then easy to arrange the parts with needles; but it is difficult to lower a second slide on top of the first without disarrang- ing these parts. An alternative method is to place the shde with its coverslips in the fingerbowl with the specimen, to arrange its parts under water and to place the second sUde on top. Whichever process is adopted, the slides are then tied or clipped, together and transferred to a jar of 95% alcohol, where they may remain for a week, or until next required. Each speci- men is treated in this manner; and it is better not to try to flatten two or three specimens on one slide. When it is desired to continue mounting the specimens, each slide is taken and placed in a fingerbowl of 95 % alcohol be- fore the cords which bind them together are cut, or the clips removed. Getting the two sUdes apart without damaging the specimen is not easy, particularly if the specimen tends to stick to one or the other of the sfides. The simplest method is to in- sert the blade of a scalpel into the gap between the shdes and, twisting it slightly sideways, see if the specimen is free. If the specimen shows signs of sticking to one slide, the other may be removed and the specimen washed from the shde to which it is stuck with a jet of 95 % alcohol from a pipet. If it shows signs of sticking to both shdes, it is still possible, bj' projecting a jet of 95% alcohol between them, to free it from both. Each shde is treated in due order until one has accumulated the whole of the flattened specimens in a dish of 95% alcohol. It must be understood that these specimens have been hardened flat so that no amount of subsequent treat- ment will ever swell them out again or prevent them from remaining in the re- quired position. It is recommended, if there are several specimens to handle, that a series of the Uttle cloth-ended tubes shown in Fig. 23 be used. The only alternative is to handle each specimen with the aid of a section lifter with the consequent risk of damage. Though not nearly so satisfactory, it is also possible, at least for the process of staining and dehydration, to place the specimens all together in a small vial in which the different fluids used may be suc- cessively placed. A wholemount of this type is best stained in carmine and the choice would lie between Mayer's carmalum (a detailed description of the use of which is given in Chapter 20), Grenadier's borax-carmine (also described in Chapter 20), and May- er's paracarmine, which will accordingly be selected. Preparation of this stain (the formula for which is given in Chapter 20 under the heading DS 11.22 Mayer 1892) does not present any difficulty, but it should be noted that the differentiating solution is 0.2% solution of strontium chloride in 70% alcohol. Adequate sup- plies of this should be available before one starts staining. The specimens are passed from 95% al- cohol to 70% alcohol. They will naturally float, but as soon as they have sunk to the bottom it may be presumed that they are sufficiently rehydrated and either the cloth-ended tube containing them may be transferred to the dish of stain or the 70 % alcohol may be poured out of the tube and stain substituted for it. One of the advan- tages of this stain is that it is relatively rapid in action — very few specimens will not be adequately stained in five to ten minutes — but it does not matter how long the materials remain in it. It is, therefore, often convenient to leave the specimens in overnight and to start differentiation the next morning. They are then either re- moved to the differentiating solution or, alternatively, the stain is poured off and the differentiating solution substituted for it. In the latter case three or four changes will be required, owing to the necessity of leaving some stain in the bottom of the tube to avoid pouring the specimens out with it. Unless the operator is quite ex- perienced, it is safer to shake the tube so as to distribute the specimens thoroughly in the stain, and then to tip this into a large fingerbowl of dilferentiating solution from which the specimens may be subse- quently picked out and transferred to a new tube of differentiator. It is tragically easy, in pouring off stain, to pour speci- mens with it down the sink. As soon as the stain has been washed off with the dif- 62 THE ART OF MAKING MICROSCOPE SLIDES Pectinatella ferentiating solution, a single specimen should be transferred to a watch glass and examined under a low power of the micro- scope. It is more than probable that little differentiation will be required, so that a simple rinse may be adequate. It is difficult to judge the exact degree of differentia- tion, but it must be remembered that the object will appear darker after clearing than it does in the differentiating solution. The internal organs should be sharply de- marcated when the outer surface of the specimen is relatively free from stain. This may be judged in Pectinatella by placing a covershp on the specimen and examining one of the branches of the lophophore under the high power of the microscope. Differentiation may be considered com- plete when only the nuclei in the cells of the lophophore are stained. The speci- mens are then washed in four or five changes of 70% alcohol, to remove the strontium chloride, before being placed for at least a day in 96% alcohol as the first stage of dehydration. They should then be transferred to two changes, with at least six hours in each, to a considerable volume of fresh 95% alcohol and may then be cleared. Absolute alcohol is not necessary if terpineol is the clearing agent. There is some danger, if the specimens are transferred directly from 95% alcohol to a fluid as viscous as terpineol, that the specimens will become distorted through the very violent diffusion current. This may be avoided in the following manner: one takes a fairly wide (about an inch) glass vial and fills it about half full of terpineol. Ninety-five per cent alcohol is then carefully poured down the side of the vial (or on to a spoon held in the vial in the manner of a bartender making a pousse-cafe) so as to float a layer of alco- hol on top of the terpineol. The specimens are now dropped into the alcohol and sink through it, coming to rest on the surface of the layer of terpineol into which they sink slowly without any strong diffusion currents. They will have sunk to the bottom after a little while, but there will still be alcohol diffusing upward from them. As soon as the diffusion currents have ceased, the alcohol should be drawn from the top of the tube with a pipet and the specimens transferred to clean ter- pineol. When they are in fresh terpineol, they should be examined carefully under a microscope to make sure that they are glass-clear without the least trace of milki- ness. If they appear shghtly milky they have either been insufficiently dehydrated, or the alcohol used for the dehydration has become contaminated with water. In either case they must be transferred to a tube of fresh alcohol for complete dehy- dration and then put back into terpineol. It is a waste of time to endeavor to pre- pare a balsam mount from a specimen which is not perfectly transparent in the clearing medium. When all the specimens are in terpin- eol, take some clean shdes, some clean %-inch circular coverslips, and a balsam bottle containing natural balsam. (The author's preference for the natural balsam rather than a solution of this material in some solvent has already been explained.) Place a drop of the natural balsam on each of, say, six slides, and then one at a time lift out six specimens from the terpineol and place them on top of the balsam. They will sink through the balsam slowly so that these six slides should be pushed on one side while a further six slides have drops of balsam put on them, and so on. As soon as the specimens have sunk to the bottom of the drop of balsam, a covershp is held horizontally above, touched to the top of the drop, and then pushed down with a needle until the specimen is flat- tened firmly against the slide. As these specimens have been properly hardened and flattened there is no risk of their being damaged by drying the mount under pres- sure; therefore one can then apply a clip (see Fig. 26) and place the shdes on a warm table to harden. Each slide is then cleaned, finished, and labeled as usual. Preparation of a Skeleton of an Insect in Balsam Two methods have already been de- gum media given in Chapter 4, and the scribed for preparing small insects as simple, though very little-known, method wholemounts. These are the use of the of dropping the insect directly into glacial Insect skeleton WHOLEMOUNTS IN RESINOUS MEDIA 63 acetic acid and then transferring it from this acid to balsam. Many small insects, or other arthropods, are too opaque for this method of preparation to render ap- parent any details of the endoskeleton, which is so frequently necessary for diag- nostic purposes. These forms must, there- fore, be skeletonized and rendered par- tially transparent before mounting. Insects are skeletonized witli 10% po- tassium hydroxide, which dissolves and removes the internal organs while at the same time softening the skeleton suffi- ciently for the specimen to be flattened and made into a wholemount. Some very skilled mounters of the past made a spe- cialty of preparing whole insects without pressure, but these specimens are chiefly valuable as exhibits, and not as sUdes for study. Let us suppose that we have an ant which is to be made into a transparent wholemount. It does not matter whether this specimen has been freshly collected or has been dried for some time; in either case it must be soaked for at least three or four days in 95 % alcohol. Unless this pre- caution is taken, the strong alkali will dis- solve the thin membrane which holds the joints together and the specimen will fall to pieces. Disregard of this simple precau- tion is responsible for more failures in this type of mount than anything else. After the specimen has been satisfactorily hard- ened in alcohol, it is transferred to water until it is rehydrated and then placed in the alkali. In the case of old and hardened specimens it is desirable first to drill a fine hole at the tip of the abdomen with a sharp needle. After 24 hours in the alkali, the specimen should be removed and stranded on a glass shde with the legs more or less in the position in which they will be mounted. The s{)ecimen is then gently stroked with a brush from the point of the head toward the tip of the abdomen, care being taken not to break off any of the appendages. The purpose of this stroking is to expel any of the viscera which may have been dissolved, either through the natural vent, or the small one which has been made with a needle. It has been stated that the specimen which we are examining, an ant, may be left for 24 hours before this is done, but in the case of thinner-walled and more delicate speci- mens, stroking must be done two or three times a day. The reason for this is that the hydrolysis of the internal organs causes great swelling and, unless some of this fluid is expelled at frequent intervals, the abdomen, thorax, and even the head will be swollen and stretched into an un- natural appearance. The process of gently stroking out the contained material either once or twice a day is continued until it is apparent that no further viscera remain in the animal. It must be emphasized again that it is quite impossible to make a good preparation if the specimen is just thrown into alkali and left there until it is sufficiently transparent. When all the vis- cera have been removed, the appendages are carefully arranged with needles as the specimen lies stranded on the slide, and a few drops of glacial acid dropped onto the specimen from a pipet. This instantly ren- ders the specimen transparent and at the same time partially hardens the append- ages in place. The specimen, however, has yet to be flattened and properly hardened before it can be mounted. A couple of thick covershps, or a couple of pieces of cardboard of the same thickness as that desired in the final specimen, are laid one on each side of the specimen to support a second shde which is then placed on top. It is essential that the specimen should be flattened without producing any wrinkles in the softened chitinous exoskeleton and, unless the insect is naturally flat, this can- not be done merely by dropping a shde on top of it. Instead, the brush which was previously used for stroking is turned side- ways, and rolled backward and forward along the insect, pressing out any wrinkles which may appear. A second slide is placed on top to hold the insect flat and, with the appendages in the position de- sired, two shdes are tied or chpped to- gether and placed in a jar of 95% alcohol until they are next required. They should not remain in alcohol for less than a week and may remain for an indefinite period without damage. After the specimen has been hardened and dehydrated in this manner, the two sUdes are very carefully separated, with the use of a fine pipet to 04 THE ART OF MAKING MICROSCOPE SLIDES Insect skeleton squirt in jets of alcohol to free the speci- men. It does not matter if tlie specimen should stick to one of the two shdes for it may then be mounted on this slide. Whether, however, the specimen is free in alcohol or adherent to one shde, it must now be cleared, and the use of turpentine for this purpose is strongly recommended. As soon as the specimen has cleared, it is placed in the center of the shde and a considerable quantity of natural balsam poured on top of it. Since this specimen will not be danaged by heat, it should now be warmed until the balsam is hot and as hquid as water. Tliis drives off the turpen- tine, as well as most of the natural solvent of the balsam, so that when the covershp has been placed and the slide cooled it will be finished. Excess balsam may then be scraped and washed off in the usual man- ner and the specimen labeled. The description just given is of the con- ventional method of preparing mounts of this type and may be equally well apphed to parts as well as to entire insects. The preparation of demonstration mounts with- out "pressure is now a lost art, and there appears to be no one alive to duplicate the feats of the old mounters of the last cen- tury who were able to turn an entire housefly into what appeared to be an am- ber glass model of itself. For the benefit, however, of those who may wish to resur- rect this art, the author would hke to offer a few suggestions as to a method by which he has prepared passable, but not good, shdes of this type. The insect, re- laxed exactly as if it were being prepared for a museum specimen, is then " set" on a glass slide, with the legs arranged in the required position and held in place by cementing the tip of each leg to the shde with a small drop of molten gelatin. A piece of wood is whittled down until it can be slid between the insect and the glass, thus stopping the legs from con- tracting and pulling the insect down in the next stage of hardening. The glass with its attached insect should then be placed in 95% alcohol and left for a week to harden. If, at this stage, it is placed directly in alkah, the ligaments of the legs will be softened and the specimen will no longer be set in a natural position. It is, however, possible, after the specimen has been re- hj'dratod, to drill a very small hole in the back of the ajjdomen and to work a hypo- dermic needle forward until the tip of it reaches to the head. A minute quantit}^ of 10% potassium hydroxide is then injected and the specimen left in a moist chamber for two or three hours. The needle is then reinserted and a further quantity of po- tassium hydroxide injected. By this time some of the viscera will have been softened sufficiently to come out of the hole on the edge of the needle. Care must be taken that none of the hydroxide gets onto the legs, or onto any of the fine appendages, and particularly that it does not run down and dissolve the gelatin which is holding the specimen in place. After three or four days of making injections daily, the vis- cera of the insect will have been washed out and the body itself commencing to soften. The slide is then put into alkali, where it remains until the legs of the specimen start to soften and to be trans- parent. The specimen is then taken to glacial acetic acid, and from glacial acetic acid to xylene which is used to wash the acetic acid from it. When all the acid has been removed the specimen is transferred to a weak solution of Canada balsam in xylene, in which it remains until it is thoroughly impregnated. A deep cell (see Chapter 1) is next ce- mented to a glass shde, and the specimen transferred to the cell which is filled to the brim with a solution of balsam in xylene. This is placed in the desiccator to evapo- rate, the cell being filled up as often as is necessary. The cell, with its contained in- sect, is then slowly heated to drive off the remainder of the solvent and finally closed with a covershp. The writer must again confess, reluctantly, that such specimens make magnificent show pieces that are of very doubtful scientific value. Preparation of an Alga in Venice Turpentine Venice turpentine is the natural balsam cidua). It is a thick balsam, of about the which is exuded by the larch {Larix de- consistency of natural Canada balsam. Alga WHOLEMOUNTS IN RESINOUS MEDIA 65 and takes its name, as do so many other resins, from the place from whicli it was first exported to England. The commonest commercial use of this material today is in the preparation of artist's pigments, so that it is usually better secured from an artists', than from a scientific, supply house. Man}' substitutes and impure speci- mens are on the market and, as the only value of Venice turpentine lies in its per- fect miscibility with alcohol, any specimen should be tested b}^ seeing whether an equal volume of tlie balsam and of 95% alcohol will make a perfectly clear mix- ture. If they do not, the specimen is not true Venice turpentine and is worthless for the technique which follows. Zimmerman 1896, p. 18, recommends that, in any case, the raw resin should be diluted with twice its own volume of 95% alcohol, filtered, and then heated until the alcohol is evaporated from it. Tliis method is, however, dangerous, unless the atmos- pheric humidity is practically nil. It is much simpler to get a first-class specimen of Venice turpentine in the first place. This medium was first recommended for the preparation of botanical wholemounts by Pfeiffer and Wellheim 1892 (23632, 8:29) but did not come into general use until it was reintroduced by Chamberlain 1915 (p. 97). This writer, however, com- plains that the original directions of Pfeif- fer and Wellheim were diffuse; he cites, not their original paper on the mounting of objects in Venice turpentine, but an- other paper on the preparation of fresh- water algae (Pfeiffer and Wellheim 1894; 10606, 26:674). The present description is drawn from all the sources cited. We will assume that we are dealing with Spirogyra, a form notoriously difficult to prepare as a satisfactory wholemount. It may be collected in quantity almost any- where in the world, and should be trans- ferred immediately after collection to a large volume of any chromic-acetic fixa- tive; the formula of Lavdowsky 1894 (Chapter 18, F 6000.0010) is excellent for the purpose. After the masses of algae have been in the solution for a day or two, they should be washed in running water for 24 hours and then pieces should bo selected for mounting. These pieces should be relatively straight, and about y> inch long, for it is a waste of time to take great masses of algae through the compUcated l)rocesses which follow, and then to select finally only the few pieces which one de- sires to mount. The selected pieces should be carefully passed through 15%, 30%, 50% and 70% alcohol and finally into 90% alcohol in which they are to be stained. Some specimens are so delicate that they will not stand transference from water to alcohol, no matter how gradual the transition phases may be, and for these the method of Chamberlain may be adopted. The algae are transferred from water to 10% glycerol antl the water then evapoiated until they are in pure glycerol. The glycerol is then washed out with 95% alcohol without risk of tlie specimen col- lapsing. This washing must, however, be thorough. Assuming that we now have the speci- mens in 95% alcohol, it is recommended that they be stained by the technique of Chamberlain (Chapter 20, DS 13.5 Cham- berlain 1915). For this there will be re- quired a 1% solution of phloxine in 95% alcohol, a similar strength solution of ani- lin blue in the same solvent, and a 0.1% dilution of hydrochloric acid, also in 95% alcohol. The specimens are transferred from alcohol to the 1 % phloxine solution , where they remain for about 24 hours. They are then rinsed in alcohol for a min- ute or two, or until most of the excess has been removed, and transferred to the ani- lin blue solution. They should remain in this until sufficient blue color is showing in the cytoplasm and until the cell walls themselves have just started to take this blue. They should not, however, remain in the blue for sufficiently long to obscure the bright red color of the nuclei. Cham- berlain suggests that from three to thirty minutes may be necessaiy, but the writer has never had to use a longer period than five. It is obviously desirable to experi- ment witli a few filaments until one has esta])lished the correct time and then stain all the rest of the filaments together. After the filaments have been stained in the blue, they should be transferred to the acid alcohol where they should remain 66 THE ART OF MAKING MICROSCOPE SLIDES Alga until the excess blue has been removed from them. The object of this acid is not to remove blue from tissues which have already taken it, which it will not do, but to rinse off the outside and thus leave the red nuclei bright and clear. If, through any accident, the material has been left in the blue stain too long, it should be transferred to a large volume of 95 % alco- hol and left there until the whole of the blue color has been removed. This will also remove most of the red from the nu- clei, and one must, therefore, start the whole process over again. After the specimens have been stained they must be put into Venice turpentine. The reason for the selection of this mate- rial is that they may be passed directly to it from alcohol without the intervention of any clearing agent which would cause them to collapse. They cannot, of course, be passed directly from the alcohol to full- strength balsam but must be got into it by a process of evaporation. The exact strength to which one transfers them is immaterial, but the strongest which can be safely used is a mixture of ten parts of 95% alcohol to one part of the balsam. No difficulty at all will be occasioned in evaporating off the alcohol, provided it is done in an absolutely dry place. A large desiccator is, therefore, charged with silica gel. This material was not available at the time of the descriptions already cited but it provides what the earlier workers lacked — a material to be used in a desiccator which both provides an adequately dry atmosphere and absorbs a certain amount of alcohol vapor. The weak Venice turpen- tine containing the specimens is, therefore, placed in an evaporating basin, or crystal- lizing dish, and placed in a desiccator con- taining siUca gel. As the alcohol is ab- sorbed relatively slowly by the silica gel, it is preferable to have two desiccators and to transfer the dish from one to the other daily, thus avoiding saturating the atmosphere with alcohol. The evaporation of the alcohol should l^e continued until the Venice turpentine is as thick as the original specimen; and it may be neces- sary, after about two or three days have been spent in the evaporation, to transfer it to a desiccator containing a fresh batch of siUca gel in an oven at 30° to 40°C. This also renders the Venice turpentine more fluid and permits the mount to be more easily made. When the Venice turpentine is suffi- ciently thick, a perfectly clean shde is taken and a small drop of Venice turpen- tine is placed on it. A needle is then dipped into the Venice turpentine and, with an- other needle, one of the short filaments of alga maneuvered alongside it; the first needle is then drawn slowly at an angle about 45 degrees from the Venice turpen- tine, so that the filament will remain lying along the side of it. The needle is then laid flat on the Venice turpentine on the slide and, by a roUing movement, the alga is transferred to the drop. As many fila- ments as are required should be trans- ferred and the slide containing the speci- mens in Venice turpentine should then be placed in a desiccator. One of the easiest errors to make in this technique is to leave a small quantity of alcohol in the Venice turpentine before starting to mount. If this is done, it is al- most impossible to prevent the moisture in the breath from clouding the resin and ruining the whole long complex operation that has already been undertaken. When as many mounts have been prepared as are required, they are removed one at a time from the desiccator, a little fresh Venice turpentine placed on top of each and the coverslip apphed. They may then be placed on a warm stage at a temperature of 30° to 40°C. and permitted to harden for a week or two. It is difficult to clean a Venice turpen- tine mount for if one tries to dissolve off the excess Venice turpentine with alcohol, the covershp may be dislodged. It is better to use only as much Venice turpentine as will exactly come to the edge of the cover- shp to avoid having to clean at all. Vos- seler 1889 (23632, 6:292) recommends that the mount, as soon as it is made, should be ringed (as described in Chapter 2) with a solution of Canada balsam in xylene, and that the Canada balsam should then be hardened. This gives a very attractive and clean mount and is strongly to be recommended. Gray's method WHOLEMOUNTS IN RESINOUS MEDIA 67 Preparation of Minute Fresh- water Organisms by the Method of Gray 1932 The technique here given, which is abridged from the original description of Gray 1932 (11360, 52:370), permits one to prepare permanent mounts of individ- ual microscopic organisms. It may be used equally well to mount an individual i)ro- tozoan or an individual alga. It consists essentiall}' of utiUzing a special fixative which renders a layer of albumen on the slide intensely sticky so that the selected object, immediately after fixation, adheres to it. The range of possible application of this method is very wide, the notable excep- tions to its use being for rotifers, stalked ciliates (wliicli cannot satisfactorily be narcotized), and nematode worms which cannot be mounted in balsam without great distortion. Almost anything else can be mounted, provided that its size lies be- tween a total length of about three milli- meters, and that of the smallest object which can be seen under a wide-field bi- nocular microscope. The reagents required, wloich can most conveniently be kept in drop bottles, are 70% alcohol, ether, 40% formaldehyde, glacial acetic acid, and the stock fixative solution (Chapter 18, F 3500.1010 Gray 1932) which consists of 1% each of picric acid and mercuric chloride in 90% alcohol. Before commencing to mount a series of specimens, it is also necessary to have some Mayer's albumen (Chapter 28, V 21.1 Mayer 1889), some clean shdes, sev- eral small specimen tubes or vials for the preparation of the fixative, some' strips of filter paper, a writing diamond, and, lastly, one or more coplin jars of 70% alco- hol in which the mounts may be accumu- lated as they are made. The required fixative is made up in small quantities according to the speci- mens which one desires to mount. When examining a sample of water without knowing what to expect, it is as well to accumulate three mixtures, each for spe- cific organisms. These are: A. for 'protozoans stock fixative 10 ether 3 acetic acid 2 40% formaldehyde 5 B. for heavily cidicularized forms (e.g. Gastrotricha) stock fixative 10 ether 1 acetic acid 4 40% formaldehyde 5 C. for delicate larvae {e.g. Miracidia) stock fixative 10 ether 2 acetic acid 1 40% formaldehyde 5 The parts given are by volume and the author usually uses drops in making up these mixtures since only a very small quantity is required even in making many slides. Half a dozen clean slides are now taken and smeared with a quantity of Mayer's albumen in the center of each. The layer should be considerably thicker than that which would be applied were one prepar- ing to attach paraffin ribbons. The collec- tion is now examined and any small object which it is desired to mount is taken up in a pipet with the least possible quantity of water and placed on the patch of albumen, beyond the limits of which the water should not run. The surplus fluid is then drawn off, sufficient, however, be- ing left for the animal to swim naturally. The animal is watched until it is in a fairly normal position and a large drop of fixa- tive then allowed to fall on it from above. Immediately the fixative has reached the water, diffusion currents of almost explo- sive intensity result, and considerable care nuist be taken to keep the rapidly moving object within the field of the dissecting microscope. If the object leaves the area of albumen, it must be guided back with a fine glass needle, the point of which will collect a capillary droplet of the fluid. When the animal is in the desired position over the albumen smear, all surplus fluid, which by now will have collected into drops moving slowly over the surface of the slide, is removed l)y the filter |)aper. The object is now closely watched under the dissecting microscope until the droplet of fluid, which will have collected round it, has so far evai)orated as clearly to show the outlines of the object. When evapora- 68 THE ART OF MAKING MICROSCOPE SLIDES Gray's method tion has proceeded this far, the sUde is gently flooded with 70% alcohol The correct point at which to do this is easily recognizable with practice but is difficult to describe in words other than the above. If evaporation be allowed to proceed too far, the animal, especially if it be spherical, is liable to become distorted; if it be ar- rested too soon, the animal is liable to be- come detached. A Uttle practice will, how- ever, readily allow one to determine the exact moment at which the alcohol must be added. After the alcohol has been added to the shde and left for a few moments, a small circle is cut around the object with a writ- ing diamond, as, once lost from the field of the dissecting microscope, such objects as small protozoans are almost impossible to distinguish from specks of dust acquired in the fixing process. The slide is then transferred to a coplin jar of 70% alcohol, for the specimen is firmly fixed in position and will not be detached through any sub- sequent process of staining and mounting. Staining may be carried out by any method. It is customary to stain most small fresh-water animals in alum hema- toxyhn, most small fresh-water plants in some safranin solution, while the writer prefers, for gastrotrichans, one of the alum carmines which must be allowed to act over a long period. It is possible with the aid of this method to mount 20 or 30 selected forms from a collection of fresh-water plankton in less than half an hour, and it will be found very useful to be able to examine a collec- tion of fresh-water plankton with the as- surance that any unknown form may be permanently attached to a shde for subse- quent identification. Smear PreparaUofis from Fluid Material General Principles Every chapter up to the present has been concerned with the preparation of microscope shdes from whole objects pre- served in as nearly as possible their natu- ral shape. Chapters 10 through 15 will be concerned with the preparation of thin slices or sedio^is of objects. Between these extremes of a whole object and a thin slice there are two types of preparation, which are discussed in the next three chapters. Smears, discussed in this and the next chapter, are exactly what their name indi- cate : they are prepared by smearing some substance on a clean glass slide where it may be fixed, stained, and mounted. Squashes, the name of which is also self- explanatory, are prepared by squeezing either animal or plant materials in such a way that thej^ disintegrate into their com- ponent cells, which may then be studied without reference to the relations which they previously had with each other. Smears may either be prepared from fluids or from soUd objects. A separate chapter is devoted to each. The present chapter deals with the preparation of thin layers of fluid so that the cells contained in them may be studied. Three operations are necessary in the preparation of smears of fluids: first, the smearing of the material itself into a layer of the required thickness; second, fixing this layer both to insure its adherence to the slide and to make sure that the contained cells remain in their normal shape; third, staining and mount- ing the fixed smear. Each of these opera- tions will be discussed successively. Preparation of the Smear The first thing to do in the preparation of a smear is to make sure that chemically clean slides are available. The adherence of the smeared material to the slide will be excellent if it is a fluid containing con- siderable quantities of protein (as blood), even if the shde be not clean, but there are many fluids which are used in the pro- duction of smears which will not adhere at all save to an absolutely clean glass surface. Any method may be used for cleaning slides, but for this particular purpose the author prefers to use a house- hold scouring powder, which consists of a soft abrasive together with some detergent agent. This powder is made into a thin cream with water and each slide is then dipped into this cream and stood in a rack to dry. As soon as it has dried the shdes may be returned to a box, preferably each being separated from the other with a thin paper insert. As slides are commonly sold with these paper separators, it is only necessary to take a box and to save the separators when one dips the slide, return- ing them after they are dried to the same box with the same separators. Two or three hundred slides may easily be prepared in this manner in a short time and stored against future use. For use the slide is polished with a clean linen or silk cloth. Smears often have to be made at un- expected moments, therefore it is a con- venience to have slides at hand which may be rendered fit for use in a few moments. The actual method of smearing the ma- terial varies greatly according to what is being used. Probably more smears are made of blood than of any other fluid, and the technique for the preparation of these is so well established that it will be de- scribed as a type. The material itself may either be taken from the puncture wound G9 70 THE ART OF MAKING MICROSCOPE SLIDES Smearing directly onto the slide, or (as in Fig. 27) behind the first slide and distributed more removed from the puncture wound with a or less uniformly on the under shde. A few pipet and transferred to the slide. The people still try to conduct the operation drop is placed about }'i of an inch from in the reverse manner: by placing the sec- one end of the slide, and a second shde (as ond slide on top of the first, sloping it at a Fig. 27. Making a smear preparation, a. Place the drop about an inch from the end of the slide. Fig. 28. Maicing a smear preparation — (continued), b. Apply a second slide just in front of the drop. Fig. 29. Making a smear preparation — (continued), c. Push the slide smoothly forward to spread the smear. shown in Fig. 28) placed on the drop. Capillary attraction will naturally dis- tribute the fluid along the edge of the sec- ond slide which is then (Fig. 29) pushed sharply forward until it reaches the end of the bottom slide. The material of which the smear is being made is thus spread out reverse angle to that shown, and then en- deavoring to push rather than drag the material across the lower shde. The objec- tion to this is that it results in crushing cells, though it must be admitted that it frequently gives a more uniform distribu- tion of the material. The use of a glass shde Fixation SMEAR PREPARATIONS FROM FLUID MATERIAL 71 for spreading has a great deal against it. There is a risk tliat the edge of the upper slide will scratch the lower, and though these scratches are not apparent in smears which are to be examined under an oil im- mersion objective, they are objectionable in a dry slide. It is also difficult to secure a sUde which is entirely flat and which will thus make a layer of uniform tliickness, for the tliickness of the laj^er obviously depends on the degree of contact between the upper sHde and the lower. The writer has recently been using a thin sheet of transparent plastic (methyl methacrylate) in place of glass and has obtained very much better results. To prepare such a sheet for use one cuts, or saws, a 3" X 1" rectangle from it and then polishes one of the edges by rubbing it briskly backward and forward on a sharpening stone or on a ^ piece of fine sandpaper. This turns up a feather edge on both sides of the edge which has been flattened. This is removed by taking the shp and holding it at just about the same angle at which it will be used for preparing the smear and giving it one or two quick strokes on the finest sandpaper. The use of a soft material like this not only insures that there will be no scratches on the slide, but also guarantees that the edge used for smearing will always remain in contact with the slide. After two or three hundred smears have been made the piece of plastic may be thrown away and a new one taken. The method described is the standard procedure for producing thin smears. These are necessary for those fluids, such as vertebrate blood or mammalian seminal fluid, which contain very large numbers of objects which must be separated as widely as possible if they are satisfactorily to be studied. There are a number of fluids, however, from which thick smears must be made either because, as in the case of inverte- brate blood, they contain relatively few cells or because, as in the case of malarial diagnostic smears, one is seeking for a parasite which is relatively sparsely dis- tributed through the material. These thick smears are made with the aid of a loop of wire held in a needle-holder of the type found in bacteriological laboratories. This loop is dipped into the fluid to be ex- amined, and used to spread it with a rotary motion in tlie center of the shde. This is very similar to the preparation of smears of bacterial material which is de- scribed in some detail in Chapter 21. Fixing Smears Smears may be fixed by drying, by alcohol, or in one of the conventional fixa- tives. When a smear is to be fixed by dry- ing it is, as soon as it has been made, waved in the air and then set on one side for subsequent treatment. This procedure is excellent in the case of objects such as bacteria or erythrocytes, which do not change their shape after drying, or for materials such as white blood corpuscles, which it is not desired to preserve in their normal shape. No other object can, how- ever, be considered satisfactory unless it has been fixed, and the simplest method of doing this is to pass the smear, just as it is drjdng, through a jet of steam. This technique has been described in Chapter 6 for mounting amebas and need not be re- peated here. All other smears should be fixed before they are dried and it is something of a problem to fix them without removing the material from the shde. It is obvious that if the material is freshly smeared onto a glass shde and then dropped into a fixative of some kind, it will be washed off. The logical solution to the problem is to use a fixative in a vapor phase, and nothing is better for this purpose than osmic acid. To use this material, a couple of glass rods are placed in a petri dish sufficiently far apart to permit the sUde to rest on them without the smear touching them. A drop or two of a solution of osmic acid, usually of 2 % strength is put on the bottom of the petri dish and the cover replaced. It must be emphasized that osmic acid fixes the mucous membrane of the nose and throat just as readily as it does a smear and every precaution must be taken to avoid inhal- ing the vapors. As soon as the smear is made, and before it has time to dry, it is placed face down across the two glass rods so that it is exposed to the vapor but not to the liquid. The cover is then replaced on the petri dish, and the slide left in place for 72 THE ART OF MAKING MICROSCOPE SLIDES Staining about three or four minutes in the case of a thin smear, or for five to ten minutes in the case of a thick one. It is then trans- ferred to distilled water to await staining. It occasionally happens that a slide must be fixed in one of the conventional fluid fixatives. This is done with the same petri-dish and glass-rod setup as is used for vapor fixation, but in this instance the fixative is carefully poured into the petri dish, which must be level, until it has reached such a depth that, when the slide is laid across the glass rods, the under side of the slide with the smear on it is in con- tact with the fluid while the upper part is free from fluid. If the smear is reasonably tliin and is laid carefully in place, it usu- ally will not become detached. Thick smears, particularly those made with fluids containing very little protein, will not stand this treatment; they must either be fixed in the vapor phase, or else the fluid itself must be mixed with a small quantity of an adhesive, such as Mayer's albumen (Chapter 28, V 21.1 Mayer 1884). Staining Smears Blood smears are so universally stained with one or another of the methylene blue- eosinate mixtures (Chapter 20, DS 13.1 and 13.2) that it comes as something of a surprise to most people to learn that any stain which is suitable for sections may also be employed for smears. The ad- vantage of methylene blue-eosinates for blood films is that the solvent methanol acts as a fixative so that they are stained and fixed in the same operation. When a blood smear is to be used for diagnostic purposes, these techniques are excellent, because the appearance of the various types of white corpuscle under this treat- ment is known to every technician. For research studies on the blood, however, it is strongly recommended that the worker experiment, first by fixing the blood film in osmic vapor in the manner described, and secondly by applying to it some other of the complex techniques described in Chapter 20. For materials other than blood there is no limit to the type of stain- ing which may be employed, though it must be remembered that very thin films require a stain of considerable intensity if the finer structures are to be made out. Thus, for example, a thin smear of mam- mahan spermatozoa is best stained by one of the very dense iron hematoxylin tech- niques such as that of Biitschli (Chapter 20, DS 11.111 Butschh 1892). The method for the application of these stains to smears differs very little, in most cases, from the method for the application of the same stains to slides, and no specific in- struction need be given. Bacteriological staining methods, which differ from those used in botany and zoology, are given in Chapter 23 under the heading DS 23.2. Typical Example Demonstration of Monocystis from the Seminal Vesicle of an Earthworm Few sporozoans are available for class demonstration purposes and the choice is practically limited to the inhabitants of the intestines of a cockroach or to the specimen at present under discussion, Monocystis. The advantage of Monocystis is that all the forms from the sporozoite to the trophozoite occur in the seminal vesicle of the earthworm, and may therefore be made available on a single smear. The degree of infection among earthworms varies greatly, but it has been the author's experience that the larger the earthworm the more hkely the chance of a heavy in- festation. But it is no use making a whole lot of smears for class demonstration pur- poses until one has satisfied oneself by a preliminary survey of a single smear that the material will be satisfactory. There is no need to kill or anesthetize the earthworm, which' is simply pinned down in a dissecting tray and sUt from the anterior end to about the 16th or 17th seg- ment. The edges of this slit are pulled back and pinned into place disclosing the large white seminal vesicles. There should be available, before mak- ing the smear, a petri dish in which are a couple of short lengths of glass rod, a sup- Monocystis SMEAR PREPARATIONS FROM FLUID MATERIAL 73 ply of Schauclinn's fixative (Chapter IS, F 3000.0000 Schaudiiiu 1893), an ade- quate supply of clean glass slides, an eye- dropper-type pipet, some 0.8% sodium chloride, and some cophn jars of distilled water. Enough fixative is poured into the petri dish so that when a sHde is laid on the pair of glass rods, its lower, but 7}ot its upper surface will be in contact with the fixative. This level is best established with a plain glass slide before the smears are started. The seminal vesicle of the earthworm is slit and a drop of the contained fluid re- moved with the pipet. This pipet is then used to smear a relatively thick layer of the material on the center of one of the clean shdes and, before it has time to dry, this slide is laid face down in tlie fixative for about two minutes. The sUde is then removed, rinsed under the tap, and ex- amined under a high power of the micro- scope after a covershp has been placed over the smear. It is rather difficult to see the trophozoite stages in an unstained preparation, but no difficulty will be ex- perienced in picking out the spore cases (pseudo-navicellae) owing to their rela- tively high index of refraction. It may be taken that adequate numbers of the para- sites are present if not less than three or four of these spore cases occur within the field of a four-millimeter objective in a thick smear of this nature. As soon as a satisfactorily infected worm has been found, the remainder of the material from the pipet is placed in a watch glass and diluted with 0.8% sodium chloride until it forms a dispersion about intermediate in thickness between cream and milk. As many smears as are required are made from this cream as rapidly as possible. The dilution in question will not retain the parasites in good condition for more than about five minutes, but if in- sufficient smears have been made in this time, it is easy to take a fresh supply of the seminal fluid from another vesicle and to dilute it in a fresh watch glass. The cream should be spread with two slides in the manner described above, and each shde placed face downward in Schaudinn's fixative for three or four minutes before being removed to a cophn jar of distilled water. After having been washed in water the smears should be transferred to 70% alcohol where they can remain until they are ready for staining. Any stain may be used but it is conventional to employ a hematoxylin mixture. The author prefers to use the old "triacid" stain of Biondi which is given in Chapter 20 under the heading DS 13.33 Biondi (1888). The ad- vantage of this solution is that the orange G is picked up by the cases of the sporo- cysts, while the trophozoites are red with clear green nuclei. Nuclei of the sperma- tozoa and spermatids of the earthworm occupy so much of the cells in which they are found that they give the whole stain a greenish cast. This green background shows up the red trophozoites and the brilliant orange sporozoites. The method of staining is easy. The solution, made in accordance with the directions given, is diluted to the extent of about 2% with distilled water. The slides are then placed in this diluted solu- tion and left until examination under the low power of the microscope shows them to have been adequately stained and differentiated. They are then briefly rinsed in distilled water and dried. There is no necessity to use any dehydrating agent, such as alcohol, which will interfere with the staining, because the smears should be sufficiently thin and the objects in them suflticiently well fixed that drying will not distort them. To complete the mount, a drop of the mountant selected is added and a coverslip applied. Balsam may be used, but the writer considers it to have too high a refractive index, and for that reason prefers one of the "neutral" mountants, based on gum sandarac, the formulas for which are given in Chapter 26 under the heading M 23.1. 8 Smear Preparations from Cut Surfaces General Principles The last chapter described the prepara- tion of sniears from materials which were fluid; the present chapter deals with the method by which smears may be obtained from the surface of materials which have been cut into blocks. Preparation of the Smears There are only two methods of produc- ing smears from the cut surface of sohd bodies. Either the cut surface is rubbed on a clean slide,- leaving behind a few cells which have been detached from it, or the cells are squeezed from a cut, and subse- quently pressed out in the form of a smear. The former method is used for animal tissues and the latter for plant specimens. The difficulty in preparing smears from the cut surface of animal tissue is not so much to secure material as to avoid secur- ing unwanted cells. The blood content of the majority of organs is so high that, if a freshly cut surface be smeared on a piece of glass, the few cells which become de- tached will be obscured by the red blood corpuscles. The technique is, therefore, usually applied to the central nervous system, from the cut surfaces of which cells not only detach themselves very readily but which has also the advantage of being only shghtly vascularized. The only difficulty in preparing a smear from a freshly cut surface of the central nervous system lies in finding a pair of forceps sufficiently wide and sufficiently blunt to hold the material without it disintegrat- ing. Apart from this the technique is essen- tially that described in the last chapter. A perfectly clean slide is taken and rubbed with the cut surface of the mate- rial. These smears cannot be dried satis- factorily but must always be fixed, and it is necessary to use the technique, dis- cussed in the last chapter, of placing the sUde face down across a pair of glass rods lying in the bottom of the petri dish in such a manner that the lower surface, but not the upper surface, is in contact with the fixative. The fixative to be selected naturally varies according to the material to be studied, but fixatives containing mercuric chloride are usually to be pre- ferred. Smears may also be fixed in osmic vapor, though they are not usually as satisfactory w'hen prepared by this method as are smears prepared from fluid material. The smear technique in plant micro- technique is largely confined to securing sporogenous tissue from anthers at various stages of their development. This tech- nique, which was introduced by Taylor 1924 (3430, 78:236), has the advantage that it permits the examination of chromo- some material without the trouble of de- hydrating, embedding, and sectioning. This smear technique must, however, be differentiated from the squash techniques, described in the next chapter, in which cells are dissociated. The smear technique can only be used for materials which can be squeezed out. There is some division of opinion as to whether a small quantity of adhesive (such as Mayer's albumen) should first be smeared on the sUde, or whether the material extruded from the anther has enough protein to cause ad- hesion. In either case the anther is cut with a very sharp scalpel about one third of the distance from its base and placed on the sUde with the cut end in the region where one wants the smear. The back of a scalpel is then rolled from a position about a milfimeter from the cut edge, toward the cut edge. The material which is thus ex- truded is rapidly smeared with the back of the scalpel, the crushed anther removed, and the slide inverted on glass rods in a fixative. It has been found (Kauffman 1927, 20540b, 2:88) that these smears are best stained with an iron hematoxyUn technique (Chapter 20, DS 11.111). 74 Negri bodies SMEAR PREPARATIONS FROM CUT SURFACES 75 In every other respect these smears are treated as though they had been prepared from a fluid material (see last chapter) but a single typical preparation applying one of these techniques will be given. Typical Example Demonstration of Negri Bodies by the Method of Dawson 1934 The detection of Negri bodies is, of course, used in the diagnosis of rabies. A method for the demonstration of these bodies in sections is described in Chapter 21, and the method here described is in- tended less for permanent preparations than for a rapid diagnostic procedure. The description referred to contains de- tailed directions for the dissection of the horn of Amnion, which is the portion of the brain usually selected for these tests. We will assume, therefore, that the worker has dissected, following the necessary pre- cautions as to his own safety, the brain of the diagnostic guinea pig in such a manner as to expose the horn of Ammon. For the preparation of paraffin sections, the horn of Ammon is divided into small pieces, but for the preparation of smears it must be kept whole and a sharp razor used to trim away about the lower third leaving a freshly cut surface. If there is any quantity of extravasated blood pres- ent, it must be washed off with a gentle stream of normal saline or it will tend to obscure the picture. To prepare the smear preparations there are required an adequate quantity of clean shdes, a supply of methanol, a 2 % solution of phloxine, and a quantity of Lofiler's polychrome methylene blue. The prepara- tion of the polychrome methylene blue solution is described in Chapter 20 (DS 11.44 Lofiier 1890) and need not be re- peated here. It is convenient to have the methanol and methj'lene blue solutions in drop bottles and to have the 2% phloxine and the 20% alcohol in coplin jars. If the slides are to be filed for reference, rather than used immediately for diagnosis, it is also desirable to have some neutral mountant (Chapter 26, M 23.1) The entire horn of Ammon is then taken and dabbed once or twice in the center of one of the clean shdes. If one endeavors to smear it, too much material usually (;omes off; but the vertical dabbing motion will provide a sufficiently thick film which, when moist, should appear as no more than a slight clouding of the surface of the slide. The shde is then waved gently back- ward and forward until it appears to be just about to dry. The material will be lost if methanol is added to it while it is completely wet, and the material will be distorted if it is permitted to become en- tirely dry; but only a little experience is necessary to enable one to adjudge the exact moment at which methanol should be dropped on the smear from the drop bottle. The shde may then be placed on one side to dry, if it is to be used immedi- ately; it should be dropped into a cophn jar of methanol if it is not to be stained for, say, half an hour. When the slides are to be stained they are waved in the air until the methanol has evaporated and then dropped into the 2 % phloxine where they remain from two to five minutes. Upon being withdrawn from the phloxine, a stream of water from a wash bottle should be used very gently to remove the excess phloxine, the slide drained by its corner onto filter paper, and the polychrome methylene blue dropped onto the surface from a drop bottle. The methylene blue is left in place for 15 seconds, washed off with a stream of water from the wash bottle, and the slide then dropped into 20% alcohol where it may be left until no more color comes away. It may remain in the alcohol for 10 or 15 minutes without damage. If the slide is required for immediate examination, nothing remains to be done save to remove it from the 20% alcohol, wave it about in the air until it is dry, and then examine it under the microscope with an oil immersion lens. If, however, the shde is to be filed for permanent refer- ence, a drop of neutral mountant should be placed on the smear as soon as it has been dried and a covershp added. Squash Preparations from Solid Bodies General Principles Nature of the Process Squash preparations are not, as their name might cause one to suppose, ob- tained merely by crushing an organ or animal in order that it may become thin enough to examine under the microscope. This would result in the hopeless distor- tion of the cells and their contents. A squash preparation, properly prepared, is obtained by causing the cells of animal or plant material to become separated one from the other without losing their indi- vidual shape in order that they may be spread out on a shde in a single layer for examination. This process is today much better known in botany than in zoology, though it was at one time the standard method of preparing histological speci- mens. Another fundamental difference be- tween the smear and squash technique is that the former always employs fresh un- fixed material while the latter should always employ- a material which has been fixed previously. Process of Maceration The separation of cells of fixed plant or animal material through the hydrolysis of their interstitial tissue of cement is known as maceration. It does not matter what fixative has been used, though in the author's experience fixatives containing cln-omic or osmic acid, or mixtures of these, are best. Tissues are fixed in the ordinary way and the fixative thoroughly removed ]:)y washing before maceration commences. The two most common metli- ods of maceration, either for animal or plant material, are acid hydrolysis and enzyme hydrolysis. Each will be described separately. Acid hydrolysis of plant tissues is almost always carried out in 10 % hydro- 7G chloric acid, in ^vhich the fixed tissue is soaked until a sample of it, placed under a coversUp, is found to disintegrate into its constituent cells when the coverslip is tapped hghtly with a needle. Acid hydrol- ysis of animal tissues, however, has been carried out with almost any acid used for microscop}^ and reference should be made to Chapter 19 (V 40) where many sug- gested mixtures are given. It may be pointed out that almost any acid fixative solution, if diluted with from 50 to 100 times its own volume of water, will act as a macerating agent. Enzyme hydrolysis of animal tissues may be conducted either in an alkahne or an acid environment, and reference should be made to the methods of Jonsset 1903 and Langeron 1942 for examples of each of these. Abbre\dated techniques for these methods are to be found in Chapter 19 under V 40 and need not be expanded here. Enzyme hydrolysis of plant tissues is of quite recent origin, and depends on the extraction of enzymes from sources which are customarily used in the digestion of plant material. Ensweller 1944 (20540b, 19, 109) suggests the extraction of various fungi but the method of Faberge 1945 (Chapter 19, V 41.1), of which a detailed description is given in one of the typical preparations following this chapter, is much to be preferred. The terminal point of enzyme maceration may be detected, exactly as is that of acid maceration, b}'' whether or not the organism or tissue unrler examination disintegrates into its constituent cells. Staining and Mounting Macerated Specimens Though the process of maceration is it- self quite easy, staining and mounting of Chromosomes SQUASH PREPARATIONS FROM SOLID BODIES 77 the products of maceration present many difficulties. It' the preparation is broken up under a covershp, it is difficult to remove it witliout losing the cells, or to stain them with the coverslip in place; if the macera- tion is carried out in a small tube, it is difficult to concentrate the cells readily on the slide after they have been stained. Probably the simplest method in most cases is to treat the individual cells as though they were a culture of protozoans: that is, to stain them, dehydrate them, and get them into a small quantity of balsam, and then to place a drop of this balsam under the coverslip. As an alterna- tive to this, the macerated material may 1)6 smeared over the surface of a slide which has had an adhesive applied to it, or it ma}' be diluted with an adhesive material such as Mayer's albumen and then treated as though it were a fluid smear. The ol)jection to this treatment is that the majority of fixed cells are very brittle and will be damaged when the smear is made. The selection of a stain is not difficult since each dissociated cell may be treated as a small wholemount. It is not worth while to double-stain macerated speci- mens, the true function of which is to present a clear picture of the shape, not the nature, of individual cells. Typical Examples Preparation of Microsporocytes of Crocus for Chromosome Examination In the last chapter it was pointed out that material of this nature could be squeezed from the anther and spread over a sfide with the back of a scalpel. This method inevitably leads to distortion both of the cells and of the chromosomes, and in the writer's opinion the method here de- scribed gives a better preparation. There is first the collection and fixation of the anthers, and second, the separation of the microsporocj'tes from the other cells of the anther by maceration. The advantage of the crocus for this jDreparation is that it maj^ be brought into flower in the laboratory at any season of the year. The anthers may be taken at any stage of their development, the most useful stage for demonstration prepara- tions being that which is reached when the flower is just beginning to color. The flower is removed from the corm, the petals stripped away, and the anthers placed in fixative. Many fixatives may be used, though one of the best is Navashin (Chapter 18, F 6000.1010 Navashin 1912). The anthei's are placed in several hundred times their own volume of this fluid which is, by convention, but probal^ly unneces- sarily, kept in the icebox. The anthers are removed after 24 hours and waslicd over- night in running water. The method of maceration selected for this example is that of Faberge 1945; the abbre\aated directions are in Chapter 19 under the heading V 41.1. This method uses the stomach of the edible snail {Helix pomatia) which dissolves the intei'stitial tissue of plant cells. Edible snails are ol)- tainable either by collection in the field o r from restaurant supply houses. Snails ob- tained from the latter are usually in a state of liibernation from having been kept on ice and must be revived by being kept at room temperature for a day or two, after which they may be given a meal of lettuce and used on the day following. The snails are killed by drowning in warm water overnight, which leaves them fully ex- panded, and the stomach is then dissected out. The snail is removed from the shell and pinned down through the foot. The mantle cavity is then lifted in a pair of forceps and sfit. As soon as the edges of this sfit have been pulled back the crop (often miscalled the stomach) will be seen as a carrot-shaped body filled with brown- ish fluid. The fluid within the crop must now be withdrawn, either by the insertion of a hypodermic syringe, or by figaturing the crop at each end, removing it, and then squeezing the contents into a tube. It is simplest to handle a good many snails at the same time, since the material removed may be preserved in an icebox with a drop of toluene on top. The anthers, taken either from the water in which they have been washed or the alcohol in which they are stored, are placed in a drop or two of the enzyme solu- tion. If the maximum number of sfides 78 THE ART OF MAKING MICROSCOPE SLIDES Hydra are required, it is desirable to cut the anthers into pieces before placing them in the fluid. Maceration will usually be com- plete in about eight hours so that it is con- venient to start the preparation in the evening and to examine individual pieces at intervals in the course of the next morning until one has determined that maceration is complete. The completion of maceration may be judged by the fact that the materials should be flabby, but not completely disintegrated. At this stage the microsporocytes are extracted by gently squeezing either the anther, or each individual piece of anther, into a small drop of water. The micro- sporocytes themselves will not appear to be distinct but will appear as a gelatinous mass. This mass should be accumulated in a drop of water on a single slide and then small drops taken from it and made into smears on other clean slides. These smears need not be fixed, but may be permitted to dry on the shde to which they will ad- here so well that they can be stained by any nuclear staining method. Aceto-car- mine is in general use for temporary prep- arations, and a description of the use of this stain in plant material is given in Chapter 20. Preparation of a Dissociated Hydra It is a little pathetic that the majority of elementary textbooks of biology should include an illustration showing the types of cell to be found in hydra and that in- structors should then issue to the student a series of sections in which these cells are not visible. The illustrations have mostly been taken from older textbooks dating from the period when disassociation tech- niques for animal tissues were common. It would surely be more reasonable to show students shdes wliich agree with the illustrations in the books they use. Fixation is necessary before dissocia- tion, and the only question to be settled is whether or not the finished slide should show muscle cells in an expanded condi- tion, in which case the hydra will have to be narcotized before fixation, or whether it will be sufficient to kill the hydra with- out narcotization. It seems better, how- ever, to show cells in the expanded condi- tion and the hydra should therefore be collected from the tank or pond where they are growing, and accumulated in a watch glass of water which is kept in a cool place in subdued fight so that the hydra may expand. A drop of 2% chloral hydrate is then added for each five milfi- liters of water in the watch glass. This should be mixed with the water by suck- ing in and out of a pipet. This is likely to cause partial retraction of the hydra but they will expand again. After about 10 minutes two or three more drops per five milUliters of water may be added and mixed in, as the hydra are usually by this time sufficiently narcotized not to con- tract. The hydra should then be watched until touching with a hair causes no retrac- tion. The watch glass is then very care- fully picked up and placed in a fingerbowl. The reason for this is that hydra can most satisfactorily be fixed in large cjuantities of hot fixative. The solution of Perenyi (Chapter 18, F 6000.0040 Perenyi 1888) is excellent for this purpose. Tliis fixative, which has few other uses, is made by dis- solving % of a gram of chromic acid in 135 milHliters of water and then adding to this 100 millihters of ethanol. Seventeen and a half millihters of strong nitric acid are then added and the solution placed on one side until it has turned violet. The fixative should be heated to 70°C. and then flooded suddenly over the narcotized hydra which should remain in the fixative for about two days before being removed for dissociation. An individual dissociated hydra may be prepared as a smear, but it is presumed in this case that a number of shdes are being made for class issue, so that it is better to proceed by a different technique. The hydra are taken from the fixative (it is unnecessary to wash them) and placed in a few drops of the selected dissociating agent in the bottom of a small tube. The selection among the acid, dis- sociating media given in Chapter 19 under the heading AF 41.1 is not important, but the writer has been successful in the pres- ent preparation by the method of Hopkins as quoted by Roberts [Chapter 28, VJ41.1 Hopkins (1895)]. This method requires first, 20% nitric acid, second, a saturated solution of potassium alum. Hydra SQUASH PREPARATIONS FROM SOLID BODIES 79 The tube containing the hydra is half filled with 20% nitric acid. The specimens may be left overnight for treatment the next morning or, if one is in a hurry, the tube may be very gently warmed (to a maximum of about 50°C.) for twenty minutes. In either case it will be found that the hydra, which had become hard in the fixative, are now flaccid and tender. The acid is removed, either by pouring or by withdrawing it with a pipet, and the tube filled with the saturated solution of potassium alum. The specimens will float for a brief time but, as soon as they have sunk to the bottom, the alum solution is poured off and replaced with fresh solu- tion. The tube is now shaken gently until such time as each hydra has dissociated into its constituent cells. If this does not take place after shaking for a few minutes, it is necessary to withdraw the alum solu- tion, replace it with nitric acid, and to continue macerating for a further period. It is not to be anticipated that all hydra out of a batch of 20 or 30 will disintegrate at the same time, but any large cell masses which remain may easily be picked out with the point of a needle after the tube containing the cells has been emptied into a watch glass. * Having thus secured a suspension of the cells in a solution of alum, it is necessary ,, first to wash most of the alum from them, ^' and then to get them into stain. For this purpose rinse the tube into a larger one which is filled with water, stir up, and allow the cells to settle. Any of the alum- carmine stains given in Chapter 20 (DS 11.21) may be used. Whichever one is chosen is poured over the cells after the supernatant water used in the last wash has been poured off. The time for staining is not important, three or four days being usually enough. Though the cells cannot be seen in the stain, it may be assumed that they have fallen to the bottom. The upper two-thirds of the stain is poured off before filUng the tube with a weak (1 %) solution of w^hatever alum was used in the prepara- tion of the stain selected. The cells are again allowed to settle, the supernatant liquid poured off, the tube refilled with alum solution, and so on, until the wash solution is practically colorless. The cells now have to be dehydrated; this is done by pouring off the alum solu- tion, replacing it with, say, 30% alcohol, which is itself replaced with 70% alcohol, as soon as the cells have fallen to the bottom. At this stage a few cells should be withdrawn with a pipet, placed on a sUde, covered, and examined under a high power of the microscope. Each cell should show the nucleus clearly stained dark red with a faint pink cytoplasm; if they appear too dark, a small drop of acetic acid should be added to the tube, mixed with the alcohol, and poured off after five minutes. The washing is continued until the alcohol no longer smells of acetic acid. It is easy to overdifferentiate at this point and unless the cells are grossly overstained, it is better to take them through without fur- ther differentiation occasioned by the wash in alum solution which thej^ had im- mediately after staining. The 70% alcohol is now replaced with absolute alcohol in which the cells should be thoroughly stirred and then left overnight. A second change of absolute alcohol should be given; this should not be poured off but should be withdrawn with a pipet, so as to leave the cells accumulated in the least possible quantity of absolute alcohol at the bottom of the tube. The tube is then filled with benzene and left until the cells have again fallen to the bottom, when the benzene is withdrawn and replaced with fresh benzene, which is again replaced. The cells, which are now lying at the bottom of the tube in not more than a drop or two of benzene, should be covered with three or four drops of a strong solution of Canada balsam in benzene. The specimens should now be stirred up and left for an hour or two until they are thoroughly permeated with the balsam, a drop of which may then be removed, placed on a sUde, and covered. By this method, as many slides may be made as there are drops of balsam; and, if the cell concentra- tion has been kept reasonaljly heavy, it is usually better to use a very small {% inch) covershp in order to get as many slides as possible. A preparation of this size should contain two or three hundred cells and will give the student an excellent picture of all of the cell types found in hydra. 10 Ground Sections General Principles Nature of the Process Previous chapters have dealt with the preparation of whole specimens either mounted individually, smeared, or squashed on a slide. There are many speci- mens which, in order that their micro- scopic structure may be examined, have to be cut into thin structures. The more usual methods of cutting such materials are given in this and the next four chap- ters. The present chapter, which describes the preparation of sections of materials too hard to be cut by anj^ conventional method, had better be ignored by anyone not specifically interested in this process. Sections of hard materials such as bone, the calcareous skeletons of coral, and even some of the hardest vegetable materials, must be prepared in two stages. The first of these stages is the preparation of a crude section from a half to one millimeter in thickness, while the second stage con- sists in grinding this down while leaving both sides polished. Preparation of the Crude Section It is presumed that we are dealing with material which cannot be cut with a knife and must, therefore, be cut with a saw. Most woodcutting saws are not hard enough for the purpose and the choice lies between the ordinary hacksaw, intended for cutting metal, and a jewelers' saw. The disadvantage of a hacksaAV is that it is very coarse, so that only thick sections may be cut; the disadvantage of the jewelers' saw is that, unless it is guided by an expert hand, it will not cut a parallel- sided section. Whichever saw is selected, however, it should have relatively coarse teeth, particularly if bone, or material containing very much animal matter, or material which has been embedded in resin, is to be cut. These materials choke the teeth of a fine saw; the tooth marks left by a coarse saw do not matter, for they will be ground and pohshed out. For very hard substances, such as teeth, it is often necessary to use a circular diamond saw, which is usually available in depart- ments of geology Avhere rock sections are cut. Even teeth, however, may be cut with a jewelers' saw provided that the blade be changed at intervals and that the entire operation be conducted under the surface of water. Another type of preparation is that in which it is desired to preserve both the hard and the soft portions at the same time. A standard example of this is the preparation of a coral, of which it may be desired not only to section the calcareous skeleton but also to retain in position the soft parts of the animal within. This can only be done by embedding the material in some substance nearly as hard as the skeleton itself. A number of resins have been proposed for this purpose. The au- thor always prefers, however, to use Canada balsam because, though it is gummy in the final grinding, it has the ad- vantage that it need not be removed for mounting, and thus obviates one rather laborious stage of the process. As an alternative one may employ the process of Henrichi 1916 (4349, 6:45) in wliich gum damar is substituted for balsam, though the method which is described in- volves the use of machinery not normally available in laboratories. The technique of 80 Grinding GROUND SECTIONS 81 oml)e(l(ling in balsam is described in some detail iu the .second example wiiich follows the chapter and need not be repeated here. Selection of Grinding and Polishing Agents Once the initial sections have been pre- pared it is necessary that they should have one face pohshed, that this polished face should then be attached to some mate- rial, and that tlie other face be j^round away and brought to a pohsh when the section is of the correct tliickness. One technique for doing this is described in the first example at the end of this chap- ter, but some discussion must take place at this point as to the selection of grinding and pohshing agents. The initial flattening of one side of the section is best done with the aid of carbo- rundum powder, using a grit of about 100- mesh. The 100-mesh carborundum itself is far too coarse to leave a surface wliich may be polished and an intermediate stage is required. In the writer's opinion, pumice is best used for this intermediate stage. We are now speaking of relatively soft material, such as bone or coral, and not of thin slices of rock which would require several stages of carborundum between 100-grit and pumice. One of the most diffi- cult things to determine is when the scratches have finally been removed; this can never be seen when the sections are wet from grinding. It is, therefore, neces- sary at intervals to wash the surface of the section which is being ground, to dip it into alcohol, and then to warm it until dry. The surface of the section is then ex- amined with a strong hand lens by re- flected light, and grinding is ceased when it presents a uniform, dull surface un- broken by scratches. The next stage is to bring this flattened surface to a high degree of polish. All the old directions recommend the use of rouge . The objection to rouge is its color, and the fact that it gets over everything on the bench and around the bench. The author most warmly recommends the substitu- tion of white rouge (eerie oxide), which is rapidly replacing ordinary rouge in the polisliing of glass, and is also excellent for microscopical specimens. It does not mat- ter very much against what surface the abrasive has up to this time been rubbed, though glass is conventional. It is, how- ever, impossible to get a fine surface with rouge on glass and one should, therefore, use a leather strop for the purpose. This does not mean a loose leather strop of the type used by barbers but rather a flat surface of horsehide which has been at- tached to a hardwood block. Rubbing the section up and down on this, while it is well lubricated with a slurry of white rouge, will soon bring it to a fine pohsh. There is no reason to get discouraged if, after polishing, the surface is found still to have a few fine scratches on it. The sec- tions are going to be mounted in balsam which will hide most of the scratches. The sections are then cemented, pol- ished side down, to a shde and ground on some flat surface with coarse carborundum until they are of the required thickness. It is unfortunate that there is no adequate method of judging this thickness except by eye; experience is the only guide which may be reasoriably followed. As soon as the section has been ground down to the required thickness it is then smoothed with pumice, polished with white rouge, and finally mounted. Practical appUcation of the principles here discussed will now be given in the form of two typical preparations. Typical Examples Preparation of a Section of Bone If the worker is interested only in the production of a section which will show the existence of Haversian canals, it is better to decalcify the bone (in one of the solutions given under AF 20 in Chapter 19) and to prepare sections by the paraffin technique described in Chapter 12. These sections, however, show neither the lamel- lae, lacunae, or canalicuh, which can only be demonstrated in a section prepared by grinding, in which all the calcareous parts remain intact. 82 THE ART OF MAKING MICROSCOPE SLIDES Bone The first thing is to secure a piece of dry bone. The majority of museums have old broken specimens from which they are only too glad to give away a bone or two. The example shown being sectioned in Fig. 30 is part of the femur of a horse which became accidentally broken. If no dried specimen of bone is available, and one is, therefore, forced to start with raw material, it is fii'st necessary to boil the bone for four or five hours in water in order to remove as much of the protein material from it as possible. If, moreover, handUng bone because it combines the stiffness of a hacksaw with the thinness of a jewelers' saw. The first cut is made at right angles to the direction of the cut shown and a second cut (as seen in the illustration) is then made, parallel to the free surface that has been cut, and at right angles to the present position of the saw in the figure. Several slabs are cut, as uni- formly as possible, but the saw kerf is stopped about a millimeter above the horizontal cut, and a second cut made, until (as is seen in the figure) as many Fig. 30. Sawing slabs of bone for sectioning. Note that the vertical kerfs have not been extended to meet the horizontal kerf. one is dealing with a long bone containing marrow, it is necessary that it should be cut with a hacksaw into short lengths in order that the marrow may be removed by boiUng. The bone is then taken from the boiling water, dried for a day or two, and then defatted by being soaked in any fat solvent. About the cheapest and most convenient solvent available is naphtha, though if price is a secondary considera- tion one can, of course, use xylene. Three or four changes, each lasting a week, in a considerable volume of solvent must be made, and the bone should then be baked in a low-temperature oven until the sol- vent has been removed. A series of thin slabs is then cut, as shown in Fig. 30. The saw there shown, which is a cheap imitation of a hacksaw, is the best that the author has found for slabs as are required have been outHned. Each is then successively cut off. A con- venient size slab for preparation of a microscope shde is approximately }^i inch square, which is the size shown in the illustration. Single sections may be handled without being attached to anything. Several blocks may, however, be ground down at the same time by cementing them to a slide as shown in Fig. 31. The hot table (shown in its entirety in Fig. 8) is heated to about 100°C. The slide is then hberally covered with natural balsam — not a solution in xylene — and the slabs laid in place. Each block of bone will have a jagged corner sticking from it, where it broke away just before the saw cut was complete, and these little jagged corners must be placed upper- most. One must also be careful that the Bone GROUND SECTIONS 83 thickest sections of l)oiie are placed at tlie outside of the piepaiatioii, or it will \)v im- possible to avoid the slide's \vol)blinf; while being ground. As soon as all the slabs of bone have been placed, the balsam is heated until it boils rapidly. The balsam usually catches fire during this process, but it may be extinguished by blowing on it. A pair of forceps is finally used to push each piece of bone into contact with the glass and the sUde is cooled. The scratches from the earl)orundum nuist now be polished out with pumice powder. One secures either another piece of glass or a flat hardwood board — they are etiually good -and prepares on this a paste of pumice and water exactly as the carborundum paste was prepared. The slide is washed to remove the carborun- dum grains and then rubl)ed, with exactly tiie same motion, in a slurry of pumice un- til each section of bone is uniformly Fig. 31. Mounting bone slabs on glass slide. While the section is cooling, a pool of water is poured on a slab of plate glass, and carborundum powder, of about 100 mesh, is sprinkled in until a thick cream is produced. The slide is then turned upside down in this cream, as shown in Fig. 32, and rubbed backward and forward with a circular movement, so as to grind down the bone. The grinding should be con- tinued until the pieces of bone have been reduced to a uniform thickness. It may be necessary, from time to time, to add more water, and the specimen should ))0 lifted at intervals to make sure that the abrasive fluid is underneath it, and not being pushed out by a wall of balsam. smooth on the surface. Some people prefer to use a hardwood, rather than a glass, slab for the pumice. It is best to dry the surface of the bone and to examine it un- der a lens by strong reflected light to make sure that the scratches have been removed. The sections are now polished on a horsehide strop cemented to a wood block. The strop is lubricated with a thick cream of either rouge or white rouge (eerie ox- ide). The shde should be rubbed rapidly with very little ])ressure; too much pres- sure is liable to dry the sections. If dried rouge is forced into the surface under pressure it is almost impossible to remove 84 THE ART OF MAKING MICROSCOPE SLIDES Bone unless the specimens are reground with pumice. The final polish can be judged by eye and should be such as would be acceptable on, say, a pohshed ivory ornament. The slide is then returned to the hot table seen in Fig. 31 and heated until the balsam is molten. Another slide is placed alongside the first, liberally smeared with balsam, and the sections transferred from the first shde to the second with the pol- ished side down. They must be pressed hard to make sure that the polished side necessary that the shde should not rock from side to side as it is pushed about, or the covershps will become ground at the end before the sections of bone in the mid- dle are thin enough. Experienced experts can often grind a section of bone until it is as thin as a number 1 coverslip, but this is not recommended to the beginner, for if the section is ground too thin it will sud- denly disintegrate and all the work done so far will be lost. When the sections are judged to be thin enough, the slide is very carefully washed under the tap to remove Fig. 32, Grinding down bone slabs. is in contact with the glass. The shde is then cooled and returned to the glass plate (Fig. 32) containing the carborundum paste, on which it is now slowly and stead- ily ground until the sections are thin enough. The thickness may partly be esti- mated by holding the specimen up to a Ught and seeing how transparent it is be- coming; the correct thickness has the transparency of a rather thin sheet of oiled paper. If the technician does not care to trust his judgment in the matter it is pos- sible to take two number 3 coverslips and to cement one on each end of the slide. A thick number 3 covershp is about the thickness of a good section of bone so that, if the bone is ground down until the cover- shp just starts to be affected by the grind- ing compound, the sections may be pre- sumed to be of the right thickness. To use this method satisfactorily, however, it is all traces of carborundum grit and the sec- tions then smoothed with pumice, as was done before. A certain amount of reduc- tion in thickness may also be produced by the pumice though it is usually better to use carborundum. As soon as the scratch marks of the carborundum have been re- moved, the sections are again washed care- fully under the tap and then pohshed. A coverslip is then placed on top of the prep- aration and the slide examined under vari- ous powers of the microscope, which will verj^ soon disclose whether or not it is thin enough. If it is not thin enough to show the required structures it should be taken back to the carborundum, ground some more, then resmoothed and repohshed in the manner described above. Several trials are often required before all the sections are found to be satisfactory. There are two schools of thought as to Coral GROUND SECTIONS 85 the mounting of these l)one sections. Some people prefer to mount them dry, in which case tlie shde is pkiced in a jar of benzene and left until all the Canada balsam has been removed. Each individual section is then picked up on a section lifter, trans- ferred to two or three fresh changes of benzene to remove the last of the balsam, and then dried under pressure between two glass slides; when it is dry it is then treated as any other dry wholeniount (see Chapter 1). This method undoubtedly makes it easier to see the finer structure of the bone, but it is applicable only to very thin sections, for the additional transpar- ency imparted b}' the balsam will be lost. It is usually more satisfactory to melt the balsam, to lift each section up, to place it in fresh balsam on its individual slide un- der a coverslip, and to heat it until all the air bubbles have been expelled. The coverslip is then held down with one of the clips shown in Fig. 25 and cooled. All these operations can be conducted much more conveniently on the machinery made for grinding and poUshing rock sec- tions, but this description has been given for the benefit of those who lack such machinery. It has from time to time been recommended in the literature that one should grind sections of this kind down on microtome sharpening stones, using oil as a lubricant. The writer has had the most wretched results by this method ; it is men- tioned only in order that it may be avoided. Preparation op a Transverse Section of a Coral with Polyp in Place It must be understood, first of all, that the preparation here to be described will give a very much less satisfactory trans- verse section of a coral polyp than will the paraffin method described in Chapter 12. This method is intended onl}^ as a compromise between a paraffin section of a polyp and a ground section of a hard structure of the type described in the last example. The living animal must first be narco- tized, fixed, and hardened. It does not matter what coral be selected. The north- ern coral {Astrangia danae) is convenient both because of its wide distribution and because it ma}- be obtained from biological supply houses. Supposing, however, it is to be collected fresh, a piece should be secured of about the size of an orange, or smaller, and brought back to the laboratory and left to expand in plenty of well-aerated sea water. It must, of course, be narcotized before fixation and a double process is best for this type of specimen. About 5% of its volume of a saturated solution of mag- nesium sulfate is therefore added to the water and the action of this narcotic is enhanced by sprinkhng menthol on the surface. After about half an hour the pol- yps will be extruded from the coral in a partially narcotized condition and should be fixed. Perfect narcosis will result in such a small quantity of the polj'p remaining in the coral that it is scarcely worth while sectioning it, whereas fixing an unnarco- tized coral causes such a contraction of the polyp that the sections will be hard to interpret. The fixative selected should be one which will harden the poh'p as much as possible without having any effect on the calcareous structure surrounding it. The copper sulfate-mercuric chloride of Lo Bi- anco (Chapter 18, F 3400.0000 Lo Bianco 1890) is excellenth' suited for the purpose and about a gallon will be required for the fixation of a specimen of the size described. As much of the water as possible is now siphoned off from the vessel containing the coral specimen and the fixative added. The fixative should be stirred at intervals for the next two or three days and then the specimen should be transferred to fresh fixative for a period of about another week. The coral is then washed in running water overnight and j^laced in 4 % formal- dehyde, which should be changed daily until the whole of the fixative has been washed out of the specimen. A coarse hacksaw is then used to cut the specimen into cubes of about an inch on a side, with due regard to the selection of pieces which will subsequently give good sections. These inch cubes are now washed in running water overnight, to get rid of the formaldehyde, and suspended in a con- siderable volume of 70% alcohol. It will be necessary to impregnate them with a resin ; 86 THE ART OF MAKING MICROSCOPE SLIDES Coral they must, therefore, be completely dehj'- drated and cleared as though whole- mounts were to be made of them (Chap- ter 6). This dehydration is very slow so that, after about two weeks in 70% alco- hol, they should be placed for a further two weeks in 95% alcohol before being transferred for still another week into ab- solute alcohol. As considerable volumes are required it may be found more eco- nomical to substitute anhydrous acetone for the absolute alcohol. The writer prefers to embed in Canada balsam but this must be freed of its con- tained essential oils if it is to become hard enough for grinding. It is very difficult to melt dry Canada balsam without obtain- ing a mass full of air bubbles and it is, therefore, better to take a pound or two of the natural balsam, place it in a wide evajjorating dish and heat it to about 120°C., with due precautions against fire, until such time as a small drop placed on a cold plate hardens rapidly to a material which will crack and chip, rather than bend, when a knife is applied to it. A solution of about 30% by weight of this essential-oil-free Canada resin in ben- zene is also required but may be made up from the commercial dried product. After the specimen has been completely dehydrated it must, of course, be de-alco- holized in some material which is miscible with the resin and it is suggested that benzene be used. Three changes of ben- zene, with about a week in each, will be required ; and it is desirable that some de- hydrating agent, preferably silica gel or calcium sulfate should be kept in the ves- sel to remove the last traces of water. When the specimen is completely impreg- nated with benzene it is transferred to the solution of drj' Canada balsam in benzene and left there for a week or two until it is imi:)regnated. The solution is then trans- ferred to an open vessel and warmed gently until as much as possible of the solvent has been removed. Care must be taken never to raise it to the boiling point of the solvent or bubbles may occur in the actual specimen which will wreck the sub- sequent preparation. The specimen is then immersed in molten balsam, which is maintained at about 100°C. until no fur- ther diffusion currents are seen. The beaker is then cooled until the balsam is completely hardened. The only practical method of removing this hardened block of clear balsam containing the specimen is to crack the beaker away from it with a hammer. One is now left with a solid block of bal- sam containing the coral, and a saw is used to trim the specimen to shape. This trim- ming should result in a rectangular block, if we are dealing with Astrangia danae, of about a 3'^-inch side, by one inch long, with the polyp protruding from one end. Canada balsam is not easy to saw and it is recommended that the blade be kept lu- bricated at all times with a weak soap solution. This rectangular block is now treated exactly as though it were a piece of bone and a series of slices of from 3^^ to one mil- limeter thick cut from it. These slices can- not, however, be handled all at one time in the manner described in the last example, but must be handled individually. Each slice is therefore taken and ground flat with carborundum used in the manner described in the last example. Instead, however, of having the section cemented on a slide, it is held on the ball of the first finger and rubbed backward and forward until it is flattened. When it is flat, it will not be satisfactory to wash it under the tap, because many of the carborundum grains will be embedded in the balsam and one should, therefore, take a rag moist- ened with benzene and wipe the surface carefully until the carborundum grains are seen to be removed. Great care should be taken to do this in such a manner that the flatness of the center of the section, where there are only the soft parts em- bedded in balsam, is not disturbed. The section is then rubl^ed up and down with the finger using pumice on wood, and again washed and cleaned. These speci- mens do not polish well on leather and a sheet of velvet (which may be gummed to a wooden block) should be substituted. This velvet is Uberally lubricated with white rouge in water and a polish put on the lower surface of the section. If the white rouge is sufficiently diluted with water, it is unlikely to become embedded Coral GROUND SECTIONS 87 in tlie balsam; therefore washing in water will remove it. The section must now l)e mounted on the slide which it is finally to occupy, but this is done in exactly the opposite manner to that described in the last example, in which the shde was covered in balsam and the object pressed to it. In this case the section must be placed on a flat surface, a slide warnred rather above the melting point of the balsam and pressed down on to the specimen so as to melt the minimum possible quantity of balsam to permit per- fect adherence. Only in this manner can one avoid disturbing the soft parts. The other side of the section is now ground down, smoothed, and polished exactly as described in the last examj^le. It will, how- ever, be much easier to estimate the thick- ness of a section of this type, for one can always observe the soft parts under the microscope. When the section has become sufficiently thin, it is only necessary to ])lace a drop of natural l)alsam on top, ap- ply a coverslip, and warm the whole while applying jiressure. Though this description has api^fied to the preparation of a coral, it may equally well be applied to the production of sec- tions of bone with the bone marrow and blood cells retained in position. Those who prefer to grind their sections on oilstones with an oil lubricant should consult the description of Henrichi 1916 (4349, 6:45). 11 Sections of Free Material General Principles Nature of the Process A section is a thin slice cut from biolog- ical materials with a view to studying either the cells themselves, or their ar- rangement, neither of which can well be made out from a wholemount. If the Though sections may be cut at any an- gle, they are usually taken through one of three planes (see Fig. 33), known as trans- verse, sagittal, and frontal. The purpose of this orientation of the material with rela- tion to the plane of the section is to permit Fig. 33. Standard section planes. worker's only interest is, in the shape of the cells, as distinct from their structure, he should attempt some of the prepara- tions described in Chapter 9, which will often be found better than sections for his purpose. a better visuahzation of the structure of the whole, from an examination of sec- tions. When the relation of organs is to be reproduced from sections, the process is known as reconstruction and is referred to briefly in Chapter 14. In theory a section 88 Microtomes SECTIONS OF FREE MATERIAL 89 can be made by cutting a thin slice from the object with a sharp knife. Few mate- rials, however, are suitable for this, nor does this procedure yield sections of the same thickness. It is, therefore, customary to use an instrument known as a micro- tome: a device for advancing a block of tissue a given amount, cutting a slice from it, and then re-advancing it for the same amount, and so on. A full account of all the types of mechanism by which this re- sult can be produced is to be found in Richards 1949 and need not be repeated here. Another objection to the mere cutting of slices from an object is the nature of biological specimens themselves. Few of these are stiff enough to withstand the ac- tion of the knife without bending, and many contain cavities which would be crushed out of recognition as the section was taken. This makes it necessary, for most biological work, to surround and sup- port the object to be cut with some mate- rial which will impregnate it. The medium most commonly used is wax. The tech- nique for cutting wax sections is described fully in the next chapter. Nitrocellulose is also employed and is described in Chapter 13. Another method of stiffening the ma- terial, so that sections may be cut from it without crushing, is to freeze the speci- men. This techniciue is described in Chap- ter 15. There are however, materials which may be cut without either the complicated microtomes described in these chapters or the support of impregnating substances. Sections which are so cut are known as free or freehand sections. These form the subject of the present chapter. Microtomes for Free Sections Even though the material itself is of the correct consistency to withstand the ac- tion of the knife, it is still necessary to have some mechanism which will produce sections of known thickness. The tj^pe of microtome usually employed in liard so(;- tioning is sliown in Fig. 34 and consists essentially of a disk, usually of polished plate glass, supported on a cylinder grip- ped in the hand. Within this cylinder there is some mechanism for holding specimens Avhich terminates at its lower end on a micrometer screw. When this screw is turned, the object in the holder is pushed above the surface of the glass plate. The collar of the micrometer screw is gradu- ated, sometimes in thousandths of an inch, but more usually in hundredths of a millimeter. The unit commonly used to describe the thickness of a section is a Fig. 34. Hand microtome. micron which is one-thousandth of a milli- meter; but hand sections are very rarely cut of less than 10-micron thickness and are usually better at two or three times this. Methods of Holding the Material Though the material itself may be suit- able for cutting, it is rarely of a size and shape which may be gripped in the holder of the hand microtome without additional support. It must, therefore, be held in some substance which will itself cut read- ily and which may be easily shaped to sup- port what is being cut. It is possible to embed the material either in wax or nitro- cellulose before cutting a hand section, but if one is to go to this amount of trouble, it is usually better to use a comi)lex micro- tome of the type described in Chapters 12 and 13. Vegetable tissues are usually em- ployed to support objects for hand section- ing and the two best known are elder i)ith and carrots. Klder pith has tlie advantage that it may be stored indefinitely and cuts with a clean crisp action. Unfortunately the pith of the American elderberry (Sam- bucus caiuidensis) does not ai)pear to he as suitable for the purpose as the pith of the 90 THE ART OF MAKING MICROSCOPE SLIDES Fixation European elderberry {S. nigra). This dif- ference between the two species may ac- count for the disfavor in which elder pith is held in the United States, but in the writer's experience it is far more conven- ient than the carrot. The disadvantage of the carrot is that it must be absolutely fresh, and even if it is kept in water over- night it loses much of that crispness which is necessary for the production of a good section. Almost all hand sections are cut from plant material, and most of them from leaves or stems. To support a leaf, a cylinder, of the right diameter to fit in the microtome is cut either from elder pith or carrot, split down the middle, and the leaf inserted. The holding screw is then tight- ened. Stems, however, cannot be held by this means and a hollow cjdinder must be prepared with an outer diameter con- venient to the microtome and an inner diameter which is slightlj' less than that of the stem to be gripped. This hollow cylinder is then split, the stem inserted, and the section cut. Of course, a few sul)- stances, such as cork or stiff plant stems, may be cut without any other support; these are, however, in the minority. Hardening and Fixing Materials for Cutting Many objects, which are in themselves unsuitable for sectioning by hand, may be made more suitable if they are fixed and hardened. A general discussion of the principles governing the selection of a fixa- tive is given in Chapters 6 and 12; the formulas which have been suggested for the purpose are given in Chapter 18. If, however, one is to go to the trouble of hardening and fixing material in a formula designed to preserve the structure of the cells, it is usually worth while to go to the additional trouble of embedding the ma- terial and cutting sections as described in the next two chapters. For material to be hand sectioned it is sufficient that it be preserved in 90% alcohol. This process is equally appUcable to the stems and leaves of the botanists, or to the verj' few animal materials, such as cartilage, which are suit- able for the production of hand sections. It must not be thought that objects em- bedded in nitrocellulose or wax should not, or cannot, be cut on a hand microtome. The study of the embryology of the frog is rendered much easier to elementary students if they are allowed to cut hand sections of blastulae, gastrulae and young larvae for themselves; and a word might be said at this point as to the method by which such blocks maj^ be readily pre- pared in quantity. The larvae are fixed, dehydrated, cleared, and impregnated with wax in the manner described in Chap- ter 12. Instead of casting each into an individual block, however, a large slab of wax — the cheapest paraffin is suitable — is cast in a tray. A heated iron rod of about I'^-inch diameter is then driven into the slab so as to make a little pool of molten wax. An impregnated larva, or egg, is then dropped into the hole and the process repeated. By this means a couple of hundred frog eggs may be embedded in ten minutes. The large block of wax, con- taining numerous eggs, is then cut with a saw into rectangles, the edges of which are trimmed with a knife until they will fit into a hand microtome. These blocks may even be cut without a hand microtome by placing the block on a bench and shaving off successive sections with a sharp scalpel. Staining and Mounting Sections Sections, wliich are taken individually from the knife and accumulated in a dish of 70% alcohol, should be treated as wholemounts rather than as sections. They may, that is, be directly mounted in either gum media (Chapter 4) or jelly media (Chapter 5) or they may be stained and mounted in resinous media in the manner described in Chapter 6. It might be pointed out, however, that many sections may be double- or triple- stained (a process which is impossible with wholemounts) and that in theory any method of staining described in Chapters 20, 21, or 23 for wax or nitrocellulose sec- tions may also be applied to a hand sec- tion. These are, however, much better applied to sections after they have been attached to a slide. If they are to be at- tempted, reference should be made to Leaf SECTIONS OF FREE MATERIAL 01 Chapter 28 (V 21.3), wliore are ftivon formulas and methods whicli may l)e used to attach individual sections. Sections which are not attached to shdes must he transferred from one fluid to anotlier witli the device known as a section lifter, wliicli is merely a small flattened sheet of metal held in a wooden handle. Typical Examples Preparation of a Transverse Section of the Leaf of Ligustrum The leaf of the privet is, in the opinion It is necessary to have a hand micro- of the writer, the easiest biological speci- tome of the typo already desci'ihed, an old- men from which a section maj' be pre- fashioned luuul razor, some freshly picketl Fig. 35. Inserting a leaf into a split cylinder of carrot. Fig. 36. Cutting a hand section. The razor is drawn across the plate with gentle pressure and the section then washed into a stender dish. pared and is included in this place as an introduction to the art of cutting sections. The leaves should be collected in sum- mer and stored in a jar of 90% alcohol which must be changed as often as it be- comes diluted from the extracted water or discolored by the extracted chlorophyll. If large quantities of alcohol are used in the first place it will be unnecessary to change it, but it must be agitated from time to time to avoid the accumulation of water at the bottom. carrots, and a stender dish of 70% alcohol. The razor shown in the illustration (Fig. 35) is flat on one side and hollow-ground on the other. This is the best kind for hand sectioning, but if it is not obtainable a double hollow-ground razor may be used. Next take a coi'k borer of such a size that it will bore out a cylinder which will fit reasonably well into the holder of the microtome. Then (Fig. 35) cut from a fresh carrot as many cylinders as are required. A leaf is then trimmed until it is the same 92 THE ART OF MAKING MICROSCOPE SLIDES Wood width as the cylinder. The cyUnder of carrot is spht, inserted into the holder of the microtome, and the leaf pushed down into the center of the cyUnder (Fig. 35). The holder of the cj'hnder is now tight- ened and the razor used to slice off as much of the leaf and cork as projects above the level of the plate (Fig. 36). The micrometer screw at the bottom of the cylinder is then turned as far as is neces- sary to advance the carrot and leaf the thickness of the required section above the glass plate, and a section shaved off. The razor must not be pushed straight across the material, but must be drawn side- ways, so that the whole length of the razor is used to cut the section. In Fig. 36 the razor is placed in the correct position for the beginning of the cut; but by the time it has passed through the block, the oppo- site end of the razor will be opposite the section. Notice also that the material is being sectioned with its thin edge, not its breadth, against the knife. This is neces- sary whether one is cutting sections by hand or by any other means, because the less material cut at the same time, the less is the chance that it will be torn from its support. The section on the knife is now brushed off into one of the stender dishes of alco- hol, the micrometer screw advanced the same amount, another section cut, and so on. About twice as many sections should be cut as are ultimately required, for at least half of them will either be damaged or will have one end thicker than the other. The sections are therefore examined un- der the low power of a microscope and those which are not considered satisfac- tory are thrown away. Stains used for materials of this type are given in Chapter 21 (DS 21.15), to- gether with a specific example. After the sections have been stained as there de- scribed, they should be dehydrated ex- actly as if they were wholemounts and mounted in balsam in the manner de- scribed in Chapter 6. Preparation of a Section of Wood The last process described is one of the easiest preparations which may be made in microtomy, whereas the present is one of the most difficult. The sectioning of wood belongs properly in this chapter on the preparation of freehand sections, be- cause wood does not need to be embedded. The difficulty of cutting it hes in the fact that it is too hard to be cut by regular methods while being in general too soft to be cut by the method for ground sections given in Chapter 10. Some plant materials, such as the wood of the fignum \'itae, or the ivory nut, may be cut by grinding techniques and make better sections b}' this method than by any other. A few woods, such as white pine cut parallel to the grain, are sufficiently soft to be cut in a hand microtome without any preparation. The present example deals with such woods as maple or oak, which fall between these two extremes. The first thing to be done in the prepa- ration of any section of wood is to cut a block, one end of which is of the size of the required section. A section size about ^i inch square is usually adequate to demon- strate the structure of the wood, and a number of blocks this size should be pre- pared with due regard to the plane of the section. The wood must next be softened. This would be relatively simple if mechanical means alone could be emploj'ed. Unfor- tunately, however, many woods, particu- larly oak and teak, contain silica, which can only be removed by treatment with hydrofluoric acid, and this acid naturally cannot be made to penetrate the wood until all air has been removed. The blocks of wood are therefore boiled in distilled water for about ten minutes. A disk of glass — or of wire gauze — which will just fit inside the beaker, is placed on top of the pieces of wood, and a sufficient weight added on top to cause the blocks to sink to the bottom. The beaker is now trans- ferred to some type of vacuum equipment and exhausted until bubbles are seen to cease leaving the wood. The vacuum is then released, the beaker returned to the flame, and again boiled for ten minutes. This process of alternate boiling and evac- uation is continued until the wood no Wood SECTIONS OF FREE MATERIAL 93 longer floats, a state showing that the greater part of air has been removed. The blocks are now transferred to 50 % commercial hydrofluoric acid, and a word of warning must be issued as to the danger of this material. Since it dissolves sihca, it cannot be handled in glass vessels, and the choice lies between hard rubber and lead. Not only is the vapor extremely corrosive, but a burn from hydrofluoric acid on the skin is worse than that from any other chemical known to the writer. Extreme care should be taken, therefore, in transferring blocks to hydrofluoric acid, where they may remain until the silica has been dissolved from them. A block of 1.^-inch oak will be satisfactorily desihci- fied in one day, but teak should remain for at least three or four. Observing the same precautions as before, the blocks of wood are removed from the hydrofluoric acid to running water and must be washed for at least three or four hours before thej' are safe to handle with the hand. A block ma}' now be removed from the water and mounted in any convenient mi- crotome for cutting. The harder woods can never be satisfactorily cut on a hand microtome and the best mechanism is un- doubtedly the sliding microtome described in Chapter 12. Exactly the same precau- tions in cutting a wood block on this mi- crotome should be taken as in cutting a block of anything else. That is, the knife must be sloped at an angle towards the block so that the greatest possible length of knife is used to cut a single section, and the block must be so orientated that the knife enters at one corner rather than flat on the side. The sections as they are cut ma}'- be removed to water, and any tendency which they have to curl may be counteracted by warming the water. An interesting variation of this stand- ard technique was proposed at the same time by Crowell 1928 (20540b, 5:149) and b}' Kisser (cited from Crowell). This method consists of mounting a dry block of wood in any tj^pe of microtome and di- recting onto its surface a jet of high-pres- sure steam. After the steam has acted for a moment or two, a single section is cut and the steam again apphed for another few moments before cutting the next section, and so on. It is stated that by this means sections of the hardest material may be taken without the use of hydrofluoric acid. Sections of wood are usuallj- mounted in balsam, dehydrated, and cleared as de- scribed in Chapter 6. Sections which tend to curl may be tied between sUdes. Sections of wood containing some natu- ral color, as oak and mahoganj', are best mounted unstained, but thin sections of colorless wood may become too transpar- ent under this treatment. Almost any dj'e may be used, since the purpose is not to differentiate the parts but only to render them Wsible. 12 Paraffin Sections General Principles Nature of the Process The last chapter discussed freehand sec- tions, that is, sections of material which is in itself sufficiently strong and sufficient!}' coherent to hold together when cut in thin slices. The majority of objects to be sec- tioned, however, contain cavities which would collapse under the action of the knife, or are not of a shape or consistency which would enable one of them to be cut by hand. Objects of this nature must, therefore, be supported in a matrix which ■will itself section well, and those contain- ing cavities must be impregnated through- out their whole substance with the embed- ding medium. Wax, nitrocellulose, and a variety of water-soluble materials have from time to time been suggested as im- pregnating and supporting agents, but the use of wax is so convenient and simple that only in special cases should any other material be emploj'ed. The advantage of wax is not only that it readily passes from a solid to a molten state at temperatures which do not dam- age the material, but also that it is some- what sticky, so that ribbons of sections may be prepared, each section being in the ribbon in the same order as it was cut from the object. Thus, if a rectangular block of wax is mounted in some kind of holder and then brought sharply down on a hori- zontal knife, the thin slice of wax which is cut off will adhere by its edge to the edge of the knife. If the block is then advanced by some mechanical device — such as a microtome — a small distance and again brought down on the knife, a second sec- tion will be cut off which will displace the first section, to which it will adhere on one 94 edge, while the other edge remains at- tached to the knife. By the repetition of these movements a long ribbon may be produced. A ribbon of this type is seen in all the stages of its preparation in Figs. 65-70. Preparation of paraffin sections is quite a complex operation and in\'olves the following stages: 1. Fixation of the material. 2. Dehydration, in order that the ma- terial may be impregnated with a fluid capable of dissolving wax. 3. The removal of the dehydrating agent with a material solvent of, or miscible with, molten wax. 4. The soaking of the cleared specimen in a molten wax for sufficiently long to insure that it shall become com- pletely impregnated. 5. Casting the now impregnated speci- men into a rectangular block of wax. 6. Attaching this block of wax to some holder which itself may be inserted into a suitable microtome. 7. The actual cutting of the sections of the block into ribbons. 8. The placing of these ribbons on a glass shde in such a manner that they will lie flat and that the con- tained section will be adherent after the wax has been dissolved away. 9. The removal of the wax solvent. 10. Staining and mounting. I^ach of these operations will be dealt with in due order. This chapter terminates with a series of examples which describe in de- tail the application of the principles dis- cussed to actual preparations. Fixation PARAFFIN SECTIONS 95 Selection of a Fixative Fixative formulas are given in Chapter 18; and the selection of the fixative for small invertebrates has already been dis- cussed in Chapter 6. When one intends to section a small invertebrate, with the pri- mary function of preservinj^ its parts in as natural as possible a relation to each other, the same fixative should be employed as is recommended for those invertebrates in- tended to be made into wholemounts. The purpose in each case is to preserve the ob- ject in as natural a shape as possible with- out special regard to the preservation of the fine details of the cells themselves. Something of the same consideration applies to blocks of tissue which are to be fixed in such a manner that their general structure, or histology will be displayed. In this case, however, there is no problem of contraction of parts, so that fixatives which would be quite useless for a whole animal may safely be applied to a block of tissue. Reference to Chapter 18 will show that there are between 600 and 800 solu- tions recommended for fixation, and there is no general agreement as to which is the best for any particular purpose. These notes are therefore written only for the benefit of the beginner who, presented with this bewildering display, lacks the ex- , perience on which to base his choice. The selections given below are modified from Gray 1933 (11360, 53:15). The figures ' following the name and date refer to the decimal classification of Chapter 18 in which the formulas for these fixatives will be found. Specific suggestions for the em- ployment of fixatives are to be found in many of the examples of the preparation of shdes which occur in this book. A. Recommended Fixatives for Em- bryos OR Whole Organs Exceeding 5 Mm in Thickness. 1. FOR USE when the PRESERVATION OF SHAPE IS OF PRIMARY IMPOR- TANCE. Bensley 1915 F 1700.0010 Erhtzkv 1877 F 4700.0000 Hoyer 1899 F 3700.0000 Lavdowski 1894 F 6000.0010 Maximov 1909 F 1700.1000 Miiller 1859 F 7000.1000 Orth 1S9() F 7000.1000 R^gaud 1910 F 7000.1000 2. WHEN IT IS DESIRED, AS FAR AS POS- SIBLE, TO PRESERVE BOTH SHAPE AND PROTOPLASMIC DETAIL. a. When shape is of greater impor- t(l7lC€ IlcUy 1903 F 3700.1000 Petrunkewitsch 1933 F 4900.0010 Rawitz 1895 F 5600.0040 Smith 1902 F 7000.1010 Zenker 1894 F 3700.0010 b. When protoplasmic detail is of greater importance Fol 1896 F 1560.0000 Gilson 1898 F 3000.00 14 Kohn 1907 F 3700.0010 Maver 1880 F 5000.0050 Rabl 1894 F 2300.0000 Tellysniczky 1898 F 3500.0010 B. Recommended Fixatives for Small Portions of Organs or Whole Or- gans OR Embryos Not Exceeding 5 Mm. in Thickness. 1. when a general-purpose fix.\- tive is required Carleton and Leach 1938 F 3000.1000 Gatenby 1937 F 6700.0040 Gerhardt 1901 F 3600.1010 Schaudinn 1900 F 3000.0000 2. WHEN PROTOPLASMIC DETAIL IS OF GREATER IMPORT.\NCE a. When nuclear fixation is espe- cially required Allen 1929 F 5600.1010 van Beneden (1905) F 3000.0010 Carnoy 1887 F 0000.0010 Carnoy and Lebrun 1887 F 3000.0010 Sanson (1928) F 0000.0010 b. When cytoplasmic detail is espe- cially required Champv 1911 F 1670.0000 Flemming 1884 F 1600.0010 Kultschitzkv 1887 F 4700.0010 Mann 1894 F 1300.0000 Smith 1935 F 1670.0010 There are only two general precautions to be observed in the practical application on THE ART OF MAKING MICROSCOPE SLIDES Dehydration of fixatives: first, that adequate volumes (at least 100 times the volume of the part to be fixed) be employed; second, that mixtures containing either chromic acid or potassium dichromate with formaldehyde be used in the dark. After fixation, tissues should be thoroughly washed in water if this is the solvent for the fixative, or in alcohol if the fixative is based on the lat- ter. Objects are usually stored, after fixa- tion and washing, in 70 % alcohol ; though if they are to be kept a long time before dehydration, it is recommended that 5% of glycerol be added to the alcohol. This glycerol musi,, however, be very thor- oughly washed out before dehydration commences. Choice of a Dehydrating Agent Chapter 25 discusses the numerous or- ganic solvents wliich from time to time have been proposed for the dehydration of biological specimens and the selection between them is not usually of great im- portance. The classic method of dehydra- tion is to soak the object in a graded series of alcohols, usually 10 or 15% apart. De- hydration through gradually increasing strengths of alcohol may be vital when one is dealing with delicate objects con- taining easily collapsible cavities, such as chicken and pig embryos, but a block of tissue may be taken from water to 95% alcohol without any apparent damage. Even though one uses increasing strengths of alcohol, the series normally in employ- ment at the present time is by no means satisfactory. It is customary, for example, to pass from water to 30% alcohol at one end of the series and to pass from 85% to 95% alcohol at the other. The diffusion currents between water and 30% alcohol are far greater and far more intense than those between 85 and 95%, and an inteUigently graded series for delicate ob- jects should run from water to 10% to 20% to 50% to 95% alcohol rather than through the conventionally spaced grada- tions. This is not at all in accordance with the recommendations in most textbooks but is based on the author's experience over a long time. In using this classic method of dehydration, it is not necessary to confine the technique to ethanol. Meth- anol or acetone will dehj-drate just as effectively, though they are rather more volatile. There is a considerable vogue nowadays for the substitution for a straight dehy- drating agent of some solvent which is both miscible with water and also with molten wax. The best known of these is dioxane, though n-butanol has also been recommended. The writer is not in love with these methods for, though the sol- vents involved are excellent dehydrating agents, they are relatively poor solvents of paraffin and frequently cause great shrinkage of delicate objects in the final transition between the solvent and the wax. For such purposes as the routine ex- amination of the tissue blocks in a patho- logical laboratory, or for sectioning rela- tively sturdy plant materials, they may justifiably be employed. For sections in- tended, however, to retain intact struc- tures on which research is subsequently to be conducted, it is most stronglj^ recom- mended that the standard routine of pass- ing from a dehydrating to a clearing reagent be retained. Selection of a Clearing Agent Reference should again be made to Chapter 25 for a list of the materials which have from time to time been recommended for de-alcohohzing, or clearing, biological specimens. The choice of a clearing agent in section cutting is of far more impor- tance than the choice of a dehj^drant, for there is not the shghtest doubt that pro- longed immersion in some of the volatile hydrocarbons, particularly xylene, leads to a hardening of the tissue with subse- quent difficulty in sectioning. The classic method is to pass from alcohol to xylene, but the only apparent reason for the choice of xylene over toluene or benzene lies in the work of Squire (1892, page 80) who timed the evaporation rate of these three solvents from an open watch glass and found xylene to evaporate the most slowly. There is little choice in the solvent power of any of these three hydrocarbons on wax; the writer's preference is for ben- zene, though it seems impossible to shake the faith of the conventional that the more expensive xylene is a necessity both as a Embedding PARAFFIN SECTIONS 97 solvent for emheddiiig media and as a clearing agent before them. These three hydrocarbons are so cheap, and are ob- tainable in such a pure form, that there seems no necessity to use any other clear- ing agent, unless one prefers the reagents which are supposed to combine the func- tions of both dehydration and clearing. It is still occasionally recommended that essential oils, such as cedar oil, be used for clearing objects for embedding. There is no justification for this unless it is vital that the object be rendered transparent (rather than alcohol-free) in order that some feature of its internal anatomy may be oriented in relation to tlie knife. Essen- tial oils are excellent for wholemounts, l)ut they are not readily removed from the specimen by molten wax; therefore, if they must be used, they should always be washed out with a hydrocarbon before the wax bath. Relatively small traces of any essential oil will destroy the cutting prop- erties of any wax mixture and, as they are nonvolatile, there is no chance of getting rid of them in the embedding oven. Choice of an Embedding Medium Formulas for the various wax mixtures used in the preparation of ribbons of sec- tions will be found in Chapter 27 (E 21.1). It is to be presumed at the present time that no one will endeavor to use a plain paraffin but will use one of these mixtures. If, for some strange reason, a pure paraffin is preferred, then it is necessary to buy (in the United States by importation) a care- fully fractionated and very expensive wax. Ordinar}' cheap paraffin is a mixture of a great variety of compounds of slightly dif- ferent melting points, and it is essential in the use of jnire wax that a wax of a veiy sharp melting point should be obtained. The choice of an embedding medium should be dictated less by the nature of the specimen than by the conditions under which it should be cut. If pure paraffin is to be employed, it sliould be seloct('(l with sucli a melting point, that tlie hardened wax will give a crisp section at the re- quired room temperature. In the Europe of twenty years ago, when many writers were recommending a wax with a melting point of 52°C., the average laboratory temperature in winter was between 50° and GO^F. A wax of 52°C. melting point, in an American laboratory kept between 70° and SO°F., is far too soft to cut any but the thickest sections. The use of waxes of 58°C., which are quite hard enough for cutting sections in an American labora- tory, is unfortunate, since such use re- quires an oven temperature of at least 60°C. which results in many tissues be- coming hard and brittle. As the introduc- tion of any foreign substance automati- cally lowers the melting point of the wax, it is obviously desirable to use mixtures rather than the pure material. The writer's preference is naturally for his own compo- sition (E 21.1 Gray 1944). The advantage of the mixtures there specified is that they have a relatively low melting point but soften ver}' little before reaching the melt- ing point. The degree of hardness (that is the thinness of the section which may be cut) may be controlled accurately by the proportion of resin added; and the writer has once secured, on a demonstration, a paraflftn ribbon more than 20 feet long of 1 -micron-thick sections. Media of this hardness, however, impregnate very slowly and should only be used for mi- nute objects. For ordinary routine prepa- rations the writer's jircference is for any of the paraffin-rubl)er-bayberry-wax mix- tures. The introduction of rubber un- doubtedly increases the stickiness of the wax and makes it easier to secure continu- ous ribbons, while the bayberry wax not only prevents the crystalUzation of the paraffin but also lowers its melting point. The beginner is strongly recommended to experiment with several of the rubber- bayberry-wax compositions and to select after exj)eriment that which gives him uni- formly successful results in his own la])oratory. Technique of Dehydrating, Clearing, and Embedding Before passing to tlic choice of a micro- tome and tiie method of using it, it is necessary to discuss briefly tlie actual op- erations which are involved in using the dehydrating, clearing, and embedding me- dia selected. The techniques of dehydra- tion and dc-alcoholization do not differ 98 THE ART OF MAKING MICROSCOPE SLIDES Embedding materially from those used in the prepara- tion of wholemounts which have been de- scribed in Chapter 6. The whole process could, however, be much simplified if people would only remember that water is heavier than the majority of dehydrat- ing agents, and that the majority of de- hydrating agents are lighter than most The first prerequisite is some device which will maintain wax just at its melt- ing point. Most people employ complex thermostatically controlled ovens for this purpose, but the exceedingly simple device shown in Fig. 37 has a great deal to recom- mend it. As will be seen, this consists essentially of a series of incandescent elec- i.'-,te Fig. 37. Simple radiant heat embedding oven. Height of the hood should be adjusted until the wax is vielted for about one-half its depth. clearing agents. Translating this theory into practice it must be obvious that the object to be dehydrated should be sus- pended toward the top of a tall cylinder of dehydrant in order that the water ex- tracted from it may fall toward the bot- tom of the vessel, and that an object for clearing should be held at the bottom of the vessel for the reverse reason. It is, indeed, practically impossible to dehy- drate a large object unless it is so sus- pended. The process of impregnating the tissues with wax lias not, however, provi- ousl}' been discussed and will be tlealt with fully. trie bulbs held, at a distance which may be varied, above a series of glass \'ials. Before commencing to embed one fills as many vials as one will require with wax, places them under the reflector, and turns on the current. After a little while it will be oI> served that the absorbed heat has melted the wax. The wax may be melted only at a small surface layer; it may be melted throughout the entire vial; or it may, as is required, be melted in the upper % of the vial. If this last is not achieved the height of the lamp must be varied until, after an hour or two, each of the vials contains about yi of unmolten, opaque wax at the Embedding PARAFFIN SECTIONS 99 bottom and 2j^ of the clear molten mate- rial above. Thus, when the object is placed in one of these vials it will droj) until it reaches the solidified layer, where it will remain in contact with molten wax at exactly the melting point of the wax. It is obvious that the room in which this opera- tion is to be conducted must be at a fairly constant temperature and be free of drafts, but only a very large volume of embedding work justifies the purchase of an expensive thermostatically controlled oven. If such an oven is to be purchased it is liighly de- sirable to avoid one in which the heat is distributed bv convection. Such an old- Vacuum ovens are occasionally required for the impregnation of the most dilficult material but should he avoided whenever possible. If a vacuum oven is to be em- ployed, moreover, it is necessary that all volatile solvents be removed from the material before it is placed in the vacuum so that it is always desirable to precede exposure in a vacuum oven by a consider- able period of embedding in an ordinary oven. Assuming that the material has been passed through dehydrating and clearing agents, and is now awaiting embedding, there are two main methods by which this Am INIAKC Fig. 38. Circulating air embedding oven. fashioned convection oven is seen in Fig. 52 and is to be found all too frequently in laboratories. Unfortunately these ovens, as any cook could tell any microtomist, vary enormously in temperature from top to bottom. The thermostat is usually placed at the top and, in a fairlj^ large oven, there may be as much as a ten- degree differential between the lowest shelf and the top one. The oven shown in Fig. 38 in which a circulating fan continu- ously moves the air and thus maintains a uniform temperature throughout the whole oven, is infinitely to be preferred. It is the high cost of such circulating-air ovens which leads the writer to believe that much more use should be made of the very simple rachant-heat embedding de- vice discussed previouslj'. may be done. luther be the oliject may transferred directly to a bath of molten wax, or it may be passed through a graded series of wax-solvent mixtures. The writer is strongh' in favor of the latter course. Let us suppose benzene has been selected as the clearing agent and that the object is in a vial containing a few milliliters of this solvent. Chips are then shaved from the block of embedding agent and add('(l to the vial. These usually dissolve very slowly and form a thickened layer at the bottom of the tube through which the object to be embedded sinks. The average object will be satisfactory if left overnight. The tube is then placed in the embedding oven, maintained at a temperature slightly abt)ve the melting point of the wax, and as many further shavings as possible are 100 THE ART OF MAKING MICROSCOPE SLIDES Casting block Fig. 39. Folding a cardboard box. a. The two long edges of a rectangular card are folded to meet in the center. Fig. 40. Folding a cardboard box — {continued), h. Folds are flattened out and the short edges are folded not quite to the center. Fig. 41. Folding a cardboard box — {continued), c. Corners are folded over. crammed into the tube. When these are completely molten, and most of the vola- tile solvent has evaporated, the object is removed with a pipet, or forceps, and placed in a dish of pure wax for an hour or two before being transferred to a second dish of pure wax for the time necessar}^ to secure complete impregnation. There is no method of forecasting how long an object will take to l^ecome com- pletely impregnated with wax. It is very easy to find out, when one has started to cut sections, that the impregnation is not complete; but there is no basis save ex- perience on which to base the timing in the different baths. If the object is to be transferred directly from solvent to wax, at least three baths should be employed, for nothing is more destructive to a good section than the presence of a small quan- tity of the clearing agent in the embedding medium. To an absolute beginner seeking Casting block PARAFFIN SECTIONS 101 Fig. 42. Folding a cardboard box — (continued), d. Kdije of the fold in folded back over the creased corners. Fig. 43. Folding a cardboard box — {continued), e. Box is opened and the corners pinched. Fig. 44. Folding a cardboard box — (continued), f. Finished box. a rough guess, it may be said that a block of hver tissue of three-to-five-millimeter side will be satisfactorily impregnated with wax after 30 minutes in each of three baths, while a 96-hour chicken embryo will require at least two hours in each of three baths for its successful impregnation. While the object is being impregnated with the wax it is necessary to decide what type of vessel will be used to cast the final block. This will depend more on tlie size of the object than on the preference of the worker. Very small objects may be most satisfactorily embedded in ordinary watch glasses (that is, ordinary thin-walled watch glasses not Syracuse watch glasses of the laboratory type) or in any other thin-walled glass vessel. Very large objects are often embedded with the aid of two thick L-shaped pieces of metal, which by being slid against each other may be caused to form a rectangular mold of vary- ing dimensions. The writer himself regards these as very clumsy, and always prefers to prepare a cardboard or paper box than to endeavor to maneuver metal molds which are always getting jarred out of place at the wrong moment. The prepara- tion of a paper or cardboard box is easy; the method preferred by the writer for large boxes is shown in Figs. 39-44. Take a rectangular sheet of thin card or stout paper approximately twice as long as it is wide. The area of the floor of the 102 THE ART OF MAKING MICROSCOPE SLIDES Casting block l)ox will be aliout J4 that of the sheet taken, but a little experience will soon show what size sheet to take for the box required. The sheet is laid on a fiat surface and the long sides folded inwards (Fig. 39) until they very nearly meet in the middle. These folds are well creased with the thumbnail. The sheet of paper is then flattened again and the other two edges (Fig. 40) folded in the same manner. It is necessary, however, that this fold be much larger than the first fold made. These folds are also well creased with the thumbnail. The folded sheet is then laid out (Fig. 41) Fig. 45 as there are boxes to be made. Center the sheet between the finger and the thumb (Fig. 46) and then fold up the sides (Fig. 47) creasing the paper where it is in contact with the edges of the block. Push up the end with the forefinger (Fig. 48) creasing both the paper in contact with the block and the flaps. Fold the flaps to the center (Fig. 49), being careful to get them straight and creasing them up the sides. Fold down the projecting flap (Fig. 50) and crease it firmly. Repeat these operations with the other side of the block and then slide the box off the end of the Length of box + twice height of box + twice length of flaps XI o o Xi i Fig. 45. Dimensions of sheet for folding a paper box. and the corners folded in the manner shown. Since these end folds are larger than the side folds there will be an over- hanging flap of paper at the top. After all four corners have been folded in, this overhanging flap (Fig. 42) is folded back over the triangular folded corner sections and this crease particularly firmly pressed with the thumbnail. When this has been done at each end, the box is finished and may be opened out as shown in Fig. 43. It will be found that the corners are not scjuare but may be squared by pressing with the thumb and forefinger in the manner shown. The finished box is shown in Fig. 44. There is a very convenient method of folding small boxes wliich requires a series of wooden blocks of cross-section ecjual to that of tlie boxes recjuired. Take such a block (Figs. 46-51) and as many sheets of bond paper of the dimensions shown in block (Fig. 51). It is well to have a series of these blocks made both in square and rectangular shapes. An additional advan- tage of this type of box is that one can put the data about the block on the flaps. Boxes cannot be made by this method much larger than 1" X H". Some people prefer to cast a series of rectangular boxes from plaster of Paris. This can be done by any competent crafts- man, but will not be described at this point. After the box has been prepared we come to the actual process of embedding which is shown in detail in Figs. 52-55. Before starting it is necessary to make sure that the following items are available: (1) a dish of water of sufficient size that the finished block may be immersed in it (in the illustration an ordinary laboratory fingerl)owl is in use); (2) some form of heat, an alcohol lamp being just as effec- Casting block PARAFFIN SECTIONS 103 tive as a bunsen burner; (3) a slab of plate glass; (4) a wide-moutli, oye-di-oppor ty])e pipet. It is presumed that tlie object itself is in the oven, which also contains a sup- ply of molten medium. It must l)e empha- sized that an object cannot successfully be impregnated with one kind of wax and embedded in another. Next wet the under- side of the bottom of the paper l)()x and press it into contact with the plate-glass slab. Then take from the oven (Fig. 52) a beaker of molten embedding material and fill the little paper box to the brim. The eye dropper is then heated in the flame to a temperature well above that at which the wax will melt, and is used to pick up the object from its own dish (Fig. 53) and to transfer it to the paper box. By the time this has been done, a layer of hardened wax will have been formed at the bottom of the paper box, so that the ob- ject will rest on the layer of solidified wax with a molten layer above. It will almost invariably happen that the surface has also cooled, so that a crust of cool wax will have been carried down with the object in the box. It is essential to get rid of this if the wax is to adhere through section cut- ting, and the pipet is again heated, used to melt the entire surface of the wax (Fig. 54), and to maneuver the object into the approximate position in which it is re- quired to lie in the finished block. Then blow on the surface until the wax is suffi- . ciently solidified to enable you to pick up the box carefully and (as shown in Fig. 55) to hold it on the surface of the water used for cooling. With most wax media it is desirable to cool the block as rapidly as possible; it should never be permitted to cool in air. It cannot, however, be pushed under the surface of the water, or the molten center is liable to break through the surface crust and thus destroy the block. After it has been held in the position indicated until it is fairly firm throughout, it may be pushed under the surface to complete the cooUng. The block may be left in water for an>- reasonable length of time; but if it is to be stored for days or weeks it is better kept in a 5% solution of glycerol in 70' t alco- hol. There seems to be a widespread de- lusion that because an object must be perfectly dehydrated before being impreg- nated with wax, it must subse(iuently be kept out of contact with fiuids. Nothing could l)e further from the truth. As will be discussed later, when dealing with the actual techiiiciue of sectioning, it is often desiral)le to expose a portion of the object to be sectioned and leave it under the sur- face of water for some days, in order to get rid of the brittleness which has been im- parted through the embedding process. Blocks which have been stored dry for a long jjeriod of time should always be soaked in a glycerol-alcohol mixture for at least a day before sectioning. It is, in any case, undesirable to section a block as soon as it has been made, for it is necessary for successful sectioning that the block should be the same temper- ature throughout. If a block is made in the evening, it is better to take it out of the water and to leave it lying on the bench overnight in order that the temperature may be stabilized. Assuming, however, that we have such a block at hand, the next thing to do is to mount it in what- ever holder is to be used. Choice of a Microtome Microtomes may be broadly divided into two classes. In the first of these the block remains stationary while the knife is moved past it; in the second group are those in which the block moves past a stationary knife. The first class (an exam- ple is shown in Fig. 56) is made by several manufacturers but is rarely used for the preparation of serial sections. They have the advantage that relatively large blocks may be cut, but thej' have the disadvan- tage that no ribbon can be ol)tained which is broader than the width of the knife. This microtome will not be discussed fur- ther in the i)resent place, for a detailed description of its use is given in the next chapter on nitrocellulose sections, with which this type of microtome is often to be preferred. A Minot, or rotary microtome, is shown in Fig. 57. In this type of microtome the rotation of the large wheel causes the block holder to move vertically up and down, in most instances tlirough a dis- tance of about three inches. The portion Fig. 46. Folding a paper box. a. The block is centered on the sheet. Fig. 47. Folding a paper box — {continued), b. The sides are folded up. Fig. 48. Folding a paper box — (continued), c. The end is folded up. 104 Fig. 49. Folding a paper box — (continued), d. The flaps are folded in. Fig. 50. Folding a paper box — {continued), e. The end is folded down and creased. Fig. 51. Folding a paper box — (continued), f. The cycle is repeated with the other end and the finished box removed. 105 106 THE ART OF MAKING MICROSCOPE SLIDES Casting block 1""-/^ W"*^'' ' " !^ " '- " ^ ^ " " -'■" '■,' , -*i^ Fig. 52. Filling with wax an embedding box which has been attached with water to a glass slide. which slides up and down has, at the end opposite to the block, a rectangular plate of hardened steel inclined at an angle of about 45°. This plate bears, under the pressure of a powerful spring, against a hardened steel knob which is itself con- nected to a micrometer screw. As the han- dle is rotated a pawl works against a ratchet to move the micrometer screw, and thus the knob connected with it, through a given distance for each rotation. As the knob moves forward, it moves the block the required distance forward at each revolution by bearing on the diag- onal plate. This mechanism is very costly to make and is liable to a large number of minor defects which are not always appar- ent until one has started section cutting. One of the most important things that must be watched is that the knob which PARAFFIN SECTIONS 107 Fig. 53. (Top) Transferring the object from the embedding dish to the wax-filled paper box. Fig. 54. (Middle) Remelting the wax around the object with a heated pipette. Fig. 55. (Bottom) Cooling the wax block. 108 THE ART OF MAKING MICROSCOPE SLIDES Microtomes Fig. 56. Sliding microtome. Fig. 57. Rotary microtome. controls the section tliickness must be so moved that an exact number of microns is indicated. If, for example, the knob is so moved that the indicator Une hes between 9 and 10 microns, the pawl will not engage the ratchet perfectly but ^\dll chip off a small portion of brass at each revolution. It only requires a few weeks' operation under these careless conditions to destroy the ratchet wheel which will have to be replaced at the factory. No inexperienced student should ever be trusted with one of these machines until the mechanism of it has l)een explained to him and clearly demonstrated. Knives and Knife Sharpening The most important single factor in the production of good sections is the knife used in cutting. It does not matter how Knives PARAFFIN SECTIONS 109 much care has l^een taken in the prepara- tion of the block or liow complex a micro- tome is used, if the knife-edge is not per- fect there is no chance of securing a perfect section. Ordinary razors are not satisfac- tory for the production of fine sections, and it is necessary to secure a microtome knife, preferably from the manufacturer of the microtome. Another type of micro- tome knife employs the edge of a safety- razor blade in a special holder; these do not, in the writer's hands, give such good results as a sohd blade. Three types of solid blade are available : first those which are square-ground, that is, in which the main portion of the knife is a straight wedge; second, those which are hollow-ground, that is, in which both sides of the knife have been ground away to a concave surface, which results in a relatively long region of thin metal to- wards the edge; third, knives which are half-ground, that is, knives of which one side is square- or flat-ground and the other side hollow-ground. This last type of knife, which the writer prefers, is a compromise. There is no doubt that a square-ground knife is sturdier than a hollow-ground knife, a point of some importance when cutting large areas of relatively hard tis- sues; but there is equallj^ no doubt that a hollow-ground knife can be brought more readih' to a fine edge. Microtome knives must be sharpened frequenth^; but it is necessary, before discussing how to do this, to give a clear understanding of the nature of the cutting edge itself. If a wedge of hardened steel were to be ground continuously to a fine edge, as in Fig. 58, it would be utterly worthless for cutting. After only a few strokes the fine feather-edge, which would be produced by this type of grinding, would break down into a series of jagged saw teeth. A micro- tome knife, or for that matter any other cutting tool, requires to have ground on its cutting edge a facet of a relatively ob- tuse angle, whether it be a square-ground knife, as in Fig. 59, or a hollow-ground knife as Fig. 60. The process of applying this cutting facet to the tip is known as setting; it is an exceedingly difficult opera- tion to conduct, but one which must be learned by every user of a microtome knife. The actual grinding of the blade itself to the correct angle, or to the correct degree of hoUowness, cannot be done in a laboratory; the knife must be returned to the manufacturer or to some scientific sup- j)ly house adetiuatcl}^ equipped with the special machinery necessary. The cutting facet, however, must be set at least once a day if the blade is in continuous use. The nature and purpose of this cutting facet is best explained by reference to the mechanism of cutting shown in Fig. 61. Notice first that the knife blade itself must be inclined at" such an angle to the block that the cutting facet is not quite parallel to the face of the block. There must be left a clearance angle to prevent the knife from scraping the surface every time that it removes a section. This clear- ance angle should, in cutting wax, be as little as possible, and it is for this reason that the blade holder of a microtome is furnished with a device for setting the knife angle. The knife angle should not be set with reference to any theoretical con- sideration, but with regard only to secur- ing this small clearance angle. The only way to judge whether or not a satisfactory clearance angle has been obtained is to observe the sections as they come from the knife. If the clearance angle is too large, so that the section is not being cut from the block but is being scraped from it, the section will have a wrinkled appear- ance and will also usually roll up into a small cylinder. If the clearance angle is too small, so that the lower angle of the facet is scraping the block after the tip has passed, the whole ribbon of sections will be picked up on the top of the block, which will itself crack off when the knife point reaches it. It is obvious that the knife angle will be changed as the angle of the cutting facet is changed, so that it is desirable to maintain the cutting facet of as uniform an angle as possible. This angle is set onto the knife in the man- ner shown in Fig. 62. Notice that the knife has been furnished with a handle and also that a small spHt cyhnder of steel has been sUpped over the back of the blade. This spht cylinder rests flat on the stone, as does the edge of the blade, so that when the knife is pushed forward (the figure 110 THE ART OF MAKING MICROSCOPE SLIDES Knives CUTTING >i FACET BLADE Fig. 58 Fig. 59 CUTTING TIP CLEARANCE ANGLE KNIFE ANGLE BLOCK Fig. 61 Fig. 58. Knife ground as simple wedge without cutting facet. Fig. 59. Flat ground knife showing cutting facet. Fig. 60. Hollow ground knife showing cutting facet. Fig. 61. Cutting action of knife on wax block. shows it at the beginning of the stroke) the cutting facet is produced as the angle between the cutting edge lying on the stone and the enlarged temporarj' back which has been placed on the knife. Since a much blunter cutting facet is required for hard materials than for soft, it is strongly to be recommended that either two knives, or at least two sharpening backs, be secured. It does not matter what kind of stone is used for sharpening pro- vided that it is of the finest obtainable grit, that it is dead fiat, and that under no circumstancs wliatever is it used for any purpose except the sharpening of micro- tome knives. It does not matter whether Knives PARAFFIN SECTIONS 111 it be a water stone, to be lubricated witli soap and water like the yellow Belf^ian stones commonly emjjloyed in l']uro[)e, or an oilstone, to he lubricated with mineral oil like the pike stones so commonly eni- ployed in the United States. It does mat- ter, however, that it should be flooded with lubricant before staitinj;-, and that the knife should be drawn with a lij;lit pressure (notice that tlie fiii,<;er is behind and t}ot on top of the knife in the illustra- tion) the entire length of the stone at each itself. If the knife-edge is nicked to a deeper extent than about a quarter of a millimeter, the only thing to do is either to return the knife to the maiuifacturer to l)e reground, or try to avoid that por- tion of the blade containing the nick when cutting sections. It must be emphasized that the only {purpose of setting is to pro- duce a cutting facet, and that grinding, which cannot be done in the ordinary laboratory, is required for the removal of knife impeifections. Fig. 62. Setting the cutting facet. operation. If onlj- the central portion of the stone is used, it soon becomes hol- lowed out and it thus becomes impossible to maintain a uniform angle. About three strokes on each side of the knife are quite enough to produce a perfectly sharp cut- ting facet; to continue beyond three strokes will have no effect other than to diminish the length of life of the knife. This direction for the use of three strokes in setting applies, of course, only to knives which have been reasonably treated and not to those which through carelessness have acquired a nick in their edge. Where the nick is large it is almost impossible to remove it in setting, for the continual repetition of setting merely grinds away the edge of the knife and ultimately alters the thickness of the blade The next question to arise is that of stropping the blade of the knife by pulhng it backward across a leather surface in the manner shown in Fig. 63. If the knife has been set properly, stropping (the only pur- pose of which is to poUsh the facet) is quite unnecessary. The nature of the leather surface which is used for stropping makes it obviously impossible to pull the knife blade forward and thei-e is a grave risk in pulhng it backward, lest the facet, instead of becoming polished on its flat surfaces, will become rounded on its edges, and thus the work of setting be undone. Certainly no beginner should be permitted to use a strop until he has demonstrated his ability to set a knife-edge to tiie point where it will cut an excellent section without strop- ping. It is also strongly recommended to 112 THE ART OF MAKING MICROSCOPE SLIDES Mounting block the beginner that he should examine the edge of a knife under the low power of a microscope before setting, after setting, and after stropping. Mounting the Block The knife being sharpened and the microtome selected, it now remains to trim the block to the correct shape and to attach it to the object holder of the micro- tome. The rough block of wax containing the object must be first removed from the mold or, if a paper box was used, the box and it is essential that these should be exactly parallel to each other. A skilled microtomist can cut these edges parallel with a safety-razor blade without very much difficulty, but numerous devices have been described from time to time in the literature to enable one to do this mechanically. It does not matter if these two edges are exactly parallel with the plane of the object; it is only essential that they be parallel with each other. At this stage plenty of wax should be left both in front of, and behind, the object. Fig. 63. Stropping a microtome knife. cut away roughly with a knife. The block should now be held against a hght so that the outlines of the contained object can be clearly seen. The block is then trimmed until the object lies in the center of a per- fect rectangle, with the major axis of the object exactly parallel to the long sides. This is best achieved by finding first the major axis, at right angles to which the sections are to be cut, and trimming down one side of the block with a sharp safetj'- razor blade, taking off only a little wax at a time. If one tries to remove a large quantity of wax there is danger of crack- ing the block. When one side has been shaved to a flat surface, the other side is shaved parallel to it. The top and bottom surfaces of the block may now be shaved. This trimmed block has now to be at- tached to some holder which can itself be inserted into the microtome. Since the majority of sections today are cut on a Spencer rotary microtome, we will de- scribe the use of one of the holders sup- phed with this machine, though the in- genuity of man has not yet succeeded in devising a worse method of attaching a paraffin block to a microtome. The holder, which is seen in Fig. 64, consists of a disk of metal with a roughened surface at- tached to a cyhndrical shank. This disk must first of all be covered with a layer of wax and it is extraordinarily difficult to get wax to adhere to these chromium- plated surfaces. If the worker is not en- tirely bound by convention, it would be \ Mounting block PARAFFIN SECTIONS 113 much better for him to secure a series of small rectangular blocks of some hard wood like maple and to soak these for a day or two in molten wax. After they are removed, drained, and cooled it is the sim- plest thing in the world to atta(;li a paraf- fin block to them and to hold them in the jaws of the microtome. Whether the metal holder or the wooden one are used, the technique is essentially the same. A layer bring these buttresses so far up the block that thoy reach the tip of the ol)ject to be cut. The metal should now be placed on one side and allowed to reach room tem- peratui-o. Many people at this point throw the block and holder into a fingerbowl of water, which is all right provided the wa- ter is at room temperature. But there is no more fruitful source of trouble in cut- ting sections than to have the knife, the Fig. 64. Mounting the wax block on the block holder. of molten wax is built up on the surface and allowed to cool. The block (see Fig. 64) is then pressed lightl}' onto this hard- ened wax and fused with it with the aid of a piece of heated metal. Some people use old scalpels but the writer prefers the homemade brass tool shown in the figure. Care must be taken to press only very lightly with the forefinger and to perform the whole operation as speedil}^ as possible to avoid softening the wax in which the object is embedded. The metal tool should be heated to a relatively high temperature and touched hghtly to -the base of the block. If the block is very long, it is also desirable to build up small buttresses of wax against each side, being careful not to block, and the microtome at different temperatures. It is much better to mount the blocks the day before one intends to cut them and to leave them on the bench to await treatment. A final inspection is then made of the block to make certain that its upper and lower surfaces are flat, smooth, and parallel. Many people do not make the final cuts on these surfaces until after the block has been mounted in the block holder. The block and the block holder, after insertion in the jaws of the microtome, are seen in Fig. 65 and it will be noticed that setscrews on the apparatus permit universal motion to be imparted to the block so that it can be correctly orien- tated in relation to the knife. It is easy to lU THE ART OF MAKING MICROSCOPE SLIDES Cutting discover whether or not the edges are parallel by lowering the V)lock until it does not quite touch the edge of the knife, ad- justing it until the lower edge is parallel, then lowering the block again and com- paring the relation of the upper edge with the edge of the knife. Cutting Paraffin Ribbons Tlie first step in cutting sections on this type of microtome is to make sure that causes the two movable holding arms to hold the knife near its edge. The knife is now held in a pair of hemicyhnders which may be moved so as to adjust the knife angle (see Fig. 61). The knife should be set at that angle which experience has shown to be desirable — no guide other than experience can be used — and the two setscrews which lock these inclinable hemi- cyhnders in place then tightened. The two original setscrews, which hold the knife in Fig. 65. Starting the paraffin ribbon. every one of the setscrews seen in Fig. 65 is fully tight. The setscrews holding the block holder may be tightened in any or- der, provided that the result leaves the block correctly orientated, l)ut those con- nected with the knife must be done in the correct order. First the knife is inserted into the holder and fixed firmly, but not tightly, in place by the two bearings at each end. The tightening of these screws place, are now screwed up as tightly as the thumb can bear. This leaves two set- screws which come through the inclinable hemicyhnders and bear on the bottom edge of the knife. These two setscrews should then be tightened simultaneously and uniformly. The effect of this is to force the knife upward and thus wedge it with extreme firmness in the knife holder. Now that everything is tight the handle Cutting PARAFFIN SECTIONS 115 on tlie hack of tlie inierotoiue is turned until tlie block is as far back as possible, and the entire knife is moved on its car- riage until the edge of the blade is about }i inch in front of the block. A last-miiuite check is now made to make sure that the divisions of the setting device exactly co- incide with the thickness desired ; then the handle is raj^idly rotated until the block in the left hand, is slipped under the rib- bon which is then raised in the manner shown in Fig. 65. Care should be taken that a few sections always remain in contact with the blade of the knife, for if the ribbon is hfted till only the edge of the section lies on the edge of the knife, the ribbon will usualh^ break. As the handle is turned, the brush in the left Fig. 66, Laying out the ribbon. starts cutting. The front face will rarely be parallel to the blade of the knife, there- fore a considerable number of sections will have to be cut until the entire width of the block is coming against the knife. No particular attention need be paid to the quahty of this initial ribbon, which may be thrown away. We will assume that all is going well and that the ribbon is coming off in a perfect condition; if it is not, refer to Table 1. The remaining operations of preparing and mounting the ribbon are far more clearly seen in illustration than by descrip- tion. As soon as the ribbon is the width of the knife in length a dry soft brush, held hand is moved away until the ribbon is the same length as the sheet of paper on which it is to be received. Legal size (fools- cap) paper is quite commonly employed and is shown in Fig. 66. Notice that the left-hand edge of the ribbon has been laid flat some distance from the edge of the paper and that a loop, sufficiently large to avoid strain on the ribbon attached to the knife, is retained with the brush, wdiile the ribbon is cut with a rocking motion with an ordinary scalpel or cartilage knife. The larger and colder this scalpel is, the less likelihood there will be of the section adhering to it. The purpose of leaving a good margin around the edge of the paper no THE ART OF MAKING MICROSCOPE SLIDES Cutting is tliat it may he desiralile to interrujit ribbon cutting for some time and to con- tinue later. In this case the worker should furnish himself with a Httle glass-topped frame which is laid over the jiaper to pre- vent the sections from being blown about. As the inexperienced worker will soon find out, the least draft of air, particularly the explosive draft occasioned by some not he cut up until a samjile has been flattened on a slide in order to determine the degree of expansion. Though the sec- tions shown in the illustration are being mounted on an ordinary 3" X 1" slide, it would be moi'e practical (for a ribbon as wide as this) to use a 3" X IH^' or even a 3" X 2" sUde. The sections should never occupy the whole area of the slide. Fig. 67. Cutting tlie ribbon in lengths. fool opening the door, is quite sufficient to scatter the ribbons all over the room. These operations of carrying the ribbon out with the left hand, transferring the Inrush to the right hand, and cutting the ribbon off, are continued until the whole of the required portion of the block has been cut and lies on the paper. The ribbon must then be divided into suitable lengths for mounting on a slide (Fig. 67). Though in theory a section should be of the same size as the block from which it came, this practically never occurs in practice and it is usually safe to allow at least ten and sometimes twenty per cent for expansion when the sections are finally flattened. The ribbon should but at least j-i of an inch should be left at one end for subsequent labeUng. When the decision has been made as to how many sections shall be left in each seg- ment of ribbon, the first row of ribbons is then cut into the required lengths (Fig. 67). Then the worker must decide what shall be used to make them adhere to the shde. It is conventional to use the albu- men adhesive of Mayer 1880 (Chapter 28, V 21.1), and to apply a thin smear of this on a clean sUde with the tip of the httle finger. The author prefers to dilute the selected adhesive two or three hundred- fold with water, and to use this dilute ad- hesive in the next operation of flattening the sections. Mounting PARAFFIN SECTIONS 117 Fig. 68. Mounting the dry ribbon. It will have been apparent to the worker from the moment that he started cutting the sections that the}^ are not absolutely flat. They may be slightly crinkled, or slightly distorted, and must be flattened by being warmed on water heated just be- low the melting point of the wax. Some people place this water on the slide and then add the sections to it, but the writer prefers to lay the ribbons on the slide as shown in Fig. 08. This is not nearly so easy as it looks. Two brushes must be moistened with the tongue just enough to bring the hairs to a point. The two moist points are then delicately touched down (too much pressure will cause the ribbon to adhere to the paper) on each end of the selected piece of section. This l)iece is tluMi lifted as shown in the illus- tration and placed on the shde. When a sufficient number have been accumulated the slide is then picked up carefully, re- versed, and laid 'on top of the last three fingers of the left hand as shown in Fig. 69. 118 THE ART OF MAKING MICROSCOPE SLIDES Flattening It is fatal to grasp the slide by the sides; if this is done, when the water is flooded on from the pipet, the meniscus coming to the edge of the sUdes will break against the fingers, to which the sections will permanently adhere. The technique shown is quite safe and the water containing the adhesive (if none has been applied to the shde) is then flooded on froni a jiij^et in tened, the slide is gently tilted backward towards the hand so as to run off the excess water against the thumb, leaving the sections stranded in place. The sUde is now usually placed on a thermostati- cally controlled hot plate (seen at the back of Fig. 78) and dried. Most people leave their shdes overnight but frequently an hour would be sufficient. Dryness can be Fig. 69. Flooding the ribbons. the manner shown. Enough fluid should be appHed to raise a sharp meniscus at the edge of the shde. The sections must now be flattened, and this is better done rapidly with a flame than slowly on a hot plate. Fig. 70 shows the shde being held over a small alcohol lamp, but a micro-bunsen can be em- ployed equally well. The shde should be exposed to heat for a moment, withdrawn to give time for the heat to jiass from the glass to the fluid, warmed again, and so on, until the sections are observed to be flat. The utmost care must be taken at this point for, if the paraffin is i)ermitted to melt, the sections will not stick to the glass. As soon as the sections are flat- gauged without the least trouble by the fact that a moist shde shows the wax to be more or less opalescent, while on a prop- erly dried shde it is almost glass-clear. The method just described is susceptible of several variations which may be briefly noticed. Some people do not drain the water from the slide, nor do they heat the slide over the lamp; they merely place the slide, as soon as the water has been added to it, on the thermostatically con- trolled hot plate so that the sections dry and flatten at the same time. The objec- tion to this procedure is that dissolved air in the water used for flattening usually comes out in the form of bubbles which accumulate under the section, either cans- Staining PARAFFIN SECTIONS 119 ing it to fall off or at least making it very difficult to observe jwoperly when mounted. There is also the risk in this procedure that the water will not stop at the edge of the shde, but will unexpectedly flood off, (;arrying the sections with it onto the surface of the hot plate. Another procedure, frequently used by the author but not recommended for the inexperienced, is to blot the sections be- fore putting them on the hot plate. A water-saturated piece of coarse filter paper appearance, cause, and cure of the more conunon defects are shown in the pages which follow. These are by no means the only defects or the only cures which may be api^lied. Every user of the microtome should have in his hands a copy of Rich- ards 1949, which lists many suggestions beyond those here given. Staining and Mounting Sections Assuming that all difficulties have been overcome, and tliat one now has a series of i_j._^ ' .-J... i i J-i~i Fig. 70. Warming tlie flooded ribbons in order to flatten them. is placed on the drained slide and pressed hard with a rubber roller, which squeezes much of the water out of both the paper and the sections. This makes sure that the sections are perfectly flattened in contact with the slide, but requires a strong nerve to try for the first time, because most people fear that the sections will stick to the paper. This has never happened in a good many thousands of shdes which the author has made by this means. Shdes so prepared are always free of air bubbles. Before proceeding to a discussion of the next steps to be taken, it may be as well to revert to the moment when section cutting started, and to discuss the innum- erable things that may happen, other than the production of a perfect ribbon. The shdes bearing consecutive ribbons, the paraffin must next be removed in order that the sections may be stained. It is con- ventional, though probably not necessary, to warm each sUde over a flame (holding it as shown in Fig. 70) until the paraffin is molten. The shde is then dropped (as shown in Fig. 78) into a jar containing xylene, benzene, or some other suitable paraffin solvent. The jars shown in this figure are of the type known as coplin jars, which are usually employed when a rela- tively small number of 1" X 3" shdes are to be handled. Larger numbers of small slides are more conveniently handled by being placed in racks, which may be moved from one rectangular jar to an- other. Individual slides may, of course, be 120 THE ART OF MAKING MICROSCOPE .SLIDES^ Defects handled in a coi)lin jar, hut it is more con- venient for tliese to have an' ordinary round specimen tube, or vial, of just over 1-inch diameter, which maintains a single shde in an upright position without the necessity of using the relatively large quantities of fluid involved in a copUn-jar set. Coplin jars are not available for slides larger than 3" by 1"; for these one is forced to use the rectangular jars. It is necessary through the subsequent proceedings to be able to recognize in- stantly on which side of the shde the sec- tion hes. This is not nearly as easy as it sounds; a lot of good shdes have been lost by having the sections rubbed off. The simplest thing to do is to incline the shde at such an angle to the light that, if the section is on top, a reflection of the section is seen on the lower side of the shde. A diamond scratch placed in the corner is of little use because it becomes invisible when the slide is in xjdene. The greatest care should be taken to remove the whole of the wax from the slide before proceed- ing further. It is usuall}^ a wise precaution to have two successive jars of xylene, pass- ing the second jar to the position of the Table 1 DEFECTS APPEARING IN RIBBONS WHILE BEING CUT Fig. 71. Ribbon curved. Fig. 72. Sections compressed. Possible Causes 1. Edges of block not parallel 2. Knife not uniformly sharp, causing more compression on one side of block than other 3. One side of lilock warmer than other Remedies 1. Trim block 2. Try another portion of knife-edge or resharpen knife 3. Let block cool. Check possible causes of heating or cooling, such as lamps or drafts Possible Causes 1. Knife blunt 2. Wax too soft at room temperature for sections of thickness required 3. Wax warmer than room temper- ature Remedies 1. Try another portion of knife-edge or resharpen knife. Compression often occurs through a rounded cuttiing facet (see Fig. 60) produced by overstropping 2. Re-embed in suitable wax or cut thicker sections. Cooling block is rarely successful 3. Cool block to room temperature Defects PARAFFIN SECTIONS Table 1 — {Continued) 121 Fig. 73. Sections alternately thick and thin, usually with compression of thin sections. Possible Causes 1. Block, or wax holding hlock to holder, still warm from mounting 2. Block, or wax holding block to holder, cracked or loose 3. Knife loose 4. Knife cracked 5. Microtome faulty Remedies 1. Cool block and holder to room temperature 2. Check all holding screws, Remove block from holder and holder from microtome. Melt wax off holder and make sure holder is dry. Re- coat holder and remount block. Cool to room tcMiiperature 3. Release all holding screws and check for dirt, grit, or soft wax. Check knife carriage for wax chips on bearing 4. Throw knife awav Fig. 74. Sections bulge in middle. Possible Causes 1 . Wax cool in center, warm on outside 2. Only sharp portion of knife is that which cuts center of block 3. Object impregnated with hard wax and embedded in soft, or some clearing agent remains in object Remedies 1. Let block adjust to room temper- ature. This is the frequent result of cooling blocks in ice water 2. Try another portion of knife-edge or resharpen knife 3. Re-embed object 5. Return microtome to maker fur overhaul 122 THE ART OF MAKING MICROSCOPE SLIDES Defects Table 1 — (Continued) Fig. 75. Object breaks away from wax or is shattered by knife. Fig. 76. Ribbon splits. Possible Causes 1. If object appears chalky and shatters under knife blade, it is not impregnated 2. If object shatters under knife but is not chalky, it is too hard for wax sectioning 3. If object pulls away from wax but does not shatter, the wrong dehy- drant, clearing agent, or wax has been used Remedies 1. Throw block away and start again. If object irreplaceable, try dis- solving off wax, redehydrating, re- clearing and re-embedding 2. Soak block overnight in pheno- glycerol mixture, rinse thoroughly, and dry, or spray section between each cut with celloidin, or dissolve wax and re-embed in nitrocellulose 3. Re-embed in suitable medium, preferably a wax-rubber-resin mix- ture. Avoid xylene in clearing mus- cular structures Possible Causes 1. Nick in blade of knife 2. Grit in object Remedies 1. Try another portion of knife-edge 2. Examine cut edge of block. If face is grooved to top, grit has probably been pushed out. Try another por- tion of knife-edge. If grit still in place, dissect out with needles. If much grit, throw block away Defects PARAFFIN SECTIONS 123 Table 1 — {Continued) Fig. 77. Block lifts ribbon. Possible Causes 1. Ribbon electrified. (Check by test- ing whether or not ribbon sticks to everything else) 2. No clearance angle (see Fig. 60) 3. Upper edge of block has fragments of wax on it (a common result of 2) 4. Edge of knife (either front or back) has fragments of wax on it Remedies 1. Increase room humidity. Ionize air, either with high frequency dis- charge or bunsen flame a short dis- tance from knife 2. Alter knife angle to give clearance angle 3. Scrape upper surface of block with safety-razor blade 4. Clean knife with xylene No ribbon forms Defect (1) Because wax crumbles (2) Because sections, though indi- Possible Causes vidually perfect, do not adhere (3) Because sections roll into cylin- ders 1. Wax contaminated with clearing agent 2. Very hard, pure paraffin used for embedding 3a. Wax too hard at room temper- ature for sections of thickness required 3b. Knife angle wrong Remedies 1. Re-embed. {Note: Wax very readily absorbs hydrocarbon vapors) - 2. Dip block in soft wax or wax-rubber medium. Trim off sides before cutting 3a. Re-embed in suitable wax. If the section is cut very slowly, and the edge of the section held flat with a brush, ribbons may sometimes be formed 3b. Adjust knife angle first, and replacing it with fresh xylene, after about ten or a dozen slides have passed through. It must be remembered that paraffin is insoluble in the alcohol which is used to remove the xylene, so that it is no use soaking a sHde in a solution of xylene in wax and imagining that it will be sufficiently free from wax for subse- quent staining. Some people go further than this and have the first two jars con- taining xylene, and then a third containing a mixture of equal parts of absolute alco- 124 THE ART OF MAKING MICROSCOPE SLIDES Staining hoi and xylene, to make sure that the whole of the wax is removed. If even a small trace of wax remains, it will prevent the penetration of stains. Assuming that one is proceeding along tlie classic xylene- alcohol series, the shde is transferred from either the fresh xylene or the xylene-abso- lute-alcohol mixture, to a coplin jar of ab- solute alcohol. It is unfortunate that no- body seems yet to have placed on the market a coplin jar, or slide-staining dish, the lid of which is satisfactorily ground into position so that absolute alcohol, soon, however, as the slide has been in water long enough to remove the alcohol, it should be withdrawn and examined carefully to make sure that it has been sufficiently dewaxed. If the water flows freely over the whole surface, including the sections, it is safe to proceed to stain- ing by what ever manner is desired. If, however, the sections appear to repel the water, or if tliere is even a meniscus formed round the edge of the section, it is an indication that the wax has not been removed, and that the slide must again be Fig. 78. Starting a slide tlirougli the reagent series. which is very hygroscopic, remains uncon- taminated. It does not matter if xylene is carried over into the absolute alcohol, but as soon as the first trace of a white floc- culent precipitate appears in the alcohol — indicating that some wax is being carried over — the alcohol must be replaced. The writer never l)others to use a series of graded alcohols between absolute al- cohol and water. These graded series are necessary, of course, when one is dealing with the dehydration of whole objects which may be distorted, l)ut the author has never been able to find the slightest difference between thin sections which have been passed from absolute alcohol to water, and those which have laboriously been downgraded through a series. As dehydrated in absolute alcohol, passed back into a xylene-alcohol mixture, and thence again into pure xylene. In the specific examples which conclude this chapter, and in numerous places throughout Chapters 20, 21, and 23, de- scriptions are given of indi\'idual staining methods. The purpose of this chapter is to discuss only the general principles in- volved in the preparation of paraffin sec- tions, so that we may presume the section to have been already stained and returned (through such dehj'drants as are specified in the method used) to xylene, and to be ready for mounting. It is again assumed that the section will be mounted in one of the resinous media described in Chapter 26 (M 30), and Mounting PARAFFIN SECTIONS 125 Canada balsam is so conventional that it may be taken as an example. The shde is removed from the xylene and drained (Fig. 78) and then placed on any convenient flat surface. A drop of the mountant is then placed on the surface of the sections. A covershp of suitable size (Fig. 79) is then held at an incUned angle with a bent needle and slowly lowered so as to exclude all air bubbles. The edges of the slide are then roughly wiped and it is returned to the hot table shown in Fig. 78 to evapo- rate the solvent used for the resin. Though This custom of evaporating the solvents from the surface of the sUde rather than from the edge of the coverslip is nowadays considered old-fashioned; but there is no doubt that it produces a better and more durable slide than does the more usual procedure. One very common accident, which maj'' occur in the course of staining or dewaxing a slide, is that the individual sections show signs of l)ecoming detached, either through not having been perfectly in contact with the shde when dried, or through having Fig. 79. Placing the coverslip on serial section slide. this is the conventional method of opera- tion it is by no means always the best. In particular there is a tendency to ha\'e a higher concentration of solvent along the edges of the coverslip than in the center, and it also takes a surprisingly long time for the whole of the solvent to be removed. It is much bettei', if one can si)are the time, to place a relatively thin coat of mounting medium on top of the slide and then to leave the solvent to evaporate from this on the surface of a hot plate. There is no risk that the slide will dry out, for the mount- ant will act as a varnish. On the next day the slide is examined and, if it appears to be sufficiently varnished, the coverslip is placed on the surface and warmed while maintaining steady pressure. The slide will then be hardened as soon as it is cooled and may be cleaned and put away. been exposed to some reagent which has a solvent action on the adhesive. The effects of this unfortunate accident may be mini- mized by having always on hand a coplin jar of the solution of Claoue 1920 (Chap- ter 28, V 21.1). This is a most admirable lacquer into which the slide may be dipped rapidly and withdrawn. This transparent lacquer hardens readily in place and holds the section attached without seriously interfering with subsequent observation. Cleaning and Labeling Slides No slide can be considered complete un- til it has been properly labeled, cleaned, and stored. Failure to clean a shde can cause rather serious damage for, if un- wanted portions of Canada balsam are left lying about close to the edges or on the surface, and if the slide be then used 126 THE ART OF MAKING MICROSCOPE SLIDES Defects Table 2 DEFECTS APPEARING IN SECTIONS DURING COURSE OF MOUNTING Defect Cause Remedy Method of prevention Sections appear wrinkled Sections have bubbles under them 1. Blunt knife used for 1. None cutting 2. Water used for flat- 2. None tening too hot, so that folds in sec- tions fused into position 3. Sections unable to 3. None expand sufficiently: (a) because water used for flattening too cold (b) because area of water too small 1. Sections insuffi- ciently flattened, so that air is trapped 2. Air dissolved in water used for flattening has come out and is trapped under sections in drying 1. Sharpen knife and cut new sections 2. Watch temperature of water used for flattening 3(a) Watch tempera- ture of water used for flattening (b) Make sure that slide is clean, so that water flows uniformly over it Sections fall 1. Wax melted in off slide flattening 1. If sections still wet, reflood slide with water and reheat to complete flattening 2. If sections still wet, reflood slide with water, work out bubbles, and reheat to complete flatten- ing 1. None 1. Check flatness sections before draining slide of 2. Slide greasy 2. 3. Alkaline reagents 3. dissolve albumen adhesive. (Sections start to work loose in course of staining or dehydrating) 4. Sections not flat- tened into perfect contact with slide None Treat slides with Claoue's solution (Chapter 28, V 21.) 4. None 2. Use air-free (boiled) water for flattening Drain slide thor- oughly and blot off excess moisture. Squeeze sections to slide 1. Watch temperature of water used for flattening 2. Use clean slides 3. See Chapter 28. V 21 for other section adhesives not alkali-sensitive with an oil-immersion lens, the oil will dis- solve a portion of the balsam which will be found very difficult to remove either from the surface of the coverslip or from the front lens of the immersion objective. The slide cannot be cleaned until it is thoroughly dried and if it has been mounted in a solution of resin, it will re- quire several days on a hot plate or ^veeks 4. Sometimes caused by swelling of sec- tions which causes center to lift. Squeeze sections to slide and dry as rapidly as possible at room temperature before the solvent has been removed. When, however, it is' finally dried, which is when the resin on the edge can be cracked, the surface resin should be removed carefully with a blunt knife and the slide left overnight. If on examination the freshly cut edge is then found to be sticky, it is evident that more solvent has moved out and that the slide Defects PARAFFIN SECTIONS 127 Table 3 DEFECTS APPEARING IN SECTIONS AFTER STAINING AND MOUNTING Defect Cause Remedy Method of Prevention Sections distorted Sections appear opaque or have highlj' refrac- tive lines out- lining cells and tissues Sections will not take stain, or stain irregularly Sections con- tain fine opaque needles or granules 1. Blunt knife and soft wax 2. Ribbon stretched when picked up on hot day 3. Tissues not properly hardened before embedding 1. Clearing agent evaporated before mountant added 2. Sections insuffi- ciently cleared or cleared in agent not miscible with mountant 1. Wax not perfectly removed before staining 2. Section not uniform thickness 3. Tissue "old" (has been stored for a long time in alcohol or, worse still, fLxa- tive) 4. Fixative not suit- able before staining technique employed 5. Fixative not fully removed 1. Imperfect removal of mercuric fixatives None, though pro- longed flattening on warm water may help As (1) above 3. None 1. None 2. Soak off cover. Clear properly 1. Return sections through proper se- quence of reagents to xylene. Leave un- til wax removed. Restain 2. None 3. Return sections through proper se- quence of reagents to water. Wash overnight. If not effective try a "tis- sue reviver" (Chapter 22, ADS 11) 4. Try mordanting sections in recom- mended fixative 5. Consult Chapter 10, ADS 11 1. Return sections through proper se- quence of reagents to water. Treat 30 min. with Lugol's iodine, rinse, and bleach in 5 % so- dium thiosulfate. Restain 1. Use suitable knife and embedding medium Handle ribbons in short lengths or use harder wax Use more suitable fixative or fix longer. Take extra care in dehydrating, clear- ing, and embedding Obvious 2. Check quality and nature of clearing agents and mountants 1. Change first jar of xylene frequently 2. See table 1 3. Store all tissues embedded in paraf- fin blocks — never liquids 4. Obvious 5. Treat tissues as in- dicated 1. Treat tissues as in- dicated in Chapter 10, ADS 11 128 THE ART OF MAKING MICROSCOPE SLIDES T. S. intestine Table 3 — (Continued) Defect Cause Remedy Method of Prevention 2. Long storage in formaldehyde 2. None 2. Never store tissues in formaldehyde — always in paraffin blocks is not }et ready. If, however, the removal of the dried balsam leaves no sticky resi- due, it is necessary to provide two finger bowls: one of 90% alcohol and the second of a moderately strong solution of soap and water. The whole slide is then dipped in the 90% alcohol and rubbed briskh' until the excess balsam is I'emoved. It is immediately (to avoid softening the bal- sam) rinsed in the soap solution, and then polished. If the slides are unsatisfactory, tables 2 and 3 above may help to locate the trouble. When dealing with valuable series of sections it is ahvays as w'ell to write the serial numl^er of the slide, and some indi- cation of its nature, on the glass in dia- mond before attaching the label. Labels are constantly becoming detached from sUdes and it is well to have a permanent record underneath them. It has already been pointed out that no two people agree as to what label adhesive to use. The author would only reiterate the counsel he has given in previous chapters: that both sides of the label be licked thoroughly, that it then be pressed into position on the slide, allowed to dry slowdy, and the requi- site information written with waterproof India ink. The violent objections which the author has expressed in previous chapters to stor- ing wholemounts in vertical grooved filing cabinets, do not, of course, apply to sec- tions, since the section is attached to the slide and cannot drift through the mount- ant. Storing in grooved trays, which hold the shde vertically, is undoubtedly the simplest method of storing such sections, but much space can be saved if they are placed in pouches of ordinary indexing cards. If two 5 by 3 index cards be taken, and one be cut down to 5 by 2, the smaller may then be stapled to the first card in such a manner as to leave a pocket into which a slide may be inserted. The full data may then be written on the index card, and these tw^o-card pockets bearing the slides can be accumulated in ordinary card-file drawers. Typical Examples The Preparation of a Transverse Section of the Small Intestine of the Frog Stained with Hematoxylin-eosin This is the simi)lest exam]:)le of paraffin sectioning which can be imagined, and it may well serve as an introduction to this type of technique, either for a class or for an individual. The intestine of a fi'og has been selected, owing to tlie usual avail- abiUty of this form in laboratories; but any small animal may be substituted in its place. Before killing the frog it is necessary to have on hand a selected fixative and, since this is intended to be an example of the ut- most simplicity, it is suggested that the cupric-nitric-paranitrophenol mixture of Petrunkewitsch (Chapter 18 F 4900.0040 Petrunkewitsch 1933) be employed. This fixative is entirely foolproof: objects may remain in it for weeks without damage, and it also permits excellent afterstaining by almost any known technique. If only a piece of intestine is to be fixed, 100 milli- liters of fixative will be sufficient; but there is no reason why any other organ in the animal (with the exception of the cen- T. S. intestine PARAFFIN SECTIONS 129 tral nervous system) should not be pre- served in this fluid for subsequent investigation. The frog is killed by any convenient method, but it is usually best for histo- logical purposes to sever a large blood vessel and permit as much l)lood as possi- ble to drain out from the heart before opening the abdominal cavity and remov- ing the intestine. One or more lengths of about 3'3 of an inch should then be cut from the intestine and transferred directly to fixative where they may remain from a few hours to several weeks. When they are next required the speci- mens should be removed from fixative, washed in running water for a few hours, and then transferred directly to 70 'o alco- hol. The easiest method of washing objects of this size in running water is to take one of the coplin jars previoush' described, to fill it with water, insert the specimen, and then to attach a cover of coarse cheese- cloth with a rubber band. This is then placed in the sink and a narrow stream of water permitted to fall on it from the tap. It will be found that the specimen will swirl round and round in the jar in a most satisfactory manner. This simple device saves all the trouble of rigging up glass tubes and boring corks to make the cum- bersome apparatus sometimes recom- mended for the purpose. The specimen is transferred, after twentj'-four hours in 70% alcohol, to 95% alcohol. It is better to use a large volume of alcohol and to suspend the object in it than to use relatively small volumes which have to l^e frequently changed. It is recommended that a wide- mouthed stoppered jar of about 500 milli- liters capacity be fitted witli a hook in the center of its stopper, from which the ob- ject can then be suspended. The majority of stoi)pers for wide-mouthed glass jars have a hollowed undersurface which may be filled with plaster of Paris, and a glass hook (which is verj^ easily bent from thin glass rod) may be inserted in the litiuid plaster. This must naturally be done some days beforehand, and the plaster must finally thoroughly be dried out in an oven before the jar is usetl for dehydrating. If the worker does not wish to go to this much trouble, it is also easy to screw a small metal "pot hook" into the under surface of a plastic screw cover for a jar of the same size. Alcohol is, however, so hygroscopic that it is better to employ a glass-stoppered jar, the stopper being greased with stoj)C()ck grease, or petro- latum, for a permanent setup. An object as coarse as the one under discussion may be sus])ended in a loop of thread or cotton directly from the liook; or if this is not desirable, it may be enclosed in a small fold of cheese cloth for suspension. After twentj'-four hours in this volume of alco- hol, the ol)ject will be completely pene- trated, and should then be transferred to absolute alcohol using the same volume in a jar of similar construction. It is useful to place about a (luarter-inch layer of an- hydrous copper sulfate at the bottom of the absolute alcohol jar, not only to make sure that the alcohol is absolute, but also to indicate, as it changes to blue, when this jar should be removed from service. Of the mau}^ de-alcoholizing (clearing) agents which may be used, the writer would in the present case select benzene because it is less liable to harden the circular muscles of the intestine than is xylene. As benzene is fighter than an ab- solute alcohol, it is not possible to employ the hanging technique for clearing, and the object should be placed in about 25 milliliters of benzene which should be changed when diffusion currents are seen to have ceased to rise from the object. This will take about six hours for an object of the size under discussion and a secoufl bath of at least six houis should also be given. It is now necessai-y to select the medium in which em])edding is to be done and the writer would recommend the rubber par- affin of Hance (Chapter 17— E 21.1 Hance 1933) which must, of course, have been prepared some time before. The melting l)oint of this medium is about 56°C. so (hat ail oven should be availal)le whi(Oi is lliermostatically controlied at about 5S"('. This oven should contain three stender dishes as well as a 500 cc beaker contain- ing about a pound of the embedding me- dium. The ol)ject is removed from ben- zene, drained briefly on a piece of filter 130 THE ART OF MAKING MICROSCOPE SLIDES T. S. intestine paper, and placed in one of the stender dishes which has been filled to the brim with the molten embedding medium. Under no circumstances should a hd be placed on the stender dish since it is desir- able that as much as possible of the benzol should evaporate while the process of em- bedding is going on. After about an hour the specimen should be removed to fresh wax in the second stender dish, where it may remain another hour, and then to the third stender dish where it should not re- main for more than thirty minutes. Shortly before the end of this last hour a decision should be made as to what type of vessel is to be used for casting the block, and it would be difficult to improve on a paper box (Fig. 50) for this object. The box having been made (it should be of ample size) it is moistened at the bottom and placed on a slab of glass in the manner described earlier in this chapter. The box should be about half filled with embedding material from the beaker and allowed to remain until the layer of wax has con- gealed on the bottom. An object like the one under discussion is best handled with an old pair of forceps rather than with a pipet. The forceps should be warmed in a flame to well above the melting point of the wax, and moved backward and for- ward across the surface so as to melt the surface film which has formed. The object is then rapidly picked up from its stender dish, placed in the wax, and enough fresh wax from the beaker added to make sure that there will be as much solid wax above as there is underneath the specimen. Blocks of this nature shrink greatly, and it will probably be best to fill the box en- tirely full. As soon as the box has been filled, the forceps should again be warmed and passed backward and forward around the object to make sure that no film of un- molten wax (which would cause it conse- quently to cut badly) remains. The wax in its box should now be blown on until it starts to congeal on the surface, then very carefully picked up with the fingers and lowered into a dish of water at room tem- perature until the water does not quite reach the top of the box. If it be thrust under the surface at this point, all of the molten wax will come out and the block be rendered useless. As soon, however, as the block is seen to be congealed through- out, it is thrust under the surface of the water and something laid on it to keep it at the bottom. It should be left in the water for at least five or six hours and much better overnight. One now sets up the microtome, and makes sure that the knife is sharpened in the manner previously described, and then mounts the block. The block having been trimmed to size and mounted as noted earlier, there remains only the actual cut- ting. The block should be trimmed so there is at least as much wax on each side of the object as there is in object itself. This amount of wax would be excessive were we preparing serial sections, but for the preparation of individual sections of this type, in an example given for the benefit of the beginner, this quantity is desirable. The handle of the microtome should now be rapidly rotated and the be- ginnings of the sections observed. There is no need to worry if the section curls to one side or the other during this preliminary period, since the entire area of the block will not be cut until twenty or thirty sec- tions have been removed. As soon, how- ever, as the knife is seen to be approaching the object, and the block in its entirety is being cut, the ribbon must be observed most carefully to see that it is suffering from none of those defects indicated in the table of defects. Should the ribbon not be coming perfectly, various suggestions given in the table may be tried until a perfect ribbon is secured. Since we are not, in this case, preparing a series of sections, it is unnecessary to cut a longer ribbon than will contain the actual number of sections required, with a few left over for emergencies. It is, however, a great mis- take to throw i)artially cut blocks away, since they may be stored in a glycerol- alcohol mixture ciuite indefinitely, and one never knows when further sections may be required. The block, however, should be labeled before being placed in its solu- tion by writing the appropriate informa- tion on a piece of paper and fusing this with a hot needle into an unwanted por- tion of the block. Each section is now cut individually T. S. intestine PARAFFIN SECTIONS 131 from the ribbon and mounted on the shde in whatever manner has been selected. Since the use of the conventional Mayer's egg albumen (Chapter XXVIII V 21.1 Mayer 1880) has already been discussed, another medium will be used. The hydro- lyzed starch of McDowell and Vassos (Chapter 8 V 21.1 McDowell and Vassos 1940) is very little known and well worth using. The directions given in the i)lace just cjuoted should be used in the prepara- tion of this thick, viscous hquid of which about four or five drops may then be added to about 50 cc of distilled water in an Erlenmeyer flask or beaker. The slides must be cleaned before the sections are mounted, and no two people have ever agreed as to what is the most desirable method of doing this. One way is first to rub the shde briskly with 1 % acetic acid in 70% alcohol and dry it by waving in the air. Other methods of cleaning the slide, which yield equally good results can be found from the index. Several drops of the diluted adhesive are placed in the center of each slide and one of the individual sections then taken up with the tip of a moistened brush and placed on the ad- hesive. As soon as the section has been placed on the fluid, the slide is lifted up, warmed carefully over a spirit lamp until the section is flat but the paraffin not melted, and then the superfluous liquid removed carefully with the edge of a filter paper. The slide is then placed on a warm table to dry and, if the drying period is to be prolonged, it is as well to place a dust cover over it, since grains of dust falling upon the shde will adhere just as tena- ciously to the adhesive used as will the specimen itself. It is pi'ojwsed in the present example to stain the slide in the simplest possible manner with coelestin blue B followed by phloxine. Various formulas for stains of the coelestin blue B type will be found in Chapter 20 under the heachng DS 11.41; that preferred by the writer for its sim- phcity is recorded as "Anonymous 1936." There will also be required a solution of phloxine for counterstaining. Phloxine ap- pears to work best from a weak alcohol solution. In Chapter 20 under the heading of DS 12,2 will be found the suggestion that it be used in 0.2% solution in 10% alcohol. Any of the other dyes thei-e rec- ommended may, of course, be substituted. Assuming the section now to be per- fectly dry, it is turned upside down and the light is reflected from it to see whether or not the section is adherent to the glass. If there is any air gap between the section and the glass, a brilliant mirror will be formed and, in a preparation as simple as this, the shde had better be thrown away. Having selected those slides which are per- fectly adherent, they are then warmed over a flame until the wax is melted and diopped into a jar of xylene, where they remain until the paraffin appears to have been removed. They are then passed to another jar of xylene where they remain for at least five minutes, and then to a jar of equal parts xylene and absolute alcohol where they remain for a further five minutes. This treatment is followed by five minutes in absolute alcohol and then by direct transference to distilled water. After they have been in distilled water for a few minutes, each slide should be lifted and inspected to make sure that the water is flowing uniformly over both the slide and section. If it tends to be repelled by the section, or a meniscus is formed around the section, this is evidence that the wax has not been completely removed, and the slide must be transferred first to 95% alcohol to remove the excess water, then to absolute alcohol until perfectly dehy- drated, and then through absolute to xylene, where it remains until the wax has been completely removed before being brought down again as previously indi- cated. The slides may be taken down one at. a time and accumulated in distilled water until they are required. When all the slides have been accumulated in dis- tilled water, they are transferred to the coelestin B staining solution. The time in this varies, but ten to fifteen minutes will probably be sufficient to stain the nuclei. One of the most useful features of this stain is that it is almost imp()ssil)le to over- stain in it. Sections may be left overnight without staining the cytoplasm to a degree which requires differentiation. After the mu'lei are blue-black, therefore, or after a time convenient to the operator hag 132 THE ART OF MAKING MICROSCOPE SLIDES T. S. intestine elapsed, the sections are transferred to fresh distilled water where they are thor- oughly washed. Each slide is then taken individually and dipped up and down in the phloxine solution until a casual inspec- tion shows the background to be yellow- pink. The intensity of stain for the back- ground, in a case like this, is a matter of choice, some people preferring a faint stain and others a darker stain; it must be remembered, in judging the color, that the section will seem darker after it has been cleared than it does in water. As soon as it has been found from a single shde what is the time required to produce the desired degree of staining, the remainder of the slides are placed in the phloxine solution together, left the ap- propriate time, and then transferred to distilled water until no more color comes away. The shdes are then passed from dis- tilled water to 95 % alcohol where they are left for about five minutes, then to fresh 95% alcohol, where they are left for five or six minutes before being passed to ab- solute alcohol. The purpose of using the 95% alcohol is not to diminish diffusion currents but simply to save diluting the absolute alcohol by passing slides directly from water to it. After the slides have been for two or three minutes in absolute alcohol, a single slide is taken and passed into the absolute alcohol-xylene mix- ture for perhaps two minutes and then passed to xylene. This slide is then ex- amined by reflected hght against a black background and should be as nearly as possible transparent with only a faint opalescence. One of the commonest faults in mounting sections is dehydrating them imperfectly, for if there is any water which has been carried through the proc- ess into the xylene (in which water is solu- ble in the extent of about }i of 1 %) this water will be extracted by the section which is in itself an excellent dehydrating agent. There is a world of difference be- tween a i^erfectly cleared (that is glass- clear) slide and one which is only more or less dehydrated so that it appears faintly cloudy. If the shde does not appear to be sufficiently dehydrated the whole of the remaining slides should be transferred to fresh absolute alcohol and another one tried. When it has become apparent from the examination of the test slide that de- hydration is complete, the remaining slides may be run up through absolute alcohol and xylene and accumulated in the final jar of xylene. As balsam was discussed in the body of this chapter, we suggest using at the pres- ent time the medium of Kirkpatrick and Lendrun (Chapter 26 M 34.1 Kirkpatrick and Lendrun 1939). Next clean the ap- propriate number of coverslips: in the present instance a %-inch circle would be admirable. The author cleans his cover- slips in the same manner as he cleans his slides: by mping with a weakly acid, alco- hol solution. Each slide is taken individu- ally, drained by its corner, laid on flat surface, and a drop of mounting medium placed on top. The coverslip is then placed on the mounting medium and pressed down with a needle. It should not be pressed absolutely into contact with the shde or too thin a layer of mounting medium will be left; some experience is recjuired to judge when the coverslip has been pushed down far enough. If this is done skillfully the surplus mounting medium will form a neat ring around the outer surface of the cover. If it does not do so, care should at least be taken that no ])ortion of the cover is devoid of surplus mountant which will be sucked under the coverslip as the solvent evaporates. These vslides should be left to dry at room tem- perature for about one day and then placed on a warm plate for about a week. After they are dried the surplus dry mounting medium should be scraped off with a knife and the excess remaining after scraping removed carefully with a rag moistened in 90% alcohol. The shdes are again dried overnight and then should be ringed with some colored varnish using the technique described in Chapter 2. This ring does not assist the preservation of the mounting medium l)ut it has always, in the writer's experience, assured that the sUde when placed in the hands of students will be treated with more respect than a non-ringed slide. The slide, after labeling, is now complete. Frog embryos PARAFFIN SECTIOKS 133 Preparation of. Serial Sections of an Amphibian Embryo Heavily yolked embryos are among the most difficult objects from which to pre- pare satisfactory serial sections, as may be witnessed by the photographs which illustrate the work of many experimental embrj'ologists. Though the example spe- cificall}' taken is that of an amphibian embryo, the methods to be discussed may l)e used for any heavily 3'olked material such as fish, or even in the preparation of sections of the early stages (segmentation and tlie like) of bird embryos. The crux of the entire matter lies in the selection of a fixative and it is doubtful whether a worse fixative for the purpose could be found than the picro-acetic- formaldehj'de of Bouin, so generally em- ployed. Two fixatives have been devel- oped specifically for heavily yolked mate- rial: that of Gregg and Puckett (Chapter 18 F 3000.1010 Gregg and Puckett 1943) which is designed specifically for the eggs of frogs, and that of Smith (Chapter IS F 7000.1010 Smith 1912) which was originally develoiied for the eggs of Crypto- hranchus, but which the author has used successfull}^ for a large variety of heavily yolked material. The author has used Smith for so many years that he is prej- udiced in favor of this formula, and the fact that he has not been so successful with the formula of Gregg and Puckett may be due to the fact that he is less ex- perienced with it and not that it is in- herently incapable of giving equally good results. As the techniciues of fixation in- volved are altogether different they will be discussed separately. Let us assume first of all that we are using the fluid of Gregg and Puckett, which has been made up according to the formula given in Chapter 18. The mass of embryos and eggs, together with their gelatinous surrounding envelopes, are taken and placed in at least 50 times their own volume of the solution. The jar centaining them should be turned upside down at intervals to insui'e that the fluid around them does not become diluted and they are then left for 24 hours. If the eggs are to be embedded and sectioned at once, they may then be removed and waslied in running water for 24 hours or, if they are to be stored for long periods, they may be moved to 2% formaldehyde directly from the fixative and may remain in this fluid, changed possibly after the first 48 hours, until they are required for use. The mass is then removed and broken into small clus- ters which are placed in a test tul)e or small flask about one-third filled with water. The flask is then shaken vigorously, which will remove a portion of the albu- minous envelope, the dirty water poured off, fresh added, and the flask again shaken. After half a dozen treatments of this type the greater part of the albumen will have been removed. When as much of the jelly as possible has been removed by this mechanical treatment the eggs are transferred to a flask of 1 % sodium hyi)0- chlorite and shaken gently and carefully. At intervals eggs will be found to detach themselves. These should be removed either with a section lifter or a small pipet to another flask containing water; b\^ tliis means the whole of the remaining jelly will be removed. The eggs, which are now in water, should be carefully examined to see whether or not the vitelhne membrane is still adherent. If it is still adherent, those which have it should be transferred back to fresh 1% sodium hypochlorite and stirred very gently, being examined at intervals under a binocular microscope, until the membrane has been removed. The eggs are then washed thoroughly to remove all traces of hypochlorite. It is better to do this with half a dozen changes of water rather than in running water, be- cause the eggs at this stage are lirittle and portions may be flaked off the outside if they are subjected to the ))umping which seems to be an inevital)le part of washing with running water. Let us now examine Smith's method. The fixative must be made up immediately before use and large volumes are required in relation to the size of the objects. The author once fixed the entire yolk of a hen's egg, using two gallons of solution; and at least 500 milliliters should be em- 134 THE ART OF MAKING MICROSCOPE SLIDES Frog embryos ployed for a cluster of a dozen or two amphibian eggs. The eggs are transferred to the solution which is immediately placed in a dark cupboard where it re- mains for about 48 hours. The original specifies that low temperature should be employed, but the writer has been unable to find the least difference in performance between heavily yolked eggs fixed at room temperature and those which have been fixed in a refrigerator. The solution should be changed once or twice during the forty-eight hours, or at least as often as it becomes dark green. It is inevitable that it should become greenish, but by chang- ing solutions before a dark-green color ap- pears, the deposition of chromium oxides on the surface of the egg and its mem- branes may be avoided. At the end of forty-eight hours the eggs are removed to large volumes of 2% formaldehyde in the dark; the solution must be changed as often as it becomes discolored and wash- ing must continue until no further color comes away. After all possible color has been removed by the 2% formaldehyde, the jars may be taken from the cupboard and stored indefinitely at room tempera- ture in the light. Most of the eggs or embryos will be found to have become de- tached from their gelatinous membranes in the course of this treatment, but the vitelHne membrane is frequently left. It becomes brittle, however, and may be re- moved without difficulty with the aid of a couple of needles. Whichever method of fixation has been employed, we are now left with the eggs or embryos in 2% formaldehyde. The process of embedding is different according to the technique to be used. By the technique of Gregg and Puckett the eggs are dehy- drated through graded alcohols, allowing in the case of frog eggs two hours each at 35%, 50%, and 60%. It is also necessary to treat them with iodine to remove the mercuric chloride, and for this purpose they are placed in 70% alcohol, to which has been added al^out 5% of Lugol's iodine (Chap. 22 ADS 12.2 Lugol (1905)). The exact proportion of iodine is not impor- tant and technical directions usually read "add iodine until the fluid is the color of port"; but the author sees no reason why this insult to a noble wine should be per- petuated. It- requires a treatment of at least 48 hours to make sure that the mercuric residues are removed and the eggs are then transferred to 80% alcohol where they are washed until no further color comes away. They are then dehy- drated for one or two hours each in 95% and absolute alcohol. Gregg and Puckett specify clearing in xylene for 30 minutes but the writer prefers cleaning in benzene, which does not seem to render the eggs so brittle. After complete clearing, they are then transferred to a mixture of one part of the hydrocarbon employed and two parts of soft (48°C.) paraffin at room tem- perature. They may remain in this mix- ture until next required. When embedding is finally to be completed this mixture is placed in an oven at 58°C. and allowed to remain until completely liquified. The eggs are then transferred to 52° paraffin for one hour and finally to whatever medium is chosen for embedding (Gregg and Puckett prefer 55° paraffin ; the writer prefers rubber-paraffin) for another 3 or 4 hours. In the alternative technique of Smith, the procedure is rather different. After the eggs are taken from formaldehyde they are passed through 35% and 50% alcohol for two or three hours each and then placed in Grenadier's alcoholic borax carmine for about two days. The formula for this fluid is given in Chapter 20 (DS 11.22 Grenadier 1879) and it must be most strongly recommended that one use the dry stock dissolved in 70% alcohol rather than the usual solution prepared direct. The eggs are removed from the stain to one-quarter of 1 % hydrochloric acid in 70% alcohol and left there for about two hours or until the first rapid color clouds have died down. It is not in- tended to complete differentiation by this process, but only to remove the excess stain. They are then, exactly as in the pre- vious technique, dehydrated through 95% and absolute alcohol before being cleared in whatever hydrocarbon is preferred. Smith prefers embedding in 52° paraffin but this, in the writer's experience, is too soft and will only permit the tliickest sec- tions. It is again recommended that one Frog embryos PARAFFIN SECTIONS 135 of the rubber paraffins with a melting point of from 50° to 55°C. be employed. In any case we now have amphibian embryos and eggs accumulated in the embedding oven in whatever medium has been decided to use. It is usually necessary in cutting sections of this type that the orientation of the embryo in relation to the knife should be known; this is difficult to estabhsh by ordinary means when one is deahng with a more or less spherical embryo. The method preferred by the writer for indicating one of the planes is to embed at the same time and alongside the spherical embryo a little rectangular block of liver or some other soft tissue. It is easy to dehydrate clear and impregnate with wax a piece of liver and keep this permanently in the embedding oven in paraffin. When one is ready to embed the eggs a small strip (in the case of the frog embryo about 3 mm. X 1 mm. X 1 mm.) is cut from this slab of hver, and is first of all laid in the paper box to which one has added the wax in the manner already described. The egg is then transferred to the same box and is most carefuUj^ ori- entated with regard to the strip of liver, so that if the liver be cut exactly at right angles, the egg will be cut in the desired plane. A strip of hver this size does not greatly increase the total area of this block, nor does it in any way interfere with whatever staining technique is em- ployed for the sections. An identical pro- ' cedure is followed, whether one is dealing with eggs fixed by the technique of Gregg and Puckett, or fixed and prestained by the technique of Smith. After the block has been hardened under the surface of water (Smith specifies 70 % alcohol for the purpose, but it does not appear to matter) it is removed, allowed to attain room temperature, and the sides trimmed away until the strip of liver is clearly seen. The block is then attached to the holder, mounted in the block holder of a microtome, and a ribbon is prepared in the usual manner. The only difficulty that is hkely to arise is that, after the ribbons have been flattened and are dry- ing, the entire yolky center of the embryo may rise in a dome. This event usually indicates that the vitelline membrane has remained on the egg and there is nothing whatever tliat can l)e done about it. Such sections always become detached in the course of staining and are in any case worthless, for if they are varnished in place they will still be so domed as to render microscopic examination almost imi)ossi- ble. If, however, several successive batches of sections, in which one is quite certain that the vitelline membrane has been re- moved from the embryo, behave in this manner, it is sometimes possible to stop the trouble by drilling a little liole in the block until one just comes to the egg itself, and then soaking the block in glycerol- alcohol. Blocks so treated will, when cut, usually be found to lie flat on the slide. If this dexice fails it is strongly recom- mended that 70% alcohol be substituted for water used to flatten the sections (any of the customary adhesives may be mixed with alcohol of this strength just as readily as with water) and that the technique of using a wet blotter and a rubber roller be used, as described in the body of this chapter. It must be emphasized that these defects are uncommon in materials fixed in the manner described; they are men- tioned only because they occur so fre- quently in the handling of amphibian embryos fixed and prepared by other methods. After the ribbons have been flattened and dried, they are then put through the ordinary series of reagents until they are ready to be stained. The technique differs very greatly according to whether one is dealing with the technique of Gregg and Puckett or that of Smith. In the technique of the latter the slides are removed from absolute alcohol and flooded with the Lyons blue and picric acid mixture of Smith (Chapter 20 DS 12.221) for a period of about one minute. They are then returned to absolute alcohol until no color comes away, and cleared in xylene before being mounted in a resinous me- dium. The writer prefers this technique to any other because of the gross swelling which occurs in yolk when exposed to aqueous solutions of stains. The rising up of the center of the section, commented on in the last paragraph, is not confined to the time when the sections are flattening. 136 THE ART OF MAKING MICROSCOPE SLIDES S. S. mouse but may also occur during staining; the center of many an excellent section has become detached from the slide simply for the reason that it has been handled in aqueous stains. The nuclei are not so clearly shown by carmine as by many other stains, but for valuable material the author has always used Smith as an in- surance policy against losses. It is also easy with this stain to distinguish the various cells since the cell membranes themselves pick up the blue while the mass of the^^yolk retains the yellow of the picric acid. Gregg and Puckett, on the contrary, take the sections down to water in the usual manner and then stain them in Delafield's hematoxyhn [Chapter 20 DS 11.122 Delafield (1885)] differentiating in acid alcohol in the manner indicated until the nuclei alone remain clearly stained. They are then counterstained in eosin- orange (Chapter 20 DS 12.222 Gregg and Puckett 1943) before returning through the alcohols to xjdene and then to the mountant. The author has never had the least success in staining amphibian em- bryos with hematoxylin because of the very strong affinity of this stain for the albumen granules in the j^olk. After staining the sUdes are cleaned and labeled in the usual manner and will show almost incredible improvement over the usual "Bouin's fixative-hematoxylin-eo- sin" technique which most modern em- bryologists appear to employ. Preparation of a Sagittal Section of an Entire Mouse This preparation is not recommended to the stern and dedicated research worker, whose onh' interest in the preparation of microscope slides is to demonstrate a the- sis, but has been included for the benefit of those who, like the author, enjoy mak- ing a beautiful slide for its own sake. It may, of course, be argued by those who have to justify themselves that such sec- tions form an admiral^le method of demon- strating the main relationships of mam- malian anatomy to a large class. It is proposed, in fact, to prepare a sagittal (vertical-longitudinal) section of an entire mouse from the tip of its nose to the very last joint of its tail. This is a feat of great technical difficulty and requires atten- tion, at odd moments, during several months. The author does not think that the end can justify the means: the beautiful preparation must be its own justification. The mouse selected for the preparation should be of such a size that the section will fit onto a standard 3-inch by 1-inch slide, but sufficiently old to be covered in hair. A litter of freshly born white mice should, therefore, be watched until the young arc completely clothed with hair; this will be between one and two weeks after birth. The next problem is to kill and fix the mouse in such a manner as to fulfill the conditions that the tail shall \)v straight, so that it can be included in a sagittal section, and that the fixative shall shall be able to penetrate to all parts. The tail must, of course, be curled under the body if the section is to be placed on a slide, and it must also be attached to some rigid structure so as to remain straight. It is desirable to kill the mouse in a relaxed condition: and injection of sodium amytal is probably the best. For those who do not have access to h3'perdermic syringes and reagents of this tj^pe, however, it is quite satisfactory to kill with ether (not chloro- form which stiffens the animal rapidly) though one has less time to work before rigor mortis sets in. Before kilUng the mouse one should have secured the finest possible needle obtainable and some very fine silk, not Hnen or cotton, thread. There is only one way of insuring that the tail shall coincide with the nose and that is to sew the two together. Therefore, as soon as the mouse is dead, open the jaws and insert a little wedge of wood so that they are partly opened (at the tip of the jaws the gap should be about 2 mm.) and then proceed to pull the tail around until the tip of it projects just beyond the nose. Using the fine needle and the fine silk sew the skin from each side of the tail to each side of the nose. It must be remem- bered that we are concerned only with getting half a dozen perfect sagittal sec- tions and that anything outside the exact central plane will not show. Now take a S. S. mouse PARAFFIN SECTIONS 137 glass rod, or a piece of plastic, and bind the tail to it with silk, being careful to bind only loosely lest the imprint of the silk show in the final section. Great care must be tak(>n to keep the tail straight along the glass; it is then attached in front and in back to the body of the mouse exactly parallel to the spine. If this is done skilfully a median sagittal section will cut through the central portion of the central nervous system for its entire length and will show a central section of the tail to its very tip. It is just as well to kill the whole htter and to prepare them in this manner, since one or two specimens are bound to get out of alignment in the course of the subsequent operations. The writer thought at one time that the simplest method of maintaining the tail straight would be to hang the mouse from a loop of tape passed through its tail with a weight attached to the nose, but the ob- jection to this is that though the tail re- mains dead straight, it does not remain exactly parallel to the spinal cord. Before all this has been done, one should have decided on the fixative to be em- ployed and have made up a sufficient quantity of it. Fixatives containing picric acid should be avoided at all costs, since the prolonged soaking in water Avhich must inevitably accompanj^ decalcification will cause the grossest sweUing and vacuolation of picric-fixed materials. The writer's pref- erence is for the dichromate-formalde- hj'de-acetic mixtures, preference being given to those which are based on the original solution of Miiller and which thus contain sodium sulfate. Numerous foi-- mulas for these mixtures will be found in Chapter 18 under the general hea(Ung F 7000.1010, the writer has employed the mixture Bohm and Opel 1907 with success in a preparation of the present tj'pe. It must not be imagined that this, or any other fixative, will penetrate rapidly enough to fix an entire mouse before con- siderable autolysis has taken place in the internal organs; it will be necessary to make small openings in the sides if we are to have a successful preparation. As soon, therefore, as the mouse has been firmly fixed in the manner described, a sharp scalpel should be taken and a series of slits made through the skin and l)eritoneum along each side of the ab- domen. These slits should not be more than a milhmeter or so in extent, or there may be a protrusion of the internal oi'gans through them. Care must also be taken not to cut the liver, or any major blood vessel, or the entire abdominal cavity will fill up with blood, and the appearance of the finished preparation will be coni- l)letely ruined. In addition to these shts down the side, about one third of each side of the head must be cut off with a fine saw so as to expose the outer surface of the brain. The use of bone forceps or wire cutters will cause distortion; a fine jewel- ers' saw is much better for the purpose. The cutting of blood vessels in this case does not make very much difference since there are few cavities into which the blood can flow. The mouse, having thus been bound to its supports and a few small openings made, is wrapped in a fold of cheesecloth and suspended at about the center of at least one liter of the selected fixative. The jar containing the mouse and fixative should then be placed in a dark place for about two days. At the end of this time the mouse is removed and the fixative is replaced with new fixative. At this time also extend the size of the openings which have been made, since all of the blood will now be coagulated and the internal organs will be more or less firmly fixed in place. In extending these openings, re- member actually that no more than the center half-millimeter of the mouse will ultimately be required, and certainly all of the limbs and considerable areas of both flanks may be removed. Some experience is necessary in deciding how^ much to remove. This is another reason why the entire litter of young mice should have been sacrificed at the same time rather than reliance placed on a single specimen. The mouse should now be placed in fresh fixative and left in the dark for a further period of about a week, the jar being ex- amined at intervals to make sure that the fixative is not turning green. It will always turn green-brown, but should it become of a fairly dark-green color, it must immedi- ately be replaced with new fixative. It is 138 THE ART OF MAKING MICROSCOPE SLIDES S. S. mouse very difficult to overharden or overfix a specimen of this kind, and at least a month should be allowed to make sure that there is perfect fixation throughout while an exposure of six months will cause no damage. After fixation is complete, the mouse (or mice) is removed from the fixative, and without removing from the cheese- cloth bags, hung in a jar through which running water flows from a tube reaching to the bottom. It (or they) should be washed for at least three days in running water before being removed and hung in a large jar of 20% alcohol or other de- hydrant. For those who find alcohol diffi- cult to olitain, either acetone, isopropanol, or methanol are equally good for dehydra- tion, but must in each instance be used in a fairly close series. Small objects may, as has been stated elsewhere, be passed with- out danger from water into absolute al- cohol, but as large an object as this mouse will have to be dehydrated very slowly. The mouse should be left in 20% alcohol for about five days and subsequently for about five days each in 50%, 70% and 90%. The experienced reader will have noticed that we have so far said nothing of decalcification which must obviously take place before the sections can be made. On the basis of the writer's experi- ence, born out by the earlier workers but apparently nowadays ignored, it would ap- pear that hardening in alcohol after fixa- tion yields a specimen which behaves very much better under the knife than does one which has been fixed only without the sub- sequent alcohol hardening. It is for this reason that he recommends that it be taken up in the manner described to 90 % alcohol, left there for a week or two, and then brought down through the same series to water, where it is left until the whole of the alcohol has been re- moved. The specimen is now ready for decalcification. For as large an object as this, particu- larly one which has been fixed in a di- chromate mixture, the method of von Ebner would be desirable. This solution, the formula for which is given in Chapter 19 under the heading AF 21.1 von Ebner (1891), employs a strong solution of sodium chloride to diminish the swelling of the tissues caused by the nitric acid used. It is also possible to use phloroglucin for the same purpose of diminishing the swelhng; a typical formula is given in the same chapter as AF 21.1 Ferreri (1895). It has, however, been the writer's experi- ence that these phloroglucin formulas work better on smaller objects and he strongly recommends the formula of von Ebner in the present case. Following this formula, the specimen is hung in a large volume of the solution and left for three or four days. At the end of this time a further 1 % of nitric acid is added and the whole stirred up. This proc- ess of adding a milliliter of nitric acid per 100 milliliters is continued every third or fourth day until decalcification is com- plete. It is as undesirable to decalcify for too long a period as it is to leave patches of hard bone to wreck the knife, hence the worker will often find himself in a quan- dary as to how to determine when de- calcification is complete. The only way this can be done with complete success is by x-ray examination, for the least trace of undissolved calcium remaining will show clearly upon the x-ray plate or fluorescent screen. It is often possible to find some friendly dentist who will prepare an x-ray of the mouse at intervals, but if this is impossible one must judge on deU- cate probing with a needle. The two places which are usuallj^, but by no means always, the last to decalicify are the inner ear and the molar teeth, and it is a reasonably safe assumption that if a fine needle can be passed through these with- out meeting more resistance than would be occasioned by tough leather, it is safe to continue. It is not safe to probe in the direction of the vertebrae, which are often slow in decalcification, because they are too close to the central area which will subsequently be sectioned. In the ab- sence of x-ray information it is much safer to decalcify too long than too short a time, and it will be suggested later that a solution be used to mordant the sections; this will undo, to a certain extent, any excessive hydrolysis which has taken place. The decalcified mouse must now be thoroughly washed to remove all traces of acid, but water should not be used for this S. S. mouse PARAFFIN SECTIONS 139 purpose since the removal of the salt is more rapid than the acid and bad hy- drolysis may occur at this moment. The mouse should, therefore, be washed with weak (2%) formaldehyde, which should be changed at daily intervals for about a week. At the end of this time the mouse may be washed in running water overnight with safety, and may then be considered ready to be dehydrated and embedded. Before dehydration it is well to trim away as much of the material as can be removed without risk of displacing the remaining internal organs. The larger the piece to be dehydrated and embedded, the longer will the process take, and it is usually per- fectly safe to reduce the preparation at this point to a slab of about yi of an inch thick. Do not hesitate to use fine ligatures of silk to hold in place any organ showing signs of diaplacement, for these ligatures will be missed by the knife as it takes the central section desired. Dehydration and clearing can follow the ordinary procedure hanging the slab of tissue at the top of considerable vol- umes of 20%, 50%, 70%, 95%, and ab- solute alcohol before laying it at the bot- tom of a jar containing benzene, which may be changed once or twice. If the mouse has been reduced, as suggested, to a slab, possibly two or three days in each of these alcohols will be sufficient to pro- vide perfect dehydration. As the block is going to be large, plain paraffin would be a most unsuitable embedding medium, the writer warmly recommends one of the rubber-paraffin media, the formula for which is given in Chapter 27 under the heading of E 21.1. In view of the large size of the specimen, the ordinary stender dishes used for embedding will have to be abandoned in favor either of beakers or crystalhzing dishes. It is essential that the specimen should lie flat during the course of embedding, or it will inevitably become distorted, and the care thus far taken to maintain the tail in a straight line with the spine will be wasted. The specimen should first be placed in a crystallizing dish filled with benzene, and about a lialf-iiich layer of cliips of the em- bedding medium should be i)laced on to]) of the specimen. The crystallizing dish should be left at room temperature for about a day — naturally covered with a plain sheet of glass — and should then be placed in a paraffin oven or warmed to about 50°C. It is necessary to use a special oven for this purpose, because the large quantity of benzol which evaporates from the preparation will be absorbed in any other wax in the oven and render it relativel}^ useless for subsequent embed- ding. Al)out three or four hours later, after the wax has become fluid, this mixture of benzene-paraffin may be enriched by pour- ing molten paraffin into it and carefully stirring it up. One should then at intervals of a few hours — it does not matter leaving it overnight — pour off about half of the fluid contained in the dish and replace it with fresh, molten paraffin. By this means, over a space of a day or two, the specimen may be passed by reasonable gradations from benzene to paraffin. This whole proc- ess should be watched and controlled with the utmost care, for it is easy for these slabs to twist out of shape in the course of impregnation. After the changes described the specimen should finally be removed very carefully to another crystallizing dish containing clean paraffin and left for at least a day to complete the impregnation with wax. Casting of the block, which will be too large for a paper box, is one of the few cases in which L-shapecl blocks of brass can profitably be employed. Alternativelj', if L-shaped blocks are not available, take two 1-inch lengths of 1-inch-square brass to form the ends of the box which one is making and attach to them with sealing wax two thin sheets of brass along each side, thus making a metal box. This should be stood on a slab of plate glass. Now pour into this box, which should be at least three inches long by one inch wide and one-and-one-half inches deep, about a half an inch of wax and allow it to cool until it is solid. Then heat, in a small beaker, aliout a teaspoonful of wax to a temperature well above its melting j)oint — it is probabl}' safest to raise it to smok- ing heat — and then pour this suddenly onto the surface of the now hardened wax at the bottom of the box. By this means the surface is again molten and the box can be filled with wax which has been maintained in the oven at about its melt- 140 THE ART OF MAKING MICROSCOPE SLIDES S. S. mouse ing point. The object is then carefully placed in the box. At this point one may detach the tail from the rod of glass or plastic to which it has been attached. Then wait until the wax in which the specimen is lying commences to solidify and carefully fill the box to the very brim with molten wax. The whole must now be cooled as rapidly as possible. When the block has completely hardened it is slid out of its metal 1)0X and placed in a large jar of water at room temperature, where it may remain overnight or until one is prepared to deal with it. In the examples previously given use has been made of the ordinaiy rotary mi- crotome, but such an instrument is useless for the very large sections wliich we are about to cut. No microtome will serve save one of the slider type shown in Fig. 55. As these sliding microtomes have no possible justifiable use in the cutting of paraffin sections, save for very large ob- jects, it is curious that so many of them (including the one shown in the illustra- tion) have relatively small object holders provided. The jaws which are nonnally used to hold the metal object holder, how- ever, can be adapted to hold a large piece of wood. The block under discussion had better be attached by melting the wax to a piece of hard wood, previously steeped in paraffin, of a size very little smaller than the block itself. After the block has been attached — it is very dangerous to trj' it before — it must be trimmed to the shape which will be used for actual cut- ting, which differs in every particular from the shape which must be used when cut- ting serial sections of small objects. For ribbon-cutting the block is always rec- tangular and the two sides must be ex- actly parallel. In the case of a very large l)lock from which single sections are to be cut in one of these sliding microtomes, three sides of it may be left more or less rectangular, but the fourth side must come to an aiigl(> pointing to the ))lade of the knife. This angle is not important but should be between 40*^ and 60"^. It does not matter whether the sloping side extends beyond the beginning of the object or not, and it is actually of no ini|)oitance what shape the other sides are i)rovided there is a 40° to 60° angle pointing towards the knife. This angle is for the purpose of pre- senting a small area of wax to the first cut. Large sections on a microtome of this type invariably roll themselves up into a cylin- der which is \'ery difficult subsequently to unroll. If, however, there is a sloping angle pointing towards the knife, the flat portion may be held with a brush against the knife and the whole section, therefore, retained more or less flat as it comes off. Now take an old microtome knife and cut 25 or 30 micron sections from the top until one gets down t(^ that part of the ob- ject which one wishes to cut. This pre- liminary flattening of the top surface of the block, and cutting away of the un- wanted portions of the specimen, also shows how this particular block is behav- ing in I'elation to the microtome itself. If the sections curl hopelessly, in spite of the point of wax, it is evident that the knife is striking at the specimen too squarely and it should be adjusted to cut at an an- gle more like that of the knife shown in Fig. 83 which is, however, set for celloidin. A certain amount of maneuvering of the knife angle l:)ackward and forward will en- able one to secure a cut in which at least half an inch of the pointed end of the wax remains straight, thus permitting a brush held in the left hand to be pressed down while the right hand completes the move- ment of the knife. Do not imagine that sec- tions of this size will ever come off flat: it is enough if they are reasonably flat. Each section will have to be flattened independ- ently in a bowl of water heated to from 5 to 10 degrees below the melting point of the embedding medium employed. The temperature is rather critical, but it may be established by experiments on un- wanted sections, so that when the block has finallj' been trimmed down to the point where the 10 or 15 essential sections can be taken, all difficulties will have been ironed out. It is inevitable, as one cuts farther and farther into the object, that fine readjustments of the orientation will have to he made. These can only be nuule by trial and error, and one should never cut off too many sections until one has finally got the block lying in the exact plane reciuired. It may be said that this l)lane may be determined reasonably when some portion of the vertebrae are being S. S. mouse PARAFFIN SECTIONS 141 out at the same time tliat tlic skin of the tail is being cut. Remember that after fine adjustment, the knife blade will have to be used as a plane to render flat the whole surface of the block before further com- plete sections can be taken. Finally, how- ever, the moment comes which culminates all the months of work. The block is lying completely flat. One places a freshlj^ sharp- ened knife in position and prepares to take the sections required. Before this final operation it will be nec- essary to have cleaned, in any manner de- sirable, the required number of slides, and to have laid these at hand alongside the vessel of water which will be used for flat- tening. Now take the brush in the left hand, the knife in the right hand, shde the knife forward, grab the little curling tail of paraffin coming from the pointed end of the block, and with one smooth, con- tinuous movement complete the section. This section, in a more or less wrinkled condition, will now be l3'ing on the knife blade, from which it may be removed with the aid of two brushes. One l:)rush held in the left hand is moistened with the lips and applied to the upper surface of the end of the section farthest from the edge of the blade, while the other is very gently slid under the section to loosen its attach- ment from the edge of the blade. Using one brush b}^ adhesion from above and one brush to balance the other end of the sec- tion from below, now drop the section onto ' the warm water where it will completely expand. A slide is then taken in the right hand and a needle in the left, with a view to stranding the section in the right posi- tion on the shde. The slide should be placed in the water and left for a moment or two until it reaches approximately the same tempera- ture and then, while held pointing down- wards at an angle of about 45°, ap- proached to the section until that side of the section which is intended to be to- wards the upper end of the slide just touches the glass. The slide is tlien very shghtly raised so as to strand the ui)per portion, which is then held in place with a needle while the whole shde is withdrawn at an angle of from 45 to 30° from the water. It is quite impossible to pass the slide horizontally under the section and then to I'aise it so that the section remains in place. It is only by withdrawing the shde at an angle, in the manner described, that one can hope to strand the section in the correct position. If tlie section is not ill correc't position on tiie slide, no attempt can be made to rearrange it. It is only pos- sible to replace the slide in water with the hope that the section will float off so that a second attempt can be made. If the sec- tion is only slightly out of position on the slide it is much better to leave it alone, since the section usually breaks at the sec- ond attempt to strand it. As soon as the section has been stranded on the slide, the slide is removed from water, laid on a flat surface, a sheet of water-saturated coarse filter paper laid on top of it, and a rubber roller of the type used by photograjjhers pressed down with considerable force so as to squeeze the water from the paper and the section at the same time. The section is then placed on a warm table to dry. Notliing has been said about the use of an adhesive for attaching the section to the shde. Provided that the slide is per- fectly clean and that the section is pressed firmly into contact with it, there should be no necessity for any adhesive at all. For those, however, who do not care to run this risk any adhesive mentioned in Chapter 2S under tlie heading of V 21.1 may be used either by smearing it on the slide, or mixed, to the extent of about 2%, in the water used for flattening. As soon as the slides are dried they may be stained in any manner desired. For a specimen of this nature the wn-iter's first ])reference is for the stain for Patay 1934 (ab])reviated directions for wiiich will be found in Chai)ter 20 under the heading DS 12.32 Patay 1934) or for the stain of Mallory (which will be found in the same chapter under the heading DS 13.41). De- tailed descriptions of the use of both of these stains are given elsewhere. If 'the slides have to be stored for any great length of time, or if the process of decalcification has been unduly prolonged, treat each shde before staining according to the method of Mullen and McCarter, which is given in Chapter 22 under the heading ADS 12.1 Mullen and McCarter 1941. 13 Nitrocellulose Sections General Principles Nature of the Process As the name indicates, this chapter is concerned with the preparations of sec- tions of material which has been impreg- nated with a solution of nitrocellulose. This process is not to be regarded as a substitute for the paraffin method de- scribed in the last chapter; it should be used only when paraffin will not give a satisfactory result. This is usually used either for exceedingly minute objects, the orientation of which in paraffin, or the re- tention of which in paraffin sections, is almost impossible, or for very large ob- jects with numerous cavities which cannot well be supported with paraffin. Paraffin in large cavities tends to shrink away, while nitrocellulose solutions do not. There are numerous disadvantages in the use of nitrocellulose. The worst for the research worker is the difficulty of preparing serial sections with the sections in their due order. Tliis may be overcome to a certain extent by the process of double embedding (see the next chapter) but tliis itself is less satisfactory than straight em- bedding and should be used only for very small or very difficult objects. One of the advantages of nitrocellulose embedding is that the process does not in- volve the use of heat; the materials are impregnated in solutions of increasing strength at room temperature, and these solutions are subsequentl}^ hardened either by evaporation or by chemical means. The size of the nitrocellulose molecules in the dispersions (usually called solutions) em- ploj'ed is so great that the material dif- fuses slowly and the the process is a long one. Various methods have been put for- ward for using nitrocellulose at high tem- peratures, but there appears to be fittle justification for them, because, if the ma- terial to be embedded will stand boiUng, it will most certainly stand embedding in paraffin. These processes appear to have been introduced by those who are so ac- customed to celloidin embedding that they do not wish to use anything else. Materials Employed Cellulose nitrate is not, as its name might indicate, a pure chemical, but is a mixture of a great number of different compounds, the relative proportions of which depend upon the method of manufacture. Few of these mixtures are suitable for cutting sec- tions; and one should alwaj's be used which is specifically prepared for the pur- pose. The best known in the world, and for many years the only one known, was celloidin, supplied by Schering. Its place has been taken in the United States today by Parlodion, marketed by Mallinkrodt. It is unfortunate that the trade names of both of these should be so closely allied to collodion, which is a pharmaceutical solu- tion of pyroxylin unsuitable for section cutting. Cellulose nitrates, other than those marketed under brand names, are broadly classified according to the viscos- ity of the standard solution. This viscosity is expressed in terms of the number of seconds taken by a steel ball of standard size to fall a standard distance through a standard column of the solution. The low- est viscosity normally marketed is that known as 5-second nitrocellulose and is the only one which may be employed in micro- technique. Another point to be watched, 142 Solutions NITROCELLULOSE SECTIONS 143 in using other than a proprietary product, is the fact that some nitrocelhilose mix- tures are quite ^^olentl3' explosive when dry, whereas both celloidin and Parlodion, though they burn briskly if given the opportunity, do not ignite with explosive violence. Cellulose nitrates other than those indicated under brand names are always marketed in solution and should never be stored in the dry state. Many of the older books suggest that chips of nitro- cellulose material, to be used for embed- ding, be stored under water. This advice is given not to lengthen the life of the chips, but only to avoid the risk of explosion. Celloidin, which term will be used throughout the rest of this chapter when- ever a nitrocellulose-embedding medium is meant, is soluble in a great variety of modern solvents, but most techniques are based on its use in a solution of a mixture of alcohol and ether. Preparation of Solutions Celloidin is not easily soluble in the al- cohol-ether mixture usually employed, therefore a special method must be used to prepare the solutions. The chips of dried material are first removed from the bottle in which they have been kept and placed in a desiccator overnight. The only real enemy of the success of embedding in cel- loidin is water, and at no stage in the pro- ceedings may one risk contamination. It ' is usual to carry in stock a 16% solution of celloidin, therefore, 16 grams of these dried chips should be weighed. These chips are placed in a dry bottle fitted -with a glass stopper which has been tested for fit. Fifty parts of absolute alcohol are then poured over them. If this alcohol is not taken from an unopened new bottle, it is desirable that it be carefully dehydrated either with calcium sulfate or copper sul- fate before being used for this purpose. The bottle is left at room temperature overnight, in order that the celloidin may swell, and when this swelling is complete 50 parts of anhydrous ether are added. The ordinary ether of commerce and the ether used for anesthetic purposes are worthless; one must employ the variety sold as ether anhydrous by sodium. Any attempt on the part of the worker to re- move water from commercial ether with sodium in his own laboratory will produce nothing but a serious explosion. The an- hydrous ether should always be taken from a freshly opened can. The bottle is rotated slowly until the celloidin is com- jiletely dispersed through the mass. The selection of a solution of this strength is based on the fact that it is about the thick- est solution which may reasonably be poured from a bottle. Not too much of the material should be prepared at one time, since ether always evaporates through even the best fitting stopper. The only method known to the writer of keeping the material satisfactorily is to secure one of the bottles, once common in pharmacies but now difficult to obtain, in which the ground glass stopper is itself covered with a domed cap — like that on a balsam bottle — ground to the neck. If such a bottle can be obtained, the outer cap, but not the inner, may be greased with glycerol and a relatively ether-tight seal thus secured. Under no circumstances should celloidin solutions be stored in an icebox in the hope of diminisliing the rate of evaporation. If these cold solutions are then brought out into a warm room, moisture will condense all over the bottle and over the solution as it is being poured. In Chapter 27 under the heading E 22.1 will be found sugges- tions for various other solutions which have from time to time been made. Infiltration of Objects with Nitrocellu- lose Solutions In the course of embedding in paraffin, as described in the last chapter, it is just possible to get away with slightly im- perfect dehydration. In impregnation with celloidin it is absolutely impossible. The prime prerequisite to the successful infil- tration of a specimen is that it be per- fectly dehydrated. For this purpose the specimen should be brought up in the con- ventional manner through such series of alcohols as may be necessary until abso- lute alcohol is reached. It should continue dehydration for some considerable time in at least two changes of absolute alcohol, the last of which has either been drawn from a sealed bottle, or from a bottle in 144 THE ART OF MAKING MICROSCOPE SLIDES Infiltration Fig. 80. Tying paper collar round wood block. Fig. 81. Putting in first layer of celloidin. which a considerable quantity of calcium sulfate or copper sulfate has been placed as adehydrant. When the specimen is com- pletely dehydrated, it is transferred to a mixture of equal parts of absolute alcohol and ether. The worker must remember always to use anhydrous ether and not the commercial variety. Specimens should re- main in this mixture until they have been completely impregnated. The actual process of getting the mate- rial impregnated with 16% celloidin may 1)6 done in two ways. Either the ol)ject may l)e i^laced in a considerable volume of a dilute solution, and evaporated, or it may be passed through solutions of in- creasing strength. The writer prefers the latter method since it is very difficult to evaporate an alcohol-ether mixture slowly under dry conditions. Most people employ solutions of 2, 4, 8, and 16% celloidin prepared bj- dilution of the 16% stock. Every vessel used for dilution, as well as the diluent itself, must be absolutely dry. The object should be taken from the ab- solute alcohol-ether mixture, and placed in about 50 times its own volume of a 2 % dilution in a glass-stoppered bottle, which should in turn be kept in a sealed desicca- tor while the imjiregnation is going on. The time of impregnation naturall}' varies according to both the size and the nature of the object, but it is a rough and ready rule for relative time that the material should spend proportionately as long in each solution as the concentration of the Casting blocks NITROCELLULOSE SECTIONS 145 Fig. 82. Transferring object in thick celloidin. celloidin; that is, if the object were to take one hour in 2 % it should have eight hours in 16%. As a measure of absolute time it may be said that an object the size of a frog's egg will require about one day in the 2 % while a flower bud the size of a walnut should remain at least ten days. The writer prefers not to endeavor to transfer the object from one solution to another, but to pour off the weak solution and re- place it ^\'ith a stronger. There is no means of telling when impregnation is complete until one comes to cut the section; but two things must be remembered: one, that the specimen cannot be damaged no mat- ter how long it be immersed in celloidin, two, that the most frequent cause of faulty sections is imperfect impregnation. When impregnation is complete it is necessary to prepare a block for cutting. Casting Celloidin Blocks Two methods may be employed to transform the celloidin from a hquid to a solid state. Either the alcohol-ether mix- ture may be permitted to evaporate, or it may be removed with another solvent, usually chloroform, in which celloidin is not itself soluble. The removal of the sol- vent with chloroform may also be done either in the hquid or in the vapor phase. It is difficult to mount blocks of celloi- din once they have been cast on an object holder, though the solution of Apathy (Chapter 27, E 21.1 Apathy (1942)) has been specifically developed for that pur- pose. It is, moreover, very nearly im- possible to mount a celloidin block on any of the metal block holders supplied with standard microtomes. The worker is, tlierefore, advised to prepare for himself a series of small wooden blocks on which the object may be directly cast. These wooden blocks should be of hardwood and a whole series should be provided accord- ing to the size of the object which is to be cut. The smallest practical size is about J2" X H" X 1" and it should be under- stood that the block will be cast on the J-^-inch end. The largest practical size is dependent entirely upon the size of the object to be cut. These blocks should be cut, sanded as smooth as possible, baked in an oven at about 80°C. to remove as much water as possible, and then thrown directly into absolute alcohol. The abso- lute alcohol is replaced with absolute alco- hol-ether and then with 2% celloidin. The blocks are transferred for se\'eral daj's to a solution of 4% celloidin and then with- 146 THE ART OF MAKING MICROSCOPE SLIDES Cutting drawn, stood on their ends in a desiccator, and dried. Once prepared, they may be used an indefinite number of times. The process of casting the block is not difficult. First of all take a paper collar (ordinary bond paper is excellent) and tie it firmly round the edge of the block so that it projects upwards for a distance of about M inch (Fig. 80). This makes a box the floor of which is the end of the wooden block and the sides of which are of paper. Then pour into the bottom of this box about K of an inch of 16% celloidin (Fig. 81) and place it in a desiccator (at the left in Fig. 81) where the alcohol and ether are allowed to evaporate until the surface of the block is firm when touched with a blunt needle. It is not required to be hard; it is only required to be sufficiently firm that an object placed on it will not sink. Remove the block from the desiccator and fill it to the brim with the 16% celloidin containing the object (Fig. 82). The object will sink through the hquid celloidin until it comes to the firm layer underneath. Needles are used to orientate it in the desired position, and it is then either placed in a desiccator to evaporate, or, better, placed in a desiccator in the base of which the desiccant has been replaced by a quantity of chloroform (at the right. Figs. 81 and 82). There is relatively rapid vapor exchange between the chloroform and the alcohol-ether, and the block by this means may be completely hardened overnight. If speed is vital, place the whole block in liquid chloroform as soon as the object has been oriented. By this means the block will be hardened in a few hours, but with some risk that the rapid diffusion currents set up may displace the object. The block should be stored in chloroform until required. If the block is to be prepared by the method of evaporation, a box of paper is made and a layer of hardened celloidin set on the bottom. After the object has been oriented in the 16% celloidin, however, the block is placed in a desiccator to evap- orate, and is filled up from time to time with 16% celloidin as it shrinks. Blocks hardened by evaporation are denser and tougher than those hardened with chloro- form. The chloroform technique is usually more satisfactory. Cutting Sections in Celloidin There are as many methods of cutting sections from celloidin blocks as there are workers who have done it, and space does not permit all the variations to be given here. Broadly speaking they fall into two classes: those in which the celloidin is cut dry, and those in which it is cut wet. Celloidin may be cut on any kind of mi- crotome, but unless an attempt is to be made to serialize sections (which is far better done by the double embedding technique described in the next chapter) a shding microtome should be used. Celloidin cannot be cut by bringing a square edge of the block against the knife. Not only must the knife be set at an angle of about 30° to the direction of travel (Fig. 83) but the corner of the block must, as shown, be trimmed to an acute angle. The block, therefore, after being trimmed to the shape shown, is clamped by its wooden base in the holder and oriented in the desired position. If the block is being cut dry the knife is now shd forward and the sections removed to a watch glass. Do not worry if they are, as is more than prob- able, considerably curled. When enough sections have been accumulated they may be dealt with in the manner to be de- scribed later. The author much prefers to cut his blocks after they have been moistened with oil of cedar. There is a double reason for this. Not only do the sections tend to stay flatter, but if the block is thoroughly impregnated with oil it will become glass- clear so that last-minute adjustments of orientation are easy. By this technique the block, which should have been chloro- form-hardened, is transferred directly to oil of cedarwood and left until it is glass- clear. When it is removed, as much as the cedar oil as possible should be wiped off with a cloth and the block mounted in the appropriate holder. Then take a finger- bowl, or beaker, of oil of cedarwood and, after having adjusted the knife to approxi- mately the correct angle, moisten the blade of the knife with the oil. The knife is then shd forward to remove a section, Cutting NITROCELLULOSE SECTIONS 147 Fig. 83. Cutting celloidin sections. and each section is received (Fig. 83) on a brush saturated with oiL From time to time the blade of the knife should be moistened with more cedar oil, and the sections as they come onto the knife may be left to accumulate — they will be held to the blade by the cedarwood — until a considerable area of the blade is covered. These sections may then be picked up with a brush moistened with oil of cedar and transferred to a container of the same fluid; or, if there are enough of them, they may merely be washed off the knife, which has been removed, and placed in the beaker or watch glass. Both the methods just described i:)re- suppose that the object has been stained, as should usually be the case, before em- bedding, and that the sections will require no further manipulation beyond flattening and mounting. When the sections are be- ing cut with a view to staining them subse- quently, the method of cutting a block moistened with 70% alcohol is to be pre- ferred. By this method the block, which must have been hardened in chloroform, is placed directly in a considerable volume of 70% alcohol. Stronger alcohol should not be employed because it will tend to soften the block. After a day or tw'o in 70% alcohol, most of the chloroform will have been removed, the block is then mounted in the usual manner, and sections are cut from it with a knife which is moistened with 70% alcohol. The blade must be moistened with a brush dipped in 148 THE ART OF MAKING MICROSCOPE SLIDES Mounting 70 % alcohol each time a section is cut, and each section must be individually removed to a beaker of 70% alcohol in order to avoid evaporation and drying. Staining Sections It is usually desirable to stain objects before celloidin sections are cut, but when it is necessary to stain subsequently, and the sections have been prepared in 70% alcohol as described, they may be sub- mitted to the action of any staining fluids exactly as though they were freehand sec- tions. That is, they may be passed from one solution to another with the aid of a section hfter, washed in water, and in general handled with considerable rough- ness without any risk of damaging them. It must be remembered, of course, that alcohol solutions may not be employed or the celloidin will be hopelessly softened. The chief objection to this procedure is the tendency of some stains to be absorbed by the celloidin; it is cUfficult to find a plasma stain which will stain tissues with- out coloring celloidin. Nuclear staining is relatively easy, as is also metal staining, which is the process most usually applied to celloidin sections. No attempt should be made to flatten the section before it has been stained, but it should be passed through all the required techniques and then returned to 70% alcohol before mounting. A method of double staining a botanical specimen is given in the typical preparation which concludes this chapter. Mounting Celloidin Sections on Slides If the section has been cut in cedar oil from an object which has been prestained, nothing further is required than to remove the section from cedar oil, place it in the center of a clean slide, add a drop of the resinous mounting medium selected, and apply the coverslip. Anj^ sUght curl Avhich tends to lift the coverslip may be easily pressed out, either by leaving a weight on the coverslip overnight or by using a small spring clip to hold the covership down. If the section has been cut dry it will usually be found too curled to mount satis- factorily, and a different technique must be followed. In this case the section is })laced in the approximate position on the slide, and the slide, with the section, placed in a small dish containing a little ether. Within a relatively short time the celloidin will have been softened suffi- ciently to be pressed flat on the slide and mounted in balsam under the coverslip. If the section has been cut in 70% alco- hol, and subsequently subjected to various staining procedures, it is necessary that it should be dehydrated before being mounted in balsam. It may be removed from 70% alcohol to a mixture of equal parts of absolute alcohol and chloroform, the former to dehydrate the specimen, the latter to prevent the dissolution of the cel- loidin. This mixture will often appear cloudy when the section is first put in, in which case it is only necessary to replace it with fresh solution and so on until both the section and the specimen remain un- clouded. When an unclouded condition has been reached, the specimen may be dehydrated in oil of cedar, placed on the slide, and mounted as previously described. It is occasionall}^ necessary, though usu- ally undesirable, to attach a number of celloidin sections to sHdes and then to stain them in position. The reason this is unsatisfactory is that it is hard enough to remove staining dyes from the celloidin matrix when both sides of the section are free in a watchglass, and nearly impossible when one side has been pressed against a glass surface. This method, however, must be employed if it is desired to serialize celloidin sections and some cogent reason prevents the use of the double technique described in the next chapter. Of the vari- ous methods given in Chapter 28 under the heading V 21.2, the writer prefers that of Heringa and ten Berge 1923 in which clean slides are coated with a 3 % solution of gelatin and dried. When these slides are required they are soaked for a couple of hours in 5% sodium sulfate, rinsed, and again dried. The section is taken from 70% alcohol, pressed firmly to the sUde — or the sections are lined up in their order and pressed firmly to the slide — and then dipped as soon as they are partially dry once or twice in absolute alcohol and chloroform. This gives a very reasonable T.S. Lily bud NITROCELLULOSE SECTIONS 149 adhesion. Another useful method of han- dhng large numbers of celloidin sUdes is that of Linstaedt 1912, also described in Chapter 28, V 21.2. The method there given can be followed; it results, in effect, in the fusing of a large quantity of cel- loidin sections into a single sheet of celluloid, which may then be handled through stains, etc. as if it were a simple section. It will be noticed that the sheet itself is made of celluloid — not celloidin— which is a material which does not readily pick up stains. The two methods of Lon- geron involve the removal of the celloidin after the sections have been mounted, and leaves one to wonder why (celloidin should have been used, instead of paraffin, in the first place. Typical Example Preparation of a Transverse Section of a Lily Bud It has already been pointed out that one of the best uses to which celloidin maj^ be put is the preparation of sections of fine structures containing cavities which would not be held b}' paraffin. The example here selected is a case in point, for it would be almost impossible by the ordinary paraffin section technique to take a transverse section of a large flower and to maintain all the different parts in relation to each other. It would indeed be almost impos- sible to secure a section at all without gross collapse of the parts. The bud of a lily has been selected be- cause it is such admirable teaching mate- rial. Sections may be taken through a level which will show both the stamen and the pistil, and the material is sufficiently large to permit an elementary botany class to get a clear idea of the arrangement of the different parts with the use of mag- nifications no higher than those provided by a hand lens. The method of staining selected, however, is sufficienth^ good to permit the examination of the individual parts, bj- an advanced class, under the high power of the microscope. The exact species of hly, provided it is one of the trumpet varieties known to florists, is quite immaterial, and the bud should be taken about a week before it is open. The best fixative to use for this kind of thing is one of the chromic- formaldehyde-acetic mixtures known to botanists under the general term of C RAF. Several formulas for these mixtures are given in Chapter 18 under the heading F 6000.1010; the ffiiids of Navashin 1912, Belling 1930, and Randolph 1935 are the ones widely used by botanists. It makes little difference which of these formulas is employed, but they must be made up immediately before use to prevent the reduction of the chromic acid by the formaldehyde. A hly bud ly/' long X }i" in diameter will need to be fixed for about four days in one of these fluids, which should be changed daily and kept in the dark. As soon as the bud is cut from the plant it should be immersed in the fixative and the extreme tip cut off to permit the contained air to leave. After fixation in these fluids, the bud should be washed for 24 hours in running water and then transferred through 20%, 50%, 70% and 90% alcohol (about a daj- or two in each) to 95% alco- hol, which should be changed as often as it becomes discolored. It is necessary to remove the chlorophyll, or else this will subsequently diffuse into the celloidin, from which it is almost impossible to re- move it. If the process of decolorization in 95 % alcohol is too slow for the worker, he may transfer the bud to absolute alcohol until it is dehydrated, and then to chloro- form where the remaining chlorophyll will be extracted very rapidly. The risk in this procedure, however, is that the chloroform will not subsequently be sufficiently re- moved, and will thus prevent proper infil- tration by the celloidin. If chloroform is used, the bud must be removed as soon as bleached to absolute alcohol, which is changed as often as the least smell of chloroform remains. It is then put through at least 6 changes of 95 % alcohol, with one day in each, before being transferred to fresh absolute alcohol to complete the dehydration. The writer's preferred method of dehy- dration for large objects has already been 150 THE ART OF MAKING MICROSCOPE SLIDES T.S. Lily bud described (Chapter 12) and should be em- ployed in the case of the lily bud. Remem- ber that almost nothing can prevent the production of a perfect celloidin section except imperfect dehydration of the speci- men. One is only safe when the specimen has remained in a large vessel of absolute alcohol, containing copper sulfate at the bottom, for a period of 24 hours, at the end of which time not the slightest trace of color shall have been accjuired by the copper sulfate. A 16% solution of celloidin is then di- luted to a strength of 2%. If the lily bud contains very large cavities — that is, if it was taken quite late in its development — it may be necessary, in order to avoid diffusion currents and some consecjuent bending of the internal structures, to start with a solution as weak as M% celloidin rather than with the conventional 2%. It is best to fix and dehydrate several buds at one time and to take the first of these up through the conventional process. If this fails a slower method must be used. The bud is then passed to a mixture of equal parts of absolute alcohol and anhydrous ether until diffusion currents are no longer apparent. If only 20 or 30 milliliters of the fluid are used for a specimen of this size, it should be changed after about 3 hours and then left overnight in a fresh solution. There is a risk, if an object of this size is picked from alcohol-ether mixture and placed in another fluid, that the rapid evaporation will leave air bubbles; there- fore, it is best to place it in a vessel with just enough alcohol-ether mixture to cover it and then to fill this vessel with 2% celloidin. The container is then rocked gently backward and forward to mix the celloidin, and the specimen is left for about 24 hours. This weak celloidin is now poured off, leaving enough of it to cover the object, and 4% celloidin is poured in. The 4:% celloidin should be left for three or four days and then replaced in the same manner by 8% celloidin, in which the specimen should be left for at least a week. Eight per cent celloidin is sufficiently vis- cous to inhibit air bubbles when the speci- men is transferred, and it should now be lifted from this thickish celloidin and put into the 16% solution. All these operations should have been conducted in glass-stop- pered bottles kept in a desiccator. The period of time in 16% celloidin is not crit- ical but two or three weeks would be a safe period. The whole process is so long drawn out, that an extra week or two makes little difference; any endeavor to save even a few days in the final impregnation may undo all the previous work. Now take a wooden block about 1" X 1" X 2" and tie onto it a paper collar (Fig. 80) at least an inch taller than the bud. This is naturally a somewhat cum- bersome arrangement; it will probably be best to use an ordinary 5" X 3" indexing card, rather than a piece of paper, in order to get the necessary stiffness. A large box of this kind will inevitably leak so that the overlapped edges should be held together with gum arable, permitted to harden, and then dried in a desiccator. After this block, with its paper walls rising from it, has been thoroughlj^ dried in a desiccator, the paper, and about half of the wooden block, is dipped in 8% celloidin and placed back in the desiccator. This procedure not only holds the paper more firmly in position but also provides an additional assurance against leakage. When this initial coat of celloidin has hardened, about '^2 inch of 16% celloidin is poured into the bottom of the box, which is then returned to the desiccator and examined at intervals until the celloidin is found to have hardened sufficiently to bear the weight of the bud. The box is then filled with 16% celloidin and the bud is inserted. It does not matter in the least if the celloidin flows up over the side, but it will be very unfortunate if not enough of it is used. The writer finds that the best method of holding a large object like this in place in the box is to take a couple of entomological pins and drive them clear through the box and the specimen, in areas from which sections are not required. Using a long needle it is then possible to reach down and adjust both the bottom and the top of the long bud so that it lies essentially in the center of the box, held in place by the entomological pins driven through it. Fine pins of this nature do not make a sufficiently large hole to permit any leakage of celloidin. If the box is now not full of celloidin, it is T.S. Lily bud NITROCELLULOSE SECTIONS 151 topped off with the 16% solution and placed in a closed vessel (a desiccator is very convenient) in the bottom of which a small quantit}' of chloroform has l)een placed. After about a day it will be found that the block has set to a rather opaque jelly-like consistency and the whole thing — block, wood, pins, and paper — -is then thrown into a large container of anhydrous chloroform. It should remain for a few days in the chloroform and then be trans- ferred to a considerable volume of 70% alcohol, which is changed daily until no smell of chloroform is observed. The block — pins, paper, and all — may be kept in 70% alcohol until it is required. When it is decided to start sectioning, the block is removed, the pins withdrawn (a pair of phers will probably be neces- sary), and the paper shaved from the sides of the block with a sharp knife or razor. It has been presumed in the directions which have so far been given that the re- quired sections will lie about one-third of the distance from the base of the block, since it is obvious that a block of this size will not have the stability to permit cut- ting at the top. Under these circumstances the upper two-thirds of the block should be removed, and it is safer to do this with a fine saw than with a knife. If it is decided that the portions of the upper block are also required, the block may be cut with a saw into as many pieces as are wanted, , and each piece mounted on a celloicUn- impregnated wooden block with the solu- tion of Apathy given in Chapter 27 under the heading E 22.1. Blocks mounted with Apathy's cement are never as satisfactory, however, as those which have been cast directly onto a wooden block, as in the first case. The reason for the retention of the entire bud through embedding, is, of course, to avoid disturbing the exceedingly delicate relationships of the parts. This would certainly ha^Dpen if one were to en- deavor to embed one third of the bud without leaving the remainder of it at- tached for support. The lower third of the block on its wooden holder is now mounted in the object holder of a sUding microtome and oriented roughly in the position de- sired. The block will be found to be suffi- ciently clear, after one has planed off the surface with a i-azor, to see down into it and select that iM)int from which the de- sired sections will be cut. No difficult}'' will be e.xperienced in cutting these sec- tions provided the knife slopes back away from the block at an angle of about 30° and hits the corner rather than the edge of the block. Before cutting, provide a beaker containing 70% alcohol, in which the sections are to be accumulated, and another beaker and brush containing 70% alcohol, with which the knife blade and the surface of the block are to be liberally anointed while sectioning is in process. As each section comes off, it should be re- moved to the dish of 70% alcohol in which the sections may be stored until they are to be stained. It is excellent practice to accumulate a large number of these sec- tions and then to issue them to a class for staining. Celloidin embedding is such a prolonged process that it is difficult to use in class periods, but there is nothing to prevent blocks or sections from being issued to classes to whom a detailed de- scription of the manner in which they have been prepared is given. A good combination for staining these sections is Delafield's hematoxylin and saf- ranin. However, the ordinary Delafield's hematoxylin solution — the formula for which is given in Chapter 20 as DS 11.122 Delafield (1885) — cannot be used at full strength or it will be difficult to remove from the celloidin matrix. It is better to dilute the original solution \\dth about 10 times its own volume of a 1 % solution of ammonium alum. One-tenth of 1 % hydro- chloric acid in 70% alcohol and one of the solutions of safranin given in Chapter 20 under the heading DS 11.42, are also re- quired. The safranin should be either in water or in an alcohol not stronger than 50%. The solution of Johansen 1940, for example, contains enough Cellosolve to soften a celloidin section undesirably. The formula of Chamberlain 1915 is that com- monly employed. Having accumulated these reagents in three dishes, and a spare dish of 70% alco- hol, the sections are placed in safranin. It is difficult to overstain in this fluid and it is probably most convenient to leave it overnight. It should be left at least until 152 THE ART OF MAKING MICROSCOPE SLIDES T.S. Lily bud it appears to be deeplj^ stained. This will take not less than three or four hours. The next morning, if staining has taken place overnight, each section is transferred sep- arately to acid alcohol and examined un- der a low power of the microscope until the safranin is observed to be almost re- moved from the cell walls. The sections are then transferred to distilled water, preferably through two changes, to re- move the acid. As soon as the acid has been removed, they are placed in the di- luted Delafield's hematoxylin and left there until the cell walls are deeply stained. This will take from five minutes to half an hour, depending on both the thickness of the section and on the nature of the specimen. The sections are then re- moved, one at a time, to acid alcohol, and left there until the stain has been removed from the celloidin matrix but not from the cell walls. As soon as this result has been achieved the sections are transferred to 70% alcohol, in which the\" are rinsed in several changes to remove the acid. Now collect as many slides as are re- quired, a mixture of equal parts absolute alcohol and anhydrous chloroform, and a bottle of whatever resinous medium has been selected as the mountant. The sec- tions are transferred from 70 % alcohol di- rect to absolute alcohol and chloroform, in which they are allowed to remain until dehydrated. Each section is then passed to cedar oil where it remains until it is clear. The sections are now taken one at a time and drained by the corner against a piece of filter paper. A drop of the resinous medium is placed on the slide, the section placed on this, another drop placed on top, and a coverslip applied. If the sections curl to a slight extent, this may be over- come, as has already been pointed out, by placing either a weight or a clip on the cover. If, however, the sections are badly curled, it is desirable to soften the celloi- din somewhat. This may be done by using a mixture of cedar oil and clove oil (cel- loidin is readily soluble in the latter) in place of the pure cedar oil for the clearing. It is as well to try 10% clove oil in cedar oil at first and, if this does not render the sections flexible enough, to increase the quantity of clove oil until they can be flattened. 14 Sections from Double-embedded Material General Principles The onl}' purpose of embedding objects first in celloidin and then in paraffin is to secure serial sections of material which cannot be handled by the paraffin method alone. This limits its utility to small ob- jects which cannot with ease be oriented in paraffin, or alternatively, to small ob- jects of which it is quite essential to obtain series, and which like many small arthro- pods, cannot be retained sufficiently firmly in a wax matrix to permit of sections being obtained. It is possible to impregnate an object with celloidin (as described in the last chapter) and then to embed the impreg- nated object in paraffin. There are, how- ever, much better ways available which shorten to a considerable extent this long process. These methods are based on the original suggestion of Peterfi 1921 (23632, 38:342) that a solution of celloi- din in methyl benzoate could profitalily be substituted for the more conventional solutions. By this method the small objects are dehydrated in the manner described in the last chapter, just as much care being necessary as though one were running a straight celloidin impregnation, but are then passed from the absolute alcohol- ether mixture to a celloidin solution con- taining methyl benzoate or methyl saU- cylate. These solutions mostly contain 1 % of celloidin. Formulas for some of them will be found in Chapter 17 under the heading E 22.1. The advantage of methyl ester solutions is that the celloidin does not contract so much on hardening and the solutions may be dropped directly into a solution of chloroform to produce a solid block. Minute objects — usually small arthropods, invertebrate larvae, or proto- zoans— are first impregnated with the methyl ester solution, and then dropped into a beaker of anhydrous chloroform. Solidification is almost instantaneous and the little globule containing the object may be removed after five or ten minutes. As there is usually difficulty in orienting these minute objects, the author prefers to color the celloidin-embedding medium with the addition of 0.1% of eosin. This enables the block to be trimmed to a rec- tangular shape which may itself be orien- tated without difficulty, for it is now clearlj^ visible in paraffin. Chips of the embedding medium are added to the chloroform, and this mixture is transferred to the oven until it is fluid. The small block is then removed, placed in i)ure embedding medium for as long as is necessary, and then made into a paraffin block as described in Chapter 12. These generahties are sufficient to introduce the process which is much better described in the form of the typical preparation which follows. Typical Example Prep.\r.\tion of a Series of Sections, Intended for Reconstruction, of a Pluteus Larva of Echinus Echinoderm larvae are among the most sections, and tlic only method by wliicli difficult objects from which to cut perfect their anatomy may reasonably be studied 153 154 THE ART OF MAKING MICROCOPE SLIDES Pluteus larva is in the form of reconstructions. The pres- ent example, since reconstruction has not been previously described, must be pre- fixed by a discussion of this process. Reconstruction involves reproduction, either as a side view or as a solid model, of greatly enlarged areas of a section. This book is not the right place to discuss the process in detail, since it is no part of the making of microscope slides. A general un- derstanding of the processes is, however, necessary in order to explain the steps which are taken in preparing the sections. To make a wax reconstruction, camera lucida drawings of the parts of the re- quired sections are transferred to sheets of wax which have been cast of the same thickness as the section would be if it were magnified to the size of the drawings. The relevant portions of the wax are now cut out and piled on top of each other until a solid model, representing an en- largement of the section, has been built. Graphic reconstruction, on the contrarj^, is done on sheets of graph paj^er. Lines of the thickness which correspond to the magnification at which one is studying the section are drawn, these lines represent a side view of the section which is being re- constructed. It must be obvious that in both cases it is necessarj^ to have one fixed, straight line running from front to back of the object in order to relate either the wax blocks or the lines drawn to some stable point. Were this not done, any curve, when reconstructed, would appear as a straight line. In reconstructing a ver- tebrate embryo the problem is simplified because either the center of the notochord, or the dorsal aorta, may be used as a point of reference. Invertebrate larvae, how- ever, suffer from the disadvantage that they rarely have any structure which runs in a straight line through them and some straight lines must be synthesized. This is best done by embedding alongside the ob- ject a hair which has been thickl}' covered with lampblack. After the block has been cast the hair is withdrawn with a sharp jerk (it might really just as well be left in place) leaving a line of lamp black run- ning from front to back. This line will ap- pear as a dot in each successive section and each structure seen in the section may be orientated with regard to the lampblack line. The first problem in dealing with echino- derm larvae is that of fixation. In the author's experience nothing is to be com- pared, for this purpose, with the "strong fluid" of Flemming (Chapter 18, F 1600.0010 Flemming 1884). Larvae from plankton samples or from breeding tanks, are accu- mulated in a small fingerbowl of clean sea water, each one then taken up in a pipet with the smallest possible quantity of sea water, and squirted rapidly into a large volume of fixative. They will be perfectly fixed in about 10 minutes and must im- mediately be removed to distilled water, in several changes of which they are washed. It is necessary to remove the fixative as rapidlj' as possible since the osmic acid is liable to deposit osmium hydroxides as a blackened layer over the tissue. Since these objects are to be embedded in cel- loidin and paraffin, it is necessary for them to be stained before embedding, or end- less difficulties will result. The writer prefers, for echinoderm larvae, Mayer's "paracarmine," the formula for which will be found in Chapter 20 as DS 11.22 Mayer 1892a. Fixed specimens should be passed from 70% alcohol to 50% alcohol, which is then replaced with the stain in which the larvae are left from 24 to 48 hours. It is easy to lose small larvae in chang- ing the staining solutions. It is recom- mended, therefore, that the tube in which the staining is done be tipped out into a large fingerbowl of the differentiating solu- tion, since the resultant dilution is fight enough in color to enable one to see the small larvae floating about. These larvae are then picked out one at a time and placed in a clean tube of the differentiating solution for about half a day. They are then removed to distilled water for two changes of about one hour each, and from this passed through 30% alcohol, 50% al- cohol, and back to 70% alcohol, in which they are well washed, and in which they may be preserved until required for sec- tioning. It may be added that the cal- careous sjiines contained in later pluteus larvae are dissolved by the acetic acid in the fixative and no decalcification is necessary. Pluteus larva SECTIONS FROM DOURLE-EMBEDDED MATERIAL 155 The specimens should now be looked over in order to select those in which the "arms" are relatively straight, and these should then be transfei'retl to a tube of absolute alcohol and changed at intervals until they are completely dehydrated. The absolute alcohol is then replaced withamix- ture of equal parts of absolute alcohol and anhydrous ether, which is changed once or twice. About three hours between changes will be enough to dehydrate ol:)- jects of this size. The embedding solution is prepared by taking 16% stock solution of celloidin in alcohol-ether and adjusting it with addi- tions of alcohol-ether and methyl sali- cylate to the composition selected. The solution of Heinz 1923, for example (Chapter 17, 21.1 Heinz 1923), with which the writer has had excellent results, is obtained by diluting 12 milliUters of the 16% stock solution of celloidin to a total volume of 50 with a mixture of ecjual parts absolute alcohol and anhydrous ether, and then mixing with this 50 milliliters of methyl salicylate. The larvae are trans- ferred directly from the alcohol-ether so- lution to this mixture and may remain for as long as required, but at least for 48 hours. The author prefers, at this stage, to add a drop or two of a saturated solution of ethyl eosin in absolute alcohol to the medium, in order that the block cast from it may be sufhciently colored to be visible when embedded in wax. After the larvae have remained in this mixture long enough to become impregnated, the tube contain- ing them is tipped into a watch glass. Each individual larva is then taken uj) in a pipet with a consideralile amount of its embedding medium, and the pipet is held vertically until the larva has sunk to the bottom. The largest possible drop con- taining the larva is then extruded from the pipet into a beaker of anhydrous chloro- form and finally shaken off the tip, so that a spherical globule of the embedding me- dium containing the larva falls into the chloroform, where it coagulates instantly. This is repeated with each of the succes- sive larvae until the batch is finislied. If orientation were not of importance, it would now be possible to proceed to paraf- fin embedding. In a case like this, how- ever, orientation is of primary imjwrtance, and the author prefers to transfer, after about an houi' in chloroform, each of the little globules to a tube of cedar oil where they remain until they are clear. Each glo- bule is then trinnned, under cedar oil, until the larva lies in a rectangular block of celloidin with the long axis of the larva exactly parallel to the long sides of the rectangular block. It is now necessary to prepare the hairs which will be used to jirovide the guide lines used in reconstruction. Take a num- ber of human hairs and dip them into any sticky substance before rolling them back- ward and forward in a watch glass of lamjiblack. Remove each hair, shake off as much lampblack as possible, and lay the hair aside until required. When it is re- quired for use, a bow of wood, or plastic, is strung with the hair, which therefore re- mains straight and under tension. Now return the celloidin blocks to chlo- roform where they remain until most of the cedar oil has been removed. Then change to fresh chloroform and add enough chips of the embedding medium to cover the blocks. Now place the tube in the oven until the wax has dissolved in the chloroform. Pour off the chloroform-wax mixture and replace it with fresh wax, in which the objects msiy continue to impreg- nate for another hour before being cast into blocks. Objects as small as this may be con- veniently cast in a watch glass. Place a pipet full of wax in a watch glass, hold this touching the surface of a fingerbowl of water, and, ag soon as the underside has haixlened, transfer a celloidin block to it and orient the block roughly. Now lay one of the stretched hairs alongside the celloi- din block, not quite touching, but abso- lutely parallel. Return the watch glass to the surface of the water so that the block will harden. Then detach the block from the watch glass and tiim it, first cutting away the ends of the hair from the bow, and then jerking out the hair to leave a straight line of lampblack behind it. The block is then mounted and cut into serial sections as in Chapter 12. The only difficult}^ which is Ukely to be 156 THE ART OF MAKING MICROSCOPE SLIDES Pluteus larva encountered is in making sure that the celloidin blocks and their contents adhere firmly to the glass sHde. It will usually be found that when the ribbon is flattened on water there is a tendency for the cel- loidin block to bow up, and it will inevi- tably become detached in subsequent staining operations. The writer has found, however, that if a saturated solution of ether in water, instead of pure water, is used for flattening, and if each section is pressed firmly in place with wet filter paper and a rubber roller as described in Chapter 12, adhesion Avill be perfect. Any standard adhesive may be used. The writ- er's preference in this instance is for the egg albumen of Mayer (Chapter 28, V 21.1 Mayer 1884). As the sections have already been stained, it is only necessary to remove the wax with xylene and to mount in whatever resinous medium is selected. 15 Frozen Sections General Principles Nature of the Process The last three chapters have dealt with sectioning specimens which have been impregnated with some material to pro- vide support, either through solidification (wax) or through the evaporation of the solvent (nitrocellulose). There are two circumstances under which neither of these processes may be used: first, when it is desired to preserve in the tissues some fatty material which would be dissolved by the reagents used prior to wax impreg- nation; second, when speed is of primary importance, as in the productions of quick sections from tumors for diagnostic pur- poses. In both cases recourse may be had to the method of frozen sections in which material is rapidly frozen until it is of a consistency which may be cut. Frozen sec- tions should not, however, be employed on any occasion when the normal processes of embedding may be used. Choice of a Microtome Any of the microtomes previously dis- cussed may be used for frozen sections with the aid of special attachments. The type of microtome shown in Fig. 84 is, however, specially made for the purpose and will be taken as the basis for the pres- ent discussion. It is essential in cutting •frozen sections that the knife should slice rather than push, and this type of micro- tome produces this movement without the expensive sUding mechanism of the micro- tome shown in Fig. 5G. The slicing effect is produced by mounting the knife to swing through the object when the handle on top is turned. This type of microtome is not as accurate, either as to. the thickness of sec- tion cut, or as to the repetition of this thickness, as is the big shder; but it is pre- sumed that no one would cut frozen sec- tions if thickness and reproducibility were primary objectives. The method of freez- ing the object will be discussed after we ToCO^ CYUNDER^ Fig. 84. Spencer clinical microtome fitted for freezing. liave dealt with the cjuestion of embedding the material in a supporting substance. Choice of a Supporting Medium Biopsy material delivered to a tech- nician from the operating theater is usu- 157 158 THE ART OF MAKING MICROSCOPE SLIDES Refrigerants ally cut without having been infiltrated with any material at all. The fact that this procedure gives sections which may be used for diagnostic purposes does not mean that it should be used for any other purpose. The sections so produced are of bad quality compared with sections cut in celloidin or paraffin; l^ut if the material is first embedded in one of the media given in Chapter 27 under the heading E 10, it is possible to produce sections nearly as good which it is desired to obtain the best possible section, and if time is of secondary importance, the method of Clark 1947 gives sections that are very nearly the equal of those which may be obtained by the paraffin method. Choice of a Refrigerant Blocks are nowadays usuall}' frozen with carbon dioxide from cylinders. The cylinder is connectel through a needle Fig. 85. Applying Anderson's medium to tissue about to be frozen. as those obtained by the paraffin or nitro- cellulose technic}ues. The choice between the formulas there gi\-en should be based on the length of time one is prepared to spend on the preparation. If only a few moments are available in excess of the ab- solute minimum time required to cut with- out embedding, better results will still be obtained if the o]:)ject is smothered in several layers of the solution of Anderson 1929. Much better results will be obtained, however, if the specimen, after it has been fixed in some material which will not alter its chemical nature, is soaked in this medium overnight in order to become im- pregnated. In dealing with materials of valve to the object holder of the micro- tome, so that a jet of supercooled carbon dioxide may be projected against the underside of the object. Other methods are available to those who lack carbon-dioxide cylinders. The standard method, prior to the introduc- tion of carbon dioxide, was to replace the tube leading from the carbon-dioxide cj-l- inder with a tube entering a bottle con- taining ether. Air was then blown through the bottle by a tube, which dipped under • the surface of the ether, so that the vapor was projected onto the underside of the block. The head absorbed by the evapora- tion of the ether was very great and blocks Cutting FROZEN SECTIONS 159 could be frozen by this means almost as rapidly as witl: carl)()n dioxide. The stench and fire hazard which accompany this procedure, however, limit it to those who do not have access to carbon-dioxide cylinders. Process of Cutting The prime necessity for jjroducinfj; a good section is, of course, a sliarp micro- assumed that a carbon-dioxide cylinder has been attached to the tube leading to the microtome, and that a brief trial has shown the gas to be flowing satisfactoi'ily. Pick up with the pi])et about half a cubic centimeter of tlio syrup, place this on the freezing table of the microtome, and turn on a small jet of carbon dioxide. Within a moment or two the gum will con- geal and the cai'bon dioxide mav be tui'ned Fig. 86. Removing section from Icnife. tome knife. The nature, care, and sharpen- ing of microtome knives has already been discussed in Chapter 12. It may be pointed out here, however, that no provision is made for altering the cutting angle of the knife in most freezing microtomes, there- fore, a knife, which is intended for use with a given microtome should be secured from the manufacturer. Let us assume that we have slightly more than the minimum time, and that a section is to be made with the syrup of Anderson. Before cutting, set out a bottle of this syrup, a pipet of the eye-dropper type, and a jar of 70% alcohol in which to receive the sections as they are cut. It is off. The object to be sectioned is then (Fig. 85) placed on top of this congealed layer of gum, and more gum is poured over the surface. Care must be taken that there is a layer of uncongealed gum l>eneath the object or it may loosen. Turn on the carbon dioxide and, as soon as the gum covering the object is congeahng, pour on a little more gum so that the object be- comes thoroughly covered. Next, turn off the carbon dioxide and insert the knife. Take an experimental cut across the top of the material and continue to shave it down with the thickness control set at 40 microns until the specimen is reached. Now reset the thickness control. No at- 160 THE ART OF MAKING MICROSCOPE SLIDES T.S. Fat tempt should be made to cut sections thinner than 20 microns by this method; sections of 30 microns are usually good enough for diagnostic purposes. The sections must be observed closely as they come from the block. If they crumble under the action of the knife, while the gum melts instantly on contact with it, it may be presumed that the block has not been frozen sufficiently hard, and the carbon dioxide should again be turned on for a few moments. It will only take a moment or two to establish the optimum condition under which only slightly curled sections appear on the blade of the knife. These prehminary cuts will, however, have soiled the blade which must now be washed with a drop of warm water to re- move the gum and then used to cut as manj^ sections as are required. As each section is cut it is removed from the blade of the knife to 70% alcohol. Most people working under pressure use the little finger (Fig. 86) for the removal of the sec- tion, though a number of very competent technicians prefer to use a brush. The gum quickly dissolves from the sections in 70% alcohol. The sections may then either be handled with a section lifter or attached to a slide with one of the adhesives given in Chapter 28 under the heading V 21.3. Staining and Mounting Sections Every pathological laboratory has its own well-established routine for staining frozen sections obtained from biopsy. Without wishing to suggest that this rou- tine be altered, the writer would like to draw attention to the existence of the techniques of Kingsley (Chapter 20, DS 13.13 Kingsley 1935) which, standard- ized as a routine, permit the comparison of diagnostic frozen sections, if necessary, with permanent paraffin sections stained in an identical manner. Typical Example Peeparation of a Section of Fatty Material Embedded in Gelatin by the Method of Clark 1947 The discussion of general principles given above was centered around the as- sumption that a freezing microtome is to be used for the production of diagnostic sections under pressure of time. The pres- ent description is of a method whereby a section of research quality may be ob- tained of material which cannot be sec- tioned by any other means. Let us suppose that it is necessary to section some fatty bodies, either from vertebrate or inverte- brate material, with a view to making permanent slides in order to record such changes as may have taken place under varying conditions. The fatty bodies are dissected out from wherever it is decided to secure them, and are placed in 4% formaldehyde until re- quired. They may be left indefinitely but they should be hardened for at least a week. Before embedding tlie pieces are washed in running water overniglit. The technique of Clark 1947 (11431, 59:337) requires a 25% dispersion of gelatin in water (made by soaking 25 grams of gelatin in water until it is thoroughly swollen, melted, and chluted to 100) and a 12.5% gelatin (which may either be prepared fresh or by the dilution of the 25% gelatin stock). The tissue, after having been washed for at least 24 hours in running water, is placed in 12.5% gelatin in an oven at 37.5°C. A block of tissue of about 3'^-inch cube requires 24 hours in this solution before being transferred to 25% gelatin maintained at the same temperature. It should spend from 24 to 36 hours in this thick gelatin bath and should then be em- bedded in 25% gelatin exactly as though it were a paraffin block. That is, a paper box should be prepared in the manner described in Chapter 12. Molten gelatin is poured onto the bottom of the box and, as soon as it has sohdified, the specimen is inserted and surrounded with further gelatin. The block is now placed in a re- frigerator to harden and left there until required. When it is taken out for cutting, the paper is stripped from the outside, and the block trimmed until the object lies in the required orientation. This trimmed T.S. Fat FROZEN SECTIONS 161 block is then left to harden in 2% formal- dehj'de for 24 hours. The hardened block is now taken and placed on the freezing stage of a micro- tome, preferably a slider of the type shown in Fig. 55. The block is carefully oriented on the stage and chilled with the aid of a stream of carbon dioxide until sections may be taken from it. If the block has been properly made, and is cooled to the correct extent, it behaves almost exactly as though it were a nitrocellulose block. Each section, as it is cut, is removed from the knife with a brush, and the sec- tions are accumulated in 50% alcohol. Each section is then taken individually in a pair of forceps, dipped for a moment in 5% gelatin, and then placed in the center of a clean slide. These sections are flexible and may be flattened to the slide without difficulty. As soon as the surplus fluid has been drained off, the shde is placed in a coplin jar at the bottom of which are a few millihters of 40% formaldehyde. Exposure to formaldehyde for about an hour will fix the section firmly; the process may be ac- celerated in an oven at a temperature of 37°C. The slides, after hardening in formaldehyde vapor, may be stored in 2 % formaldehyde. The next problem is that of staining and mounting. In the writer's opinion the best stain for the demonstration of fat in sec- tions of this type is that of Lillie 1945 (Chapter 21 DS 22.4). This method re- quires a saturated solution of oil blue in 60% isopropanol, a 0.1% solution of Bismark brown R in water and some 5% acetic acid. The slides to be stained are removed from 2% formaldehyde, rinsed briefly, and tiansferred to the blue solu- tion where they should remain from five to ten minutes, or until examination under the low power of the microscope shows that the individual fat granules have ab- sorbed the blue dye. They are then briefly rinsed in water and transferred to Bismark brown solution for one to two minutes. When they are removed from this solution they will appear to be a muddy brown, and should then be rinsed in 5% acetic acid until examination under the micro- scope shows the fat granules to be brilliant blue while the supi)orting connective tis- sues are stained brown. The slide should then be rinsed in water to remove the acetic acid and return to 2 % formaldehyde to await mounting. It is obvious that slides of this nature cannot be mounted in a resinous medium, for the necessary dehydrating and de- alcoholizing agents would remove the fat from the sections. It is therefore necessary that they be either mounted in glycerol jelly in the manner described in Chapter 5, or (in the author's opinion this is better) in one of the gum media described in Chapter 4. The author has naturally a preference for his own formula (Chapter 26, M 13.1 Gray and Wess 1950). The sec- tions can be mounted permanently in this media by removing them from 2% form- aldehyde, rinsing them briefly in water, draining them, placing a drop of the mounting medium on top of each section, and applying a coverslip. The slides Avill be hard enough to handle in less than an hour, and will be as nearly permanent as anv section of fatty material can be. 16 Injections General Principles Nature of the Process Injection is the art of filling cavities in materials intended for microscopical ex- amination, with some material which will render them more visible. In materials in- tended for gross dissection, the prepara- tion of which is not described in this book, onlj- the blood vessels are so treated and the idea has, therefore, developed that the process may only be applied to blood vessels. From the point of view^ of the microscopist any cavity may be filled whether it be blood, lymph, or bile vessels, or even the caniculi of bone (see Chapter 28, V 31.1 Altmann 1870). The material to be injected, if it is to be readily visible, must be either brightly colored or densely opaque. Though the term injection usually brings to mind the insertion of materials under pressure, there are some cases, as in the example just mentioned, in which a vacuum may be used to displace a fluid or air and then replaced with the material being used. Injections may be prepared either as sections or as whole- mounts, but different methods and mate- rials should be used for each. It will be necessary, therefore, to discuss first the selection of the injection medium, second the methods by which this medium may be inserted into the cavity which one desires to demonstrate, and lastly the method of preparing a permanent mount from the tissues so injected. Before proceeding with this discussion, however, it is necessary to point out that injection is not the only, or even the best, means of showing fine vessels in a micro- scopic preparation. In Chapters 21 and 23 'y\'ill be found methods of demonstrating both blood capillaries and bile capillaries by differential staining. These methods are usually less laborious than is the process of injection and should always be tried before an injection is undertaken. « Selection of an Injection Medium The recjuirements of an injection mass are that it shall run readily into the vessels being injected, that it be readily visible when it is in the \-essels, and that it remain in place through subsequent manipula- tions. The requirement that it should go into the vessels easily is met by providing a fluid medium of low surface tension which does not itself cause contraction of the muscular coats of the blood vessels. The condition of low surface tension is usually met by including in the medium a certain quantity of glycerol, though Hag- mann 1940 (2d540b, 15:115) has recom- mended the inclusion of a wetting agent in a medium intended for injection of insect trachea. There is no reason why similar wetting agents should not be included in media intended for blood vessels. The recjuirement that the injection should be visible when in position may be met in two ways. First one may incorpo- rate with the injection mass a pigment fineh' enough divided to be apparently in- visible but of a sufficiently large molecular size not to pass through the wall of the capillaries. The majority of injection masses are of this type and the detailed directions for preparation of them (Chap- ter 28, V 31.1) are largely devoted to the jirecipitation of a pigment of such fine grain size that it fulfills the required con- ditions. The second method of achieving 162 Media INJECTIONS 163 the same result is to inject some material which can, with ease, be sulisceiuently stained differential!}'. Thus Fischer 1902 (23681, 13:277) recommended the injec- tion of milk, the fat in which could sub- sequently be stained by any of the fat- staining "methods (Chapter 21, DS 22.4). The objection to this type of mount, how- ever, is that it cannot be dehydrated with- out the removal of fat, and must, there- fore, be mounted in one of the aqueous mounting media discussed in Chapters 4 and 5, few of which ha\'e an index of re- fraction high enough to show the speci- mens properly. There is also the objection that, if the specimens are to be sectioned, a freezing technique must be used rather than the more con\-entional parafhn or nitrocellulose methods. A far better ap- proach to this prol)lem is that of Altmann 1878 (23632, 10:191) who suggested the injection of dilute olive oil which could be stained, before embedding, with osmic acid. Various methods liave from time to time been proposed for the injections of solutions of stains of such low penetrating power that they will not pass beyond the walls of the capillaries. Robin 1871, p. 40 and Hoyer 1882 (2981, 2:19) suggested the injection of media containing silver nitrate which was subsequently "devel- oped" with one of the solutions intended for the development of silver by metal- staining techniques (see Chapter 23, AMS21.1). The problem of ensuring that the blood vessels remain open while the injection medium is inserted into them is more diffi- cult to solve. Glycerol in particular causes the blood vessels to contract, but this difficulty can be overcome in two waj^s. Either the animal may be killed with some substance which causes the greatest pos- sible vasodilation, such as alcohol or amyl nitrite, or blood can be washed from the vessels with a normal citrate-saline, and then this fluid followed by some material, such as foimaldehyde, which will fix and harden the blood \'essels in an open condi- tion. This was the universal practice of the older workeis Init is rarely done today. The final difficulty is that of persuading materials to stay in place after they have been injected. This is not difficult in the preparation of wholemounts, in which cut surfaces of blood vessels are unlikely to be exposed. It is very difficult, however, in the case of materials which have to be sec- tioned and from the cut blood vessels of which material is likely to bo washed, in the course of manipulation, if it is not specifically held in position. It is, there- fore, desirable that the injection pigment should be incorporated in some mass which may be hardened in position. This again points up one of the advantages of using milk, which may be readily coag- ulated. Egg white, which may also be easily coagulated in position, is unsatis- factory for the reason that it is difficult to incorporate pigments with it. Gelatin, which may be set in position by coohng, and subsequently hardened by formalde- hyde to a condition which cuts readily, is of almost universal employment. An interesting method for obviating all of these difficulties is one of the oldest in microtomy. This consists of first injecting a solution of some material and, second, injecting after it a material which will cause a precipitation of a relatively gross pigment in situ. This method is of great antiquity and was apparently discovered by Doyere sometime before 1839 (see Cooper 1847, 156). Doyere himself recom- mended several solutions, but the author has only seen successful preparations pre- pared by the injection of 2% potassium dichromate followed by 2% lead acetate. This method, which is described in detail in one of the typical preparations follow- ing this chapter, has recently been re- discovered by various authors and yields finer injections of capillaries than are ob- tainable by any other method. It is not, of course, satisfactory for thin sections from which the material would be readily washed, but a thick section of a human kidney in the writer's collection, prepared by this method sometime before 1847, shows a better demonstration of the glomeruli than is obtainable by any other method. Methods of Injection There are two metliods Ijy which injec- tion media may be inserted into fine blood vessels. In the first method an inert mate- 164 THE ART OF MAKING MICROSCOPE SLIDES Method Fig. 87. Injecting a rat through the carotid artery. rial, such as milk or a suspension of India ink, is injected into the blood stream, and the natural i)iimi)ing action of the heart is utiUzed to carrj- it to the finest capillaries. The second method is to force the injec- tion medium into the blood vessels of a dead animal in such a manner that it dis- places the fluid already there. One method of auto-injection is described in consider- al)lc detail in one of the examples which terminate this chapter. Forcing an injection material into the blood vessel of a dead animal is by no means as easy as it sounds. In the first place one has to secure some hollow tube which may be inserted into the blood Method INJECTIONS 165 vessel and held there firmly in spite of pressure being applied. An ordinary hyiio- dermic needle cannot be used because it cannot be held firmly in the blood vessel; it is too smooth, even when tied in place, to withstand the pressure of tlie injection. The older books are filled with descrip- tions of injection "pipes" which had a little ridge of metal turned on their ends, ai'ound which a thread might be tied to hold them in position. These pipes were, however, mostly very large and could rarely be used with anything smaller than tlie dorsal aorta of a rabbit. It is very easy to turn a hypodermic needle into a good injection pipe by putting a httle ridge of fine silk on it immediately liehind the orifice. The only silk which is suitable is that used for tying trout flies, and is sold under the designation six 0. The hypo- dermic should be held in a convenient holder, the end of the silk attached with a half-hitch just behind the orifice, wrapped around a half-dozen times, and then built uj) into a little ridge of four or five layers before being finished with a tohip finish. A drop of any good lacquer or varnish is then used to impregnate the silk, and the minute injection pipe is read}'. If the reader cannot understand this description it is recommended that he apply to the nearest tier of trout flies who will be able in ten minutes to prepare for him a dozen needles. The next difficulty which must be faced is to attach to the other end of the needle some apparatus through which pressure ma}' be maintained in order to drive in the injection medium. The ordinary h3'po- dermic syringe, for which these needles are made, is singularly ill-adapted to the purpose. Pressure will have to be applied for a considerable time, and various de- vices have from time to time been pro- posed for leaning a weight of some kind upon the upper end of the i)lunger. The writer, however, has always had more success with the device shown in Fig. 87 in which a bottle containing the injection medium is suspended, at a height which may be varied, above the object being in- jected. The difficulty here is to secure a satisfactory attachment between the bottle and the hypodermic needle. In the illustration, a piece of heavy-walled glass tube lias l)een di'awn out to a])proxi- mately the right size and then ground at the tip, in the manner in which the tip of a hypodermic syringe is ground, to fit the needle. This can easil}' be done l)v any competent glass blower, but the process is beyond the facilities of most laboratories. It is, however, so necessary and so desir- al)le, that the worker who intends to con- duct a number of injections is well advised to have some of these glass tubes made. It must be emphasized that a method of de- taching the needle from the supply must be available, since in almost all injection methods two or more fluids must be suc- cessively used. It is highly desirable to wash the excess blood from the animal by running a saline solution through the lilood vessels before the insertion of the injection medium, and this obviously can- not be done unless the attachment to the needle may be readily changed. Equip- ment of the type shown is used, however, only when one desires to inject most of the capillaries of an entire animal and it can- not be used satisfactorily with a gelatin medium which has to be kept molten throughout the course of the injection. If such a warm injection is to be made, one must provide a heating jacket to surround the bottle containing the medium, and also immerse the entire animal in a tray of hot water until the injection is completed. Gelatin media, in fact, are far better adapted for the injection of small parts of animals (as described in one of the specific examples following this chapter) in which a hypodermic syringe may be used under the surface of warm water. There are three ver}^ common causes of the failure which attends the first attempts of almost every individual to make an in- jection. The first of these is the failure to provide some means for the blood to get out when the injection medium is inserted. It is ridiculous to insert a needle into an arter}' and to expect an injection medium to be pushed through, unless some pro- vision is made for the removal of tlie ])lood through the corresponding vein. One cannot, moreover, merely sever an artery and insert the needle into its end, or the bleeding from the other cut end will 166 THE ART OF MAKING MICROSCOPE SLIDES Mounting provide too ready a means of egress for the material inserted. One should, therefore, always slit the artery, slide the injection needle into the slit, and then ligate it in two places, the proximal ligature being for the purpose of holding the needle in place, and the distal ligature being for the pur- pose of sealing the other end of the slit. The second great cause for failure lies in the stopping up of the blood vessels before an injection has reached them. This stop- page may be produced either by the natural contractions of the vessels them- selves, or by the presence in the injection medium of some particle too coarse to pass through the finer capillaries. The con- traction of the vessels themselves may be prevented by killing the animal with a vasodilating material, or by waiting before injection until rigor mortis has passed off. If the latter course is adopted one must, of course, wash the contained blood out of the blood vessels with a saline solution in order to prevent coagulation. The last, and most frequent, cause of failure is that the blood vessels will burst before the medium has penetrated them. This is obviously due to the appUcation of too great pressure, and is commonest among workers wiio use a hypodermic syringe and endeavor to push in the injec- tion as if they were making a hypodermic injection of a drug. Tlie rat shown in Fig. 87 was perfused, using the pressure shown, for a period of six hours with a glycerol- carmine mixture before the injection could be considered complete. It is occasionally possible, with very small organs, to com- plete an injection in a few moments, but it is usually far better to use a low pres- sure and take a long time, not only to avoid bursting of the vessels but also to l^revent a gross swelhng and distortion of them. Mounting Injections Injections may either be mounted as a wholemount in the manner described in Chapter 6, or prepared as sections by the methods described in Chapters 12, 13, and 14. In either case the material, after injec- tion, should be fixed in a medium which will coagulate the injection material, if sections are to be cut, or which will remove the unwanted portions of the injection medium, such as glycerol, if wholemounts are to be made. The processes of dehy- drating, clearing, and mounting, or em- bedding need not be described further since they have already been dealt with in the chapters indicated; but attention should be drawn to the fact that sections, if the}^ are to be cut, should be relatively thick. The only purpose of an injection is to show the course of blood vessels and this cannot be done in an ordinary histo- logical section of 10 or 12 microns in thickness. It is much better to cut a sec- tion of from }/[o- to J-^-millimeter in thick- ness, and then to treat that as though it were a wholemount, being particularly careful that it is perfectly dehydrated and perfectly cleared so that the uninjected portions may be as transparent as glass. In sections of this type one may easily follow the course of even the finest blood vessels with the aid of a binocular dissect- ing microscope. The general principles discussed in this chapter are illustrated by three quite different injections which will now be described. Specific Examples Injection of the Blood Vascular System of a 60-hour Chick Embryo with India Ink The trick of making these preparations, which are so widely used for teaching pur- poses, appears to be known to very few people and Is, therefore, worthwhile de- scribing here. The age of 60 hours for the chick has been specified in this example because this is the easiest size on which to learn the technique; but the method to be described can equally well, after some practice, be applied to any chicken embryo between the time when the heart is first formed and the end of the 96th hour, when the specimen becomes too big to inject conveniently. A description is given else- where (Chapter 20) of the removal of an embryo from the yolk and need not be repeated. To prepare the medium, take any com- Chicken embryo INJECTIONS 167 mercial India ink and dilute it with about ten times its volume of ilistilled water, to each 100 milliliters of which has been added one droj) of ammonia. The use of tap water, pai'ticulai'ly if slightly acid, tends to agglomerate the carl)on jiarticles and thus to make injection impossible. Next make a series of hollow glass needles of a size suitable for the injection. For these take short lengths of ordinary, soft- glass, 3-millimeter tubing, heat them in a flame until they are soft, remove them from the flame, and draw them out to about 1 millimeter in diameter; then re- turn them to the flame, pulling sharply, when they are soft so as to secure a fine tip. This tip should be almost exactly the size of the Cuverian sinus of the embrj'o which is to be injected. It is simplest to prepare two or three dozens of these little injection needles, approximatelj' the right size and then to select the best. One needle may be used for the entire batch but the exact size is vital to the success of the operation. In case the worker is unac- quainted with the Cuverian sinus, he is recommended to look at the body of the embryo just along the side of the heart, where he will see that a large anterior and a large posterior vein come together to form the cross piece of a T, the stem of which passes downward towards the heart. This stem is the Cuverian sinus. Now secure two stender dishes, one filled with the diluted India ink, and the other with phj'siological saline. Syracuse watch glasses, one for each embryo to be injected, should be placed in a dish of water at about 40°C., so that they may be warmed through, and a small beaker of any fixative, with an eye-dropper type pipet in it, should be at hand. Two pairs of fine forceps will be required, as well as the large scissors and blunt-nosed forceps used to remove the embryo. The injection is best made under a wide-angled dissecting binocular which is set up to transmit light through the embryo. Now remove an embryo from the yolk and, after washing it in the manner de- scribed in Chapter 20, transfer it to one of the warmed watch glasses containing physiological saline. Drain out most of the saline so as to leave the embryo and its surrounding extra-embryonic areas stranded on the bottom, and transfer the dish to the stage of the binocular micro- scope. A needle of the required size is now selected and attached to a short length of rubber tubing, terminating in a mouth- piece of the type sold for use with blood pipets. Dip this needle under the surface of the injection medium and suck until a consideraljle portion of the glass tube is filled. Remove the needle from the injec- tion medium and transfer it to the dish of normal saline. Now give a very slight suck on the tube in such a manner as to fill the fine capillary portion of the tube with saline without di'awing in enough to dilute the India ink in the body of the tube. This is the most vital stage in the proceedings, for if the fine capillary is left filled with India ink, this ink will flow out as soon as the needle touches the heart and the operator's view w'ill be obscured. After each injection is done, return the injection tube to the dish of normal saline and again refill the capillar}' tip with saline. The only dissection of the embryo that is necessary is to split a hole through the pericardium. This may be readily done by tearing with two pairs of fine forceps, one held in each hand. Utmost care should be taken not to break any blood vessels in doing this, but a few minutes' practice on some embryos will be far more instructive than any amount of description. Now pick up the injection needle and take the mouthpiece at the end of the rubber tube between the teeth. Provided the mouthpiece is kept in the teeth and the mouth kept open, no pi'es- sure can possibly be applied before it is re- quired. Assuming the operator to be right- handed, so that the tube is in his right hand, the embryo is now turned so that its anterior-posterior axis is at about 45° to the operator with the head lying, as it were, to the northwest. The tip of the in- jection needle, which it is to be remem- bered is filled with saline and not with India ink, is now applied to the heart just at the bend. A sharj), shoi't stal) is used to drive the needle through until the end of the needle lies free in the cavity of the ^•entricle; a very slight and careful pressure 168 THE ART OF MAKING MICROSCOPE SLIDES Rabbit kidney is now aiDplied with the mouth, while one watches the stream of sahne coming out of the end to make sure that it is going into the ventricle. If the needle does not lie in the ventricle it may be withdrawn and a further stab made. A skilled operator can stab so as to bring the aperture of the needle into the ventricle nine out of ten times; this is the only skill which is re- quired in the whole operation. As soon as the tip of the needle is seen to lie in the right place, blow very gently until a small quantity of India ink enters the hearts Even though the heart has stopped beat- ing it will now usually start again under the action of the foreign substance in- serted. Continue to blow gently so as to keep the ventricle at all times filled with India ink. It is practically impossible to inject into the blood vessels of the chick bj- pressure; one is forced to rely on the movements of the heart to drive the mate- rial round. If the ventricle is filled with India ink and emptied by contraction a half-dozen times, the injection will be found to be perfectly satisfactory, even to the tip of the finest arteries. The injection of the veins is slightly more difficult, since it must be undertaken by pressure and in this case the tip of the needle is inserted exactly into the junction of the anterior and posterior cardinal sinuses with the Cuverian sinus. In this case, as soon as the needle is in position, blow with a gentle continuous pressure, watching the other parts of the embryo, and cease instantl_y when the India ink has distributed itself to the ends of the veins. A really successful preparation will fill most of the veins as well as the arteries b}' injection through the ventricle. Now remove the needle and drop a small quantity of the selected fixative onto the embryo. If this is done rapidly there will be no leakage of India ink from the tip of the ventricle, and the injection may then be turned into a wholemount by any of the methods described in Chapter 6. Precipitation of Lead Chromate in the Glomeruli of a Rabbit Kidney This is a preparation of great difficulty, not lightly to be undertaken by the in- experienced, but of which the beauty more than justifies the effort. The method requires three solutions, each in the quantity of several hundred milliliters. The first solution is a phj-sio- logical saline which has been saturated, by shaking, with amyl nitrite. Amyl nitrite is a vasodilator of great strength and should be handled carefully. The second solution is a 2% solution of potassium or sodium dichromate, and the third is a 2% solution of lead acetate. All three of these solutions should be passed through a filter immedi- ately before use. There are also required three aspirator bottles, with tubes and glass tips set up in the manner shown in Fig. 87. Lastly a hypodermic needle will be required, with a silk ridge raised on it in the manner described above, and with a diameter approximately two-thirds that of the renal artery of a rabbit. It is also necessary to have a dish of such a depth that the kidney may be covered with physiological saline. Now kill the rabbit, preferably with the aid of ether to which has been added a considerable quantity of amyl nitrite. Ether produces unconsciousness quickly but death very slowly, and great care should be taken that the rabbit is com- pletely dead before proceeding further. Stretch the rabbit on a board, open the abdomen, push the intestines to one side to expose the kidney, and then carefully trace the course both of the renal artery and of the renal vein. These two are then ligated in two places with surgical silk and severed between the ligatures. Now dissect out the rest of the kidney until it is free from the body. It is far more impor- tant that the surface of the kidney should not be damaged than that it should be free of adherent tissues. Remove the kidney from the rabbit and transfer it to a dish of physiological sahne, which is then placed under a binocular chssecting microscope. Insert the hypodermic needle into the renal artery and tie it firmly in place. Take care that the hypodermic needle is entirely filled with saline and that no air entei's. This is why it is desir- able to. work under the sui'face of saline. An aspirator bottle filled with amyl- nitrite-saturated saUne is now arranged as Rabbit kidney INJECTIONS 169 shown ill Fig. 87, and the ground end of the ghiss conuet'tion inserted into the hypodermic needle. Tliis should be done under the surface of saline in order to avoid an)' possibility of air bubbles. Cut the renal vein above the first ligature and remove the clip froni the rubl)or tul)e leading to the aspirator bottle. A stream of blood will immediately leave the renal vein as it is displaced by the saline being injected into the artery, and the perfusion should be continued until this stream of blood stops. Now replace the clij) on the rubber tube and withdraw the glass con- nection from the hypodermic needle. Change the bloody saline in the dish for clean saline, being careful that at no time does the open end of the hypodermic needle have any opportunity to acquire an air bubble. Now take the second aspirator bottle which contains the 2% potassium dichro- mate, remove the clip until a clear stream of chromate is flowing through the glass connection, and then attach the glass con- nection to the hypodermic needle. Open the clip again and allow the potassium dichromate to flow through until it is seen to have rej^laced the saline in its entirety. Replace the chp again, remove the dichro- niate-filled aspirator bottle, and replace the dichromate-contaminated saline in the dish with fresh saline. It is better to re- place it two or three times to be quite certain that no dichromate remains in the dish. Before the last change of saline, briefly connect the aspirator bottle con- taining the saline and the hypodermic needle, and permit the saline to flow for about a couple of seconds to make sure that the hypodermic needle and its con- nections are again rendered free of dichro- mate. If this precaution is not taken, the needle is almost certain to become choked with lead chromate when the lead acetate is injected, and the solution in the dish will become so cloudy that one can no longer see what is going on. As soon as there is no potassium dichro- mate anywhere except in the kidney, take the third bottle containing the 2% lead acetate and connect it to the hypodermic needle. Remove the chp and permit the lead acetate to flow until the entire kidney has assumed a dense, opaque yellow ap- pearance. It is very improbable that any of the precipitated lead acetate will be forced through the fine capillaries of the glomeruli and out through the renal vein. The time necessary to precipitate the material within the glomeruli of the kid- ney may vary from five minutes to two or three hours but can readily be judged by eye. Even though only certain areas of the kidney go densely opaque yellow, the preparation should not be rejected since many sections may be obtained from even a small, properly injected, area. The kid- ney is now removed from the saline and thrown into 10% formaldehyde until it is next required. The kidney will be sufficiently hardened to permit sectioning in about a week. At the end of this time, therefore, the kidney is transferred to a sahne solution and cut into sUces about two millimeters thick. Do not be alarmed that there will be hberated at this time considerable quan- tities of lead dichromate. The lead dichro- mate \v\\\ not come out of the fine capil- laries of the glomeruU, and each slice should be washed until it ceases to give rise to the clouds of yellow pigment which are, by this process, removed from almost all vessels except the finest ones. These two-millimeter slices should now be cut into sections about 100 microns thick (J^o milhmeter). This may be done either bj^ embedding them in a very soft wax in the manner described in Chapter 12, or, far better, by embedding them in nitrocellu- lose in the manner described in Chapter 13. Whichever method is adopted, the sec- tions should be mounted in balsam, and the utmost care should be taken to de- hydrate and clear them thoroughly, so that the uninjected tissues appear glass- clear. After they are mounted these sec- tions may be studied by transmitted light, in which case the glomeruli will be seen only as an opaque shadow; or they may be studied by reflected light, either after the slide has been placed on a black back- ground, or after a piece of black paper has been attached to the undersurface. The examination of these specimens by re- flected light will show the relationships of the glomeruli, and their attendant arteri- oles, better than any other method known to the author. 170 THE ART OF MAKING MICROSCOPE SLIDES Intestine Injection of the Intestinal Capillaries of a Rabbit with Carmine-gelatin The capillaries of the intestine may, of course, be injected with lead chromate in the manner described in the last example. This illustration is given, however, for the benefit of those who prefer the more con- ventional method of filling the fine capil- laries with red gelatin for study. The same rabbit may be used as was used in the last example provided that there are available two more aspirator bottles, one containing normal saline and the other 4% formaldehyde. While the kidney is being perfused with saline, in the manner described in the last example, remove a length of about two inches from the intestine, including a portion in which a branch of the mesenteric artery is seen to enter the intestine. Now place this in the dish with the kidney and attach it to its own aspirator system of normal saline, after having inserted a hypodermic needle into the artery exactly as described in the case of the kidney. Perfuse this specimen until all the blood has been removed. Now transfer it to another dish and perfuse, after the normal saline, 4% formaldeh3'de. It will be noticed that in this preparation, no precautions have been taken to open a reheving vein; the purpose of the per- fusion \\T.th 4 % formaldehyde is to expand the fine capillaries and permit them to set in this expanded condition. After the per- fusion has gone on for about a couple of hours, remove the piece of intestine and transfer it to a jar of 4% formaldehyde, where it may remain until next required. The writer has never found it practical to set up an elaborate arrangement of hot- water baths with a view to injecting fresh material with a gelatin mixture and, there- fore, has alwaj's recommended that the material be fixed and hardened with the capillaries widely expanded. By this means it is possible to secure a specimen which may be injected relatively rapidly with a carmine-gelatin material held in a hypo- dermic sjainge. Of the many carmine-gelatin injection media, the formulas for which are given in Chapter 28 under the heading V 32.1, the writer prefers that of Moore 1929. About 100 milliliters of the selected mass will be required. Before the injection can be made, the formaldehyde must be removed from the specimen. This may be done by washing the specimen for two or three days in running water, though it is usually safer in addition to perfuse it through the needle — which has, of course, remained attached to the artery throughout this period — -using an aspirator system set on a shelf above the sink in which the specimen is being washed. When all of the formalde- hyde has been removed, secure a vessel large enough to hold both hands, a one- or two-milliliter hypodermic syringe, and the specimen. Fill the dish with water heated to about 35°C. before placing in it the kidney and the hypodermic syringe. The temperature is not of importance, pro- vided that it is above the melting point of the gelatin mixture. Melt the gelatin on a water bath and, when the kidney and needle have both been warmed through, remove the hypodermic syringe from the water bath, fill it in the ordinary manner with the injection mass, and then lower it into the water bath and attach it to the hypodermic needle. Now apply a slow and gentle pressure to the end of the plunger until a sufficient quantity of gelatin has been injected into the capillaries. The terminal point of the operation may be judged when reasonably large areas have become bright pink to red in color. Do not expect the injection to go into the whole length of the intestine ; be satisfied if }i of an inch is well injected. Now remove the hypodermic syringe and then drop the piece of intestine into ice water in order to chill and set the gelatin. As soon as the kidney is chilled it is transferred back to 10% formalde- hj'de, where it may remain until wanted for the production of sections of about 100 microns in thickness. In the present case excellent sections may be prepared by embedding in any low-melting-point wax, and cutting single sections on any kind of microtome. These sections are then attached to the slides by using very considerable quantities of adhesive, be- fore being dewaxed and transferred to balsam in the manner described in Chapter 12. Part II Methods and Formulas Used in Making Microscope Slides Foreword to Part II Part I of this book described the methods of making microscope shdes and contained specific information as to tech- nicjues and formukis onh' in the typical preparations which were appended to each tains specific information both as to tech- niciues and formnkis, but does not give general instructions as to the preparation of the sUdes except in a few typical prepa- rations inserted into Chapters 20, 21, and chapter. This second part of the book con- 23, which deal with staining. Classification The 3500 specific formulas and methods which follow have been arranged accord- ing to a system of classification designed to bring similar methods and formulas to- gether, and to make it easy for the worker to find that which he requires. The primary division of this mass of material has' been into chapters; within each chapter there is a decimal division of the contents. Each chapter is designated by one, two, or three letters which indi- cate the nature of the formulas and tech- niques contained in it. Thus, DS stands for dye stains, ADS for accessory dye stains, and so on. The complete decimal classification emploj'ed in each chapter is given at the beginning of each chapter, and, for purposes of cross reference, each formula or method is assigned the letter of its chapter and its own decimal designa- tion. Thus a reference to DS 11.41 indi- cates division 11.41 of Chapter 20. The addition of the author's name and date to the decimal classification renders clear the identity of the method referred to, and permits it to be given in the least possible space. By this method it has been possible to ensure that no formula for a solution is given more than once, and that each solu- tion is placed with other solutions of similar composition or properties. The method by which journal references have been added to these designations is ex- plained in the Introduction. Measurements and Units Employed With the exception of the fixatives (Chapter 18) all formulas for solutions have been adjusted to give approximately 100 parts. The fixatives have been ar- ranged to give either 250 parts when pre- pared from pure reagents, or 100 parts when prepared from the stock solutions described in the second part of Chapter 18. This exception, in the case of the fixa- tives, has been made since the writer has convinced himself, by inquiring among numerous workers, that 250 milliliters is the quantity of fixative most frequenth' prepared. It is to be presumed, in the preparation of all the solutions, that fluids ai'e meas- ured by volume and solids by weight. The words milliliter and gram have, therefore, l:)een omitted since their inclusion would have added 30 or 40 pages to the total length of the book. In a few cases where the context does not make it entirely clear that fluids are to be measured in volumes and solids by weight, the ap- propriate units have been given. Saturated solutions are assumed to mean saturated solutions at room temperature, unless otherwise specified. It is also to be understood that the solvent of a saturated solution is water unless a specific state- ment to the contrary is made. In a few cases which might cause confusion, as in the case of dyes which are equally fre- cjuently dissolved in alcohol or water, the 173 174 METHODS AND FORMULAS USED IN MAKING MICROSCOPE SLIDES designation sat. aq. sol. has been used to indicate that water is the solvent. The words "in water" have also been omitted following the words "wash," "rinse," etc. unless the context does not render the meaning of the instruction clear. It is also to be presumed, unless a specific designation to the contrary is made, that distilled water will be used in all operations. Certain pharmaceutical abbreviations, used to shorten the technical directions for making up certain solutions, will be found expanded in the List of Abbreviations immediately preceding the "List of Books and Journals cited." 17 Preservatives Decimal Divisions Used in Chapter P 00 GENERALITIES 01 General observations 02 Method of arrangement of formulas P 10 FORMULAS 11 Solutions of inorganic reagents 11.1 Formulas 12 Organic- reagents 12.1 Alcohols, aldehydes, ketones 12.2 Phenols, and mixtures containing phenol and phenol derivatives 12.3 Other organic reagents, including mixtures 12.1, 12.2 13 Other preservatives, including mixtures of 11 and 12 13.1 Formulas P 00 Generalities P 01 General Observations The distinction between preservatives, fixatives, and mountants is difficult to draw and has not always been observed in biological literature. As used in the present work, a preservative is distin- guished from a mountant only in that the latter will hold the coverslip in place of its own accord, while the former requires that the coversHp be sealed in position by one of the methods described in Chapters 2 and 3. It does not, for example, appear justifiable to the writer to refer to the well-known lactophenol of Amman (P 12.2 Amman 1898) as a mountant, since it can- not be used for the preparation of jierma- nent slides unless the edge of the coverslip is sealed. The distinction between preserva- tives and fixatives is more difficult to draw, but the author has endeavored to group in the present place all those fluids which are most usually employed for the actual preparation of a wholemount, or for the storage of material, as distinct from those fluids which are employed for the fixation of material before mounting or sectioning. When the writer has been in doubt he has given a cross reference both in Chapter 18 and in the present chapter. Probably the most widely used pre- servative today is a simple dilution of formaldehyde, opinion varying as to the exact concentration which may be em- ployed. Certainly the 4% formaldehyde solutions (the so-called 10% formalin so commonly referred to in the literature) is far too strong for the preparation of a microscope slide, and a dilution to at least H 0 of this concentration is perfectly ade- quate to prevent the growth of micro- organisms in a sealed preparation. Alcohol by itself is practically worthless because of the difficulty of sealing it under a cover- shp. Various mixtures of alcohol, glycerol, and formaldehyde have, from time to time been employed, however, and are given below. P 02 Method of Classification of Formulas The fornudas given below have been grouped according to their usage as much 175 176 METHODS AND FORMULAS P 11.1-P 12.1 as their composition. In the first group (P 11.1) there are the few surviving simple solutions of inorganic reagents. These were once the most widely used class of preservatives, but they are now obsolete and scarcely ever employed, al- though they are the easiest of all fluids to seal. P 12.1 contains the organic reagents as distinct from the inorganic and is divided according to whether or not it contains phenol (P 12.1 are the alcohols, aldehydes, glycerine, etc., without phenol; 12.2 contains the phenol mixtures). A third group, P 12.3 below, contains miscellaneous organic reagents including complex mixtures of materials which, had they been used singly, would have been placed in groups P 12.1 or P 12.2. The classification P 13.1 contains all the other water-miscible preservatives which could not justifiably have been placed in either of the previous two classes. P 10 Formulas 11 SOLUTIONS OF INORGANIC REAGENTS 11.1 Formulas 11.1 Assier test. 1882 Chevalier Chevalier 1882, 290 formula: water 100, sodium chloride 10, acetic acid 1 11.1 Boitard 1921a Boitard 1921, 49 formula: water 100, ammonium alum 10, sodium chloride 11.5, mercuric chloride 1.2 11.1 Boitard 1921b Boitard 1921, 379 formula: water 86, sodium borate 8, boric acid 2, potassium nitrate 3, sodium chloride 1 11.1 Goadby trst. 1855a Queckett Queckett 1855, 300 formula: water 100, sodium chloride 6, potassium alum 3, mercuric chloride 0.15 11.1 Goadby test. 1855b Queckett Queckett 1855, 301 formula: water 100, mercuric chloride 0.08, ammonium alum 2.4, sodium chloride 4.8 note: Several other solutions were published by Goadby (Queckett, loc. cit.). These, however, appear to be the only ones widely used (cf. Robin 1871, 377 and Frey 1877, 136) for microscopial, as distinct from anatomical, preservation. 11.1 Kronecker test. 1907 Bohm and Oppel Bohm and Oppel 1907, 9 formula: water 100, sodium chloride 0.6, sodium carbonate 0.006 11.1 Ralf test. 1872 Martin Martin 1872, 189 formula: water 100, potassium alum 0.2, sodium chloride 0.2 11.1 Woods 1929 19938,70:637 formula: water 100, copper acetate 0.5, acetic acid 4 method: Fix 4 hrs. in above. Pour off solution and add ammonia till color changes to purple. Return objects and leave 1 hr. Transfer to 5% glycerol, concentrate by evaporation, and mount in M 12.1 Kaiser 1880 RECOMMENDED FOR: algae. 12 ORGANIC REAGENTS 12.1 Alcohols, Aldehydes, Ketones 23635, 30 :442 12.1 Caberla 1878 formula: water 30, glycerol 10, 95% ale. 20 12.1 Francotte test. 1942 Langeron formula: water 50, 95% ale. 20, glycerol 30 12.1 Gatenby and Painter 1937 formula: water 50, glycerol 25, 95% ale. 25 Langeron 1942, 1011 Gatenby and Painter 1937, 222 P 12.1-P 12.2 PRESERVATIVES 177 12.1 Hantzch (est. 1882 Chevalier cif. Rade Chevalier 1882, 325 kormula: water 60, ^0% ill''- 30, j^lycerol 10 12.1 Jager test. 1937 Gatenby and Painter Gatenhy and Painter 1937, 222 formii.a: s(>a water SO, ^lyrorul 8, U59(; Jili^- 8 12.1 Moleschott (csl. 1871 Robin Robin 1871, 289 formula: water 50, 95% ale. 25, acetic acid 25 12.1 Monnig 1930 18G40, 16:199 formula: water 90, 40% formaldehyde 10, chloroform ^..s. to sat. RECOMMENDED FOR: preservation of gorged ticks. 12.1 Newmarch 1938 Microscope, 2 :r.i2 formula: water 70, glycerol 25, 40% formaldehyde 5 12.1 Oudemann test. 1939 Scott cit. Morgenthaler Microscope, 3:163 formula: 95% ale. 60, water 27, glycerol 5, acetic acid 8 12.1 Pampel 1914 23635, 108:290 formula: water 60, 95% ale. 30, 40% formaldehyde 12, acetic acid 8 RECOMMENDED FOR: preservation of arthropoda for external studies. 12.1 Puppe 1899 test. 1922 Silvester 4349, 8:54 formula: water 42, glycerol 42, alcohol 16 12.1 Railliet test. 1942 Langeron Langeron 1942, 871 formula: water 93, 40% formaldehyde 5, acetic acid 2, sodium chloride 0.9 12.1 Robin 1871 Robin 1871, 289 formula: water 20, 95% ale. 40, glycerol 40, acetic acid 4.5, nitric acid 2 12.1 Woods 1897 3430, 24:206 REAGENTS REQUIRED: A. 95% alc. ; B. water 95, glycerol 5, copper acetate 0.5, 40% formaldehyde 1; C. water 95, glycerol 5, 40% formaldehyde 1 method: [fresh plants] — » A, few moments —^ water, few moments — > [repeat cycle till adherent air removed] -^ B, till blue-green — * C, till no more color comes away -^ M 12.1 Wood 1897 12.2. Phenols and Mixtures Containing Phenol and Phenol Derivatives , 12.2 Alcorn and Yeager 1937 20540b, 12 :157 formula: water 20, glycerol 40, lactic acid 20, phenol 10, acetic acid 0.3, orseillin BB 0.025 RECOMMENDED FOR: preserving and staining fungus-infected plant tissues. 12.2 Amann 1896 23632, 13:18 formula: lactic acid 20, phenol 20, glycerol 40, water 20 note: This mixture is widely used in continental Europe but was ignored in England and the United States until the formula was reprinted by Linder 1929 (19938, 70 :430). Although Linder correctly acknowledged the source of the formula, many subsequent writers refer to "Linder's Medium" (cf. Carpenter and Nebel 1931; 19938, 70:154). In 1933 (19938, 77:23) Moore suggested the addition of 0.5 phenosafranin to "Lin- der's Medium." This may account for the curious statement in Gatenby and Painter 1937, p. 692, that "Linder's Medium is [here is given Amman's mixture] . . . w^ith a small amount of carmine." Some additional confusion has been occasioned by the fact that Linder (loc. cit.) cites an undated text by Satory as his source for Amman's formula; Moore (loc. cit.) then cites the composition as " Satory 's formula as given by Linder." The stains of Maneval 1936 (DS 12.15) are designed for use before this medium. 12.2 Amann 1899 23632, 16 :38 formula: chloral hydrate 50, lactic acid 25, phenol 25 178 METHODS AND FORMULAS P 12.2-P 12.3 12.2 Amann 1899 see also S 41.1 Amann 1899a and b 12.2 Archibald and Marshall 1931 16035, 23:272 formula: water 25, lactic acid 25, glycerol 25, phenol 25 RECOMMENDED FOR: fixation and preservation of cercaria. 12.2 Bastian test. 1877 Frey Frey 1877, 135 formula: glycerol 90, phenol 6 12.2 Langeron 1942 Langeron 1942, 660 formula: chloral hydrate 40, phenol 40, lactic acid 20, sodium salicylate 10 12.2 Lepik 1928 16233, 18:869 formula: 95% ale. 40, phenol 20, lactic acid 40, glycerol 20 12.2 Mukerjil931 9940,19:281 formula: water 20, glycerol 20, lactic acid 40, chloral hydrate 10, 40% formaldehyde 10, acetic acid 4 12.2 Linder 1929 see P 12.2 Amann 1896 (note) 12.2 Moore 1933 see P 12.2 Amann 1896 (note) 12.2 Priestly 1917a 13034,2:471 formula: phenol 20, glycerol 40, lactic acid, 20, water 20 12.2 Priestly 1917b 13034, 2 :471 formula: chloral hydrate 50, lactic acid, 25 phenol 25 12.2 Semmens 1937 Microscope, 1 :5 formula: water 50, lactic acid 40, glycerol 39, phenol 25 12.3 Other Organic Reagents, Including Mixtures of 12.1, and 12.2 12.3 Amann 1899 23632, 16:38 formula: chloral hydrate 50, lactic acid 50 12.3 Andre see V 51.1 Andre 12.3 Brazille 1908 5401, 33:114 formula: water 50, 95% ale. 30, phenol 0.6, acetic aicid 10 12.3 Bruin test. 1930 Guyer Guyer 1930, 71 formula: water 20, glucose 70, 95% ale. 5, camphor 0.5, glycerol 5 NOTE : the camphor is dissolved in the ale. and the solution added to the other ingredients. 12.3 Brumpt test. 1942 Langeron Langeron 1942, 819 formula: water 97, 40% formaldehyde 7.5, picric acid 0.1, acetic acid 0.5 RECOMME.VDED FOR: mounting and preserving parasitic protozoans. 12.3 Brun 1889 test. 1937 Gatenby and Cowdry Gatenby and Cowdry 1937, 221 formula: water 70, glucose 20, glycerol 10, 95% ale. 5, camphor 0.5 12.3 Cole 1903 Cross and Cole 1903, 267 formula: water 30, glycerol 30, 95% ale. 30, acetic acid 0.3 12.3 Eckert 1931 20540b (abstr. 1932)7:68 formula: water 95, P 12.2 Amann 1896 5, copper chloride 0.2, copper acetate 0.2 RECOMMENDED FOR: Either alone, or diluted 1:5 with P 12.3 Pfeiffer (1921) as a pre- servative for green algae. 12.3 Fabre-Domergue 1889 14901 (1889) formula: water 60, glucose 40, methanol 20, glycerol 10, camphor q.s. to sat. 12.3 Gater 1928 4184, 19:367 formula: water 40, acetic acid 6, chloral hydrate 60 P 12.3-P 13.1 PRESERVATIVES 179 12.3 Gerlach 1885 11360,5:511 formula: sat. aq. sol. {circ. 1.5%) arsenic trioxide 100, gelatin 20, glycerol 60 12.3 Kaiserling 1897 22575, 147 :389 FORMULA i: water 100, 40% formaldehyde 20, potassium nitrate 1.5, potassium acetate 3 FORMULA II : water 100, potassium acetate 10, glycerol 20 note: The first solution is intended for the fixation of slabs of pathological material, the color of which is redeveloped in 80% ale, before final preservation in the second solu- tion. Both solutions are, however, excellent general-purpose preservatives. 12.3 Ordonez 1865 test. 1882 Chevalier Chevalier 1882, 323 formula: water 30, glycerol 75, tannin, 0.5 12.3 Pfeiffer test. 1931 Eckert 20540b (abstr. 1932) 7 :G8 formula: acetic acid 30, methanol 30, 40% formaldehyde 30 RECOMMENDED FOR: preservation of green algae. 13 OTHER PRESERVATIVES INCLUDING MIXTURES OF 11 AND 12 13.1 Beale 1880 Beale 1880, 66 formula: methanol 6, acetone 3, creosote 0.5, chalk q.s. to sat., water 95, camphor 1 13.1 Blaydes 1937 19938, 85:126 formula i: water 45, 95% ale. 45, 40% formaldehyde 5, acetic acid 5, copper sulfate 0.2 formula II : water 25, 95% ale. 60, 40% formaldehyde 10, acetic acid 5, copper sulfate 0.2 recommended for: preservation of color in green plants. 13.1 Chevalier 1882 Chevalier 1882, 319 formula: water 60, glycerol 30, calcium chloride 6 13.1 Cole 1903 Cross and Cole 1903, 181 formula: water 50, camphor to sat. glycerol 50, acetic acid 0.2, copper acetate 0.2, mercuric chloride 0.01 13.1 Frey 1877 Frey 1877, 136 formula: water 90, glycerol 10, sodium chloride 2, mercuric chloride 1 13.1 Craig 1916 4349, 6:56 formula: water 80, glycerol 20, 40% formaldehyde 8, potassium acetate 8 13.1 Frost 1913 4349, 4:41 formula: sat. sol. thymol 100, cane sugar 44, potassium acetate 2, chloral hydrate 1, sodium fluoride 1 13.1 Gardiner 1898 test. 1931 Crafts 20540b, 6:127 formula: water 60, glycerol 30, zinc chloride 2, iodine 0.2, potassium iodide "a trace" 13.1 Gibson test. 1905 Lee Lee 1905, 268 formula: water 60, 95% ale. 30, glycerol 30, mercuric chloride 2, acetic acid 0.15 13.1 Grawitz 1887 7276,27:604 formula: water 100, sodium chloride 15, sugar 4, boric acid 3, potassium nitrate 2 13.1 Haythorn 1915 4349, 6 :66 formula: water 100, sugar 44, potassium acetate 4, arsenic trioxide 0.5 13.1 Jackson 1919 test. 1922 ips. 4349, 8:75 formula: water 100, 40% formaldehyde 2, cane sugar 10 13.1 Jores 1896 23681, 7:134 formula: water 100, 40% formaldehyde 10, sodium sulfate 2, magnesium sulfate 2, sodium chloride 1 13.1 Jores 1913 22264, 16:357 formula: P 13.1 Peck 1900 100, chloral hydrate 1 180 METHODS AND FORMULAS P 13.1 13.1 Kaiserling 1896 2813, 33:775 roRMiii,A: water 57, 40% formaldehyde 43, potassium acetate 1.7, potassium nitrate 0.5 13.1 Kaiserling 1899 22264 (1899) 203 formula: water 84, 40% formaldehyde 16, potassium nitrate 0.9 potassium acetate 1.8 13.1 Keefe 1926 19938, 64:332 formula: water 45, 95% ale. 45, 40% formaldehyde 5, glycerol 2.5 acetic acid 2.5, copper chloride 10, uranium nitrate 1.5 note: Jirouch 1929 (20540b, 4:17) makes frozen sections of leaves preserved by this method and mounts in glycerol jelly. 13.1 Kirchner 1885 test. 1896 Zimmerman Zimmerman 1890, 41 formula: water 90, glycerol 10, chrome alum 0.5 RECOMMENDED FOR: preservation of color in algae. 13.1 Klotz and Coburn 1916a 4349, 6:51 formula: water 100, 40% formaldehyde 0.5, chloral hydrate 1.0, sodium sulfate 0.55, sodium bicarbonate 0.5, sodium chloride 0.45, potassium nitrate 1.0, potassium sulfate 0.05 13.1 Klotz and Coburn 1916b 4349, 6:53 formula: water 100, 40% formaldehyde 0.5, chloral hydrate 1, sodium sulfate 1.6, potassium sulfate 0.025, sodium chloride 0.3, sodium bicarbonate 0.6 13.1 Klotz and MacLachan 1915 4349, 5:59 formula: water 100, 40% formaldehyde 3, chloral hydrate 3, sodium sulfate 0.7, sodium bicarbonate 0.6, sodium chloride 0.55, potassium sulfate 0.06, potassium nitrate 1.2 13.1 Mall 1902 590, 5:433 formula: water 80, glycerol 20, potassium hydroxide 1 recommended for: preservation of mammalian embryos in which ossification studies are to be made in wholemounts. 13.1 Manesse test. 1921 Boitard Boitard 1921, 49 formula: water 80, 95% ale. 20, potassium alum 10, potassium nitrate 10, sodium chloride 10 13.1 Melnikow-Raswedenkow 1896 23681, 7:49 formula: water 62.5, glycerol 37.5, potassium acetate 19 13.1 Melnikow-Raswedenkow 1895 test. 1922 Silvester 4349, 8 :54 formula: water 50, 40% formaldehyde 50, sodium acetate 3, potassium chlorate 0.5 13.1 Ordonez 1866a Chevalier 1882, 323 formula: water 60, sat. aq. sol. camphor 20, glycerol 20, acetic acid 1 13.1 Ordonez 1865b test. 1882 Chevalier Chevalier 1882, 323 formula: water 100, glycerol 4, 95% ale. 4, mercuric chloride 0.03 13.1 Ordonez 1865c test. 1882 Chevalier Chevalier 1882, 323 formula: water 75, sat. aq. sol. camphor 15, glycerol 3, chromic acid 0.3 13.1 Pacini test. 1871 Robin Robin 1871, 376 STOCK solutions: I. water 90, glycerol 10, mercuric chloride 0.9, sodium chloride 1.7; II. water 85, glycerol 15, mercuric chloride 0.4, acetic acid 0.75 WORKING solutions: either of above diluted 1:3 13.1 Peck 1900 2813, 42 formula: water 100, 40% formaldehyde 5, sodium sulfate 3.2, sodium bicarbonate 1.2, potassium chloride 0.6, potassium sulfate 0.05 13.1 Ripart and Petit 1884 see F 4000.0010 Ripart and Petit P 13.1 Formulas PRESERVATIVES 181 13.1 Robin 1871 Robin 1871, 373 formula: F 7000.0000 Midler 1859, 50, glycerol 50 13.1 Thwaite test. 1880 Beale Beale 1880, 65 formula: 95% ale. 6, wood creosote 0.5, chalk in fine powder q.s. to sat., water 50, camphor water 50 13.1 Topping test. 1871 Robin Robin 1871, 375 formula: water 50, glycerol 50, aluminum acetate 10 18 Fixatives Decimal Divisions Used in Chapter F 01 Method of classification F 02 General observations F 03 Formulas arranged by classes F 0000 Solutions without primary or secondary fixative agents 0000.0010 Acetic alone .0012 Acetic-trichloracetic .0014 Acetic-nitric .0016 Acetic-hydrochloric .0023 Trichloracetic-formic .1000 Formaldehyde alone .1010 Formaldehyde-acetic .1020 Formaldehyde-trichloroacetic .1030 Formaldehyde-formic .1040 Formaldehyde-nitric .1080 Formaldehyde- (other inorganic acids) .1200 Formaldehyde-acetaldehyde .1300 Formaldehyde-acetone .4000 (Other modifiers) .4010 (Other modifiers)-acetic F 1000 Osmic acid in all combinations 1000.0000 Osmic alone .0010 Osmic-acetic .0019 Osmic-acetic- (other organic acid) .0030 Osmic-formic .0040 Osmic-nitrie .1010 Osmic-formaldehyde-acetic 1200.0000 Osmic-platinic .0010 Osmic-platinic-acetic 1230.00 10 Osmic-platinic-mercuric-acetic 1236.0010 Osmic-platinic-mercuric-chromic-acetic 1250.0010 Osmic-platinic-picric-acetic 1260.0000 Osmic-platinic-chromic .0010 Osmic-platinic-chromic-acetic .0030 Osmic-platinic-chromic-formic 1268.0000 Osmic-platinic-chromic-(other inorganic salts) .0020 Osmic-platinic-chromic- (other inorganic salt)-trichloro- acetic 1270.0000 Osmic-platinic-dichromate .0010 Osmic-platinic-dichromate-acetic 1300.0000 Osmic-mercuric .0010 Osmic-mercuric-acetic 1340.0000 Osmic-copper-mercuric 1350.0010 Osmic-mercuric-picric-acetic 1360.0010 Osmic-mercuric-chromic-acetic 182 Decimal Divisions Used in Chapter FIXATIVES 183 1 370.0030 Osmic-mercuric-dichromate-f ormic .1000 Osmie-mercuric-dichromate-formaldehyde 1378.0000 Osmic-mercuric-dichromate- (other inorganic salts) 1380.0010 Osmic-mcrcuric- (other inorganic salts)-acetic .0030 Osmic-mercuric- (other inorganic sal ts)-f ormic 1400.0000 Osmic-cupric .0010 Osmic-cupric-acetic 1500.0010 Osmic-picric-acetic .0040 Osmic-picric-nitric .0050 Osmic-picric-sulfuric 1560.0000 Osmic-pieric-chromic .1010 Osniic-picric-chromic-fornialdehyde-acetic 1580.0000 Osraic-picric-(other inorganic salt) 1600.0000 Osmic-chromic .0010 Osmic-chromic-acetic .0020 Osmic-chromic-trichloracetic .0030 Osmic-chromic-formic .0060 Osmic-chromic-hydrochloric .1010 Osmic-chromic-acetic-formaldehyde 1670.0000 Osnmic-chromic-dichromate .0010 Osmic-chromic-dichromate-acetic .0019 Osmic-chromic-dichromate-acetic-(other organic acids) .0090 Osmic-chromic-dichromate-(other organic acids) 1700.0000 Osmic-dichromate .0010 Osmic-dichromate-acetic .0030 Osmic-dichromate-formic .0040 Osmic-dichromate-nitric .1000 Osmic-dichromate-formaldehyde .1010 Osmic-dichromate-formaldehyde-acetic 1780.0000 Osmic-dichromate- (other inorganic salt) 1800.0000 Osmic- (other inorganic salt) .0010 Osmic-(other inorganic salt)-acetic .0030 Osmic- (other inorganic salt)-f ormic F 2000 Platinic chloride in combination with fixative agents of higher numerical rank 2000.1000 Platinic-formaldehyde .1010 Platinic-formaldehyde-acetic 2300.0000 Platinic-mercuric .0010 Platinic-mercuric-acetic .1000 Platinic-mercuric-formaldehyde .1030 Platinic-mercuric-formaldehyde-f ormic 2356.0000 Platinic-mercuric-picric-chromic 2470.0000 Platinic-cupric-dichromate 2500.0010 Platinic-picric-acetic 1030 Platinic-picric-formaldehyde-f ormic 2600.0000 Platinic-chromic .0010 Platinic-chromic-acetic 2700.0000 Platinic-dichromate F 3000 Mercuric chloride in combination with fixative agents of higher numerical rank 3000.0000 Mercuric alone .0010 Mercuric-acetic .0012 Mercuric-acetic-trichloroacetic .0014 Mercuric-acetic-nitric .0020 Mercuric-trichloroacetic ,0030 Mercuric-formic .0040 Mercuric-nitric .0060 Mercuric-hydrochloric .1000 Mercuric-formaldehyde .1010 Mercuric-formaldehyde-acetic 184 METHODS AND FORMULAS Decimal Divisions Used in Chapter .1012 Mercuric-formaldehyde-acetic-trichloroacetic . 1020 Mercuric-formaldehyde-trichloracetic .1310 Mercuric-fonnaldehyde-acetone-acetic .3000 Mercuric-acetone .3010 Mercuric-acetone-acetic 3400.0000 Mercuric-cupric .1010 Mercuric-cupric-formaldehyde-acetic .1014 Mercuric-cupric-formaldehyde-acetic-nitric 3470. 1000 Mercuric-cupric-dichromate-formaldehyde 3500.0000 Mercuric-picric .0010 Mercuric-picric-acetic .0015 Mercuric-picric-acetic-sulfuric . 1000 Mercuric-picric-formaldehyde .1010 Mercuric-picric-formaldehyde-acetic 35G0.0040 Mercuric-picric-chromic-nitric 3600.0000 Mercuric-chromic .0010 Mercuric-chromic-acetic .0040 Mercuric-chromic-nitric .1010 Mercuric-chromic-formaldehyde-acetic 3670.0000 Mercuric-chromic-dichromate .0010 Mercuric-chroniic-dichromate-acetic 3700.0000 Mercuric-dichromate .0010 Mercuric-dichromate-acetic .0030 Mercuric-dichromate-formic .0040 Mercuric-dichromate-nitric . 1000 Mercuric-dichromate-formaldehyde .1010 Mercuric-dichromate-formaldehyde-acetic 3780.0000 Mercuric-dichromate- (other inorganic salts) .1000 Mercuric-dichromate- (other inorganic salts)-formaIde- hyde 3800.1000 Mercuric- (other inorganic salts) -formaldehyde F 4000 Cupric salts in combination with fixative agents of higher numerical rank 4000.0010 Cupric-acetic .0020 Cupric-trichloracetic .0040 Cupric-nitric .1000 Cupric-formaldehyde .1010 Cupric formaldehyde acetic .1090 Cupric-formaldehyde- (other organic acids) 4500.1010 Cupric-picric-formaldehyde-acetic 4600.1000 Cupric-chromic-formaldehyde . 1010 Cupric-chromic-formaldehyde-acetic 4700.0000 Cupric-dichromate .0010 Cupric-dichromate-acetic . 1 000 Cupric-dichromate-f ormaldehy de 4900.0040 Cupric- (other organic agent)-nitric F 5000 Picric acid in combination with fixative agents of higher numerical rank 5000.0000 Picric alone .0010 Picric-acetic .0015 Picric-acetic-sulfuric .0020 Picric-trichloroacetic .0040 Picric-nitric .0050 Picric-sulfuric .0060 Picric-hydrochloric .1000 Picric-formaldehyde .1010 Picric-formaldehyde-acetic .1020 Picric-formaldehyde-trichloroacetic .1030 Picric-formaldehyde-formic .1040 Picric-formaldehyde-nitric .1090 Picric-formaldehyde- (other organic acid) .1310 Picric-formaldehyde-acetone F 01 FIXATIVES 185 5600.0000 Picric-chromic .0010 Picric-chromic-acetic .0040 Picric-chromic-nitric .0050 Picric-chromic-sulfuric .1000 Picric-chromic-formaldehyde .1010 Picric-chromic-formaldehyde-acetic 5670. 1000 Picric-chromic-dichromate-formaldehyde 5700.0000 Picric-dichromate .0050 Picric-dichromate-sulfuric . 1010 Picric-dichromate-formaldehyde-acetic 5800.1010 Picric- (other inorganic salts)-formaldehyde F 6000 Chromic acid in combination with fixative agents of higher numerical rank 6000.0000 Chromic alone .0010 Chromic-acetic .0030 Chromic-formic .0040 Chromic-nitric .0060 Chromic-hydrochloric .0070 Chromic-oxalic .1000 Chromic-formaldehyde .1010 Chromic-formaldehyde-acetic . 1040 Chromic-formaldehyde-nitric 6700.0000 Chromic-dichromate .0010 Chromic-dichromate-acetic .0040 Chromic-dichromate-nitric .1010 Chromic-dichromate-formaldehyde-acetic 6800.0000 Chromic- (other inorganic salts) .0030 Chromic-(other inorganic salts)-nitric F 7000 Dichromates without other primary fixative agents 7000.0000 Bichromate alone .0010 Dichromate-acetic .0040 Dichromate-nitric .1000 Dichromate-formaldehyde . 1010 Dichromate-formaldehyde-acetic .1030 Dichromate-formaldehyde-formic .2000 Dichromate-acetaldehyde 7800.0010 Dichromate- (other inorganic salt)-acetic .1000 Dichromate-(other inorganic salts)-formaldehyde .1012 Dichromate-(other inorganic salts)-formaldehyde-tri- chloroacetic F 8000 Solutions with "other inorganic" primary fixative agents 8000.0010 (Other inorganic agent)-acetic .0014 (Other inorganic agent)-acetic-nitric .0030 (Other inorganic agent)-formic .1000 (Other inorganic agent)-formaldehyde .1010 (Other inorganic agent)-formaldeliyde-acctic F 9000 Solutions with "other organic" primary fixative agents. 9000.0010 (Other organic agent)-acetic .4000 (Other organic agcnt)-(othcr modifier) F 04 Basic fixative solutions 04.0 Explanation 04.1 List of solutions 04.2 Formulas arranged alphabetically F 01 Method of Classification No arrangement of formulas based on modification of a fixative intended by one the intended application to tissues would writer for the preparation of brain tissue appear to be in any way practical. The for subsequent gold staining; (Smith 1930) most diverse ingredients are used for the is considered by another writer (Smith most diverse purposes, thus only a slight 1904) to be ideal for the preparation of 186 METHODS AND FORMULAS FOl large-yolked eggs. Under these circum- stances one is forced to fall back on the ingredients themselves, which are sur- prisingly few in number and which may be conveniently classified into groups ac- cording to the presumptive role which they play in fixation. The first of these great groups contains mostly metalhc salts, presumably intended to denature, or to form compounds with, the proto- plasm. One or more of these ingredients is found in almost all fixative solutions, and they are, therefore, referred to in this classification as 'primary fixative agents. PRIMARY FIXATIVE AGENTS 1. "Osmic acid" (osmium tetroxide) 2. Platinic chloride 3. Mercuric chloride 4. Cupric sulfate, nitrate, or chloride 5. "Picric acid" (2, 4, 6 trinitrophenol) 6. "Chromic acid" (chromium trioxide) 7. Potassium or sodium dichromate 8. Other inorganic salts 9. Other organic reagents Since no one has yet proposed, nor is it to be hoped that anyone will propose, a mixture containing more than four of these agents, it is possible to indicate mixtures of them by using four numbers from the list above. Thus, 1000 represents a fixative containing only osmic acid, 1300, a fixative combining osmic with mercuric, 1360, an osmic-mercuric-chromic mixture, and so on. These symbols are always written in their proper numerical order. Thus the combination 3600 would indicate a mer- curic-chromic mixture without any other primary agent. The addition of osmic would produce the symbol 1360. The numerical rank assigned to these varying agents has been designed, as far as pos- sible, to render this classification easy to handle. The vast majority of fixatives are modi- fied by the addition of one, but never more than two, fixative modifiers, which is the term here used to describe the commonly employed aldehydes and ketones. These are incorporated in the symbohc classifica- tion of a fixative by placing a decimal point after the symbol for the primary fixative agents and employing the first two positions to the right of this decimal point for the fixative modifiers. The fol- lowing list of fixative modifiers appear at present to be sufficient: FIXATIVE MODIFIERS (.) 1. formaldehyde (.) 2. acetaldehyde (.) 3. acetone (.) 4. all other fixative modifiers The only other common additions to fixative solutions are acids, of which never more than two appear to be employed in any one composition. It is, therefore, pos- sible to indicate the acids as two further figures placed to the right of the modifier. For this purpose the fixative acids may be listed as: FIXATIVE ACIDS (.00) 1. acetic (.00) 2. trichloroacetic (.00) 3. formic (.00) 4. nitric (.00) 5. sulfuric (.00) 6. hydrochloric (.00) 7. oxalic (.00) 8. all other inorganic acids (.00) 9. all other organic acids The combination of the agents with the modifiers and acids thus gives an eight- figure description which will indicate the composition of the fixative. This will be rendered clearer by a few further ex- amples. Thus: 0000.1000, formaldehyde without other admixture; 0000.1010, for- maldehyde-acetic; 0000.1012, formalde- hyde-acetic-trichloroacetic; 1670.0010, os- mic-chromic-dichromate-acetic. • The advantage of this method of classi- fication is that it permits an indefinite ex- pansion as new formulas appear in the hterature. It also brings logically together formulas of similar composition, but of gradually increasing complexity. That some system of classification is necessary, is indicated by the fact that more than 300 classes of fixatives are at present in existence. Additional difficulties are provided by two-solution formulas: that is, such a formula as those in which osmic acid and F02 FIXATIVES 187 potassium dichromate, each in its own solution, are used successively in the course of fixation. These have been classi- fied for the sake of simplicity as thouj^h both reagents were included in the same solution. The last case of ambiguity which occurs is that in which a complex salt, involving two primary fixative agents as one compound, is utihzed. There are several formulas, for example, in which copper dichromate is specified as an in- gredient. In these cases the formulas have been classified as though they were com- posed of a mixture of copper and dichro- mate rather than as prepared from the compound. F 02 General Observations The great confusion and diversity of opinion which occurs in the literature as to what constitutes a desirable quality for a fixative ingredient, is probably due to a similar disagreement as to what consti- tutes a desirable quality in a fixative mix- ture. In general, those who study the form of small invertebrates require first that a fixative should not interfere with the recognition of the object after fixation, that is, that its general shape shall not be distorted by the fluid applied to it. Cytol- ogists, histologists, and pathologists, on the contrary, require as a first considera- tion that the inner structure of the cell constituent shall remain unaltered, or shall at least present on microscopic ex- amination only those features which are thought to have been present in the original hving cell. It is obvious that the first group of workers are dependent for their effect largely upon the osmotic pressure shown by the material or, alter- natively, they require a solution that so rapidly and thoroughly hardens the outer coat that it will not become distorted by the passage of solutions through it. Thus Young (1935) gives directions for adjust- ing the osmotic pressure of solutions by the addition of sodium chloride, and Gray 1933 (11360, 53:13) pointed out that the addition of sodium sulfate to potassium dichromate to give the solution of Miiller improved the osmotic performance of the mixture. Heat is considered by many to be a desirable characteristic; Pantin (1946, 8) recommended heating fixatives for small marine invertebrate larvae. Heat should also possibly be apphed to fixation of internal constituents of cells, since Thomas and Morris 1925 (10996, 48:501) showed that the dichromates do not pre- cipitate albumin unless they are heated either at the same time, or immediately after, the apphcation of the reagent. The principal opponents of the view that the osmotic pressure of the solution is of primary importance are Baker and Craw- ford {test. Langeron 1942, 362) who showed that solutions with high osmotic pressure do not of necessity cause distortion; and Hirsch and Jacobs 1926 (1820, 18:7) who denied emphatically that any improve- ment in fixing qualities could be found by the addition of sodium chloride to fixative solutions. Those who desire to fix only the con- stituents of the living cell have been them- selves sharply divided into two schools of thought. The first of these schools con- sidered that the precipitating, or coagu- lating powers of the reagents employed were of primary importance. The second considered penetration and pH to be the most critical characteristic of a fixing fluid. The school which depended upon coagulation for fixation appears nowadays to be so thoroughly discredited that one can only refer to the summary of their work given in B5hm and Oppel (1907, 14). Here will be found a complex classification of fixatives based entirely upon the mate- rials which were precipitated and rendered insoluble by the apphcation of various reagents. There is, indeed, a fatal objec- tion to this work which was pointed out by Bolles Lee (1905, p. 22). This is that the more organic dead materials are pre- cipitated by the solution employed, the greater will be the artifacts produced. The case for cytologists and histologists is probably best given by Cretin (1925, LeMans) who considers that for neutral solutions, the total concentration of salts is important, but that for any solution containing acid, the pH must be carefully 188 METHODS AND FORMULAS F02 adjusted so that it is not below that of the isoelectric point of the proteins to be fixed. He considered a pll in the general vicinity of 4.2 to be the most desirable. His work was pubhshed at the same time as that of Gihei 1925 (3432, 39:164), who gives a table showing the pH of a few fixatives, none of which, however, were as alkaline as the upper Hmit indicated by Cretin. This work was followed in 1928 by the de- tailed study of Zirkle 1928 (17191a, 4:201) who came to the conclusion that there was a critical pH between 4.2 and 5.2. Fixative solutions more acid than this critical point were required for nuclear fixation, while those more alkaUne gave better results in cytoplasmic studies. Jacquiert 1930 (6630, 104:483) considered that a low pH with a suitable osmotic adjust- ment would give the more generally desir- able fixative. This last study confirmed the work of Burchardt 1897 (6011, 12:337) who recommended alkahne di- chromate for cytoplasmic fixation and acid dichromate for nuclear fixation. This uni- versal preference for acid fixatives must not cause one to lose sight of the work of Barnabo 1904 (3389, 13:198) and 1905 (3389, 14:139, 205) who stated that mer- curic fixatives buffered with bicarbonate gave, in general, very much better pic- tures both of nuclear and cytoplasmic fixation than did the more usual acid mix- tures. Another characteristic which may be mentioned is that the majority of mate- rials used for fixation are either strongly reducing or strongly oxidizing materials. This was first brought forward as a basis for the construction of formulas by Unna and Golodetz 1912 (7175, 22:10). They based their results on previous studies by Unna 1911 (1739, Fest. Waldeyer, 78) on the oxidizing and reducing properties of hving tissues. This view, that oxidizing and reducing qualities should first be sought, is advocated very strongly by Langeron in the 1942 edition of his Precis de Microscopic. The Uterature is so confused that no specific recommendations can be made for the theoretical production of a perfect fixative. The confusion which exists, however, with regard to the desirable qualities of a formula is very httle com- pared to the confusion which has arisen ill the jiractical i)reparation of these solu- tions. Authors have, in many instances, made no attempt to check the literature for the existence of a solution before they have invented one of their own. This duplication is all the more difficult to check for the reason that authors prepare their own solutions from any stock solu- tion wliich happens to be on their shelves at the time. Thus various authors have recommended a few milUhters of a 2%, 3%, 4%, or 5% solution of potassium dichromate to be mixed with a few other milhhters of 0.25%, 0.5%, 1%, or 2%, osmic acid. One of the most laborious features of the preparation of this work has been the reduction of all these form- ulas to a standard volume as though they had been prepared from dry salts. These standardized formulas are offered in two ways. First, together with the full citation to the original appearance, have been given the ingredients required to prepare 250 milUhters of solution from standard materials. This figure of 250 milhliters, rather than 100, has been taken as a matter of convenience since this is the quantity most usually made up. The names of chemicals have been given in full rather than in symbolic form for the reason that it is difficult to know where to draw the fine. It is to be presumed that any biologist would not have the slightest difficulty in preparing the solution made up of HgClz and HNO3. It is felt, how- ever, that the majority would be stopped cold by the directions to mix (N02)3- CeHiOH, HCHO and CH3COOH. To avoid the usual confusion between formol, formaldehyde, and formalm, the author has used the term 40% formaldehyde throughout, intending to indicate by this the fluid which comes in the bottle so labeled. Formulas are also presented in an abbreviated, alphabetical fist, permitting the preparation of the formulas referred to from standard solutions, which act, as it were, as the lowest common denomi- nator of about 80% of fixatives employed. The explanation of this method, together with the list of fixatives, forms the second half of the present chapter. F 0000.0010 FIXATIVES 189 F 03 Formulas Arranged by Classes F 0000 SOLUTIONS WITHOUT PRIMARY OR SECONDARY FIXATIVE AGENTS OOOO.OniO Acetic Alone Acetic acid is never used nowadays in simple aqueous dilution because it causes rapid hydrolj'sis of the protoplasm. This hydrolysis is diminished by the presence of alcohol, there- fore, the alcoholic dilutions given below can alone be recommended. These hydrolize cyto- plasm rapidly, but do not greatly affect the nucleus. They are, therefore, mostly employed for the demonstration of nuclei or of materials containing considerable nucleic acid. Acetic acid is regarded by Gatenby and Cowdry (1928, 52) as "a substance most injurious to the finer elements of the cytoi)lasni, but in some cases it is indicated for a study of nuclear ele- ments." Langeron (1942, 73), on the contrary, regards it as one of the most important fixa- tive agents. Unless a rapid hydrolysis of the cytoplasm is required to bring nuclear elements into sharp contrast, it is strongly recommended that it be employed in mixture either with chromic acid or with mercuric chloride. Spiess 1953 {in verb.) states that the substitution of proprionic acid for acetic improves cytological fixation. 0000.0010 Bartelmez 1915a 1135,25:87 formula: abs. ale. 225, acetic acid 25 0000.0010 Bartelmez 1915b 1135, 25:87 formula: abs. ale. 237.5, acetic acid 12.5 0000.0010 Bartelmez 1915c 1135, 25:87 formula: ale. 152, chloroform 80, acetic acid 16 0000.0010 van Beneden and Heyt 1877 3678, 14:218 formula: abs. ale. 125, acetic acid 125 0000.0010 Bradley 1948 20540b, 23 :29 formula: abs. ale. 95, acetic acid 31, chloroform 125 0000.0010 Carnoy 1887 6011, 3:6 formula: abs. ale. 180, acetic acid 60 0000.0010 Carnoy 1887 6011,3:276 formula: abs. ale. 150, acetic acid 25, chloroform 75 note: Langeron 1934, p. 349 refers to this as "Carnoy or van Gehuchten." 0000.0010 Farmer and Shove 1905a 17510, 48:559 formula: abs. ale. 214, acetic acid 36 0000.0010 Farmer and Shove 1905b 17510, 48:559 formula: abs. ale. 167, acetic acid 73 0000.0010 Favorsky 1930 766, 70: 376 formula: 50%-80% ale. 235 to 250, acetic acid 15 to 1 note: Use strong acid /weak ale. for tough tissues and vice versa. 0000.0010 Gilson 1897 formula: abs. ale. 80, acetic acid 80, chloroform 80 0000.0010 Hetherington 1922 11428,9:102 formula: abs. ale. 100, chloroform 75, acetic acid 25, phenol q.s. to make 2.50 RECOMMENDED FOR: fixation and dehydration of nematodes. 0000.0010 Lendrum 1935 11431,40:416 FORMi la: water 10, abs. ale. 75, chloroform 10, phenol 4, acetic acid 3 0000.0010 Moleschott 1871 see P 12.1 Moleschott 1871 190 METHODS AND FORMULAS F 0000.0010-F 0000.1000 0000.0010 Potenza 1939 test. 1942 Langeron Langeron 1942, 419 formula; methanol 50, dioxane 200, paraldehyde 5, acetic acid 12.5 0000.0010 Sansom test. 1928 Gatenby and Cowdry formula: abs. ale. 165, chloroform 75, acetic acid 7.5 0000.0010 Strauss 1909 23053a, 20 :3 formula: 95% ale. 242.5, acetic acid 7.5 000.0012 Acetic-trichloroacetic 0000.0012 Davenport and Kline 1938 20540b, 13:160 formula: n-butyl ale. 150, n-propyl ale. 50, acetic acid 25, trichloroacetic acid 25 0000.0012 Hofker 1921 23632, 38:130 formula: abs. ale. 200, acetic acid 25, trichloroacetic acid 25 0000.0014 Acetic-nitric 0000.0014 Robin 1871 see P 12.2 Robin 1871 0000.001 6 A cetic-hydrochloric 0000.0016 Jenkin 1920 see AF 21.1 Jenkin 1920 0000.0023 Trichloroacetic-formic 0000.0023 Davenport and KUne 1938 20540b, 13:160 formula: n-butanol 150, n-propyl ale. 50, trichloroacetic acid 25, formic acid 12.5 0000.0023 Davenport, McArthur and Bruesch 1939 20540b, 14 :22 formula: n-butanol 160, n-propyl ale. 65, trichloroacetic acid 12.5, formic acid 12.5 0000.1000 Formaldehyde Alone Formaldehyde in aqueous solution can scarcely be regarded as a fixative, though it is widely used as a preservative fluid. All figures given for the alcoholic dilutions which follow refer to cubic centimeters of the ordinary commercial so-called 40 % formaldehyde. This solu- tion becomes acid on standing, but may be buffered to a desirable pH, usually about 8.5, by the addition of borax. Another common procedure is to keep small chips of marble in the stock bottle ; though this tends to cause a cloudy precipitate, if in no way interferes with its fixative value. Burke 1933 (608b, 9:915) prefers pyridine (3 to 5%) as a neutralizing agent but this cannot (Warbritton 1937, 20540b, 12 :125) be used in mercuric mixtures. Koenig, Groat, and Windle 1945 (20540b, 20:13) recommend the addition of from 2.5% to 5.5% of gum arable to counteract the gross cell shrinkage produced by diluted formaldehyde. 0000.1000 Baker 1944 17510, 85:1 formula: water 225, 40% formaldehyde 25, calcium chloride 1 0000.1000 Benario 1894 7276, 20 :572 formula: 90% ale. 250, 40% formaldehyde 2.5 0000.1000 Boeke 1910 766,35:193 formula: water 100, 95% ale. 125, 40% formaldehyde 10 0000.1000 Bujor 1901 see AF 51.1 Bujor 1901 0000.1000 Burke 1933 608b, 9 :915 formula: water 187.5, 40% formaldehyde 67.5, pyridine 12.5 0000.1000 de Castro 1916 21344, 14:83 formula: water 250, 40% formaldehyde 37.5, urea nitrate 3.75 0000.1000 Gulland 1900 23632, 17:222 formula: 95% ale. 225, 40% formaldehyde 25 F 0000.1000-F 0000.1010 FIXATIVES 191 0000.1000 Hampert 1926 22575, 259:179 formula: 95% ale. 130, water 35, 40% formaldehyde 85, potassium acetate 10 0000.1000 Hornell 1900 11976,5:86 formula: 90% ale. 225, 40% formaldehyde 25 0000.1000 Jones test. 1915 Chamberlain Chamberlain 1915, 20 formula: water 75, 95% ale. 170, 40% formaldehyde 5 0000.1000 Kaiserling see P 12.3 Kaiserling 1928 0000.1000 Parker and Floyd 1895 766, 11:150 formula: 95% ale. 256, 40% formaldehyde 5 0000.1000 Schaffer 1908 23635, 89:1 formula: water 32, 95% ale. 128, 40% formaldehyde 80 0000.1000 Schaffer 1918 766, 51 :373 formula: 80% ale. 160, 40% formaldehyde 80 0000.1010 Formaldehyde-acetic These mixtures are valuable when it is necessary to avoid any metallic constituent, or to prevent the formation of the yellow color of picric mixtures. They are reasonably good gen- eral-purpose fixatives. 0000.1010 Armitage 1939 Microscope, 3 :213 formula: water 125, dioxane 100, 40% formaldehyde 20, acetic acid 15 0000.1010 Becher and Demoll 1913 Becher and DemoU 1913, 43 formula: water 150, 95% ale. 75, 40% formaldehyde 22, acetic acid 4 0000.1010 Bles 1905 21652, 41 :792 formula: 70% ale. 225, 40% formaldehyde 17.5, acetic acid 7.5 0000.1010 Boule 1908a 15063, 10:15 formula: water 235, 40% formaldehyde 55, acetic acid 12.5 0000.1010 Boule 1908b 15063, 10:15 formula: 95% ale. 200, 40% formaldehyde 50, acetic acid 10, ammonia 1 0000.1010 Dietrich test. 1946 Roskin Roskin 1946, 91 formula: water 150, 95% ale. 75, 40% formaldehyde 25, acetic acid 5 0000.1010 Dietrich see also F 0000.1010 Kahle 1908 (note) 0000.1010 Fontana 1912 7176,55:1003 formula: water 200, 40% formaldehyde 40, acetic acid 2 RECOMMENDED FOR: prior to MS 34.5 Fontana 1912 {q.v.). 0000.1010 Hosokawa 1934 test. 1942 Langeron Langeron 1942, 841 formula: methanol 250, 40% formaldehyde 12.5, glacial acetic acid 2.5 0000.1010 Jackson 1922 4349, 8:125 formula: 90% ale. 200, 40% formaldehyde 25, acetic acid 25 0000.1010 Kahle 1908 23820, 21:10 formula: water 150, 95% ale. 65, 40% formaldehyde 30, acetic acid 5 note: Kingsbury and Johannsen 1927, p. 8, refer this formula, without reference to "Dietrich or Kahle." 0000.1010 Lavdowsky 1894 7936a, 1:361 formula: 30% ale. 225, 40% formaldehyde 25, acetic acid 5 note: Kupperman and Noback 1945 (1887a, 40:78) recommend adding 1% ferric alum when tissues are to be hematoxylin stained. 192 METHODS AND FORMULAS F 0000.1010-F 0000.1040 0000.1010 Liiko 1910 lest. 1910 Tellyesniczky Ehrlich, Krause, et al. 1910, 1:472 formula: 75% ale. 250, 40% formaldehyde 25, acetic acid 12.5 note: Mayer 1920 (p. 30) refers to this formula as "Tellyesniczky." Tellyesniczky, loc. cil. says clearly "... eine Mischung, eingefuhrt vovi Assistenten Bela V. Luko." 0000.1010 Mahdissan 1935 1798,85:61 formi'la: ahs. air. 150, chloroform 75, 40% formaldehyde 25, acetic acid 12.5 0000.1010 Murray and Fielding test. 1937 Findlay 11360, 57:138 formula: water 115, abs. ale. 115, 40% formaldehyde 12.5, acetic acid 7.5 0000.1010 Podhradszky 1934 23632,50:285 formula: water 225, 40% formaldehyde 25, acetic acid 12.5 0000.1010 Railliet see P 12.1 Railliet (1942) 0000.1010 Roskin 1946 Roskin 1946, 292 formula: water 200, 40% formaldehyde 50, acetic acid 7.5 0000.1010 Romeis 1948 Romeis 1948, 49 formula: water 108, 95% ale. 112, 40% formaldehyde 25, acetic acid 5 0000.1010 Ruge 1942 test. Langeron 1942 Langeron 1942, 636 formula: water 250, 40% formaldehyde 5, acetic acid 2.5 0000.1010 Scheuring 1913 test. 1948 Romeis Romeis 1948, 528 formula: 95% ale. 120, 40% formaldehyde 120, acetic acid 10 0000.1010 Sikora 1917 23632, 34:161 formula: abs. ale. 175, acetic acid 30, 40% formaldehyde 30, chloroform 15 note: With the addition of 1% of picric acid this becomes F 5000.1010 Leeuwen 1907 (q.v.). 0000.1010 Tellyesniczky see F 0000.1010 Luko 1910 (note) 0000. 1 020 Formaldehyde-trichlor acetic 0000.1020 Champy 1913 1915, 52:18 formula: sat. aq. sol. phenol 200, 40% formaldehyde 48, trichloroacetic acid 3.6 0000.1020 Davenport, Windle, and Beach 1934 20540b, 9:10 formula: water 225, 40% formaldehyde 25, trichloroacetic acid 1.25 0000.1020 Heidenhain 1916 23632,32:365 formula: water 200, 40% formaldehyde 37.5, trichloroacetic acid 12.5 0000.1030 Formaldehyde-formic 0000.1030 Szepsenwol 1935 6630, 120:689 formula: water 220, 40% formaldehyde 20, formic acid 10 recommended for: prior to Szepsenwol 1935 MS 33.21 (q.v.) via 1% and 3% silver nitrate. 0000.1040 Formaldehijde-nitric 0000.1040 Hoskins 1907 11689, 4:176 formula: water 225, 40% formaldehyde 20, nitric acid 6 0000.1040 McCIung and Allen 1929 McClung 1929, 422 formula: water 225, 40% formaldehyde 19, nitric acid 6.3 0000.1040 Wilhelmi 1909 test. 1920 Mayer Mayer 1920, 33 formula: 90% ale. 195, 40% formaldehyde 18.75, nitric acid 7.5 F 0000.1080-F 1000.0000 FIXATIVES 193 0000.1080 Fornialdehyde-{other inorganic acids) 0000.1080 Cohen 1934 20540b, 9:104 formula: water 225, metaphosphoric acid 9, 40% formaldehyde 25 0000.1200 Formaldehyde-acetaldehyde 0000.1200 Besta 1910 766,36:477 formulas: a. water 200, 40% formaldehyde 50, acetaldehyde 5; B. water 250, am- monium molybdate 10 method: [fix A, 2 days] — * distilled water, 24 hrs. with frequent changes —> B, 2 days 0000.1300 Formaldehyde-acetone 0000.1300 Bing and Ellermann 1901 1739, 3:260 formula: 40% formaldehyde 25, acetone 225 0000.4000 (Othek Modifiers) 0000.4000 MacFarland and Davenport 1941 20540b, 16:53 formula: water 112, 95% ale. 112, chloral hydrate 12.5, formamide 25 0000.4010 {Other modifier s)-acetic 0000.4010 Grapnuer and Weissberger 1933 23833, 102 :39 formula: methanol 50, dioxane 200, paraldehyde 5, acetic acid 12.5 F 1000 OSMIC ACID IN ALL COMBINATIONS 1000.0000 OsMic Alone It cannot be too strongly emphasized that osmic acid is a dangerous material to handle. The vapor causes the death of tissues with which it comes in contact almost instantly and, indeed, it may be employed for fixation in the vapor phase. This vapor, applied to the cornea of the eye or to the membranes of the nose, can cause the most grave damage in a short space of time. Since osmic acid is very easily reduced in the presence of almost all organic material, solutions are difficult to keep. The writer prefers the addition of about .01% of potassium permanganate. This gives a pink color, which may easily be judged by the eye, so that the oxidation of the permanganate can be corrected by the addition of a few drops of a stronger solution. The product of the reduction, which is a dead black in color, appears to be some of the lower oxides. These lower oxides may again be easily oxidized (bleached) and it is customary to use various mixtures of hydrogen peroxide (see Chapter 19, AF 31.1 Overton 1890) for the purpose, but the specimen must be washed thoroughly or the oxides will be reprecipitated on the tissues. Osmic acid alone is rarely used save for very small objects. It has the great advantage that it gives a faithful picture of cytological detail. Olney 1953 (Turtox News, 31 :29) states that nitric acid may be substituted for osmic acid in fixative formulas. 1000.0000 Bizzozero 1885 1789a, 102 formula: water 250, osmic acid 0.625, sodium chloride 0.19 1000.0000 Ewald 1897 23354,34:257 formula: water 250, osmic acid 0.375, sodium chloride 1.25 note: The above is recommended for amphibian and reptilian blood; for mammals the NaCl is increased to 0.65%. 1000.0000 Mann 1894 23632, 11 :479 formula: water 250, osmic acid 2.5, sodium chloride 1.875 1000.0010 Osmic-acetic These are excellent mixtures, and are probably as good as the osmic-chromic-acetic more usually recommended. The rapid fixing and hardening power of the osmic acid counteract.s the swelling properties of the acetic. These fixatives ciin be recommended both for cytological and nuclear fixation. 194 METHODS AND FORMULAS F 1000.0010-F 1000.1010 1000.0010 Faber-Domergue 1889 Ann. Micr., 1 :1, Paris formula: water 200, osmic acid 2.5, acetic acid 50 1000.0010 Fol 1896 test. Poll 1910 Ehrlich, Krause, et al. 1910, 2, 345 formula: water 250, osmic acid 0.25, acetic acid .25 note: The fixative of Schmidt 1896 (1780, 47:47) is identical. 1000.0010 Hamann 1885 test. 1910 Poll Ehrlich, Krause, et al. 1910, 2 :345 formula: water 250, osmic acid 1.25, acetic acid 1.25 1000.0010 Hertwig 1879 10899, 13 :457 formula: water 250, osmic acid 0.06, acetic acid 0.25 1000.0010 Hertwig 1885 14555, 10 :337 formula: water 250, osmic acid 1, acetic acid 2.5 1000.0010 Orr 1900 11431,6:387 formula: water 250, osmic acid 4, acetic acid 0.5 1000.0010 Rawitz 1895 Rawitz 1895, 19 formula: water or sea water 250, osmic acid 0.06, acetic acid 0.25 1000.0010 Schmidt 1896 see F 1000.0010 Fol 1896 (note) 1000.0010 Schwarz 1888 2701, 18:3 formula: water 250, osmic acid 0.5, acetic acid 2.5 1000.0010 Zacharias 1887 1780,30:111 formula: abs. ale. 200, osmic acid 0.025, acetic acid 50, chloroform 0.25 note: Mayer 1920, p. 37, refers this formula (without the chloroform) to 1888 (766, 3:24), but see below. 1000.0010 Zacharias 1888 766, 3 :24 formula: abs. ale. 200, osmic acid 0.04, acetic acid 50 1000.0019 Osmic-acetic-{other organic acid) 1000,0019 van Ermengen 1894 23684, 15 :969 formula: water 250, osmic acid 1.5, acetic acid 2.5, tannic acid 12 RECOMMENDED FOR: fixation of bacterial smears before silver staining of flagella. 1000.0030 Osmic-formic 1000.0030 Henking 1891 23632, 8:156 formula: water 200, glycerol 40, osmic acid 0.025, formic acid 7.5, dahlia violet 0.1 1000.0030 Kerschner 1908 1780, 71 :522 formula: water 225, osmic acid 0.5, formic acid 50 1000.0030 Viallane 1883 test. 1928 Gatenby and Cowdry Gatenby and Cowdry 1928, 210 formula: a. water 250, osmic acid 2.5; B. water 160, formic acid 80 method: [fresh tissues] — > A. till lightly browned -^ B, 10 mins. — > MS 23.21 Viallane 1883 1000.0040 Osmic-nitric 1000.0040 Kolossow 1892 see AMS 11.1 Kolossow 1892 1000.0040 Nicolas 1891 10157,8:3 formula: water 250, osmic acid 1.25, nitric acid 7.5 1 000.1 01 0 Osmic-formaldehyde-acetic 1000.1010 Swank and Davenport 1934 20540b, 9:11 formula: A. water 225, 40% formaldehyde 25; B. water 250, osmic acid 0.82, acetic acid 2.5, potassium chlorate 0.625 method: [fresh tissue] — » A, 24 hrs. -^ B, directly without washing, 1 wk. -^ [sections.] F 1000.1010-F 1260.0010 FIXATIVES 195 1000.1010 Swank and Davenport 1935 20540b, 10:88 formula: water 200, osmic acid 0.025, 40% formaldehyde 30, acetic acid 2.5, potassium chlorate 1.5 1200.0000 OSMIC-PLATINIC 1200.0000 van der Stricht 1895 1825, 14:469 formula: water 250, osmic acid 1, platinic chloride 2 1200.0010 Osmic-platinic-acetic These once widely recommended cytological fixatives are doubtfully any better than plain osmic-acetic mixtures. 1200.0010 Hermann 1889 1780, 34 :59 formula: water 250, osmic acid 1, platinic chloride 1.8, acetic acid 12.5 1200.0010 Meves 1908 1780, 72:816 formula: water 250, osmic acid 0.15, platinic chloride 0.56, acetic acid 3.75 1200.0010 Niessing 1895 1780,46:147 formula: water 225, osmic acid 1, platinic chloride 13, acetic acid 25 1230.0010 OSMIC-PLATINIC-MERCURIC- ACETIC 1230.0010 Cox 1895 23632, 13 :498 formula: water 240, osmic acid 0.6, platinic chloride 4.5, mercuric chloride 6.5, acetic acid 36 1230.0010 Niessing 1895 1780, 46:147 formula: water 235, osmic acid 0.5, platinic chloride 6.25, mercuric chloride 8.75, acetic acid 12.5 1236.0010 OSMIC-PLATINIC-MERCURIC-CHROMIC- ACETIC 1236,0010 Marchoux and Simond 1906 857, 20:105 formula: water 250, osmic acid 0.75, platinic chloride 1.5, mercuric chloride 15, chromic acid 1, acetic acid 7 1250.0010 OSMIC-PLATINIC-PICRIC- ACETIC 1250.0010 vom Rath 1895 766, 11 :285 formula: water 250, osmic acid 0.5, platinic chloride 2.0, picric acid 3.0, acetic acid 4.0 1260.0000 OSMIC-PLATINIC-CHROMIC 1260.0000 Whitman 1883 651, 17:1204 formula: a. 0.25% osmic in sea water; B. F 2600.0000 Eisig 1879 method: pelagic fish ova — > A, 5-10 mins. — > B, 2 days 1 260.001 0 Osmic-platinic-chromic-acetic These mixtures are now rarely seen. The addition of the platinic acid is of very doubtful value. 1260.0010 Besson 1904 Besson 1904, 750 formula: water 250, osmic acid 1.4, platinic chloride 1.4, chromic acid 2.1, acetic acid 14 note: Besson attributes this to "Borrel" but gives no reference. 1260.0010 "Borrel" see F 1260.0010 CauUery and Mesnil 1905, or F 1260.0010 Marchoux and Simond 1906, or F 1260.0010 Besson 1904 1260.0010 Brass 1884 23632, 1 :39 formula: water 250, osmic acid 0.2 to 0.3, platinic chloride 0.3 to 0.8, chromic acid 0.3 to 0.8, acetic acid 0.3 to 0.8 1260.0010 CauUery and Mesnil 1905 1789, 6:281 formula : water 240, osmic acid 1.3, platinic chloride 1.3, chromic acid 2.0, acetic acid 13 note: Laugeron 1934, p. 339 refers to this mixture as "MHanqe de Borrel {1905)" but offers no reference. 196 METHODS AND FORMULAS F 1260.00I0-F 1300.0000 1260.0010 Marchoux and Simond 1906 857, 20:105 formula: water 250, osmic acid 1.5, platinic chloride 1.5, chromic acid 2, acetic acid 14 note: Poll 1910 (Ehrlich, Krause, et al. 1910, 2, 348) refers to this formula (without ref- erence) as "Borrel's mixture." 1 260.0030 Osmic-platinic-chromic-formic 1260.0030 Pianese 1889 1886, 2 :412 formula: water 250, osmic acid 1, platinic chloride 1.5, chromic acid 0.13, formic acid 0.5 1268.0000 OSMIC-PLATINIC-CHROMIC- (other INORGANIC SALTs) 1268.0000 Nebel 1934 7033a, 5:1 REAGENTS REQUIRED: A. 0.1% ammonium hydroxide; B. 0.5% thorium nitrate; C. water 230, osmic acid 5.75, platinic chloride 2.3, chromic acid 0.2 method: [root tips] -^ A, 5 mins. -^ 5, 3 hrs. -^ C, 24 hrs. 1268.0020 Osmic-platinic-chro7nic-{other inorganic salt) -trichloroacetic acid 1268.0020 Friedenthal 1908 test. circ. 1938 Wellings Wellings circ. 1938, 25 formula: trichloroacetic acid 156, osmic acid 4, platinic chloride 4, chromic acid 8, uranium acetate 78 1270.0000 osmic-platinic-dichromate 1270.0000 Veratti test. 1900 Golgi Golgi 1900, 2 :687 formula: water 250, osmic acid 0.6, platinic chloride 0.08, potassium dichromate 4.0 1270.0010 Osmic-'platinic-dichr ornate-acetic 1270.0010 Johnson 1895 test. 1905 Lee Lee, 1905, 43 formula: water 250, osmic acid 0.5, platinic chloride 0.4, potassium dichromate 4.3, acetic acid 12.5 1300.0000 OSMIC-MERCURIC These mixtures are cytological fixatives, and are worthless for nuclear preservation. They are best applied to small invertebrates in which the rapid killing action of the osmic is backed up by the hardening and mordanting action of the mercuric chloride. 1300.0000 Apathy 1893 23632, 10 :349 formula: water 250, osmic acid 2.5, mercuric chloride 9 1300.0000 Apathy 1897 14246, 12 :495 formula: water 250, osmic acid 1.25, mercuric chloride 8.75, sodium chloride 0.7 1300.0000 Braun 1895 test. 1910 Poll Ehrlich, Krause, et al. 1910, 2 :351 formula: water 250, osmic acid 0.013, mercuric chloride 17.5 1300.0000 Biihler 1898 22302,31:316 formula: water 250, mercuric chloride 17.5, osmic acid 0.125 1300.0000 Heidenhain 1896 23632, 13:186 formula: water 250, osmic acid 1.25, mercuric chloride 8.75 1300.0000 Hirschler 1918 1780, 91 :140 formula: water 250, osmic acid 2.5, mercuric chloride 8.75 1300.0000 Kolster test. 1910 Poll Ehrlich, Krause, et al. 1910, 2:351 formula: water 250, osmic acid 7.5, mercuric chloride 17.5, sodium chloride 1.25 1300.0000 Mann 1894 23632, 11 :481 formula: water 250, osmic acid 1.25, mercuric chloride 10, sodium chloride 2.25 note: The original method calls for equal parts of 1% osmic and a sat. sol. mercuric chloride in 0.9% NaCl. F 1300.0000-F 1370.0030 FIXATIVES 197 1300.0000 Pappenheim 1896 1789a, 145 :587 formula: water 250, osmic acid 2.5, niorcuric chloride 8.75 1300.0010 Osmic-mercuric-acetic These are general-purpose fixatives, giving good preservation both of cytoplasmic and nuclear elements, as well as being much easier for after-staining than are osmic-acetic mix- tures alone. The mercuric chloride maj' assist in preserving the osmic acid against reduction. 1300.0010 Colombo 1903 23632,20:282 formula: water 250, osmic acid 1, mercuric chloride 7, acetic acid 0.5 1300.0010 Cox 1896 23632, 13 :498 formula: water 200, osmic acid, 1.0, mercuric chloride 7.0, acetic acid 50.0 1300.0010 Druner 1894 10899,28:296 formula: water 2-40, osmic acid 0.125, mercuric chloride 12.5, acetic acid 12.5 1300.0010 Tschassownikow test. 1910 Spuler Ehrlich, Krause, et al. 1910, 2:526 formula: water 245, osmic acid 1.25, mercuric chloride 13, acetic acid 6.25 1300.0010 Zieglwallner 1911 test. 1928 Schmorl Schmorl 1928, 217 formul.\: water 100, abs. ale. 125, osmic acid 1, mercuric chloride 3, acetic acid 25 1340.0000 OSMIC-CUPRIC-MERCURIC 1340.0000 Merton 1932 1798, 76:171 STOCK, solutions: I. 2% copper sulfate; II. 2% osmic acid; III. sat. sol. mercuric chloride. method: [Paramecium on smear of V 21.1 Mayer 1884] —> I, added to fluid on slide, 5 mins. -> drain -^ vapor of II, 1 min. -^ III, on slide 10 mins. — » 70% ale. 1350.0010 OSMIC-MERCURIC-PICRIC-ACETIC 1350.0010 Becher and Demoll 1913 Becher and Demoll 1913, 43 formula: water 245, osmic acid 0.5, mercuric chloride 3.75, picric acid 0.75, acetic acid 5 1350.0010 vom Rath 1895 766, 11 :285 formula: water 250, osmic acid 0.5, mercuric chloride 8, picric acid 1.4, acetic acid 2.2 1360.0010 OSMIC-MERCURIC-CHROMIC- ACETIC 1360.0010 Brouha test. 1930 Guyer Guyer 1930, 150 formula: water 240, osmic acid 5, mercuric chloride 11.5, chromic acid 5, acetic acid 12.5 1360.0010 Cori lest. 1910 Spuler Ehrlich, Krause, ei al. 1910, 2 :526 formula: water 250, osmic acid 0.003, mercuric chloride 15.3, chromic acid 0.08, acetic acid 0.03 note: This may also be prepared by mixing 7 parts of 7% mercuric chloride with 1 part of F 1600.0010 Cori 1890. 1360.0010 Galescu 1908 6630, 65:429 formula: water 250, osmic acid 0.04, mercuric chloride 4.4, chromic acid 0.5, acetic acid 0.2 1360.0010 Podwyssozki 1886 2526, 1 :287 formula: water 250, osmic acid 0.5, mercuric chloride 1, chromic acid 2, acetic acid 5 1370.0030 OSMIC-MERCURIC-DICHROMATE-FORMIC 1370.0030 Charipper 1928 763, 38:401 formula: water 240, osmic acid 1, mercuric chloride 10, potassium dichromate 5, sodium sulfate 2, formic acid 10 198 METHODS AND FORMULAS F 1370.1000-F 1500.0050 1S70.1000 Osmic-mercuric-dichromate-formaldehyde 1370.1000 Levi 1918 1820, 11 :515 formula: water 250, osmic acid 0.5, mercuric chloride 5.6, potassium dichromate 2.75, 40% formaldehyde 2.5 1378.0000 OSMIC-MERCURIC-DICHKOMATE-(oTHER INORGANIC SAI,Ts) 1378.0000 Benoit 1922 6630,86:1101 formula: water 250, osmic acid 1.25, mercuric chloride 3.1, potassium dichromate 3.75, uranium nitrate 2.0, sodium chloride 0.5 1380.0010 OSMIC-MERCURIC-(OTHER INORGANIC SALTS)-ACETIC 1380.0010 Farkas 1914 23833,46:143 formula: water 100, 95% ale. 125, osmic acid 1.25, mercuric chloride 4, acetic acid 25, sodium iodide 0.005 1380.0030 Osmic-mercuric-{other inorganic salts) -for )nic 1380.0030 Nebel 1932 23639b, 16:251 formula: water 225, osmic acid 2.5, mercuric chloride 3, uranium chloride 1, formic acid 25 1400.0000 OsMic-cuPRic 1400.0000 Gardner 1897 2981, 17 :398 formula: water 250, osmic acid 0.2, copper acetate 14.0 1400.0000 de Waele 1899 test. 1920 Mayer Mayer 1920, 61 formula: water 250, osmic acid 0.5, copper acetate 12.5 1400.0010 Osmic-cupric-acetic 1400.0010 Duboscq 1899 see DS 21.3 Duboscq 1899 1500.0010 OSMIC-PICRIC-ACETIC No advantage can be secured by the addition of picric acid, which gives water-soluble pre- cipitates, to such an excellent fixative as osmic-acetic alone. These formulas are, however, widely recommended in the literature. 1500.0010 Flemming 1882 test. 1920 Mayer Mayer 1920, 40 formula: water 250, osmic acid 0.25, picric acid 0.6, acetic acid 0.25 1500.0010 Giesbrecht test. 1910 Poll cit. Lee and Mayer Ehrlich, Ivrause, et al. 1910, 1 :345 formula: sea water 250, osmic acid 0.1, picric acid 3, acetic acid 10 1500.0010 vom Rath 1895 766, 11 :289 formula: water 250, osmic acid 0.3, picric acid 3, acetic acid 2.5 1500.0010 Schuberg test. 1920 Mayer Mayer 1920, 40 formula: water 240, osmic acid 0.25, picric acid 2.7, acetic acid 7.5 1500.0010 Spiiler 1892 1780,40:530 formula: water 250, osmic acid 0.125, picric acid 3, acetic acid 1.5 1500.0040 Osmic-picric-nitric 1600.0040 Hill 1910 17510, 64:1 formula: water 250, osmic acid 0.05, picric acid 3, nitric acid 12.5 1500.0040 Rawitz 1895 Rawitz 1895, 24 formula: water 250, osmic acid 0.7, picric acid 3, nitric acid 12.5 1500.0050 Osmic-picric-sulfuric 1500.0050 Erlanger 1895 14555, 22 :493 formula: water 250, osmic acid 0.1, picric acid 3, sulfuric acid 5 F 1500.0050-F 1600.0000 FIXATIVES 199 1500.0050 Francotte circa 1890 Francotte, 197 formula: water 250, osmic acid 0.1, picric acid 0.5, sulfuric acid 5 1500.0050 Schuberg 1903 23635, 74:155 formula: water 250, osmic acid 0.4, picric acid 3, sulfuric acid 5 1560.0000 OSMIC-PICRIC-CHEOMIC Attention must again be drawn to the improbability that picric acid will impart additional qualities to an already good mixture, such as osmic-chromic alone. 1560.0000 Fol 1896 test. 1910 Poll Ehrlich, Krause, et al. 1910, 2:348 formula: water 250, osmic acid 0.01, picric acid 0.5, chromic acid 0.6 1560.1010 Osmic-picric-chromic-acetic-formaldehyde 1560.1010 Skovstedl933 1032,47:227 formula: water 190, osmic acid 1, picric acid 1.5, chromic acid 2.8, acetic acid 9, 40% formaldehyde 50, urea 3.8 1580.0000 0SMIC-PICRIC-(0THER INORGANIC SALT) 1580.0000 Perriraz 1905 5392, 61 :213 formula: water 175, 95% ale. 75, osmic acid 0.12, picric acid 0.75, silver nitrate 0.75 preparation: Dissolve the osmic acid in the ale, and the salts separately in water. Add heated (50°C.) picric solution to silver nitrate, then osmic. 1600.0000 Osmic-chromic The osmic-chromic mixtures share with the following class the distinction of being the best general-purpose fixatives. They give better cytological fixation than do the more strongly acid osmic-chromic-acetic, but the considerable number of formulas included under each class bear witness to their good qualities. When in doubt as to what fixative to choose for an object with which one has had no previous experience, it cannot be too strongly recom- mended that one from either this or the following class be employed. 1600.0000 Barret 1886a 17510, 26:607 formula: 50% ale. 250, osmic acid 0.5, chromic acid 0.5 1600.0000 Barret 1886b 17510, 26:607 formula: water 250, osmic acid 0.5, chromic acid 0.4 1600.0000 Barret 1886c 17510, 26:607 formula: water 250, osmic acid 0.25, chromic acid 0.65 1600.0000 Bohm and Oppel 1896 see F 1600.0000 Flesch 1879 1600.0000 Brock 1886 23635, 44 :333 formula: water 250, osmic acid 0.04, chromic acid 0.25 1600.0000 Drew 1920 see ADS 12.1 Drew 1920 1600.0000 Flesch 1879 1780, 16 :300 formula: water 250, osmic acid 0.25, chromic acid 0.6 note: The formula given (without reference) by Bohm and Oppel in the 1896 edition of their Taschenbuch does not differ significantly from this. 1600.0000 Haug 1891 see AF 21.1 Haug 1891 1600.0000 Lo Bianco 1890 14246, 9:443 formula: water 250, osmic acid 0.05, chromic acid 2.5 1600.0010 Osmic-chromic-acetic Flemming 1882 was the first of these excellent mixtures. The whole group is often referred to as "Flemming." "Flemming without acetic" (FWA) is just that, and belongs in the last class. 200 METHODS AND FORMULAS F 1600.0010 1600.0010 Baker 1945 Baker 1945, 97 formula: water 237, osmic acid 1, chromic acid 2, acetic acid 13 1600.0010 Belar 1929 Meth. wiss. Biol, 1 :638 formula: water 250, osmic acid 1, chromic acid 2, acetic acid 1.5 1600.0010 Bonn test. 1937 Gatenby and Painter Gatenby and Painter 1937, 675 formula: water 240, osmic acid 0.3, chromic acid 1, acetic acid 7.5 1600.0010 Bonner test. Meyer 1915 Meyer 1915, 198 formula: water 243, osmic acid 0.3, chromic acid 1.1, acetic acid 7 1600.0010 Burkhardt 1892 test. 1910 Poll Ehrlich, Krause, et al. 1910, 2 :248 formula: water 250, osmic acid 1.7, chromic acid 2.5, acetic acid 20 1600.0010 Catcheside 1934 1032,48:601 formula: water 250, osmic acid 0.4, chromic acid 4.5, acetic acid 3, maltose 3 1600.0010 Chamberlain 1906 3430, 42:321 formula: water 220, osmic acid 0.05, chromic acid 2.5, acetic acid 25 1600.0010 Cori 1890 23632, 6 :441 formula: water 250, osmic acid 0.025, chromic acid 0.6, acetic acid 0.25 1600.0010 Duggar 1909 test. 1937 Gatenby and Painter Gatenby and Painter 1937, 702 formula: water 250, osmic acid 0.5, chromic acid 1.25, acetic acid 0.5 1600.0010 Ferguson 1904 see F 1600.0010 Mottier 1897 (note) 1600.0010 Fischler 1906 10606, 48 :42 formula: water 250, osmic acid 0.27, chromic acid 1.0, acetic acid 6.5 1600.0010 Flemming 1882 Flemming 1882, 381 formula: water 250, osmic acid 0.25, chromic acid 0.6, acetic acid 0.25 note: This is "Flemming's first or weak mixture" (Gatenby and Cowdry 1928, 37). 1600.0010 Flemming 1884 23632, 1 :349 formula: water 250, osmic acid 1, chromic acid 1.8, acetic acid 12.5 note: This is "Flemming's second or strong formula" (Gatenby and Cowdry 1928, 37). 1600.0010 Flemming test. 1910 Meves Ehrlich, Krause, et al. 1910, 1:476 formula: water 250, osmic acid 0.5, chromic acid 1.8, acetic acid 12.5 1600.0010 Fol 1884 test. 1928 Gatenby and Cowdry Gatenby and Cowdry, 37 formula: water 250, osmic acid 0.05, chromic acid 0.6, acetic acid 0.25 1600.0010 Friedmann test. 1900 Pollack Pollack 1900, 28 formula: water 240, osmic acid 0.2, chromic acid 2.25, acetic 10 1600.0010 Gates 1907 3430, 43 :81 formula: water 250, osmic acid 0.25, chromic acid 1.7, acetic acid 1.25 1600.0010 Jolly 1907 1823, 9:142 formula: water 250, osmic acid 0.75, chromic acid 1.5, acetic acid 2.5 1600.0010 Laguesse 1901 test. 1907 Bohm and Oppel Bohm and Oppel 1907, 358 formi'la: water 250, osmic acid 3, chromic acid 0.8, acetic acid 0.6 1600.0010 Laguesse test. 1933 Cajal and de Castro Cajal and de Castro 1933, 28 formula: water 240, osmic acid 1.6, chromic acid 1.6, acetic acid 1 1600.0010 Langendorf lest. 1910 Poll Ehriich, Krause, et al. 1910, 2:347 FOHMUL.\: water 250, osmic acid 0.5, chromic acid 1.25, acetic acid 75 1600.0010 Lehrmitte and Guccione 1909 20080, 19 :205 formula: water 250, osmic acid 0.1, chromic acid 0.9, acetic acid 0.4 F 1600.0010-F 1670.0000 FIXATIVES 201 1600.0010 Lillie test. 1929 McClung and Allen IMcClung 1929, 423 fohmula: water 250, osiiiic acid 0.95, chromic acid 1, acetic acid 2 1600.0010 Meves 1910 Ehrlich, Krause, ct al. 1910, 1:476 formula: water 250, osmic acid 1, chromic acid 0.9, acetic acid 12.5 1600.0010 Meves and Duesberg 1908 1780, 71:571 formula: water 250, osmic acid 0.25, chromic acid 1.0, acetic acid 6.25 1600.0010 Mobius 1887 14555,12:174 formula: sea water 250, osmic acid 0.25, chromic acid 0.7, acetic acid 0.25 1600.0010 Mottier 1897 10006,30:109 formula: water 250, osmic acid 0.75, chromic acid 2.0, acetic acid 12.5 note: Ferguson 1904 (16953, 6) is identical. 1600.0010 Newton and Darlington 1919 11211, 21:1 formula: water 250, osmic acid 1, chromic acid 1.5, acetic acid 7 1600.0010 Oguma and Kihara 1923 1825, 33 :493 REAGENTS REQUIRED: A. F 0000.0010 Camoy 1887; B. F 1600.0010 Flemming 1882 method: [small pieces] — » A, 1 min. —* B, 24 hrs. 1600.0010 Schmorl 1928 Schmorl 1928, 217 formula: water 120, 95% ale. 120, osmic acid 1, chromic acid 1.9, acetic acid 12.5 1600.0010 Showalter 1926 1032, 40:713 formula: water 225, osmic acid 0.06 chromic acid 1, acetic acid 3 1600.0010 Sypkens 1904 17770a, 2. formula: water 250, osmic acid 1, chromic acid 1.9, acetic acid 10 1600.0010 Taylor 1924 3430, 78 :236 formula: water 240, osmic acid 0.6, chromic acid 1, acetic acid 4, maltose 3 1600.0010 Yamanouchi 1908 3430,45:145 formula: water 250, osmic acid 0.2, chromic acid 1.0, acetic acid 27 1600.0020 Osmic-chromic-trichloroacetic 1600.0020 Winiwarter test. 1930 Guyer Guyer 1930, 217 formula: water 250, osmic acid 1, chromic acid 2, trichloracetic acid 4 1 600.0030 Osmic-chromic-formic 1600.0030 Guthrie 1928 test. 1928 Gatenby and Cowdry Gatenby and Cowdry, 1928, 66 formula: water 240, osmic acid 1.0, chromic acid 1.9, formic acid 12.5 1 600.0060 Osmic-chromic-hydrochloric 1600.0060 Takahashi 1908 11135, 18:167 formula: water 250, osmic acid 1.25, chromic acid 0.2, hydrochloric acid 0.05 1600.1000 Osmic-chromic-formaldehyde 1600.1000 Kaufman 1929 763, 42 :365 formula: water 237.50, osmic acid 1, chromic acid 1.9, 40% formaldehyde 12.5 1670.0000 OSMIC-CHROMIC-DICHROMATE These are well-known cytological fixatives, particularly for protoplasmic inclusions. The next class (i.e., the present fixatives acidified) give reasonable nuclear fixation as well, but cannot compare with formulas specifically designed for that purpose. 1670.0000 Benda 1901 test. 1948 Romeis Romeis 1948, 231 formula: water 250, osmic acid 1, chromic acid 1.9, acetic acid 2.5 202 METHODS AND FORMULAS F 1670.0000-F 1700.0000 1670.0000 Champy 1911 test. 1942 Langeron Langeron 1942, 384 formula: water 250, osmic acid 1.1, chromic acid 1.0, potassium dichromate 2.9 1670.0000 Nakamura 1928 13367,55:1 formula: water 250, osmic acid 1, chromic acid 2, potassium dichromate 5 1670.0000 Nassanow 1923 1780, 97:136 formula: water 250, osmic acid 1.7, chromic acid 0.85, potassium dichromate 5 1670.0000 Severinghaus 1923 763, 53 :3 formula: water 250, osmic acid 1.2, chromic acid 0.6, potassium dichromate 3.75 1670.0000 Zweibaum 1933 4285a, 10:210 formula: water 250, osmic acid 0.3, chromic acid 1, potassium dichromate 2.7 1 670.001 0 Osmic-chromic-dichr ornate-acetic 1670.0010 LaCour 1929 LaCour's 2B—auct. 14900,124:127 formula: water 250, osmic acid 0.5, chromic acid 1.5, potassium dichromate 1.5, acetic acid 0.8, urea 1.5, sodium sulfate 0.8 1670.0010 LaCour 131 LaCour' s 3BD—aucL 11360,51:119 formula: water 250, osmic acid 0.6, chromic acid 1.0, potassium dichromate 1.0, acetic acid 1.5, saponin 0.1 1670.0010 LaCour 1931c LaCour' s 2BE—auct. 11360,51:124 formula: water 240, osmic acid 0.5, chromic acid 1.4, potassium dichromate 1.5, acetic acid 0.75, saponin 0.05 1670.0010 Smith 1935a Smith's SI— auct. 11211,49:119 formula: water 250, osmic acid 0.8, chromic acid 1.25, potassium dichromate 0.6, acetic acid 1.5, saponin 0.06 1670.0010 Smith 1935b Smith's S2— auct. 11211,49:119 formula: water 250, osmic acid 0.8, chromic acid 1.2, potassium dichromate 1.6, acetic acid 1.0, saponin 0.1 1670.0019 Osmic-chromic-dichr omate-acetic-{other organic acids) 1670.0019 Benda 1903 764, 12 :752 formula: a. water 250, osmic acid 1, potassium dichromate 1.8, acetic acid 6.35; B. water 125, chromic acid 1.25, pyroligneous acid 1.25; C. water 250, potassium dichromate 5 method: a, 8 days -^ water, 1 hr. -^ B, 24 hrs. — > C, 24 hrs. — > running water, 24 hrs. 1670.0090 Osmic-chro7nic-dichromate-{other organic acid) 1670.0090 Champy 1913 1915, 54:307 formula: a. water 250, osmic acid 1.25, chromic acid 1.15, potassium dichromate 3; B. water 175, chromic acid 2.5, pyroligneous acid 35; C. water 250, potassium dichro- mate 7.5 method: a, 24 hrs. -^ distilled water ,J>^ hr. — > B, 20 hrs. -^ distilled water, }i hr. — > C, 3 days -^ running water, 24 hrs. 1700.0000 OSMIC-DICHROMATE These are the best known of all the cytoplasmic fixatives, particularly for the demonstra- tion of mitochondria. They should not, however, be employed as general-purpose fixatives, for which use they were not intended. 1700.0000 Altmann 1890 test. 1928 Gatenby Gatenby and Cowdry formula: water 250, osmic acid 2.5, potassium dichromate 6.25 note: Langeron 1942, 384 assigns an identical formula, without reference, to Altmann 1894. F 1700.0000 FIXATIVES 203 1700.0000 Andriezen 1894 3579, 1 :909 formula: a. water 250, osmic acid 0.13, potassium dichromate 5; B. water 250, osmic acid 0.25, potassium dichromate 4.6 method: [thin slices, suspended by waxed threads] -^ A, 24 hrs. in dark —y B, 48 hrs. — » F 1700.0000 Golgi 1900 3}^ to 6 days 1700.0000 Baker and Thomas 1933 Baker 1945, 97 formula: water 250, osmic acid 2.5, potassium dichromate 4.75 1700.0000 Berkely 1897 10920, 6:1 formula: water 250, osmic acid 0.6, potassium dichromate 6 1700.0000 Cajal 1890 23632, 7 :332 formula: water 250, osmic acid 0.4, potassium dichromate 6 1700.0000 Cajal 1891 6011,8:130 formula: water 250, osmic 0.25, potassium dichromate 6.5 1700.0000 Dekhuyzen 1903 6628, 137:415 formula: sea water 250, osmic acid 0.68, potassium dichromate 5.4 1700.0000 Gedoelst 1889 see F 1700.0000 Golgi 1880 (note) 1700.0000 van Gehuchten test. 1927 Kingsbury and Johannsen Kingsbury and Johannsen 1927, 89 formula: water 250, osmic acid 0.5, potassium dichromate 6 1700.0000 Golgi 1880 test. 1903 ips. Golgi 1903, 1:102 formula: water 250, osmic acid 0.4, potassium dichromate 4 note: This method was republished by Golgi in 1883 (test, ipsi, loc. cit. 2:504). An identical formula is given by Gedoelst 1889 (6011, 5:131). 1700.0000 Golgi 1900 test. 1903 ips. Golgi 1903, 2 :685 formula: water 250, osmic acid 0.7, potassium dichromate 5 note: This formula is identical with the third solution of Andriezen 1894 (see above) but is listed as Golgi 1900 for convenience. 1700.0000 Kolossow 1897 test. 1928 da Fano Gatenby and Cowdry 1928, 608 formula: water 250, osmic acid 0.63, potassium dichromate 10 1700.0000 Lo Bianco 1890 14246, 9:435 formula: water 250, osmic acid 0.05, potassium dichromate 12.5 1700.0000 Lowenthal 1893 23632, 10 :309 formula: water 250, osmic acid 0.5, potassium dichromate 5 1700.0000 Marchi 1886 19460, 12 :50 formula: water 250, osmic acid 0.88, potassium dichromate 1.7, potassium sulfate 0.8 1700.0000 Mettler 1932 20540b, 7:102 formula: water 250, osmic acid 0.5, potassium dichromate 6 1700.0000 Metzner 1907 test. 1920 Mayer Mayer 1920, 53 formula: water 250, osmic acid 9.4, potassium dichromate 4.5 • note: The formula is originally given in terms of a "sat. sol. dichromate." This is here taken to represent a 7.5% sol.; the original may have used anything between 7% and 12%. 1700.0000 Roskin 1946 Roskin 1946, 97 formula: water 165, osmic acid 3.3, potassium dichromate 2.1 1700.0000 Schultze 1904 23632, 21:7 formula: water 250, osmic acid 1.25, potassium dichromate 5.6 1700.0000 Timofecheff test. 1933 Cajal and de Castro Cajal and de Castro 1933, 125 formula: water 240, osmic acid 0.8, potassium dichromate 8 204 METHODS AND FORMULAS F 1700.0000-F 1700.1000 1700.0000 Windle 1926 11135, 40:229 formula: water 250, osmic acid 0.4, potassium dichromate 7.5 1700.0000 Wlassow 1894 2526, 15 :543 formula: water 250, osmic acid 0.1, potassium dichromate 0.6, sod. chloride 2.0 1700.0000 Zietschmann 1903 23635,74:1 formula: water 250, osmic acid 0.75, potassium dichromate 3.2, potassium sulfate 1.6 1 700.001 0 Osmic-dichr ornate-acetic This is a much neglected group of general-purpose fixatives, indicated for histological study in which it is desired to preserve the lipoid constituents of the cell. 1700.0010 Bensley 1911 590, 12 :297 formula: water 250, osmic acid 1, potassium dichromate 4, acetic acid 0.75 1700.0010 Bensley and Bensley 1938 Bensley and Bensley 1938, 45 formula: water 250, osmic acid 1, potassium dichromate 5, acetic acid 1.25 1700.0010 Hoehl 1896 1739, (1896) :32 formula: water 250, osmic acid 0.5, potassium dichromate 6, acetic acid 5 1700.0010 Oxner 1905 10899, 40 :589 formula: water 255, osmic acid 0.6, potassium dichromate 5.25, acetic acid 12.5 1 700.0030 Osmic-dichromate-jorviic 1700.0030 Oxner 1905 10899, 40 :589 formula: water 255, osmic acid 0.6, potassium dichromate 5.25, formic acid 15 1 700.0040 Osmic-dichr omate-nitric 1700.0040 Dekhuyzen 1903 6628, 137:415 formula: sea water 250, osmic acid 0.9, potassium dichromate 6.25, nitric acid 1.2 note: The original formula, which is said to be isotonic with sea water, requires 250 ml. of a 2.5% solution of potassium dichromate in sea water, to be mixed with 25 ml. of N nitric acid and 54 ml. of 2% osmic acid. 1700.1000 Osmic-dichr omate-forvialdehrjde The apparently irrational mixtures that comprise this and the next few classes are in reality of the utmost value, not only for the cytological and neurological studies for which they were originally intended, but also for the fixation of embryos and of small invertebrates. Where nuclear fixation is as important as cytoplasmic fixation, the acidified mixtures should be used. 1700.1000 Fish 1895 21400a, 17:319 formula: water 250, osmic acid 0.025, potassium dichromate 2.5, 40% formaldehyde 0.5, potassium sulfate 2.5 1700.1000 deirisola 1895 test. 1928 da Fano Gatenby and Cowdry 1928, 609 formula: water 235, osmic acid 0.25, potassium dichromate 12.5, 40% formaldehyde 37.5 1700.1000 Maximow 1909 23623,26:179 formula: water 225, osmic acid 0.5, potassium dichromate 5, potassium sulfate 2.5, 40% formaldehyde 25 1700.1000 Mislawsky 1913 23632, 81 :394 formula: water 200, osmic acid 0.125, potassium dichromate 6, 40% formaldehyde 50 1700.1000 Murray 1919 1200, 6:77 formula: a. water 225, potassium dichromate 5.6, potassium sulfate 2.25, 40% formal- dehyde 25; B. water 250, potassium dichromate 6.5, potasshim sulfate 2.5; C. water 250, osmic acid 5 method: [whole organ or large piece] -^ A, overnight — + B, thin slices, 2-7 days -^ C, 2 days -^ running water, 12 hrs. F 1700.1000-F 2000.1010 FIXATIVES 205 1700.1000 Schridder (est. 1928 Gatenby and Cowdry Gatenby and Cowdry 1028, 330 fcjumula: .1. water 225, potassium dichroiiiatc 5."), i)otassiuiii siilfato 2.25, 40% formal- dehyde 25; B. water 250, potassium dichromate 0, potassiinu sulfate 2.5; ('. water 250, osmic acid 5 METHOD FOR MITOCHONDRIA AND FATS IN BIRDS AND M\MMAI,s: A, 2 days -* B, 2-4 days -^ (7, 2 days > running water, 24 hrs. 1700.1000 Smirnow 1895 1780, 52:202 formula: water 250, osmic acid 0.375, potassium dichroraate 9.5 1 700.101 0 Osmic-dichromate-formaldehyde-ncetic 1700.1010 Swank and Davenport 1934 20540b, 9:11 formula: .1. distilled water 225, 40% formaldehyde 25; B. water 250, osmic aeitl 0.85, potassium dichromate 0.34, potassium sulfate 0.8, acetic acid 5 1780.0000 OSMIC-DICHROMATE-(oTHER INORGANIC SALTS) 1780.0000 Cajal 1933a Cajal and de Castro 1933, 29 formula: water 250, osmic acid 0.5, potassium dichromate 6, ferric chloride 12 1780.0000 Cajal 1933b Cajal and de Castro 1933, 29 formula: w^ater 240, osmic acid 0,4, potassium dichromate 4.8, potassium ferricyanide 1.2 ISOO.OOOO 0SMIC-(0THER INORGANIC SALTs) 1800.0000 Bensley and Bensley 1929 590, 44:79 formula: water 250, osmic acid 5, ferric chloride 1.25 1800.0000 Busch 1898 15058, 17:476 formula: water 250, osmic acid 0.75, sodium iodate 2.5 1800.0000 Kolossow 1898 1780, 52 :1 formula: water 250, osmic acid 1.25, uranium nitrate 6.25 1800.0010 (ind .0030 Osmic (icid-{othcr inorganic salts) -{acids) 1800.0010 Frenkel 1893 766, 8 :539 formula: water 250, osmic acid 1.25, acetic acid 1.25, palladium chloride 1.9 1800.0030 Pianese 1899 1886, 2:412 formula: water 250, osmic acid 1, cobalt chloride 2, formic acid 0.5 F 2000 PLATINIC CHLORIDE IN COMBINATIOX WITH FIXATIVE AGENTS OF HIGHER NUMERICAL RANK Platinic chloride is a valuable and neglected fixative agent. Its main value lies in its mordanting power for after-staining, for it appears to interfere less with the staining proper- ties of the tissues than any other reagent. It is stated by Langeron (1942, 372) to have all the advantages of chromic acid wath none of the disadvantages resulting from the discoloration of the tissue. It is particularly valuable in mixtures with chromic acid where it inhibits the production of ehroniic oxide, which causes such gross discoloration and prevents good after- staining. 2000. ] 000 Platinic-formaldehyde 2000.1000 Cajal 1893 test. Pollack 1900 Pollack 1900, 133 formula: water 150, platinic chloride 0.08, 40% formaldehyde 100 2000.1010 Platinic-formaldehyde-acetic 2000.1010 Bouin 1900 1825, 17:211 formula: water 200, platinic rhioride 1.8, 40% formaldehyde 50, acetic 12.5 2000.1010 Retterer 1900 11024, 36:508 formula: water 125, platinic chloride 6.25, 40% formaldehyde 125, acetic 7.5 206 METHODS AND FORMULAS F 2300.0000-F 2500.1030 2300.0000 Platinic-mercuric This is an admirable fixative, particularly before complex staining techniques. Where nuclear fixation is of importance the acidified mixtures of the next class should be used. 2300.0000 Becher and Demoll 1913 Becher and Demoll 1913, 43 formula: water 250, platinic chloride 0.7, mercuric chloride 3.75 2300.0000 Rabl 1894 23632, 11:165 formula: water 250, platinic chloride 0.625, mercuric chloride 4 2300.001 0 Platinic-mer curie-acetic 2300.0010 Bouin test. 1910 Spuler EhrUch, Krause, et al. 1910, 2:523 formula: water 240, platinic chloride 1.6, mercuric chloride 5.6, acetic acid 24 2300.0010 Hoffmann 1908 23635, 89 :598 formula: 25% ale. 250, platinic chloride 1.25, mercuric chloride 4.5, acetic acid 12.5 2300.0010 Lenhossek 1898 1780, 51 :220 formula: water 250, platinic chloride 1.25, mercuric chloride 6.4, acetic acid 12.5 2300.1 000 Platinic-mercurie-formaldehyde 2300.1000 Sziits 1913 23632,29:290 formula: water 190, platinic chloride .625, mercuric chloride 7.6, 40% formaldehyde 62.5 2300.1 030 Platinic-mereurie-formaldehyde-formic It is unfortunate that the name of Bouin should be so widely associated with picric- formaldehyde-acetic mixtures. Undoubtedly the best general-purpose fixative which he con- tributed is the only present occupant of this class. It gives a better general-purpose fixation than does the more generally employed "Bouin's fluid," and permits better and more billiant after-staining. 2300.1030 Bouin 1898 2844, 6:54 formula: water 180, platinic chloride 1, mercuric chloride 4.2, 40% formaldehyde 60, formic acid 30 2356.0000 platinic-mercuric-picric-chromic 2356.0000 von Pacaut 1906 1823, 8 :438 formula: water 250, platinic chloride 0.1, mercuric chloride 17.5, picric acid 3.5, chro- mic acid 5 note: This formula is referred by Mayer 1920 to Pacaut 1905 see 6593 (1905) :407. Spuler 1910 (Ehrlich, Krause, et al. 1910, 2 :522) refers to Pacaut 1905 but gives the 1906 journal reference. 2470.0000 Platinic-cupric-dichromate ' 2470.0000 Burkhardt 1897 6011, 12:335 formula : water 240, platinic chloride, copper dichromate 9 2500.0010 Platinic-picric-acetic 2500.0010 vom Rath 1895 766, 11 :282 formula: water 250, platinic chloride 1.25, picric acid 3, acetic acid 2.5 2500.1030 Platinic-pieric-formaldehjde-formic 2500.1030 Bouin and Bouin 1898 test circ. 1938 Wellings Wellings circ. 1938, 31 formula: water 180, platinic chloride 0.9, picric acid 1, 40% formaldehyde 45, formic acid 23 F 2600.0000-F 3000.0000 FIXATIVES 207 2600.0000 Platinic-chkomic The purpose of the platinic constituent of these mixtures is to prevent the discoloration of tissues by the chromic acid. The fixative picture obtained does not differ from chromic alone, or chromic-acetic, in the two classes here given. 2600.0000 Eisig 1878 14246, 1 :341 formula: water 250, platinic chloride 0.32, chromic acid 1.25 note: This solution is frequently referred to Whitman who republished it (Whitman 1885, 153, 238) without reference. 2600.0000 Merkel 1870 test. 1910 Poll Ehrlich, Krause, et al. 1910, 1 :224 formula: water 250, chromic acid 0.5, platinic chloride 0.5 2600.0000 Whitman 1885 see F 2600.0000 Eisig 1878 (note) 2600.001 0 Platinic-chromic-acetic 2600.0010 Brass 1884 23632, 1 :39 formula: water 250, platinic chloride 0.35 to 0.85, chromic acid 0.35 to 0.85, acetic acid 0.35 to 0.85 2600.0010 Lavdowsky 1894 764, 4:355 formula: water 250, platinic chloride 0.12, chromic acid 2.4, acetic acid 12.5 2700.0000 Platinic-dichromate 2700.0000 Roncoroni test. 1900 Pollack Pollack 1900, 106 REAGENTS REQUIRED: A. V 7000.0000 Midler 1859; B. 0.8% platinic chloride F 3000 MERCURIC CHLORIDE IN COMBINATION WITH FIXATIVE AGENTS OF HIGHER NUMERICAL RANK Mercuric chloride is probably the best known and most widely used fixative agent. It has many disadvantages. In the first place, it is a dangerous poison, which can be absorbed through the skin (of those sensitive to it) and produce chronic, cumulative mercury poison- ing. Unless the individual desiring to use these solutions is satisfied that he is not sensitive to mercury, he should wear rubber gloves when handling the solution. Even those not sensitive should take the utmost precautions to prevent contact of this dangerous material with the bare skin. The second disadvantage is that once a material has been placed in mercuric solu- tion, it cannot be handled with any metal instrument, but must be manipulated entirely with instruments of glass, wood, or plastic. Mercuric chloride has also a tendency to render materials brittle. The last disadvantage is that this reagent tends to cause a precipitate of small crystals in the tissues unless it is washed out either by very prolonged washing in water, or by relatively prolonged washing in solutions of iodine, which must then themselves be removed by subsequent washing in alcohol. Against these disadvantages is the fact that mercuric fixatives permit the most brilliant after-staining of almost any class. Mercuric chloride is not usefully employed in simple aqueous solution, but is occasionally useful, in the formulas which follow, in alcohol solution. 3000.0000 Mercuric Alone 3000.0000 Apathy test. 1920 Mayer Mayer 1920, 56 formula: 50% ale. 250, mercuric chloride 9, sodium chloride 1.25 3000.0000 Giemsa 1909 test. 1938 Mallory MaUory 1938, 41 formula: water 165, 95% ale. 85, mercuric chloride 11.5 3000.0000 Heidenhain 1888 16155, 43 (Suppl.): 40 formula: water 250, mercuric chloride 22, sodium chloride 1.25 3000.0000 Lenhossek 1899 test. circ. 1938 Welling Welling 1938, 29 formula: water 115, 95% ale. 135, mercuric chloride 9, sodium chloride 11 3000.0000 Lowit 1887 20170, 95:144 formula: water 250, mercuric chloride 0.2, sodium sulfate 6, sodium chloride 2.5 3000.0000 Neukirch 1909 see DS 22.6 Xeukirch lUOO 208 METHODS AND FORMULAS F 3000.0000-F 3000.0010 3000.0000 Pietschmann 1905 1683, 16 :63 formula: 90% ale. 250, mercuric chloride 0.9 3000.0000 Prowazeko 1906 see F 3000.0000 Schaudinn 1893 (note) 3000.0000 Rothig 1900 1780, 36 :354 formula: water 225, 95% ale. 25, mercuric chloride 15.7 3000.0000 Schaudinn 1893 13635, 57:19 formula: 60% ale. 250, mercuric chloride 5.5 note: Prowazeko 1906 (23632, 23:1) specifies 90% ale. for the above solutions. 3000.0000 Schaudinn 1900 23831, 13:197 formula: 30 %> ale. 250, mercuric chloride 10 3000.0000 Schmorl 1928 Schmorl 1928, 29 formula: water 235, mercuric chloride 11, sodium chloride 1.25 3000.0010 Mercuric-acetic The mercuric-acetic mixtures share with the chromic-acetic mixtures the honor of being the most popular fixatives. It is probable that they deserve this. They are simple to make up, stable in sohition, easy to handle, and do not harden as badly as does mercuric chloride alone. They permit the most brilliant after-staining, and are widely used for the fixation of marine forms. They can be confidently recommended to the inexperienced worker, provided that he remembers to wash out the mercury thoroughlj' and not to handle the fixed object with any metal instrument until washing is complete. 3000.0010 Altman 1890 test. 1920 Mayer IMayer 1920, 59 formula: water 200, mercuric chloride 3.5, acetic acid 50 3000.0010 Apathy 1896 14246,12:495 formula: water 150, ale. 100, mercuric chloride 5, acetic acid 1.25 3000.0010 Beguin test. 1910 Spuler Ehrlich, Krause, et al. 1910, 521 formula: water 225, mercuric chloride 16, acetic acid 25 3000.0010 van Beneden test. 1905 Lee Lee 1905, 54 formula: water 190, mercuric chloride to sat. (circ. 20), acetic acid 60 note: Both Lee 1905, 54 and Mayer 1920, 53 attribute this solution to van Beneden, but neither quotes a reference. 3000.0010 Bignami test. 1896 Kahlden and Laurent Kahlden and Laurent 1896, 127 formula: water 250, mercuric chloride 2.5, acetic acid 2.5, sodium chloride 2 3000.0010 Borgert 1900 23831, 14:207 formula: water 190, mercuric chloride 13.5, acetic acid 60 3000.0010 Carazzi test. 1920 Mayer Mayer 1920, 56 formula: 30% ale. 240, mercuric chloride 5.2, acetic acid 8.3 3000.0010 Carnoy and Lebrun 1887 6011,13:68 formula: abs. ale. 80, acetic acid 80, chloroform 80, mercuric chloride to sat. {circ. 60) note: The original authors attribute this mixture to Gilson, by whom it was never pub- lished. It is, however, frequently referred to as "Gilson's Mixture." Langeron 1934, 344 calls it "Gilson (1897)" but gives no reference. 3000.0010 Carter 1919 1032,13:213 formula: water 125, 95% ale. 125, mercuric chloride 7.5, acetic acid 7.5 3000.0010 Cholodkowsky test. 1910 Spuler Ehrlich, Krause, et al. 1910, 2:521 formula: water 250, mercuric chloride 17.5, acetic acid 1.25 3000.0010 Coker 1902 3430, 33 :90 formula: water 250, mercuric chloride 0.9, acetic acid 2.5 F 3000.0010 FIXATIVES 209 3000.0010 Cornwall 1937 Microscope, 1:137 KORMUi.A: water 250, clinniiic iicid 1.25, acetic acid 2.5 3000.0010 Davidoff 1889 112IG, 9:118 K()HMUL.\: water 190, mercuric chloride 13.2, acetic acid (10 3000.0010 Eisig 1898 14240, 13:89 i'()RMi't,,\: sea water CIO, mercuric cliloride 0.5, acetic acid (iO 3000.0010 Eltringham 1930 fixative B—auct. Eltringham 1930, 44 formula: water 145, 95% ale. 100, mercuric chloride 10, acetic acid 9 RECOMMENDED FOR: whole iiisccts iiot larger than a housefly. 3000.0010 Gilson 1897 see F 3000.0010 Carnoy and Lebrun 1887 (note) 3000.0010 Goto 1898 11130, 10:239 formula: water 250, mercuric chloride 4.2, acetic acid 3.75, glycerol 11.25 3000.0010 Hein test. 1910 Spuler Ehrlich, Krausc, et at. 1910, 2:521 formula: sea water 245, mercuric chloride 20, (or to sat.), acetic acid 5 3000.0010 Jager test. 1928 Schmorl Schmorl 1928, 432 formula: water 165, 95% ale. 85, mercuric chloride 1.7, acetic acid 0.3 3000.0010 Kaiser 1891 2842, 7:4 formula: water 250, mercuric chloride 8.3, acetic acid 7.5 3000.0010 Kolster test. 1910 Spuler Ehrlich, Krause, et al. 1910, 2:521 formula: water 250, mercuric chloride 20, acetic acid 2.5, sodium chloride 1.25 3000.0010 Laidlaw test. 1936 Pappenheimer and Hawthorne 608b, 12:627 formula: water 250, mercuric chloride 10, acetic acid 12.5 3000.0010 Lang 1878 766, 1:14 formula: water 250, mercuric chloride 15, acetic acid 17.5, sodium chloride 20 3000.0010 Langeron 1942 Langeron 1942, 387 formula: water 22.5, mercuric chloride 13.5, acetic acid 7.5 3000.0010 Lapp test. 1910 Spuler Ehrlich, Krause, et al. 1910, 2:251 formula: 50% ale 250, mercuric chloride 9, acetic acid 5 3000.0010 Lenhossek 1897 1780, 51:215 formula: 25% ale. 250, mercuric chloride 12.5, acetic acid 12.5 3000.0010 Lo Bianco 1890 14246, 9:443 formula: water 210, mercuric chloride 11.5, acetic acid 42.5 3000.0010 Mingazzini 1893 19353, 3 :47 formula: 30% ale. 190, mercuric chloride 9, acetic acid 62.5 3000.0010 Ohlmacher 1897 11189,3:671 formula: abs. ale. 200, chloroform 37.5, mercuric chloride 50, acetic acid 12.5 note: This formula is referred by Spuler 1910 (Ehrlich, Krause, et al. 1910, 2:521) to 1899. 3000.0010 Oxner test. 1910 Spuler Ehrlich, Krause, et al. 1910, 2:521 formula: water 240, mercuric chloride 17.5, acetic acid 7.5 3000.0010 vom Rath 1895 766, 11:286 formula: abs. ale. 250, mercuric chloride 1.25, acetic acid 5 3000.0010 Pearl 1903 test. circ. 1938 Wellings Wellings circ. 1938, 29 formula: water 225, mercuric chloride 16, acetic acid 25 3000.0010 Roskin 1946 Roskin 1946, 94 formula: water 235, mercuric chloride 14, acetic acid 15 210 METHODS AND FORMULAS F 3000.0010-F 3000.0040 3000.0010 Schmorl 1928 Schmorl 1928, 29 formula: water 250, mercuric chloride 7.5, acetic acid 1.5 3000.0010 Sherlock test. 1930 Eltringham Eltringham 1930, 46 formula: water 238, mercuric chloride 17, acetic acid 12 3000.0010 Spuler 1910 EhrHch, Krause, et al. 1910, 2 :521 formula: abs. ale. 250, mercuric chloride 10, acetic acid 7.5 3000.0010 Wenrich and Geiman 1933 20540b, 8:158 formula: water 200, abs. ale. 50, mercuric chloride 8, acetic acid 5 3000.0010 Woltereck test. 1910 Spuler Ehrlich, Krause, et al. 1910, 2:251 formula: water 100, 90% ale. 150, mercuric chloride 9, acetic acid 25 3000.0012 Mercuric-acetic-trichloroacetic This mixture is thought by many to be superior to mercuric-acetic, but the author has never seen any evidence, as apart from opinion, that this is true. 3000.0012 Heidenhain 1909 23632, 25 :405 formula: water 250, mercuric chloride 17.5, acetic acid 2.5, trichloroacetic acid 5 note: This is Heidenhain' s Subtriessig or Heidenhain' s suh-tri-acetic of some authors. It is given by Cajal and de Castro 1933 as a decalcifying solution, for which purpose it is probably excellent. 3000.0014 Mercuric-acetic-nitric The formula of Gilson 1898 below is, in the author's opinion, one of the finest general- purpose fixatives which can be employed by those without previous experience of fixation. Objects may be left in it for weeks at a time without becoming unduly hardened. It holds a very reasonable balance between cytoplasmic and nuclear fixation, and it is particularly good before any of the more complex triple and quadruple stains. It was once more widely em- ployed than it is at present, but it seems to have been removed from popular esteem through the present passion for Bouin's picro-formol-acetic. 3000.0014 Carazzi test. 1910 Spuler Ehrlich, Krause, et al. 1910, 2:522 formula: water 250, 70% ale. 25, mercuric chloride 5, acetic acid 1.25, nitric acid 3.75, sodium chloride 2.5 3000.0014 Duggar 1909 test. 1937 Gatenby and Painter Gatenby and Painter 1937, 701 formula: water 225, 96% ale. 25, mercuric chloride 8.5, acetic acid 1.7 nitric acid 4 3000.0014 Gilson 1898 6011,14:374 formula: water 220, 60% ale. 25, mercuric chloride 5, acetic acid 1, nitric acid 4.5 3000.0014 Petrunkewitsch 1901 23831,14:576 formula: water 150, 95% ale. 100, mercuric chloride to sat., acetic acid 45, nitric acid 5 SOOO . 0020 Mercuric-trichloroacetic 3000.0020 Huber test. 1943 Cowdry cit. Addison Cowdry 1943, 95 formula: 95% ale. 250, mercuric chloride 7.5, trichloroacetic acid 3.75 3000.0030 Mercuric-formic 3000.0030 Altmann 1890 test. 1920 Mayer Mayer 1920, 09 formula: water 200, mercuric chloride 3.5, formic acid 50 3000.0030 Ruffini 1905 23635, 79:150 formula: water 235, mercuric chloride 6.25, formic acid 16 3000.0040 Mercuric-nitric 3000.0040 Apathy and Boeke test. 1910a Spuler Ehrlich, Krause, et al. 1910, 2 :524 formula: water 240, mercuric chloride 15, nitric acid 10 F3000.0040-F 3000.1000 FIXATIVES 211 3000.0040 Apathy and Boeke test. 1910b Spuler Ehrlich, Krause, et al. 1910, 2 :524 formula: water 240, inercurit,' chloride 25, nitric acid 10 3000.0040 Frenzel 1886 1780, 26 :232 formula: 80% ale. 125, sat. sol. mercuric chloride in 80% ale. 125, nitric acid 10 note: Mercuric chloride is about 20% soluble in 80% ale.; the 125 parts quoted above therefore contain about 25 of the dry salt. 3000.0040 Kostanecki and Siedlecki 1896 1780, 48:184 formula: water 250, mercuric chloride 9, nitric acid 7.5 3000.0040 Saling 1906 test. 1937 Gatenby and Painter Gatenby and Painter 1937, 389 formula: water 125, 957o ale. 100, mercuric chloride 5, nitric acid 10 3000.0060 Mercuric-hydrochloric 3000.0060 Meyer 1912 test. 1915 ips. Meyer 1915, 198 formula: 95% ale. 250, mercuric chloride 12.5, hydrochloric acid 1 3000.1000 Mercuric-formaldehyde These are very bad fixatives, save for a few specialized purposes for which some have been recommended by their inventors. 3000.1000 Bouin 1900 1825, 17:211 formi'la: water 190, mercuric chloride 11.25, 40% formaldehyde 62.5 , 3000.1000 Brinkmann 1903 14246, 16:367 formula: water 220, mercuric chloride 8.8, 40% formaldehyde 30, sodium chloride 2.2 3000.1000 Carleton and Leach 1938 Carleton and Leach 1938, 33 formula: water 225, mercuric chloride 16, 40% formaldehyde 25 3000.1000 Dawson and Friedgood 1938 20540b, 13:17 formula: water 225, mercuric chloride 9, 40% formaldehyde 25, sodium chloride 1.8 3000.1000 Downey 1913 8545, 15:25 formula: water 225, mercuric chloride 20, 40% formaldehyde 25, sodium chloride 2.2 3000.1000 Eltringham 1930 fixative A — auct. Eltringham 1930, 44 formula: water 225, mercuric chloride 15, 40% formaldehyde 25 recommended for: soft parts of insects free of chitin. 3000.1000 Gilson (1905) see P 13.1 Gilson (1905) 3000.1000 Heidenhain 1916a Heidenhain's weak mixture — compl. script. 23632, 32 :365 formula: water 200, mercuric chloride 10, 40% formaldehyde 50, sodium chloride 1.25 3000.1000 Heidenhain 1916b Heidenhain's strong mixture — compl. script. 23632, 32 :365 formula: water 125, mercuric chloride 10, 40% formaldehyde 125, sodium chloride 1.25 3000.1000 "J.A." test. 1937 Findlay 11360, 57:294 formula: water 225, mercuric chloride 6.25, 40% formaldehyde 25 3000.1000 Rosenthal 1900 test. 1907 Bohm and Oppel Bohm and Oppel 1907, 202 formula: sat. sol. picric acid 250, 40 7o formaldehyde 12.5 use: fixation of adipose tissues before DS 22.4 techniques. 3000.1000 Schmorl 1928 Schmorl 1928, 29 formula: water 200, mercuric chloride 11.25, sodium chloride 1.25, 40% formaldehyde 50 3000.1 01 0 Mercuric-formnldeh^jde-aceiic These are slightly better than the preceding class, but they are not nearly as good as straight mercuric-acetic mixtures. There seems to be no reason for the inclusion of the formaldehyde in a general formula of this nature. 212 METHODS AND FORMULAS F 3000.1010-F 3000.3000 3000.1010 Bohm and Oppel 1907 Bohm and Oppel 1907, 114 formula: water 200, mercuric chloride 12.5, 40% formaldehyde 25, acetic acid 12.5 3000.1010 Bouin 1900 1825, 17:211 formula: water 190, mercuric chloride 1.8, 40% formaldehyde 50, acetic acid 12.5 3000.1010 Cox 1891 1780, 37:16 formula: water 150, mercuric chloride 10.5, 40% formaldehyde 50, acetic acid 25 3000.1010 Destin test. 1943 Cowdry Cowdry 1943, 61 formula: water 230, chromic acid 2.3, 40% formaldehyde 15, acetic acid 5 3000.1010 Gregg and Puckett 1943 20540b, 18:179 formula: water 225, 40% formaldehyde 20, acetic acid 5, mercuric chloride 12.5 3000.1010 Gough and Fulton 1929 see ADS 12.2 Gough and Fulton 1929 3000.1010 Heinz 1910 2526,29:369 formula: water 200, mercuric chloride 8.8, 40% formaldehyde 50, acetic acid 1.25 3000.1010 Spuler 1910 Ehrlich, Krause, et al. 1910, 2 :521 formula: water 225, mercuric chloride 6.75, 40% formaldehyde 25, acetic acid 0.25 3000.1010 Stieve test. 1948 Romeis Romeis 1948, 74 formula: water 190, mercuric chloride 11.5, 40% formaldehyde 50, acetic acid 10 3000.1010 Stieve test. 1946 Roskin Roskin 1946, 94 formula: water 190, mercuric chloride 2, 40% formaldehyde 50, acetic acid 10 3000.1010 Worcester test. 1929 McClung and Allen McClung 1929, 420 formula: water 200, mercuric chloride 14, 40% formaldehyde 22.5, acetic acid 25 SOOO.l 01 2 Mercuric-formaldehyde-acetic-trichloroacetic 3000.1012 Heidenhain 1916 Susa—compl. script. 23632,32:365 formula: water 200, mercuric chloride 11.25, 40% formaldehyde 50, acetic acid 10, trichloroacetic acid 5, sodium chloride 1.25 note: This mixture, named by its inventor, Susa, was first published in 1916 (loc. cit.) but the formula involved using Heidenhain 1916 F 3000.1000; the rationalized formula from dry salts did not appear till 1917 (23632, 33:233). This formula is still beloved of some pathologists. 3000.1012 McNamara, Murphy, and Gore 1940 see AF 21.1 McNamara, et at. 1941. 3000.1012 Romeis 1918 23422,6:101 formula: water 176, mercuric chloride 6, 40% formaldehyde 60, trichloroacetic acid 4 SOOO.l 020 Mercuric-formaldehyde-trichloroacetic 3000.1020 Hartz 1950 Tech. Bull, 20 :77 formula: water 200, mercuric chloride 12, 40% formaldehyde 60, trichloroacetic acid 0.2 3000.1020 Romeis 1918 test. 1920 Mayer Mayer 1920, 56 formula: water 220, mercuric chloride 12.5, 40% formaldehyde 12, trichloroacetic acid 5 8000.1310 Mercuric-formaldehyde-acetone-acetic 3000.1310 Sz.-Gyorgyi 1914 23632,31:23 formula: acetone 185, mercuric chloride 6, 40% formaldehyde 60, acetic acid 7.5 8000.3000 Mercuric-acetone 3000.3000 Held 1897 1739, (1897) :227 formula: water 150, acetone 100, mercuric chloride 2.5 F 3000.3010-F 3500.0010 FIXATIVES 213 SOOO.SOl 0 Mercuric-acetone-acetic 3000.3010 Lepine and Santter 1936 42852, 13 :287 formula: abs. ale. 80, mercuric chloride 20, acetone 80, acetic acid 80 3400.0000 Mercuric-cupric Mercuric-cupric mixtures are excellent fixatives for small invertebrates, and have been widely used for the fixation of marine forms. As histological fixatives they are inferior. 3400.0000 Lo Bianco 1890 14246, 9:443 formula: water 250, mercuric chloride 1.75, copper sulfate 25 3400. 1010 Mercuric-cupric-forma Idehyde-acetic 3400.1010 Becher and DemoU 1913 Becher and Demoll 1913, 48 formula: water 230, copper sulfate 5, mercuric chloride to sat. 40% formaldehyde 20, acetic acid 1.25 3400.1010 NeUs 1900 3678,72:6 formula: water 210, mercuric chloride 17.5, 40% formaldehyde 35, acetic acid 1.25, copper sulfate 5 3400.1014 Mercuric-cwpric-Jormaldehyde-acetic-nitric 3400.1014 Stappers 1909 6011,25:356 formula: water 220, 60% ale. 25, mercuric chloride 5, copper nitrate 5, 40% formalde- hyde 12.5, acetic acid 1, nitric acid 4.5 3470.1000 MERCURIC-CUPRIC-DICHROAIATE-FORMALDEHYDE 3470.1000 Kingsbury 1912 763,6:48 formula: water 225, mercuric chloride 7.5, copper sulfate 2.5, copper dichromate 6.25, 40% formaldehyde 25 3500.0000 Mercuric-picric Mercuric-picric mixtures, whether acidified or modified with formaldehyde, are doubtfully an improvement over mercuric alone. There seems to be no reason for incorporating picric acid with its attendant disadvantages of color. These mixtures have, however, been widely used in the past and are still sometimes recommended for embryological purposes. 3500.0000 Baumgairtel 1918 see DS 23.5 Baumgiirtel 1918 3500.0000 Imhof test. 1910 Spuler Ehrlieh, Krause, et al. 1910, 1:251 formula: water 250, mercuric chloride 6.3, picric acid 1.0 3500.0000 Jeffry test. 1915 Chamberlain Chamberlain 1915, 29 formula: sat. sol. mercuric chloride in 30% ale. 187, sat. sol. picric acid in 30% ale. 63 3500.0000 Lenhossek 1897 1780, 51:215 formula: water 250, mercuric chloride 8.75, picric acid 1.6 3500.0000 Mann 1894 23632,11:479 formula: water 250, mercuric chloride 20, picric acid 2.5, sodium chloride 2.0 3500.0000 Rabll894 23632,11:165 formula: water 250, mercuric chloride 4.5, picric acid 0.75 note: Ehrlieh, Krause, et al. 1910, 1:478, attribute an identical formula to Zilliacus 1905 3500.0000 Zilliacus 1906 see F 3500.0000 Rabl 1894 (note) S500.0010 Mercuric-picric-acetic 3500.0010 Becher and Demoll 1913 Becher and Demoll 1913, 43 formula: water 245, mercuric chloride 3.75, picric acid 0.75, acetic acid 5 214 METHODS AND FORMULAS F 3500.0010-F 3500.1010 3500.0010 Bohm and Oppel test. 1910 Spuler Ehrlich, Krause, et al. 1910, 2 :523 formula: a. water 237.5, mercuric chloride 6.5, acetic acid 12.5; B. water 250, picric acid 3 method: [reptile eggs] -♦ A, 2-3 hrs, -^ B, 12-24 hrs. -y 70% ale, 3500.0010 Fish 1896 test. 1920 Mayer Mayer 1920, 57 formula: water 250, mercuric chloride 1.25, picric acid 0.25, acetic acid 2.5 3500.0010 Lenhossek 1907 10157, 24:293 formula: water 210, 95% ale. 30, mercuric chloride 12.5, picric acid 3 (to sat.), acetic acid 12.5 3500.0010 Michaelis test. 1948 Romeis Romeis 1948, 71 formula: water 250, mercuric chloride 3.6, picric acid 0.6, acetic acid 3 3500.0010 Tellyesniczky 1898 1780, 52 :242 formula: water 250, mercuric chloride 8.75, picric acid 1.6, acetic acid 2.5 3500.0010 Volker test. 1910 Spuler Ehrlich, Krause, et al. 1910, 2:251 formula: water 240, mercuric chloride 8, picric acid 1.25, acetic acid 12.5 3500.0010 Winge 1930 23639b, 10 :699 formula: abs. ale. 150, mercuric chloride 2, picric acid 2.5, acetic acid 25, urea 7.5, chloroform 75 3500.0015 Mercuric-picric-acetic-sulfuric 3500.0015 Lang test. 1910 Spuler Ehrlich, Krause, et al. 1910, 2 :523 formula: water 250, mercuric chloride 15, picric acid 3, acetic acid 12.5, sulfuric acid 5 3500.1 000 Mercuric-picric-formaldehyde 3500.1000 Cretin 1938 Le Mans, 48 formula: water 225, mercuric chloride 6.5, picric acid 1.5, 40% formaldehyde 25 3500.1000 Kingsley 1937 8545, 57 :87 formula: water 230, mercuric chloride 15, picric acid 0.5, 40 % formaldehyde 20, sodium chloride 1.75 3500.1000 Mann 1894 23632, 11:479 formula: water 250, mercuric chloride 6.25, picric acid 2.5, 40% formaldehyde 25 3500.1000 Mann 1898 22246, 12 :39 formula: water 250, mercuric chloride 6.25, picric acid 2.5, 40% formaldehyde 12.5 3500.1000 "Mann" see F 3500.1000 Spuler 1910 (note) 3500.1000 Spuler 1910 Ehriich, Krause, et al. 1910, 2 :522 formula: water 180, mercuric chloride 6.3, picric acid 1.0, 40% formaldehyde 60 note: Spuler {loc. cit.) attributes this, without citing his source, to Mann. 3500 .1010 Mercuric-picric-formaldehyde-acetic These are, to all intents and purposes, a picro-formaldehyde-acetic improved by the addi- tion of mercury. This improvement doubtfully justifies the use of the mixture for general purposes. 3500.1010 Branca 1899 11024,35:767 formula: water 210, mercuric chloride 15, picric acid 2.5, 40% formaldehyde 40, acetic acid 3.75 3500.1010 Gray 1932 11360, 52:370 stock solution: 95% ale. 250, mercuric chloride 2.5, picric acid 2.5 WORKING solutions: .\. stock 125, 40% formaldehyde 62.5, acetic acid 25, ether 37.5 B. stock 125, 40% formaldehyde 62.5, acetic acid 50, ether 12.5 C. stock 140, 40% formaldehyde 70, acetic acid 14, ether 28 note: a detailed description of the uses of these fluids is given in Chapter 16. F 3500. 101 OF 3670.0000 FIXATIVES 215 3500.1010 Pfuhl 1932 Supifonneis—aud. 23507a, 31:18 formula: water 200, mercuric chloride 6, picric acid 1, 40% formaldehyde 50, acetic acid 12.5 3500.1010 Yokum 1918 22084, 18:337 formula: 95% ale. 140, ether 25, picric acid 1.25, mercuric chloride 2.5, 40% formalde- hyde 62, acetic acid 25 3560.0040 MERCURIC-PICRIC-CHROMIC-NITEIC 3560.0040 Hennings 1900 23632, 17:311 formula: water 86, abs. ale. 128, mercuric chloride 8, picric acid 0.3, chromic acid 0.2, nitric acid 36 note: This is stated by the author to soften chitin. 3600.0000 Mercuric-chromic Mercuric-chromic mixtures are good fixatives, though the mercuric constituent tends to make small forms more brittle than do the simple chromic fixatives. They are in general to be recommended for cytoplasmic rather than nuclear fixation. 3600.0000 Lo Bianco 1890 14246, 9 :443 formula: water 250, mercuric chloride 1.2, chromic acid 0.7 3600.0000 Mann 1898 22246, 12 :39 formula: water 250, mercuric chloride 6.25, chromic acid 6.25 8600.001 0 Mercuric-chromic-acetic 3600.0010 Novak 1902 test. 1910 Poll Ehrlich, Krause, et al. 1910, 1:225 formula: water 200, mercuric chloride 2, chromic acid 0.25, acetic acid 50 3600. 0040 Mercuric-chromic-nitric 3600.0040 Kingsbury and Johannsen 1927 Kingsbury and Johannsen 1927, 75 formula: water 185, mercuric chloride 7.5, F 6000.0040 Perenyi 1882 65 recommended for: stated to prevent hardening of chitinized structure. 3600.1010 Mercuric-chromic-Jormaldehyde-acetic 3600.1010 Gerhardt 1901 766, 20 :244 formula: water 225, mercuric chloride 4.25, chromic acid 0.7, acetic acid 6.25, 40% formaldehyde 62.5 3600.1010 Hertwig 1905 test. circ. 1938 Wellings Wellings circ. 1938, 29 formula: water 220, mercuric chloride 5.25, chromic acid 0.75, 40% formaldehyde 25, acetic acid 7.5 3600.1010 Novak 1901 766, 20 :244 formula: water 225, mercuric chloride 5.3, chromic acid 0.75, 40% formaldehyde 25, acetic acid 7.5 3600.1010 Rothig 1904 test. 1948 Romeis Romeis 1948, 552 formula: water 217, mercuric chloride 5.4, chromic acid 0.75, acetic acid 7.5, 40% formaldehyde 25 3670.0000 mercuric-chromic-dichromate 3670.0000 Williamson and Pearse 1923 11025, 57:193 formula: water 250, mercuric cliloride 12.5, potassium dichromate 5, chromium fluoride 5 preparation: Boil the dichromate and fluoride 30 minutes. Cool, filter, and add mercuric chloride to filtrate. Boil till solution is complete. 216 METHODS AND FORMULAS F 3670.0010-F 3700.0010 3670.001 0 Mercuric-chromic-dichromate-acetic 3670.0010 Ruffini 1927 Ruffini 1927, 7 formula: water 250, mercuric chloride 3, chromic acid 1.8, potassium dichromate 1.2, sodium sulfate 0.6, acetic acid 12 note: a second formula (loc. cit. p. 63) reduces the acetic acid to 3. 3700.0000 Mercuric-dichromate Mercuric-dichromate fixatives are among the better known cytoplasmic reagents. They are probably better for this purpose than the mercuric-chromics of the previous classes. 3700.0000 Bensley 1910 763,4:379 formula: water 125, 95% ale. 125, mercuric chloride 32, potassium dichromate 2.5 note: The original formula calls for a mixture of equal parts 2% dichromate and a sat. sol. IIgCl2 in abs. ale. Spuler 1910 (Ehrlich, Krause, ei al. 1.910, 2:254), quotes this formula as "Bensley 1896" but gives no further reference. 3700.0000 Bensley and Bensley 1938 Bensley and Bensley 1938, 37 formula: water 250, mercuric chloride 12.5, potassium dichromate 6.25 3700.0000 Cox test. 1895 Rawitz Rawitz 1895, 17 formula: water 250, mercuric chloride 2.5, potassium dichromate ■ 2.5, potassium chromate 2 3700.0000 Duthie 1937 see F 3700.0000 Lane (1910) (note) 3700.0000 Foa 1891 test. 1920 Mayer Mayer 1920, 52 formula: water 250, mercuric chloride 5, potassium dichromate 5, potassium sulfate 2.5 3700.0000 Foa 1895 11360,37:287 formula: water 250, mercuric chloride 10, potassium dichromate 6.25, sodium chloride 1.25 note: Spuler 1910 (Ehrlich, Krause, et al. 1910, 2 :254) refers to this formula as "Nicki- forow-Foa" but gives no reference. 3700.0000 Hoyer 1899 1780, 54 :97 formula: water 240, mercuric chloride 4, potassium dichromate 3.2 3700.0000 Lane test. 1910 Spuler Ehrlich, Krause, et al. 1910, 2:254 formula: water 125, 96% ale. 125, mercuric chloride 32, potassium dichromate 4.4 note: The original calls for a mixture of equal parts 33^^% dichromate and a sat. sol. HgCl2 in 96%. Duthie 1937 (Gatenby and Cowdry 1937, 420) calls for 2,^% dichromate. 3700.0000 Marrassini lest. 1910 Spuler Ehrlich, Krause, et al. 1910, 2:254 formula: water 250, mercuric chloride 2.5, potassium dichromate 5.0 3700.0000 Nickiforow see F 3700.0000 Foa 1895 (note) 3700.0000 Salkind 1917 23632, 29 :540 formula: water 250, mercuric chloride 10, potassium dichromate 6.25, chloral hydrate 10 3700.0000 Wlassow test. 1910 Spuler Ehrlich, Krause, et al. 1910, 2:254 formula: water 200, mercuric chloride 0.3, potassium dichromate 0.12, sodium chloride 6.0 3700.0000 Wolbach 1919 11343,41:75 formula: water 250, mercuric chloride 15, potassium dichi'omate 6.7 3700.0010 Mer cur ic-dichr ornate-acetic These are some of the better general-purpose fixatives. The addition of acetic acid adds a reasonable nuclear fixation to the excellent cytoplasmic fixation afforded by the principal constituent. 3700.0010 Bensley and Bensley 1938 Bensley and Bensley 1938, 44 formula: water 237.5, mercuric chloride 12, potassium dichromate 6, acetic acid 12.5 F 3700.0010-F 3700.1000 FIXATIVES 217 3700.0010 Bowie 1925 763, 29 :57 formula: water 250, mercuric chloride 12.5, potassium dichromate 6.25, acetic acid 5 3700.0010 Dahlgrens 1897 766, 13:149 formula: water 240, mercuric chloride 8, potassium dichromate 2.5, acetic acid 12.5, potassium sulfate 1.25 3700.0010 Kohn 1907 1780, 70:273 formula: water 250, mercuric chloride 3.2, potassium dichromate 5.8, acetic acid 12.5 3700.0010 Kultschitzky 1897 1780, 49:8 formula: 50% ale. 215, mercuric chloride 0.625, potassium dichromate 5, acetic acid 2.5 3700.0010 Lavdowsky 1893 23632, 10 :4 formula: water 250, mercuric chloride 0.35, potassium dichromate 12.5, acetic acid 5 3700.0010 Leigh-Sharpe 1921 in verb 1921 formula: water 250, mercuric chloride 12.5, potassium dichromate 7.5, acetic acid 12.5 3700.0010 Long and Mark 1912 test. 1937 Gatenby and Painter Gatenby and Painter 1937, 366 formula: water 225, mercuric chloride 5, potassium dichromate 5, acetic acid 25 3700.0010 Sonnenbrodt 1908 1780, 72 :416 formula: water 240, mercuric chloride 1.6, calcium dichromate 1.4, acetic acid 8 3700.0010 Spuler 1910 Ehrlich, Krause, et al. 1910, 2:527 formula: water 250, mercuric chloride 6.5, potassium dichromate 4.25, acetic acid 3.75, potassium sulfate 2.125 3700.0010 Zenker 1894 14674, 41 :533 formula: water 240, mercuric chloride 12.3, potassium dichromate 5, acetic acid 12.5, potassium sulfate 2.5 note: The "Zenker-formol" of most authors is F 3700.1010 Heidenhain 1916. Ralston and Wells 1939 (591b, 3:72) double the quantity of acid and use the resultant fluid for decalcification. 3700.0030 Mercuric-dichromate-formic 3700.0030 Guthrie 1928 test. Gatenby and Cowdry Gatenby and Cowdry 1928, 65 formula: water 240, mercuric chloride 12.5, potassium dichromate 5, formic acid 12.5, potassium sulfate 2.5 3700.0040 Mercuric-dichromate-nitric 3700.0040 Angelucci lest. 1895 Rawitz Rawitz 1895, 17 formulas: a. 3% nitric acid; B. F 7000.0000 Miiller 1850 method: [vertebrate retina] — » A, 3-^ to 2 hrs. — > B, 10 days 3700.0040 Whitney test. circ. 1938 Wellings Wellings 1938, 35 formula: water 238, mercuric chloride 12.5, potassium dichromate 6.25, sodium sulfate 2.5, nitric acid 12.5 3700.1 000 Mercuric-dichromate-formaldehyde 3700.1000 Baley 1937 11431, 44:272 formula: water 225, mercuric chloride 3.4, potassium dichromate 3.4, 40% formalde- hyde 25 3700.1000 Bensley 1910 2975, 29:3 formula: water 225, mercuric chloride 12.5, potassium dichromate 6.25, 40% formalde- hyde 25 3700.1000 Bensley and Bensley 1938 Bensley and Bensley 1938 formula: water 225, mercuric chloride 11, potassium dichromate 5.5, 40% fonnaide- hyde 25 218 METHODS AND FORMULAS F 3700.1000-F 3780.0000 3700.1000 Danchakoff test. 1930 Guyer Guyer 1930, 215 formula: a. water 250, mercuric chloride 12.5, potassium dichromate 6.25, sodium sulfate 3; B. 40% formaldehyde note: Add 5 to 10% B to A immediately before use. 3700.1000 Ellermann 1919 23632,36:56 formula: water 230, mercuric chloride 12.5, potassium dichromate 12.5, 40% formalde- hyde 25, potassium sulfate 2.5 3700.1000 Harvey 1907a 590,6:207 formula: water 165, mercuric chloride 3, potassium dichromate 4, 40% formaldehyde 85 3700.1000 Harvey 1907b 590, 6 :208 formula: water 190, mercuric chloride 4.5, potassium dichromate 1.9, 40% formalde- hyde 60 3700.1000 Helly 1903 23632,20:414 formula: water 240, mercuric chloride 12.5, potassium dichromate 5, 40% formalde- hyde 12.5, potassium sulfate 2.5 3700.1000 Klein 1906 590,5:323 formula: water 100, 96% ale. 125, mercuric chloride 30, potassium dichromate 4, 40% formaldehyde 25 3700.1000 Maximov 1909 23632,26:177 formula: water 250, mercuric chloride 12.5, potassium dichromate 6.25, 40% formalde- hyde 25, sodium sulfate 2.5 3700.1010 Mercuric-dichromate-formaldehyde-acetic 3700.1010 Hamazaki 1935 22575,295:703 REAGENTS REQUIRED: A. Water 250, mercuric chloride 7.5, potassium dichromate 5, acetic acid 12.5; B. 4% formaldehyde method: [small pieces of muscle] -^ A, 3 days — > S, 1 day 3700.1010 Heidenhain 1916 23635,32:365 formula: water 225, mercuric chloride 11.25, potassium dichromate 4.5, 40% formal- dehyde 25, acetic acid 11.5, potassium sulfate 2.15 note: This is the Zenker-formol of most authors. 3700.1010 Held 1909 test. 1933 Cajal and de Castro Cajal and de Castro 1933, 245 formula: water 250, mercuric chloride 7.5, potassium dichromate 6.25, sodium sulfate 2.5, 40% formaldehyde 1.25, acetic acid 7.5 3700.1010 Kolmer 1912 766, 42:47 formula: water 215, mercuric chloride 3, potassium dichromate 9.75, 40% formalde- hyde 10, acetic acid 25 3700.1010 Krueger 1911 1820, 6:173 formula: water 215, mercuric chloride 11, potassium dichromate 4.5, acetic acid 11, 40% formaldehyde 25, potassium sulfate 2.2 3700.1010 Meeker and Cook 1928 1883,57:185 formula: water 200, mercuric chloride 10, potassium dichromate 5, sodium sulfate 5, acetic acid 25, 40% formaldehyde 25 3780.0000 mercuric-dichromate-(other inorganic salts) 3780.0000 Schiller 1930 solution 131—auct. 23639b, 11 :63 formula: water 250, mercuric chloride 0.15, potassium dichromate 5, uranium acetate 0.2, magnesium acetate 2.5 F 3780. 1000-F 4000.1090 FIXATIVES 219 S780.1000 Mercuric-dichromate-iother inorganic snUs)-forrnaldehyde 3780.1000 Benoit 1922 G630, 86:1101 formula: a. water 250, mercuric chloride 3.1, potassium dichromate 3.75, uranium nitrate 2.0, sodium chloride 0.5; B. 2% formaldehyde method: [fix A, 4 hrs.] — > B, 12 hrs. — > wash 8S00.1000 Mercuric-(other inorganic salts) 3800.1000 Dominici 1905 8545, 2:219 formula: water 220, mercuric chloride 15, 40% formaldehyde 30. Add sufficient tincture of iodine to produce a deep wine color F 4000 CUPRIC SALTS IN COMBINATION WITH FIXATIVE SALTS OF HIGHER NUMERICAL RANK Cupric salts are never used alone for fixation. They are good fixatives in general, owing to their power of mordanting tissues, particularly those which it is desired subsequently to stain in hematoxylin. 4000.0010 CUPRIC-ACETIC 4000.0010 Behrens 1898 test. 1920 Mayer Mayer 1920, 232 formula: water 250, acetic acid 2.5, copper chloride 0.5, copper nitrate 0.5, phenol 2.5 4000.0010 Ripart and Petit 1884 test. 1884 Carney Carnoy 1884, 94 formula: water 125, camphor water 125, acetic acid 1.8, copper acetate 0.5, copper chloride 0.5 note: The camphor water employed is the pharmaceutical saturated solution of camphor in water; Mayer (Mayer, 1920, 232) suggests the substitution of thymol. 4000.0020 Cupric-trichloroacetic 4000.0020 Friedenthal 1907 20189, 209 formula: water 190, copper acetate 8.75, trichloroacetic acid 60 4000.0040 Cupric-nitric 4000.0040 Petrunkewitsch 1933 19938, 77:117 STOCK solutions: I. water 250, copper nitrate 30, nitric acid 20; II. water 250, phenol 10, ether 15 WORKING solution: stock I 60, stock II 180 4000.1000 Cupric-formaldehyde 4000.1000 Gelderd 1909 6011,25:12 formula: sea water 250, copper nitrate 5, 40% formaldehyde 12.5 4000.1000 Stappers 1909 6011,25:356 formula: water 220, copper nitrate 5, 40% formaldehyde 30 4000.1010 Cupric-formaldehyde-acetic 4000.1010 Emig 1941 Emig 1941, 71 formula: methanol 75, water 67, copper acetate 0.75, 40% formaldehyde 75, acetic acid 7.5 recommended for: algae. 4000.1090 Cupric-formaJdehyde-{other organic acids) 4000.1090 Zirkle 1931 7033a, 2 :85 formula: water 217.5, 40% formaldehyde 25, proprionic acid 7.5, copper hydroxide to excess 4500.1010 CUPRIC-PICRIC-FORMALDEHYDE-ACETIC The original purpose of Hollande in the mixture given below was to increase the picric acid content of the well-known picric-formaldehyde-acetic of Bouin. The characteristics of the fixative are, however, changed by the addition of copper, and it cannot be regarded as a modified Bouin though it is found in some textbooks under this name. 220 METHODS AND FORMULAS F 4500.1010-F 4700.0010 4500.1010 Hollande 1918 6630, 81:17 formula: water 250, copper acetate 6.25, picric acid 10, 40% formaldehyde 25, acetic acid 2.5 preparation: Add picric acid to the copper solution. Filter and add other ingredients. 4500.1010 Kostoff and Kendall 1929 11211, 21:113 formula: water 187, copper oxide 7.5, picric acid 2, 40% formaldehyde 37.5, acetic acid 25, urea 2.5 4600.1000 CUPRIC-CHROMIC-FORMALDEHYDE 4600.1000 Zirkle 1928 17191a, 4:201 formula: water 225, chromium sulfate 12.5, 40% formaldehyde 25, copper hydroxide to excess 4600.1010 Cupric-chromic-formaldehyde-acetic 4600.1010 Benda test. 1911 Mallory and Wright Mallory and Wright 1911, 386 formula: water 225, copper acetate 12.5, chrome alum 6.25, acetic acid 12.5, 40% formaldehyde 25 note: This is an adaptation of ADS 12.1 Weigert 1891 to fixative purposes. See also DS 21.22 Jakob 1913. 4600.1010 Jakob 1913 see DS 21.22 Jakob 1913 4700.0000 Cupric-dichromate The formula of Erlitzky here given was one of the first fixatives to be widely employed. These mixtures are primarily cytoplasmic fixatives and are not very useful, even after the addition of acid, for the preservation of nuclei. 4700.0000 Erlicki see F 4700 Erlitzky 1877 4700.0000 Erlitzky 1877 17035, 5 :739 formula: water 250, potassium dichromate 6.25, copper sulfate 1.25 note: This solution is to be found in almost every text as "Erlicki," presumably copy- ing the statement in Lee 1885, 403 that "This modification of MuUer is known in Germany as Erlicki's solution." The second edition of Lee (1890) gives a reference to a Polish journal. The fifth edition (1900) adds a reference to 1897 (Progres MMicale, No. 39). This incorrect "1897" was continued to the 7th edition where a printer's error changed 39 to 31. This double error has continued to the 11th edition (Gatenby and Beams 1950, 35). These compound errors have been copied by many other texts in which 39 and 31 have been treated both as volume and page numbers. Moreover, the French paper gives the name as Erlitzky. 4700.0000 de la Iglesia 1904 test. 1910 Poll. Ehrlich, &ause, et al. 1910, 1:234 formula: 50% ale. 250, potassium dichromate to sat., copper acetate to sat. 4700.0000 Zirkle 1934 17191a, 20:169 formula: water 250, copper sulfate 2.5, potassium dichromate 3.1, ammonium di- chromate 3.1 4700.0010 Cupric-dichromate-acetic 4700.0010 Aigner 1900 20170,38:109. formula: water 250, copper sulfate 2.9, potassium dichromate 6.25, acetic acid 2.5 4700.0010 Kultschitzky 1887 23632, 4:348 formula: 50% ale. 250, copper sulfate to sat., potassium dichromate to sat., acetic acid 0.5 cc. 4700.0010 Rubaschkin 1904 1780, 63 :577 formula: water 250, copper acetate 2, potassium dichromate 7, acetic acid 12, 40% formaldehyde 25 F 4700.0010-F 5000.0010 FIXATIVES 221 4700.0010 Wolters test. 1896 Kahlden and Laurent Kahlden and Laurent 1896, 73 formula: water 125, 95% ale. 125, cupric sulfate, potassium dichromate, a.a. to sat., acetic acid 0.75 4700.1000 Cupric-dichromate-formaldehyde 4700.1000 Kenyon 1896 11135, 6:133 formula: water 200, copper sulfate 5, potassium dichromate 9.5, 40% formaldehyde 50 4700.1000 Strong 1903 11135, 13:290 formula: water 125, 40% formaldehyde 125, copper dichromate 6.25 4900.0040 CUPRIC-(0THER ORGANIC AGENT)-NITRIC 4900.0040 Petrunkewitsch 1933 19938,77:117 formula: water 100, 96% ale. 150, ether 12.5, copper nitrate 5, nitric acid 7.5, paranitro- phenol 12.5 note: See also Roskin 1946. 4900.0040 Roskin 1946 Roskin 1946, 97 formula: water 100, dioxane 150, copper nitrate 5, nitric acid 7.5, paranitrophenol 12.5 note: This is stated by Roskin {loc. cit.) to be a modification of Petrunkewitsch 1933. 4900.0040 Waterman 1937 20540b, 12 :21 formula: water 100, dioxane 150, copper nitrate 5, nitric acid 7.5, paranitrophenol 12.5, ether 12.5 (added after other ingredients have stood 2-3 days and been filtered) F 5000 PICRIC ACID IN COMBINATION WITH FIXATIVE AGENTS OF HIGHER NUMERICAL RANK 5000.0000 Picric Alone Picric acid is not a good fixative. It forms, with the cytoplasmic constituents of the cell, compounds which are easily water-soluble, so that fixation in any of the mixtures containing no other primary fixative constituent than picric acid always shows huge vacuoles. Special after-treatment is also required for the removal of picric acid, and it is notoriously almost impossible to secure differentiation of histological structures after fixation in picric mixtures. These considerations do not, however, prevent their being the most popular class of fixatives in use at the present time. 6000.0000 Gage test. 1920 Mayer Mayer 1920, 39 formula: water 125, 95% ale. 125, picric acid 0.5 5000.0010 Picric-acetic The acidified picric mixtures given in this class and the next three classes have nothing to recommend them for any purpose save an occasional special technique. They give wretched preservation of cytoplasmic constituents, and there are many better fixatives for nuclei. 5000.0010 Armitage 1939 Microscope, 3 :213 formula: water 100, picric acid 1.1, acetic acid 25, dioxane 125 50000.0010 Allen and McClung 1950 Jones 1950, 60 formula: water 125, dioxane 100, picric acid 1.2, acetic acid 25 5000.0010 Bigelow 1902 4604, 40 :66 formula: water 240, picric acid 3, acetic 12.5 note: The original formula calls for a saturated solution of picric acid in 5% acetic. 5000.0010 Boveri 1887 10899, 21 :433 formula: water 250, picric acid 3, acetic acid 2.5 5000.0010 Davidoff 1899 14246, 9:118 formula: water 190, picric acid 2,4, acetic acid 60 222 METHODS AND FORMULAS F 5000.0010-F 5000.0050 5000.0010 Deegener 1909 23831,27:634 formula: 45% ale. 240, picric acid 1.6, acetic acid 12.5 note: Deegener {loc. cit.) refers to this as "Zimmer's mixture," but quotes no reference. Mayer 1920 indexes it as "Zimmer's Gemisch" but quotes the journal reference of Deegener's paper. 5000.0010 Dobell 1914 1798,34:133 formula: 90% ale. 180, picric acid 40, acetic acid 67.5 5000.0010 Eltringham fixative D—auct. Eltringham 1930, 46 formula: 95% ale. 200, chloroform 37.5, picric acid 2, acetic acid 12.5 RECOMMENDED FOR: general insect histology. 5000.0010 Gulick 1911 1820, 6:339 formula: water 250, picric acid 1, acetic acid 7.5 5000.0010 Hertwig lest. 1895 Rawitz Rawitz 1895, 22 formula: water 100, picric acid 1.1 (to sat.), acetic acid 3-5 5000.0010 Kingsbury and Johannsen 1927 Kingsbury and Johannsen 1927, 9 formula: abs. ale. 200, chloroform 35, picric acid 2, acetic acid 15 5000.0010 Potenza 1939 test. 1942 Langeron Langeron 1942, 419 formula: water 125, picric acid 1.5, acetic acid 25, dioxane 100 5000.0010 Zimmer 1909 see F 5000.0010 Deegener 1909 5000 .0015 Picric-acetic-s u Ifu ric 5000.0015 Blanc test. 1895 Maggi Maggi 1895, 112 formula: water 100, picric acid 0.1, acetic acid 0.6, sulfuric acid 0.3 5000.0015 Nemec 1899 10606,33:314 formula: sat. sol. picric acid 250, acetic acid 1.25, sulfuric acid 1.25 6000.0015 Patterson 1907 2975, 13 :252 formula: F 5000.0050 Kleinenberg 1879 230, acetic acid 20 5000.0020 Picric-trichloroacetic 5000.0020 Cretin 1941 6630, 135 :355 stock: I. dioxane 100, picric acid 25; II. water 35, trichloroacetic acid 5, methanol to make 100 WORKING solution: stock I 50, stock II 60 RECOMMENDED FOR: fixation of starch and glycogen in tissues held at 1-3°C. 5000.0040 Picric-nitric 5000.0040 Eltringham 1930 Eltringham 1930, 94 formula: water 250, picric acid 2.6, nitric acid 10 5000.0040 Kingsbury and Johannsen 1927 Kingsbury and Johannsen 1927, 9 formula: water 237, nitric acid 13, picric acid q.s. to sat. 5000.0040 Mayer 1880 14246,2:5 formula: water 250, picric acid 4, nitric acid 6.25 5000.0040 Zimmermann 1896 test. 1922 Schneider Schneider 1922, 323 formula: a. 3% nitric acid; B. 1% picric acid in 95% ale. method: [plant material]—* A, 24 hrs. -^ water, 24 hrs. — * B, 12 hrs. -> 96% ale., thorough wash 5000.0050 Picric-sulfuric 5000.0050 Kleinenberg 1879 17510, 19:208 formula: water 250, picric acid 0.75, sulfuric acid 5 note: Langenbeck 1898 (11373, 14:303) is this solution made with sea water. Mayer 1880 (14246, 2 :2) is the same solution as Kleinenberg, made another way; Mayer 1920 (p. 40) is incorrect in supposing it to be "stronger." F 5000.0050-F 5000.1010 FIXATIVES 22-^ 5000.0050 Langenbeck 1898 see F 5000.0050 Kleinenberg 1879 (note) 5000.0050 Mayer 1880 see F 5000.0050 Kleinenberg 1879 (note) 6000.0050 Wistinghausen 1891 1424G, 10 :47 formula: water 250, picric acid 0.75, sulfuric acid 1.25 5000.0060 Picric-hydrochloric 5000.0060 Becher and Demoil 1913 Becher and Demoll 1913, 43 formula: water 250, hydrochloric acid 20, picric acid q.s. to sat. 6000.0060 Mayer 1880 1424G, 2 :5 formula: water 250, picric acid 5, hydrochloric acid 5 5000.1 000 Picric-formaldehyde These mixtures appear to have nothing whatever to recommend them but are still quoted in the literature. 6000.1000 Aniline 1903 test. 1920 Mayer Mayer 1920, 39 formula: 70% ale. 225, picric acid to sat. (circ. 15), 40% formaldehyde 25 6000.1000 Graf 1897 test. 1910 Blum Ehrlich, Krause, et al. 1910, 1 :489 formula: water 225, picric acid 2.8, 40% formaldehyde 25 note: Blum 1910 (Ehrlich, Krause, el al. 1910, 1:488) quotes four other "picric-formal- dehydes of Graf"; this is the most usual. 6000.1000 Mayer 1920 Mayer 1920, 31 formula: sea water 200, picric acid 0.25, 40% formaldehyde 25 5000.1000 Rossman test. 1947 Davenport cit. Deane, et at. 20540b, 63, 401 formula: abs. ale. 225, picric acid 22.5, 40% formaldehyde 25 5000.1000 Verhoeff 1926 test. 1938 Mallory Mallory 1938, 258 formula: water 95, 95% ale. 130, picric acid 2.7, 40% formaldehyde 25 5000.1010 Picric-formaldehyde-acetic These are today the most widely used general-purpose fixatives. They have nothing what- ever to recommend them for any purpose save the demonstration of nuclei in meiosis, for which the original Bouin 1897, undoubtedly the best-known fixative at present employed, was developed. Bouin himself recommended his fluid for no other purpose than the demon- stration of nuclear figures in the testis of the rat, and it is diflScult to understand how its use has become so widespread. It is almost impossible to secure sharp cytoplasmic staining after any of these mixtures. Their use should be confined entirely to that for which they were orig- inally proposed, that is, the demonstration of nuclear figures. 5000.1010 Allen 1929a Allen's PFAi—auct. McClung 1929, 425 formula: water 190, picric acid 2.4, 40% formaldehyde 25, acetic acid 25 5000.1010 Allen 1929b Allen's PFA<,—auct. MeClung 1929, 425 formula: water 225, picric acid 2.8, 40% formaldehyde 12.5, acetic acid 12.5 5000.1010 Allen 1929c Allen's PFA^—auct. McClung 1929, 425 formula: water 190, picric acid 2.4, 40% formaldehyde 35, acetic acid 25, urea 2.5 5000.1010 Bauer 1933 23639b, 33:143 formula: dioxane 212.5, picric acid 75, 40% formaldehyde 25, acetic acid 12.5 RECOMMENDED FOR: preservation of glycogen in tissues. 5000.1010 Becher and Demoll 1913 Becher and Demoll 1913, 48 formula: water 25, 95% ale. 140, picric acid 2.6, 40% formaldehyde 67.5, acetic acid 17.5 224 METHODS AND FORMULAS F 5000.1010 5000.1010 Belar 1929 23639b, 10 :76 formula: water 30, 95% ale. 120, picric acid 1, 40% formaldehyde 60, acetic acid 15 note: Belar refers this solution, without reference, to "Bouin-Duboscq." 6000.1010 Bouin 1897 Bouin's Fluid— compl. script. 1823, 1 :229 formula: water 190, picric acid 2.4, 40% formaldehyde 60, acetic acid 12.5 note: This is the "Bouin's Fluid" of most authors, but see also Bouin F 2000.1010, F 2300.0010, F 2300.1030, F 3000.1000 and F 3000.1010. For "alcoholic Bouin" (see F 5000.1010 Brasil 1905. See also note under Roskin 1946 who also recommends (Roskin 1946, 97), following the suggestion of Waterman 1937 (20540b, 2:23) that dioxane be substituted for water in the above; for partial dioxane substitution see Puckett 1937. Kupperman and Noback 1945 (1887a, 40:75) recommend adding 1% ferric alum when tissues are subsequently to be stained in hematoxylin. 5000.1010 Brasil 1904 alcoholic Bouin— compl. script. 1915, 4:74 formula: 80% ale, 150, picric acid 1, 40% formaldehyde 60, acetic acid 15 note: Many authors refer this, without reference, to "Duboscq-Brasil." 5000.1010 Brumpt test. 1942 Langeron see P 12.3 Brumpt 5000.1010 Carothers 1916 11373,2:445 formula: water 225, picric acid 2.5, acetic acid 25, 40% formaldehyde 62, urea 2.5 5000.1010 Claverdon 1943 19938,97:168 formula: isopropyl ale. 137.5, acetone 75, picric acid 12.5, 40% formaldehyde 12.5, acetic acid 12.5 5000.1010 Dammin 1937 see DS 23.32 Dammin 1937 6000.1010 Debaisieux 1935 6011, 44:273 formula: 80% ale. 167, picric acid 3.3, 40% formaldehyde 67, acetic acid 16 5000.1010 Dobell 1923 16035, 16:365 formula: 95% ale. 225, picric aeid 18, 40% formaldehyde 62.5, acetic acid 12.5, chloroform 0.1 6000.1010 Duboscq-Brasil 1905 see F 5000.1010 Brasil 1904 6000.1010 Gendre 1937 4285a, 14:262 formula: sat. sol. picric acid in 95% ale. 200, 40% formaldehyde 37.5, acetic aeid 12.5 6000.1010 GUbert 1935 591, 22:52 formula: sat. sol. {circ. 6%) picric acid in 70% ale. 190, acetic acid 7.5, 40% formalde- hyde 38 6000.1010 Ingelby 1925 11360, 1925:423 formula: water 180, picric acid 3, 40% formaldehyde 63, acetic acid 5, potassium bromide 2 5000.1010 Langeron 1934 Langeron 1934 p. 344 STOCK formula: water 180, 40% formaldehyde 60, picric acid to sat. WORKING formula: stock 95, acetic acid 5 6000.1010 van Leeuwen 1907 23833, 32:316 formula: abs. ale. 175, picric acid 2.5, acetic acid 30, 40% formaldehyde 30, chloro- form 15 note: For "Leeuwen without picric" see F 0000.1010 Sikora 1917. 6000.1010 Lenoir 1929 6630,101:1203 formula: stock formula I. water 180, 40% formaldehyde 60, picric acid to sat. at 50°C.; stock formula IL water 180, acetic acid 60, picric acid to sat. at 50°C. working formula: stock I 75, stock II 25 note: The original specifies "neutral formol" but this appears to be quite unnecessary. 6000.1010 Lillie 1944 4349, 24:35 formula: water 212.5, picric acid 2.2, 40% formaldehyde 25, acetic acid 12.5 F 5000.1010-F 5000.1310 FIXATIVES 225 5000.1010 Puckett 1937 20540b, 12:97 formula: water 125, dioxane 85, picric acid 1.6, 40% formaldehyde 40, acetic acid 10.6 5000.1010 Roskin 1946 Roskin 1946, 92 formula: water 250, picric acid 1.9, 40% formaldehyde 60, acetic acid 12 note: Roskin {loc. cit.) refers to this as "Boiiin's mixture" without reference. It is certainly not the mixture of Bouin 1897 {q.v.), which is given by Roskin as "a good modification of Bouin's mixture." 5000.1010 Schweitzer 1942 test. 1946 Roskin Roskin 1946, 200 formula: methanol 175, picric acid 0.8, 40% formaldehyde 37, acetic acid 15, chloro- form 37 5000.1010 Smith test. 1930 Guyer Guyer 1930, 223 formula: water 110, 95% ale. 110, picric acid 1.5, 40% formaldehyde 12.5, acetic acid 12.5 5000.1010 Waterman 1937 see F 5000.1010 Bouin 1897 (note) 5000.1010 Wetzel 1925 1798, 51:209 formula: water 145, 95% ale. 35, picric acid 1.2, 40% formaldehyde 72, acetic acid 18 5000 . 1 020 Picric-formaldehyde-trichloroacetic 5000.1020 Bouin test. 1934 Langeron Langeron 1934, 345 formula: water 190, picric acid 2.4, 40% formaldehyde 60, trichloroacetic acid 0.25 5000.1020 Cretin 1937 4285a, 14:163 formula: 95% ale. 45, water 10, picric acid 4, 40% formaldehyde 45, trichloroacetic acid 2 5000.1020 Moreaux 1910 2844, 19 :265 formula: water 210, picric acid 3 (to sat.), 40% formaldehyde 37, trichloroacetic acid 6 5000.1020 Roskin 1946 Roskin 1946, 93 formula: water 240, picric acid 1.9, 40% formaldehyde 60, trichloroacetic acid 0.1 5000.1020 Stieve test. 1948 Romeis Tripiform—auct. Romeis 1948, 76 formula: water 210, picric acid 2.2, 40% formaldehyde 15, trichloroacetic acid 0.75 5000.1030 Picric-formaldehyde-jormic 5000.1030 Lillie 1944 4349,24:35 formula: water 85, picric acid 0.9, 40% formaldehyde 10, formic acid 5 5000.1 Olf-O Picric-formaldehyde-nitric 5000.1040 Hollande 1911 1833, 13:133 formula: abs. ale. 81, 40% formaldehyde, sat. with picric acid 18, nitric acid 1.5, benzene 4.5 5000.1090 Picric-formaldehyde-ipther organic acids) 5000.1090 Foley 1938 20540b, 13:5 formula: abs. ale. 245, picric acid 0.5, 40% formaldehyde 2.5, monochloroacetic acid 2.5 5000.1090 Schabadasch 1939 test. 1948 Romeis Romeis 1948, 256 formula: 95% ale. 137.50, calcium picrate 87.50, 40% formaldehyde 12.5, mono- chloroacetic acid 12.5 recommended for: fixation of glycogen in tissues. 5000.1310 Picric-formaldehyde-acetone-cLcetic 5000.1310 Claverdon 1943 19938,97:168 formula: isopropyl ale. 137, picric acid 12.5, 40% formaldehyde 12.5, acetone 75, acetic acid 12.5 226 METHODS AND FORMULAS F 5000.1310-F 5600.1010 5000.1310 van Walsen 1925 23632, 42 :439 formula: water 130, picric acid 1.5, 40% formaldehyde 35, acetic acid 2.5, acetone 85 5600.0000 Picric-chromic The addition of chromic acid to picric-acid mixtures probably undoes some of the damage occasioned by the picric, in that the tissues after sectioning appear less vacuolated. The presence of the picric acid, however, continues to cause diffused staining. 5600.0000 Fol test. 1885 Whitman Whitman 1885, 237 formula: water 250, picric acid 0.3, chromic acid 0.63 note: Gatenby and Cowdry 1928, 57 state that they have seen this formula "with the addition of a trace of acetic acid, quoted as 'liquid of Haensel'"; they do not say where or when. 5600.0000 Haensel see F 5600.0000 Fol (1885) (note) 5600.0000 Kahlden and Laurent 1896 Kahlden and Laurent 1896, 70 formula: water 250, picric acid 0.25, chromic acid 0.375 5600.001 0 Picric-chromic-acetic 5600.0010 Haensel see F 5600.0000 Fol (1885) (note) 5600 . 00I^0 Picric-chro mic-nitric 5600.0040 Rawitz 1895 Rawitz 1895, 24 formula: water 250, picric acid 1, chromic acid 1.25, nitric acid 2 5600.0050 Picric-chroviic-sulfuric 5600.0050 Fol lest. 1910 Mayer Ehrlich, Krause, et al. 1910, 2:401 formula: water 250, picric acid 0.5, chromic acid 0.88, sulfuric acid 3.3 5600.0050 Lo Bianco 1890 14246, 9:443 formula: water 250, picric acid 1.5, chromic acid 1.25, sulfuric acid 5 5600.1 0000 Picric-chromic-formaldehyde 5600.1000 Cohen 1935 20540b, 10:25 formula: water 225, chromium sulfate 11.25, 40% formaldehyde 25, picric acid to sat. note: Salicylic acid may be substituted for picric acid. 5600.1000 Masson test. 1947 Paquin and Goddard cit. Warren 4349, 27:195 formula: water 187.5, picric acid 2.2, chrome alum 7.5, 40% formaldehyde 67.5 preparation: Soak the alum in the formaldehyde for 1 hour before adding the picric acid dissolved in the water. Leave for 24 hours and filter. 5600.1010 Picric-chromic-formaldehyde-aceiic These are some of the best nuclear fixatives that have ever been developed, but they should be confined exclusively to the fixation of tissues for the demonstration of nuclear figures. They give very poor cytoplasmic fixation. 5600.1010 Allen 1918 Allen's PF A,,— auct. 11373,31,135 formula: water 190, picric acid 2.4, chromic acid 3.8, 40% formaldehyde 60, acetic acid 7.5, urea 5 5600.1010 Allen 1929a test. 1929 McClung Allen's Bx—auct. McClung 1929, 425 formula: water 190, picric acid 2.4, chromic acid 2.5, 40% formaldehyde 35, acetic acid 25, urea 2.5 5600.1010 Allen 1929b lest. 1929 McClung Allen's PFAi^ or B,^—aucl. McClung 1929, 425 formula: water 190, picric acid 1.6, chromic acid 2.5, 40% formaldehyde 50, acetic acid 12.5, urea 2.5 F 5600.1010-F 6000.0000 FIXATIVES 227 6600.1010 Bachuber 1916 2975, 30:294 formvla: water 190, chromic acid 3.75, picric acid 2.4, 40% formaldehyde 37, acetic acid 25 5600.1010 Painter 1924 763,27:77 FORMULA : water 225, picric acid 2.5, chromic acid 3.75, acetic acid 25, 40% formaldehyde 62, urea 5 5670.1000 PICRIC-CHROMIC-DICHROMATE-FORMALDEHYDE 6670.1000 Chura 1925 23632, 42 :55 formula: water 200, picric acid 0.16, chromium fluoride 1, chrome alum 1, potassium dichromate 4.75, formaldehyde 50 RECOMMENDED FOR: fix 24 hours. Then transfer to ADS 12. 1 Chura 1925a, if chromosomes are to be stained, or to ADS 12.1 Chura 1925b for mitochondria. 5700.0000 PiCRIC-DICHROMATE 5700.0000 Schultze 1904 23632, 21 :7 formula: water 250, picric acid 0.125, potassium dichromate 7.5 5700.0050 Picric-dichromate-sulfuric 6700.0050 Vialleton 1887 915, 6:168 formula: water 250, picric acid 0.4, potassium dichromate 1.25, sulfuric acid 2.5 5700.1 01 0 Picric-dichromate-formaldehyde-acetic 5700.1010 Lenoir 1930 6630, 103:1253 formula: water 50, 95% ale. 100, ammonium dichromate 6, 40% formaldehyde 50, water 38, acetic acid 12, picric acid to sat. 5800.1010 Picric-(other inorganic salts)-pormaldehyde 5800.1010 Goodrich 1919 17510, 64:38 formula: water 190, picric acid 2, water 1, potassium iodide 0.25, iodine 0.125, 40% formaldehyde 56, acetic acid 11 5800.1010 Turchini 1919 6630, 82:1131 formula: water 250, picric acid 2, ammonium molybdate 17, 40% formaldehjale 50 recommended for: fixation of thiazin dyes in tissues. F 6000 CHROMIC ACID IN COMBINATION WITH FIXATIVE AGENTS OF HIGHER NUMERICAL RANK 6000.0000 Chromic Alone Chromic acid used alone can scarcely be considered a fixative; it is rather a hardening agent. It has a very low penetration and is used as a fixative only for marine forms containing large quantities of w^ater. 6000.000 Lo Bianco 1890 14246, 9 :443 formula: 35% ale. 250, chromic acid 1.25 6000.0000 Howell 1884 test. 1910 Poll Ehrlich, Krause, et al. 1910, 1:222 formula: 30% ale. 250, chromic acid 0.125 6000.0000 Klein 1878 17510, 18:315 formula: 30% ale. 250, chromic acid 0.275 6000.0000 Pritchard 1873 17510, 13:427 formula: 75%, ale. 250, chromic acid 2.5 6000.0000 Vlakovic test. 1926 E. W. B. at. Wilkie 11360, 46:287 formula: water 30, 95% ale. 220, chromic acid 0.7 228 METHODS AND FORMULAS F 6000.0010 6000.0010 Chromic-acetic These are magnificent fixatives. The cytoplasmic fixation afforded by the chromic acid is coupled with nuclear fixation by the acetic. The only disadvantage is that they tend to dis- color the tissues, which sometimes renders after-staining difficult. They are as standard in botanical practice as are the osmic-chromic-acetic mixtures for zoological purposes. 6000.0010 Ammerman 1950 20540b, 25:197 formula: water 238, chrome alum 3, 40% formaldehyde 30, acetic acid 2 RECOMMENDED FOR: heavily yolked embryos and insect larvae. 6000.0010 Arnold 1888 1780, 31 :541 formula: water 250, chromic acid 0.75, acetic acid 1.25 6000.0010 Bataillon 1904 1756,18:1 formula: water 225, chromic acid 2.25, acetic acid 25 6000.0010 Becher and DemoU 1913 Becher and DemoU 1913, 24 formula: water 100, chromic acid 0.25, acetic acid 0.1 6000.0010 Claussen 1908 2626,26:144 formula: water 250, chromic acid 1.25, acetic acid 2.5 6000.0010 Czermak 1893 1823, 42 :581 formula: water 250, chromic acid 0.625, acetic acid 2.5 note: M'llroy and Hamilton 1901 (11024, 40) is identical. Yamanouchi 1906 (3430, 41 :425) requires the same ingredients dissolved in sea water. 6000.0010 Demarbaix 1889 6011, 5:25 formula: water 250, chromic acid 1.3, acetic acid 7.5 6000.0010 Ehler test. 1910 Poll cit. Lee und Mayer Ehrlich, Krause, et al. 1910, 1:223 formula: water 250, chromic acid 2, acetic acid 0.5 note: Gatenby and Cowdry (1928, 36) quote, without reference, an "Ehler's Chrom- acetic" which is identical with F 6000.0010 Lo Bianco 1890. 6000.0010 Ehler see F 6000.0010 Lo Bianco 1890a (note) 6000.0010 Faussek 1900 14246, 14:88 formula: water 250, chromic acid 2.5, acetic acid 0.6 6000.0010 Felix 1891 test. Poll 1910 Ehrlich, Krause, et al. 1910, 1:223 formula: water 250, chromic acid 6.25, acetic acid 2.5 6000.0010 Fick 1893 23635, 56 :529 formula: water 250, chromic acid 1.25, acetic acid 0.25 6000.0010 Flamming 1882 test. 1928 Gatenby and Cowdry Gatenby and Cowdry 1928, 35 formula: water 250, chromic acid 0.50, acetic acid 0.25 6000.0010 Fol test. 1910 Poll Ehrlich, Krause, et al. 1910, 1:224 formula: water 270, chromic acid 0.45, acetic acid 2.5 6000.0010 Friedlander 1889 Friedlander 1889, 33 formula: water 250, chromic acid 0.4, acetic acid 2.5 6000.0010 Gates 1908 3430, 43 :81 formula: water 250, chromic acid 1.75, acetic acid 1.25 6000.0010 Gates and Latter 1927 11360, 47:280 formula: water 250, chromic acid 2.5, acetic acid 2.5 note: Another formula in the same paper reduces the chromic acid to 1.5. 6000.0010 Hertwig 1892 1780, 39 :353 formula: water 250, chromic acid 2.5, acetic acid 0.5 6000.0010 King test. 1910 Poll Ehrlich, Krause, et al. 1910, 1 :223 formula: water 225, chromic acid 0.625, acetic acid 25 F 6000.00 10-F 6000.0060 FIXATIVES 229 6000.0010 Kopsch 1897 see F 6000.0010 Virchow (1897) (note) 6000.0010 Lavdowsky 1894 764,4:355 formula: 10% ale. 250, chromie acid 0.5, acetic acid 1.25 6000.0010 Lo Bianco 1890a Bianco's Chromacetic 1 — aud. 14246, 9 :443 formula: water 250, chromic acid 2.5, acetic acid 12.5 note: Ehler test. 1928 Gatenby and Cowdry (Gatenby and Cowdry 1928, 30) is identical with this, but see also Ehler (1910). 6000.0010 Lo Bianco 1890b Bianco's Chromacetic 2 — aucl. 1424G, 9:443 formula: water 25, chromic acid 0.25, acetic acid 250 6000.0010 M'llroy and Hamilton 1901 see F 6000.0010 Czennak 1893 (note) 6000.0010 Miiche 1908 2626, 26 :8 formula: water 250, chromic acid 2.5, acetic acid 2.5 6000.0010 Schaffner 1906 3430, 41:183 formula: water 250, chromic acid 0.75, acetic acid 1.75 6000.0010 Simons 1906 3430,41:183 formula: water 250, chromic acid 0.625, acetic acid 0.25 6000.0010 Virchow test. 1897 Kopsch 1780, 51:184 formula: water 225, chromic acid 0.5, acetic acid 25 note: This formula is often attributed to Kopsch {loc. cit.) but must not be confused with F 7000.1000 Kopsch 1896. 6000.0010 Yamanouchi 1906 see F 6000.0010 Czermak 1893 (note) 6000.0010 Zimmermann 1893 1780, 41 :367 formula: sea water 240, chromic acid 0.62, acetic acid 12.5 6000.0030 Chromic-formic 6000.0030 Rabl 1884 1455, 10:215 formula: water 250, chromic acid 0.8, formic acid 0.25 6000.0030 Rohl test. 1910 Poll Ehrlich, ICrause, et al. 1910, 1:223. formula: water 250, chromic acid 0.75, formic acid 0.25 6OOO.OO4O Chromic-nitric 6000.0040 Haug 1891 see AF 21.1 Haug 1891 6000.0040 Perenyi 1882 23833, 5 :459 formula: water 165, 90% ale. 75, chromic acid 0.6, nitric acid 10 6000.0040 Perenyi 1888 23833, 11:139 formula: water 135, abs. ale. 100, chromic acid 0.75, nitric acid 17.5 6000.0040 Robin 1871 Robin 1871, 305 formula: water 250, potassium chromate 5, nitric acid, 1.25 6000.0060 Chromic-hydrochloric 6000.0060 Busch 1877 see AF 21.1 Busch 1877 6000.0060 Calvet 1900 test. 1910 Poll Ehrlich, Krause, et at. 1910, 1:224 formula: water 250, chromic acid 0.425, hydrochloric acid 0.31 230 METHODS AND FORMULAS F 6000.0070-F 6000.1010 6000.0070 Chromic-oxalic 6000.0070 Graf 1898 test. 1910 Poll Ehrlich, Krause, el al. 1910, 1 :224 formula: 30% ale. 250, chromic acid 0.75, oxalic acid 8 6000.1000 Chromic-formaldehyde Chromic-formaldehyde mixtures must be prepared immediately before use, and it is desirable to use them in the dark. Chromic-formaldehyde alone, without acidification, is of no value save for cytoplasmic fixation. 6000.1000 Braus 1896 7137, 6:307 formula: water 180, chromic acid 0.625, 40% formaldehyde 60 6000.1000 Levitsky 1910 2G26 28 :540 formula: water 225, chromic acid 0.4, 40% formaldehyde 25 6000.1000 Lo Bianco 1890 14246, 9:443 formula: water 125, sea water 110, chromic acid 1.25, 40%, formaldehyde 12.5 6000.1000 Marina 1897 19443, 2 :20 formula: 90%, ale. 250, chromic acid 0.25, 40%, formaldehyde 12.5 6000.1000 Nemec test. 1937 Gatenby and Painter Gatenby and Painter 1937, 678 formula: water 232, chromic acid 2.3, 40% formaldehyde 18 6000.1000 Prokofieva 1934 7033a, 5 :498 formula: water 190, chromic acid 6.25, 40%, formaldehyde 60 6000.1 01 0 Chromic-formaldehyde-acetic This class of fixative is widely shown to botanists under the generic name GRAF. 6000.1010 Belling 1928 20540b (abstr.) 4:94 formula: water 185, chromic acid 2.5, acetic acid 25, 40% formaldehyde 40 6000.1010 Belling 1930 BeUing 1930, 244 formula: stock I. water 212, chromic acid 3.5, acetic acid 38; stock II. water 117, 40% formaldehyde 133; stock III. water 185, 40% formaldehyde 65 working solutions: A. stock I 125, stock II 125; B. stock I 125, stock III 125 RECOMMENDED FOR: Usc solution A for general nuclear fixation; solution B for metaphase preparations only. 6000.1010 Destin test. 1929 McClung and Allen McClung 1929, 422 formula: water 250, chromic acid 2.5, 40% formaldehyde 15, acetic acid 5 6000.1010 Gatenby and Painter 1937 see F 6000.1010 Navashin 1912 (note) 6000.1010 Guyer 1930 Guyer 1930, 218 formula: water 160, chromic acid 1.6, acetic acid 10, 40% formaldehyde 85 6000.1010 Karpenchenko 1924 14,387:11211 formula: water 235, chromic acid 1, acetic acid 7.5, 40% formaldehyde 7.5 note: This formula is attributed to "Navashin 1924" by Semmens 1939 {Microscope, 3:4). 6000.1010 Marchoux 1910 test. 1928 Gatenby and Cowdry Gatenby and Cowdry 1928, 65 formula: water 120, chromic acid 0.8, 40% formaldehyde 120, acetic acid 75. note: Gatenby and Cowdry (lac. cit.) cite this formula as "from Perez 1910 (1915, 5:11)." The journal reference is incorrect and the writer has never found any other source for the formula. 6000.1010 Navashin 1912 23253, 42, 28 formula: water 188, chromic acid 2, 40% formaldehyde 50, acetic acid 12 note: Gatenby and Painter 1937, 677 recommend the reduction of the chromic acid to 1.6. F 6000.1010-F 6800.0000 FIXATIVES 231 6000.1010 Perez 1910 see F 6000.1010 Marchoux 1910 (note) 6000.1010 Randolf 1935 20540b, 10 :95 formula: stock I. water 230, chromic acid 2.5, acetic acid 20; stock II. water 175, 40% formaldehyde 75 WORKING solution: stock I 125, stock II 125 6000.1010 Retterer 1900 11024,36:508 formula: water 160, chromic acid 4.8, acetic acid 20, 40% formaldehyde 80 6000.1010 Weber 1930 22073, 9:319 formula: stock I. water 220, chromic acid 3.3, acetic acid 30; stock II. water 120, 40% formaldehyde 130 working solution: stock I 125, stock II 125 6000.1010 Winge 1930 23039b, 10:699 formula: water 205, chromic acid 1.9, 40% formaldehyde 26, acetic acid 18.5 6000. 1040 Chrojnic-formnldehyde-nitric 6000.1040 Yao-Nan 1927 4285a, 4:71 formula: water 88, chromic acid 0.15 40% formaldehj-de 12, nitric acid 0.2, sodium chloride 0.75 6700.0000 Chromic-dichromate 6700.0000 Rutherford (est. 1878 Marsh Marsh 1878, 68 formula: water 250, chromic acid 0.5, potassium dichromate 1 6700.0000 Weigert 1896 7936a, 6:10 formula: water 250, potassium dichromate 12.5, chromium fluoride 5 6700.0010 Chrojnic-dichr ornate-acetic Chromic-dichromate mixtures are intended for cytoplasmic fixation; both the chromic and the dichromate fix specific elements of cytoplasm. 6700.0010 Burckhardt 1897 6011, 12:335 formula: water 230, chromic acid 1.5, potassium dichromate 3.8, acetic acid 12.5 6700.0010 Goldsmith test. 1930 Guyer Guyer 1930, 218 formula: water 237.5, chromic acid 1.9, potassium dichromate 2, acetic acid 12.5 6700.0010 Maximow 1916 6630, 79:462 formula: a. Champy 1913 F 16700.0090; B. water 60, chromic acid 30, acetic acid 0.6; C 3% potassium dichromate method: fix A, 3-4 days —* wash -^ B, 24 hrs. -^ wash — * C, 3 days —* wash — > [sections stained by DS 13.22 KuU 1913 or DS 22.21 Baker and Thomas 1933] 6700.0010 Stockwell 1934 see ADS 12.2 StockweU 1934 6700.0040 Chromic-dichroynate-nitric 6700.0040 Gatenby 1937 Gatenby and Painter 1937, 380 formula: water 235, chromic acid 2.25, potassium dichromate 4.5, nitric acid 15 6700.0040 Kollmann 1885 1739, 51 :296 formula: water 250, chromic acid 5, potassium dichromate 12.5, nitric acid 5 6700.101 0 Chromic-dichromate-fonnaldehyde-acetic 6700.1010 Semmens 1939 CS33 — auct. Microscope, 3 :5 formula: water 238, chromic acid 3.75, ammonium dichromate 2.5, 40% formaldehyde 12.5, acetic acid 0.6 6800.0000 Chr()Mic-(other inorganic salt.s) 6800.0000 Bhaduri and Semmens 1942 11360, 62:21 formula: water 250, chromic acid 2.5, sodium uranate 2.5 232 METHODS AND FORMULAS F 6800.0000-F 7000.0040 6800.0000 Lenoir 1926 19076, 38 :720 formula: water 250, chromic acid 2.5, potassium iodide 2.5 6S00.0010 Chromic- {other inorganic salts)-acetic 6800.0010 Semmens 1939a CSl9—auct. Microscope, Z -A formula: water 249, chromic acid 2, ammonium uranate 0.65, acetic acid 1 note: The ammonium uranate is prepared as follows. Wash 10 uranium trioxide by- stirring and decantation with 20 5% hydrochloric acid. Dissolve residue in 20 cone, hydrochloric acid, filter and add ammonia to filtrate until precipitation complete. Wash and dry ppt. 6800.0010 Semmens 1939b Flemming-uranic — auct. Microscope, 3:5 formula: water 238, chromic acid 1.9, sodium uranate 2.5, acetic acid 12.5 note: The sodium uranate is prepared as the ammonium uranate of "a," with the substitution of N sodium hydroxide for ammonia. 6800.0010 Semmens 1939c Benda-uranic — auct. Microscope, 3:5 formula: water 250, chromic acid 1.5, sodium uranate 2, acetic acid 2.5 6800-0030 Chro7nic- (other inorganic saUs)-formic 6800.0030 Pianese test. 1904 Besson Besson 1904, 750 formula: water 250, chromic acid 1, cobalt chloride 20, formic acid 0.3 F 7000 BICHROMATE WITHOUT OTHER PRIMARY FIXATIVE AGENTS 7000.0000 Dichromate Alone Bichromate alone is an adequate general-purpose bulk fixative when it is desired to preserve large masses of material. The cytological and histological pictures obtained from this fixative, however, are not so good as those obtained with other classes. 7000.0000 Cajal 1890 23632, 7 :332 formula: water 250, potassium dichromate 9 7000.0000 Cole 1884 Cole 1884b, 21 formula: water 185, 95% ale. 65, potassium dichromate 3.6, sodium sulfate 2 7000.0000 Gerlach 1872 test. 1928 Gatenby and Cowdry Gatenby and Cowdry 212 formula: water 250, ammonium bichromate 5 7000.0000 Golgi 1903 Golgi 1903, 1:163 formula: water 250, potassium dichromate 5 7000.0000 Hamilton 1878 11025, 12:254 formula: 25% ale. 250, potassium dichromate 4.8, potassium sulfate 2.4 7000.0000 Miiller 1859 Miiller's Fluid— compl. script. 22302, 10 :80 formula: water 250, potassium bichromate 6.25, potassium sulfate 2.5 7000.0010 Dichromate-acetic 7000.0010 Tellyesniczky 1898 1780, 52 :242 formula: water 250, potassium dichromate 7.5, acetic acid 12.5 note: This is the " Tellyesniczky 's Fluid" of most authors, but see also F 0000.1010 and F 3500.0010. 7000.0040 Dichromate-nitric 7000.0040 Benda test. 1900 Pollack Pollack 1900, 30 REAGENTS REQUIRED: A. 10% nitric acid; B. 1% potassium dichromate method: [fresh tissue] — > A 24 hrs. — > B 14 days 7000.1000 Dichromate-formaldehyde The apparently irrational dichromate-formaldehyde mixtures, including the next class with the addition of acetic acid, are excellent fixatives for heavily yolked material. They exercise less hardening effect, and render this material less brittle, than any other group. Though the majority have been proposed for the purpose of cytoplasmic fixation only, they are to be widely recommended in embryological practice. These mixtures must be prepared F 7000.1000 FIXATIVES 233 immediately before use and fixation should always take place in the dark. It is desirable to wash them out with a weak solution of formaldehyde, also in the dark, until such time as no further color comes away. 7000.1000 Bock 1924 23032,40:318 formula: water 240, potassium dichromate 5, sodium sulfate 2, 40% formaldehyde 10 7000.1000 Braus 1896 7137, 5:307 formula: water 190, potassium dichromate 4.8, 40% formaldehyde 60, potassium sul- fate 2.4 7000.1000 Bubenaite 1929 23632, 46 :359 formula: a. water 225, 40% formaldehyde 25, B. water 250, potassium dichromate 6 7000.1000 Cajal test. 1933 ips. Cajal and de Castro 1933, 282 formula: water 250, potassium dichromate 10, 40% formaldehyde 37.5 7000.1000 Durig 1895 766, 10 :659 formula: water 225, potassium dichromate 7.5, 40% formaldehyde 25 7000.1000 Eisath 1911 14370, 20:3 formula: water 250, potassium dichromate 7, sodium sulfate 4, 40% formaldehyde 37 7000.1000 Fish 1895 21400a, 17:319 formula: water 250, potassium dichromate 7.5, 40% formaldehyde 2.51 7000.1000 Hultgren and Andersson test. 1910 Poll Ehrlich, Krause, et al. 1910, 1:232 formula: water 250, potassium dichromate 5, potassium sulfate 2.5, 40% formalde- hyde 10 7000.1000 Kopsch 1896 766, 11 :727 formula: a. water 200, potassium dichromate 7.5, 40% formaldehyde 50; B. water 250, potassium dichromate 8.75 note: See MS 34.1 Kopsch 1896. 7000.1000 Kose 1898 see F 7000.1000 Stilling 1898 (note) 7000.1000 Lachi 1895 14425, 5:15 formula: water 187, potassium dichromate 7.5, 40% formaldehyde 63 7000.1000 Landsteiner 1903 2526,33:237 formula: water 240, potassium dichromate 3, potassium sulfate 1.5, 40% formalde- hyde 12.5 7000.1000 Mettler 1932 20540b, 7:102 formula: a. water 125, 40% formaldehyde 125; B. water 250, potassium dichromate 7.5 method: [brain exposed but not removed from body] -^ A, 3-5 days — > [brain re- moved to 5 1 wk. in dark] -^ thin slices to fresh B, changed when discolored, 2 wks. in dark ->" F 1700.0000 Mettler 1932 note: Before metal staining, neutral formaldehyde must be employed. 7000.1000 MoUer 1899 23635, 66 :85 formula: water 200, potassium dichromate 6, 40% formaldehyde 50 7000.1000 Miiller 1899 Muller-formol — compl. script. 1780,55:11 formula: water 225, potassium dichromate 1.5, 40% formaldehyde 25 7000.1000 von Orth 1896 23632, 13:316 formula: water 225, potassium dichromate 5.6, 40% formaldehyde 25, potassium sulfate 2.25 note: The liquid of Schridde 1910 {test. 1937 Gatenby and Painter, 396) is identical. 7000.1000 Pfeiffer and Jarisch 1919 23422, 10:1 formula: water 225, potassium dichromate 8, 40% formaldehyde 25 7000.1000 Regaud 1910 1823, 11:291 formula: a. water 200, potassium dichromate 5, 40% formaldehyde 50; B. water 250, potassium dichromate 7.5 method: a. 5 days in dark, changing every 24 hrs. -* B,S days, changing every 24 hrs. 234 METHODS AND FORMULAS F 7000.1000-F 7000.1010 7000.1000 del Rio-Hortega 1928 test. 1933 Cajal and de Castro Cajal and de Castro 1933, 281 formula: water 225, potassium dichromate 15, chloral hydrate 15, 40% formalde- hyde 25 7000.1000 del Rio-Hortega 1929 3231, 9:21 formula: water 225, potassium dichromate 4.5, 40% formaldehyde 25 note: This solution must be made with pure distilled water and borax-neutralized formaldehyde if used before metal staining. 7000.1000 Sanchez test. 1933 Cajal and de Castro Cajal and de Castro 1933, 128 formula: water 225, potassium dichromate 5, 40% formaldehyde 25 7000.1000 Schreiber 1898 766, 14:275 formula: a. water 237, potassium dichromate 0.9, 40% formaldehyde 16; B. water 225, potassium dichromate 4.4, 40% formaldehyde 3.8 7000.1000 Schridde 1910 see F 7000.1000 von Orth 1896 (note) 7000.1000 Smirnow 1898 1780, 52 :202 formula: water 200, potassium dichromate 10, 40% formaldehyde 50 7000.1000 Smith 1930 Turtox News, 8:91 formula: a. water 225, 40% formaldehyde 25; B. water 250, potassium dichromate 10 method: [fresh tissues] —y A, till required -^ thin slices, B 3-5 days -^ distilled water, thorough wash -^ blot -^ MS 34.1. Andriezen 1884 7000.1000 Stilling 1898 1780, 52:176 formula: water 225, potassium dichromate 6.25, 40% formaldehyde 25 note: The formula of Kose 1898 (20181, 6:224) is identical. 7000.1000 Strong 1895 766, 10:494 formula: A. water 250, potassium dichromate 8.75, 40% formaldehyde 8.75; B. water 163, potassium dichromate 7.5, 40% formaldehyde 87 note: See MS 34.1 Strong 1895. 7000.1000 Whitehead 1932 11431,35:415 formula: water 220, potassium dichromate 6, 40% formaldehyde 30 7000.1000 Wiesel 1902 764, 19:481 formula: a. water 225, potassium dichromate 2.5, 40% formaldehyde 25; B. 2% potas- sium dichromate method: [fresh tissue] -^ A, 4: days — > B, 2 days 7000.1010 Dichromate-formoldehyde-acetic This class of fixatives is warmly recommended by Romeis 1948, p. 58, under the generic name of Kaformacet. 7000.1010 Bohm and Davidoff 1905 Bohm and Davidoff 1905, 27 formula: water 225, potassium dichromate 5, 40% formaldehyde 25 preparation: Add the formaldehyde after complete solution of dichromate. 7000.1010 Bohm and Oppel 1907 Bohm and Oppel 1907, 114 formula: water 225, potassium dichromate 2.25, sodium sulfate 1.25, 40% formalde- hyde 12.5, acetic acid 12.5 7000.1010 Cummings 1925 11135,38:401 formula: water 250 potassium dichromate 8.5, 40% formaldehyde 10, acetic acid 12.5 7000.1010 Haver 1927 11431,30:621 formula: water 225, potassium dichromate 5.6, sodium sulfate 2.25, 40% formaldehyde 12.5, acetic acid 12.5 7000.1010 Held 1909 65, 31:196 formula: water 240, potassium dichromate 2.5, 40% formaldehyde 8.8, acetic acid 4.0 7000.1010 Joseph 1918 1798, 38:164 formula: water 175, potassium dichromate 5.4, 40% formaldehyde 50, acetic acid 25 F 7000.1010-F 8000.0010 FIXATIVES 235 7000.1010 Lillie 1948 Lillie 1948, 35 formula: water 220, potassium dichromate 3.3, 40% formaldehyde 22, acetic acid 11 7000.1010 Romeis 1948 Romeis 1948, 58 formula: water 212.5, potassium dichromate 6.5, 40% formaldehyde 25, acetic acid 12.5 7000.1010 Semmens 1939 CS32—auct. Microscope, 3 :5 formula: water 238, ammonium chromate 10, methenamine 10, acetic acid 2.5 note: The methenamine is decomposed to formaldehyde by the acid. 7000.1010 Smith 1912 11373,23:91 formula: water 230, potassium dichromate 2.5, acetic acid 6.25, 40% formaldehyde 12.5 recommended for: heavily yolked embryos. 7000.1010 Wittmaack /<.s/. 1910 Poll l-]lirlic]i, Krause, et al. 1910, 1:233 formula: water 210, potassium dichromate 12.5, 40% formaldehyde 25, acetic acid 7.5 7000.1030 Dichromate-formaldehyde-formic 7000.1030 Ciaccio 1910 22575, 199:381 formula: water 210, potassium dichromate 9.8, 40% formaldehyde 40, formic acid 1 7000.2000 Dichromate-acetaldehyde 7000.2000 Vassale and Donaggi 1895 14425, 6:82 formula: water 250, potassium dichromate 8.75, acetaldehyde 12.5 7800.0010 DlCHROMATE-(OTHER INORGANIC SALT)-ACETIC 7800.0010 Russell 1941 4349, 21 :47 formula: water 250, potassium dichromate 6.25, zinc chloride 15, acetic acid 12.5 7800.1000 Dichromate-{other inorganic salts) -formaldehyde 7800.1000 Gross and Lohaus 1932 23632, 49:168 formula: sodium borate-HCl buffer, pH 7.6 250, potassium dichromate 2.5, calcium chloride 2.5, 40% formaldehyde 10 7800.1000 La Manna 1937 23632, 54:257 formula: a. 4% formaldehyde; B. water 250, potassium dichromate 15, zinc chloride 12.5 method: a, 3 days -^ B, 1 day, 56°C. 7800.1012 Dichromate-{other inorganic salts)-formaldehyde-acetic-trichloroacetic 7800.1012 Kolmer test. 1938 Walls 20540b, 13 :69 formula: water 217, potassium dichromate 4.5, uranium acetate 1.9, 40% formaldehyde 9, acetic acid 23, trichloroacetic acid 12 RECOMMENDED FOR: whole eyes. note: Walls (loc. cit.) refers to this formula, without reference, as "Kolmer's Fluid." This term is more usually applied to F 3700.1010 Kolmer 1912. F 8000 SOLUTIONS WITH "OTHER INORGANIC" PRIMARY FIXATIVE AGENTS 8000.0010 (Other inorganic agent)-acetic 8000.0010 Belar 1929 23639b, 10:76 formula: water 125, 95% ale. 125, zinc chloride 13, acetic acid 13 8000.0010 Juel test. 1915 Meyer Meyer 1915, 198 formula: water 125, 95% ale. 125, zinc chloride 5, acetic acid 5 8000.0010 Rawitz 1909 23632, 25 :385 formula: water 100, 95% ale. 125, phosphotungstic acid 10, acetic acid 25 236 METHODS AND FORMULAS F 8000.0014-F 9000.0010 8000.001 4 {Other inorganic agent) -acetic-nitric 8000.0014 Gilson 1890 6011,6:122 formula: water 80, 95% ale. 20, zinc chloride 5, acetic acid 1.25, nitric acid 1 8000.0030 {Other inorganic agent) -formic 8000.0030 Carpenter and Nebel 1931 19938, 74:154 formula: water 250, ruthenium tetroxide 0.1, formic acid 1 8000.1000 {Other inorganic agent)-Jor maldehijde 8000.1000 Aoyama 1930 23632, 46:490 formula: water 210, 40% formaldehyde 40, cadmium chloride 2.5 8000.1000 Baker 1944 17510, 85:1 formula: water 225, calcium chloride 1, 40% formaldehyde 25 8000.1000 Besta 1905 19460, 39:1 formula: water 190, 40% formaldehyde 60, tin ammonium chloride 10 8000.1000 Cajal 1914 21344, 12:127 formula: water 187, abs. ale. 63, 40% formaldehyde 37, uranium nitrate 2.5 8000.1000 Drew 1920 11360, 40:295 formula: water 200, cobalt nitrate 5, sodium chloride 2, 40% formaldehyde 50 8000.1000 da Fano 1920 11360, 40:157 formula: water 250, 40% formaldehyde 37.5, cobalt nitrate 2.5 8000.1000 Fish 1896 21400a, 17:319 formula: water 200, 40% formaldehyde 50, zinc chloride 15, sodium chloride 88 8000.1000 Krohntal 1899 23632, 16:235 formula: water 235, lead formate 2, 40% formaldehyde 15 8000.1000 Lawrentjew test. 1933 Cajal and de Castro Cajal and de Castro 1933, 360 formula: water 80, 95% ale. 80, 40% formaldehyde 80, arsenic trioxide 1 8000.1000 Lison 1931 1825, 41:343 formula: water 250, lead acetate 10, 40% formaldehyde 25 8000.1000 Merland 1935 4285a, 12:290 formula: water 250, neutralized 40% formaldehyde 37.5, sodium bromide 25, cobalt nitrate 5 8000.1000 Michmanns 1946 11431,58:93 formula: water 225, cobalt nitrate 2.5, calcium chloride 1, 40%, formaldehyde 25 8000.1000 Penfield 1930 608b, 6:445 formula: water 200, 40% formaldehyde 50, potassium iodide 15, urea 10 8000.1000 del Rio-Hortega 1925 test. 1928 da Fano Gatenby and Cowdry 1928, 644 formula: water 225, 40% formaldehyde 25, uranium nitrate 3.75 8000.1000 Slonimski and Cunge test. 1948 Romeis Romeis 1948, 470 formula: water 250, potassium ferricyanide 4, 40% formaldehyde 25, sodium chloride 10 8000.1000 Warthin 1916 4349, 6:71 formula: water 250, 40% formaldehyde 25, calcium chloride 0.05, potassium chloride 0.05, sodium bicarbonate 0.025, sodium chloride 2.25 8000.1010 (Other inorganic a gent) -formaldehyde-acetic 8000.1010 Salkind 1916 6630,79:16 formula: acetic acid 30, lead subacetate 30, water 150, 40% formaldehyde 30 F 9000 SOLUTIONS WITH "OTHER ORGANIC" PRIAURY FIXATIVE AGENTS 9000.0010 Sannomiya 1926 8542a, 4 :363 formula: abs. ale. 250, acetic acid 12.5, sulfosalieylic acid 7.5 F 04 Basal Fixative Solutions FIXATIVES 237 9000.0000 Bank and Davenport 1940 20540b, 15 :9 formula: water 125, 95% ale. 125, formamide 10, chloral hydrate 6 9000.1000 Huseby 1946 16913,61:122 formula: water 225, resorcinol 12 to 20, 40% formaldehyde 25 9000.4000 Davenport, Windle, and Rhines 1947 Conn and Darrow 1947, Id, 24 formula: paranitrophenol 12.5, water 112, 95% ale. 112, formamide 25 F 04 Basal Fixative Solutions 04.0 EXPLANATION These basal fixative solutions were originally suggested by Gray 1933 (11360, 63:13) where he published a brief list of the more common fixatives which could be prepared by their aid. This list was slightly amplified, and formed an appendix to the tenth and eleventh editions of Lee's Microtomisi's Vade Mecum. Briefly, the method involves having available standardized solutions, through the mixture and dilution of which it is possible to prepare about 80% of commonly employed fixatives. The original method of Gray involved the preparation of two series of such solutions, known as the aqueous and alcoholic series, but the latter has now been suppressed, since experience has shown it to be impractical. The system of numbering the solutions used in this volume differs from that originally proposed and is based on the numerical classification used in the first part of this chapter. That is, the number designating the stock solution corresponds to the number used to designate this ingredient in the list of formulas which have already been given. All the solutions employed are stable except the osmic acid, the preparation of which is described in the next paragraph. References to the original citations of the fixatives, indicated in the following list by the author's name and date only, will be found in the preceding section of this chapter. They may be traced either from the alphabetical index or by taking the first four numbers at the head of the column. This will give, in that order, the numerical designation of the class in which the fixative belongs. The addition of the symbols for modifier and acid is readily made by simple inspection. 04.1 LIST OF SOLUTIONS The following solutions are of sufficient strength to require dilution for the preparation of any of the fixatives shown. The first two columns in this tabular material show the two most commonly employed diluents. The last column shows such other ingredients as must be added to secure the required fixative. It is understood that when the ingredient is liquid, a volume is indicated and when the ingredient is solid, a weight is indicated. Solution 1 — 2% osmic acid note: This should be prepared in chemically clean glassware in filtered, triple-distilled water, to which has been added enough potassium permanganate (approximately 0.01%) to give a faint pink color. This pink color should be maintained b}^ the addition of a few drops of potassium permanganate solution whenever necessary. Solution 2 — 1% platinic chloride Solution 3 — 7% mercuric chloride Solution 4 — 5% copper sulfate Solution 5 — a saturated aqueous solution of picric acid. Solution 6 — 2% chromic acid Solution 7 — 7.5% potassium dichromate Solution 8 — 7.5% potassium dichromate and 3% sodium sulfate note: This is a triple-strength Miiller's 1859 fixative. The previous solution (7) may be substituted for it by those who do not believe in the efficacy of the sulfate content of Miiller. Solution 9 — 40% formaldehyde Solution 10 — glacial acetic acid 238 METHODS AND FORMULAS Aigner-Bartelmez ca a o CS c^ ^ c2 c2 (U o o ^ lO iC o »o o (N 1-1 cq rt rt lO c^ lO o H ^ o rr) 1— 1 z O W S w D <1 O EC Ph iJ >— -< <3 CO -< Q m H O ^ -i3 « P4 « ^- CO ^ • 8+1 ^ ^<^co M coco .^ lO 00 (D ^O -, ^ lO "^ o O CO CO >a 02 lOiOOiOiOOO o t^ t^ lO t^ t^ O lO •* ^02 CO O m « -5 o o O CI O O iC lO (M iC lO 00 O p5 O O N ^O OO ■ ^ -O -lO OO -OOiC • *^0 O-O 0--0 *S '3 -rt* ■ lO lO--Ci § w CO o O 00 •rH OS o o >o "^ r^ o lO CO ;o ^ o 00 00 00 OS OS OS OS OS (N * OS OS OS OS 00 00 00 00 ca CIS 6 S o OS oj »-l 00 c -• 2 c S I' 4, >^CS O CU < < o o T-l 00 o o OS OS to t- OS OS 00 00 ^CS ^CS < < a> o o m OS "O T-i d >> >. Jl XI -*-• ■4-» ^C8 ^CS 00 o OS PQ w •O ,H 00 « Si-" >> CS t:3 j3 ti -- ■<-> 'S o < < < O lO iC (N O (N (M OS CO (N lO CO C: OS CO CO a» CO OS iH Tjl lO J_ ^ ^ (i> OS OS _Q T-I ,H 13 IH ll j3 > 4> 4) In (1 Ih -<-> _< IH Ui Vh iH C3 cS ^ cS cS PQ PQ PQ PQ PQ Bartelmez-Besta FIXATIVES 239 a a o u O CO o u 03 O a • (N O '^. »C lO CO 1— t Q 1-^ 1—1 a m 3 O '3 o O O o o 111? CO "^ 00 d d d d O CO d d O 03 '4< (N ^ 4) ■* o^ • 00 o • o lO o 00 _• CO ^t, O _|_ e3 m o O 2 CO o ■ CO CO O lO o o o z Cfa «C '^ lO o "^ c^ -f t^ CO lO O LO 00 lO eo eo w eo so T-( ,-1 1-1 tH »H o o> ^ ^ ^ J2 (J >a to T-l •tH Tt< a» o» o tH tH o> N N tH a> u o ID d) PQ PQ o o a e QQ c c CS c8 Ih U f\ -^ ■ cS »0 ^ t-j rt4S oS2J'-'«oeo 0JOClS00OOO'0 050J0S'" qi-I»HtH c .5 (-■ *ih w cs rt M^HiS — z3 c c a a ^ Q> ^ i> pQnpQpqpqpQPQpqpq h- CO CO • • t^ • CO C LO o eo t- ir. ■ ■ • lO lO t- 00 O) 00 00 00 00 CO CO eo eo eo o a O) o o >1 t-4 1-1 •1-1 tH i-t V >.>.>>>>>. n 4> 4> 43 4> 4> c OS to 05 (n U] U) c c c c c y-t cB ta rt c8 rt ,- O 1-4 C iH iH > -,>,>,>,>>>>> OS o» ") 1—4 iH 0) t- •4-1 ■4-1 4 4> 4) 4> 4> 4> -J m (D C<3 CQ on 0 C tr C/1 !/3 t/5 Ifl t/I v •^-» 4-* r c c C c n c (3 c >- c/i c/: m 41 4, 4 4> 4> 4) 43 4) 4J 43 43 > PQ PC PQ pq pq P3 pi) PQ PQ PQ PQ 240 METHODS AND FORMULAS Besta-Cajal en a o 40% forma] dehyd 00 1— « CO e^ 9 4- =« o t- H t-.;r P A ►J O M m (O ^P < ^e W < ^ M lO at. pier aci 02 "* ■*!< .-H 3 NaCl' cetone NaCl H2SO 00 03 "-^ C^ • r~^ t^~ '""' •r5 -O o o u o 10 ■ a> ^ o lO 1—1 lO 10 (M fO (M '-' ■ 00 • t^ -* O O O lO 10 (M O (M O CO o I> (N »0 ■^ CO CO O (M 10 o « ^O bO '^ o i^ C4 ^ O W co' ^ ^9 c^ o o 10 10 Cq CO >o 00 10 6? 10 ■ CO z 03 W2 o C8 '^ ••OOCJiCCOiCOO -^CiiMOCOOi-Ht^ r-4 Ci I- CO o « S ii s O OS Z3 00 0> 00 rd T-t > d Ci S O 5 c3 N 10 Tj< i2 ^ o Of t- t- c o c3 Xi 00 0 OS o* o> th il; a> (V n) a> Cii Oi c:l, p, Oc CLi CLi C 0000 CO .S?.5f .S .H mmmcSnnpqpqpqpqnnpqpqpqpq;^ :0 :0 0> - — ' '^ 00 rH S; c c e bfl.3 a c tT 3 0000 OOOOiOcOOO*^ lO'^CDOOiCOOO OS OS •r-l ■^ o o pq PQ 00 Tjt 00 OS OS y-i 00 ca :S PQ PQ os OS 00 00 13 "O u l-i cs ca 3 3 PQ PQ eo 00 ^ .^ eo OS o o th CO OS 00 OS OS OS OS rH ^ 00 00 00 00 'P Jr ca rt Gj ca 3 !2 •'->•'->•»->•'-> 3 3 ca ca ca ca PQ PQO O U O Gajal-Cori FIXATIVES 241 09 a o 9 03 a> o wd -> a LO to ^^ •Offi IS 'S OS o a a CD lO d "1 rH CO CO >— 1 r^ CO rH O o CO iq CO d co' ■ iQ O O CO • (M ^ CO ■ >o o • d (M d d o d 0) O (M O 00 ^- CO '^ o 4- (S o n 2: o P iJ O OS < < n CO (M C^ O o CO c^ o lO CD O IN CO • 00 o o o o o o O -ti lO (N (M lO (N lO CO •^- ■* o eo S. bC CT> O O (N CO Cl o o iM o C4 (NQ IM 00 OOOtO(NIM IN C-q' (N (M O IS ci IN ■ t- O • lO o o o CO ■ t^ o CO lO o > 5^ ^ u, o o o ^ c a e ^ 1^ 1^ It IH c^ cd ^ cd u o o u M to (U o tH OS •a c I- o o 0> ci3 X) O "O eo eo eo CO OS OS OS OS OS lO (N OS Q4 o< o< o< &i e s E 6 e 03 cQ cd cC cC O 43 J3 XI XI 43 X! o o o o o u o 3 XI o OS o o 00 ; ■ ■ ^ S • : 'S OS Tt< e^ OS 52 o ^ -^ ^ o [2 c - -^ 6 -H ^ •^^ rt^ O •— '-^ j3 4*: ^000000 o u u o u u o 242 METHODS AND FORMULAS Cox-Ewald a o •o v o o o i-H 00 o • r3 ^ ii o • o lO >— I lO cS o ^ • • o • • F— f .2^ o3 ;z; lO °^ 1> o o lO lO . • -1- «^ o p ■ o -t< CO CO o c^ >o lO CO o p o m Hi < CO ^O ^ (M lO O o O (M' CO O CO (M lO Tfi O O o 00 CO o o o o m ^O O Tt^ O O • • lO • lO • (M O (M 05 00 1> •<*< 00 C9 CI w CD P4 (N Q o o IN (N »o O "^ O (N CO CO (M O to ^ E:; CO CO 00 t3 a CIS . . O CD 05 O 00 ^ '^ 00 ^ 00 "3 : t- S eo o» 2 S 00 '-I 00 '-" '^ eo C OS 00 CO ■ '— I O CO o . t^ lO Tt^ CO O <35 00 00 00 00 1-i 1-1 !tt itl o o > > t3 O o a> a O 00 to eo . OS 0*0^ — , OS '-I OS '-' M <^ ^^ .3 OS 4) S W (d ci 0) Si E '-I Faber-Friedmann FIXATIVES 243 a o •a « It a> O K CO ^§ o o •J:2o 1^- CO O a o d >0 ~. (M iC ^ "^ --O^ ^ d (N >o >o o o o o ^ o ^A O -c w CO Ph H-c lO 00 Mh t- 1— 1 IM O H 00 >c o o a> i^ • lO C^ IQ O d C lO lO 1^ 1^ (N CO CO rt (N ci lo lO ^ --1^ 00 o (N O iC I-H O oo o IM o o CO lO o o* S 1-1 «* V CA CO cS cd d, (14 loc^toiooio •tOictOiciC'Oi^iocoiccor^ --fcoic — c^ l^t^OiOOOOOC3t^05'^iOOOCO 00 00 00 00 s s V 01 00 3 (14 pL4 (X4 pb| •^ t3 rrt ca U3 ca ^ to to CO (O 0)0)0 0) 00 00 00 00 1-1 T-t tH 1-1 K K J3 J3 J3 ^ S 6 (A ui ca ca V a> ■ ^H 'vH ivH 'w^ ^^ ^i^ pL4 (l4 (X4 (Z4 (X4 (X4 o> .g .3 .H .S .3 " to o> o> la ca ^ 00 «o CO s ca ^ o> '-• o o '^ « c^ S eo '-' I CO *_? O) Ui o o 00 o 00 o> 2 0> 0> e 2 iH jH 5 1—1 -^^ -^ — ' J_» ^^OOOOOOOOOw»»M P^(X4(I^t^p^(X((X4pE4pE4(]H(I4(I4(X4(X4(]4 00 di 1-1 T3 _ c a a ca ., E 244 METHODS AND FORMULAS Gage-IIaver CO c o a> J3 O CO o ■ d d b4 i OS W) o »o (N t CO lO c3 03 a a O O in lo 4) o3 O d J - iC CO o o CO lid o PS W CO lO O iC lO O 00 CO CD CO to CO iQ lO ■ lO • CO o o o 00 '^ O) tr- 3 '-' O 0) [fl C to a> Oil cd ^ cd o o o o Spa U Ci C/l (A O N OS t- «H 00 m CO o 00 CO "^ CO '^ t~ lO 1:^ l^ O o> ^- o •^ B O cQ 0) (1) O O > >oooo J3 u O) (U 00 00 *: SP t- o o eo 00 <=> o o> >. 00 o> oj S g> ^ tH T-l «E «? tfl ._ ._ ._ bfl bO tuD O >4-i o o o o to ^ :3 o O O O O O O O lO O CO lO CO CO CO Ph ^ O 5? j^-s-g CO CO liO O O O I- 3 t^ lO "-I ■* CO CO 00 00 efl CO o> OS lO 00 ^ "^ <= J3 00 00 O 00 eo csj 6 U c ^ d> OJ l-i > > «i iH iH > rt 03 rt c8 cS CrtflJ^H^X+J+J>'-^t-lt-tlHMW^ iHi-33S3ScSc8c^ o e« • O ^-1 O o UJ o • . D trichlor acid IS o O a _3 O rt c o o o o (N 1— i o iC (N f >o 1— 1 o o 3 . 1—1 •o -* lO w cc rci • ic '-. _ (N 'V^ ■ (M IC o rH r^ <^ 1— i o o T-H T— ( a O) ^ 1 S o ^*^ o lO o o lO >0 . lO • • o 5 "o (>4 o '"' CJ (N re o ■ • • u o 03 O o lO C3 lO (N --H ^^ t- '--^o p M hJ o ■j: to ^C < ^6 tn < j * K o o lO CO eS a "^ C/J Tjt ^02 to O CO ^O 0 3 3 1-3 (^ bC 3 to 3 O iC 3 lO lO i-H 3 I-- O • iC CO "-I iC • c^ 04 C4 >c lO 3 ^ r" — 3 O Utl 1.1 3 (M 00 O o I- z ti fa p: W CO O ■ 1:0 00 ■ " 3 CO -f S " OOCDOiCOCOCOCOCO .S .S .3 .S .S .S .S .3 cccccccc •Q *0 T3 'O *t3 "O "O 'O • -^ .-^ tv^ .— ^ ..-» •.-< .^^ ..^ r- 3 >C 3 (M I- CO 3 00 iC Tf 1- "> •" n rr\ t- 2? 5? eo g 05 o a> o o o 1-1 CO o O) 0 N C -C "O "O ^ O 1^ 4^ c^ ^ 00 T-l 10 CI 00 05 00 00 iH ^ 3 'O C5 (M •O .^ C ■*-» ♦J +-» 4-» ^M ^^ '*P^ ^^ ^^ ^^ ^^ ^^ ^^ ^^^ ^^ ^^ ^^ ^^ ^^ WW 10 o a> ^o b£^ Ui — WW 00 .-I OS .- O* ^S J3 ^ .t: o WW o 3 00 o OS 1-1 p4 □ ^ c3 i-i >5 -^ -< 00 tH — . »-l -^ '*3 *- !r, c « JJ 003 WWW W 10 10 OS 00 a OS ■" c ** -5 -o 246 METHODS AND FORMULAS 'J. A."-Lachi 09 c o TS U O '5 a o o o o to g O -c 'C O ^ o {2W § ic (N CO CO iC o ^. 1-1 OJ CO .O o O 00 J. « (MO ■ IM • CO lO CO o o ■ ^ 00-; co^ 9 4- «« lO N CO z o Ha* H P h3 O U2 «o M ^ =^o Tf! < HT* m o o _ m -T* u «o . S -S ^a=^ 02 CO 00 ^ (N '^ CO lO (N CO O l^ t- o c CO LO O 00 (M CO 20 00 o o rH 3 CO ^ O lO o t^ f_ bCCO rH Tt< 00 I-- C5 C5 O CO IC i-O o CI --Pi O lO o o 4) w M .5 .3 S 6 6 •^ C (U ^ o o (A VI tn ^3 ay tj c ,H O «0 o> o* o^ •-* ^ 00 00 .^ 4J ti -' +:? in 00 LO 1—1 CO 00 o» .^ la •a 00 K/> '^ K/ K/ K/ ^ ^ PH W PH HH W HH l-l o o o o '^ u, (/] CO LaCour-Lo Bianco FIXATIVES 247 ca u o CO d c o to O •J3 lO O 00 O CO (N "-I ffQ lO "^ "^l CO o d •o , , o . . o , , , (Tt . -^-> , [ ej o OS •1-4 a C CI lo w ^ K '* ^ lO t (N CO ■ 00 »c • 1^^ . o o s^ »o .-H O o o lO CQ z o o DQ < n ^O 00 lO o e«3 CO lO 00 to t>^'»^ O O O C^ CC (M T-H CO lO o o ■ 00 o • • t^ ■ lO lO >o >o (N ■ • »— I ■ (N (N (N to o 00 o CO ^o bC ffi o o t-- o t- O tH Ofl w o> o> T-K »-l IH IH 3 3 O o CJ o eo eo o at [A Cfl a> bC buO.S C3 cl c^ •-1 )-) I-) 4) t- •— o 9. -r^ lO o CO iM a» O ^ s cS CO o o o o o CO CO O 00 00 v c o o u o • C O O O (N ■ Tf lO lO 10 00 OS _ t-< o go 1_. ^^ o» OS 00 00 0) o CO.;* c^ cd cd hj »-) )-4 1^ )-) XI O a> Oi _ 00 00 ^ > •a w >-) t- t- 00 o> OS OS OS OS 00 00 00 00 o > CU 1-1 (A CA a> »4 O t^ t^ lO CO ^ ^ (N O iC lO ■* (N IM CO OS 00 ,-1 '-t . OS O O O o o "=> OS OS OS 05 OS OS 00 00 00 00 00 00 OSmcS .l-tiH'r-t^THi-l 1-t cq Ti< 00 •s---"^ • J ^ .2 .i m 03 PQ m « M vSSSoooooo ,^_1_1h-1)-1i-)i-)i-)»-)i-) o O O O o o u O o o (J o ■ o CO 00 lO e^ o u rn Z ;< o 00 00 00 at ^ CO CS 00 o o c C8 ^-•S-* PQ n n o o o h-) l-j »J TO t3 C > O :o •J 1-) t- 00 00 :o O O iCi CO (N lO (N O •a G o s c ctf X O J3 u IM G O Xfi c o> o iCOcDOOOOOOOiCO'-^ o o 03 iH X x =^ r^ S o ^ o XI «S 43 ui a> ui 00 — OS t- O 0> iH OS J3 «D ^ ■* ■* S2 O o» O 00 ::J 00 00 '-I O* iH tH .^ S i I i in .3 £ cS oi C o tfl tfl X OS ^ 00 00 OS o o £? » OS OS OS « iH ^ ^ OS bo M nd Allen id Cook 1 Ih Ih esbe esbe 00 00 eo OS n X! U 3 3 og 00 o M M eg QQ o tH OS 1-i TO e nn OS OS OS •o •« iH > o •n c hfi ^^ iH 1-i »H 1- c c OS UJ "= fr. u l-i IH OS OS 3 2 0) 0. (1 a Cfl w [/ OS — ' ^ U 0) ^ Vd -4^ -*- > > > > o bO O 4) 4) 0) (U n3 Mislawsky-Pianese FIXATIVES 249 CO a o at u o O o o *i TS lO lO tH ^ (LI O 03 1 "cfl -a (N rT) s >> ^ o o ^ c^ (^^ 00 T^ lO o t^ CO lo CO CO O Tf CD O o o lO (N O O O oo_:co^ O 4- o3 lO ■ o »c CO • CO CO o CO z o H ►J o CO < < n CO "'. o ^ ^ lO 00 CO CO o» <» eo ^ _i _, :0 O :3 0) a> 03 3 s :3 :3 3 3 3 -^ lO t- CO ■^ I- O lO n « C« O lO 1- lO 00 00 lO CO -t •+ lO 00 13 o lO lO o o 05 0> 0) (U c c X X oo OJ CO a> O 00 th -M n 0) 4) 4> OS « 4> > PU pL, Pk , >. 03 c a l-i va> MU I-. 73 — ■« 1-1 o CO l-l CS •a c 03 l-l >o o ^ 00 03 Xi 00 00 1) 0) P, P^ P, Pk P, P, PL< 250 METHODS AND FORMULAS Pietschmann-Rothig xa a o i-i 03 lO (M O 00 o 0) a O id • ■© ,-1 o O 03 hi 1-1 to c3 0) o o O O CO o c3 o o3 O (N O (N lO lO CO o g ^ O o o O O '^ "* i-H 1-1 (M (M O CO Z O o CO iJ -0 •* O (N o to iCi CO CO CO iC lO «o O CO o 00 o o C4 lO (M O (N Q • O o o o o iC CO CO to i* tC to to O z OS w b to O iC lO o • 00 CO 00 a s u 00 00 00 OS t> > O O O to CO o to bC ^~. a X> o> Lr ■rH tH ^ W N "O "O o 3 3"? •- 03 03 P5 ? bi) fcuO cS Ml) CI d o> o> CO 03 oj bD to 0) 0) 03 00 »H t- .,_ O O '-' 2 \»H \f^ ^ — ^ *r: iJ ii ,s ^ •O "O « Ph J3 00 O) s o CO to CO OS 00 o» «0 «£> O ^ ^ o o> 0» oj lO Ui s o S ■^ 2; -22 C CO (A CA Ifl ■k' O O O O p :p p4 pti p4 pa, Pi Pi Rothig-Smirnow FIXATIVES 251 I It « o o o3 o CO lis »f5 .—I 0) ■♦a c3 >> I" O o u o o CO . *r> O d O si d O d ^ . CO • d • CO -! d • (N '* CO CO 00 • . o lO lO O "* • • (N d i-H ^ S 4- =3 O iC CO CO Ifi z o o in •«! DQ < «o to o m OQ ^-d o3 d CO CO CO lO CO »0 Tt< O O TtH CO ■ o o o lO 1-1 (M iH iH S 4> O !tl s -^ CO u W 1/1 ^ 2 CD ^ 00 o" N Xi XI X 3 3 3 ■n ja 43 u u u N CO CO CO CO CO CO C4 eo o> iH CO «-^ .3 c8 > o V .la CO CO eo 00 OS '"I U) O 5 o M 6 CO CO 252 METHODS AND FORMULAS Smith-Sziits CD e o '■^ •O cii i-i a> -^ 3 "^ ei (N c ■;: o &, S3 O O o d o CO 0) o o n:2 d d d iQ -+< Tt< o o o o CO d s o ,9 c3 o> si O lO -^ CO O O iC lO o Ci O 03 • -^ iC >— I 00 00 o>o>o>2222'-''-''-' O O lO o TjH lO IM O CO ^ J3 ^ ^ ^ 0) 0) 0) 0) "3 '3 "3 "S 'S 3 =» 3 3 .*J .2 .2 G C C G C ciiO<0-t->-i-> 00 00 ■!-> *-> O I-. 2 C/3 "> !r! to *> CO > o c > "9 'O .— OS o» ^ 00 00 C ^ tH Ctf ^ c c c 2 o o oj 2 !_, iH !> iH ■4-> •!-• !> CO Ol C/3 O a > Q a cS 13 < ^' CO 1^ I-I o a. c > c8 Q •d ci c« , CO CO O 00 05 ■* 00 to CO OS o c > Q • o CO OS 00 o OS T-l to a iH ^ — N o «co d ©2 0) c SS o tfl Is) rs] N ? >, N N N CO CO CO CO CO Takahashi-Zweibaum FIXATIVES 253 SB a o U (N '^ O (N O ^ -iJ 12 • '^ -w lO « >5 tS ^ o • o» 2 00 00 «2 .-t T-1 2 >.!>.-' •^ K it! o . >> O H H H CC o O lO OS ;-^00 a> o 00 03 X> eo eo 00 00 00 00 0) c 00 2 00 00 o ? ■^^ o CC lO CO eo OS s (N (M O CO 10 O lie O O iQ -r CC !■- (M CC o — ■ 00 > > > > — 4) £ c CO a> 4) > C C O HJ kH *J •S 6 CA i: CO ^ o o 00 en u <1> CC CI o CO CC 1-1 '^ to c 5 c a XI u 6 3 JO N Nl N N Nl 19 Accessory Fixative Formulas Decimal Divisions Used in Chapter AF 00 General observations and explanation of decimal divisions AF 10 Fixative removers 11 For use after picric mixtures 11.1 Formulas 12 For use after mercuric mixtures 12.1 Formulas 13 For use after other fixatives 13.1 Formulas AF 20 Decalcifying agents and methods for softening chitin 21 Acid media for decalcification 21.1 Formulas 22 Alkaline or neutral media for decalcification 22. 1 Formulas 23 Methods for softening chitin 23.1 Formulas AF 30 Bleaching agents 31.1 Formulas AF 40 Macerating and digesting agents 41 Methods involving use of hydrolysis 41.1 Formulas for acid hydrolyzing solutions 41.2 Formulas for alkaline hydrolyzing solutions 41.3 Formulas for neutral hydrolyzing solutions 42 Methods using enzyme digestion 42.1 Formulas AF 50 Narcotizing agents 51.1 Formulas AF 00 General Observations and Classification of Formulas The formulas included in this chapter solutions. The formulas are arranged ac- are those which are used either immedi- cording to the use for which they are in- ately before, or immediately after, fix- tended, and therefore fall logicall}' into ation, and which would therefore seem the classification indicated above, to warrant the title Accessory fixative AF 10 Fixative Removers Most fixatives may be removed from nation of picric acid with various of the tissues by the simple process of washing proteins present. Some of these picric either in alcohol or in water. The principal compounds are soluble in water, and if exceptions to this statement are fixatives picricmaterialbe washed for a long period, containing picric acid or mercuric chloride, many of the cells will be found to be Much of the yellow picric stain cannot be heavily vacuolated. This may be required removed, since it results from the combi- if studies of the nucleus only are being 254 AF 11.1-12.1 ACCESSORY FIXATIVE FORMULAS 255 made and, in this case, even more of the cytoplasm can be dissolved by washing in weakly alkahne solutions. Lithium car- bonate solutions are often suggested, though they are no more effective than sodium carbonate solutions of the same molar concentration. The three special formulas given below (AF 11.1) for the removal of picric from material have specific functions indicated under each. The removal of fixatives which contain mercuric chloride is a far more difficult and \atal matter. It has long been known that sections or wholemounts prepared from mercuric-fixed materials tend to de- velop needlehke crystals, or black gran- ules, presumably of mercuric chloride. The formation of these crystals cannot always be prevented, even by prolonged washing in water or alcohol, and it has become customary to treat sections with a solution of iodine in potassium iodide, on the ground that the mercuric iodide so formed will itself form a complex with the excess potassium iodide and thus be re- moved. The exact composition of the solution employed is not a matter of any great importance, and the several for- mulas given! below (AF 12.1) may be diluted or increased in strength at the option of the worker. The last division of fixative removers (AF 13) gives two formulas for the prevention of precipi- tates, which occasionally occur in mate- rials which have been fixed and stored in formaldehyde for a long period. One formula, which might more properly be- long in the divisions on bleaches (AF 30), is used to remove the black material depos- ited by osmic acid on the outside of small specimens. AF 11 FOR USE AFTER PICRIC MIXTURES AF 11.1 Formulas 11.1 Bolcek 1930 23632, 47:334 formula: abs. ale. 80, nitric acid 10, origanum oil 20, cedarwood oil 10 RECOMMENDED FOR: treatment of picric-fixed materials before celloidin embedding. 11.1 Cappell test. Carleton and Leach 1938 Carleton and Leach 1938, 31 reagents required: A.' 70% ale; B. 5% sodium thiosulfate method: alternate sections between A and B recommended for: removal of yellow color from mounted sections before staining. 11.1 Lenoir 1930 6630, 103:1253 formula: water 70, 95% ale. 30, ammonium acetate 10 recommended for: removal of yellow color from picric-fixed specimens before embed- ding or making wholemounts. AF 12 FOR USE AFTER MERCURIC MIXTURES AF 12.1 Formulas 12.1 Gram 1884 Gram' s iodine — compl. script. 8645,2:6 formula: water 5, iodine 1, potassium iodide 2, water to 300 12.1 Haug 1889 23632,8:11 formula: water 50, glycerol 50, ADS 12.2 Lugol (1905) 2, potassium iodide 1 recommended for: removal of mercury fixatives. 12.1 La Cour 1931 11360, 51:123 formula: 80% ale. 100, potassium iodide 1, iodine 1 recommended for: removal of mercuric chloride fixatives. 12.1 Lugol (1905) test. 1905 Lee Lugol's iodine — compl. script. Lee 1905, 62 formula: water 110, iodine 6, potassium iodide 4 preparation: Dissolve iodine and potassium iodide in 10 water. Dilute to 100. 256 METHODS AND FORMULAS AF 13.1-21.1 AF 13 FOR OTHER USES AF 13.1 Formulas 13.1 Lhotka and Ferreira 1950 20540b, 25 :27 formula: water 100, chloral hydrate 20 RECOMMENDED FOR: removal of "bound" formaldehyde from tissues. 13.1 Overton 1890 23632, 7:10 formula: ale. 70% 100-250, hydrogen peroxide 10 RECOMMENDED FOR: bleaching overfixed or blackened osmic acid preparations. 13.1 Schridde 1906 h-sl. 1938 Mallory Mallory 1938, 40 formula: water 70, 95% ale. 30, ammonium hydroxide 0.5 RECOMMENDED FOR: removal of precipitates from formaldehyde-fixed tissues before embedding. 13.1 Verocay 1908 23681, 19:769 formula: water 80, 96% ale. 20, potassium hydroxide 0.01 RECOMMENDED FOR: prevention of precipitate in formaldehyde-fixed tissues before embedding. AF 20 Decalcifying Fluids and Methods for Softening Chitin These two groups of methods have been thrown together for the reason that they solve essentially the same problem, that is, to soften material normally so hard that it cannot be sectioned. The removal of calcium has for many years been achieved principally with acids in which the calcium carbonate and phosphate are soluble. These acids naturally lead to a gross hydrolysis of the tissues, unless this hydrolysis is restrained by the addition of some other agent. The agents used to restrain swelling are very varied and will be noticed as a part of all the formulas given under the heading AF 21.1. Fairly recently attempts have been made to remove the calcium in solutions that are either neutral or very slightly alkaline. In 1938 Wilkes (14900, 142:958) proposed the removal of the calcium through a base exchange similar to that used in the softening of water, and employed for this purpose a 30% solution of -sodium hexametaphosphate. Dotti, Taparo, and Clarke 1951 (Tech. Bull., 21 :475) used a base exchange resin (Win — 3000) in combination with 10 to 20% formic acid; Birge and Imhoff 1952 {Tech. Bull, 22:16) use a slightly alkaline chelating agent. The softening of chitin (AF 23.1 below) is a problem which has not yet satisfactorily been solved. Diaphanol was once recommended for the purpose, but is no longer on the market, and the preparation of its principal constituent (chlorodioxyacetic acid) is so dangerous that it cannot be recommended in the ordinary laboratory. The technique of Jurray 1937 has given better results in the author's hands than anj^ other, but it will only work on certain insects, and then its reaction cannot always be forecast accurately. 21 EMPLOYING ACID MEDIA FOR DECALCIFICATION 21.1 Formulas 21.1 Andeer 1884 23730,33:193 formula: water 100, phloroglucinol 0.01, hydrochloric acid 3 21.1 Anonymous 1946 4349,26:13 formula: water 85, 40% formaldehyde 10, nitric acid 5 21.1 Bayerl 1885 1780, 23:35 formula: water 100, chromic acid 1.5, hydrochloric acid 0.5 21.1 Belloni 1939 test. 1943 Cowdry Cowdry 1943, 35 formula: water 100, formaldehyde 6, formic acid 50 21.1 von Beust test. cire. 1938 Wellings Wellings circ. 1938, 230 formula: water 100, 40% formaldehyde 10, formic acid 10 RECOMMENDED FOR: embryonic teeth. AF 21.1 ACCESSORY FIXATIVE FORMULAS 257 21.1 "Bielschowsky-Agduhr" see ADS 21.1 Cajal 1933a (note) 21.1 Bodecker 1937 11147,16:143 formula: methanol 100, celloidin 6, nitric acid 4.5 RECOMMENDED KOR: euaniol of teeth. 21.1 Brain 1950 11300, 70:313 formula: water 100, formic acid 5, calcium phosphate 2 RECOMMENDED FOR: teeth (sec E 24.1 Brain 1950, Chapter 27). 21.1 Busch 1877a 1780,14:481 formula: water 100, chromic acid 2, hydrochloric acid 3 21.1 Busch 1877b 1780,14:481 REAGENTS REQUIRED: A. watcr 100, chromic acid 0.1, potassium dichromate 1; B. water 100, potassium dichromate 1, nitric acid 2 method: [fixed tissues] — > A, several days — > B, till decalcified — > rinse — > 95% ale. 21.1 Cajal 1933a Cajal and de Castro 1933, 39 reagents required: A. 20% formaldehyde; B. 4% nitric acid; C. 80% pyridine method: [fresh tissue] —> A, 1-3 days -> B, till decalcified-^ wash, 24 hrs. running water -^ C, 24 hrs. — > 95% ale, till pyridine removed. note: The method attributed, without reference, to "Bielschowsky-Agduhr" by Cajal 1933, 40 differs from the above only in the omission of C. 21.1 Cajal 1933b Cajal and de Castro 1933, 40 formula: water 86, 40% formaldehyde 11, nitric acid 3 21.1 Carleton and Leach 1938 Carleton and Leach 1938, 211 REAGENTS REQUIRED: A. Water 80, nitric acid 10, 40% formaldehyde 10; B. 5% sodium sulfate method: [fresh or fixed bones] -^ A, till decalcified -+ B, till free from acid — > [sections] 21.1 de Castro 1925a 21344,23:427 formula: water 65, ale. 35, chloral hydrate 5, nitric acid 3 21.1 de Castro 1925b 21344,23:427 formula: water 40, 95% ale. 60, urethan 2, nitric acid 3 21.1 de Castro 1925c 21344, 23:427 formula: water 40, 95% ale. 60, aprobarbital 2, phenobarbital 2, nitric acid 4 note: Those solutions are primarily intended for materials subsequently to be metal stained. Tlie author has translated the hypnotics and sedatives from European proprietary trade names to the terms preferred in contemporary American usage. 21.1 de Castro 1926 21344, 23:427 formula: water 50, 95% ale. 50, chloral hydrate 2.5, nitric acid 3.4 RECOMMENDED FOR: use before silver staining of nerves in bone or teeth. ^ note: 1 urethan or 0.6 diethylamine barbiturate may be substituted for chloral hydrate, in which case the alcohol should be increased to 60%. 21.1 Cretin 1925 Le Mans, 48 formula: water 90, potassium iodide 22.5, iodine 25, trichloroacetic acid 10 recommended for: decalcification after F 3500.1000 Cretin 1925 fixation. 21.1 David 1935 19938, 82:179 reagents keqiured: A. 10%, nitric acid; B. 5% sodium sulfate; method: [fresh bone] -^ A, till decalcified^ B, 24 hrs. — > running water, 24 hrs. 21.1 von Ebner test. 1891a Haug 23632, 8:3 formula: water 100, nitric acid 2, sodium chloride 18 note: Add 1 nitric acid daily until decalcification complete. : ( 21.1 von Ebner test. 1891b Haug 23632, 8; formula: water 15, 95% ale. 85, hydrochloric acid 0.4, sodium chloride 0.4 258 METHODS AND FORMULAS AF 21.1 21.1 Evans and Krajian 1930 1789a, 10:477 formula: water 75, sodium citrate 10, formic acid 25 21.1 Ferreri test. 1895 Rawitz Rawitz 1895, 28 formula: water 100, nitric acid 10, phloroglucinol 1 21.1 Fieandt and Sazen 1936 23632, 53:125 REAGENTS REQUIRED: .4. water 90, 40% formaldehyde 10, sulfuric acid 5, ; B. 4% formal- dehyde; C. water 90, 40% formaldehyde 10, lithium sulfate 5; D. 5% lithium sulfate METHOD [chrome-fixed material] -^ A, 4-6 wks. -^ B, 1 wk. -^ C, 1 wk. ^^ D, 1 wk. — ♦ wash RECOMMENDED FOR: inner ear. 21.1 Fischer test. circ. 1938a Wellings Wellings circ. 1938, 42 formula: water 225, 40% formaldehyde 12.5, trichloroacetic acid 2.5, sodium chloride 25 RECOMMENDED FOR: teeth. 21.1 Fischer test. circ. 1938b Wellings Wellings circ. 1938, 43 formula: water 67, formic acid 33, 40% formaldehyde 5 RECOMMENDED FOR: teeth. 21.1 Gairns 1944 20540b, 19:127 REAGENTS REQUIRED: A. 10% nitric acid; B. 2% potassium alum; C. 5% sodium bicarbonate method: [formol-fixed teeth and bones] —>■ 70% ale. 1 day — > A, changed alternate days, till decalcified —> rinse -^ B, 12 hrs. —> rinse — » C, 24 hrs. — > [paraffin sections] 21.1 Gage test. 1937 Gatenby and Painter cii. Fish Gatenby and Painter 1937, 252 formula: water 100, ammonium alum 2.5, nitric acid 5 21.1 de Galantha 1937 4349, 17:72 formula: water 25, nitric acid 10, 95% ale. 100, sat. sol. picric acid 5, olive oil 10 21.1 Gomori 1933 see MS 33.1 Gomori 1933, sol. D. 21.1 Gooding and Stewart test. 1938 Carleton and Leach Carleton and Leach 1938, 211 formula: water 75, formic acid 25, 40% formaldehyde 5 21.1 Haug 1891a 23632, 8:3 formula: water 100, chromic acid 1, hydrochloric acid 1 21.1 Haug 1891b 23632, 8:3 formula: water 100, osmic acid 0.1, chromic acid 0.25 21.1 Haug 1891c 23632, 8:7 formula: water 30, 96% ale. 70, hydrochloric acid 3, sodium chloride 0.5 21.1 Haug 1891d 23632,8:7 formula: water 30, 96% ale. 70, nitric acid 3, sodium nitrate 0.25 21.1 Haug 1891e (Haug's slow decalcifier — compl. script.) 23681, 2:193 STOCK solution: nitric acid 20, phlorogucinol 2, water 80 PREPARATION OF STOCK: Dissolvc phloroglucinol in warm (not hot) acid and dUute to 100. WORKING solution: water 100, stock solution 20 21.1 Haug 1891f {Haug's rapid decalcifier — compl. script.) 23632,8:11 formula: water 30, 95% ale. 70, phlorogulcinol 1, nitric acid 5 preparation: Dissolve phloroglucinol in warm {not hot) acid. Cool, add water and alcohol. 21.1 Heidenhain test. 1933 Cajal and de Castro see F 3000.0012 Heidenhain 1909 AF21.1 ACCESSORY FIXATIVE FORMULAS 259 21.1 Hennings 1900 see F 3560.0040 Hennings 1900 21.1 Hopewell-Smith test. circ. 1938 Wellings Wellings circ. 1938, 42 REAGENTS REQUIRED: A. 10% hydrochloric acid; B. nitric acid; C. sat. sol. lithium carbonate method: [fixed teeth varnished with celloidon] ^ 100 A, 2 days ^ add 1.5 B, leave 2 days — + add further 1.5 B, leave 2 days — > wash — » C, 20 mins. note: Wellings {loc. cit.) states that Choquet recommends adding 1% palladium chlo- ride to A. 21.1 Jenkin 1926 11431,24:166 formula: water 10, 95% ale. 73, acetic acid 3, hydrochloric acid, 4, chloroform 10 note: This is also a fixing and dehydrating agent. 21.1 Katz test. 1895a Rawitz Rawitz 1895, 28 formula: water 100, chromic acid 0.4, nitric acid 5 21.1 Katz test. 1895b Rawitz Rawitz 1895, 28 formula: water 100, chromic acid 0.4, nitric acid 10, palladium chloride 0.01 21.1 Kingsbury and Johannsen 1927 Kingsbury and Johannsen 1927, 78 formula: water 100, potassium alum 3.5, nitric acid 5 21.1 Kristensen 1948 20540b, 23:151 formula: water 82, formic acid 18, sodium formate 3.5 21.1 Langeron 1942 Langeron 1942, 357 . REAGENTS REQUIRED: A. water 45, 40% formaldehyde 5, formic acid 50; B. 4% formalde- hyde method: [formaldehyde-fixed objects]—* A, till decalcified—* B, thorough wash 21.1 Marsh 1878 Marsh 1878, 67 formula: water 100, chromic acid 1, nitric acid 0.75 21.1 McNamara, Murphy, and Gore 1940 11284, 25:874 formula: water 80, 95% ale. 10, trichloroacetic acid 6, nitric acid 1, 40% formaldehyde 8, mercuric chloride 2 21.1 Ralston and Wells 1939 591b, 3:72 formula: water 100, potassium dichromate 2, mercuric chloride 5, sodium sulfate 1, acetic acid 10 note: This is F 3700.0010 Zenker 1894 with the acetic acid content doubled. 21.1 Richman, Gelfand, and Hill 1947 1887a, 44:92 formula: water 100, nitric acid 8, formic acid 10 method: The object is placed in a perforated porcelain capsule suspended in the liquid. One platinum electrode is inserted in the object, the other in the free liquid. An EMF of +6 volts is applied to the object. At 30°C. large objects arc completely decalcified in 2-4 hrs. 21.1 Rosbach and Leavitt 1952 Tech. Bull, 22:198 formula: water 95, trifluoroacetic acid 5 note: This is said to give complete decalcification of teeth in 5 or 6 days without loss of cytological detail. 21.1 Rousseau 1897 23632, 14:207 formula: water 20, 96% ale. 80, nitric acid 20 note: It is recommended that the object be embedded in celloidin before decalcification. 21.1 Schaffer 1903 23632,19:460 reagents required: A. 3% nitric acid; B. b% sodium sulfate method: [fixed material, embedded in celloidin] —> waters A, till decalcified -^ wash — * B, 24 hrs. -^ B, fresh solution, 24 hrs. — * wash. 260 METHODS AND FORMULAS AF 21.1-22.1 21.1 Schmorl 1928a Schmorl 1928, 44 formula: water 90, 40% formaldehyde 10, nitric acid 10 21.1 Schmorl 1928b Schmorl 1928, 48 formula: water 100, potassium dichromate 2, sodium sulfate 1, nitric acid 0.3 21.1 Schridde 1910 test. 1937 Gatenby and Painter Gatenby and Painter 1937, 252 formula: water 90, 40% formaldehyde 10, nitric acid 10 21.1 Sailer 1881 Seller 1881, 43 formula: water 100, chromic acid 0.5, nitric acid 1 note: Gatenby and Cowdry 1937, 253, attribute to "Seller" (without reference) a fluid containing half the quantity of chromic and three times the quantity of nitric. 21.1 Tello 1932 lest. 1933 Cajal and de Castro Cajal and de Castro 1933, 369 formula: water 95, nitric acid 5, chloral hydrate 15 21.1 Thoma 1891 23632, 8:191 reagents required: A. 1% nitric acid in 95% ale. B. 95% ale. rendered milky with chalk method: [fixed tissues]^ A, changed daily till decalcified—^ B, changed daily, till acid-free 21.1 Waldeyer test. 1891 Haug ' 23632, 8:4 REAGENTS REQUIRED: A. 0.17% chromic acid; B. 0.25% chromic acid; C. 0.5% chromic acid; D. water 100, chromic acid 0.5, nitric acid 2 method: [fresh tissue] -^ A, 2 days — » B, 2 days —> C, 4 days -^ C, till decalcified 21.1 Welling circ. 1938 Welling circ. 1938, 37 REAGENTS REQUIRED: A. water 100, potassium dichromate 1, chromic acid 0.1; B. water 100, chromic acid 0.1, nitric acid 2 method: [embryos] —> A, frequently charged, several wks. — > B, frequently changed, till decalcified 21.1 Wilson 1934 11431,39:531 REAGENTS REQUIRED: A. 4% formaldehyde in 0.9% sodium chloride; B. ether 50, abs. ale. 50; C. ether; D. 20% nitric acid; E. sat. aq. sol. lithium carbonate method: [tissue blocks] — > A, 2-3 days — > thorough wash abs. ale. via graded ale. series -^ B, 1 hr. — > C, 2 changes, 1 hr. each -^ B, I hr. — » water, via graded ale. series — > D, in vacuo, till effervescence ceases (20 mins.-3 hrs.) -^ E, large volume, in vacuo, till effervescence ceases (few niins.) — > wash — > [paraffin sections] AF 22 EMPLOYING ALKALINE OR NEUTRAL MEDIA FOR DECALCIFICATION 22.1 Birge and Imhoff 1952 Tech. Bull, 22:16 formula: ethylene diamine tetraacetic acid, tetrasodium 10, water 100 note: This salt which is Versene, and its close relatives Calsol, Versenate, and Scqvrslrene, chelate with metal ions to form nonionized compounds. The decalcifying action is slow, but is stated by the authors to have no action on the staining quality of the tissues. 22.1 Kramer and Shipley lest. 1929 Shipley McClung 1929, 260 formula: water 70, citric acid 25, magnesium oxide 1.3, ammonia 30, hydrochloric acid q.s. for pH 7.5 preparation: Dissolve the citric acid with heat in 30 hot water, add the magnesium oxide and stir to solution. Cool, add ammonia, dilute to 100, leave 24 hours and filter. Adjust to pH 7.5 with hydrochloric acid. 22.1 White 1923 11431,26:425 formula: adjust a 6% solution of acetic acid to pH 7.5-8 with ammonia AF 23.1-31.1 ACCESSORY FIXATIVE FORMULAS 2G1 AF 23 METHODS FOR SOFTENING CHITIN 23.1 Formulas 23.1 Cox If si. 1930 Eltringham Eltringham 1930, 93 REAGENTS REQUIRED : A. 10% potassiuiii liydroxidoj B. 33% acetic acid method: [fixed insects] -^ A, 24 hrs. — > wash, G his. —* B, I day — > wash, 6 hrs. — ♦ paraflin, via usual reagents note: Eltringham {loc. cit.) finds "even the elytra of small beetles . . . sufficiently softened for sectioning in 58° paraffin." 23.1 Eltringham 1930a Eltringham 1930, 94 REAGENTS REQUIRED: A 6% sodiuiii hypoclilorito; B. F 5000.0040 Eltringham 1930 method: [fixed insects] -^ water —> /I, 30 hrs., 60°C. -^ wash, 4 hrs. — > /^, G days, 60°C. -^70% ale, boil 1 min. ^70% ale, 24 hrs. -^ paraffin, via cedar oil 23.1 Eltringham 1930b Eltringham 1930, 95 reagents required: A. 6% sodium hypochlorite; B. F 5000.0050 Kleinenberg 1879 method: [ale. fixed insects] —>■ A, 24 hrs., 60°C. -^ wash -^ B, boil, 1 min. —> B, 60°C., 4 days -^70% ale. boil -> paraffin, via cedar oil 23.1 Eltringham 1930c Eltringham 1930, 95 reagents required: A. F5000.0050 Kleinenberg 1879 method: [living insects] — ^ A, boil 10 mins. -^ A, 6 days, 60°C. — > 70% ale, boil -^ par- affin, via cedar oil 23.1 Henking 1891 23632, 8:156 formula: water 30, 95% ale. 70, hydrochloric acid 0.2, pepsin 0.1 note: This is intended to soften fixed and stained insect eggs before sectioning. 23.1 Hennings 1900 see F 35G0.0040 Hennings 1900 23.1 Kingsbury and Johannsen 1927 see F 3600.0040 Kingsbury and Johannsen 1927 23.1 Jurray 1937 11360, 57:15 formula: chloral hydrate 50, phenol 50 method: [insects fixed in F 3000.0010 Carnoy and Lebrun 1887]-^ mixture, 12-24 hrs. — * paraffin, via chloroform 23.1 Roonwall 1935 see AF 23.1 Slifer and King 1933 (note) 23.1 Slifer and King 1933 19938, 78:366 formula: water 20, 95% ale. 80, phenol 4 recommended for: insect eggs which are treated 24 hours between fixation and sectioning. note: Roonwall 1935 (23833, 110:17) prefers 1-2% phenol. AF 30 Bleaching Agents The formulas here given are designed to remove natural pigments, and particularly melanin, from specimens of which it is desired to make wholemounts. The classic method is that of Mayer 1880, which may be safely employed on almost any material. These methods mostly depend on the use of nascent chlorine, nascent hydrogen, or sulfur dioxide, which are liberated under conditions designed to prevent, as far as possible, damage to the specimens. AF 31.1 Formulas 31.1 Alfieri test. 1910 Mosse cit. Mayer Ehrlich, Krause, et al. 1910, 1:715 REAGENTS REQUIRED: A. 0.1% potassium permanganate; B. 0.3% oxalic acid method: [whole objects or sections] — » A, till bronze — * water, thorough wash -^ B, till bleached — > wash 31.1 Grenacher 1885 23632, 2:244 formula: water 15, 95% ale. 45, glycerol 30, hydrochloric acid 3 note: Though not technically a bleaching mixture, this was recommended by Grenacher for the removal of pigment from the eyes of arthropods. 262 METHODS AND FORMULAS AF 31.1-41.1 31.1 Grynfelt and Mestrezat 1906 6630, 61:87 formula: water 55, barium chlorate 25, sulfuric acid 4.25 PREPARATION OF STOCK: Dissolve barium chlorate in 35 warm water. Cool below 30°C. Mix sulfuric acid in 20 water. Add to chlorate solution in small portions with constant agitation. Filter. WORKING solution: 90% ale. 100, stock solution 15 31.1 Langeron 1942 Langeron 1942, 670 formula: water 100, sodium perborate 17, oxalic acid 6 preparation: Dissolve sodium perborate in water. Add oxalic acid to solution. note: This is equivalent to "12-vol." hydrogen peroxide. 31.1 Mayer 1880 14246,2:8 formula: potassium chlorate 0.1, hydrochloric acid 0.1, 70% ale. 100 preparation: Mix potassium chlorate and hydrochloric acid in flask. Leave till chlorine freely produced. Add ale. 31.1 Mayer 1881 14246, 2:8 formula: potassium chlorate 0.1, hydrochloric acid 0.3, 70% ale. 100 preparation: Mix potassium chlorate and hydrochloric acid. Wait few moments. Add 70% ale. 31.1 Monckeberg and Bethe 1899 1780, 54:135 formula: water 100, sodium bisulfite 2, hydrochloric acid 1 31.1 Murdock 1945 4349,25:71 formula: acetone 50, hydrogen peroxide (3%) 50, ammonia 0.06 recommended for: removal of laked blood pigments in formaldehyde-fixed tissue. 31.1 Lundvall 1927 766,62:353 formula: 95% ale. 90, 40% formaldehyde 10, oxalic acid 6 recommended for: synchronous preservation and bleaching of small vertebrates in which it is intended to stain bone or cartilage. See particularly DS 21.11 Lundvall 1927. 31.1 Tomlinson and Grocott 1944 see DS 23.33 Tomlinson and Grocott 1944 (note) AF 40 Macerating and Digesting Agents These were at one time very much more widely used than they are now, for it appears to be the present custom to endeavor either to see everything in sections, or to reconstruct from sections what the structure would have looked like had it not been cut to pieces. It is a great deal simpler, in many cases, to macerate the tissues in order that individual cells may be separated. The process of maceration involves the solution, either by acid hydrolysis or enzyme hydrolysis, of the materials which attach the cells one to another. At the same time that this solution is going on, it is necessary to provide a fixative which will prevent swelling or dissociation of the cells themselves. There is naturally a rather critical time factor, and the process can only be handled properly by trial and error. It may be added that almost any fixative, if diluted with 20 or 30 times its volume of water, will in fact become a macer- ating agent. Even 30% alcohol, the use of which is usually attributed to Ranvier (Lee 1890, 241) will produce disassociation of many tissues. AF 41 METHODS USING HYDROLYSIS AF 41.1 Formulas for Acid Hydrolyzing Solutions 41.1 Apathy 1898 23632, 10:49 formula: water 30, glycerol 30, 95% ale. 30, acetic acid 5, nitric acid 5 41.1 Becher and Demoll 1913a Becher and Demoll 1913, 24 formula: water 100, osmic acid 0.25, chloral hydrate 3 41.1 Becher and Demoll 1913b Becher and Demoll 1913, 23 formula: water 30, 95% ale. 30, glycerol 30, nitric acid 5, acetic acid 5 AF41.1 ACCESSORY FIXATIVE FORMULAS 263 41.1 Drost test. 1895 Rawitz Rawitz 1895, 7 formula: sea water 100, osmic acid 0.05, chromic acid 0.125, acetic acid 0.05 41.1 Felix test. 1942 Langeron cit. Mann Langcron 1942, 351 formula: 30% ale. 100, salicylic acid to sat. 41.1 Freude 1879 20170, 78:102 formula: water 50, glycerol 35, hydrochloric acid 15 41.1 Gage 1892 21400a, 14:120 formula: water 75, 95% ale. 25, picric acid 1 41.1 Goodrich 1942 17510,83:245 formula: water 100, ADS 12.2 Lugol 0.25, sodium chloride 0.9, boric acid to sat. 41.1 Haller 1887 14555, 11 :321 formula: water 50, glycerol 25, acetic acid 25 41.1 Harlow 1928 3420,85:226 REAGENTS REQUIRED: A. sat. aq. sol. chlorine; B. 3% sodium sulfite method: [match-size pieces of wood] -^ boil -^ A, 2 hrs. —^ B, 80°-90°C., 15 mins. -^ [repeat A-^ B cycle till wood fails to turn red in B] —* wash —y section or tease recommended for: separation of wood fibers. 41.1 Hertwig 1879 see F 1000.0010 Hertwig 1879 (note) This is given as a macerating agent by Gatenby and Painter 1937, 246. It was intended in the original as a fixative. 41.1 Hertwig test. 1895 Rawitz Rawitz 1895, 7 formula: water 100, osmic acid 0.025, acetic acid 1 41.1 Hopkins test. 1895 Rawitz Rawitz 1895, 7 reagents required: A. 20% nitric acid; B. sat. aq. sol. potassium alum method: [fresh tissues] -^ A, till soft, — » wash -^ B, till sufficiently disintegrated 41.1 Klebs test. 1895 Rawitz Rawitz 1895, 9 formula: water 100, sucrose 5, sulfuric acid 5 41.1 Konigstein 1895 20170 (1895) 71 formula: water 30, glycerol 30, hydrochloric acid 30 41.1 Kuhne 1862 test. 1895 Ranvier Ranvier 1895, 79 note : Under this reference, also cited by Gatenby and Painter 1937, 246, a method of pro- ducing a serious explosion is described. The original, which must surely be misquoted, is not available to the author. 41.1 Ludwig 1872 test. 1883 Cole Cole 1883, 135 formula: 90% ale. 80, hydrochloric acid 20 method: [vertical slices of fresh kidney] -^ macerant, boil with reflux, 1-3 hrs. — > leave to settle -^ decant — > add water, leave 24 hrs. -^ shake — * mount selected tubules. recommended for: isolation of renal tubules. 41.1 MacCallum 1900 test. 1905 Bohm and Davidoff Bohm and Davidoff 1905, 23 formula: water 40, glycerol 40, nitric acid 20 41.1 Masson 1929 4349, 12:81 formula: 95% ale. 90, nitric acid 10 41.1 Mobius 1887 see F 1600.0010 Mobius 1887 (note) 41.1 Rogers test. 1927 Kingsbury and Johannsen Kingsbury and Johannsen 1927, 82 formula: water 80, picric acitl I, acetic acid 20 recommended for: disassociation of striped muscle. 264 METHODS AND FORMULAS AF 41.2-42.1 AF 41.2 Formulas for Alkaline Hydrolyzing Solutions 41.2 Behrens, Kossel and Schiefferdecker 1889 Behrens, Kossel and Schieffer- decker 1889, 156 REAGENTS REQUIRED: A. water 67.5, potassium hydroxide 32.5; B. water 50, acetic acid 50 method: [fresh tissue] — » A, on shde, till macerated — * drain -^ B, dropped on slide — > DS 11.2 stain if required -^^ M 11 or M 12 mountant. 41.2 Reinke 1892 766, 8:582 formula: water 50, glycerol 20, 95% ale. 20, lysol 10 AF 41.3 Formulas as for Neutral Hydrolyzing Solutions 41.3 Arnold 1898 1780, 52:135 formula: water 100, potassium iodide 10, iodine 0.15 41.3 Gage 1895 test. 1896 ips. Gage 1896, 177 formula: water 99.5, 40% formaldehyde 0.5, sodium chloride 0.6 41.3 Gage 1897 21400a, 19:179 formula: water 100, potassium dichromate 0.25, sodium sulfate 0.1, sodium chloride 0.9 41.3 Landois 1885 tt^st. 1937 Gatenby and Painter Gatenby and Painter 1937, 245 REAGENTS REQUIRED: .4. Water 100, ammonium chromate 1.25, potassium phosphate tribasic 0.5, sodium sulfate 0.6; B. solution A. 50, DS 11.26 Beale 1857 50 note: Gatenby and Painter give a reference to "1780, n.v., 445" which is incorrect. 41.3 Moleshott and Borine 1872 test. 1875 Ranvier Ranvier 1875, 242 formula: water 100, 95% ale. 20, sodium chloride 1 41.3 Schiefferdecker 1886 1780, 28 :305 formula: water 60, glycerol 30, methanol 3 note: This formula was republished by the same author in 1911 (23632, 28:318). 41.3 Soulier 1891 test. 1905 Lee Lee 1905, 302 formula: F 4000.0010 Ripart and Petit 50, 2% sodium thiocyanate 50 note: Soulier is reported (loc. cit.) to have added thiocyanates to a large number of fixatives. The mixture here given is thought by Lee to be the best. AF 42 METHODS USING ENZYME DIGESTION AF 42.1 Formulas 42.1 Faberge 1945 20540b, 20:1 preparation: Extract stomach contents of Helix pomatia. Preserve with 1 drop toluene. recommended for: removal of cell wall from plant tissues for smear preparation of chromosomes. This technique is described in detail in Chapter 9. 42.1 Hoehl 1897 1739, n.v., 136 formula: water 100, sodium carbonate 0.3, pancreatin 0.25 42.1 Jousset 1903 1863, 15 :289 formula: water 100, glycerol 1, hj'drochloric acid 1, sodium fluoride 0.3, pepsin 0.1 42.1 Kuskow 1895 1780,30:32 formula: water 100, pepsin 0.5, oxalic acid 3 42.1 Langeron 1942 Langeron 1942, 353 formula: water 100, sodium hydroxide 0.2, pancreatin 0.3, thymol to sat. note: Use at 37°C. AF 51.1 ACCESSORY FIXATIVE FORMULAS 205 AF 50 Narcotizing Agents Narcotizing agents as here given are not intended for use on animals wliicli will subse- quently recover, but are intended to leave in a relaxed condition small invertebrates and larvas which are subsequently to ho fixed. It is hoped by the worker that these relaxed speci- mens will not contract out of their normal shape on the application of fixative. One of the best methods of narcotizing small marine invertebrates is with the aid of carbon dioxide, for manj^ of these forms have a natural resi)onsc of endcavoritig to increase tlu;ir surface area when they hnd their oxygen supjily being diminished or the concentration of carbon dioxide in their environment rising. Carbon dioxide is best used for this purpose by taking one of the devices commercially sold for aerating drinks, filling it with sea water, and saturating thi.s with carbon dioxide. In some fresh-water invertebrates heat will produce the same effect. Many poisons have from time to time been regarded as narcotics and thus Zebrowski 1926 (21400a, 45:258) has recommended a 2% solution of strychnine sulfate for planaria; this same reagent was recommended for rotifers by Pritchard 1851, 39. Magnesium sulfate is widely used for marine coelenterates, since an excess of magnesium ions in the water appar- ently has the effect of inhibiting muscular contraction in these forms. Either ether or chloroform may be used in the vapor phase by the process of exposing small invertebrates swimming under a coverslip to an atmosphere of a volatile anesthetic. This process was first recommended by Beauchamp 1904 (5401, 29:26) who used it for Vorticella and other stalked ciliates. In the experience of the writer cocaine, or cocaine hydrochloride, is still, in spite of the flood of synthetic substitutes, the best narcotic for general use. AF 51.1 Formulas 51.1 Baker test. 1937 Gatenby and Painter Gatenby and Painter 1937, 10 formula: water 90, 90% ale. 10, cocaine hydrochloride 0.6 51.1 Bujor 1901 1915, 10:49 formula: water 100, sodium chloride 0.9, ether 10, 40% formaldehyde 10 RECOMMENDED FOR: anesthetizing and killing cestodes before regular fixation. 61.1 Cori 1893 23632,55:626 formula: water 90, methanol 10, chloroform 0.3, sodium chloride 0.6 51.1 Gray 1935 Micr. Rec. (1935) 35 REAGENTS required: A. Rousselet 1895 AF 51.1; B. 3% hydrogen peroxide 10, water 90 method: [stalked ciliate protozoa] -^ A, added drop by drop, till cilia start slowing —> B, added drop by drop till cilia stop — > fixative, instantly 51.1 Gray 1952 Gray 1952, 14 formula: menthol 48, chloral hydrate 52 preparation: Grind in a mortar until an oily fluid results. method: Place a few drops on the surface of water containing animals to be narcotized. 51.1 Hanley 1949 Microscope, 7:156 formula: water 90, ethylene glycol monoethyl ether 10, eucaine hydrochloride 0.3 51.1 Langeron 1942 Langeron 1942, 1013 formula: water 50, methanol 50, cocaine hydrochloride 5 note: This is called "concentrated liquid of Rousselet" by Langeron. See, however, AF 51.1 Rousselet 1895. 51.1 Lo Bianco test. 1937 Gatenby and Cowdry Gatenby and Cowdry, 1937, 11 formula: sea water 40, water 10, 95% ale. 30, glycerol 20 51.1 Morrison 1948a Turtox News, 26 :5i formula: water 93, methanol 7, cocaine hydrochloride 0.03 51.1 Morrison 1948b Turtox News, 26 :5i formula: water 70, 95% ale. 7, methanol 5, hydroxylamine hydrochloride 0.1 266 METHODS AND FORMULAS AF 51.1 51.1 Rousselet 1895 11479,5:1 formula: water 90, methanol 10, cocaine hydrochloride 0.6 note: See also AF 51.1 Langeron 1942. 51.1 Volkonsky 1933 3919,67:135 formula: isotonic saline 100, aprobarbital 0.3, phenobarbital 0.3, chlorobutanol 0.06 51.1 Waddington test. 1937 Gatenby and Painter Gatenby and Painter 1937, 11 formula: water 50, sat. sol. chloroform 50, cocaine 0.5 51.1 de Waele 1934 899a, 12:492 formula: buffer pH 6 100, sodium taurocholate 1 RECOMMENDED FOR: evagination of scolices of cysticerci. 20 Formulas and Techniques for Dye Stains of General Application Decimal Divisions Used in Chapter DS 00 GENERALITIES 01 General observations 02 Method of arrangement of formulas DS 10 DECIMAL DIVISIONS USED FOR DYE STAINING TECHNIQUES OF GENERAL APPLICATION 11 Nuclear staining techniques 11.1 Hematoxylin stains 11.10 Tj^pical examples Demonstration of spermatogenesis in the rat testis using the iron hematoxylin stain of Heidenhain 1892 Preparation of a wholemount of a 48-hour chicken embryo using the alum hematoxylin stain of Carazzi 1911 Preparation of a series of demonstration slides, each having six typical transverse sections of a 72-hour chicken embryo, using the acid alum hematoxylin stain of Ehrlich 1896 Till Mordant hematoxylin staining 11.111 After ferric alum mordants 11.112 After ferric chloride mordants 11.113 After other mordants 11.12 Direct hematoxylin staining 11.121 Formulas incorporating iron mordants 11.122 Formulas incorporating alum mordants 11.123 Formulas incorporating acid alum mordants 11.124 Formulas incorporating other mordants 11.2 Carmine stains 11.20 Typical examples Preparation of a wholemoimt of a liver fluke using the "car- malum" stain of Mayer 1897 Preparation of a wholemount of a medusa using the alcoholic borax-carmine stain of Gronacher 1879 Preparation of a smear to show chromosomes in salivary glands of Chironomus using the iron-aceto-carmine stain of Belling 1921 11.21 Alum carmines 11.22 Alcoholic carmines 11.23 Acetocarmines 11.24 Picro-carmines 11.25 Iron carmines 11.26 Ammonia carmines 11.27 Hydrochloric^ carmines 11.28 Other carmine formulas 11.3 Brazilin and other natural stains 267 268 METHODS AND FORMULAS 11.4 Synthetic nuclear stains 11.40 Typical examples Preparation of a strewn slide of pollen grains using the safranin stain of Johannsen 1940 Demonstration of chromosomes of the grasshopper in a smear preparation of the testes, using magenta by the method of Henneguy 1891 11.41 Soluble metallic lakes of the oxazines, etc. 11.42 Safranin 11.43 Magenta 11.431 Magenta as dye 11.432 Magenta as leucobase 11.44 Thiazines 11.45 Crystal violet 11.46 Other synthetic nuclear stains 12 Plasma staining techniques 12.1 Single contrast formulas 12.11 Aqueous solutions 12.12 Weak alcohol solutions 12.13 Strong alcohol solutions 12.14 Clove-oil solutions 12.15 Phenol solutions 12.16 Other solutions and mixtures 12.2 Double contrasts from one solution 12.20 Typical examples Preparation of a transverse section of a Squahis embryo using the picro-carmine stain of Rarivier 1899 followed by the picro- indigo-carmine of Cajal 1905 Preparation of the transverse section of the tongue of a rat using coelestin blue followed by the picro-acid-fuchsin of van Geisen, 1896 12.21 Contrasts for red nuclei 12.211 Formulas containing picric acid 12.212 Other formulas 12.22 Contrasts for blue nuclei 12.221 Formulas containing picric acid 12.222 Other formulas 12.3 Complex contrast formulas 12.30 Typical examples Preparation of a transverse section of an earthworm using the iron hematoxylin stain of Regaud 1910 followed by the acid fuchsin-aniline blue of Masson 1912 Preparation of a transverse section of the head of a mouse using an acid alum hematoxylin stain (Masson 1934) followed by ponceau 2R-light green (Patay 1934) 12.31 Techniques employing the phosphotungstic (molybdic) reaction with fuchsin 12.32 Techniques employing the phosphotungstic (molylxlic) reaction with other dyes 12.33 Other complex contrasts 13 Complex techniques involving both nuclear and plasma staining 13.1 T<'chiii(iuo8 employing the oosinates of the thiazins without other admixture 13.10 Typical example Preparation of a blood smear using the methylene blue-azur A- methylene violet-eosin Y stain of Kingsley 1935 13.11 Methylene blue eosinates 13.12 Polychrome methylene blue eosinates 13.13 Other thiazine eosinates DSOO DYE STAIN'S OF GENEHAL APPLlfATION 2G9 13.2 13.3 13.4 13.5 13.6 13.7 13.31 13.32 Techniques employing thiazine.s, and their eosinates, in combination with other dyes. 13.21 In combination with orange (j 13.22 In combination with other dyes Techniques employing methyl green as the nuclear stain 13.30 Typical example Staining a section of the suprarenal body in the methyl green- acid fuchsin-orangc (i stain of Foley 193',) In comI)ination with pyronin In combination with other dyes Techniques employing acid fuchsin as the nuclear stain 13.40 Tj'pical examples Preparation of a transverse section of Amphioxus using the acid fuchsin-aniline blue-orange Ci stain of Mallory 1901 Involving the acid fuchsin-phosphomolybdic reaction Involving the acid fuchsin-phosphotungstic reaction Not involving either reaction. Techniques employing safranin as the nucl(;ar stain 13.50 Typical example 13.51 Other techniques Techniques employing hematoxylin as the nuclear stain Other complex techniques of general application 13.41 13.42 13.43 DS 00 Generalities DS 00 GENERAL OBSERVATIONS The term dye staining as used in the present work applies to those methods by which objects or parts of objects are colored. It is distinguished from metal staining (Chapter 23) more by convention than by any scientific actuality, for some of the methods emi)loyed involve tlie ap- plication of salts of metals for the produc- tion of color, while some of the metal- staining techniques involve the deposition of colored compounds in the same manner. The terms dye, pigment, stain, and lake have become so hopelessly- confused in biological literature as almost to have lost their original meaning. Technically a stain (and the process of staining) involves only the arldition of color to an othei'wise color- less material. The term dye should be aj)- ])lied to those stains which remain on the material on which the}^ are deposited and cannot subsequently be removed by nor- mal techniques. A pigment, as tlie term is customarily used in other than biological literature, I'cfers to a solid color matter not in a state of solution. But the use of the term dye for ruthenium I'ed is widely current in biological literature even though the material itself is a finely di- vided suspension of an insf)luble material. A lake, in nonbiological literature, is an insoluble compound of a dye and some other material, usually a metal, which is used to hold it in place upon the dyed material. Though still technically cor- rectly emploj^ed with regard to the mor- dant staining with hematoxylin, carmine, and the like, the term should also be ap- l)lied to many othei- methods of staining, such as the eosin-azur stains. The necessitj^ for staining specimens in- tended for microscopical examination is not nearly as great as the widespread use of the process would lead one to supi)ose. Many writers have stressed the un(le.'?ir- ability of the current habit of staining every object which is to be examined under the microscoi)e, and one cannot do better in this respect than quote the words (in the writer's translation) of Langeron 1942, 485: "Histological science has cer- tainly not progressed in pioportion to the enoruKHis luunber of staining reagents which liave been placed at its disposal. One cannot too often repeat that staining is not the whole of histology and that the first duty of the microtoinist is not to be a dyer but to know how to study through a microscope. That is the art which the former inastei's jjossessod fundamentally and which it would l)e desirai)le to see a little better cultivated today." It would 270 METHODS AND FORMULAS DSOO be to the great advantage of students pre- paring microscope slides if they were de- prived of access to coloring reagents until they had made at least a hundred shdes without the aid of the reagents. This is particularly true in the preparation of wholemounts, in many of which more de- tails of structure are brought out by the varying refractive index of the organs, than they do by soaking them in red and blue solutions which tend in many in- stances only to obscure the finer struc- tures. These remarks do not apply with nearly so much force to the staining of sections, though it is probable that such structures as ciliated epithelium can be better distinguished in an unstained than in a stained preparation. The true value of staining is realized when it provides either a specific coloration of an organ or cellular structure which is to be studied, or alternatively when it provides a con- trast between two such structures. A lesser, and a less necessary, purpose is to render apparent through the introduction of color those very few structures, the re- fractive index of which so closely approxi- mates either that of the mounting medium or of their neighboring structures as to render them indistinguishable as un- colored objects. Contemporary opinion on the theoretical composition of materials used for staining has altered very httle since the original account of Witt 1876 (2627, 9:522) who first advanced the now widely held theory that the presence of color in a chemical is conditioned by the presence of certain groups or radicals known as chromophores, and that materials known to contain these groups should be called chromogene. The power of imparting this color to other substances is given to a chromogen by the presence of an auxochrome. The ma- jority of auxochromes are either alkali or acid radicals which impart the property of solubility to the materials under dis- cussion. It is unfortunate that the partial absorption of these theories by biologists should have left them with the almost universal habit of classifying dyes either as basic or acidic, according to the nature of the auxochrome, and left them also en- deavoring theoretically to forecast the performance of such a dye on the basis of its alleged physical reaction. There are in point of fact four main ways in which a color may be caused to remain diffused throughout or adherent to, a particular structure. The first of these, surface adsorption, is a physical re- action and is dependent both on the charge upon the ionized dye and upon the materials on which this dj-e is precipitated. It might be imagined, and has indeed been widely stated, that it is thus only neces- sary to know the isoelectric point of the protein involved and the pH of the dye solution to be able to secure a perfect differential absorption at all times. This is to a considerable extent true in the case of vital staining or of proteins whose nature has not been altered. In the latter instance, for example, the dried smears, used either in the staining of blood or the staining of bacteria, fulfill the require- ments. In these circumstances the control of the pH of the staining solution is all that is required to secure reproducible and perfect results. As soon, however, as a fixative is employed which denatures the proteins, the problem becomes more com- plex; and there is no method save that of trial and error by which the desirable pH of a dye solution may be determined. This does not mean that pH should not be more widely controlled, for there is little doubt that many of the troubles of the early microtomists, who were forced to specify a particular source for a particular dye, were due to the influence on the pH of the final solution of various impurities left in the commercial product by different manufacturers. Another physical reaction involved in the staining of tissues is the saturation of a material with a dye and the subsequent precipitation of the dye in place by the use of solvents for dehydrating in which the dye is not soluble. These methods are little used and difficult to control. Lastly, in a few cases, a definite chemical com- bination is entered into between the dye- stuff and the tissue which is being differ- entially stained. A common and obvious case of this is the use of alizarin red S to stain boney structures or the calcareous plates of invertebrates. In this case a DS 00-DS 10 DYE STAINS OF GENERAL APPLICATION 271 colored lake ("calcium alizarinate") is formed between the bone and dye, result- ing in a (juite sharp differential staining after tlie extraction of the excess dye with alkalis in which the lake is insoluble. It is difficult to maintain today the old distinction between direct and indirect staining. In the former instance the stain was applied from a very weak solution on the assumption that it would be differ- entially absorbed by varjdng structures and tissues. Actually, however, in the majority of these methods, the density of the tissue controls the degree of absorp- tion. Indirect methods are those in which the dye is appHed from a relatively strong solution and is subsequently dissolved away, or extracted, from the unwanted structures either by a solvent or b}' some additional chemical reagent. The last method of staining which is commonly employed is that of mordant staining, in which the tissue is first caused differentially to absorb a substance with wliich the d3'e subsequently makes an in- soluble compound. Nuclear stains are the most widely used by this method, al- though it is probable that in many in- stances the metals employed in fixative solutions do themselves act as mordants. In the majority of cases tissues fixed by methods other than those specified for a particular staining technique may be rendered available for this technique if the sections attached to the slide are mor- danted in the required fixative before staining. DS 02 METHOD OF ARRANGE- MENT OF FORMULAS The original method of classification for stains was to divide them into basic {i.e., nuclear staining), acid (i.e., plasma stain- DS 10 Dye Staining Techniq It cannot be too strongly emphasized that the selection of a dye staining tech- nique for the general purposes of histology, embryology, and cytology is a matter in which the investigator should use his own initiative rather than rely on his knowl- edge of what has been done before. In too many cases a relatively worthless tech- ing), and neutral groups. This method has been rendered obsolete by the employ- ment of d\-es in formulas which alter their basic staining reactions. The method of classification adopted by Langeron in the numerous editions of his Precis de Micro- scopic is based upon the chemical nature of the dyes themselves, and would appear at first sight to be the most logical. This is not, however, as good in practice as in theor}', for it results in the widespread separation of formulas which, in lab- oratory practice, are, or should be, interchangeable. Tlie classification given at the begin- ning of this chapter has been erected on the principal that those solutions which may be profitably interchanged for each other upon the laboratory bench should have their formulas published under the same heading. The first step has, there- fore, been the removal of "Techniques of Special AppUcation" to Chapter 21. This chapter does not contain all those stains which have been proposed for special pur- poses, but only those which experience has shown to be otherwise valueless. The triple stain of Kull 1913, for example, which is widely employed for the demonstration of mitrochondria, has many other uses, and is accordingly placed with Rhamy 1930, which it resembles. Both are, however, mentioned as cross references in the divi- sion of the special section for which they were proposed. Each of these two chapters has been further divided into convenient subdiAd- sions, which in some cases are founded upon the components of the solutions, in other cases upon their practical use. The result is that every formula is accom- panied either by those formulas or by references to those formulas, which may be employed as a substitute. ues of General Application nique has been applied j^ear after year for no other reason than that it has become conventional to do so. Were this reasoning to be taken to its logical conclusion and techniques utilized only for the original purpose for which they had been invented, we sliould find tliat the use of Bouin's picro-formaldehytle-acetic fixative would 272 METHODS AND FORMULAS DS 10-DS 11.10 be entirely confined to the study of the spermatogenesis of rats, and that the majority of the carmine stains, now widely emploj-ed for staining whole- mounts, would l)e confined to the pre- staining of tissues prior to embedding, so that the nuclei might be stained in sec- tions subsecjuently to be cut from them. The dj-e staining techniques of general application are here divided into the three broad headings of those ])rimarily in- tended for nuclear staining (DS 11), those primarily intended for providing a con- trast to a jM-eviously ai)i)lied nuclear stain- ing (DS 12), and those intended for ap- plication to sections in which both the nuclei and background are to be stained in the same operation (DS 13). DS 11 NUCLEAR STAINING TECHNIQUES This section includes those techniques customarily em])loyed for staining nuclei in general, and they are to be kept distinct from those special techniques intended for the studv of the nucleus as such, which are to be found in Chapter 21 (DS 22.1). Nuclear staining techniques are here classified solely on the liasis of the re- agents employed and of the manner of their employment. Prior to the use of stains in microtomy, hematoxylin was widely employed in the dye industry for obtaining purples and blacks, the former with the aid of alum mordants, and the latter with the aid of iron mordants. This division has been fairly closely followed in microtomy and is used as the basis for the division into mordant staining and direct staining which follows. The selection be- tween these two methods must be made entirely on the purpose for which the method is intended. The use of carmine is of greater antiq- uity than is the use of hematoxylin. The original use of carmine in textile dyeing was with the aid of a whole range of mor- dants, which enabled the dyer to select colors ranging from a brilliant scarlet to a dead black. In general the methods of microtomy utilize only the red aluminum mordants and to a lesser extent, the black iron mordants. The main use of carmine in microtomy, however, is in direct stain- ing with the aid of solutions containing aluminum mordants. There is consider- able misapprehension about the degree of alkalinity of the various formulas. Early workers tended to say that a solution was too alkaline on the sole basis of the fact tliat it was prepared with a sodium salt rather tliaii \\ith the more expensive lithium compounds or with borax. The rediscovery in German}' in the 1930's of some of the older sodium-carmines has led to their inclusion in the present work. Brazilin and saffron are also to a minor extent employed in the staining of nuclei; and they terminate the list of tlie so-called natural dyes. The synthetic dyes form a large group which are here subdivided according to which dye is used. Too much attention cannot be drawn to the oxazine dyes de- scribed by Becher 1921, which have been rediscovered at intervals. The}' have never replaced hematoxylin, but it must appear to most microtomists they are ultimately destined to do so. They have the ad- A-antage over hematoxylin stains in that the}' are relatively acid-fast once they have adhered to the nucleus, and therefore give a whole range of dark blue and black nuclear stains which can be employed before counterstaining methods involving acid materials. There is some confusion between the use of nuclear stains in zoology and botany and the use of the same stains for the preparation of bacteria. Nuclei and bacteria have essentially the same staining reactions, and a method which may be utilized for one can without difficulty be adapted to the other. DS 11.1 Hematoxylin Stains DS 11.10 TYPICAL EXAMPLES Demonstration of spermatogenesis in the rat testis using the iron hematoxy- lin stain of Heidenhain 1892 The laboratory white rat is one of the best forms in which to show spermato- genesis, for the reason that it has a con- tinuous breeding period and all stages are therefore available in almost every section examined. Baker 1945, 180 recommends the salamander Triturus for this purpose, DS 11.10 DYE STAINS OF GENERAL APPLICATION 273 ])ecauso of the very large cliromosonios wliicli it possesses. It is a disadvantage, however, that spermatogenesis in Triturus occurs only in the summer months im- mediately following the breeding cycle. The rats selected should bo young males, and are most conveniently killed with chloroform. The scrotal sac is then opened by a median inci.sion, the testes are removed, and the epididymis trimmed away. The testes should be placed on a clean glass plate and slashed with a sharp scalpel or razor about two-thirds of the way thi-ough. The slashes should be spaced a few millimeters apart and should be made before the testes are thrown into the selected fixative solution. Few fixatives for this i)urpose can improve on the fluid originally suggested by Bouin 1897 (Chap- ter 18, F 5000. 1010) ."This fixative today has many uses of doul:)tful validity; but for this, the material which it was origi- nally designed to preserve, it has rarely been surpassed. At least 100 milliliters of fixative should be emploj-ed for a normal size testis, and the bottle containing it should be reversed once or tw'ice during the first few hours to avoid tlie accumula- tion of diluted fluid at the bottom. The length of time allowed for fixation is not of any great importance, but it should, in any case be overnight, and should not in general exceed several weeks. 'After the object is removed from the fixative, it may be washed for about an hour in running water befoie being transferred to 70% al- cohol to complete the removal of the picric acid. It must be emphasized that washing it in water after putting it in picric-acid fixatives results in considerable vacuola- tion of the cytoplasm. This does not in the present instance interfere with the study of the nucleus. After three or four changes of 70% alcohol, leaving the testes in a considerable volume of solution for at least two or three days between changes, the final removal of the picric acid may be completed by adding a small quantity of dry Hthium carbonate to the alcohol used for washing. It will be impossible to re- move the whole of the yellow color, some of which is caused by combination of the picric acid with the albuminoids present, but the last alcohol used for washing should be only very faintly tinted with yellow. It is not the color of the fixed mate- rial to which objection is raised during the l)assage of the material through paraffin; it is the fact that unless most of the picric- acid be removed, there will l)e a crystal- lization which will damage the tissues. Small i)ieccs may now be removed from the testis itself for embedding. It is best to select a jjiece about one millimeter in from the surface and of about a two-milli- meter side. These should be embedded in paraffin and cut about five microns thick in the usual manner. These sections should then be attached to a slide. Particular attention must be paid to the fact that the slides are clean and that not too much of whatever adhesive is employed be al- lowed to remain. The dry sections attached to the slide should now be warmed on the underside until the paraffin melts, placed in xylene until the paraffin is completely removed, and then run down in the ordinary way through absolute alcohol and lower-per- centage alcohols to distilled water. Thej' should then be lifted from the distilled water and examined carefully. If there is a tendency for the water to gather in droplets on the slide, or if upon shaking the water from the slide each section ap- pears to retain around itself an adherent coat of water, it is an indication that the wax was not properly removed in the xylene or that the X3'lene itself is so old as to have a wax content too high to be use- ful. Such sHdes must be returned through the alcohols to absolute alcohol and thence to clean x.ylene, in which they should be left for a few minutes before again being brought down to water and reexamined. There is no more common cause of the failure of the stains to take than the im- perfect removal of the wax. Only tw^o solutions are required for staining. These are a 2>^% solution of ferric alum and a ^2% solution of hema- toxylin. The onlj' difficulty in making the ferric alum solution is to secure a pure and unoxidized sample of the reagent. Most of the crystals in a new bottle are of a clear violet color, but after it has been opened for some time, particularly if the stopper be loose, most of the crystals be- 274 METHODS AND FORMULAS DS 11.10 come covered with a brownish deposit which must be scraped off with a knife before the solution is prepared for stain- ing. If the brown powder on the outside of the crystal forms a layer of any thick- ness, it is better to reject the whole and secure a fresh supply of the reagent. He- matoxyhn itself has little staining effect, the color being produced by the formation of lakes with hematin, an oxidation prod- uct of hematoxylin. It was customary in former times to prepare considerable quantities of solution, which was kept with the stopper loose in the bottle for a period of at least one month before use. For the purpose of Heidenhain's tech- nique, however, it is far more important that a small quantity of the ferric alum be carried over into the hematoxylin solu- tion than that the latter should be aged. The staining will be both simpler and more effective if a few drops of hematox- ylin be placed in the iron alum solution and a few drops of the iron alum solution be placed in the hematoxyhn. Both solu- tions should, of course, be filtered immedi- ately before use if the finest shdes are re- quired for the reason that chromosome figures in a rat can be obscured by even a very small particle of dust. The slides are now taken from distilled water and placed in the mordant solution. It matters very little how long they remain in this solution, although the usual direc- tions call for overnight. The ideal time varies with every type of tissue studied and is greatly dependent on temperature. If the solutions be heated to 50°C., with the understanding that this will cause a swelhng of the section and a general ob- scuring of the finer details, the period may be shortened to as httle as 10 minutes. But the finest stains are those secured by leaving the sections in the mordant solu- tions at room temperatures. On removal from the mordant solution the sections should be rinsed very briefly in distilled water. The purpose of the rinse is te re- move the surplus mordant from the sur- face of the slide without extracting it from the tissues. The shdes are then placed in the hematoxylin solution, in which they should remain for approximately the same length of time as they have been in the mordant. It is not of importance how long this be though from three to 24 hours is customary period. Sections may be re- moved from time to time from the staining solution and examined with the naked eye. A successful preparation shows the whole section to have become completely black- ened, although a slight bluish tinge in the black is permissible. If the sections have not become completely blackened in 24 hours, it is only necessary to replace them, after a brief rinse, in the mordant solution and leave them there, say a fur- ther period of 24 hours, before returning them to the stain. If, however, the sections are sufficiently blackened on removal from the staining solution, it remains only to differentiate them, that is, to extract the color from all portions of the sections except the chromosomes. This is customarily done with the same solution in which they were mordanted, though, of course, a fresh solution or a stronger solution may be employed if desired. Differentiation at the commencement of the process goes relatively slowly so that all of the slides, which are presumable being carried in a glass rack, may be removed and placed in the ferric alum solution. The actual time in which differentiation takes place cannot be forecast because it de- pends on a large number of uncontrollable factors. But it is never less than five minutes nor very often more than a few hours. Sections should therefore be with- drawn from the ferric alum every four or five minutes and examined briefly under the low-power of a microscope. It is a mat- ter of great convenience in controUing dif- ferentiation of chromosomes in this type of preparation if an ordinary student mi- croscope can be fitted with a glass plate over the stage so that a slide wet with ferric alum can be placed, without fear of damage to the instrument, on the surface of the stage for examination. It is not un- common for a beginner to place the slide upside down on the surface of the stage, with the subsequent loss of all the sections. This can readily be avoided if the worker will make it a matter of routine, as he lifts the slide from the mordant, to hold it at an angle between himself and a light DS 11.10 FORMULAS AND TECHNIQUES 275 source so that the hght is reflected from the surface. If the sections are, as they should be, on the upper surface of the shde when it is placed on the stage, they will ap- pear to be double through the reflection from the luider surface as well as the upper surface of the glass. A good rule is never to place an unmounted slide on the stage of the microscope until the double reflec- tion has been seen. If a low-power examination of the sec- tion shows the nuclei to be standing out clearh', the entire tray should be removed to distilled water, because from this time on dififerentiation is very rapid and each slide must be controlled separately. If, however, the nuclei are not sharply de- fined and a considerable degree of black or bluish color remains in the background, then the entire tray may be left in the iron alum for as long as is necessary. When this preliminary differentiation, down to the distinction of the nuclei under low power, has been completed, it is necessary to continue differentiation while examin- ing the slides at frequent intervals under a very high power of the microscope. It is a matter of convenience if a water immer- sion objective is available. It is obviously impossible to place immersion oil on a wet slide, while the short working distance of a high-dry objective renders it particu- larly liable to cloud from the evaporation and recondensation of the water. Water immersion objectives are usually of three millimeter equivalent focus. This provides a sufficiently wide field to permit differ- entiation to be observed, while at the same time it has a magnification sufficiently high for satisfactory control. Each slide should now be taken separately and re- turned to the ferric alum for a few minutes and then reexamined. The various phases of mitosis and meiosis do not retain the stain to the same degree and care must be taken that the color is not washed com- pletely out of the other chromosomes by examining only metaphase figures in which the color is retained longer than in any other. Considerable practice is re- quired to gauge accurately the exact moment at which to cease the differentia- tion, which may be stopped almost in- stantly by placing the sUdes in a sUghtly alkaUno solution. In ]']urope most tap waters are sufficiently alkafine for this purpose and are generally specified; but in the cities of the United States it is often best to add a very small quantity of lith- ium carbonate or sodium bicarbonate to the water which is used to stop differentia- tion. Slides may be left in water for any reasonable period of time; the process is complete when the slide turns from a brown to a blue color. The sHdes are then rinsed in distilled water, upgraded through the various per- centages of alcohols, dehydrated, cleared, and mounted in balsam in the usual man- ner. SUdes which have on them sections required for examination over a long pe- riod of time should have the sections some distance from the edge of the coversfip because, as the balsam oxidizes inward from the edge, it tends to remove the color of the stain from the chromosomes, leav- ing them a rather unpleasant shade of brown. If this happens to a valuable slide, however, the matter can be remedied by the utilization of a green fight which will make the chromosomes again appear black. Preparation of a wholemount of a 48-hour chicken embryo, using the alum hematoxylin stain of Carazzi 1911 Fertile eggs are relatively easy to secure and should be incubated at a temperature of 103°F. for the required period of time. The term 4S-hour chick is relatively mean- ingless, because the exact stage of devel- opment which will have been reached after two days in the incubator depends not only on the temperature of the latter, but also on the temperature at which the egg was stored prior to its incubation, and even on the age of the hen. It is therefore desirable, if any very specific age of devel- opment be required, to start a series of eggs in the incubator at three- or four-hour intervals and then to fix and mount them at the same time. For the removal of the embryos from the egg there are required first a number of fingerbowls, or any kind of circular glass dishes of five to six inches diameter and 276 METHODS AND FORMULAS DS 11.10 two to three inches depth, a number of Syracuse watch glasses, a large quantity of a 0.9% solution of sodium chloride, a pair of large dissecting scissors, a pair of fairly fine forceps, a pipet of the eye-drop- per t.ype, some coarse filter paper, and a pencil. No ver}' great accuracy is required in making up the normal salt solution, although it is customarily specified that the temperature of the solution should be 102 to 103°F. Anywhere within 10 degrees on either side of these figures, however, is relatively safe. The egg is removed from the incubator and placed in one of the fingerbowls which is then filled with enough warm normal saUne to immerse the egg completely. If the operator is rather skilled, it is, of course, possible to break the egg into the warm saline as though one were breaking it into a frying pan. But it is recom- mended that the inexperienced worker prepare several hundred embryos before attempting to do this. The method by which he can be assured of securing a per- fect embryo on every occasion is first to crack open the air space which lies at the large end of the egg and then to let the air which hes within it bubble out through the warm saUne. This permits the yolk to fall down out of contact with the upper sur- face of the shell, which may now be re- moved, as he works from the air space toward the center with a pair of blunt-nose forceps. Again a matter of practice is in- volved, for a skilled operator can remove this shell in large portions, while the inex- perienced one should work very carefully to avoid puncturing the yolk. If the yolk is punctured it is simpler to throw the egg away and start with another one. After about half of the shell has been re- moved, it will be quite easy to tip the yolk with the embryo lying on top of it out into the saline. The next operation is to cut the embryo from the yolk by a series of cuts made well outside the blood vessel sinus terminale which marks the limits of the developing embryonic structures. To do this with suc- cess requires more courage than experi- ence. Just as soon as the vitelline mem- brane is punctured, the yolk starts squirting out through the hole and render- ing the fluid milky so that the embryo is obscured. The smaller the hole which is cut, the more violently does the yolk squirt out. Thus, the larger the scissors which are employed, the more easily will the embryo be removed. The easiest method is to take a pair of blunt forceps in the left hand and grip the extra-embry- onic areas of the chick well outside the sinus terminale. Use a certain amount of drag, so that the vitelline membrane is wrinkled, and then make a transverse cut with a large pair of scissors directly away from you, about a third of an inch outside the sinus terminale on the side of the em- bryo opposite to that which is held by the forceps. This initial cut should be at least an inch long and should be made firmly. Two cuts at right angles to the first, each an inch in length, should then be run on each side of the embryo. The part gripped with the forceps should then be released, and the free edge where the first cut was made should be gripped so that the em- bryo can be folded back away from the yolk. It is now relatively easy by a fourth cut to sever all connections between the embryo and the underlying materials. The embryo, held by the forceps in the left hand, will now be free in the saline solu- tion. The embryo is much stronger than it looks and will not be damaged provided the tip of the forceps is kept under the surface of the solution. The embryo must now be transferred to clean saline, preferably in another fin- ger-bowl. This transfer may be made either with a very wide-mouthed pipet of the eye-dropper type or by scooping it up in a smaller watch glass with plenty of saline and transferring it to the fresh solution. Here it should be picked up again by one corner with the forceps and waved gently backward and forward. This is to remove from it the adherent viteUine membrane (which may, however, already have fallen off) as well as to wash from it such yolk as may remain. At this stage the embryo should be examined to make sure that the heart is beating and that it is in a fit condition for fixation. The embryo is now scooped out on one of the Syracuse watch glasses with as Uttle water as possible. Next it is necessary to DS 11.10 DYE STAINS OF GENERAL APPLICATION 277 persuade it to flatten out on the bottom in an upside down position, that is, so that that portion of the embryo which was jne- viously in contact with the yolk is now di- rected toward the operator. To determine which side of the embryo is uppermost requires considerable practice, unless the primary curvature of the head toward the right has already started. The best point of examination is the heart, which lies, of course, on the lower surface of the embryo. Having maneuvered the embryo in the saline in the watch glass until it is upside down, the water should now be drained off with the aid of a pipet which is run rapidly with a circular motion round and round the outside of the blastoderm while the water is being drawn up. As experience will soon show, any attempt to drain the water up a stationery- pipet will result in the embryo being drawn out in the direc- tion in which the water is being sucked. A little practice with the pipet being run round and round the outside the blasto- derm and about a milUmeter away from it ■will enable the operator to strand the em- bryo so that it is perfectly stretched in all directions. Under no circumstances should a needle be used to arrange the embryo, or the point will adhere to the blastoderm from which it cannot be detached without damage. If the embrj-o is not flattened and spread out satisfactorily, it is only necessar}^ to add a little clean saline with a pipet and repeat the operation. A piece of coarse filter paper or paper towel is now taken and cut into a rec- tangle of such size that it will fit easily into a Syracuse w^atch glass. An oval or circular hole is then cut in the middle of this (most easily done by bending it in two and cutting from it a semicircle) of such a size as will exactly cover those areas of the embryo which it is desired to retain. That is, if only the embryo is required, the hole may be relatively small; if it is neces- sary to retain the whole of the area vasculosa with its sinus terminale, the hole must be correspondingly enlarged. The hole must not, however, be larger than the blastoderm removed from the egg, be- cause the next stej) causes the unwanted extraembryonic regions to adhere to the paper, leaving the embryo clear in the center. By this means alone will the em- bryo be prevented from contracting and distorting when fixative is applied to it. Having prepared a rectangle of a suitable size with a suitable hole in the center, such data as are pertinent may be written on the edge in pencil. The paper is then dipped in clean saline. If the saline used has already become contaminated with egg white, a sharp puff should be directed at the whole to make quite certain that a film of moisture does not extend across it because tlie bubbles so produced always disrupt the embryo if this film is left. The rectangle of filter paper is now dropped on top of the stretched embryo in such a manner that the embryo does not become distorted. That is a great deal easier than it sounds, though a few false trials may be made bj- the beginner. The writer's pro- cedure is to place one end of the rectangle on the edge of the watch glass nearest to him, taking care that it does not touch the blastoderm, and then to let it down sharply. The edges of the blastoderm must be in contact with at least two-thirds of the periphery of the hole if it is to remain stretched. As soon as the paper has been let down, the end of a pipet or a needle should be used to press lightly on the edges of the paper where it is in contact with the blastoderm, to make sure that it will adhere. The embryo is now ready to be fixed, and the choice of a fixative must naturally be left to the discretion of the operator. The author's preference, when hematox- ylin is subsequently to be employed for staining, is for the mercuric-chromic-form- aldehyde mixture of Gerhardt (Chapter 18, F 3600.1010 Gerhardt 1901). The dis- advantage of the customarily used picric formulas is that they interfere seriously with subsequent staining by hematoxj-lin. The fixative should be applied from an eye-dropper type pipet in the following manner. First a few drops are placed on the center of the embryo so that a thin film of fixative is spread over it. After a moment or two a little more may be added with a circular motion on the paper which surrounds the embryo. The j)aper sliould again, at this point, be pressed onto the periphery of the blastoderm with a needle, 278 METHODS AND FORMULAS DS 11.10 or the end of the pipet, to make sure that adherence is perfect. The whole should be left for a moment or two before being very gently shaken from side to side to make quite certain that the embryo is not stick- ing to the watch glass. If it is sticking, the end of the pipet containing the fixative should be slid under the edge of the paper and a very gentle jet of fixative used to free the embryo. As soon as the embryo is floating freely in fixative the Syracuse watch glass may be filled with fixative and placed on one side while the same cycle of events is repeated with the next embryo. After about 10 minutes in the fixative, the paper may be picked up by one corner and moved from reagent to reagent with- out the shghtest risk of the embryo be- coming either detached or damaged. The paper must not be picked up with a pair of metal forceps unless these have been waxed, or the mercuric chloride in the fixative will damage the metal. It is the writer's custom to leave the embryos in the watch glass for about 30 minutes be- fore picking them out and transferring them to a large jar of the fixative which is l^referably kept in a dark cupboard. The total time of fixation is not important, but it should be not less than one day nor more than one week. When the embryos are removed from the fixative they should be washed in running water overnight and then be stored in 70% alcohol. When one is ready to stain a batch of embryos it is only necessary to transfer them from alcohol back to distilled water, leaving them there until they are thor- oughly rehydrated, and then to transfer them to a reasonably large volume of Carazzi's hematoxylin (DS 11.122 Ca- razzi 1911) in which they may remain overnight. It is a mistake to stain them initially for too short a period, for the result will be that the outer surface of the embryo becomes adequately stained while the inner structures do not. This defect, however, is very difficult to detect until the embryo is finally cleared for mounting. When the embryos are removed from the stain, at which time they should appear a deep purple, they should be transferred to a large fingerl)owl of distilled water and rocked gently backward and forward until most of the stain has been removed from the papers to which they are at- tached. Each embryo should now be taken separately and placed in 0.1% hydro- chloric acid in 70% alcohol. The color will immediately start to change from a deep purple to a pale bluish-pink. They should remain in this solution until, on examina- tion under a low power of the microscope, all the required internal structures appear clearly differentiated. Most people differ- entiate too little, forgetting that the pale pink of the embryo will be changed back to a deep blue by subsequent treatment and that the apparent color will also in- crease in density when the embryo is cleared. No specific directions for the ex- tent of the differentiation can be given beyond the general ad\'ice to differentiate far more than you anticipate to be neces- sary. After the embryos have been suffi- ciently differentiated each one should be placed in alkaline tap water, either as it occurs in nature or as it is rendered alka- line with the addition of sodium bicar- bonate. Here it should remain until all the acid has been neutralized and the embryo itself has changed from a pink back to a blue coloration. It may then be dehy- drated in the ordinary manner through successive alcohols, and it is the author's custom to remove it from its paper only w^hen it is in the last alcohol and before it is placed in the clearing reagent. Some persons place it in the clearing reagent attached to its paper and remove it only before mounting. Any clearing reagent may be tried at the choice of the operator. The author's preference for chicken em- bryos is terpineol which has the advantage of not rendering these delicate structures as brittle as do many other reagents. The mountant may be Canada balsam or any of its synthetic substitutes. Preparation of a series of demonstra- tion slides, each having six typical transverse sections of a 72-hour chicken embryo, using the acid alum hematoxylin stain of Ehrlich 1896 The last example described in some de- tail the manner in which a cliicken embryo DS 11.10 DYE STAINS OF GENERAL APPLICATION 279 may be removed from the yolk and fixed in a Syracuse watch glass where it is stretcliod by a collar of filter paper. Ex- actly the same procedure should be fol- lowed in the present instance, save that it is not necessary to make the hole in the paper a size larger than will accommodate the embryo itself. The same fixative rec- ommended there should be employed, but after the removal of the fixative, the embryo should be embedded in paraffin and cut by the methods described in Chapter 12. A complete ribbon of serial sections should be taken from the whole embryo. It is presumed for the purpose of this example that the reader wishes to have a series of slides for class use, on each of which will be arranged, in order, trans- verse sections through the region of the eye, the ear, the heart, and the anterior, middle and posterior abdominal regions. These regions will be found all that is re- quired for teaching an elementary class the development of the eye, ear, and heart, and the closure of the amnion and neural folds. It is first necessary to identify those sections which will show the required structure and to isolate the portions of ribbon containing them. Provided the sec- tions are placed against a background of black paper, this is relatively simple with the aid of a long-arm binocular dissecting microscope. This microscope may be swung over the ribbons and will supply sufficient magnification to enable the re- gions of the ribbon to be identified by a competent microtomist. If the operator has had little practice at this, it might be desirable to stain the embryo in carmine before embedding, preferably with one of the alum-carmines given in section DS 11.21 below. Each portion which contains the selected sections is then cut from the ribbon with a sharp scalpel moved with a rocking motion, picked up on a camel's- hair brush, and transferred to another sheet of black paper. The rest of the ribbon may now be thrown away. The sections in each of the selected strips of ribbon are then counted to determine the maximum number of slides which may be made — the ear sections are usually the limiting factor — and the pieces of ribbon are trimmed so that each contains approximately the same number of sections. The required num- ber of slides are then cleaned and a few drops of the usual adhesive added to an ounce or so of filtered distilled water in a small flask. The only difficulty of this procedure is in fixing each section in its correct place on the shde. A single shde is taken, placed in front of the operator, and covered lightly with the dilute adhesive. The fluid should extend to the edge of the sUde, but should not be raised in a meniscus sufficiently high to cause any appreciable slope of the fluid from the center of the sUde toward the edges. The end section is then cut from each of the ribbons with a sharp scalpel with a rocking motion. These sec- tions are then placed in the correct order (but without any regard to symmetry) on the surface of the fluid on the shde. To secure these sections in the required posi- tion it is now necessary to have two fine brushes, a mounted needle, and a bunsen or spirit lamp. The last section, that is, that section which is required to he farthest from the label on the sUde, is now secured in posi- tion with a brush held in the left hand, wliile the second section is maneuvered with a brush held in the right hand until its edges touch those of the first section. Both sections will be held together by capillary attraction when the brush is removed. The needle is then warmed in the flame and used to fuse the edges of the sections together in two spots. If the en- tire edge is melted it will create a ridge which will prevent the compound ribbon from lying flat against the shde. Two minute spots fused together with the point of the needle are quite sufficient to hold the section in place. The brush is again picked up with the right hand and used to guide the next section into its appropriate place. This section is then spotted into position with the tip of the w^arm needle, and so on, until all the sections have been fused into a continuous ribbon. The rib- bons are then flattened w-ith heat and drained as described in Chapter 12. The sections are now, in the ordinary course of events, left on the warm table until they 280 METHODS AND FORMULAS DS ll.lO-DS 11.11 are entirely dry before they are dewaxed in xylene and brought down to 90% alcohol through absolute alcohol in the usual manner. Ehrlich's acid alum hematoxylin (DS 11.123 Ehrhch 1896) has been selected for this typical example because it is one of the best, though at the same time one of the most frequently misused, of the hema- toxylin stains. The method given for its preparation should be rigorously followed, that is, the hematoxylin should be dis- solved in a mixture of acetic acid and absolute alcohol, and the glycerine, water, and ammonium alum should be added to the bottle. The bottle should then be shaken vigorously and the mixture al- lowed to ripen with the bottle stopper loose for some months. "Artificially" rip- ened hematoxyhn does not give as good a preparation, but there is no reason why this stain should not be prepared in half- gallon lots at routine intervals so that a sufficiently ripened solution is always available. When it has once been ripened, which can be told both by the "fruity" smell and by its dark color, it remains in a fit condition to use for many years. One of the most frequently omitted precautions is that of maintaining the concentration of the ammonium alum by adding about 100 grams per liter to the bottle in which the hematoxylin is kept after it has been sufficiently ripened. This stain should never be diluted but should always be used full strength by the method now to be described. Each slide, or all the shdes together in a glass tray, are taken from the 95 % alcohol and placed in full-strength Ehrhch's hema- toxyhn solution for a few minutes. The exact time is not important, but they should be examined at intervals to make sure that they are not becoming over- stained. If the technician is inexperienced, it is recommended that a period of one minute be used, and that they should then be examined under a low power of the microscope. The nuclei should appear quite densely stained, the background be- ing only hghtly stained. Each shde is then removed individually from the tray, wiped on the underside with a clean cloth, and then differentiated with 95% alcohol (never with acid alcohol) dropped onto it from a drop bottle or from a pipet. It will be observed at once that the drops of the viscous hematoxylin solution are rolled back from the section by the 95% alcohol, and that after this has continued for a short time the nuclei become more distinct and the background less distinct. The ex- act point at which differentiation should cease is determined by the operator, but it is better, in general, since the sections are not to be counterstained, to discontinue differentiation when the nuclei are clearly defined against the background. Each sec- tion is then transferred directly to a saturated solution of lithium chloride in 70% alcohol, in which it turns from pink to blue. If the conventional method of differentiating these stains with acid alco- hol is followed, it results in a hopelessly diffuse stain. The purpose of the 95% alco- hol is to utilize the surface tension of the stain to hold it in the nuclei. If the slide is placed in acid 70% alcohol, it will be found that the stain diffuses out from the nuclei which, instead of appearing clear and sharp, appear blurred around the edges as does an out-of-focus photograph. Differentiation by rolhng back the stain with 95% alcohol gives a clear, sharp stain which is as well differentiated as any of the ferric alum mordant stains, but which has the advantage of giving a greater transparency and also of staining the background sufficiently to render it apparent for class demonstration purposes. The slides may remain in the saturated solution of lithium chloride in 70 % alcohol for as long as is required. They are subse- quently passed directly through the higher alcohols to xylene and mounted in balsam or some synthetic substitute. 11.11. MORDANT HEMATOXYLIN STAINING Mordant staining with hematoxylin results, in general, in black nuclei, heavily and densely stained, from which the stain is with difficulty removed by subsequent treatment. It is therefore to be recommended in those cases in which it is desired to follow with a complex counterstaining, particularly those which involve the use of an acid rinse. The method most DS ll.II-DS 11,111 DYE STAINS OF GENERAL APPLICATION 281 commonly employed is still that of Heidenhain 1892, from which the majority of the other formulas have been derived. These variations are for the most part in the concentration of the solutions employed or in the temperature at which they are used. The technique of R^gaud 1910 is very frequently specified in Europe as a prior staining for complex after- staining methods. During the early part of the present century, the substitution of ferric chloride for ferric alum as a mordant was followed in many circles. Mordants other than iron are rarely employed, although the introduction of copper by Faure 1924 has provided histologists with a staining method considered by many people to be a definite improvement. Diamond 1945 {Tech. Bull., 6:08) finds that the addition of .1% of a wetting agent (Tergitol 7) improves the stain. 11.111 After Ferric Alum Mordants 11.111 Benda 1893 2246, 7:161 REAGENTS REQUIRED: A. ADS 12.1 Benda 1893 35, water 65; B. 1% hematoxylin; C. ADS 12.1 Benda 1893 5, water 95 method: [sections] —>■ water -^ A, 24 hrs. — > rinse -^ B, till black -+ C, till differentiated — » balsam, via usual reagents 11.111 Biitschli 1892 Butschli 1892, 80 REAGENTS REQUIRED: A. 2% fernc acetate; B. 0.5% hematoxylin method: [sections of protozoans] — > A, 24 hrs. — > rinse — > B, 3 hrs. —>■ distilled water —> balsam, via usual reagents 11.111 Diamond 1945 519b, 15:68 reagents required: A. 4% ferric alum; B. water 100, hematoxylin 0.5, Tergitol 7 0.1; C. sat. aq. sol. picric acid method: [sections or smears] -^ water —> A, 5 mins. -^ rinse — » B, 5 mins. -^ rinse — > C, till differentiated, 3-5 mins. —y tap water, till blue — > balsam, via carbol-xylene 11.111 Dobell 1914 1798,34:139 reagents required: A.1% ferric alum in 70% alcohol; B. 1% hematin in 70% alcohol; C. 0.1% HCI in 70% alcohol method: [thin sections or protozoan smears]-^ 70% alcohol—* A, 10 mins. ^ B, 10 mins. — > 70% alcohol, quick rinse —> C, till differentiated—* 70% alcohol, wash -^ counterstain — » balsam, via usual reagents 11.111 Freitas 1936 1345, 31 :707 reagents required: A. 0.5% ferric alum in 70% ale; B. 1% hematin in 70% ale. 60 phosphate buffer pH 7.6 30; C. 0.3% picric acid method: [sections] -^ A, 1 hr. -^ fi, 1 hr. -* wash -^ C, till differentiated -^ wash -» balsam, via usual reagents 11.111 French 1923 626,3:213 REAGENTS REQUIRED: A. 3.5% ferric alum; B. 95% ale. 98, hematoxylin 1, sat. aq. sol. lithium carbonate 2; C. 1% ferric alum method: [sections or smears] -^> yl, overnight -^ rinse —♦ B, overnight -^ wash —> C, till differentiated -^ wash note: Solution B is attributed, without reference, to Rosenbush. 11.111 Galiano 1928 see DS 13.6 Galiano 1928 11.111 Haggquist 1933 23632, 60:77 REAGENTS REQUIRED: A. 5% fcrric chloride; B. 1% hematoxylin; C. 1% ferric chloride. method: [sections] -* water —» ^, 1 hr. —> quick rinse -^ B, 1 hr. — > wash — > C, till differentiated — > wash -^ balsam, via usual reagents 11.111 Hance 1933 19938,77:287 STOCK FORMULA I: 10% hematoxylin in 95% alcohol REAGENTS REQUIRED: A. 2.5% ferric alum; B. water 100 ml., stock I 10 ml., 2.5% sodium bicarbonate 0.1 method: [sections] -^ A, Yi to 2 hrs. -* distilled water, rinse — > /?, 3^ hr. — > tap water, rinse -^ .4, till differentiated — > tap water, till IjIuc -^ balsam, via usual reagents 282 METHODS AND FORMULAS DS 11.111-DS 11.112 note: In preparing B solution, the bicarbonate should be added until the color changes from yellow to plum. This color change is of more importance than the quantity of alkali required to produce it. 11.111 Heidenhain 1892 Feslchr. Kolliker, 118 REAGENTS REQUIRED: A, 2.5% ferric alum; B. 0.5% hematoxylin, "ripened" at least on month method: distilled water -^ A, 30 mins. to 24 hrs. -^ distilled water, rinse —* B, 30 mins. to 24 hrs. — > tap water, rinse, — > A, till differentiated—* tap water, till blue — » balsam, via usual reagents note: Murray 1919 (1200, 16:77) substitutes 3.5% ferric alum for A, above. Masson 1912 (4956, 87:291) substitutes 4% ferric alum for A and 1%, hematoxylin for B, mordanting and staining 5-10 minutes at 50°C. See also DS 12.31 Masson 1912. A detailed description of the use of this stain in the demonstration of chromosomes is given under DS 11.10. 11.111 Hirsch and Bretschneider see DS 21.21 Hirsch and Bretschneider 1938 11.111 Kofoid and Swegy 1915 16599, 51 :289 reagents required: A. 0.4%, ferric alum in 50 %o ale; B. 0.50% hematoxylin in 70%, ale. method: [smears] -> 50% ale. -^ A, 10 mins. -* rinse, 50% ale. — > B, 30 mins. -^ A, till differentiated —>■ wash thoroughly recommended for: protozoan smears. 11.111 Laudau see DS 21.212 Laudau 1924 11.111 Loyez see DS 21.212 Loyez ,^ 11.111 Markey, Culbertson, and Giordano 1943 see DS 23.33 Markey, et al. 1943 11.111 Masson 1912 see DS 11.111 Heidenhain 1892 (note) 11.111 Murray 1919 see DS 11.111 Heidenhain 1892 (note) 11.111 Regaud 1910 1823, 11:291 REAGENTS REQUIRED: A. 5% fcrric alum; B. water 80, hematoxylin 1, glycerol 10, 90% alcohol 10; C. ADS 21.1 Masson 1942 method: distilled water-* A, 30 mins., 50°C -^ distilled water, rinse -^ B, 30 mins., 50°C. -^ distilled water, wash -* C, till differentiated -^ tap water, till blue -^ bal- sam, via usual reagents note: a detailed description of the use of this stain before a complex contrast is given under DS 12.30 below. 11.111 Rosenbush see DS 11.111 French 1923 (note) 11.111 Shortt 1923 9940, 10:836 reagents required: A. 2.5% ferric alum; B. water 95, hematoxylin 1, phenol 5 method: identical with 11.111 Heidenhain 11.112 After Ferric Chloride Mordants 11.112 Cole 1926 19938, 64:452 stock solutions: abs. ale. 100, sodium hydrosulfite 1, hematoxylin 5 reagents required: A. ADS 12.1 Cole 1926; B. Stock 3, ammonia 0.5, water 100; C. 0.1%, hydrochloric acid; D. 0.01%, ammonia method: water -^ A, on slide, 5 mins. -* rinse -^ B, on slide, 10 mins. -^ C, till differ- entiated — » D, till blue — > balsam, via usual reagents 11.112 Mallory 1900 11189,5:18 reagents required: A. 10% ferric chloride; B, 1% hematoxylin; C. Q.2b% ferric chloride DS 11.113 DYE STAINS OF GENERAL APPLICATION 283 METiiou: [sections] > distilled water — > A, 3 5 niins. -^ li, on slide, draining and renew- ing till precipitate ceases to form, till blue-black, 3-5 niins. — > tap water, wash -^ C, till differentiated -^ tap water, wash — > balsam, via oil of cretan origanum 11.113 After Other Mordants 11.113 Apathy 1888 23632, 5:47 REAGENTS REQUIRED: A. 1% hematoxylin; B. 1% potassium dichromate method: [sections] —> water—* A, overnight-^ B, till stain first produced sufficiently differentiated — > water, till yellow color removed -^ balsam, via usual reagents 11.113 Bensley and Bensley 1938 Bensley and Bensley 1938, 79 reagents required: A. sat. sol. copper acetate; B. 5% potassium chromate; C. 1% hematoxylin; D. water 80, ADS 21.1 Weigert 1885, 20 method: [sections] — > water -^ ^, 5 mins. -^ rinse — > B, dip — * rinse — > C, 2 mins. — > A, 1 min. — > D, till differentiated — * balsam, via usual reagents 11.113 Drew 1920 see DS 22.0 Drew 1920 11.113 Fajerstajn 1901 see DS 21.212 Fajerstajn 1901 11.113 Heidenhain 1884 1780, 24:468 reagents required: A. 0.3% hematoxylin; B. 0.5% potassium chromate method: [whole objects or sections]—* waters A, overnight^ rinse -^ B, till dark stain first produced sufficiently differentiated -^ water, till yellow color removed — » balsam, via usual reagents 11.113 Knower 1930 19938, 72:172 preparation of stock solution: Dissolve 5 hematoxylin in 100 abs. ale. Add 1 sat. sol. sodium metabisulfite. REAGENTS REQUIRED: A. ADS 12.1 Colc 1916; B. stock 4, water 8, ammonia 1, 95% alcohol 100; (Mix in order given; leave first three ingredients 30 seconds before diluting); C. 0.1% hydrochloric acid; D. sat. sol. lithium carbonate in 70% alcohol method: [sections from material fixed in any copper-containing fixative (see Chapter 18, F 1400, F 2400, F 3400, F 4000)] -^ 95% alcohol -^ A, 5 mins. -* 95% alcohol, quick rinse -^ B, 5 mins. —> C, till differentiated -^ D, 2 mins. -^ balsam, via usual reagents note : Though this was originally intended as a nerve stain, it is far too good a general- purpose stain to be omitted from this section. The original calls for "sodium bi- sulphite," which is not soluble to the extent indicated; however "the bisulphite of commerce usually consists chiefly, or almost entirely, of sodium pyrosulphite" (Merck Index, 5th ed., 1940, 506). Sodium pyrosulfite, which is commonly sold as "metabi- sulfite," is soluble to the required extent and is accordingly specified in the formula given above. 11.113 Kulschitzky 1889 766, 4:223 formula: water 98, acetic acid 2, hematoxylin 1 use: For general purposes as DS 11.113 Knower 1930. For nervous elements see Chap- ter 21 DS 21.212. The formula of Welters 1890 (23632, 7:466) differs only in contain- ing 2 parts hematoxylin. 11.113 Mallory 1936 Lead hematoxylin see DS 21.212 Mallory 1936 11.113 Nissl 1894 466, 51 :245 reagents required: A. 2% ferric acetate in 90% ale; B. 1% hematoxylin in 70% ale.; C. 1% hydrochloric acid in 70% ale. method: [sections of brain] —> A, 30 mins. — > rinses B, 30 mins. ^ rinse—* C, till differentiated —> balsam, via usual reagents 284 METHODS AND FORMULAS DS 11. 113-DS 11.12 11.113 Schultze 1904 23632, 21 :5 HEAGENTs REQUIRED: A. F 1600.0010 Flemming 1882; B. sat. sol. hematoxylin in 70% ale; C. 1% potassium dichromate in 50% ale; D. bergamot oil method: [sections of material fixed, or mordanted, in /I]— » 50% ale. -^ B, 24 hrs. — > wash — > C, 1 hr. —♦95% ale. -^ D, till differentiated — > balsam 11.113 Welters 1890 see DS 11.113 Kulschitsky 1889 (note) 23632,7:466 11.12 DIRECT HEMATOXYLIN STAINING Hematoxylin cannot be used in direct staining unless some mordant is incorporated with the solution for the purpose of fixing the stain on the material to be colored. The term direct staining is used in this case in contrast to mordant staining and must not be confused with the term direct staining as opposed to indirect staining. The former indicates merely that the object is stained in a relatively strong solution for a length of time sufficient to impregnate the whole and that it is subsequently exposed either to an acid solution or to a solution of the mordant with a view to extracting it from those objects which it is desired to bring into con- trast. The term indirect staining, as used in this same sense, indicates the employment of a very weak solution in order to permit a differential absorption of the stain by those parts of the object to be stained (usually the more dense) which it is intended to bring out. As a generality it may be said that direct staining, in this sense, is usually applied to sections while indirect staining is better for the preparation of wholemounts, provided that one has the leisure to wait for the somewhat lengthy process to finish. The direct-staining formulas are divided into four classes according to the mordant which is incorporated. The first group, incorporating iron mordants, is used almost exclusively for staining the central nervous system in sections; their use and variations are more fully described in Chapter 21. It is doubtful that these stains could ever be employed for indirect staining of wholemounts, but for staining nuclei they are far better than the other three classes, though less widely employed. The next two divisions include these formulas containing alum mordants and acid-alum mordants, the separation of these two being necessitated by the large number of formulas to be found in each. Both are employed for sections and for wholemounts, the best known being unquestionably the formula of Delafield (1885). This reagent has the advantage of being almost foolproof, but it has to be ripened for a considerable period before it can be employed: Watson 1945 (11360, 63:21) recommends barium peroxide for ripening these solutions. The formula of Carazzi 1911 is almost identical but may be used as soon as it is prepared. It is strongly recommended to the attention of those whose staining has previously been confined to Delafield. The formula of Mayer 1896 was once very widely employed for staining wholemounts, but it has nowadays fallen somewhat into disuse. It also required ripening for a considerable period before employment. The formulas incorporating an acid, usually acetic, in addition to the alum mordant are among the best of the general-purpose stains. The formula of Ehrlich 1886 is the most widely known, though any of the others can be recommended. The alum-mordant formulas are the only ones which can be employed in great dilution for indirect staining. It is usually a waste of time to employ acid-alum formulas for this purpose. The diluent to be employed should have the same composition as the formula itself, without the inclusion of hematoxylin. It is a mistake to follow the very wide recommendation that 0.1% hydrochloric acid be employed. This reagent is difficult to remove from the object before its final mounting and leads ultimately to the breakdown of the color. In all cases hematoxylin stains should be "blued" after they have been differentiated, in some alkaline solution, preferably containing free ions of an alkali metal. Lithium carbonate is widely used, though a weak solution of calcium chloride, adjusted with ammonium hydrox- ide to a pH of about 8, is more satisfactory. The old exhortation to use tap water originated in Europe where most of the tap waters are alkaline. The majority of city tap waters in the United States are worthless for this purpose. The chrome-hematoxylins and copper-hematoxylins, which form the fourth class, are of comparatively recent introduction or, at least, of comparatively recent acceptance. The formulas of Hanson 1905 and of Liengme 1930 are, however, excellent reagents and should be tried for sections in those instances in which the more customary formulas do not yield DS 11.121 DYE STAINS OF GENERAL APPLICATION 285 satisfactory results. The phosphomolybdic and phosphotungstic hematoxylins of Mallory, though originally intended for staining nervous structures, are useful for a more general purpose. Mayer's 1891 "haemacalcium" was originally intended for staining wholemounts of small marine invertebrates and is admirable for the purpose. 11.121 Formulas Incorporating Iron Mordants 11.121 Anderson 1929 Anderson 1929, 129 STOCK solutions: I. 0.5% hematoxylin in 50% ale. 100, 2% calcium hypochlorite 5; II. water 100, ferric alum 3, sulfuric acid 2.5 WORKING solution: stock solution I 60, stock solution II 30 note: Overstaining of sections rarely occurs but may be corrected with 0.1% hydro- chloric acid in 70% alcohol. 11.121 Barrett 1932 see DS 22.11 Barrett 1932 11.121 Faure 1924 6639, 90:87 reagents required: .4. 90% alcohol 100, hematoxylin 3.2; B. ADS 12.1 Faure 1924; C. 1% hydrochloric acid; D, sat. aq. sol. lithium carbonate method: [distilled water] —> A + B, (equal parts), 5 sees. —> wash, tap water—* C, quick rinse -^ wash, tap water — » D, till blue — > wash -^ [counterstain, if desired] — > balsam, via usual reagents 11.121 Hansen 1905 23632, 22:55 formula: water 100, ferric alum 4.5, hematoxylin 0.75 preparation: Dissolve the alum in 65 water. Dissolve the dye in 35 water. Mix. Boil. Cool. Filter. 11.121 Held test. 1937 Gatenby and Painter Gatenby and Painter 1937, 374 reagents required: A. 5% ferric alum; B. water 95, DS 11.124 Held 1937 5 method: [water] — > A, 24 hrs. -^ B, 12-24 hrs. -* A, till differentiated — ♦ balsam, via usual reagents 11.121 Janssen 1897 6011,14:207 formula: water 70, ferric alum 5, hematoxylin 1, abs. ale. 5, glycerol 15, methanol 15 preparation: Dissolve the alum in the water and the hematoxylin in the ale. Mix. Leave 1 week. Filter and add remaining ingredients. note: This formula is recommended by Lillie and Earle 1939 (20540b, 14:53) as a sub- stitute for Weigert 1904. 11.121 Kefalas 1926 11360, 46:277 formula: acetone 100, ferric chloride 1, hydrochloric acid 0.05, hematoxylin 1 11.121 Krajian 1950 Krajian 1950, 196 formula: water 50, 95% ale. 50, hematoxylin, ferric alum, ferric chloride, potassium iodide a.a. 6 preparation: Dissolve salts in water and dye in ale. Mix. note: This formula, designed for bacteriology (see DS 23.222 Krajian 1950), is an excellent general-purpose hematoxylin. 11.121 La Manna 1937 23632, 54:257 formula: water 100, hematoxylin 1, ferric chloride 3 11.121 LilUe and Earle 1939 608b, 15:765 STOCK solutions: I. 95% ale. 50, glycerol 50, hematoxylin 1; II. water 100, ferric alum 15, ferrous sulfate 15 working stain: stock I, 50, stock II 50 11.121 Lillie 1940 1789a, 29:705 formula: water 100, ferric chloride 1.2, hematoxylin 1, hydrochloric acid 1 286 METHODS AND FORMULAS DS 11.121-DS 11.122 11.121 Morel and Bassal 1909 11024, 45:632 STOCK solutions: I. 95% ale. 100, hematoxylin 1; II. water 100, ferric chloride 2, copper acetate 0.04, hydrochloric acid, 1 woKKiNG solution: stock I 50, stock II 50 11.121 Olivecrona 1917 see DS 21.21 Olivecrona 1917 11.121 Paquin and Goddard 1947 4349, 27:198 formula: water 75, 95% ale. 25, glycerol 13, ferric alum 5, ammonium sulfate 0.7, hematoxylin 0.8 preparation: Dissolve the dye in the glycerol and ale. with gentle heat. Cool and add, with constant agitation, the other ingredients dissolved in the water. Leave 24 hours. 11.121 Rozas 1935 23632, 52:1 reagents required: A. water 74, 95% ale. 6, glycerol 20, ferric alum 1, aluminum chloride 1.2, hematoxylin 0.6; B. 20% ferric alum method: [sections] -> water -^ yl, 12-24 hrs. — > fi, till differentiated —> balsam, via usual reagents 11.121 Seidelin 1911 see DS 11.121 Weigert 1904 (note) 11.121 Thomas 1943 4285a, 20:212 formula: water 60, dioxane 40, acetic acid 6, hematoxylin 2.5, ferrous chloride 6, ferric chloride 1.5, ferric alum 3, hj'drogen peroxide 1 preparation: Dissolve the hematoxylin in the dioxane and add the hydrogen peroxide. Dissolve the salts in the water and acid. Filter into the hematoxylin solution. 11.121 Verhoeff 1908 see DS 21.13 Verhoeff 1908 11.121 Weigert 1903 test. 1910 ips. Ehrlieh, Krause, et al. 1910, 1:231 stock solutions: I. 0.4% ferric chloride; II. 1% hematoxylin in 95% alcohol reagents required: A. stock I 50, stock II 50; B. ADS 21.1 Masson 1942 method: [distilled water] -^ A, 1-2 hrs. —>■ distilled water, rinse —^ B, till differentiated —^ tap water, till blue — » balsam, via usual reagents 11.121 Weigert 1904 23632,21:1 stock solutions: I. water 95, ferric chloride 0.6, hydrochloric acid 0.75; II. 1% hema- toxylin in 95% ale. REAGENTS REQUIRED: A. StOCk I 50, StOck II 50 METHOD : [distilled water] — » stain, till sufficiently colored — > distilled water, wash — > tap water, till blue -^ balsam, via usual reagents note: See Chapter 21, DS 21.212 for use of these reagents in nerve staining. Weigert diluted his B stock from a 10% stock just before use and specified a German pharma- ceutical preparation, here reduced to terms of laboratory reagents, for his solution A. Seidelin 1911 (16035, 4:94) used 3 stock A to 2 stock B in preparing solution .4. 11.121 Yasvoyn test. 1946 Roskin Roskin 1946, 150 STOCK solutions: I. 0.1% hematoxylin; II. 2.5% ferric alum WORKING solutions: add II with constant stirring to 20 drops I until solution just remains blue method: [sections] —> stain, 2-5 mins. —♦70% ale. if differentiation required — * balsam, via usual reagents 11.122 Formulas Incorporating Alum Mordants 11.122 Belloni 1939 lest. 1943 Cowdry Cowdry 1943, 35 formula: water 100, potassium alum 3, hematoxylin 0.15, chloral hydrate 0.1, potas- sium hydroxide 0.01 11.122 Bohmer 1868 1780,4:345 STOCK solutions: I. 3.5% hematoxylin in abs. ale.; II. 0.3% potassium alum WORKING formula: stock I 10, stock II 90 DS 11.122 DYE STAINS OF GENERAL APPLICATION 287 11.122 Bullard, in verb. Harpst 1951 formitla: water 225, 95% ale. 'Mi, glycerol 'S'3, acetic acid 3.5, anunonium aliun 6, mercuric oxide 0.8, hematoxylin 0.8 I'KiorARATioN: Dissolve the dye in 15 of 50% ale. with 2 acetic acid. Heat and add 2 am- nioniinu alum in 25 water. Boil, add mercuric oxide, and filter. Add remaining in- gredients to cold filtrate. note; The author has not been able to discover any literature reference for this solution which is widely used in pathological laboratories. 11.122 Cajal and de Castro 1933 Cajal and de Castro 1933, 77 PREPARATION OF STOCK: To 100 of a 5% solution of hematoxylin add 5 ammonia. Evaporate to dryness. STAINING solution: 2% solution of above powder in 50% ale. 50; 5% ammonium alum 50 11.122 Carazzi 1911 23632, 28:273 REAGENTS REQUIRED: A. water 80, potassium alum 5, hematoxylin 0.1, potassium iodate 0.02, glycerol 20; B. 0.1% hydrochloric acid in 70% METHOD FOR WHOLE OBJECTS: water -^^ lA + 10 water, 3 to 10 mins. — > distilled water -+ B, if differentiation necessary — > tap water till blue -^ balsam, via usual reagents METHOD FOB SECTIONS: water -^ A, till sufficiently stained, 5 mins. to 12 hrs. -^ dis- tilled water, wash — * tap water, till blue — > balsam, via usual reagents NOTE : A detailed description of the use of this stain in the preparation of a wholemount is given under DS 11.10 above. 11.122 Delafield test. 1885 Prudden 23632, 2 :288 REAGENTS REQUIRED: A. Water 70, ammonium alum 3, hematoxylin 0.6, abs. ale. 4, glycerol 15, methanol 15 method: as Carazzi 1911 note: The original calls for preparation from sat. aq. sol. ammonia alum and 16% hematoxylin solution. 11.122 Friedlander 1882 Friedlander 1882, 92 formula: water 30, glycerol 30, 95% ale. 30, hematoxylin 0.6, potassium alum 0.6 11.122 Gage 1892 test. 1896 ips. Gage 1896, 178 formula: water 100, potassium alum 4, chloral hydrate 2, 95% ale. 2, hematoxylin 0.1 11.122 de Groot 1912 23632,29:182 FORMULA OF SOLVENT: 95% alc. 65, water 27, glycerol 8 formula of stain: 95% alc. 65, water 27, glycerol 8, hydrogen peroxide 0.75, hema- toxylin 0.2, calcium chloride 1.5, sodium bromide 0.75, ammonium alum 2.2, potassium ferricyanide 0.08 PREPARATION OF STAIN: Take 100 solvent. Mix 1.5 solvent with 0.75 hydrogen peroxide and dissolve 0.2 hematoxylin in this. In 25 solvent dissolve 15 calcium chloride and 0.75 sodium bromide. Add this to the hematoxylin solution and dissolve 1.1 am- monium alum in the mixture. In 40 solvent dissolve 0.08 potassium ferricyanide and add this to mixture. In the remaining solvent dissolve 1.1 ammonium alum and add to the mixture. 11.122 Harris 1900 11032,3:777 formula: 95% alc. 5, hematoxylin 0.5, potassium alum 10, water 100, mercuric oxide 0.25 preparation : Dissolve the hematoxylin in alc. Dissolve alum in water and raise to boil- ing. Pour hot solution on hematoxylin. Boil and throw mercuric oxide into boiling solution. Cool rapidly. Filter. note: Mallory 1938, 72 suggests the addition of 5% acetic acid. 11.122 Harris and Power test. 1884 Cole Cole 1884b, 42 formula: hematoxylin 20, alum 60, water 100, abs. alc. 6 preparation: Grind the hematoxylin in a mortar with the alum, adding water in small portions while grinding. Filter and add alcohol. 288 METHODS AND FORMULAS DS 11.122 11.122 Haug test. 1900 Pollock Pollock 1900, 84 formula: water 100, aluminum acetate 5, abs. ale. 5, hematoxylin 5.5 preparation: Add the hematoxylin dissolved in the ale. to the acetate dissolved in the water. 11.122 Kleinenberg 1876 test. 1907 Bohm and Oppel [Bohm and Oppel 1907, 103 STOCK solutions: I. water 30, 95% ale. 70, calcium chloride to sat., ammonium alum to sat.; II. Stock I 12, sat. sol. pot. alum in 70% ale. 88; III. sat. ale. sol. hematoxylin WORKING solution: stock II 100, stock III 3 11.122 Kleinenberg test. 1884 Cole - Cole 1884b, 42 formula: sat. sol. calcium chloride in 70% ale. 15, sat. sol. potassium alum in 70% ale. 85, sat. sol. hematoxylin in abs. ale. 1 11.122 Launoy 1904 test. 1907 Bohm and Oppel Bohm and Oppel 1907, 356 formula: water 100, potassium alum 0.5, hematin 1 11.122 Lee 1905 Lee 1905, 188 formula: water 100, hematoxylin 0.1, ammonium alum 5, sodium iodate 0.02, chloral hydrate 5 11.122 LilUe 1948 see DS 11.123 Lillie 1941 (note) 11.122 Lowenthal 1892 see DS 13.6 Lowenthal 1892 11.122 Mallory 1938 MaUory 1938, 70 formula: water 100, potassium alum 5, hematoxylin 0.25, thymol 0.25 11.122 Martinotti 1910 23632,27:31 formula: water 70, glycerol 15, methanol 15, hematin 0.2, ammonium alum 1.5 preparation: Dissolve alum in 60 water. Add hematin dissolved in 10 water. Add other ingredients to mixture. 11.122 Mayer 1891 14246,10:172 formula: water 100, 95% ale. 5, ammonium alum 5, hematoxylin 0.1 preparation: Add the hematoxylin dissolved in the ale. to the alum dissolved in the water. Ripen some months. note: Mayer {loc. cit.) also recommended the addition of 2% acetic acid to the above, when used for sections. 11.122 Mayer 1896 Mayer's Glycheaemaluvi—aud. 14246, 12 :310 formula: water 70, glycerol 30, ammonium alum 5, hematoxylin 0.4 preparation: Grind the hematoxylin to a stiff paste with a little of the glycerol. Mix the other ingredients and use the solution to wash out the mortar with successive small doses. 11.122 Mayer 1901 23632, 28:273 formula: water 100, potassium alum 5, hematoxylin 0.1, sodium iodate 0.02 note: Mayer 1903 (23632, 20:409) substitutes ammonium alum for potassium alum. 11.122 Prudden 1885 see DS 11.122 Delafield (1885) 11.122 Rawitz 1895a Rawitz 1895, 62 formula: water 65, glycerol 35, potassium alum 1, hematoxylin 1 11.122 Rawitz 1895b Rawitz 1895, 63 formula: water 50, hematin 0.25, ammonium alum 1.5, glycerol 50 11.122 Rindfleisch test. 1877 Frey Frey 1877, 100 stock solutions: I. sat. aq. sol hematoxylin; II. sat. aq. sol. {circ. 14%) ammonium alum WORKING solution: water 85, stock I 10, stock II 5 DS 11.122-DS 11.123 DYE STAINS OF GENERAL APPLICATION 289 11.122 Sass 1929 20540b, 4:127 formula: water 100, ammonium alum 5, hematoxylin 0.1, sodium iodate 0.1 note: See also DS 11.123 Sass 1929. 11.122 Tribondeau, Fichet, and Dubreuil 1916 6630, 79:288 FORMULA FOR STOCK SOLUTION: 95 alc. 100, Water 22, silver nitrate 2, sodium hydroxide 1, hematoxylin 5 PREPARATION OF STOCK SOLUTION: Add the hydroxide dissolved in 20 water to the silver nitrate dissolved in 2 water. Wash ppt. by decantation and transfer to flash with re- flux condenser. Dissolve the hematoxylin in alc. and add to silver suspension. Raise to boiling, cool, filter. WORKING solution: 5% potassium alum 100, stock 5 11.12S Forrmilas Incorporating Acid Alum Mordants 11.123 Anderson 1923 test. 1929 i-ps. Anderson 1929, 192 REAGENTS REQUIRED: A. watcr 90, abs. alc. 5, hematoxylin 0.25, calcium hypochlorite 0.4, ammonium alum 2, acetic acid 5; B. 0.1% hydrochloric acid PREPARATION OF A : Add the hypochlorite to 20 water. Leave 4 hours. Filter. Add filtrate to hematoxylin dissolved in water. Dissolve other ingredients in rest of water. Add this to dye solution. method: water -> A, 2-3 mins. — > B, till differentiated -^ tap water, till blue 11.123 Anderson 1929 Anderson 1929, 129 formula: water 70, 95% alc. 5, acetic acid 5, calcium hypochlorite 4, ammonium alum 3, hematoxylin 0.5 preparation: as Anderson 1923. 11.123 Apathy 1897 14246, 12:712 formula: water 45, 95% alc. 25, glycerol 35, hematoxylin 0.3, acetic acid 1, salicylic acid 0.03, ammonium alum 3 preparation: Dissolve the hematoxylin in 10 water, 25 alc. Allow to ripen for some months. Dissolve the alum and acids in 35 water. Add to dye solution; then add glycerol. 11.123 Cole 1903 Cross and Cole 1903, 170 formula: water 32, 95% alc. 32, glycerol 29, acetic acid 7.5, hematoxylin 0.6, am- monium alum 0.6 preparation: Add alum dissolved in water to hematoxylin dissolved in alc. Add other ingredients to mixture. 11.123 Conklin test. 1930 Guyer Guyer 1930, 232 formula: water 80, DS 11.122 Delafield 1885 20, F 5000.0050 Kleinenberg 1879 4 11.123 EhrUch 1886 23632, 3:150 reagents required: A. water 30, 95% alc. 30, glycerol 30, acetic acid 3, hematoxylin 0.7, ammonium alum to excess; B. sat. 70% alc. sol. lithium carbonate. ^ preparation of a : Dissolve hematoxylin in the alc. and acid. Dissolve 1 ammonium alum in water and glycerol. Mix with dye solution. Allow to ripen for some months. Add excess {circ. 10) ammonium alum to ripened solution. method for sections: 90% alc. — ► A, Y^ to 2 mins. -+ 90% alc. applied from drop bottle, till differentiated — > B, till blue -+ balsam, via usual reagents note: The passage of sections to water before staining, or the use of water to differ- entiate, results in a diffuse stain. A detailed description of the use of this stain for sections is given under DS 11.10 above. 11.123 Harris 1900 see DS 11.122 Harris 1900 (note) 11.123 Langeron 1942 Langeron 1942, 523 reagents required: .1. DS 11.122 Mayer 1901 100, chloral hydrate 5, citric acid 0.1; B. 0.1% HClin70% alc. method: [sections or whole objects]-^ distilled waters A, (sections) 10 mins. or A, (whole objects) 24 to 48 hrs. -^ B, till differentiated -> tap water tiU bl.ue -» balsam, yig, usual reagents 290 METHODS AND FORMULAS DS 11.12a-DS 11.124 11.123 LiUie 1941 20540b, 16 :5 formula: water 70, glycerol 30, acetic acid 20, hematoxylin 0.5, sodium iodate 0.1 preparation: Add the iodate to the dye dissolved in the water. Leave overnight. Add other ingredients. note: This formula was republished, without any reference to its previous publication, by Lillie 1942 (20540b, 17:90). 11.123 Mallory 1938 see DS 11.122 Harris 1900 (note) 11.123 Mann 1892 ksl. 1934 Langeron Langeron 1934, 475 formula: water 35, 95% ale. 32, glycerol 25, acetic acid 3, hematin 0.6, potassium alum 3.5 preparation: Dissolve the dye in the acid. Add mixed ale. and glycerol. Then add alum dissolved in water. 11.123 Masson test. 1934 Langeron Langeron 1934, 475 formula: water 100, acetic acid 2, hematin 2, potassium alum 6 preparation: Dissolve the alum in boiling water. Add dye, cool, filter. Add acid to filtrate. note: a detailed description of the use of this stain before a complex contrast is given under DS 12.30 below. 11.123 Mayer 1891 Maj/er's acid hemalum — auct. see DS 11.122 Mayer 1891 (note) 11.123 Mayer test. 1924 Langeron Langeron 1942, 525 note: This is DS 11.122 Mayer 1896 with the addition of acetic acid. Mayer, however, recommended this addition only to his 1891 formula. The present solution is, there- fore, Langeron's variant. 11.123 Sass 1929 20540b, 4:127 formula: water 100, ammonium alum to sat., hematoxylin 1, sodium iodate 1, acetic acid 3 11.123 Watson 1943 11360, 63:20 formula: water 32, abs. ale. 32, glycerol 32, acetic acid 3, ammonium alum 0.064, hematoxylin 0.64, potassium permanganate 0.032 preparation: Dissolve the alum and permanganate in water. Add dye dissolved in ale. and then other ingredients. note: This formula stains as well as DS 11.123 Ehrlich 1886, from which Watson de- veloped it, and does not require ripening. 11.134 Formulas Incorporating Other Mordants 11.124 Alzheimer 1910 lithium-heinatoxylin Nissl and Alzheimer 1910, 411 formula: water 90, 95% ale. 10, hematoxylin 1, Hthium carbonate 0.03 preparation: Add the alkali dissolved in water to the dye dissolved in ale. 11.124 Bacsich 1937 lithium-hematoxylin 11025,72:163 formula: water 100, hematoxylin 1, lithium carbonate 0.1 11.124 Clara 1933 molybdic-hematoxylin 23632,60:73 stock solution: 1% hematoxylin 50, 10% ammonium molybdate 50, molybdic acid to excess (circ. 1) , working solution: stock 0.1, water 100 11.124 Cook test. 1883 Hogg copper-hematoxylin Hogg 1883, 237 formula: water 100, "extract of logwood" 15, copper sulfate 2.5, potassium alum 15 preparation: Grind the dry powders in a mortar. Add enough water to make a paste. Leave two days; add rest of water. Leave 12 hours. Filter. note: The "extract of logwood" is the result of evaporating an aqueous extract of log- wood to dryness; in addition to hematoxylin and hematin (about 70% of the whole) it contains tannin, glucosides, and resins. DS 11.124 DYE STAINS OF GENERAL APPLICATION 291 11.124 Cretin 1925 ferricyanide-hematoxylin LeMans, 93 REAGENTS REQUIRED: A. Water 100, ferrous sulfate 4; B. water 100, potassium fcrro- cyanide 2, potassium fcrricj'anide 1; C. water 100, hematoxylin 0.5; D. water 100, ferric alum 5 method: Distilled water — > A, 24 hrs. -^ running tap water, overnight —^ B, 3-6 hrs. — » distilled water, rinse -^ C, overnight -^ D, till differentiated results: nuclei dense, opaque black 11.124 Donaggio 1904 lin-hernatoxylin 1006,22:192 formula: water 100, hematoxylin 0.5, stannic chloride diammine 10 preparation: Dissolve the dry salts each in 50 water. Mix solutions. note: Donaggio 1940 (18208, 22:171) substitutes stannic chloride for the diaminine complex. 11.124 Gomori 1941 chrome-hemaloxylin 608b, 17, 395 formula: water 100, sulfuric acid 0.1, potassium dichromate 0.1, chrome alum 1.5, hematoxylin 0.5 preparation: Dissolve the alum and dye each in 50. Mix. Add 2 5% potassium di- chromate and 2 5% sulfuric acid. Ripen 2 days. 11.124 Hansen 1905 chrome-hemaioxylin 23G32, 22 :64 formula: water 100, sulfuric acid 0.2, hematoxylin 0.3, chrome alum 3, potassium dichromate 0.2 preparation: Boil the chrome alum in 85 water until green. Dissolve hematoxylin in 5 water. Add to alum solution. Then add successively, acid in 2 water and dichromate in 7 water with constant stirring. Filter. 11.124 Harris 1900 mercuric-hematoxylin see DS 11.122 Harris 11.124 Heidenhain test. 1907 Bohm and Oppel vanadium-hematoxTjlin Bohm and Oppel 1907, 105 formula: 0.5% hematoxylin GO, 0.25% ammonium vanadate 30 11.124 Held test. 1937 Gatenby and Painter phosphottwlybdic hematoxylin Gatenby and Painter 1937, 374 formula: water 30, 95% ale. 70 hematoxylin 1, phosphomolybdic acid 15 preparation: Dissolve hematoxylin in solvents. Add acid. Leave 1 month. Decant. 11.124 Hornyold 1915 test. Gatenby and Painter 1937 iodine-he matoxylin Gatenby and Painter 1937, 158 reagents required: A. a])s. ale. 25, water 75, hematoxyhn 0.8, ammonium alum 0.5, tincture of iodine USP (see note) 0.5; B. 0.1% acetic acid in 70% ale. preparation of a : Add the alum dissolved in the water to the hematoxylin dissolved in the ale. Ripen some days. Add iodine. method: [sections] —> water -^ /I, 5-10 mins. -^ rinse -^ B, till blue —> balsam, via usual reagents note: The tincture of iodine mentioned in the original formula is the British, which contains 2li % each of I; and KI in 90% ale. The official American tincture is double this concentration and should be used in the preparation above. 11.124 Hueter 1911 see DS 11.124 Schueninoff 1908 (note) and Mallory 1891 (note) 11.124 Kleinenberg 1876 and 1879 see DS 11.124 Squire 1892 (note) 11.124 Liengme 1930 iron-copper-hematoxylin 4285a, 7:233 REAGENTS REQUIRED: A. DS 11.122 Bohmcr 1868 50, ADS 12.1 Morel & Bassal 1909 50; B. 0.1% hydrochloric acid; C. sat. sol. lithium carbonate in 70% ale. method: 70% alcohol -^^ A, 1-4 days ^ B, till differentiated -» 70% alcohol, wash -> C, till blued — » balsam, via usual reagents 11.124 Loyez test. 1938 Carleton and Leach lithium hematoxylin Carleton and Leach 1938, 259 STOCK i: abs. ale. 100, hematoxylin 10; stock ii: water 100, sat. aq. sol. lithium carbon- ate 4 WORKING solution: stock I 10, stock II 90 292 METHODS AND FORMULAS DS 11.124 11.124 Mallory 1891 phosphomolybdic hematoxylin 766,7:375 formula: water 100, hematoxylin 1, phosphomolybdic acid 1, chloral hydrate 7.5 preparation: Add the acid dissolved in 10 water to the dye dissolved in 90. Add chloral hydrate to mixture. method: water —> stain, 10 mins. to 1 hr. — > 30% ale. till differentiated note: Phenol may be substituted for chloral hydrate. Hueter {test. Schmorl 1928, 173) substitutes phosphotungstic for phosphomolybdic acid; but see note under Schuenin- off 1908. 11.124 Mallory 1900 phosphotungstic hematoxylin 11189,5:19 REAGENTS REQUIRED: A. water 100, hematoxylin 0.1, phosphotungstic acid 2, hydrogen peroxide 0.2 preparation: Add the acid dissolved in 20 water to the dye dissolved in 80. Add hydrogen peroxide to mixture. method: [sections of material fixed in F 3700.0010 Zenker 1894 or similar fixative] -* water, thorough wash -^ A, 12-24 hrs. -* 95% alcohol, about one minute-^ abs. ale. till differentiation complete — > balsam, via xylene note: This is a polychrome, general-purpose stain. 11.124 Mallory test. McClung 1929 phosphotungstic hematoxylin McClung 1929, 288 reagents required: A. 0.25% potassium permanganate; B. 5% oxalic acid; C. water 100, hematoxylin 0.1, phosphotungstic acid 2, potassium permanganate 0.025 method: [sections of material fixed in F 3700.0010 Zenker 1894 or similar fixative]-^ A, 5-10 mins. -> water, thorough rinse -^ B, 10-20 mins. -^ C, 12-24 hrs. -^ 95% ale, quick rinse -^ abs. ale, least possible time -^ balsam via xylene note: This is a polychrome general-purpose stain, 11.124 Mayer 1891 Haemacalcium—auct. 14246,10:182 REAGENTS REQUIRED: A. water 30, ale. 70, acetic acid 1.5, hematoxylin 0.15, aluminum chloride 0.15, calcium chloride 7.5; B. 95% ale. 30, water 70, aluminum chloride 2 PREPARATION OF A : Grind the dye with the aluminum chloride in a beaker. Add solvents and warm to solution. Then add calcium chloride. method: [whole objects] ^ 70% ale. -^ ^, till stained, usually overnight -+ B, till differentiated — > balsam, via usual reagents note: Mayer 1910 " Haemastrontium" {test. Gatenby and Cowdry 1937, 160) differs from above only in substitution of strontium chloride for calcium chloride, and of 0.1 citric acid for 1.5 acetic acid. 11.124 Mayer 1910 Haemastrontium — auct. see DS 11.124 Mayer 1891 (note) 11.124 Police 1909 phosphomolyhdic-hematoxylin 1949, 4:300 formula: water 70, ale. 30, chloral hydrate 10, hematoxylin 0.35, phosphomolybdic acid 0.03 11.124 Rawitz 1909 aluminum-hematoxylin 23632,25:391 formula: water 50, glycerol 50, hematin 0.2, aluminum nitrate 2 preparation: Add the aluminum nitrate dissolved in 25 water to the dye dissolved in 25 water. Then add glycerol. 11.124 Schroder 1930 lilhium-hematoxylin 23430, 166, 588 formula: water 100, hematoxylin 0.3, lithium carbonate 0.04 11.124 Schueninofi 1908 phosphomolybdic hematoxylin 23681,18:6 formula: water 100, hematoxylin 0.9, phenol 2.5, phosphomolybdic acid 0.5 note: Hueter 1911 (Romeis 1948, 351) differs from this only in the substitution of phosphotungstic acid; but see note under Mallory 1891. 11.124 Schweitzer 1942 /(.■?/. 1946 Roskin chrome-hematoxylin Roskin 1946, 200 formula: water 125, chrome alum 5, hematoxylin 0.5, 10% sulfuric acid 4, potassium dichromate 0.275 preparation: To the first three ingredients dissolved iu 90 water add the dichromate dissolved in 10. DS 11.124-DS 11.2 DYE STAINS OF GENERAL APPLICATION 293 11.124 Squire 1892 calcium-hematoxylin Squire 1892, 25 formula: water 10.5, ale. 96, hematoxylin 1, calcium chloride 8, aniinouium alum 1.2 preparation: Add the alum dissolved iu 6.5 water to the calcium chloride dissolved in 4 water. Add ale, leave 1 hour, filter. Dissolve dye in filtrate. note: In the early 1880's almost any alum-calcium chloride-hematoxylin was referred to as "Kleinenberg," who recommended this method of preparing an aluminum chloride-hematoxylin (which, in effect, this is) without recourse to the very acid salt in commerce in his time. The method given by Kleinenberg 1876 (Grundsiige der Entwickelungsgeschichte, Leipsig) proved impractical and a revised method (17510, 74:208) published in 1879, usually erroneously cited as the original, proved little better. 11.124 Thomas 1943 phosphoinolyhdic hemntoxylin 4285a, 20:49 FOR.MULA: water 44, dioxane 40, ethylene glycol 11, phosphomolybdic acid 16. 5, hema- toxylin 2.5, hydrogen peroxide 2 preparation: Dissolve the hematoxylin in the dioxane and add the hydrogen per- oxide. Dissolve the phosphomolybdic acid in the other solvents and filter into the hematoxylin. 11.2 Carmine Stains As in the case of hematoxylin, carmine was widely employed in the dyeing trade prior to the introduction of stains into microtomy. The carmine itself was com- monly obtained as a tin lake, prepared by boihng cochineal extracts with tin salts, usually the tartrate. Though it is cus- tomary to distinguish between those stain- ing formulas employing carmine lakes, those employing extracts of the raw cochi- neal, and those employing carminic acid, there is no justification for this since those formulas employing the two latter re- agents have always incorporated with them some material which will take the lake into solution. The six divisions of the carmine for- mulas here employed are based entirely on the ingredients, the most widely known being the first two classes of "alcohol carmines" (DS 11.22) and "alum car- mines" (DS 11.21). Considerable con- fusion has been occasioned by the fact that Grenacher, in 1879, published for- mulas for each of these two divisions and early workers almost invariably employed the alum carmine, while modern workers seem to prefer the "alcohohc borax carmine." The early employment of carmine for staining materials before sectioning was necessitated by the fact that no method had been worked out for attaching sec- tions to shdes, so that the fewer manipu- lations which were undertaken in the sec- tions, the more chance there was for preserving the whole. The straight alum carmines are best employed for direct staining from exceedingly dilute solu- tions, a 1 % solution of ammonia alum being the customary diluent. Alcohohc carmines, particularly that of Grenacher 1879, are most employed for the prepara- tion of wholemounts of small inverte- brates. The formulas of Mayer 1881 and Mayer 1892a, are the best devised for small marine invertebrates. Though the bora.x-carmine of Grenacher is commonly made today by the method here given for the "working formula direct" the original method of Grenacher was to prepare the dry stock and to make up working for- mulas from it in various strengths of al- cohol. This gives far better control of the process, since the solubility of the dry stock is a direct function of the concentra- tion of alcohol employed. When any of the alcoholic carmines are used, they are differentiated with a .1% solution of hydrochloric acid in 70% alcohol. The aceto-carmines, (DS 11.23) which form the next class, are more widely em- ployed in botanical than in zoological techniques, and their most valuable ap- phcations is the staining of unfixed nuclei. They should be confined if possible to this use, for their preservation as permanent objects is difficult. Their only other use, besides the counting of chromosomes, is in the diagnostic staining of parasitic platyhelminthes. Picro-carmines (DS 11.24) are warmly recommended to the beginner, for it is 294 METHODS AND FORMULAS DS 11.20 almost impossible to overstain in them. The picric acid, moreover, acts as a fixa- tive. It is possible to take a small living invertebrate, throw it into the stain for ten minutes or so, and remove it fixed and stained. The original formula of Ranvier 1889 called for a preparation of a dry stock and the preparation of a working solution from this. Until quite recent times the dry stock could be purchased. It undoubtedly makes a better solution and keeps better than do the formulas prepared as solutions directly. Iron carmines (DS 11.25) had a brief vogue in the first decade of the present century and then again fell into disfavor. They are nuclear stains strongly re- sembling the reactions of the iron hema- toxyhns. The formula of De Groot 1903 is the one most usually recommended. Am- monia carmines (DS 11.26) and hydro- chloric carmines (DS 11.26) are no longer very well known, though the formula of HoUande 1916 gives excellent nuclear staining. The final class, which contains those formulas that cannot reasonably be fitted into the previous classes, are rarely used today. The only formula finding any great acceptance is the "hthium carmine" of Orth (1892), which was rediscovered in Germany in the 1920's. It is most warmly recommended by Spielmeyer, 1924, for counterstaining sections of the nervous system. 11.20 TYPICAL EXAMPLES Preparation of a wholemount of a liver fluke using the carmalum stain of Mayer 1897 Though many persons will be forced to rely for their material on a supply house, better preparations can be made if the living flukes are secured from a slaughter house. In this case the flukes should be removed from the liver (where they will mostly be found crawling upon the surface if the animal has been dead for some time) to a vacuum flask containing physio- logical saline solution at a temperature of about 35°C. to which has been added a small quantity (approximately one tenth of a gram per liter) of gelatin. Flukes can be transported ahve for relatively long distances in this solution, and every possi- ble effort should be made to keep them alive until they have been brought to the laboratory. In the laboratory the con- tents of the thermos flask should be poured into a dish and the worms trans- ferred individually to another large dish containing warm physiological saline, where the last of the blood will be washed from them. Better preparations will be secured if time be taken to anesthetize the worms before fixing them. Most of the thick and opaque mounts which one seens in laboratories result from an en- deavor to fix an unanesthetized worm which has contracted during the course of fixation. Liver flukes are easy to anes- thetize, the simplest method being to sprinkle a few crystals of menthol on the surface of the warm saline and leave them for about half an hour. One should not, of course, permit them to die in this solu- tion, but should watch them carefully, terminating the process when their mo- tions become exceedingly slow and con- sist only of an occasional feeble contrac- tion rather than the active movements in wliich they were indulging when removed from the liver. While the worms are being anesthetized preparations for fixing them should be made. Take two sheets of quarter-inch plate glass, each of such a size as will en- able one to lay on them the number of worms which are to be fixed, and place upon the lower plate two or three thick- nesses of a rather coarse filter paper or paper toweUng. Blotting paper is too soft for tliis purpose; a good filter paper is much to be preferred to a paper towel. The selection of a fixative must, of course, rest in the hands of the operator, but the author's preference is for the mercuric- acetic-nitric mixture of Gilson 1898 (Chapter 18, F 3000.0014). This has all the advantages in sharpness of definition given by mercuric fixatives, while the addition of nitric acid appears to render the flattened worms less brittle in sub- sequent handling. Whatever fixative is selected, the sheet of filter paper is now saturated thoroughly with it and the anesthetized worms are removed from the physiological saline and laid out one by one about an inch apart on the blotting paper. This must be done as rapidly as DS 11.20 DYE STAINS OF GENERAL APPLICATION 295 possible to prevent fixation taking place before an additional layer of i)aper (satu- rated with fixative) is placed on the top, and the second sheet of glass placed on top of this. Assuming that the sheets of glass are the size of a sheet of typewriting paper, it is suggested that about a two- pound weight then be placed on the upper sheet of glass. The "sandwich" should now be left for at least 12 hours before removing the glass and upper paper. Then the worms should be picked up one by one on a glass section lifter (metal cannot be used because of the presence of mercuric chloride) and transferred to a large jar of fixative, where they may remain from another day to another week at the discre- tion of the technician. At the conclusion of fixation the worms should be washed in running water for at least 24 hours. It has not been the author's experience that this fixative, followed by such washing, requires the use of iodine for the final removal of the mercury. At this stage iodine tends to render the worms brittle and the author strongly recommends its omission. After being thoroughly washed in water, the worms may be stained. The formula selected for this example is the very well-known carmalum of Mayer (DS 11.21 Ma5^er 1897 below). Objects of this type are better stained by the additive process than by a process of differentiation. That is, they are better placed in weak solution and allowed to absorb the stain slowly than if placed in a strong solution which will require subsequent differentiation. The best diluent for the stain is a 5 % solu- tion of potassium alum. The extent of the dilution is dependent upon the choice of the operator and the size of the object which is to be stained. In the present in- stance a dilution of about one part of the stain to 100 parts of 5% potassium alum would be correct. It is far worse to have the solution too strong than it is to have it too weak and, since it is an excellent preservative, the worms can remain in it indefinitely. The worms are placed in this diluted stain and left there until their internal structures have become clearly visible. It is suggested that they be ex- amined at the end of a week and sub- sequentl}^ every three days, until such time as examination with a low-power binocular microscope, using a bright light from beneath, shows the testes to be darkly stained. At this point the worms are removed to a fresh solution of 5% potassium alum and rinsed for a short time until all the adherent color has left them. They will still, however, be pink on the outside. Since the purpose of the stain is to demonstrate the internal organs it is desirable to bleach this outer layer in order to produce bright scarlet internal organs against a white background. In the experience of the writer this may be done most readily with the aid of a potassium permanganate-oxalic acid bleach in the following manner. Prepare a solution of potassium permanganate so weak that it appears only a very faint pink. This is best done b}^ adding a few drops of a strong solution to a beaker of distilled water. Each worm is then taken individually and dropped into the solution until such time as it has turned a bronzy brow^n on the outside. This appearance can best be de- tected in reflected light and just as soon as the first bronze sheen appears on the out- side, the worm must be removed to fresh distilled water, where it can remain until all the other worms in the batch have been similarly treated. It will be necessary, of course, to renew the potassium permanga- nate from time to time by adding a few more drops of the stock solution to the beaker. The strength of oxalic acid used to bleach the worm is quite immaterial. Two or 3%, arrived at by guess work rather than by weighing, is an adequate solution. As the bleaching of the surface is not as critical as is the deposition of the potassium jier- manganate, all the worms may be bleached at the same time by pouring oflf from the beaker the distilled water in which they have been accumulated, and substituting for it the oxahc acid. One or two twists of the wrist, to rotate the worms in the beaker, will result in their turning from a bronze sheen to a dead white. The oxalic acid is then poured off without any waste of time, and the worms washed in running tap water for an hour or so, before being dehydrated in the ordinary way, cleared, and mounted in balsam (see Chapter 6). 296 METHODS AND FORMULAS DS 11.20 Little trouble will be experienced with worms curling if they have been fixed and treated as described; if they do curl, they should undergo the final dehydration in 95% alcohol pressed loosely between two sheets of glass. Preparation of a wholemount of a medusa using the alcoholic-borax- carmine stain of Grenacher 1879 The preparation of a good wholemount of a medusa, particularly of the thick- bodied type such as Sarsia or Gonionemus, is one of the most difficult operations known in microtomy and is under no cir- cumstance, to be attempted by a beginner who is liable to be discouraged. No refer- ence is here intended to those horrible travesties of wrinkled and crumpled me- dusae which are occasionally seen. A good shde of a medusa should differ from the medusa in life only in that to a trans- parency as great as that of the original should be added such staining as will bring out structures previously almost invisible. The first essential in the preparation of a good wholemount is the collection and preservation of the medusa itself. It is only with extreme rarity that well-pre- served specimens (from the microtomist's point of view) can be secured from bio- logical supply houses. It is presumed in the discussion wliich follows, therefore, that the mounter is in a position to secure his own living material. The first, and most essential, feature is that the medusa be kept in a large volume of clean sea water from the first moment of its collec- tion. It is no use securing an assorted plankton sample in which are swimming a few medusae and anticipating that these will remain for more than a few moments in an undamaged condition. The entire plankton sample should be tipped into a large bowl of clean sea water and each in- dividual medusa removed from it to an- other bowl of clean sea water. From this clean collection there should be selected and thrown away all those medusae which show the slislitest damage, together with all those which are sluggish in their move- ments or are beginning to turn opaque. Subsequent processes are so laborious that it is a waste of time to start with other than perfect specimens. Medusae need to be rather thoroughly narcotized prior to fixation and, though it is commonly stated that only cocaine will work for this purpose, the author has had considerable success either with chloral hydrate or with mixtures of menthol and chloral hydrate together. A few crystals of menthol should be placed on the surface and a few drops of a 10% solution of chloral hydrate in sea water should be added for each 100 cc. of the fluid in which the medusae are swimming. Though it would appear to be a waste of reagents at first sight, it is far better to commence the process of narcotization in a relatively large volume of sea water, for medusae actively swimming in small containers are likely continually to liit themselves on the sides of the vessel. They will not expand into that perfectly relaxed condition which is prerequisite to success in fixation. The actual fluid used for fixation is a matter of individual preference; but the author has always had such success with the mercuric-cupric mixture of Lo Bianco 1890 (Chapter 18, F 3400.0000) that he has not experimented widely with other fixatives. The technique employed in fixa- tion is more important than the fixative, and the method of Lo Bianco (loc. cit.) can scarcely be improved. First, secure a large glass bowl of the type customarily used in kitchens for mixing cakes, and fill it about one-third full of clean sea water. Then transfer each narcotized medusa to this bowl of clean sea water until there have been accumulated as many medusa as can conveniently be placed in it without their actually rubbing against each other. Take the bowl in the left hand and swing it in a circle in such a manner that the contained fluid with the medusae commences to ro- tate within the bowl. When all the me- dusae are facing in the same direction with their tentacles streaming behind them through the flow of the water, then slightly increase the speed while at the same time pouring in the fixative as a steady stream from a large jug held in the right hand. Allowance should be made for twice as much fixative as there is fluid DS 11.20 DYE STAINS OF GENERAL APPLICATION 297 in which the medusae are swimming. The rotary movement of the left hand should on no account be discontinued until at least three or four minutes have passed after the addition of the fixative. A safe criterion is that the endodermal canals shall have become opaque through the action of the fixative before the rotary- movement is discontinued. The medusae will be found to settle to the bottom of the bowl in about the same period of time as it takes the mounter to resecure the use of liis left arm. The medusae may then be removed, one at a time with a glass spoon, to a fresh large volume of the fixative in which they should remain overnight, though a period of two or three days in this fixative, will not damage them. There now commences, between this time and the time of the final mounting of the medusa in balsam, a continual struggle against the collapse of the delicate mesoglea with the subsequent wrecking of the mount. This collapse is produced by osmotic pressure, and it is sufficient to re- move the medusae to distilled water to ensure spoihng them. It is therefore neces- sary to transfer the medusae first to about a gallon of clean, filtered, sea water, to commence the process of washing, and then, through a quite finely drawn glass tube connected with the fresh water tap, to permit the sea water slowly to be replaced by fresh water. It is now necessary to rig up some form of drip mechanism by which the water in which the medusae are resting may be re- placed with increasing strengths of alco- hol by a slow and continuous process. The writer himself uses a 50 milhhter wide- mouth bottle standing upon his bench with a liter aspirator bottle standing on the shelf above. A rubber tube from the aspirator bottle passes to an ordinary pipet inserted through a hole in the cork of the bottle, from which comes a second drainage tube leading to the waste. A screw-controlled pinchcock in the middle of the rubber tube leading from the aspi- rator bottle permits a very fine control of the flow of alcohol which is estabhshed, of course, before the cork is inserted into the 50 milhhter bottle. For replacing, in the first stage, the water used for wasliing \\'ith 15% alcohol, a flow of 50 or 60 drops a minute will be found perfectly safe. The flow should be permitted to continue until the whole hter of 15% alcohol has passed out of the aspirator bottle and through the 50-milhliter bottle in which are the specimens. For the type of process which is now being described it is better to utilize Grenadier's dry powder, the method of preparation for which is given in the ab- breviated formula below (11.21 Grenacher 1879), than it is to endeavor to use the prepared solution in 70% alcohol. If this powder is obtainable, or can be made, it is recommended that about 100 millihters of a 2% solution be prepared in 30% al- cohol and run through the aspirator bottle into the specimen bottle in the manner in which the alcohol has already been used. This stain may be permitted to remain in contact with the specimens overnight or for whatever period of time is convenient to the operator. The aspirator bottle is now again filled with a hter of 30% alco- hol to wliich has been added approxi- mately one milhhter of hydrochloric acid. This acid alcohol is now used to flush out the stain from the specimen bottle. Unless a relatively pale pink solution alone re- mains after the passage of the acid alcohol, a further hter will have to be run through. The specimens may be left to differentiate in acid alcohol until inspection with the naked eye shows the mesoglea to be prac- tically free of stain. The internal organs, however, should remain a rather bright pink. It must not be forgotten that the apparent degree of staining will increase greatly when the specimens are cleared. The process of dehydration should now be completed using successive liter por- tions of 60% alcohol, 80% alcohol, and 95 % alcohol run through by the slow flow method already described. To complete the process of dehydration the author prefers, on a cost basis, to use acetone in place of absolute alcohol, though there is, of course, no reason why alcohol should not be employed. However, it usually is necessary to run through at least two liters of the last dehydrating agent in order to be quite certain that the speci- mens are perfectly dehydrated. 298 METHODS AND FORMULAS DS 11.20 As a clearing agent, tlu^ auilior ))iefers benzene, for the double reason that it can be used for the solution of balsam in the next stage and also because it leads to less hardening than almost any other reagent. The acetone or alcohol is replaced with benzene by the same slow flow process: passing through the stages of 20% ben- zene, 40% benzene, 60 7o benzene, 80% benzene, the percentage figures referring to the amount of benzene in mixture with fresh acetone or alcohol. These benzene- acetone mixtures should be run through in quantities of a Uter each, but they need not, of course, be wasted, for only the first fraction passing through will be seriously diluted and the remaining material may be led from the outlet pipe of the bottle in which the specimen is to another bottle in wliich it may be stored for future use. Though it is perhaps pointing out the obvious, attention should be drawn to the fact that either synthetic rubber tubing or alternatively solid glass joints must be employed. Transference of the perfectly cleared specimens to Canada balsam is a more difficult project than even the clearing and dehydrating already undertaken. The author prefers the method of evaporation, even though it is most tediously slow, because it has the advantage of complete safety. In this niethod the benzene in which the specimens are now resting is re- placed, using the same flow method which has been used for the application of all the other reagents, by a 1 % solution of Canada balsam in benzene. Unfortunately it is impossible to evaporate the benzene directly from the balsam, unless a tre- mendous volume of solution is employed, for even the relatively thin solution used for mounting sections should be at least 40% by weight balsam and for the mount- ing of wholemounts, 60 or 70% is not excessive. A solution stronger than 1%, even when apphed by the drip method, will inevitably result in the collapse of the specimen; therefore one is forced to sub- stitute increasingly stronger solutions be- fore the final evaporation is made. The simplest way to do tliis is to pass the specimens as described into a 1 % solution and then to transfer the specimens in this solution to a beaker of the 1% solution. The height of the 1% solution in the beaker is then measured with a millimeter scale and a red mark made halfway be- tween the top of the liquid and the bottom of the beaker. The beaker is then placed in a large desiccator, the lid of which is removed at daily intervals to liberate the benzene, and the whole is permitted to evaporate until the halfway mark is reached. A fresh solution of 2% balsam is then taken and a drop of it placed care- fully in the presumable 2 % solution which is now in the beaker. If this drop shows by its refractive index that it is now of the same concentration as that in the beaker, the solution which is to be added should be adjusted, either with the addition of balsam or with the addition of benzene, until there is httle or no difference be- tween the two solutions. The beaker is then filled up with tliis fresh 2% solution to the original level and again permitted to evaporate down to the halfway mark. At this time an additional portion of 4% solution is added and so on until the beaker is filled with a 32% solution. This is now permitted to evaporate until it is just hquid enough to permit the removal of each medusa with a considerable por- tion of the balsam either to a hollow- ground slide or a deep cell. No coverslip is placed on it at this stage, but additional solution from the beaker is piled on top of the specimen as it evaporates so that a Uttle mount of semiliquid balsam is con- stantly kept over the specimen. The ut- most care must be taken to exclude dust during these proceedings. When the balsam has, by evaporation, been thick- ened to the point where it no longer flows, a coverslip should be dipped in clean benzene and placed on top of the mound of balsam and the shde slowly heated on a hotplate until it becomes sufficiently fluid for the coverslip to settle of its own accord onto the specimen. The specimen is then cooled, permitted to set for a day or two, and then cleaned up in the ordinary manner. Though it must be admitted that the preparation of mounts of this type oc- cupies several months, only a very few hours per week are required to look after DS 11.20 DYE STAINS OF GENERAL APPLICATION 299 the preparation. The results leave nothing to be desired, whether the mount is made of the medusa, which has been taken as a typical example, or of any other exceed- ingly delicate material that is liable to collapse in mounting. Preparation of a smear to show chromo- somes in saUvary glands of Chi- ronomus using the iron-aceto- carmine stain of BelUng 1921 Aceto-carmine preparations are easy to make but their utility is limited by their short life, which rarely exceeds, even in a successful preparation, more than a week or two. They are, however, excellent for class demonstration, and the preparation which follows should be placed in the hands of every student of cytology who wants to see for himself the really startling detail which can be obtained in salivary gland chromosomes. Though this tech- nique is simpkst when the larva of the dipteran Chironomus is employed, there are many other small flies with which it may be used. The larva of Chironomus which is recog- nized by the rapidity of its movements and its bright blood-red coloration, may usually be collected from any fresh-water stream. It is a waste of time to dissect out the salivary glands since they may be ob- tained more easily by pulling the animal apart. Take the Chironomus larva and strand it on a clean glass sUde on which the final mount is to be prepared. If the animal is living in ordinary pond water containing much debris, it is desirable to transfer it for an hour or two to filtered water in order that the worst of the debris may be removed. Two mounted needles are all that are required to complete the technique. The first needle is pressed firmly upon the head of the larva which, for a right-handed individual, may be most conveniently directed toward the left. With the point of the second needle a very small hole should be torn in the upper integument immediately behind the junc- tion of the head with the thorax. Though this hole is not absolutely essential it is an insurance against the animal's breaking in an undesirable place. The second needle is then pressed firmly and horizontally across the abdomen and drawn with a slow and steady motion to the right. Nine times out of 10 the larva will break in half at the junction of the head with the tliorax, and there will be drawn out of the thorax the anterior portion of the ahmen- tary canal together with, of course, large numbers of attendant structures, includ- ing the salivary glands. The salivary glands may be recognized as a pair of flattened, transparent, pear-shaped struc- tures in which the nuclei are so large that they may be recognized with even the low power of a microscope. Everything except the salivary glands should now be dis- sected away with the needles, the glands themselves being severed from their at- tachment either with the point of the needle or with the edge of a minute scalpel. A few drops of isotonic saline may now be employed to wash away the remainder of the adherent material, leaving the salivary glands isolated on, and usually adherent to, the surface of the slide. It is assumed that a supply of Belling's aceto-carmine has already been secured, made up according to the directions given in the formula below (DS 11.23 Belling 1921). All that is now necessary is to place a few drops of this on top of the salivary glands and to add a coverslip. Care should be taken that sufficient is added to pre- vent undesirable squashing at this stage of the proceedings. The slide is now placed on one side for about five minutes, the aceto-carmine being replaced as it evapo- rates. Very little should normally be lost, however, unless the atmosphere is unduly dry. The specimen is then examined under the low power of the microscope and, if it appears to be too opaque, is flattened by gentle pressure from a needle. The ejected aceto-carmine is mopped up on a filter paper until such time as it is sufficiently thin. The specimen may then be exam- ined under the high power of the micro- scope and the remarkable structure of the chromosomes noted. There is no really satisfactory method of rendering permanent an aceto-carmine preparation, though they will kooji for some weeks if the edge of the coverslii) is cemented (sec Chaptors 2 and '^) with 300 METHODS AND FORMULAS DS 11.21 any material suitable for the purpose, media of Zirkel 1940 (see Chapter 26). Alternativel}^ recourse may be had to These are considerably more stable but the combined mounting and staining cannot be regarded as permanent. 11.21 ALUM CARMINES 11.21 Anderson 1926 11431,29:117 formula: water 95, abs. ale. 10, acetic acid 5, carmine 1, ammonium alum 3.5, calcium hypochlorite 0.1 preparation: To the carmine suspended in the ale, add the hypochlorite suspended in 5 water. Dissolve the alum in 90 water, bring to boil, add carmine mixture boil 1 minute, cool, filter. Add acid. 11.21 Arcangeli 1885a 16977,4:233 formula: water 100, ammonium alum 15, boric acid 2, carmine 0.25 preparation: Boil 10 minutes. Filter. 11.21 Arcangeli 1885b 16977,4:233 formula: water 100, ammonium alum 15, salicylic acid 0.25, carmine 0.25 preparation: As Arcangeli 1885a. 11.21 Bohm and Oppel 1907 see DS 11.21 Rabl 1894 (note) 11.21 Czokor 1880 1780, 18:413 formula: water 100, cochineal 2, potassium alum 2, phenol 0.25 preparation: Suspend the cochineal and alum in 200 water. Boil till volume is reduced to 100. Leave 2 days, filter, and add phenol to filtrate. 11.21 Fyg 1928 see DS 11.28 Fyg 1928 11.21 Gower 1939 2054b, 14:31 formula: water 100, alum 5, residue from preparation of DS 11.23 Schneider 1880 {q.v.) 0.5 note: This was originally recommended for trematodes, but is an excellent general- purpose, wholemount stain. 11.21 Grenacher 1879 1780, 16:465 , formula: water 100, carmine 1, ammonium alum 10 preparation: Add the carmine to the boiling alum solution. Cool. Filter. method: [water] —> suitable dilution of stain, till sufficiently stained -^ balsam, via usual reagents note: The larger the animal, the greater the dilution. For protozoans use full strength overnight; for a large leech use 1 :5000 for 2 months. This stain is frequently confused with 11.22 Grenacher 1879. Mahrenthal's carmine (Rawitz 1895, 60) is 4 parts of this solution with 1 of 95% ale. 11.21 Guyer 1930 Guyer 1930, 9 formula: water 100, potassium alum 6, cochineal 6 preparation: Boil 30 minutes. Dilute to 150. Boil till reduced to 100. Cool. Filter. 11.21 Kirkpatrick test. 1938 Carleton and Leach cit. Cappell Carleton and Leach 1938, 105 formula: water 100, acetic acid 2.5, ammonium alum 2.5, cochineal 2.5, salicylic acid 0.1 preparation: Soak cochineal 20 minutes in 10 water with 2.5 acetic acid. Add 40 water and boil 1 hour. Dissolve alum in 50 water, bring to boil, and add to boiling cochineal. Boil 1 hour. Cool, make up to 100, filter, add salicylic acid. 11.21 Mahrenthal see DS 11.21 Grenacher 1879 (note) 11.21 Mayer 1892 C ar malum— auct. 14246,10:482 formula: water 100, potassium alum 5, carminic acid 0.5 use: Full strength, usually for 1 to 10 days, on embryos prior to embedding and section- ing. DS 11.21 DS 11.22 DYE STAINS OF GENERAL APPLICATION 301 11.21 Mayer 1897 Carmnlum—aucl. 23032,14:29 formula: water 100, potassium alum 5, carmino 2 preparation: Boil 1 hour. Cool. Filter. note: a detailed description of the use of this stain in the preparation of a wholemount is given under 11.20 above. 11.21 Partsch 1877 1780, 14:180 formula : water 100, potassium alum 6, cochineal 3, salicylic acid 0 25 preparation: Boil alum and cochineal 2 hours. Cool. Filter. Add salicylic acid. 11.21 Rabl 1894 23G32, 11:168 formula: water 100, potassium ahim 4, cochineal 1 preparation: Suspend cochineal and alum in 130 water. Boil till reduced to 100. Cool. Filter. note: The formula of Bohm and Oppel 1907, 98 is in every way identical. 11.21 Rawitz 1895 Rawitz 1895, Gl formula: water 100, potassium alum 5, carminic acid 0.5 11.21 Rawitz 1899 766,15:438 formula: water 50, glycerol 50, ammonium alum 6.5, carminic acid 0.7 preparation: Dissolve the dye with heat in the alum solution. Filter. Add glycerol. 11.22 ALCOHOLIC CAUMINEvS 11.22 Grenacher 1879 Alcoholic borax carmine — compl. script. 1780, 16:448 preparation of dry stock: Boil 250 water, 8 carmine, 10 sodium borate for 30 minutes. Cool overnight. Filter. Evaporate filtrate to dryness. working formula from dry stock: 30%, 50%, or 70% ale. 100, dry stock to sat. preparation working formula direct: Boil 50 water, 1.5 carmine, 2 sodium borate for 30 minutes. Cool. Add 50 70% ale. Leave 2-3 days. Filter. reagents required: A. any selected working formula; B. 0.1% hydrochloric acid in 70% ale. method: [alcohol of lower concentration than selected working formula]-^ A, not less than 12 hrs. — > B, till pink and translucent — » balsam, via usual reagents note: Though it is customary to prepare the working solution direct, much better results may be obtained from dry stock in variously concentrated alcohol. The solubility de- creases very rapidly with increasing alcoholic content so that power of the stain may be accurately controlled. A detailed description of the use of this stain in the prepara- tion of a wholemount is given under 11.20 above. 11.22 von Mahrenthal see DS 11.21 Grenacher 1879 (note) 11.22 Mayer 1881 Alcoholic cochineal — compl. script. 14246,2:14 formula: water 30, 95% ale. 70, cochineal 10 preparation: Digest 1 week. Filter. method: [marine invertebrate larvae, formaldehyde or alcohol preserved] —> stain, 1-10 mins. —^70% ale, till differentiated — > balsam, via usual reagents note: For the material indicated this stain gives better results than other carmine formulas. 11.22 Mayer 1892a Paracarmine — compl. script. 14246,10:491 reagents required: A. 70% ale. 100, ahaninum chloride 0.5, strontium chloride 4, carminic acid 1; B. 0.1% strontium chloride in 70% ale. method: [small invertebrates] -^ 50% ale. — > .4, till required structures are stained, 5 mins. to 1 wk. — » i?, if differentiation necessary -^ balsam, via cedar oil 11.22 Mayer 1892b 14246, 10:498 formula: water 50, 95% ale. 50, nitric acid 0.3, cochineal 5, calcium chloride 5, alumi- num chloride 0.5 preparation: Grind the dry ingredients to a paste with the acid. Mix solvents with paste, bring to boil, leave 5 days, filter. 302 METHODS AND FORMULAS DS 11.22-DS 11.23 11.22 Schwarz 1933 23632, 50:305 formula: water 50, methanol 50, carmine 2, sodium borate 2 preparation: Boil the carmine and sodium borate I hour in 100 water. Evaporate to 25. Cool. Dilute to 50 and add 50 methanol. Leave 1 day. Filter. 11.22 Seller 1881 SeUer 1881, 62 formula: water 100, 95% ale. 50, sodium borate 1, carmine 0.6 11.22 Spuler test. 1907 Bohm and Oppel Bohm and Oppel 1907, 99 formula: water 100, cochineal 10, 95% ale. q.s. preparation: Boil the cochineal in 100 water till reduced to 50. Add 95% ale. until ppt. appears. Filter. Evaporate to 100. 11.23 aceto-carmines 11.23 Belling 1921 651, 54:573 preparation: To 50 DS 11.23 Schneider 1880 add ADS 12.2 Belling 1921 until ppt. appears. Then add 50 DS 11.23 Schneider 1880. method: [smears for chromosome counts, or fresh cestodes for diagnosis]—* stain, on slide — > examine note: a detailed description of the use of this stain is given under 11.20 above. 11.23 Gower 1939 see DS 11.21 Gower 1939 11.23 Henneguy 1887 Henneguy 1887, 88 STOCK formula: water 100, potassium alum 6, carmine 2, acetic acid 25 preparation of stock: Boil the dye and alum in the water 1 hour. Cool. Add acid; leave 10 days, filter. WORKING solution: a. water 99, stock 1 method: [95% ale] —* A, until stained —> distilled water -^ balsam, via usual reagents note: This is the only aceto-carmine well adopted for general staining of wholemounts. 11.23 Nickiforow test. 1900 Pollack Pollack 1900, 76 formula: water 100, carmine 3, sodium borate 10, ammonia q.s., acetic acid 0.5 preparation: Boil dye and sodium borate 1 minute in 200 water. Add ammonia drop by drop till all carmine is dissolved. Evaporate to 100. Add acid. 11.23 Schneider 1880 23833,3:254 formula: water 55, acetic acid 45, carmine 5 preparation: Boil 15 minutes under reflux. Cool. Filter. method: as Belling 1921 note: This formula is frequently (cf. Gatenby and Painter 1937, 685) attributed to Belling 1921 (q.v.). The residue on the filter paper may be used in the preparation of DS 11.21 Gower 1939. 11.23 Semmens 1938 see M 11.1 Semmens 1938 11.23 Semichon 1924 19227a, 11:193 REAGENTS REQUIRED: A. water 50, acetic acid 50, carmine 5; B. 70% alcohol preparation of a : Digest ingredients 1 hour at 90°C. Cool. Filter. method: [living material] -^ A, till stained -* B, till differentiated — > balsam, via usual reagents 11.23 Zacharias 1894 23833, 11 :62 REAGENTS REQUIRED: A. water 70, acetic acid 30, carmine 0.5; B. 1% acetic acid; C. 1% ferric ammonium citrate PREPARATION OF A : As Semichou 1924. method: [whole objects to be sectioned, or sections] — > water -^ A, 2-5 hrs. — > B, rinse -^ C, 2-3 hrs. -* distilled water, thorough wash —> balsam, or paraflfin, via usual reagents 11.23 Zirkle 1937 see M 12.1 Zirkle 1937 and M 13.1 Zirkle 1937 DS 11.2^DS 11.24 DYE STAINS OF GENERAL APPLICATION 303 11.23 Zirkle 1940 see M 11.1, M 12.1, M.13.1, M 22.1 and M.31.1 Zirkle 1940 11.24 PICRO-CARMINES 11.24 Arcangeli test. circ. 1890 Francotte Francotte, 217 formula: water 100, picric acid 1, carmine 0.5 prepar.\tion: Boil 10 minutes. Cool. Filter. 11.24 Bizzozero test. 1889 Friedlander Friedlander 1889, 89 formula: water 100, 95% ale. 20, picric acid 1, carmine 1, ammonia 6 preparation: Dissolve carmine in ammonia. Add 100 water. Dissolve picric in 100 water. Add to carmine and evaporate to 100. Cool; filter. Add ale. to filtrate. 11.24 Francotte circ. 1890 Francotte, 216 formula: water 100, carmine 1, ammonia 5, picric acid 1, chloral hydrate 1 preparation: Dissolve carmine in ammonia. Dissolve picric acid in 50 hot water and add to carmine. Dilute to 100. Add chloral hydrate. 11.24 Friedlander 1889 Friedlander 1889, 88 formula: water 50, carmine 1, ammonia 1, sat. sol. picric acid 100 preparation: Dissolve carmine in ammonia. Add water and picric solution. 11.24 Gage 1880 (est. circ. 1890 Francotte Francotte, 213 formula: water 100, carmine 1, ammonia 50, picric acid 1 prepakation: Dissolve carmine in ammonia. Add picric dissolved in water. Leave 1 day. Filter. Evaporate to dryness. Dissolve residue in 100 water. 11.24 Guyer 1906 test. 1930 ips. Guyer 1930, 239 formula: water 50, sat. aq. sol. picric acid 50, ammonia 5, carmine 1 preparation: Dissolve carmine in ammonia. Add water and picric solution. Leave 2 days. Filter. 11.24 Jensen 1937 23684, 139:333 STOCK solutions: I. water 100, 0.1, carmine 0.5; II. water 100, picric acid 0.5, mag- nesium oxide 2 WORKING solution: stock I 80, stock II 20 11.24 Legal 1884 14555, 8:353 bormula: DS 11.21 Grenacher 1897 90, sat. aq. sol picric acid 10 11.24 Lowenthal 1892 see DS 13.7 Lowenthal 1892 11.24 Lowenthal test. 1900 Pollack Pollack 1900, 79 formula: water 100, sodium hydroxide 0.5, carmine 2, picric acid q.s. preparation: Dissolve carmine and alkali in 50 boiling water. Boil 15 minutes. Dilute to 100 and cool. Add picric acid in excess of saturation. 11.24 Malassez test. 1877 Frey Frey 1877, 96 formula for dry stock: water 100, ammonia 2, carmine 0.5, picric acid 2.5 preparation for dry stock: Dissolve carmine in ammonia and water. Add picric. Shake; allow to settle; and decant. Evaporate supernatant to dryness. FORMULA FOR WORKING SOLUTION: water 100, dry stock 2 preparation OF WORKING SOLUTION: Mix. Leave 1 week. Filter. 11.24 Mayer 1897 23632, 14:23 formula: DS 11.28 Mayer 1897 10, 0.6% magnesium picrate 90 11.24 Neuman test. 1928 Schmorl Schmorl 1928, 120 reagents required: A. DS 11.21 Grenacher 1879 100, picric acid 1.25; B. 2% hydro- chloric acid in glycerol; C. anhydrous glycerol method: [sections] — > A, 5-10 mins. — * B, 10 mins. -^ C note: To prepare balsam mounts, substitute a sat. sol. picric acid in abs. ale. for C above and clear in clove oil. 11.24 Oppler test. 1928 Schmorl ^- Schmorl 1928, 341 formula: carmine 0.5, ammonia 0.5, picric acid 0.005, water 100 304 METHODS AND FORMULAS DS 11.24-DS 11.25 11.24 Orth test. 1904 Besson Besson 1904, 255 fokmula: DS 11.28 Orth (1892) 65, sat. aq. sol. picric acid 35 note: This same formula is given by Squire 1892 but without acknowledgment to Orth. 11.24 Ranvier 1875 Ranvier 1875, 100 PKEPARATiON OF DRY STOCK: Dissolve 5 Carmine in 50 ammonia. Add 500 sat. sq. sol. picric acid. Evaporate to 100. Cool 24 hours. Decant supernatant liquid which is evaporated to dryness. WORKING solution: water 100, dry stock 5 PREPARATION ot WORKING SOLUTION: Boil 10 minutes. Cool. Filter. method: [anything, living or dead] -^ stain, till done — > 70% ale. till color clouds cease — > balsam, via usual reagents note: The original formula called for a prolonged period of putrefaction in the course of manufacture. This stain, now practically unknown, should be placed in the hands of every beginner. A live Cyclops may be dropped into the solution and removed, fixed, and stained 10 minutes later; so may any other object. A description of a preparation utilizing this stain is given under DS 12.20 below. 11.24 Rutherford test. 1878 Marsh Marsh 1878, 79 formula: sat. sol. picric acid 100, carmine 1, ammonia 2, water preparation : Dissolve carmine in ammonia and 5 water. Raise picric solution to boiling and add carmine solution. Evaporate to dryness. Dissolve residue in 100. 11.24 Squire 1892a Squire 1892, 34 formula: anmionia 1.5, carmine 0.5, water 2.5, sat. aq. sol. picric acid 100 preparation: Dissolve dye in ammonia. Add picric solution. 11.24 Squire 1892b Squire 1892, 35 formula: water 100, sodium hydroxide 0.05, carmine 0.5, picric acid q.s. preparation: Dissolve the carmine in the boiling alkali sol. Add just enough sat. aq. sol. picric acid to redissolve the ppt. first formed. 11.24 Squire 1892c Squire 1892, 35 formula: DS 11.28 Orth (1892) 25, sat. aq. sol. picric acid 75 note: See also Orth (1904) note. 11.24 Vignal test. 1907 Bohm and Oppel cit. Henneguy Bohm and Oppel 1907, 118 formula: water 100, picric acid 2, carmine 1, ammonium hydroxide 5 PREP.\RATiON of DRY STOCK: Mix all ingredients. Leave 2-3 months in closed bottle. Evaporate at room temperatures to 80. Decant and evaporate supernatant to dryness. WORKING solution: water 100, dry stock 1 11.24 Weigert 1881 22575,84:275 formula: sat. aq. sol. picric acid 100, carmine 1, ammonia 2, acetic acid q.s. preparation: Dissolve carmine in ammonia. Add picric solution and leave 24 hours. Then add just enough acetic acid to produce permanent ppt. Filter. 11.25 IRON carmines 11.25 de Groot 1903 23632,20:21 formula: water 100, hydrochloric acid 0.05, potassium alum 2.5, ferric alum 0.05, car- minic acid 0.5 preparation: Dissolve ferric alum in 10 water. Add dye and dilute to 100. Heat to 60°C. and add potassium alum. Cool. Filter. Add acid to cold filtrate. 11.25 Hansen 1905 23632, 22:85 formula: water 100, sulfuric acid 1, ferric alum 3, cochineal 3 preparation: Boil all ingredients 10 minutes. Cool. Filter. 11.25 Lee 1902 test. 1905 ips. Lee 1905, 170 reagents required: A. ADS 12.1 Benda 1893; B. 0.5% carminic acid in 50% alcohol method: [sections] — > water -^ A, some hrs. — » rinse, 50% ale. -♦ B, 1-2 hrs. — » wash, 50% ale. — > balsam, via usual reagents DS 11.25 DS 11.27 DYE STAINS OF GENERAL APPLICATION 305 11.25 Peter 1904 23632, 21:314 REAGENTS required: A. watcr 100, cochineal G, liydrocliloric acid 0.3; li. 2.5% ferric alum PREPARATION OF a: Boll cochiiieal in 150 water till reduced to 30. Dilute with hot water to 100. Cool. Filter. Add acid. method: [whole objects to be sectioned, or sections] — > A, 48 hrs. at 37°C. — » rinse —<■ B, some hrs. -^ balsam, or paraffin, via usual reagents result: Said by Lee {Lov 1905, 171) to stain yolk granules red on a gray background. 11.25 Spuler 1901 test. 1910 ips. Ehrlich, Krause, et. al. 1910, 1:240 PREPARATION OF STOCK SOLUTION: Boil 20 cochineal 1 hour in 100 water. Filter, saving both residue and filtrate. Boil residue in 100 water 1 hour. Filter, lleject residue. Mix filtrates and reduce to 100. Add 95% ale. till ppt. appears. Filter. Reduce filtrate to 100. Filter. REAGENTS required: A. stock 35, 50% alcohol 65; B. 0.75% ferric alum method: 70% alcohol -^ A, 48 hrs. at 37°C. -^ wash -* B, 24 hrs. 11.25 Wellheim 1898 23632, 15:123 REAGENTS REQUIRED: A. 0.01% ferric chloride in 50% ale; B. 0.5% carminic acid in 50% ale; C. 0.1% hydrochloric acid in 70% ale. method: [sections] -^^ 50% ale. — + A, overnight -^ rinse, 50% ale. -+ B, 1-2 hrs. — > wash, 50% ale. -^ C, till differentiated — > balsam, via usual reagents 11.25 Zacharias 1894 see DS 11.23 Zacharias 1894 11.26 AMMONIA CARMINES 11.26 Beale 1857 test. 1880 Beale Beale 1880, 125 formula: carmine 0.5, ammonia 1.5, water 44, 95% ale. 12, glycerol 44 preparation: Dissolve carmine in ammonia. Add other ingredients. 11.26 Cole 1903 Cross and Cole 1903, 169 formula: water 100, carmine 1, ammonium hydroxide 2, sodium borate 6 preparation: As Beale 1857. 11.26 Frey 1877 Frey 1877, 94 formula: water 40, glycerol 40, 95% ale. 20, carmine 4, ammonia q.s. to dissolve preparation: As Beale 1857. 11.26 Gerlach 1858 tent. 1892 Squire Stpiire 1892, 31 formula: water 100, ammonia 1, carmine 1 note: Squire (loc. at.) and Lee (Lee 1905, 172) recommended that the solution be allowed to grow mold, evaporated to dryness, and then redissolved in distilled water, 11.26 Hoyer test. 1900 Pollack Pollack 1900, 78 preparation of DRY STAIN : Dissolvc 2 carmine in 4 ammonia and IG water. Boil till smell of ammonia not apparent. Cool. Add 80 95% ale. Filter. working solution: water 100, powder from above 0.5 11.26 Meriwether 1935 4349, 14 :64 formula: water 75, ammonia 25, potassium chloride 6.25, potassium carbonate 1.9. carmine 2.5 preparation: Boil the salts and the dye in the water 2 minutes. Cool and add ammonia. 11.26 Smith test. 1903 Cross and Cole Cross and Cole 1903, 171 formula: water 50, glycerol 25, 95% ale. 25, carmine 1, ammonia 1.5, sodium borate 1 preparation: As Beale 1857. 11.26 Squire 1892 Squire 1892, 31 formula: water 40, 95% ale. 20, glycerol 40, carmine 2.5, ammonia 5 preparation: As Beale 1857. 11.27 hydrochloric carmines 11.27 Hollande 1916 Chlorcarmin—compl. script. 6630, 79 :662 reagents required: A. water 90, 95% ale. 10, carmine 7, hydrochloric acid 2.5 water q.s. to bring filtrate to 90, 70% ale. 10; B. 3% ferric alum; C. 1% pyridine 306 METHODS AND FORMULAS DS 11. 27-DS 11.28 PREPARATION OF A'. Criiul the rarmiiip wiili iliR acid. Wasli out iiiortar while srinrling; with 12 doses each of 10 water. Boil washings till reduced to 70. Cool. Filter. Dilute to 90. Add ale. method: [sections] — * water -^ A, 2 hrs. -^ water, rinse —> B, till black, few minutes -^ B, fresh solution, till differentiated, >2~2 hrs. -^ C, thorough wash — > running water, 15 mins. -^ balsam, via usual reagents 11.27 Kingsbury and Johannsen 1927 Ivingsbury and Johannsen 1927, 44 formula: water .30, 95% ale. 70, carmine 2, hydrochloric acid 3 preparation: Boil all ingredients under reflux. Cool. Filter. 11.27 Langeron 1942 Langeron 1942, 517 REAGENTS REQUIRED: A. watcr 2.5, 90% ale. 100, carmine 2.5, hydrochloric acid 2.5; B. 0.5% hydrochloric acid in 80% ale. PREPARATION OF A i Grind carmine, acid, and water to a paste. Leave 1 hour. Transfer to reflux flask with ale. Reflux on water bath till solution complete. method: [whole objects]-^ 70% ale. — > A, till stained-^ B, till differentiated —> bal- sam, via usual reagents 11.27 Mayer 1881 14246,2:1 formula: 95% ale. 90, water 15, carmine 4, hydrochloric acid 1, ammonia q.s. to give pH7 preparation: Heat water, carmine, and acid to 80°C. Add ale. and reflux 10 minutes. Adjust to pH 7 with ammonia. note: In 1883 (14246, 4:521) Mayer directed that the carmine, acid, and water be boiled to complete solution, cooled, and the ale. added before neutralization. 11.27 Mayer 1883 see DS 11.27 Mayer 1881 (note) 11.27 Meyer 1885 2626, 10:363 formula: 90% ale. 50, carmine 1.25, hydrochloric acid 5, chloral hydrate 60 preparation: Reflux the carmine with the acid and ale. for 30 minutes. Cool. Filter. Add chloral hydrate. 11.27 Schwarz 1934 23632, 50 :305 preparation of dry stock: Boil 1 carmine and 4 ammonium alum in 150 water until reduced to 75. Filter. Evaporate filtrate to dryness. WORKING solution: water 60, methanol 40, hydrochloric acid 0.2, dry stock 8 11.28 OTHER CARMINE FORMULAS 11.28 Arcangeli 1885 boric-carmine 16977, 4:233 formula: water 100, boric acid 4, carmine 0.5 preparation: BoU 10 minutes. Cool. Filter. 11.28 Best 1906 potash-carmine see DS 22.5 Best 1906 11.28 Cuccati 1886 soda-carmine 23632, 3 :50 formula: water 90, 95% ale. 10, acetic acid 0.5, sodium carbonate 6.5, carmine 1.7, chloral hydrate 2 preparation: Boil the carmine with the carbonate in 35 water. Cool. Add the ale. to cooled solution. Leave overnight; filter, and bring filtrate to 100. Add the acid and chloral hydrate. 11.28 Cuccati test. 1889 Friedlander soda-carmine Friedlander, 1889, 90 formula: water 100, carmine 4, sodium carbonate 13, abs. ale. 7, acetic acid 1.5, chloral hydrate 2.5 preparation of dry stock: As Cuccati 1886, save that the final solution is evaporated to dryness. preparation of working solution: 80% ale. 100, powder q.s. 11.28 Francotte ciir. 1890 boric-carmine Francotte, 209 formula: water 25, 95% ale. 75, boric acid 5, carmine 0.4 preparation: Boil under reflux 15 minutes. DS 11.28 DYE STAINS OF GENERAL APPLICATION 307 11.28 Frey 1877 horax-carmine Frey 1877, 95 formula: water ;i5, sodium borate 3.5, carmine 0.6, 05% ale. 00 preparation: Boil dye with water and borax. Cool. Filter. Add ale. to filtrate; leave 24 hours. Filter. 11.28 Fyg 1928a chrome'Carmine 23032, 45 :2 12 formula: water 100, chrome alum 6, carmine 1 preparation: Add carmine to boiling alum solution. Boil 15 minutes. Cool. Filter. note: Copper alum may be substituted for chrome alum. Copper alum (Merck Index 1940, 165) is prepared by fusing together 34% potassium alum, 32% cupric sulfate, 32% potassium nitrate, and 2% camphor. 11.28 Fyg 1928b soda-carmine 23632, 45 :242 formula: water 100, sodium bicarbonate 5, carmine 0.5 preparation: Boil 5 minutes. Cool. Filter. 11.28 Haug test. 1900 Pollack ammonia-carmine Pollack 1900, 80 formula: carmine 1, ammonium chlorate 2, water 100, ammonia 0.25, lithium car- bonate 0.5 preparation: Boil carmine in chlorate solution 15 minutes. Cool. Add other ingredients. Filter. 11.28 Kahlden and Laurent 1896 lithium-carmine Kahlden and Laurent 1896, 56 formula: sat. aq. sol. lithium carbonate 100, carmine 3 preparation: Boil 15 minutes. Cool. Filter. 11.28 Linder see P 12.2 Amann 1896 (note) 11.28 Mayer 1897 magnesia-carmine 23632, 14:23 formula: magnesium oxide 0.2, carmine 2, water 100 preparation: Boil 5 minutes. Cool. Filter. note: Magnesium oxide very readily turns to the carbonate which cannot be used for this preparation. Hence the insistence on fresh magnesia {magnesia iisla — "burnt magnesia" in pharmacopeial Latin) in the original formula. 11.28 Mayer 1902 aluminum-carmine 14246,10:490 formula: water 100 aluminum chloride 1.5, carminic acid 0.5 11.28 Orth test. 1892 Squire lithium-carmine Squire 1892, 33 formula: water 100, lithium carbonate 1.5, carmine 2.5 preparation: Boil. Cool. Filter. 11.28 Rawitz 1899 aluminum-carmine 14246, 10 :489 formula: water 50, glycerol 50, aluminum nitrate 2, cochineal 2 preparation: Boil the cochineal with the water and nitrate 1 hour. Cool. Filter. Add glycerol. 11.28 Rukhadze and Blajin 1929 see DS 23.33 Rukhadze and Blajin 1929 11.28 Schmaus test. 1896 Kahlden and Laurent uranium-carmine Kahlden and Laurent 1896, 158 formula: water 100, sodium carminate 1, uranium nitrate 0.5 preparation: Boil 5 minutes. Cool. Filter. 11.28 Thiersch test. 1871 Robin ammonia-carmine Robin 1871, 318 formula: water 65, ammonium acetate 2, carmine 3, oxalic acid 3, 95% ale. 35 preparation: Dissolve acetate in 15 hot water. Add carmine, heat to solution. Add oxalic acid dissolved in 50 water slowly and with constant stirring. Cool. Filter. Add ale. to filtrate. 11.28 Thiersch test. 1877 Frey ammonia-carmine Frey 1877, 94 formula: water 40, 95% ale. 60, carmine 1.25, ammonia 1.25, oxalic acid 2 PREPARATION : Dissolve carmine in ammonia with 5 water. Filter. Add acid dissolved in 40 water. Add ale; leave 1 day; filter. 308 METHODS AND FORMULAS DS 11.3-DS 11.4 11.3 Bkazilin and Other Natural Stains 11.3 Bensley 1916 brazilin 590,29:37 foemula: water 10, brazilin 0.05, water 90, phosphotungstic acid 1 preparation : Add the acid dissolved in 90 water to the dye dissolved in 10. method: water -^ stain, 12 to 3 hrs. -^ water, wash -^ balsam, via usual reagents 11.3 Guinard 1890 alkand 11071,6:447 preparation: Infuse 20 alkanet in 60 abs. ale. I'ilter. Evaporate to dryness and dissolve residue in 10 acetic acid. Add 100 50% ale. 11.3 Hickson 1901 brazilin 17510, 44:470 REAGENTS REQUIRED: A. 1% ferric alum in 70% ale; B. 10% ale. 100, brazilin 0.5 method: 70% ale. -» A, 3 hrs. -^ 70% ale, quick rinses B, overnight -> 70% ale. thorough wash — > balsam, via usual reagents 11.3 O'Leary see DS 21.213 O'Leary 11.4 Synthetic Nuclear Stains The term synthetic nuclear stains is here used in contrast to those stains, such as hematoxylin and carmine, which are ex- tracted from natural sources. These syn- thetic dyes are widely called aniline dyes without very much justification, since by no means are all of them directly derived from aniline. The now obsolete term coal- tar dyes was far more accurate. Almost any stain can be used to color nuclei differ- entially by the adjustment either of the pH or tlie chemical composition of the solution in which it is employed. Those here fisted are those most widelj^ employed for the purpose either alone or in combina- tions in complex contrast formulas. The most interesting of these reagents, grouped in the first class, are the oxazine dyes, which are turned into their metallic lakes in the course of the preparation of the staining solutions. These were intro- duced by Becher 1921, in a book which never received a very wide circulation. In their staining reactions they strongly re- semble hematoxylin, giving eitlier black or blue nuclei, but they do so without the necessity for differentiating, and are far less liable either to fading or to altei'ation in color. They have been rediscovered at various intervals since 1921, and it is a matter of some astonishment to those who have employed them that they have not received wider acceptance as a substi- tute for hematoxylin. The author's choice among the formulas is the anonymous one, given first. Probably the one most widely employed is the formula of Proe- scher and Arkush, 1928 (20540b, 3:36). The publication of this paper in the United States started a brief vogue for these materials. The original publication of Becher quoted not only the utilization of these oxazine dyes for nuclear staining, but also for the staining of the central nervous system and for some general- purpose polychrome stains. These will be found in their appropriate divisions later. Safranin, in the Engfish-speaking coun- tries, is more widely employed in botanical tlian in zoological technique. In Europe, however, particularly in France, it re- mained the standard nuclear stain for many years, and has not yet been regu- larly supplanted by hematoxyfin. Thus a standard French technique for a histo- logical preparation will be safranin-light green, in which the corresponding British or American usage calls for hematoxyfin- eosin. This safranin-light green combina- tion has found occasional acceptance in the literature as Benda's stain. There is little to choose between any of the for- mulas, it being a matter of convention that the techniques of Chamberlain 1915, and Johansen 1940, are customarily em- ployed for plant material, while Babes, 1887 and Bohm and Oppel 1907 are com- monly employed for animal materials. Magenta is less widely employed as a single nuclear stain than it is in combina- tion with plasma stains in complex tech- niques. The formula of Ziehl 1882, though customarily confined to bacterial staining, is here included for the reason that it is just as good a nuclear stain as it is a stain DS 11.4-DS 11.40 DYE STAINS OF GENERAL APPLICATION 309 for microorganisms. Indeed, it is very nearly as specific as is the Fuelgen reaction without any of tlie difficulties attached to the latter. The Fuelgen reaction itself, here cited from the paper of Fuelgen and Rossenbeck 1924, is not now as widely em- ployed as was formerly the case. It is actually one of the oldest staining methods known, having been introduced by Schiff in ISGG (825, 93:140), as a reagent for al- dehydes. Its nuclear reactions appear to depend upon the selective staining of thy- monucleic acid, but the stain itself is so difficult and so temperamental that it is doul)tful that it has occasioned any real advance in microtomy. The next class of synthetic nuclear stains, the thiazins, comprise methylene blue and its oxidation homologs: Lauth's violet and the azurs. These are used mostly in admixtures of unknown compo- sitions, commonly referred to as poly- chrome methylene blues. These thiazins are in general excellent and specific nuclear stains and their use should not be confined to blood, as is customarily the case. They became unpopular at about the opening of the present century because of the difficulty at that time of securing mount- ing media in which they could adequately be preserved. They are hable to rapid fad- ing when mounted in balsam, and also tend to be extracted by alcohol in the course of the dehydration period. If amyl alcohol is used for dehydration, and one of the neutral mounting media in place of balsam, there is no reason why the thiazins should not again become popular. The formulas given are only those in which methylene blue is employed alone. Many more formulas will be found later in the present chapter under the headings DS 13.1 and DS 13.2, with their subdivisions, which deal with the common combinations of thiazins and various eosins. The next class, the crystal violets and gentian violets, are today almost confined to botanical microtechniques. This is un- fortunate since they are very specific for nuclei and also do not stain albuminous yolk material as does hematoxylin. It is strongly recommended that those who are handling heavily yolked embryos should try the formula of Johansen 1932 in preference to the more conventional techniques. 11.40 TYPICAL EXAMPLES Preparation of a strewn slide of pollen grains using the safranin stain of Johansen 1940 It is not intended here to give an exam- ple of the technique by which safranin is used for general nuclear staining, but only to give a method which may be employed for the demonstration of the nuclei in pol- len grains, a rather difficult subject. Pollen should be collected into a tube containing 1% acetic acid in absolute al- cohol. It is simple to do this by dipping the anther under the surface of the fluid and then resealing the tube. In this manner a vast number of pollens can be collected in a surprisingly short time. Pollen grains ma}' remain in this fluid for a day of two, but it is undesirable to leave them any longer than this before allowing them to settle. The supernatant liquid should then be poured off and replaced with fresh ab- solute alcohol. This : change should be repeated several times or until the fluid poured off no longer smells of acetic acid. Wlieu the acetic acid has been washed out, the absolute alcohol is replaced with a mixture of equal parts of absolute alcohol and ether, and the pollen grains are left in this mixture overnight. A solution should then be prepared from any nitrocellulose used for embedding, such as celloidin (see Chapter 13). About half of the alcohol-ether mixture should then be poured from the top of the tube containing the pollen and replaced with an equal quantity of the weak celloidin solu- tion. After this has been allowed to pene- trate the pollen grains for a few hours — (this will be definitelj^ established by the grains falhng to the bottom) — about three-quarters of the fluid is poured off and the tube again refilled with a fresh 0.5% solution of celloidin. When this has in its turn impregnated the pollen grains, they may be left in the tube for an indefi- nite period. When it is desired to make mounts, it is necessary to clean as many co\-erslips as the number of mounts required, taking 310 METHODS AND FORMULAS DS 11.40 rather more than the usual amount of trouble. Each tube is now agitated until the pollen grains (but not air bubbles) are dispersed throughout the celloidin, one drop of the dispersion being then taken and allowed to run over the surface of the cover. The most critical stage of the pro- ceedings is the next one, in which the cel- loidin is congealed without being per- mitted to evaporate to dryness. If it is allowed to evaporate to dryness, it will almost invariably become detached, either by curling in the dry state or in one of the solutions which is subsequently applied to it. The best method of congealing the cel- loidin is with the aid of chloroform vapor, a high concentration of which is easily maintained at the bottom of a coplin jar by pouring a few cubic centimeters into the jar, placing the hand over the top, shaking the jar once or twice, and then setting it on the bench with a loose cover in place. Chloroform vapor is so heavy that it will remain in the jar for a long period. Each drop of the suspension of pollen should be spread uniformly over the coverslip and then waved once or twice in the air until it appears about to become dry. Each coverslip is then, while held with forceps, lowered into the chloroform vapor at the bottom of the cophn jar and held there for a few moments until it ac- quires a slightly opaque appearance. The coverslip is then dropped into a rack in a jar of 70% alcohol, where it may remain until sufficient have been accumulated to permit staining. Two reagents are required for the stain of Johansen 1940, the first being the rather specially prepared solution of safra- nin given under the technique of Johan- sen 1930 (11.42 Johansen 1930) below, and the other, developed by Masson (Chapter 22, ADS 22.1 Masson 1942), a 65:35 mixture of a saturated solution of picric acid in 95% alcohol, and 95% alco- hol. The coverslips carrying the dispersion of pollen are removed from 70% alcohol when is ready to stain them, transferred to distilled water until the diffusion cur- rents have died down, and then placed in a jar filled with Johansen's safranin stain, where they may remain not less than 24 hours or until next required. Each individual coverslip is now taken sep- arately and rinsed in the differentiating solution of Masson until examination un- der the low power of a microscope shows a clear differentiation of red nuclei against the yellow background of the pollen cell. Differentiation may be stopped immedi- ately by transferring the covershp from the picric acid to water. Several courses are now open to the mounter. If it is desired only to show the nuclei, the slide may be dehydrated in the regular manner as far as 90% alcohol, cleared in terpineol to avoid dissolving the celloidin holding the pollen grains in place, and then mounted in balsam. If the gen- eral outline of the pollen cell is required as well as the nuclei, the material may be treated in the same manner up to the moment of its clearing and then counter- stained either with a solution of light green or fast green in clove oil. If the latter course is adopted, the excess hght green in clove oil should be washed off with clove oil and the oil itself subsequently washed out in xylene as a preliminary to mounting in balsam. The very common practice of passing directly from a solution of hght green in clove oil to balsam itself is most unsatisfactory and results always in the gradual removal of the light green from the tissues as the clove oil diffuses through the balsam. The intermediate washing in clove oil is also itself insufficient^ since it will result in the removal of additional dye, and necessitate the third step recom- mended, i.e. removal of clove oil with xylene. Demonstration of chromosomes of the grasshopper in a smear preparation of the testes using magenta by the method of Henneguy 1891 The grasshopper provides unusually fa- vorable material for the demonstration of chromosomes, for the reason that these are compact and small, usually with the sex chromosomes well differentiated. Any species of grasshopper may be employed, but the specimens should be collected during the early months of tlie summer, preference being given to young specimens (Baker 1945, 182) in which the wings are not yet fully grown. DS 11.40 DYE STAINS OF GENERAL APPLICATION 311 The most essential precaution to be ob- served in the dissection of testes from small insects for cytological investigations is that the insect should be dissected under the surface of physiological sahne. For this purpose the most convenient ves- sel is a deep dish or fingerbowl, to the bottom of which is fitted a circle of cork or linoleum weighted on the underside with lead sheet to which it is attached by rivets. It is undesirable in cytological in- vestigations to kill an animal by an anes- thetic. In the case of the grasshopper, the simplest method is to remove the head with a pair of sharp scissors before pinning the body down in place, after the removal of the wings and legs from one side, with the aid of four pins placed at the anterior and posterior margins of the body. Sufh- cient physiological saline is then added to cover the specimen to a depth of about a quarter of an inch, and the dissection is then conducted under a low^-power binocu- lar microscope. Before commencing the dissection it is desirable to have available the necessary fixative, which in the pres- ent instance should be the osmic-chromic- acetic of Flemming 1882 (Chapter 18, F 1600.0010) or some other fixative of the same general composition. It is also neces- sary that the slides to be employed be rigorously cleaned and that the fixative be placed in a petri dish of a diameter suffi- cient to permit the slide to be laid flat. Smears of the type which are going to be prepared cannot satisfactoril}' be fixed in such a manner that they will adhere to the slide, if the whole of the latter is immersed in the fixative. It is therefore necessary to secure two short glass rods, the ends of which are bent at right angles to prevent them from roUing, and to place them in the bottom of the petri dish into which the fixative is poured until it just reaches the upper surface of the rods. The depth of the fixative should be tested before any dissec- tion is undertaken by laying a clean slide across the two glass rocls. The whole of the lower surface of the slide should be in contact with the fixative, which should not at any point cover the ui)per surface. Three or four shdes are now laid at hand and the dissection commenced by the re- moval of the cliitinous exoskeleton from the whole side of the abdomen. It is very difficult to find the testes unless one is accjuaintcd witli the species of grasshopper under dissection, for they may either be fused into a single central mass or may be composed of a series of tubules joined to- gether at the base and lying across the upper part of the intestine at right angles to the direction of the vas deferens. It is probably easiest first to identify the in- testine, then to identify the heart, and then to look between the intestine and heart at about the central portion of the body for a series of small, curly objects attached posteriorly to a tube. These curly objects (or object, according to the spe- cies) wall be the testes. They should be removed by cutting away the adherent connective tissues and trachea, and should then be thoroughly washed in the physio- logical sahne which remains in the dish. A piece is now cut from one of the testes of about one-quarter-millimeter side and is removed with a pair of fine forceps to the surface of one of the specially cleaned slides. Excess normal saline should be re- moved from it with chemically clean blot- ting paper, and a second slide crushed down on it with a twisting movement in such a way that the smear is distributed over an area approximately equivalent to a IS-millimeter circle. The two slides are then rapidh^ pulled apart and each is laid face down on the glass rods in the fixative. The petri dish should be covered with the customary lid which one should remove only when adding slides to the dish to avoid inhaling the irritating vapors of the osmic acid. Usually, two or three shdes can be placed in a dish at one time. The smears should not remain in contact with the fixative for more than about 30 min- utes before being carefully removed and placed in distilled water. They may re- main in distilled water for at least several days but must, in any case, be thoroughly washed in several changes with not less than two or three hours in each change. When all the slides have been accumulated in distilled water, and are known to be free of both chromic and osmic acids, one may then proceed to the staining technique. In the technique originally described by Henneguy (D8 11.43 Henneguy 1891) al- most any of the synthetic nuclear stains listed in this section were recommended. 312 METHODS AND FORMULAS DS 11.40 But the results obtained on insect material with magenta are so much better than those obtainable with any other stain that it has in the present work been confined to this dye. The only two solutions re- quired are a 1 % solution of potassium per- manganate, which must be freshly pre- pared before each use, and a 1 % solution of magenta in distilled Avater. Each of these can most conveniently be handled in a standard coplin jar. The slides bearing the smears are re- moved from the distilled water and placed in 1 % potassium permanganate for about five minutes. A word of warning is neces- sary at this point. The water from wliich the slides are passed to the permanganate must be distilled, for the least trace of a reducing agent in it will cause the potas- sium permanganate to be precipitated on the surface of the smear, from wliich it cannot subsequently be removed without great trouble. If, however, the distilled water is pure and the potassium per- manganate is freshly made, the sections as they are removed from the potassium permanganate will be of a medium purple color with only a trace of brown. They must be passed very rapidly into another jar of distilled water, without giving them an opportunity to oxidize, or they will be ruined. After the briefest rinse in this sec- ond dish of distilled water, the sections are dropped directly into the 1 % magenta. As the time in which they should remain in this is variable, it is desirable to take a single smear from the batch and run this through ahead of the remainder in order to estabhsh the timing before running the remainder through in a single operation. In general, about five minutes should be sufficient, but the first slide should be re- moved from the magenta and rinsed briefly in water after about two minutes and examined with the naked eye against a white background. It should be a deep purple color: between the light purple (which it is hkely to show after three min- utes immersion) and a dense black purple (which will be almost impossible subse- quently to differentiate). If it is already sufficiently stained at three minutes, an- other sfide should be tested at one and two minutes; if it is not sufficiently stained after three minutes, it should be returned and examined at one-minute intervals un- til it shows the desired coloration. It may then be transferred to a jar of distilled water and allowed to remain there until the rest of the batch have been put through, on the timing already estab- lished, and placed in the same jar. Each sfide will have to be differentiated separately, because alcohol removes the stain rapidly while clove oil removes it slowly. Each section is therefore placed in 95% alcohol when color clouds will im- mediately be seen to come from it. After a few minutes of rinsing in the alcohol, the slide is then removed to the stage of the microscope and examined under an eight- millimeter objective, if such is available, or under a 16-milliraeter objective with a 20-X eyepiece if the eight-millimeter one cannot be used, to see if the nuclei are yet clearly differentiated from the cytoplasmic background. If the slides are left to differ- entiate in alcohol until the nuclei alone remain colored, it will be impossible to stop the process in time. They should therefore be carefully watched to deter- mine the time at which the nuclei can be clearly distinguished against a lightly stained background. Each slide is then placed in clove oil in which it may be left to differentiate until the background, which now may be examined under a high-power objective, is seen to be entirely free from stain, leaving the chromosomes briUiantly colored. It must be emphasized that the differentiation in clove oil is very slow, and if insufficient differentiation in alcohol be given, it may be necessary to wait some weeks until the clove oil has completed the job. The process sounds complicated to describe, but is, as a matter of fact, very easy to learn and yields re- sults with great certainty. As soon as the differentiation in clove oil has proceeded to the desired degree, the slides are washed in xylene to re- move the whole of the clove oil and then mounted in balsam in the ordinary man- ner. These preparations are remarkably permanent, and the stain is, if anything, more specific to chromosomes than is the iron hematoxylin which is more usually employed. DS 11.41 DYE STAINS OF GENERAL APPLICATION 313 11.41 SOLUBLE METALLIC LAKES OF THE OXAZINES 11.41 Anonymous 1936 Catalogue of Vector Mfg. Co., London, n.d. (received 1936) FORMUL.\: water 100, ferric alum 2.5, coelestin blue B 0.5, glycerol 14, sulfuric acid 2 preparation: Boil the dye 5 minutes in alum solution. Cool. Filter. Add other ingredients. method: [sections]-^ water—* stain, 5 mins. to 1 hr. -^ water, wash -^ [counterstain] — > balsam, via usual reagents note: a description of the use of this stain, before a complex contrast, is given under DS 12.20 below. This is probably derived from DS 11.41 Lendrum 1935 {q.v.). 11.41 Becher 1921a , Becher 1921, 46 formula: water 100, ferric alum 5, napthopurpurin 0.5 preparation: Boil 5 minutes. Cool. Filter. method: [sections] — ♦ water—* stain, 2 hrs. -^ water, wash — > [counterstain] — > balsam, via usual reagents result: nuclei black 11.41 Becher 1921b Becher 1921, 72 formula: water 100, chrome alum 5, gallocyanin 0.5 preparation: as Becher 1921a. method: as Becher 1921a save that 24 hrs. staining is recommended result: nuclei deep blue note: Buzaglo 1934 (4285a, 11:40) diminishes the gallocyanin to 0.1 and adds 1% formaldehyde to the filtrate. 11.41 Becher 1921c Becher 1921, 40 formula: water 100, aluminum chloride 5, napthazarin 0.5 preparation: As Becher 1921a. method: as Becher 1921a save that 24 hrs. staining is recommended result: nuclei, deep blue violet notes: Becher {loc. cit.) recommends also napthopurpurin (dark red nuclei), purpurin (scarlet nuclei), and galloflavin (yellow nuclei) in solutions of aluminum chloride. 11.41 Buzaglo 1934 see DS 11.41 Becher 1921b (note) 11.41 Cole 1947 20540b, 22:103 formula: water 100, chrome alum 5, gallocyanin 1.5 preparation: Boil the dye 5 minutes in the alum solution. 11.41 Einarson 1932 and 1935 see DS 22.3 Einarson 1932 and 1935 11.41 Lendrum 1935 11431,40:415 formula: water 84, glycerol 14, sulfuric acid 2, ferric alum 4.2, celestin blue 0.42 preparation: Boil the dye in the alum 5 minutes. Cool. Filter. Add glycerol and acid. 11.41 Lendrum and McFarlane 1940 sec DS 11.41 Proescher and Arkush 1928 (note) 11.41 Petersen 1926 23632, 43:355 formula: water 100, aluminum sulfate 10, gallocyanin 0.05 preparation: Boil 10 minutes. Cool. Filter. Make up to 100. 11.41 Proescher and Arkush 1928 20540b, 3:36 formula: water 100, ferric alum 5, coelestin blue B 0.5 preparation: As Becher 1921a. method: [sections]—* water —^ stain, till nuclei deep blue black, 3 mins. to 2 hrs. — » water, wash — * [counterstain] — * balsam, via usual reagents note: Proescher and Arkush {loc. cit.) also recommended gallocyanin blue and gallo- cyanin in ferric alum .sohition; but for the latter see DS 13.5 Becher 1921. These solu- tions are le.ss stable than coelestin blue B solutions. For coelestin blue B as a poly- chrome stain see DS 21.16 Becher 1921. Lendrum and McFarlane 1940 (11431, 50:381) add 14 glycerol to this solution. 314 METHODS AND FORMULAS DS 11.42 11.42 SAFEANIN 11.42 Babes 1887 23632, 4:470 formula: water 100, aniline 2, safranin 7 preparation: Heat to 60°C. for 1 hour stirring frequently. Cool. Filter. method: [sections of osmic fixed material] — > water -^ stain, till nuclei bright red, 1 hr. to 10 days —> water, wash —^ balsam, via usual reagents note: This is what is usually meant by Babes Safranin even though Babes had pre- viously (1883; 1780, 21:356) recommended both alcoholic and aqueous solutions. A slight modification of this is given by Langeron 1934, (100) as "Babes-Langeron- Dubosq." This method with a light green counterstain is sometimes called Benda's stain. See also DS 12.15 Land (1915). 11.42 Benda see DS 11.42 Babes (note) 11.42 Bohm and Oppel 1907a Bohm and Oppel 1907, 209 formula: water 72, ammonium carbonate 0.25, 40% formaldehyde 8, sat. ale. sol. safranin preparation: Add the dye solution mixed with the formaldehyde to the carbonate solution. method: [sections] —» water -^ stain, 24 hrs. — >95% ale, quick rinse —> water —* [counterstain] — > balsam, via usual reagents 11.42 Bohm and Oppel 1907b Bohm and Oppel 1907, 113 formula: water 85, 40% formaldehyde 5, 95% ale. 10, phenosafranin 1 11.42 Chamberlain 1916 Chamberlain 1915, 51 formula: water 100, 95% ale. 50, water soluble safranin 0.5, alcohol soluble safranin 0.5 preparation: Dissolve each dye in the appropriate solvent and mix the solutions. note: For a rationalization of this procedure see Conn 1946, 109. 11.42 "Dubosq" see DS 11.42 Babes (note) 11.42 Johansen 1940 Johansen 1940, 62 reagents required: A. 95% ale. 25, water 25, methyl cellosolve 50, safranin 0.1, sodium acetate 1, 40% formaldehyde 2; B. ADS 22.1 Masson (1942) preparation op A: Dissolve the dye in the cellosolve. Add first ale, then water. Then add remaining ingredients. method: [sections] — * water -^ A, 24 to 48 hrs. -^ B, till differentiated -^ [counterstain] — > balsam, via usual reagents note: A description of the use of this stain is given under 11.40 above. 11.42 Lowit 1891 see ADS 22.1 Lowit 1891 note: "Lowit's method" means only the use of his differentiating solution for any safranin-stained material. 11.42 Pfitzner 1881 14555,7:289 formula: water 60, 95% ale. 40, safranin 0.3 11.42 Rawitz 1895 Rawitz 1895, 76 reagents rp:quired: A. 20% tannin; B. 2% potassium antimony tartrate; C. sat. sol. safranin; D. 2.5% tannin method: [sections of chrome-fixed or mordanted material] -^ water -^ A, 24 hrs. -^ B, 24 hrs. — * rinse, D, 24 hrs. — > water, till no more color comes away — >• D, is further differentiation required — > balsam, via usual reagents 11.42 Roskin 1946 Roskin 1946, 152 STOCK solution: water 50, 95% ale. 50, safranin 3.3 preparation: Dissolve the dye in ale, mix with water. working solution: stock 20, 50% ale. 80 11.42 Semichon 1920 test. 1934 Langeron Langeron 1934, 500 formula: wat(M- 50, 90% ale. 50, safniniii 0.5, 40% formaldehyde 1 preparation: Dissolve dye in ale. then add other ingredients. DS 11. 42-DS 11.431 DYE STAINS OF GENERAL APPLICATION 315 method: [sections] -^^ 70% ale. —> stain, 30 niins. to 2 days —» absolute alcohol, till differentiated — > balsam, via xylene 11.42 Zwaademaker 1887 23632,4:212 KEAGENTS REQUIRED: A. 95% alcohol 50, safranin 1,5, sat. aq. sol. aniline 50; B. absolute ale. PREPARATION OP A: Dissolve dye in ale., then add aniline. method: [sections of F 1600.0010 fixed material] -^ 95% ale. -^ A, overnight-^ B, till differentiated -^ balsam, via xylene 11.43 MAGENTA 11431 Magenta as Dye 11.431 Albrecht test. 1943 Cowdry Cowdry 1943, 17 formula: water 100, 95% ale. 20, phenol 8, magenta 4 preparation: Dissolve the dye in phenol and ale, then add water. 11.431 Augusta 1932 see DS 11.431 Ziehl 1882 (note) 11.431 Biot 1901 see DS 11.431 Gallego 1919 (note) 11.431 Davalos test. 1894 Kahlden and Laurent Kahlden and Laurent 1894, 89 formula: water 100, abs. ale. 1, phenol 5, magenta 10 preparation: Grind the dye with the ale, wash out mortar with 10 successive portions of 5% phenol. 11.431 Dupres 1935 11425,46:77 STOCK formula: water 80, 95% ale. 20, magenta 0.75 preparation OF stock: Digest 48 hours at 40°C. Cool. Filter. reagents required: ^4. sat. aq. sol. aniline 100, stock 5, acetic acid 1.5; B. ADS 22.1 Dupres 1936 method: [sections] -^ water —> A, 1-10 mins. — > B, till differentiated, 5-10 mins. — » counterstain -^ balsam, via usual reagents 11.431 Gallego 1919 21344, 17:95 REAGENTS REQUIRED: A. DS 11.43 Ziehl 2, water 98; B. 0.5% formaldehyde method: [sections of formaldehyde material]-^ A, 5 mins. — » wash -^ B, 5 mins. -^ wash -^ balsam, via usual reagents results: nuclei, dark violet, cytoplasmic structures, polychrome NOTE : This method of converting magenta to a blue-black stain was originally proposed for bacteriological purposes by Biot 1910 (6593, Congres de Lyon, 234). 11.431 Goodpasture and Burnett 1919 14975, 13:177 formula: water 80, 95% ale. 20, phenol 1, aniline 1, magenta 0.5 preparation: Dissolve the dye in the mixture of ale. phenol and aniline. Add water. note: This solution was republished by Goodpasture 1925 (608b, 1 :550) to which paper and date reference are frequently made. See also DS 23.12 Hertig and Wolbach 1924. 11.431 Henneguy 1891 11024, 27:397 reagents required: A. 1% potassium permanganate; B. 1% magenta method: [sections or smears from F 1600.0010 Flemming 1882 fixed material] — » water — > A, 5 mins. -^ water, wash—* B, 5 mins. -^> water, rinse—* 95% ale., till partly differentiated — >• clove oil, till differentiation complete -^ balsam, via xylene note: Schneider 1922, 113 applies this method to any section by mordanting in 1% chromic acid. A description of the use of this stain for chromosomes is given under DS 11.40 above. 11.431 Huntoon 1931 591b, 1:317 formula: water 75, glycerol 25, phenol 3.75, magenta 0.6 11.431 Kinyoun test. 1946 Conn et al. Conn et al. 1946, 1 V, 6 formula: water 100, 95% ale. 20, phenol 8, magenta 4 31G METHODS AND FORMULAS DS 11. 431-DS 11.44 11.431 Krajian 1943 11284,28:1602 formula: xylene 65, creosote 35, 95% ale. 5, magenta 0.3 preparation: Dissolve dye in ale. Add to other solvents. 11.431 Manevall928 20540b, 4:21 formula: water 90, aniline 2.5, 95% ale. 7.5, acetic acid 0.2, magenta 1 11.431 Muller and Chermock 1945 11284,30:169 formula: water 100, 95% ale. 20, phenol 8, magenta 4, w'etting agent 0.1 note: The original specifies "Tergitol 7" as the wetting agent. 11.431 Pottenger 1942 test. Farber 1942 20540b, 17:183 formula: water 80, 95% ale. 15, phenol 5, magenta 1.0 11.431 Schneider 1922 see DS 11.431 Henneguy 1891 11.431 Tilden and Tanaka 1945 Tech. Bull., 9:95 formula: water 90, methanol 10, phenol 5, magenta III 0.5 11.431 Ziehl 1882 7276, 8:451 and 1890 23632, 7:39 reagents required: A. water 100, 90% ale. 10, magenta 1, phenol 5; B. 1% acetic acid preparation of a : Grind the dye with phenol in a mortar. When dissolved add the ale. in 10 successive lots while grinding. Then use water in 10 successive lots to wash out mortar. Filter accunmlated washings. method: [sections or smears] -^ water -^ .1, 10-20 mins. -^ B, till differentiated —> counterstain -^ balsam, via usual reagents note: For the use of this reagent for bacteria see DS 23.212 Neelsen 1883, and DS 23.211 Ziehl. A detailed description of the former use is given under DS 23.20 (Chapter 21). The solution of Augusta 1932 (6630, 111:719) contains 2 magenta but is otherwise identical. 1 1 .432 Magenta as Leucobase 11.432 Coleman 1928 20540b, 13:123 formula: water 100, magenta 1, A^-hydrochloric acid 4, potassium metabisulfite 1, activated charcoal 0.25 method: Dissolve dye in water. Add metabisulfite and then acid. Leave 24 hrs. in dark. Add charcoal, shake 1 min. Filter. 11.432 Feulgen and Rossenbeck 1924 23543, 135:203 formula: water 100, magenta 0.5, N-1 hydrochloric acid 20, 10% sodium bisulfite 5 preparation: Raise water to boil. Add dye. Cool to 50°C. and filter. Add acid to filtrate and cool to room temperature. Add bisulfite solution, leave 24 hours in dark. method: see DS 22.1 Feulgen and Rossenbeck 1924 note: The widely quoted method of de Tomasi 1936 (20540b, 11:137) differs only in adding 0.5 dry potassium metabisulfite in place of 5 10% sodium bisulfite. See com- ments on sodium bisulfite in note to DS 11.113 Knower 1930. 11.432 Lillie 1948 Lillie 1948, 143 note: This differs from Coleman 1928 only in the substitution of sodium metabisulfite for potassium metabisulfite. 11.432 SchifE 1886 825,140:93 formula: water 100, magenta 0.025, sulfur dioxide q.s. to decolorize solution 11.432 de Tomasi 1936 see DS 11.432 Feulgen and Rossenbeck 1924 11.44 THIAZINS 11.44 Anonymous 1946 4349,26:13 formula: water 75, glycerol 20, abs. ale. 5, toluidine blue 1, lithium carbonate 0.5 preparation: Dissolve dye and alkali in water, incubate 24 hours at 37°C. and return volume to 75. Add other ingredients. 11.44 Borrell see DS 11.44 Langeron 1908 DS 11.44 DYE STAINS OF GENERAL APPLICATION 317 11.44 Cobin 1946 Tech. Bull, 7 :92 formula: water 100, sodium phosphate, dibasic 2.94, potassium dihydrogen phosphate 3.68, methylene blue 0.76 prepar.\tion: Heat the dye and sodium pliosphate, dibasic in 30 water on a water bath for 30 minutes. Cool and add the acid phosphate dissolved in 70 water. 11.44 Gabbett 1887 see DS 23.213 Gabbett 1887 11.44 Gatenby and Cowdry 1928 see DS 11.44 Nicolle 1871 (note) 11.44 Goodpasture test. Langeron 1934 Laugeron 1934, 498 formula: water 100, methylene l^lue 0.25, potassium carbonate 0.25, acetic acid 0.75 prepar.\tion: Boil the dye with the carbonate 30 minutes under reflux. Cool. Add acid, shake to dissolve ppt. Boil 15 minutes. Cool. Filter. 11.44 Jadassohn Ust. 1928 Schmorl Schmorl 1928, 150 formula: water 100, methylene blue 1, sodium borate 1 11.44 Kingsbury and Johannsen 1927 Kingsbury and Johannsenl927, 45 formula: water 90, abs. ale. 10, methylene blue 0.2, potassium hydroxide 0.005 11.44 Kuhne test. 1904a Besson Besson 1904, 155 formula: water 100, 95% ale. 30, sodium carbonate 1, methylene blue 0.5 11.44 Kuhne test. 1904b Besson Besson 1904, 154 formula: water 100, 95% ale 10, methylene blue 2, phenol 2 11.44 Langeron 1908 1886. 12:135 preparation: To 100 0.5% silver nitrate add 3% sodium hydroxide till no further ppt. is produced. \\'ash ppt. thoroughly by decantation and add to it 100 1% methylene blue. Boil 5 minutes. Cool. Filter. use: see DS 13.13 Shortt 1918. note: This is "Borrel's Blue." The method of preparation as stated by Langeron 1942, 545, differs little from that of Laveran 1900 (6630, 52:549). 11.44 Langeron 1934 Langeron 1934, 496 stock solutio.\: water 100, phenol 0.5, azur II 1 WORKING solution: water 90, stock 10, 1% potassium carbonate 2 method: [fixed smear] -^ stain, 1 min. -^ water, rinse — > dry -^ neutral mountant note: This is also used as a mordant for some DS 13.12 techniques. 11.44 Langeron 1942 Langeron 1942, 610 reagents required: A. water 100, polychrome methylene blue 1; B. glyceric ether 25, water 75 method: [water] — > A, 5 min. to 12 hrs. -^ B, till differentiated -^ water, thorough wash — > abs. ale. least possible time -^ balsam, via cedar oil note: Langeron {loc. cit.) also recommends Beauverie and Hollande 1916 ADS 22.1 in a 1% dilution for B, above. 11.44 Largret and Aubertin 1938 see 23.12 Largret and Aubertin 1938 11.44 Laveran 1900 see DS 11.44 Langeron 1908 11.44 Lbffler 1890 23684, 7:625 formula: water 80, 95% ale. 20, methylene blue 0.3, potassium carbonate 0.8 preparation: Dissolve dye in ale. Add carbonate solution. method: see DS 11.423 SahU 1885 note: a description of a use for this stain in bacteriology is given under DS 23.20 in Chapter 21. 11.44 Manson test. 1929 Wenrich McClung 1929, 408 formula of stock solution: water 100, sodium borate 5, methylene blue 2 PREPARATION OF STOCK SOLUTION: Stir the dye into the boiling borax solution. Cool. FUter. 318 METHODS AND FORMULAS DS 11.44 WORKING solution: water 100, stock 1 method: as DS 11.44 Langeron 1934 note: When used after DS 13.12 Wright, or similar stains, the term panoptic is some- times applied. 11.44 Manwell 1945 11284,30:1078 formula: water 100, methylene blue 0.1, 1% sulfuric acid 0.6, potassium dichromate 0.1, 1% potassium hydroxide 2 preparation: Dissolve dye in water. Add acid and dichromate. Autoclave 2 hours at 3 pounds or till solution is blue. Add alkali drop by drop shaking till ppt. dissolved. Leave 48 hours. Filter. NOTE : Manwell (loc. cit.) states this method to be a modification of that of Singh, Jaswant and Bhattacharji 1944 (9943, 79:102), and refers to it as the "JBS" {sic) method. 11.44 Martinotti 1910 23632, 27:24 formula: water 75, glycerol 20, 95% alcohol 5, toluidine blue 1, lithium carbonate 0.5 11.44 Michaelis 1901 23684, 29:763 formula: water 90, methylene blue 1, 0.4% sodium hydroxide 5, 0.5% sulfuric acid 5 preparation: Add the alkali to the solution of the dye, boil 15 minutes. Cool. Add acid, filter. 11.44 Moschkowsky test. 1946 Roskin Roskin 1946, 287 formula: water 100, sodium borate 2, methylene blue 1 11.44 MuUer and Chermock 1945 11284, 30:169 formula: water 70, 95% ale. 30, potassium hydroxide 0.0007, methylene blue 0.44 11.44 Nicolle 1871 23730, 9 formula: sat. sol. thionine in 50% ale. 10, 1% phenol 90 note: Gatenby and Cowdry 1928, p. 187 use 50% ale. and increase the phenol solution to a 6: 1 ratio. Langeron 1942, 539 takes 20 thionin solution to 80 2% phenol. Conn 1936 cites "Thionin, carbol-, Nicolle's" {sic) in the index but not in the text. 11.44 Proescher and Drueger 11284, 10:153 formula: a. water 100, methylene 1, sodium peroxide 0.025, hydrochloric acid q.s. preparation: Boil dye and peroxide for 15 minutes. Cool. Adjust to pH 7 with hydro- chloric acid. 11.44 Roques and Jude 1940 test. 1949 Langeron Langeron 1949, 621 preparation: Grind 1 methylene blue with 10 anhydrous potassium sulfate. Add 100 95% ale. and shake at intervals for some hours. Filter and evaporate filtrate to dryness. 11.44 Stoughton 1930 1025, 17:162 formula: water 95, phenol 5, thionin 0.1 ^ 11.44 Terry 1922 11284, 8:157 formula: water 100, methylene blue 0.2, potassium carbonate 0.2 preparation: Boil 23^ minutes. Cool. 11.44 Raadt 1912 see DS 13.12 Raadt 1912 11.44 Sahli 1885 23632,2:14 reagents required: A. water 70, sodium borate 30, sat. sol. (arc. 4%) methylene blue 1 method: [sections of chrome-fixed or mordanted material] —> water—* A, 1-3 hrs. — > abs. ale. till differentiated — > balsam, via usual reagents 11.44 Singh, Jaswant, and Bhattacharji 1944 see DS Manwell 1945 (note) 11.44 Stevenel 1918 5310, 11:870 formula: a. water 100, methylene blue 1.3, potassium permanganate 2.0 preparation: Dissolve the dye and permanganate each in 50 water. Mix and heat on water bath tiU ppt. redissolved. Cool. Filter. method: see DS 13.13 Boy6 1940 DS 11.44 DS 12 DYE STAINS OF GENERAL APPLICATION 319 11.44 Unna 1892 23632,7:483 PREPARATION OF DRY STOCK: Water 100, 95% ale. 20, methylene blue 1, potassium carbonate 1. preparation: Simmer ingredients till volume reduced to 100. Leave 24 hours. Filter. Evaporate to dryness. 11.44 Volkonsky 1933 test. 1942 Langeron Langeron 1942, 1105 formula: water 50, glycerol 50, methylene violet 0.4, azur II 0.1, potassium carbonate 0.1 11.45 CRYSTAL VIOLET 11.45 Atkins 1920 1105G, 5:321 formula: water 75, 95% ale. 25, aniline sulfate 0.75, crystal violet 3.8 11.45 Brown and Brenn 1931 10919, 48:69 formula: water 100, phenol 0.1, sodium bicarbonate 1.25, crystal violet 0.75 11.45 Huntoon 1931 591b, 1:317 formula: water 60, glycerol 25, 95% ale. 15, crystal violet 0.45 11.45 Johansen 1932 20540b, 7:17 REAGENTS REQUIRED: A. water 100, 95% ale. 20, crystal violet 1; B. 1% picric acid in 95% alcohol method: [sections, perfectly freed from fixative] —> water —> ^4, 5 mins. — > water, rinse -^ B, till no more color comes away —* balsam, via usual reagents note: The author strongly recommends this for nuclei in heavily yolked material. 11.45 LaCour 1931 11360, 51:124 reagents required: A. 1% gentian violet; B. ADS 12.2 Lugol (1905); C. 1% chromic acid method: [sections or smears] — > water -^ A, 10 mins. — > water, quick rinse — » abs. ale, 2 sec. — » 5, 2 mins. -^ abs. ale, 2 sec. —> C, 15 sec. — > abs. ale, 5 sec. — > C, 15 sec. — » abs. ale, 15 sec. -^ clove oil, till clear — » xylene, till clove oil removed — + balsam 11.45 Nastikow test. 1904 Besson Besson 1904, 155 formula: water 100, gentian violet 0.2, mercuric chloride 0.05 11.45 Newton 1927 11295,47:346 reagents required: A. 1% gentian violet 100; B. ADS 12.2 Lugol (1905) method: [sections or smears] — > water -^ A, 3-10 mins. — * water, rinse — > B, 30 sees. — > 95% ale, quick rinse — > abs. ale, 5-10 sees. — > clove oil, till differentiated —> xylene, 3 changes, till clove oil removed — > balsam 11.45 Stirling 1890 test. Mallory 1938 Mallory 1938, 90 formula: water 88, 95% ale 10, aniline 2, gentian violet 5 11.46 OTHER SYNTHETIC NUCLEAR STAINS 11.46 Lendrum 1945 11431, 57:267 formula: water 90, 95% ale 10, dextrin 0.5, phenol 0.4, acid fuchsin 1 preparation: Mix the dye with the molten phenol and add the ale. Add to this the dextrin dissolved at 80°C. 12 PLASMA STAINING TECHNIQUES Plasma staining techniques serve a dual role. Originally intended only to act as a contrast to the nuclear staining of the color selected, they may also be adapted to a wide range of differential tissue stain- ing. Indeed, there is a very poor case to be made for their use unless they are so em- ployed, for a good hematoxylin or oxazine nuclear stain requires no contrast which cannot be more simply and readil}^ pro- vided by a color screen between the lamp and the microscope than by staining the section examined. The techniques here given are divided into two main divisions: first, those which give only a single color irrespective of the material on which they 320 METHODS AND FORMULAS DS 12.1 DS 12.14 are employed, and second, those which give a double contrast from a single solu- tion. As the latter are no more difficult to apply than are the single contrast mate- rials, there seems to be no reason for the retention of the former group save custom. The third division contains those complex stains which are designed to provide a differential staining of the cytoplasmic elements present. 12.1 Single-contrast Formulas The division of solutions designed to produce a single contrast to the nuclear staining has been based entirely on the solvent employed. It is unnecessary, in most cases, to list references, since the majority of them are recommended by so many persons that it would be a waste of time to secure the original recommenda- tion. The difference between the aqueous solutions and the weak alcohol solutions is very slight, since the alcohol in the latter is almost certainly designed as a biostatic agent. Almost any antiseptic can be used for this purpose, alcohol being retained as a matter of convenience. The strong alcohol solutions are either of dyes insoluble in water or of materials so readily extracted by alcohol that their use must be left to the very last stage of dehydration if they are not to be lost. Clove oil solutions are widely used both in zoological and botanical techniques, but it is a pity that more attention has not been paid to the formula of Johansen 1940, given under 12.16, which combines all the desirable characters of an alcohol and a clove oil solution. The phenol solu- tions are necessitated either through the desirability of having an acid solution, or by the difficulty of securing a solution of the dye without the special technique em- ployed, or by the impossibility of preserv- ing the solution without a biostatic re- agent being included. They are far more widely employed in bacteriology than in either zoology or botany. The few stains given in the last division, other than Johansen 1940 which has already been noticed, are unusual formulas specifically designed to take part in a more complex technique given later in the work. 12.11 AQUEOUS SOLUTIONS, compl. scHpt. formula: water 100, acid fuchsin 0.2 or instead of acid fuchsin either Bordeaux red 1, or Congo red 0.5, or erythrosin 1, or methyl green 1, or orange G 1, or orange II 1, or ponceau 2R 1 method: [sections, with desired nuclear staining]—* water—* stain, ^2 to 2 mins. -^ water, wash -^ balsam, via usual reagents note: Congo red and acid fuchsin are very alkali-sensitive and not safe after hematoxylin stains which have been "blued" in alkalis. Erythrosin is practically confined to botan- ical techniques, as orange II and ponceau 2R are confined to animal procedures. The writer considers the latter the best general-purpose counterstain to blue. Methyl green tends to be fugitive. 12.12 WEAK ALCOHOL SOLUTIONS, compl. script . formula: water 90, 95% alcohol 10, benzopurpurin 0.5 or instead of benzopurpurin either anilin blue 1, or Biebrich scarlet 1, or eosin B 0.5, or eosin Y 0.2, or methyl blue 1, or phloxin 0.2, or rose bengal 0.5 method: as 12.11 note: The use of alcohol i.s either to assist in forming a smooth suspension (as in Biebrich scarlet) or to discourage the growth of molds (as in the eosins). The "Magdala Red" commonly specified in botanical techniques is actually phloxine (Conn 1946, 112). 12.13 STRONG ALCOHOL SOLUTIONS, compl. script. formula: 95% ale. 100, aurantia 0.5 or instead of aurantia, either chromotrope 2R 2.0 or ethyl eosin 0.5 or light green 0.5 or fast green FCF 0.5 or spirit blue 0.5 method: [sections, with desired nuclear staining] -* 70% ale. -^ stain, }i to 2 mins. — > 95% ale, rinse — > balsam, via usual reagents 12.14 CLOVE OIL SOLUTIONS, compl. script. formula: oil of cloves 100, light green to sat. or, instead of light green, either i&st green FCF, or orange G, or safranin O DS 12.15-DS 12.16 DYE STAINS OF GENERAL APPLICATION 321 method: [sections, or smears, with desired prior staining] —» 95 % ale. ^ stain, usually dropped on slide, till sufficiently colored —> clove oil, dropped on slide, till excess stain removed -^ xylene, several changes — + balsam note: The reputation for rapid fading, which stains applied in this manner enjoy, is largely due to imperfect removal of clove oil before mounting. 12.15 PHENOL SOLUTIONS, compl. script, (but probably originated by Ziehl 1882 7276, 8:451) formula: water 100, phenol 2, 95 f'^ ale. 10, cresyl violet 1 or instead of cresijl violet, either gentian violet 1 (= "Gram's violet" — and.) or methylene blue 1.5, or magenta 1 (see also DS 11.43 Ziehl 1890), or thionin 1.5 (see also DS 11.46 Nicolle), or isamine blue 1 (see DS 23.11 Nicolau 1939), or crystal violet 1 (see DS 23.213 Carpano 1910), or crystal violet 2 (see DS 23.213 August 1932), or rose bengal (see DS 23.211 Conn 1928) prepar.\tion: Grind the dye with the phenol and ale. in a mortar. Wash out the mortar while grinding with 10 successive portions of water. Filter the accumulated washings after 24 hours. 12.16 OTHER SOLUTIONS 12.16 Bensley 1916 590, 29:37 FOR.MUL.^: water 100, anilin blue 0.2, phosphomolybdic acid 1 method: [sections with red nuclei]-* waters stain, 5 mins. — > water, quick rinse —> -^ abs. ale, till differentiated — >• balsam, via xylene note: This was originally designed by Bensley {loc. cit.) to follow his DS 11.3 brazilin. 12.16 Donaldson 1917 iodine-eosin 11995,192:571 formula: ads 12.21 Lugol 1905, 50, sat. aq. sol. eosin Y 50 method for protozoans in fece,s: Mix 10 parts stain with 1 feces — > place under cover- slip -^ seal note: This method is also applica])lc to many marine invertebrate larvae. 12.16 Fraenkel test. 1948 Romeis tannin-fuchsin Romeis 1948, 519 formula: water 60, acid fuchsin 0.12, tannin 10, ADS 22.1 Unna (1928) 33 12.16 Jensen 1937 23684, 39:333 formula: DS 11.24 Jensen 1937 (stock II) 100, phenol 0.5, eosin B 0.05 12.16 Johansen 1940 fast green Johansen 1940, 59 formula: methyl cellosolve 30, clove oil 30, abs. ale. 30, fast green FCF 0.5 note: Though intended for botanical histology (see DS 21.41 Johansen 1940) this solu- tion is of wide application as a counterstain. 12.16 Kofoid (1920) see DS 23.33 Kofoid (1920) 12.16 Krajian 1938 eosinol—aud. 1887a, 25:376 prep.\ration of dry stain: To 0.35 eosin B dissolved in 0.7 water add 0.7 acetic acid. Mix and incubate at 36°C. until dry. working solution: xylene 75, phenol 25, all dry stain from above 12.16 Maneval 1936 20540b, 11 :9 formula: water 80, acetic acid 20, anilin blue 0.01 {or, instead of anilin blue, acid fuchsin 0.1) ferric chloride 0.6 12.16 McClean 1934a New Phytologist, 33 ilUi) PREPARATION OF DRY STOCK: To 50 1 % eosin add 1.75 10% hydrochloric acid. Leave 24 hours, filter, dry ppt. WORKING solution: xylene 100, all ppt. from above note: Erythrosiii may be substituted for eosin. 12.16 McClean 1934b New Phi/tologisl, 33 •:il(i preparation of dry stock: To 50 1% nile blue sulfate add 12.5 10% sodium hydroxide. Leave 24 hours, filter, dry ppt. wOKKixc; solution: xylene 100, all ppt. fttim al)()ve 12.16 Nuttall 1908 16035,1:162 formula: xylene 100, picric acid to sat. {circ. 10%) note: Originally intended for potash-cleared arthropod endoskeletons. 322 METHODS AND FORMULAS DS 12.16-DS 12.20 12.16 Pearson 1941 Tech. Bull., 3:16 preparation: To 100 0.5% eosin Y add, drop by drop, 4 hydrochloric acid. Wash ppt. by decantation. dry, and dissolve in 40 95% ale. 12.16 Smith 1926 see DS 21.14 Smith 1926 12.16 Tonkoff 1900 test. 1907 Bohm and Oppel Bohm and Oppel 1907, 120 formula: abs. ale. 100, spirit blue 0.1, ADS 12.1 Gram 1884 0.03 12.16 Unna 1895 orange G-tannin 14352,21:540 formula: water 100, tannin 33, orange G 2 use: see DS 13.22 Langeron 1942b and DS 22.2 Volkonsky 1928. 12.2 Double Contrasts from One Solution Double contrasts from one solution are just as easy to use as are single contrasts. In those cases in which there is no histo- logical or cytological difference between the elements which it is desired to stain, they present no improvement over the more conventional techniques, but unless one is dealing with homogenous organs, or with a very young embryo, there seems to be no possible reason why the double-con- trast material should not be employed. The division of these stains into two groups is based entirely on the color of the nucleus which they are designed to set off. The first group, contrasting with red nuclei, can therefore be used either after magenta, carmine, or safranin, the picro- contrasts being by convention more com- monly employed after carmine than after the other reagents. The picro-indigo- carmine of Cajal 1895 is the oldest and best known of these mixtures, but it is probably less effective than the formula of Masson used for the same purpose. The formula of Grosso 1914 was originally developed for staining blood, but it is of much wider application. The picro-spirit blue of Smith 1912, if used after magenta as a nuclear stain, is, in the opinion of the present writer, undoubtedly the best tech- nique which has yet been developed for staining sections of heavily yoked mate- rial. The non-picric formulas, which are included in the next division of con- trasts for red nuclei, contain two of the best counterstains (Chatton 1920 and Kostowiecki 1932) which have ever been developed. The formula of Kostowiecki in particular has all the advantages of the better known Mallory-Masson poly- chrome stains, but may be applied from a single solution. Roux's blue here given in Roux's formula of 1894 was at one time well known but has now fallen into disuse. Contrasts for blue nuclei present more difficulties than do those for red since nuclei for these techniques are custom- arily stained by hematoxyhn, which is very sensitive to acid. Though many of the picro-contrasts can be employed with hematoxylin, it is strongly recommended that one of the oxazine nuclear stains given above under DS 11.42 be employed in its place. The best known of these picro-contrasts is van Gieson 1896. The non-picric contrast formulas, given under DS 12.222 below, may be safely employed after either hematoxylin or methylene blue stains. Probably the best known, and certainly one of the easiest to use, is the saffron-erythrosin of Masson 1911. 12.20 TYPICAL EXAMPLES Preparation of a transverse section of a Squalus embryo using the picro- carmine stain of Ranvier 1899 fol- lowed by the picro-indigo-carmine of Cajal 1905 This simple little preparation is very old-fasliioned, but it may well be placed in the hands of a beginning student as his introduction to the art of section cutting and staining. Moreover, it utiUzes the embryos of Squalus acanthus, which are frequently found in elementary labora- tories where this form is dissected. There is often grave doubt as to what shall be done with such embryos and they are usu- ally lost. It is presumed, of course, that we are dealing with an inland laboratory in which preserved specimens are brought from a biological supply house, so that the small embryos themselves will already DS 12.20 DYE STAINS OF GENERAL APPLICATION 323 have been fixed, if the term can properly be used in this case, in formaldehyde for a considerable period. Each embryo, after removal from the oviduct, should be thoroughly washed, using soap if neces- sary to remove the mucus from the out- side, rinsed thoroughly to remove the last traces of soap, and then thrown into a gallon jar of picro-carmine prepared by the method given under DS 11.24 Ranvier 1899. The quantity of picric acid in this solution causes it to act as fixative and preservative, as well as a nuclear stain, and it has the additional advantage that it is quite impossible to overstain in it. Em- bryos may therefore be thrown into such a jar of picro-carmine during the course of the first half of the year's work and uti- lized during the second half of the year when microtechnique is usually taught. When it is desired to cut sections, the embryos are removed from the large jar of picro-carmine and washed in running water until no further color comes away from them. Though it is immaterial how long they have remained in stain, it must be added that some weeks are usually required to secure a sufficient impregna- tion. The picric acid in a properly pre- pared Ranvier solution is usually suffi- cient to have decalcified the placoid scales of a young dogfish. These should be tested by rubbing a steel needle against the direc- tion of the skin. A hard irregularity will indicate that the calcium of the placoid scales has not been removed. In this case recourse must be had to one of the methods for decalcification given in Chap- ter 19 under AF 20. The specimens are then embedded and sectioned in the customary manner, it being perfectly possible for even a moder- ately skilled operator to cut a reasonable hand section from the paraffin block con- taining embryos of this size. It is, of course, preferable to use a microtome if one is available. The sections are then ac- cumulated and attached to the slide in the ordinary manner, deparafinized, and run down through the customary reagents until they reach 70% alcohol where the slides may be accumulated. A brief check under the low power of the microscope vUl now establish whether or not the nuclei have been satisfactorily stained scarlet throughout the body of the prepa- ration. If they have not, it is no use pro- ceeding further, for one must devote some other staining technique to these sections. Nine times out of 10, however, it will be found that the nuclei are clearly stained and quite reasonably differentiated. If the tissues other than the nuclei are stained a dark red rather than the light pink which is ine\dtable, each section should then be briefly differentiated in a 0.1 % solution of hydrochloric acid in 70% alcohol. This, however, will very rarely be necessary. The sections are now run down to water before being stained in picro- indigo-carmine (DS 12.211 Cajal 1895), which is the easiest, simplest, and most foolproof of all double staining methods. The shdes bearing the sections are placed in the solution from three to five minutes, withdrawn from the stain, rinsed briefly in water, and then placed directly in ab- solute alcohol, and left until a naked-eye examination shows that the connective tissues are clear blue. This clear blue is in distinction to the bright green of the muscles or the red of such other tissues as have retained the carmine. The moment that this change from green to blue, which is clearly and sharply seen, takes place the slide is placed in xylene which stops the differentiation and permits subsequent mounting in balsam. The results obtained by this method are not, of course, to be compared for brilliance with the more complex methods using acid fuchsin and some of the phos- photungstic reactions. But this is undoubt- edly the first double-staining method which should be tried by any beginner — to whom encouragement is probablj' of more value than the actual colors to be obtained. Preparation of a transverse section of the tongue of a rat using coelestin blue B followed by picro-acid fuchsin The chief difficulty in preparing a trans- verse section of the tongue is to avoid hardening of the muscle which tends to become brittle, either if imperfectly fixed 324 METHODS AND FORMULAS DS 12.20 or if handled with undesirable reagents in any stage of the proceedings. It is there- fore recommended that the following de- scription be followed rather closely, for it can be adapted almost without variation to any other heavily muscularized tissue which it is desired to stain. As this preparation is intended to show only the gross histological elements pres- ent, it is unnecessary to specify the man- ner in which the rat should l)e killed; but, the sacrificed animal may be used also for the staining of taste buds in the posterior lobe of the tongue by the method de- scribed in some detail in Chapter 23, "Demonstration of the nerve ending in taste buds by the method of Bielschowsky 1914," in which case the rat had better be killed by a blow on the head rather than by an anesthetic. The tongue may be easily removed by severing the articulation of the lower jaw and by removing this together with the adherent tongue which may be detached with a short scalpel or cartilage knife. A portion of the tongue approximately 5 mm. in length is now cut off and placed in a large volume of the selected fixative. Though opinions vary widely as to the most desirable fixative to use for muscular- ized tissues, it may be said at once that no alcoholic solution and no solution con- taining picric acid or osmic acid can be recommended. The author's choice would be for the cupric-acetic-phenol formula of Behrens 1898 (Chapter 18 F 4000.0010), which he has employed most successfully on a variety of heavily muscularized tis- sues. This formula would also provide an excellent prior-mordanting for the stain- ing techniques which follow. Whatever formula is selected, however, a large volume (about 100 cc. for the piece of rat tongue described) should be employed and permitted to act for no longer than is necessary to secure the complete impreg- nation of the tissues. When the piece has been successfully fixed it must be washed overnight in running water and then de- hydrated. The process of dehydrating, clearing, and embedding is the point at which most n^usculurized tissues become unmanageable. Nothing, of covn-se, can counteract the effect of improper fixation, but even with good fixation the utmost attention must be paid to the selection of dehydrating agent and clearing agent, and to the temperature at which the embed- ding takes place. It has been the experi- ence of the writer that the newer substi- tutes for alcohol in dehydrating tend to harden or render brittle muscular tissue to a greater extent tlian does the more old- fasliioned method of using ethanol. There is little choice in the matter of clearing prior to embedding, for it has been found by numerous workers that benzene has a less hardening effect on muscular tissue than have other agents. Unless it is desired to cut very thin sec- tions, a wax of no higher melting point than 52°C. should be emploj^ed. It may be stated categorically that should the tem- perature be permitted to rise above 56°C., it would be better to throw the prepara- tion away than to waste time endeavoring to section it. Paraffin sections are now cut from the block by the standard method, then flattened and attached to a slide by either gelatin or egg albumen. It is recom- mended that as soon as the sections are flattened, they should be pressed to the slide with a piece of wet filter paper, rolled into position with a rubber roller, and dried with the maximum possible speed. As soon as they are dried, the sections are deparaffinized l)}^ the usual techniques and taken down to distilled water, where they may remain until one is ready to stain them. Coelestin blue B as the nuclear stain is selected in this instance because the contrast of muscularized tissues is better brought out with the aid of a picro- contrast than by any other method. These picro-contrasts are, however, so acid that hematoxylin-stained nuclei are often de- colorized in the course of counterstaining. Any of the oxazine formulas (DS 11.41 above) may be chosen, the writer's prefer- ence being for the first (DS 11.41 Anony- mous 193G). The sohition presents no difficulties of pi'eparation and need not be rejected if it shows a shght precipitate at the bottom. The sections are passed di- rectly into it from the distilled water and allowed to remain until an examination under the low power of the microscope shows the nuclei to be clearly and deeply DS 12.21-DS 12.211 DYE STAINS OF GENERAL APPLICATION 325 stained. It is very diflicult to overstain in ground. If a small quantity of yellow is this solution and, though the time speci- picked up by the connective tissues it will fied in the formula given is from five be removed in the process of differentia- minutes to one hour, no damage will be oc- tion. The time is not critical, but that casioned should the sections remain over- given in the formula cited (from two to night in the staining solution. After re- 10 minutes) will be found to cover the moval from the staining solution the}' are range normally necessary, rinsed in distilled water and accumulated A slight difiiculty will be occasioned in in a jar either of distilled or tap water, dehydration through the tendency of the until it is desired to counterstain them. picric acid to leave the tissues in the Any of the formulas given under DS various alcohols employed. This may 12.221 below may be used for counter- either be prevented by dehj'drating them staining, but that of van Gieson (DS in a series of 1 % solutions of picric acid in 12.221 van Gieson 1896), though old, is the various alcohols, or it may be ignored still one of the best. Its only disadvantage completely, according to the depth of is that it has a tendency to remove hema- yellow color which it is required to retain, toxylin from stained nuclei, an effect If, on the contrary, it is desired to have which the present method avoids. The them a very pale yellow, they may have stain requires Uttle or no differentiation, to spend a period of time beyf)nd that so that the sections may be placed in it necessary for dehydration in 95% alcohol and examined from time to time until the to remove the unwanted picric acid. The muscles are seen to be stained yellow sections are then cleared in xylene in the against a red connective-tissue back- normal manner and mounted in balsam. 12.21 CONTRASTS FOR RED NUCLEI 12.211 Formulas Containing Picric Acid 12.211 Borrel 1901 see DS 12.211 Cajal 1895 (note) and also DS 23.11 Borrel 1901 12.211 Cajal 1895 Icsl. 1905 Lee picro-indigo-carmine Lee 1905, 20 formula: water 100, picric acid 1, iudigo-carmine 0.25 method; [sections with red nuclei] — » water — > stain, 3-5 mins. — > water, quick rinse — > abs. ale, till connective tissue clear blue result: muscle, green; most connective tissues, blue. note: See also DS 22.12 Hruby 1933. A detailed description of the use of this stain is given under 12.20 above. This stain is referred to Borrel (without reference) by Besson 190-1, 751; see, however, DS 23.11 Borrel 1901. Calleja (Cajal and de Castro 1933, 87) specifies prior staining of nuclei in DS 11.28 Orth (1892). 12.211 Calleja see DS 12.211 Cajal 1895 (note) 12.211 Curtis 1905 picro-naphthol black 6630,57:1038 STOCK solutions: I. sat. aq. sol. picric acid; II. water 80, glycerol 20, naphthol blue black 1 WORKING solution: stock I 90, stock II 10 method: [red, preferably safranin-stained, nuclei] —> stain freshly prepared, flooded on slide, 10-15 mins. -^ abs. ale. till differentiated — » toluene to stop differentiation -* balsam result: nuclei, red; cartilage, blue; other structures, yellow. 12.211 Curtis 1905b picro-naphthol black 1863,17:003 formula: water 100, glycerol 2, acetic acid 0.01, picric acid 0.9, naphthol blue black 0.1 method, etc.: as Curtis 1905a 12.211 Domagk test. 1948 Romeis picro-thiazin red Romeis 1948, 168 formula: water 100, picric acid 1, thiazin red 0.01 12.211 Dubreuil 1904 picro-methyl blue 6593,6:62 formula: water 100, methyl blue 0.1, picric acid 0.9 326 METHODS AND FORMULAS DS 12.211 12.211 Grosso 1914 picro-methyl green 8545,18:71 PREPARATION OF DRY STOCK: Add a sat. aq. sol. picric acid to a sat. aq. sol. methyl green till no further ppt. is formed. Wash and dry ppt. PREPARATION OF STOCK SOLUTION: methanol 100, dry stock 0.5 PREPARATION OF WORKING SOLUTION: stock solution 15, Water 65 method: [sections with red nuclei] -* stain, 5-10 mins. —>• water, quick rinse —> abs, ale, till red color clouds cease -^ balsam, via xylene 12.211 Grosso 1914 see DS 21.3 Grosso 1914 12.211 Hruby 1933 see DS 22.12 Hruby 1933 12.211 Krause 1911 test. 1948 Romeis picro-indigo-carmine Romeis 1948, 169 formula: water 100, picric acid 1, indigo-carmine 0.3 12.211 Lillie 1945 picro-blue 11571b, 25:1 formula: water 100, picric acid to sat., methyl blue 0.1 or anilin blue 0.1 note: Lillie 1948 p. 191 recommends this as a contrast for blue nuclei. 12.211 Lillie 1948 picro-naphthol black Lillie 1948, 191 formula: water 100, picric acid q.s. to sat., naphthol blue black 0.02 to 0.04 12.211 Masson test. 1934 Langeron picro-indigo-carmine Langeron 1934, 552 REAGENTS REQUIRED: A. 1% acetic acid; B. sat. aq. sol. picric acid 100, indigo-carmine 0.25; C. 0.2% acetic acid method: [nuclei red, preferably by some DS 11.43 method] -^ A, thorough rinse —> B, 10 mins. — » water, quick rinse -^ C, till connective tissue clear blue, about 2 mins. — > abs. ale. shortest time to complete dehydration — > balsam, via xylene result: very like Cajal 1895 but with a greater range of shades. 12.211 Minchin test. 1928 Goodrich picro-light green Gatenby and Cowdry 1928, 432 formula: 90% ale. 100, picric acid 5, light green 1 method: [red nuclei] —> 90% ale. — » stain, 10 mins. — > abs. ale. tUl differentiated—* balsam, via xylene result: similar to Smith 1912. 12.211 Neubert 1922 picro-thiazin red 23418(1), 66:424 reagents required: A. water 100, 95% ale. 10, thiazin red 0.15, picric acid 0.03; B. sat. 95% ale. sol. picric acid method: [sections with hematoxylin-stained nuclei]-^ water —> A, till collagen deeply stained — > rinse -^ B, thorough wash -^ balsam, via usual reagents 12.211 Pfitzer 1883 picro-nigrosin 2626, 1 :44 formula: sat. sol. picric acid 100, nigrosin 0.2 12.211 Pol 1908 test. 1948 Romeis picro-indigo-carmine Romeis 1948, 169 formula: water 100, picric acid 1, indigo-carmine 0.4 12.211 Roskin 1946 see DS 13.43 Roskin 1946 12.211 Shumway 1926 picro-indigo-carmine see DS 13.5 Shumway 1926 12.211 Smith 1912 picro-spiril blue 11373,23:94 formula: sat. ale. sol. {circ. 1%) spirit blue 100, picric acid 1 method: [sections of material bulk stained in carmine] — ♦ abs. ale. -^ stain, 2 mins. — > abs. ale, till differentiated — > balsam, via xylene result: nuclei, red; yolk, yellow green; yolk-free cytoplasm, clear blue. 12.211 White test. 1905 Hall and Herxheimer picro-erythrosin Hall and Herxheimer 1905, 63 formula: water 100, sat. ale. sol. erythrosin 4, picric acid 0.6, calcium carbonate to excess DS 12.212-DS 12.221 DYE STAINS OF GENERAL APPLICATION 327 12.212 Other Formulas 12.212 Chatton 1920 eosin Y -light green 1915,59:21 REAGENTS REQUIRED: A. 95% alc. 100, light green 1, eosin Y 2; B. 5% acetic acid in abs. alc. PREPARATION OF a: Dissolve with occasional agitation over period of some days. Filter. method: [sections with red nuclei] — » 95% alc. —* A,b mins. -^ B, till connective tissue clear green -^ balsam, via usual reagents result: on arthropod material, for which the stain was designed, chitin is green on a red background. On vertebrate material the picture is similar to DS 12.32 Patay 1934. 12.212 Kostowiecki 1932 orange G-anilin blue 23632, 49 :337 formula: water 100, anilin blue 0.06, orange G 0.2, phosphomolybdic acid 1 preparation: Boil dyes with water 3 minutes. Add acid to hot sokition. Cool. Filter. method: [sections with red nuclei] — > water -^ stain, till dark colored, l^ to 12 hrs. -^ water, rinse -^95% alc, 1 min. -^ balsam, via usual reagents result: procartilage, light blue; cartilage, dark blue; muscle, orange; other connective tissues, blue green. 12.212 Roux 1894 dahlia-methyl green 766,9:248 formula: water 90, abs. alc. 20, dahlia violet 0.5, methyl green 0.5 preparation: Grind each dye separately in 10 abs. alc. Wash out each mortar with 50 water in small successive doses. Collect washings; leave 24 hours; filter. Mix filtrates; leave 24 hours filter. method: [red nuclei] -^ water — > stain, 5-15 mins. -^ blot — > abs. alc, till differentiated — * balsam, via usual reagents note: The dahlia violet used by Roux may have been almost any mixture of pararo- saniline derivatives. Cheap samples of gentian violet work admirably. The result is an excellent polychrome counterstain wherever reproducibility of research results is of less importance than classroom clarity of demonstration. The working solution has been evaporated to dryness and sold as Roux's blue, (See DS 13.5 Bohm and Oppel 1907.) 12.212 Unna test. 1928 Hill anilin blue-orcein Gatenby and Cowdry 1928, 280 reagents required: A. 0.1% acetic acid; B. water 50, abs. alc. 25, acetic acid 2.5, glycerol 10, orcein 0.5, anilin blue 0.5 preparation of B: Dissolve blue in water with gentle heat. Filter. Dissolve orcein in alc. and add to it the acid and glycerol. Add this mixture to the blue. method: [red nuclei—* A, few minutes —> B, 1 to 10 hrs. -^ A, till differentiated —> balsam, via usual reagents result: bone and elastic fibers red-brown against a blue background. note: This reaction is more usually applied by the method of Pasini (1928) (DS 12.31). 12.22 CONTRASTS FOR BLUE NUCLEI 12.221 Formulas Containing Picric Acid 12.221 Curtis 1905 picro-ponceau 1863, 17 :603 formula: water 100, ponceau S 0.1, picric acid 1, acetic acid 0.04 12.221 Fite 1939 picro-fuchsin 11284,25:743 formula: water 100, picric acid 0.5, acid fuchsin 0.1 12.221 van Gieson 1896 picro-fuchsin 23632, 13 :344 formula: sat. sol. (circ. 1.2%) picric acid 100, acid fuchsin 0.05 method: [blue nuclei] —> water ^ stain, 2-10 mins. -^ water, quick rinse —> balsam, via usual reagents note: a detailed description of the use of this stain is given under 12.20 above. 12.221 Gnanamuthu 1931 picro-congo red 11360,51:401 formula: sat. sol. {circ. 1.2%) picric acid 50, ammonia 50, Congo red 2 preparation: Add the ammonia to the picric solution. Dissolve the dye in mixture and boil till no odor of ammonia is apparent. Cool. Add sufficient water to redissolve ppt. formed on cooling. 328 METHODS AND FORMULAS DS 12.221-DS 12.222 method: [blue nuclei (DS 11.123 Ehrlich 1886 specified in original)]—* waters stain, 1-2 mins. -^ blot — > abs. ale, minimum time for dehydration -^ balsam, via usual reagents result: muscle, red; other tissues, yellow and orange. Good for most heavily muscular- ized tissues. 12.221 Hansen 1898 picro-fuchsin 766,15:152 REAGEXTs required: A. sat. sol. {circ. 1.2%) picric acid 100, acetic acid 0.3, acid fuchsin 0.1; 5. water 98, A. 2 method: PdIuc nuclei] — > water —* A, some hrs. — * B, wash -^ balsam, via xylene result: selective red stain on white fibrous connective tissue. 12.221 Lillie 1948 picro-fuchsin Lillie 1948 formula: water 100, picric acid 1, acid fuchsin 0.1, hydrochloric acid 0.25 note: Lillie (loc. cit.) also recommends his DS 12.211 solution as a contrast for hema- toxylin-stained nuclei. 12.221 Ohlmacher 1897 picro-fuchsin 11189,2:675 formula: sat. sol. {circ. 1.2%) picric 50, water 50, acid fuchsin 0.5 12.221 Schaffer 1899 picro-fuchsin 23635,66:214 formula: sat. aq. sol. picric acid, acid fuchsin 0.15, acetic acid 0.05 12.221 Thompson 1945 see DS 21.3 Thompson 1945 12.221 Unna test. Lillie 1948 picro-fuchsin Lillie 1948, 190 formula: water 90, acid fuchsin 0.25, nitric acid 0.5, glycerol 10, picric acid q.s. to sat. 12.221 Weigert 1904 picro-fuchsin 23632, 21 :3 formul.^: sat. sol. {circ. 1.2%) picric acid 100, acid fuchsin 0.1 method: as van Gieson above 12.221 Wilhelmini 1909 picro-fuchsin 8338,22:18 formula: water 90, 95% ale. 10, ammonium picrate 0.8, acid fuchsin 0.2 method: as van Gieson above 12.322 Other Formulas 12.222 Delephine test. circ. 1938 Wellings fuch sin-orange Wellings 1938, 104 formula: water 100, acid fuchsin 0.04, orange G 0.2 12.222 Gray 1952 ponceau-orange Gray 1952, 24 formula: water 100, orange II 0.6, ponceau 2R 0.4 method: Palue nuclei] — > water — >■ stain, 1-2 mins. -^ blot -^ abs. ale, till differentiated —* balsam, via usual reagents 12.222 Gregg and Puckett 1943 eosin-orange 20540b, 18:179 formula: 95% ale. 100, eosin 0.2, orange G 0.01 12.222 Guyler 1932 indigo-carminc-eosin Y 11977,18:314 formula: water 100, indigo-carmine 0.25, eosin Y 1, thymol trace method: [blue nuclei (original specifies 11.122 Delafield 1885)]-^ water— > stain, over- night — > water, quick rin-se — > abs. ale, till differentiated — >• balsam, via usual reagent 12.222 Hayem test. 1896 Kahlden and Laurent eosin-aurantia Kahlden and Laurent 1896, 117 formula: 1% eosin W, 1% aurantia, a.a. q.s. to give rose-colored solution 12.222 Kingsbury and Johannsen 1927 orange-acid fuchsin Kingsbury and Johannsen 1927, 76 formula: water 100, glj'cerol 7, orange G 1, acid fuchsin 2 12.222 Langeron 1942 see DS 12.222 Masson 1911 (note) DS 12.222-DS 12,3 DYE STAINS OF GENERAL APPLICATION 329 12.222 Male 1924 fiichsin-martius yellow 11035,42:455 formula: water 80, 95% ale. 20, acid fuchsin 0.6, inaitius yellow 0.8 12.222 Masson 1911 saffron-cnjlhrosin 6630,70:573 REAGENTS REQUIRED: A. 1% erythrositi ; B. wnfcr 100, safTron 2, 5% tannin 1, 40% fonualdehyde 1 PREPARATION OF B: Extract the .saffron in (he water 1 hour OO^C. Filter. Add other ingredients to filtrate. method: [blue nuclei]-* water -> A, 5 mins. ^ water, quick rinse -> 70% ale, few sec. -> water, thorough wash -* B, b mins. -^ blot -> abs. ale, flooded over slide, till dehydrated —> balsam, via xylene note: Langeron 1942 (p. 596) substitutes eosin B for erythrosin in the above. 12.222 Semichon 1920 methyl blue-eosin-victoria yellow 5401,45:73 REAGENTS REQUIRED: A. Water 100, methyl blue 0.04, eosin Y 0.2, victoria yellow 0.1 method: [Ijlue nuclei]^ water -^ A, overnight^ drain -> abs. ale, till differentiated — > xylene — > balsam result: horn, hair, chitin, yellow; cartilage, blue; other tissues, orange, 12.222 Squire 1892 fuch sin-orange Squire 1892, 42 formula: water 80, 95% ale. 20, acid fuchsin 0.3, orange G 2.0 method: as Gray 1952 above 12.222 Sziitz 1912 polychrome alizarin 23632,29:289 REAGENTS REQUIRED: A. 5% aluminum acetate; B. sat. ale. sol. alizarin red S 1, water 100 method: [hematoxylin-stained sections of F 2300.1000 Sziitz 1912 fixed material]—* water — > ^, 5 hrs. — » rinse -^ B, 5 hrs. -^ wash — > balsam, via usual reagents result: nuclei, blue; cytoplasm, varying shades of red, cytoplasmic inclusions being generally very darkly stained. 12.3 Complex Contrast Formulas These complex contrast formulas are specifically designed to differentiate the cytoplasm into various histological com- ponents. They are more widely used in embryology than in general histology, and the first two classes, here given, are all developed from the original discovery of Mallory 1904 (11189, 5:15) that a solution of phosphomolybdic or phosphotungstic acid will remove acid fuchsin from col- lagen while allowing it to remain either in the muscle or in the nuclei. Mallory 's orig- inal method, however, involves staining the nuclei with acid fuchsin as an inherent part of the technique, and is therefore given under the subheading DS 13, in which combined nuclei and plasma stain- ing techniques are dealt with. The group here discussed is commonly associated with the name of Masson, and the term "Masson's Trichromic" is widely applied. These methods require prior staining of the nuclei with hematoxylin and the sub- sequent staining of the connecting and supporting tissues in accordance with Mal- lory's principle. This stains nuclei a color from blue to deep purple, and they are thus distinguished far more clearly from the background cytoplasm than are those stained by the original method. All of these phosphotungstic and phosphomo- lybdic methods are excellent. The second class of complex contrast formulae are those employing phosphomo- lybdic or phosphotungstic acid reaction with other dyes than fuchsin, while at the same time retaining the differential nu- clear stain. Of these, Patay 1934 is a very nearly fool-proof and brilliant method of triple staining. Indeed the whole class of stains, with tlie exception of Fleming 1891, are of recent origin, and at the present time are the most popular group of coun- terstains which have yet been developed. Though they are customarily employed in zoological technique, no one who has tried them in botanical technique has ever re- gretted it. The final class of complex contrast for- mulas contains only those mixtures which cannot otherwise be classified. Among 330 METHODS AND FORMULAS DS 12.30 them only the techniques of Margolena in 1933 and de Winwarter and Saimonte 1908 can be regarded as having any wide application. 12.30 TYPICAL EXAMPLES Preparation of a transverse section of an earthworm, using the iron-hema- toxyhn stain of R^gaud 1910, fol- lowed by the acid fuchsin- anihn blue of Masson 1912 Considering that almost every student of biology studies a transverse section of the earthworm, it is really surprising that sections commonly available from biologi- cal supply houses should have been pre- pared with little thought for their ultimate use. Quite apart from any considerations of staining, it may be pointed out that the average field of view of the 3.5X objec- tive, commonly used in elementary bio- logical laboratories, is about 4 mm., so that a worm larger than 4 mm. in diameter cannot be got into the field of view of this low power at one observation. It is un- doubtedly a technical feat of some skill to cut a transverse section of a large earth- worm, but it is also extraordinarily diffi- cult for a beginning student to envisage the relationships of the whole by suc- cessive examinations of a variety of fields. It may be taken, therefore, as the first prerequisite to a useful preparation that one should select an earthworm of approxi- mately the right size. Though tliis may prove an arduous labor, it may be pointed out that at least 100,000 sections may be obtained from a single worm. Further, the majority of sections which one sees in elementary classes have been stained by a standard hematoxylin-eosin tech- nique which gives students very little chance of envisaging the relationships of the muscles; whereas if one employs a technique of the kind here recommended, the muscles will be stained a brilliant scarlet and the connective tissues to w^hich they are attached will be stained a bright blue. There is also a much better differ- entiation of the various layers of the intes- tine and of the nervous tissue. Once the earthworm has been collected and selected, it then becomes necessary to remove from it the grit with winch the intestine is filled. The classic method of doing this, to which the writer strongly adheres, is to place the earthworm in an environment of old coffee grounds. It is not, however, sufficient, to take coffee grounds from which a single drink has been extracted. Grounds left over from the preparation of coffee should be taken, boiled for a considerable period in water, filtered, and again boiled, until fittle or no coloring matter remains to be extracted from them. The earthworm may be ec- lectic in its diet, but its constitution does not enable it to survive the glucosides and alkaloids of a partially extracted coffee bean. These depleted grounds should then be placed in a layer an inch or two thick in a clean glass jar, the selected earth- worms placed on the surface, hghtly sprinkled with coffee grounds, and per- mitted to live undisturbed for a period of about a week. The only critical factor is the degree of humidity of the grounds. This should be such that water cannot be pressed from them when they are squeezed in the hand, yet each grain should present the appearance of being moist. After a week on a diet of coffee grounds the worms will have voided most of their grit content and may now be narcotized prior to fixation. Though it is not as necessary to narcotize them for section cutting as it is for dissection, it is never- theless desirable, since it appears that muscles fixed after narcotization (at least those of the Annelida) are not nearly as liable to become brittle as are those fixed in the violent state of contraction which results from dropping the worm directly into the fixative. Earthworms may be satisfactorily narcotized under the surface of water containing almost any known narcotic agent, and the choice between chloroform, chloral hydrate, or cocaine and its substitutes must rest largely upon the availability of the reagents. The worm may be considered adequately narcotized when it does not react rapidly when placed on a flat surface of filter paper and slowly stretched with the fingers. If it does not contract at all, and particularly if its outer DS 12.30 DYE STAINS OF GENERAL APPLICATION 331 surface presents an opaque white appear- ance, the worm may be considered dead and only fit to be thrown away. If it con- tracts strongly and develops lateral twist- ings, it is insufficiently narcotized. Any stage between these two may be con- sidered sufficient. It is much more convenient if the worm be killed in an extended condition. The simplest way to do this is with the aid of two blocks of glass lying on top of each other. Capillary attraction will hold a small worm quite satisfactorily into the angle of these two blocks, or it may be forced against the angle by a heavy piece of glass rod, if capillary attraction proves insufficient. The selection of the fixative is a matter of some dispute; the author himself prefers the antique picro-sulfuric acid of Kleinenberg 1879 (Chapter 18, F 5000.0050) which was developed for the purpose of fixing earthworms and is ad- mittedly worthless for any other purpose. It lends itself excellently, however, to afterstaining, and leaves the embedded worm less brittle than any other fixative which the writer has ever tried, with the possible exception of the irrational chromic-nitric mixture of Perenyi (F 6000.- 0040 Perenyi 1882). This also has the advantage of preventing the worm from becoming brittle. Whichever fixative is chosen, the worm should now be immersed in it to just about its own depth, until such time as it has partially hardened, i.e. enough to hold it straight, and should then be placed in a large volume of the fixative for whatever period is desired. If Kleinenberg is employed, 24 hours will be sufficient, but the solution of Perenyi should be allowed to act for from one to three days. Kleinenberg's fixative should be washed out in large volumes of 70% alcohol until no more color comes away. If Peren}^i's fixative is employed, running water may be used for the washing proc- ess. In either case, mercuric fixatives should be avoided, since considerable tampering with the specimen with steel instruments remains to be done, and this cannot be satisfactorily conducted after mercuric fixatives. After it is washed, the worm should be divided into pieces about three-eighths of an inch long. It is usually desirable to retain about half an inch of the anterior region for sagittal sections. The repro- ductive regions need only be saved in the unlikely event that they may be required for advanced classes. The segments should now be dehydrated in alcohol, or any other selected reagent, in the customary manner and cleared in oil of cedar. This reagent is selected because it does not render the segments brittle, yet docs make them sufficiently transparent for exami- nation by transmitted light with a dis- secting microscope (to disclose such sand particles as may still remain in the intes- tine). These show up clearly as brightly refractive objects, and must be removed by being pushed out of position with the bent end of a sharp needle. The experi- enced technician does not need to be told that the retention of a single one of these sand grains within the body of the worm will result in the destruction not only of the microtome knife but of a considerable volume of material which might otherwise be used for good sections. If any con- siderable mass of sand grains remains on the inside, it may often be removed by filling a hypodermic syringe with cedar oil and directing the jet of cedar oil against the inside of the intestine. If this is done, several changes of cedar oil should be used to make sure that the particles of sand dislodged from the inner surface of the intestine do not become reattached to the epidermis. The cedar oil should now be thoroughly removed in benzene (xylene tends to render the muscles brittle) since cedar oil itself tends to dilute the wax or to require too long a period of embedding for such muscularized tissues as those of the earthworm. The pieces are then embedded in paraffin and cut into 10-micron sections, Avhich may then be mounted, either individually or in such groups as may be desired on clean slides, and then flattened and dried. When the sections are dried they are deparaff^inized as usual and taken down through the various reagents to water, in which they may be accumulated in batches to be stained together. The selection of tlie iron hematoxylin of R^gaud (DS U.IU R^gaud 1910) in 332 METHODS AND FORMULAS DS 12.30 preference to any other is based only on the fact that it is a quicker process. If the operator does not mind waiting the re- quired 24 hours, there is no reason why he should not employ the solution of Heidenhaim or any other of the iron mordant techniques. The solutions re- quired for this staining method consist first of a 5% solution of ferric alum, second, of a glycerol-alcohol-hematoxylin solution, and third of a picric alcohol differentiating solution recommended by Masson himself for use before his tri- chrome methods. The first step is to raise both the ferric alum and the hematoxylin solutions to approximately 50°C. This temperature is not critical, but the rate of staining is dependent on temperature and decreases rapidly as the temperature drops below 50°C. Temperatures above 50°C. may cause the sections to fall off the slide. The first batch of sections is now taken from water and placed in the heated ferric alum solution for a period of 30 minutes. It is then removed from the ferric alum solu- tion, rinsed very rapidly in distilled water, and transferred to the heated hematoxylin solution for a further period of at least 30 minutes. On removal from the hema- toxyhn solution the sections should be blue-black. If they are not deeply stained enough, it is necessary to return them to the hematoxylin. The slides may now be rinsed briefly in distilled water and trans- ferred to tap water prior to differentiation. If relatively large runs are being taken through, it will usually be desirable to bring all the sections through their nuclear staining before proceeding either with the differentiation or the counterstain. The picric alcohol of Masson (ADS 21.1 Masson 1912), which is used for differenti- ating, is a relatively slow medium in com- parison with the ferric alum differentiation frequently recommended for iron hema- toxyUn stains. As, however, a somewhat acid afterstain is being employed in this instance, care should be taken that differ- entiation does not proceed too far. It will be quite sufficient to differentiate the background to a pale straw color (it will, of course, become blue in tap water) rather than to the absolutely colorless background which would be desirable were one using this technique to demonstrate chromosomes. As soon as the required degree of differentiation has taken place, the sections are returned to tap water and washed until no further picric acid leaves the specimen. It will be convenient for this purpose to have a fairly large dish in the sink through which a current of tap water is running continuously. A certain amount of differentiation will take place while this washing is going on, and the washing should not be discontinued until the nuclei are deep blue-black and the differentiated background has changed again from the very pale straw color to which it was turned by the picric differ- entiator to a pale blue. Failure to under- take this second bluing after differenti- ation is responsible for many of the failures to retain hematoxylin in the nucleus. The acid fuchsin-anilin blue stain, which is here selected as a counterstain, is one of the easiest to apply, provided the in- structions are followed closely. The solu- tions required are given under DS 12.31 Masson 1912a below; and it will be ob- served from the technique described that an acid environment must be retained throughout, even to the mounting medium. The first solution required is a mixture of acetic acid and acid fuchsin in water, to which the sections may be passed directly from the tap water, and in which they should not remain longer than five min- utes. Each batch of sections to be stained by this method must be taken through individually, for there is no stage of the proceedings between the original staining with acid fuchsin till the sections are dehy- drated in xylene at which a pause may be made for the accumulation of separate batches. After removal from the acid- fuchsin solution the sections will be seen to be stained deeply red, and they require only the quickest rinse in tap water before being placed in 1 % phosphomolybdic acid where considerable quantities of red dye will be removed. The time in this solution is not as critical as in many of the others, and the five minutes specified may be varied from, say, tlwee to eight minutes without any great damage to the speci- DS 12.30 DYE STAINS OF GENERAL APPLICATION 333 men. On removal from the phospliomo- lybdic acid it must be emphasized that no water rinse may be given. Each slide must be taken separately, drained, and the very deep blue "C" solution poured onto the surface of the slide where it may remain from two to five minutes. It may be men- tioned in parenthesis that this deep-blue stain has just as great an affinity for the human skin as for any other protein ma- terial, and that it is far more difficult to remove from the skin than it is from any section. Rubber gloves or forceps are most warmly recommended to the operator. The D solution (1% acetic acid) should be available in two vessels, one containing a fairly large volume (a 500-ml. beaker is admirable) and the other a standard coplin jar or whatever vessel is customarily employed for slide staining. The section is rinsed rapidly in the large container to remove the adherent blue, and then is placed in the smaller container until such time as no further color is seen to leave it. One of the best features of this triple stain of Masson is that one does not require to control differentiation, for if the technique has been followed properly to this stage, the blue will not leave the connective tissues in the acid solution. Modifications in which it is recommended to differenti- ated in alcohol should be avoided, since this reagent can remove the whole of the blue color. Even when the 1% aqueous acetic acid is used at this stage of the proceedings, it is undesirable to leave the slide for too long a period after the blue color has ceased to be liberated from it, because there is a tendency to remove the hematoxylin from the nuclei, and it is for this reason that the slides cannot be accumulated in this reagent. Tlie only delicate portion of the preparation now follows: tlie dehydration of the sections without the loss of the blue. Even after mordanting. in phosphomolybdic acid, the blue is hkely to be removed from the tissues with alcohol, and dehydration should be undertaken directly in absolute alcohol to which has been added 1 % acetic acid. Either amyl alcohol with 1 % acetic acid, or acetone with 1 % acetic acid, may be substituted for absolute alcohol and will render the dehydration of the speci- men much more easy without loss of the blue. There seems to be, liowever, a tendency to adhere to absolute alcohol on the part of histologists; and the alterna- tive reagents are only mentioned for the benefit of those who might be prepared to break with tradition. The least possible time should be employed in dehydration; preferably the slide should be dipped up and down in tlie alcohol and transferred to xylene at intervals to see whether ov not it is sufficiently dehydrated to clear. After they have been cleared in xylene, the shdes may be accumulated in this reagent until the whole of the batch is ready for mounting. Many workers have specified that sali- cylic acid be dissolved in the xylene to render it acid, but this is a precaution really only necessary if large quantities of alcohol are carried over into it. The normal provision of two or three changes of xylene will render the addition of sali- cylic acid unnecessary. Each slide is now mounted; the most strongly acid balsam which can be obtained is used ; or one may employ salicylic balsam. This does not necessitate making up a special mounting medium, since the easiest method of adding the acid to the balsam is from the coverslip. A saturated solution of salicylic acid is made in xylene and each coverslip is dipped in this and permitted to dry in air so as to become coated with a thin film of salicyhc acid. Ordinary xylene balsam, or any other resin dissolved in xylene, is then placed on the sections and one of the salicylic-acid-treated coverslips is placed on the surface. Within a moment or two the salicylic acid will have dis- solved in and dispersed through the mounting medium; that remaining on the upper surface can easily be wiped off after the slide is dry. Preparations prepared in this manner, with due attention to the maintenance of an acid environment, will be found to be quite permanent enough for use for many years in class, and will be such a great improvement over the sections of earth- worm usually supplied for class teaching purposes as more than to warrant the slight additional trouble which is required for their preparation. 334 METHODS AND FORMULAS DS 12.30 Preparation of a transverse section of the head of a mouse using an acid- alum hematoxylin stain (Masson 1934) followed by ponceau 2R-light green (Patay 1934) A large section of this type is required not so much for the demonstration of his- tological detail as for the demonstration to classes of the morphological relationship of the various parts of the head region. For this reason the fixative to be employed should be selected more on the basis of its abihty to penetrate large structures than in the hope that it will permit fine histo- logical differentiation of detail. It is, more- over, less necessary that the fine detail of histological structures be preserved by im- mediate fixation than that the mouse be so arranged as to permit the penetration of fixative to all parts. These considerations must therefore dictate both the manner in which the mouse is killed and that in which it is fixed. If there are available to the worker only the cruder methods of killing, the mouse should be left until rigor mortis has passed off before the different parts are arranged. Or alternatively, and far more satisfactorily, the mouse may be killed by the injection of sodium bar- bitol or some such reagent, in order that it may die in a perfectly relaxed condi- tion. The author has already expressed in several places his conviction that a mounter should experiment for himself with fixatives, rather than follow already estabhshed recommendations. But in the present case his own preference is very strongly for the mercuric-dichromate-ace- tic mixture of Zenker 1894 (Chapter 18, F 3700.0010). It must be remembered that this specimen will have to be decalcified and the use of the mercuric-dichromate is therefore to render the tissues as hard as possible and to prevent, as far as possible, the hydrolysis of the softer parts by the acids used for decalcification. If it is desired to secure the transverse section through the region of the eyes, the head should be severed from the body as soon as the mouse is dead, and about three-quarters of the lower jaw, the an- terior end of the tongue, and the anterior end of the snout should be removed with a pair of sharp scissors. A series of holes should then be drilled, or driven with a sharp point, into the skull in as many places as possible, leaving free from holes only a band approximately one-quarter of an inch wide in the exact region of the eye where the section is required. A band of thread should then be tied tightly around both the anterior and posterior cut sur- faces so as to hold the skin in position. Otherwise the differential contraction of the latter while in the fixative will cause it to become torn loose and to give a most unsightly appearance to the section. The more (and the more deeply) the holes are drilled the better will the fixative pene- trate and the subsequent section appear. Previous to these operations one should have prepared at least a liter of the re- quired fixative and to have placed this in a tall narrow jar. A liter-measuring cylinder, with a cork to fit, is excellent, for the pur- pose, although tall museum jars of approx- imately the same shape are frequently available. A staple or bent pin, should be driven into the underside of the cork, to which a thread or piece of string may be attached, on the end of which is hung the prepared head in a cheese cloth, or any loosely-woven bag. Fixation with a di- chromate solution is always best con- ducted in the dark, so the jar with the sus- pended head may now be placed in a dark cupboard and forgotten for a period of two or three weeks. The exact time of fixation is unimportant; several months could be allowed to elapse without any great risk of damaging the piece. Nothing, however, can be done with the piece if it is under- fixed, and even if there are a relatively large number of holes in it, two weeks is not too long a period for the fixative to penetrate. The head is now removed from the fixa- tive and taken out of its bag, and then washed in running water for at least 48 hours. If this can be done in the dark it will prevent the possibiUty of the chrom- ium being deposited as a dark-green layer over the outside, an event which would render after-staining difficult. One is now faced with the problem of decalcification, for which purpose any of the formulas given in Chapter 19 under DS 12.30 DYE STAINS OF GENERAL APPLICATION 335 AF 20 may be employed. The writer has a strong preference for a simple solution of picric acid, provided that the specimen has, as in this case, been prior-fixed in a dichromate or a mercuric mixture. The specimen may remain in a saturated solu- tion of picric acid for as long as is neces- sary to secure complete decalcification. Decalcification may be conducted in the same jar used for fixation, with precisely the same technique of suspending the object in a small loosely-woven cloth bag about one-third of the way down the bottle. It is only necessary to place an excess of picric acid in the bottom of the jar, to fill it up with water, and shake it a few times to produce saturation before inserting the cork bearing the specimen. In the case of the mouse, at least two months will be required to decalcify the skull, the test object being the inner ear, the bony protection of which is thicker than that of any other part of the cranium. This may be tested at intervals by re- moving the object from the decalcifying reagent and probing dehcately in the region of the inner ear with a fine needle. It is very easy to distinguish between the tough nature of a decalcified bone and the hard, sharp reaction which one obtains from endeavoring to press the point of a pin into an actual calcified structure. It is to be recommended, however, in the interest of safety, that a period of at least one additional week in the decalcifying reagent be allowed between the time when one imagines the head to be perfectly decalcified and the time when one decides to embed and section it. The great advan- tage of picric acid as a decalcifying agent is that one cannot leave the specimen in it for too long a period; no harm whatever can be occasioned to the head of the mouse if it be left for even a year or two in the reagent. After removal from the picric acid, the head is washed in running water for two or three days until it ceases to liberate any j-ellow color. It cannot be made en- tirely white, because compounds will be formed between the picric acid and the protoplasm. Three days' washing, for an object of the size described, should be ample. The specimen is then dehydrated. embedded, and sectioned. No special pre- cautions are necessary save to remember that the dehydrating agent, the clearing agent, and the wax will penetrate slowly through so large and so tough an object. It is therefore desirable in embedding first to impregnate the object thoroughly with a saturated solution of wax in the selected clearing agent and then to evaporate the clearing agent off slowly, thus gradually increasing the concentration of the wax. A low-melting-point wax is better than a high melting point for preventing the hardening of the tissues, since it may be presumed that a 12- to 15-micron section will be cut. It is unwise to loosen the string holding the skin in place until at least the last change has been made in the clearing reagent. At this point, a sec- tion for final embedding, about a quarter to three-eights of an inch thick may be cut through the object with a fine saw, although the writer prefers to leave the final trimming of the object until it is in the paraffin block. When the paraffin block has been pre- pared, it should be trimmed down roughly with a knife until such time as one can clearly see the eyes and thus distinguish the region of which it is desired to cut a section. The whole front end to within a millimeter or two of this can now be cut off with a fine saw. It is almost impossible to trim off these large masses with a knife without either cracking the paraffin block or so dragging the object within the paraffin that the hold of the embedding agent is loosened. Section cutting presents no special diffi- culty, the more so as it is not anticipated that one intends to mount ribbons, but only to select individual sections. If these large sections have a tendency to roll up on the knife, wet the blade of the knife with 70% alcohol in order to hold down the beginning of the ribbon, and then cut as many sections as are desired. The closer one trims the block to the region which one desires to section, the less necessity will there be to substitute a freshly sharpened knife before taking off the sections from the actual regions re- quired. Sections are flattened as usual, though it is recommended that they be 336 METHODS AND FORMULAS DS 12.30-DS 12.31 rolled into position on the slide to avoid the almost inevitable curling which will result from the absorption of water by sections of this very large area and their subsequent swelling within the bounds of the paraffin which retains them. The sections are then dewaxed in the ordinary manner and passed to water to await staining. The stains which are required in the present instance are the acid alum hema- toxylin of Masson (DS 11.123 Masson 1934) and the three solutions specified by Patay 1934 for his triple staining method (DS 12.32 Patay 1934). A description has already been given (DS 11.10 typical preparations) of the method by which an acid alum hematoxylin of this kind can be utilized to secure a sharp and clear differ- ential staining of nuclei. In the present instance, however, a diffuse blue stain, particularly of cartilaginous areas, is pre- ferred to a sharp definition of nuclei. One need, therefore, have no hesitation in placing the sections directly into this stain where they may remain overnight or for such period of time as is convenient to the operator. They will usualh' be satis- factorily stained within an hour and some- times in less time ; though this depends so much upon the time which they have spent in previous solutions, that it cannot be forecast with accuracy. When the sec- tions are removed from hematoxyhn they should be differentiated in a 0.1 % solution of hydrochloric acid in 70% alcohol until the nuclei and the matrix of the cartilage, wliich will be clearly visible in large areas, alone remain blue. It is more damaging to over-differentiate than to under-diff erentiate . When differentiation is complete, the sections should be accumulated in a jar of alkaline tap water (rendered alkaline when necessary by the addition of sodium bicarbonate) until they have turned from dull purple to clear blue. The sections are now taken from the water and j^assed to the 1% solution of ponceau 2R for a period of two minutes, briefly rinsed in water, passed into 1% phosphomolybdic acid for approximately two minutes, or until such time as the cartilage is freed from the red stain, rinsed again briefly in water, and then dipped two or three times in 90*^0 alcohol to remove the exces- sive water before being placed for about 30 seconds in the ^2% solution of fight green in 90 ';o alcohol, which constitutes the final stain of the series. The section should then be passed into absolute alco- hol until such time as no further green color comes away before being passed to xylene and mounted in balsam. This method of triple staining is one of the most foolproof and satisfactory of any known to the author, and it is particularly applicable to those structures which con- tain bone as well as cartilage. 12.31 TECHNIQUES EMPLOYING THE PHOSPHOTUNGSTIC (-MOLYBDIC-) REACTION WITH ACID FUCHSIN 12.31 Brillmeyer 1929 acid fuchsin-anilin blue-orange G 11571b, 12:122 REAGENTS required: .4. 0.2% acid fuchsin; B. water 100, phosphomolybdic acid 1, anilin blue 0.5, orange G 2.0 method: [blue nuclei (original specifies DS 11.122 Delafield 1885)] -♦.4,1 min. — > drain — > B, 2-3 hrs. — » water, quick wash -^ balsam, via usual reagents note: Weiss 1932 (20540b, 7:131) differs only in the dilution of A to 0.04% and in the substitution of 4 minutes for 3 hours immersion in B. DS 11.122 Mayer 1901 is recommended for prior staining of sections from picric fixed or mordanted material. 12.31 Crossmon 1937 acid fuchsin-orange G-light green {or -anHin blue) 763, 69 :33 reagents required: A. water 100, acetic acid 1, acid fuchsin 0.3, orange G 0.13, thymol 0.06; B. 1% phosphomolybdic acid; C. either water 100, acetic acid 1, light green 1 or water 100, acetic acid 2, aniline bhxe 2; D. 1% acetic acid method: [sections, nuclei hematoxyUn-stained] — > water —> A, 1 min. — > rinse -^ B, till collagen decolorized — > quick rinse -^ C, 5 mins. — > rinse -^ D, till differentiated —* rinse -> abs. ale. — > balsam, via xylene DS 12.31 DYE STAINS OF GENERAL APPLICATION 337 12.31 Goldner 1938 acid fuchsin-ponceau 3R-oran thorough wash — » A, 5 mins. -^ B, wash — > (!, till collagen decolorized -> H, rinse — ♦ D, 5 niins. —* H, 5 mins. — > blot — > abs. ale, least possible time — ♦ balsam, via xylene result: general cytoplasm, red; erythrocytes, orange; collagen, green. note: Other color combinations may be obtained by substituting for A ai)ove either water 100, azophloxin 0.5, acetic acid 0.2 or water 100, acid fuchsin 0.025, ponceau 2R 0.075, azophloxine 0.01, acetic acid 0.2. For a further modification see I^S 13.7 Romcis 1948. 12.31 Haythorne 1916 acid fuchsin-orange G-anilin blue 4349, 6 :61 REAGENTS REQUIRED: A. water 100, hydrochloric acid 0.06, 95% ale. 4, orange G 0.8, ferric alum 5; B. 0.5% acid fuchsin; C. sat. aq. sol. phosphomolybdic acid 100, anilin blue 2.5, orange G 2.5 preparation of A: Dissolve the orange G in 70 water with the ale. and acid. Dissolve the alum in 25 water and add to the dye solution. Filter. method: [sections of F 3700.0010 Zenker 1894 fixed material after 30 mins. staining in DS 11.122 Bohmer 1868] -> water — >• A, 2 mins. -^ water, 5 mins. -^ B, 3 mins. -^ blot —> C, 20 mins. —> blot — » 95% ale, quick rinse —> abs. ale, from drop bottle, till differentiated — > balsam, via xylene result: nuclei, reddish black; cartilage, white fibrous tissue, blue; keratin, chitin, erythrocytes, bright orange; muscle, red. 12.31 Laidlaw see DS 23.11 Laidlaw (1936) 12.31 Lendrum and McFarlane 1940 picro-orange G-acid fuchsin-ponceau 2R-anilin blue 11431, 50:381 reagents required: A. water 20, 95% ale. 80, picric acid 1, orange G 0.2; B. water 99, acetic acid 1, acid fuchsin 0.5, ponceau 2R 0.5, sodium sulfate 0.25; C. 1% acetic acid; D. 1% phosphomolybdic acid; E. water 99, acetic acid 1, anilin blue 2 method: [.sections with nuclei stained by DS 11.41 technique] — > water — * A, 2 mins. — » overnight — > rinse — > B, 1-5 mins. — > C, rinse — > D, till collagen not quite decolorized — > E, 2-10 mins. -^ C, rinse -^ balsam, via usual reagents note: Fast green FCF may be substituted for anilin blue in E above. 12.31 Lillie 1940 Biebrich scarlet-fast green 20540b, 15:21 reagents required: A. water 99, acetic acid 1, Biebrich scarlet 1; B. water 100, phos- photungstic acid 2.5, phosphomolybdic acid 2.5; C. water 97.5, acetic acid 2.5, fast green FCF 2.5; D. 1% acetic acid method: [sections with blue nuclei (original specifies DS 11.121 Weigert 1903)] — > water — > A, 2 mins. — > rinse — > B, 1 min. -» C, 2 mins. -» Z), 1 min. or till differentiated -^ balsam, via acetone and xylene 12.31 Masson 1912a acid fuchsin-anilin blue 4956,87:290 reagents required: A. water 100, acetic acid 0.5, acid fuchsin 0.5; B. 1% phospho- molybdic acid; C. water 100, acetic acid 2.5, anilin blue to sat.; D. 1% acetic acid; E. 0.1% acetic acid in abs. ale. method: [sections (original required prior staining in DS 11.111 R6gaud 1910)] —^ water —* A, 5 mins. -^ water, quick rinse — » B, 5 mins. -^ drain -^ C, poured on slide, 2-5 mins. -^ D, till differentiated 5-30 mins. — > E, till dehydrated -^ salicylic-xylene —>■ salicylic balsam result: nuclei, black (if prior stained in Rdgaud) or deep red ; coUagens, light blue; bone, dark blue; epithelia, muscle, some glands, light red; erythrocytes, orange; nervous tissues, violet. note: a detailed description of the use of this stain is given under 12.30 above. 338 METHODS AND FORMULAS DS 12.31 12.31 Masson 1912b acid fuchsin-ponceau 2R-anilin blue 4956, 87 :290 REAGENTS REQUIRED: A. water 100, acetic acid 1, acid fuchsin 0.35, ponceau 2R 0.65; B, C, D, E as Masson 1912a above method: as Masson 1912a above 12.31 Masson 1912c acid fuchsin-metanil yellow 4956, 87 :290 REAGENTS REQUIRED: A. Water 1, acetic acid 1, acid fuchsin 100; B. 1% phosphomolyb- dic acid; C. sat. sol. {circ. 6%) metanil yellow; D. 1% acetic acid method: [sections (original required prior staining in DS 11.111 Regaud 1910)] — >• water -^ A, 5 mins. — > water, quick rinse -^ B, 5 mins. — > drain —> C, poured on .slide, 5 mins. -^ D, 5 mins. -^ salicylic balsam, via usual reagents 12.31 Masson 1912 see cdso DS 12.32 Masson 1912 and DS 13.41 Masson 1912 12.31 McFarlane 1944a picro-acid fuchsin-anilin blue 20540b, 19:29 reagents required: A. water 98, acetic acid 2, picric acid 0.2, phosphotungstic acid 1, acid fuchsin 1, anilin blue 2; B. 2% acetic acid; C. water 90, 95% ale. 10, picric acid 0.25, phosphotungstic acid 2.5 method: [sections with blue nuclei] -^ water ^ ^, 5 mins. — > B, rinse — > C, till differ- entiated — > D, 1 min. -^ B, wash -^ balsam, via usual reagents 12.31 McFarlane 1944b picro-acid fuchsin-anilin blue 20540b, 19 :23 reagents required: A. water 98, acetic acid 2, acid fuchsin 0.8, picric acid 0.2; B.2% acetic acid; C. water 60, 95% ale. 40, picric acid 1, phosphotungstic acid 10; D. water 97.5, acetic acid 2.5, anilin blue 2.5; E. water 90, 95% ale. 10, picric acid 0.25, phos- photungstic acid 2.5 method: [sections with DS 11.121 stained nuclei] — » water — > A, 5 mins. — » B, rinse — * C, 5 mins. -^ rinse — > D, 5-10 mins. — > B, rinse — > £", 5 mins. -^ B, wash — » balsam, via usual reagents 12.31 McFarlane 1944c picro-orange G-acid fuchsin-ponceau 2R-anilin blue 20540b, 19:23 reagents required: A. water 20, 95% ale. 80, picric acid 1, orange G 0.25; B. water 99, acetic acid 1, acid fuchsin 0.25, ponceau 2R 0.25; C. 2% acetic acid; D. water 20, 95% ale. 80, picric acid 1, phosphotungstic acid 10; E. water 97.5, acetic acid 2.5, anilin blue 2.5; F. water 80, 95% ale. 20, picric acid 0.5, phosphotungstic acid 5 method: [sections, stained but not differentiated, in DS 11.111 Regaud 1910] —> rinse — » A, till nuclei differentiated -^ wash, till only erythrocytes yellow -^ B, 5-10 mins. -^ C, rinse — > Z), 5 mins., till differentiated —>■ C, rinse — > E, 10 mins. — > C, rinse -^ F, till differentiated -^ C, wash -^ balsam, via usual reagents 12.31 Papanicolaou 1941 see DS 23.4 Papanicolaou 1941 12.31 Pasini test. 1928 Hill eosin B-acid fuchsin-anilin blue-orcein Gatenby and Cowdry 1928, 280 reagents required: A. 2% phosphotungstic acid; B. 50% ale. 35, glycerol 40, eosin B 0.7, DS 12.216 Unna (1928) 35, sat. aq. sol. acid fuchsin 4 preparation of b: Dissolve the eosin in 35 50% ale. Add, in order, the Unna's stain, fuchsin solution, and glycerol. method: [sections with blue nuclei (original specifies DS 11.123 Ehrlich 1886)] —* water — > A, 10 mins. — *• water, quick rinse -^ B, 15-20 mins. — > 70% ale. quick rinse —> abs. ale, 20 sees. -^ A, 5 sees. -^ abs. ale. till differentiated -^ balsam, via xylene result: coUagen, blue; elastic fibers, purple; erythrocytes, bright orange. note: See also DS 13.42 Walter 1930. 12.31 PoUak 1944 orange G-light green-ponceau 2R-acid fuchsin 1887a, 37 :294 reagents required: A. water 50, 95% ale. 50, acetic acid 1, phosphotungstic acid 0.5, phosphomolybdic acid 0.5, orange G 0.25, light green SF 0.15, ponceau 2R 0.33, acid fuchsin 0.17; B. 0.2% acetic acid DS 12.31-DS 12.32 DYE STAINS OF GENERAL APPLICATION 339 PREPARATION OF A'. Mix Water, ale, and acetic acid. Divide into four portions. In first dissolve ))h()si)honiolyb(lic acid with licat; in second dissolve pliosphotiingstic acid and Orange G; in third dissolve light green; in fourth dissolve acid fuchsin and ponceau 2R. Mix and filter. method: [sections with blue nuclei] -^ water -^ A, 3-7 mins. —> B, till differentiated — + 95% ale, till dehydrated — > balsam, via usual reagents 12.31 Wallart and Honette 1934 (iri C, dropped on from pii)et, 30 sees. — > abs. ale. —* xylene — > balsam result: black nuclei, red cytoplasm, yellow collagen, pink clastin. 12.31 Weiss 1932 see DS 12.31 Brillmeyer 1929 (note) 12.32 TECHNIQUES EMPLOYING THE PHOSPHOTUNGSTIC (-MOLYBDIC-) REACTION WITH OTHER DYES 12.32 Dupres 1935 toluidine blue-orange G 14425,46:46 REAGENTS REQUIRED: A. 1% phosphomolybdic acid; B. water 100, toluidine blue 0.25, orange G 4, oxalic acid 4 method: [red nuclei (original specifies DS 11.43 Dupres 1935)] -^ A, 10 mins. -^ water, thorough wash — > B, 2-5 mins. — > drain -^95% ale. till differentiated —^ balsam, via usual reagents result: nuclei, red; collagens, blue; bone, dark green; muscle, light green; erythrocytes, bright orange; epidermis, blue; layer of Malpighi, orange; hair, etc., bright red. note: Dupres 1935 {loc. cil.) also recommends methyl green in place of toluidine blue in B above. 12.32 Gomori 1950 chromolrope-fast green Tech. Bull, 20:77 reagents required: A. water 100, acetic acid 1, chromotrope 2R 0.6, fast green FCF 0.3, phosphotungstic acid 0.6; B. 0.2 acetic acid method: [smears, or sections not more than 5 /i thick, prior stained in hematoxylin] — > water — > A, 5-20 mins. —> B, rinse — > balsam, via usual reagents note: Wheatley 1951 {Tech. Bull., 21:92) recommends this technique for protozoans in intestinal smears. 12.32 Hollands 1912 magenta-orange G-lighi green 1915,10:62 reagents required: ^4. 1% magenta in 70% ale; B. 0.1% hydrochloric acid in 70% ale; C. 1% phosphomolybdic acid; D. sat. sol. {circ. 11%) orange G; E. 0.2% light green method: [blue nuclei (Langeron 1942, 606 recommends DS 11.122 methods)] — » water — > A, 6-12 hrs. — > water, 5 mins. -^ B, till color clouds cease, few seconds -^ water, thorough wash -^ C, 5 mins. -^ water, rinse — > D, 5 mins. — > E, poured on slide 3*^ to 1 min. -^ 95% ale, few sees., till differentiated —* balsam, via amyl ale and benzene result: resting nuclei, blue; mitotic figures, red; cartilage, purple; fibrous tissue, light green; erythrocytes and keratin, bright orange. 12.32 Koneff 1936 see DS 13.7 Koneff 193G 12.32 Lillie 1940 see DS 12.31 Lillie 1940 12.32 Masson 1912 ponceau 2R-anilin blue 4956, 87 :290 reagents required: A. water 100, acetic acid 1, ponceau 2R 1; B; C; I); E, as DS 12.31 Masson 1912a method: as DS 12.31 Masson 1912a 12.32 Patay 1934 ponceau 2R-light green 4285a, 11:408 reagents required: A. 1% ponceau 2R; B. 1% phosphomolybdic acid; C. 0.5% liglit green in 90% ale 340 METHODS AND FORMULAS DS 12.33 method: [blue nuclei (original recommends DS 11.123 Masson (1934) insufficiently differentiated)] -^ water—* A, 2 mins. — > water, brief rinse -^ B, 2 mins. — > water, brief rinse — * C, 30 sees. — * balsam, via usual reagents result: cartilage, blue (from hematoxylin); other coUagens, light green; bone, brilliant green; epithelia and muscle, orange; erythrocytes, yellow; nervous tissue, gray. note: In the opinion of the author this is the finest triple stain yet described. A detailed description of the use of this stain is given under 12.30 above. 12.33 OTHER COMPLEX CONTRASTS 12.33 Blank 1942 see DS 13.22 Blank 1942 12.33 Flemming 1891 gentian violet-orange G 1780, 37 :249 KEAGENTS REQUIRED: A. 1% gentian violet; B. sat. sol. (circ. 11%) orange G method: [sections of Flemming 1882 or other F 1600.0000 or F 1600.0010 fixed material nuclei stained by DS 11.42 method]-* water-* A, 3 hrs. — > quick wash — > B, few moments — > abs. ale, till no more color comes away — > balsam, via clove oil note: Johansen (Johansen 1940, 84) substitutes a saturated solution of orange G in clove oil for B, above. For Stockwell's Variation see DS 13.5 Stockwell 1934. 12.33 Hubin 1928 eosin Y-orange G-safranin 1825, 37 :25 reagents required: A. 0.1% acetic acid; B. abs. ale. 70, water 30, eosin Y 0.1, orange II 0.2, safranin 0.2; C. 0.1%, hydrochloric acid in 70%, ale. preparation of b: Dissolve the eosin in 50 ale. and 10 water. Add to this the orange dissolved in 10 water. To this add the safranin dissolved in 20 ale. and 10 water. method: [blue nuclei (original specifies DS 11.122 Carazzi 1911 used on sections of F 5000.1040 Hollande 1911 fixed and F 5000.1010 Bouin 1897 mordanted material)] -* A, 4-5 sees. — * 70% ale., 5 mins. -^ B, 1-3 mins. — > C, till differentiated, 2 to 10 sees. -^ balsam, via usual reagents result: nuclei, cartilage, bone, blue; nerves and blood vessels, yellow; muscles and ganglia, brown. 12.33 Johansen 1940 see DS 12.33 Flemming 1891 (note) 12.33 Lendrum 1939 GEEP stain— and. 11431,49:590 reagents required: A. water 50, 95% ale. 50, eosin Y 0.2, erythrosin 0.2, phloxine 0.2, gallic acid 0.5, sodium salicylate 0.5; B. sat. sol. tartrazine N.S. in ethylene glycol monoethyl ether preparation of a: Add the dyes dissolved in the ale. to the other ingredients dissolved in water. method: [sections with hematoxylin-stained nuclei] — > A, 2 hrs. ^95% ale, rinse — > B. on slide, till color balance satisfactory 12.33 Lendrum 1947 11431,59:394 REAGENTS REQUIRED: A. water 100, calcium chloride 0.5, phloxine 0.5; B. sat. sol. tartrazine in ethylene glycol monoethyl ether method: [sections with blue nuclei] — > A, 30 mins. -* rinse -^ B, from drop bottle, till differentiated -* balsam, via usual reagents 12.33 Lillie 1940 Biebrich scarlet-picro-anilin blue 1789, 29 :705 REAGENTS REQUIRED: A. water 99, acetic acid 1, Biebrich scarlet 0.1; B. water 100, picric acid 1, anilin blue 0.1; C. 1% acetic acid method: [sections, nuclei stained in hematoxylin] -^ water — » A, 4 mins. — > rinse — » B, 4 mins. -^ C, 3 mins. — » salicylic balsam, via usual reagents 12.33 Lillie 1948 see DS 13.7 LUlie 1948 12.33 Margolena 1933 phloxine-orange G 20540b, 8:157 reagents required: A. 0.5% phloxine in 20% ale; B. 0.5% orange G in 95% ale. method: [blue nuclei] —» water —> A, 1-5 mins. —» 70% ale, thorough rinse ^ B, dropped on slide, H to 1 min. — > abs. ale, till no more color comes away -^ balsam, via usual reagents 12.33 Masson 1929 metanil yellow-picro-fuchsin 11571b, 12:75 REAGENTS REQUIRED: A. water 100, acetic acid 0.5, metanil yellow 0.5; B. 0.2% acetic acid; C. 3% potassium dichromate; D. DS 11.221 van Gieson 1896; E. 1% acetic acid DS 12.33-DS 13 DYE STAINS OF GENERAL APPLICATION 341 method: [sections with blue nuclei (original requires DS 11.111 R^gaud 1910)] -^ water -^ A, 5 mins. — > B, rinse — > C, 5 mins. — >• D, poured on slide still wet with C, 2 mins. —y E, till yellow clouds cease -^ salicylic balsam, via usual reagents result: collagens, clear red. 12.33 Masson 1911 erythrosin-saffron 6630, 70:573 REAGENTS REQUIRED: A. water 100, erythrosin 1, 40% formaldehyde 0.25; B. water 100, saffron 2, 40% formaldehyde 1, 5% tannic acid 1 PREPARATION OF B : Boil saffron 1 hour in water. Cool. Filter. Add other ingredients. method: D-Aue nuclei] -^> ^, 5 mins. -^ water, quick rinse— » 70% ale, till collagens colorless -^ water, quick rinse -^ B, 5 mins. -+ water, rapid rinse — > abs. ale, mini- mum time possible — » balsam, via xylene result: nuclei, blue; collagens, yellow; muscle, red. 12.33 Maximow 1909 eosin Y-azur II 23632,26:177 STOCK FORM I las: I. 0.1% cosiu Y; II. 0.1% azur II WORKING solution: water 100, stock I 10, stock II 10 method: [sections with chromatin stained in very dilute DS 11.122 formula] -^ water, 24 hrs. -> stain, 12-24 hrs. -^ 95% ale, quick rinse -* abs. ale, till differentiated -^ neutral balsam, via X3'lene 12.33 Millet 1926 acid fuchsin-martius yellow 4285a, 3:2 REAGENTS REQUIRED: A. 5% acid fuchsin in 40% ale; B. 5% martins yellow in 40% ale. method: [blue nuclei] -* A, 5 mins. at 30°C. — » 40% ale, till no more color comes away — > fi, 5 mins. -^ balsam, via usual reagents note: This method was developed for insect histology, for which it is excellent. 12.33 Pianese 1896 malachite green-acid fuchsin-martius yellow 2526, 1:193 formula: water 75, 95%, ale 25, malachite green 0.25, acid fuchsin 0.05, martius yellow 0.005 12.33 Reinke 1894 gentian violet-orange G 1780, 44:262 formula: a. sat. sol. {circ. 1%) gentian violet 25, sat. sol. {circ. 11%) orange G 0.2, water 75 note: Use after safranin nuclear staining (DS 11.421) and differentiate with clove oil 12.33 Scriban 1924 picro-fuchsin-brilliant green 6630,90:531 reagents required: A. water 100, picric acid 0.5, acid fuchsin 0.1; B. 60% ale 100, picric acid 0.2, brilliant green 0.1. method: [sections with DS 11.111 stained nuclei] -^ thorough wash-* A, 3-4 sees. -» abs. ale, wash -^ B, 3-4 sees. -^ abs. ale, wash -^ balsam via usual reagents 12.33 Stockwell 1934 see DS 13.5 Stockwell 1934 12.33 de Winiwarter 1923 see DS 12.33 de Winiwarter and Sainmont 1908 12.33 de Winiwarter and Sainmont 1908 crystal violet-orange G 23632, 25:157 reagents required: A. 1% crystal violet; B. sat. sol. (circ. 11%) orange G; C. 0.1%, HCl in abs. ale method: [sections of F 1600.0000 or F 1600.0010 fixed material; nuclei stained by 11.42 method] -^ water -» ^, 24 hrs. -* brief rinse -y B, 1 min. -* C, 2 to 3 hrs. -* clove oil, till differentiated -^ balsam note: de Winiwarter 1923 (1825, 32 :329) recommends a process which differs only in the substitution of 0.2% orange G for B, above. 13 COMPLEX TECHNIQUES INVOLVING BOTH NUCLEAR AND PLASMA STAINING This is a large class of staining tech- elements of the background are differ- niques, involving those methods in which, entially stained. They are divided broadly, by a series of successive and interlocking for purposes of this work, into seven operations, both the nuclei and all the classes, of which the first two (DS 13.1 342 METHODS AND FORMULAS DS 13.1 and 13.2) employ the thiazins and their related compounds either in combination with eosin, or in the next class, with such other dyes as have been employed. The next class (DS 13.3) takes up the methyl green combinations and is followed by the group (DS 13.4) of complex formulas in which the Mallory reaction is employed. This is followed by a small group (DS 13.5), largely of French origin, in which safranin is employed as the nuclear stain, and another small group (DS 13.6) in which hematoxylin is employed. This still leaves for the last class (DS 13.7) a considerable miscellaneous group. Most of these complex techniques are better employed for class demonstration than for research purposes. This does not apply with any force to the meth3dene blue-eosinates, so widely employed in blood staining, in which the material to be stained is customarily an unfixed, heat- dried smear, the staining reactions of the constituents of which can be controlled by the accurate adjustment of the pH of the staining solution. When, however, as in the other classes, the staining method is intended to operate on materials which have been fixed and sectioned, there is the greatest difficulty in securing a reproduci- bihty of results, and the utmost attention should be paid to the recommendations of the original author with regard to the fixative to be employed. When, as in the case of the methylene blue-eosinates, the entire staining materials are usually ap- plied from a single solution, there is nothing save the isoelectric point of the proteins on which they act which controls the differential staining. This factor of lack of reproducibility of results tends to render dangerous a publication of research observations based only on these staining methods for the slightest variation in the material, source of material, or fixative, and would cause anyone endeavoring to check the results to come to conclude that the method described is not applicable to his case. There is no finer group of stains which may be employed for class teaching purposes, particularly in sections of em- bryos and the like where a great range of tissues are available. Even in this instance, however, care should be taken that the entire batch of sections required is pre- pared at the same time, or students may become confused by the apparent abnor- malities presented by successive batches of material. 13.1 Techniques Employing THE "EOSINATES" OF THE ThIAZINS WITHOUT Other Admixture The eosiimtes of methylene blue and its oxidation products are compound dyes which are usually utilized in solution in methanol. They may, however, be pre- pared either directly in solution or in the form of the dry powder, and in the latter state are at present widely found in com- merce. For the purposes of the present work these techniques are divided into three classes. First (DS 13.11) are the straight methylene blue-eosinates, among which the original formulas of Jenner 1899 and May-Griinwald 1902 are the best known. These two techniques are not very widely used today, save as a preliminary to staining with other mixed polychrome methylene blue-eosinates. An interesting variation of these is the method of Sabrazes 1911, in which the stain is pre- pared in actual contact with the object to be colored. The next class of these stains (DS 13.12) is usually incorrectly identified as Giemsa stains, though to the parasitologist the formulas of Leishman 1901 and Roma- nowski are possibly better known. These formulas are irrational, for they contain the eosinates of polychrome methylene blue, which is itself a mixture of varying composition, and which often widely varies in nature even though the method of preparation is specified. Giemsa 1902 prepared his mixture from ingredients of known composition. His formula is there- fore given in the next section. The most logical stains to use are those of the third class (DS 13.13), which are the eosinates of methylene blue and its varying oxidation products. They are pre- pared, however, from reagents of known composition. The formula of Giemsa 1902 is the best known of these, but the name has unfortunately been used to describe almost any azur-eosinate without specific reference to the exact azur intended. The DS 13.1-DS 13.10 DYE STAINS OF GENERAL APPLICATION 343 original formula by Giemsa is'no more than a two-lino footnote to a papor in a sonio- what obscure journal, and both tlie dry powder in commerce and the solutions recommended by subsequent wi'iters vary enormously from the original proportion specified by Cliemsa. A combination of Giemsa 1902 and May-Griinwald 1902 was published by Pappenheim 1908 and has become widely disseminated through the hterature as the panoptic method. Two other modifications of the same tech- nique, published by Pappenheim four years later, are still widely referred to in the literature as panoptic, though tlie majority of those using this term refer actually to the 1908 formula. Some of these techniques have been rationahzed, particularly one by MacNeal 1922 which lias become so widespread that the dry stains in the proportion given for Mac- Neal's solution have appeared in com- merce under the name MacNeaVs Tetra- chrome. This is the more unfortunate as there are only three stains involved. The most completel}' rationalized of all these processes is that of Kingsley 1935. He specifies not only the ingredients to be used but also the pH to whicli the solu- tions should be buffered. He has, more- over, provided techniques whereby these same solutions can be applied either to smears, paraffin sections, or frozen sec- tions, and has thus permitted for the first time the complete correlation of the temporary results obtained from a frozen section hurriedly produced in the course of an operation and a permanent paraffin section which may be secured from post- mortem material. 13.10 TYPICAL EXAMPLES Preparation of a blood smear using the methylene blue-azur A-methylene violet-eosin Y stain of Kingsley 1935 All of the azur-eosin techniques ai'e de- signed primarily to differentiate cell types one from another rather than to differ- entiate gross histological structures. There is, therefore, very little need for detailed instructions to be given. A blood film is as good an object on which to practice as any other. In spite of the numerous formu- las which are given in this section it cnn- uot be too strongly recommended that the method of Kingsley (D8 13.13 Kingsley 1935) be used exclusively, unless one is endeavoring only to follow a diagnostic method given l)y a previous writer, or endeavoring to understand how some previous author has secured a result which one is unable to dui)li('ate by a more rational metliod. The oidy diliiculty in the method of Kingsley here given is the preparation of the necessary solutions: these tnust be made with chemically pure reagents. Tlie two stock formulas are quite stable, but they must be prepared accurately. Tlie figures for the dye con- tents of the solutions, which are given in terms of milligrams, should be adhered to and should be weighed on an accurate balance. Both the formulas are, however, liable to a certain degree of biological degradation from molds, and it is, there- fore, not desirable to prepare very large quantities at one time. The reference to a buffer at pH 6.9 does not specify the buffer salts to be used. Kingsley {loc. cit.) specifies phosphate buffers, but the writer has used phthalate buffers with ecjual success. The ver}' small quantity of the l)uffer required and the necessity for having it at an accurate pH suggests that (unless very considerable facilities are at the disposal of the preparator) he purchase this buffer solution ready-prepared rather than prepare it himself. The methanol, acetone, and glycerol specified as staining ingredients should be of a reagent grade. Since the working solution C requires only half a milligram of eosin Y to be added to it, and since it is improbable that the majority of people have the facilities for weighing this accurately, 1 % solution of eosin Y in acetone can be prepared as a stock solution and a 1 % dilution of acetic acid in acetone may be prepared as another stock solution. A moment of calculation will illustrate how these can be diluted with acetone to give the re- quired staining reagent. Blood films are simple to make, pro- vided one has chemically clean glassware and remembers, if he has never made such a preparation before, tliat the smear must 344 METHODS AND FORMULAS DS 13.11 be pushed across the lower slide, not pulled across it. That is, one should lay one chemically clean slide on the bench, place a small drop of blood on it, and place a second slide in contact with the first at an angle of about 45° so that the blood is drawn out by the capillary attraction into the angle. The upper shde is now pushed forward (Fig. 29, Chapter 7) leaving be- hind it a thin smear of blood which is then air dried. The blood film is fixed either by placing it in chemically pure methanol for from one-half to one minute, or by pouring two or three doses of chemically pure methanol over it. After removal from methanol, the slide is dried, and the stock staining solution A (DS 13.13 Kingsley 1935, below) is then poured on it and left for a period of from five to eight minutes. This time is, of course, too long for ordi- nary rapid diagnostic work, but has the advantage that slides may be accumu- lated in a long fine. Then one may con- tinue the process on the first slide, when one has spent five minutes in preparing the series. After tlie required time has elapsed, the stain is washed off with a jet of distilled water directed on it from a wash bottle in such a manner as to float off the scum which has gathered on the surface as well as to rinse off the su- perfluous stain. The film may then either be air dried for examination, or it may be mounted in a neutral mounting me- dium (see Chapter 26) after dehydration through direct drying. Provided the stain- ing solution has been accurately made and pure reagents used, a perfect stain wiU in every instance result from this treatment. The methods given for paraffin sections are just as easy to follow and need not be elaborated here. 13.11 METHYLENE BLUE-EOSINATES 13.11 Assmann 1906a test. 1928 Schmorl Schmorl 1928, 241 REAGENTS REQUIRED: ^4. DS 13.11 May-Grunwald 1902 (working sol.); B. water 100, DS 11.44 Unna 1892 method: [dried smear] -^> A, 1 ml. poured on slide lying in petri dish, 3 mins. 15 ml. poured into dish around slide, 3-4 mins. -^ wash — > dry B. 13.11 Assmann 1906b test. 1928 Schmorl Schmorl 1928, 246 REAGENTS REQUIRED: A. DS 13.11 May-Grunwald 1902 (working sol.); B. 0.001% acetic acid method: [sections of F 7000.0000 Miiller 1859 or F 3700.0010 Zenker 1894 fixed ma- terial] — » A, several hrs. -^ B, till color changes to clear eosin -^ wash — > balsam, via usual reagents 13.11 Chenzinsky 1894 23632, 11 :269 formula: sat. sol. (circ. 4.5%) methylene blue 40, 0.5% eosin Y in 70% ale. 20, water 20 method: [fresh smear] — * stain, 5 mins. -^ water, rinse —>-^ dry 13.11 Ellerman 1919 23632, 36:56 reagents required: A. water 100, 40% formaldehyde 5, eosin Y 1; B. DS 13.11 May- Griinwald 1902 (working sol.) 50, water 50 method: [5 M paraffin sections of F 3700.1000 Ellerman 1919 fixed material] — > water — » A, 15 mins. — > wash, 2-4 mins. 45°C. — > B, 30 mins. -^ wash, 5-10 mins. — > blot — > abs. ale. till differentiated — ^ M 32.1 mountant via usual reagents 13.11 Held (1900) see DS 22.21 Held (1900) 13.11 Jenner 1899 11995, 6:370 preparation of dry eosinate: Mix equal parts 1.25% eosin Y and 1% methylene blue. Leave 24 hours. Filter. Wash and dry filtrate. WORKING solution: dry powder 0.5, methanol 100 method: [fresh smear] — » stain, 3 mins. — > water, rinse — > dry NOTE : The most usual employment of this formula is as a fixative before such methods as DS 13.13 Slider and Downey (1929). 13.11 Jenner test. 1905 Lee Lee 1905, 385 formula: 0.5% methylene blue 50, 0.5% eosin Y in methanol 62.5 method: as Jenner 1899 DS 13.11-DS 13.12 DYE STAINS OF GENERAL APPLICATION 345 13.11 Lim 1919 17510, 63:542 REAGENTS REQUIRED: A. 1% cthyl eosin in abs. ale; B. \% methylene blue method: [sections or smears] — > abs. ale. — > ^1, on slide, 1 min. -^ water, thorough wash —* B, on slide, 1 min. -^ blot — + abs. ale., flooded on slide, 2-3 sees. -^ benzene — > neutral mountant or — » dry 13.11 May-Griinwald 1902 23720, 11:265 PREPARATION OF DRY EosiNATE: Mlx cqual parts of 0.5% eosin Y and 0.5% methylene blue. Filter. Dry filtrate. Wasli dried filtrate and redry. WORKING SOLUTION: methanol 100, dry powder from above to sat. method: [air-dried smear] —> stain, on slide, 3 mins. ^ add equal volume distilled water, leave 1 min. — » wash — > dry note: See note under Jenner 1899 with which this technique is nearly, but not quite, identical. Zieler {Icsl. Schmorl 1928, 2-16) stains paraffin sections 2-3 minutes and de- hydrates in acetone; for another application of this stain to sections see DS 13.11 Assmann 1906b above. 13.11 Michaelis 1901 7276,27:127 formula: water 40, abs. ale. 25, acetone 35, methylene blue 0.25, eosin 0.15 preparation: Dissolve the methylene blue in 25 water, 25 ale. Dissolve the eosin in 15 water, 35 acetone. Mix. Filter. 13.11 Miiller test. 1928 Schmorl Schmorl 1928, 239 reagents required: A. 0.5% eosin Y in 70% ale.; B. A 60, 0.25% methylene blue 30 method: [blood smear fixed 3 mins. in methanol]-^ A, 3-5 mins. — > distilled water, wash -^ blot — > B, J2-I min. -^ distilled water, wash -^ dry 13.11 Nocht test. 1903 Ehrlich Ehrlich, Krauso, et al. 1903, 784 formula: water 65, acetone 17, 0.1% thiazin 10, 0.1% eosin 10, buffer pH 4.6 note: This is not a specific technique, though it is often quoted as such; it is a general direction for experiments with any thiazins and any eosin. 13.11 Sabrazes 1911 6630,70:247 reagents required: A. 30% eosin Y in 95% ale.; B. 0.2% methylene blue method : [smear] — > place 1 drop A on smear and 1 drop B on coverslip — > seal with wax for temporary examination -^ or water, quick rinse — * dry 13.11 Willebrand 1901 7276, 27 :57 formula: water 65, abs. ale. 35, eosin 0.25, methylene blue 0.5, 1% acetic acid q.s. preparation: Dissolve the eosin in 35 ale, 15 water. Dissolve the methylene blue in 50 water. Mix the solutions and add acid drop by drop till solution turns red. Filter. method: [fresh smear] -^ stain 5 mins. -^ rinse -^ dry 13.11 Zieler see DS 13.11 May-Griinwald 1902 (note) 13.12 polychrome methylene blue-eo.sinates 13.12 Brice 1930 see D. DS 22.8 Brice 1930 13.12 Diercks and Tibbs 1947 11056, 53:479 REAGENTS REQUIRED: A. Water 100, DS 13.13 MacNcal 1922 6 method: [moist smear] —^ methanol 3 to 5 mins. -^ A, 15-20 mins. — > acetone, till no more color comes away -^ blot — > dry 13.12 Gordon 1939 11284,24:405 reagents required: .1. 10% ammonia; B. ADS 12.2 Gordon 1939; C. water 100, eosin Y 0.25, phloxine 0.75; DS D. 11.44 Loffler 1890 method: [sections of formaldehyde material]-^/!, 1 min. -^ wash -^ /?, 3 mins.—* wash -* C, 2 mins. -^ wash — > b, 4 mins., 37°C. — >• wash — > abs. ale, till ditTerentiated — > balsam, via xylene 13.12 JSB stain see DS 11.44 Manwell 1945 (note) 13.12 Langeron 1934 see DS 11.44 Langeron 1934 346 METHODS AND FORMULAS DS 13.12 13.12 Leishman 1901 3579,2:757 PREPARATION OF DRY EOSiNATE: To 100 0.5% methylene blue add 0.25 sodium carbon- ate Digest 12 hours at 65°C. followed by 10 days at 15°C. Filter. Add 50 0.5% eosin B to filtrate. Leave 12 hours. Filter. Wash and dry filtrate. WORKING solution: methanol 100, dry stain 0.15 method: [air-dried smear] —> stain, on slide, 5-10 mins. — > water, 1 min. — > blot dry note: This method is stated by Leishman {loc. cit.) to be a modification of a stain pro- posed by Rowmanowski 1891 {St. Petersburg Med. Wschr., 16 :297) to the original of which the writer has never had access. 13.12 Lillie and Pasternack 1936 4349, 15:65 preparation of dry stock: Dissolve 0.25 silver nitrate in 12.5 water. Add 1-2 5% sodium hydroxide, wash ppt. five times by decantation. Add 50 1% methylene blue to wet ppt. Shake at intervals for 11 days. Filter and add 45 1% eosin. Leave overnight, filter, and dry ppt. formula of stock solution: methanol (acetone-free) 75, glycerol 25, dry stock 0.6 working solution: citric acid-sodium phosphate, dibasic buffer (see note) 70, acetone 6, methanol 6, stock solution 4 note: The buffer must be so adjusted that after the addition of the other ingredients the pH is 5.3. 13.12 Mallory test. Langeron 1942 Langeron 1942, 614 reagents required: A. water 100, eosin Y 5; B. water 85, DS 11.44 Sahli 1885 15; C. ADS 22.1 Wolbach 1911 method: [water] -^ A, 20 mins. -^ rinse — > fi, 5 mins. -^ B, fresh solution, 15 mins. — » B, fresh solution, 30 mins. -* wash -^ C, till differentiated -^ abs. ale. least possible time — > balsam, via cedar oil result: nuclei, blue; other structures, polychrome. 13.12 Manwell 1945 11284,30:1078 reagents required: A. DS 11.44 Manwell 1945; B. water, adjusted to pH 6.5 with acetic acid; C. 0.2% eosin Y method: [methanol fixed smear] -* dry -^ A, 30 sees. -^ B, wash -> A, 30 sees. -^ dry — + neutral mountant 13.12 Marie and Raleigh 1924 11284, 10:250 preparation of dry stock: Dissolve 1 methylene blue in 100 0.5 sodium bicarbonate and expose to U.V. arc 30 mins. in shallow dish. Cool and add 500 0.1% eosin Y. Filter, wash, and dry ppt. working solution: methanol 100, dry stock 0.16 13.12 Michelson 1942 11284,27:551 preparation of dry stock: Dissolve 1 methylene blue in 100 N/100 sodium hydroxide. Heat 2\i hours, 55°C. shaking for 1 minute at half-hour intervals. Add 1 sodium bromide, continue heating 2^2 hours. Cool. Filter. Add 60 1% eosin Y and mix. Add further eosin in doses of 5 till solution is reddish. Leave 24 hours. Filter. Dry filtrate. WORKING solution: 0.017% dry stock in methanol 13.12 Raadt 1912 23632, 29:236 STOCK solution: water 100, methylene blue 1, lithium carbonate 0.5 reagents required: .4. stock 10, water 90; B. DS 13.11 Jenner 1899 25, water 75 method: [methanol fixed smears] -^ A, on slide, 5-10 mins. -^ rinse -» blot -^ B. 5-10. mins. — > wash -^ dry 13.12 Roques and Jude 1940 /(.s7. 1949 Langeron Langeron 1949, 621 formula of STOCK SOLUTION: methanol 50, glycerol 50, eosin B 0.2, methylene blue 0.2, DS 11.44 Roques and Jude 1940 0.5 preparation of STOCK SOLUTION: Dissolve dyes in methanol, leave 24 hours, filter, and add glycerol. working solution: water 95, stock 5 13.12 Rowmanowsky 1891 see DS 13.12 Leishman 1901 (note) DS 13.12-DS 13.13 DYE STAINS OF GENERAL APPLICATION 347 13.12 Schmorl 1928 see DS 23.33 Schmorl 1928 13.12 Senevet 1917 5310, 10:510 STOCK solutions: I. methylene l)lue 1, sodium borate 3, water 100; II. eosiii Y 1, water 100 WOKKING solution: water 100, stock II 0.2, stock I 0.25 to 0.4 method: [metham>l-fixed smears] — > A, 2-3 hrs. —> wash -^ dry 13.12 Stafford 1934 1091'.), 55:229 REAGENTS uequired: A. Water 100, potassium dichromate 1, eosiu 1; B. DS 11.44 Good- pasture (1934); C. acetone 100, abs. ale. 10 method: [frozen sections of formaldehyde-ale. material] —r water -^ yl, 1 sec. — > wash — > B, 30 sees. -^ wash — ♦ blot on slide — > C, dropped on section till dehydrated -+ balsam, via xylene 13.12 Singh, Jaswart, and Bhattacharji 1944 see DS 11.44 Manwell 1945 (note) 13.12 Wright 1910 Rep. Mass. Gen. Hosp., 3:1 FORMULA OF METHYLENE BLUE STOCK: Water 100, methylene blue 1, sodium bicarbonate 0.5 PREPARATION OF STOCK SOLUTION: Digest at 100°C. 1\^ hours. WORKING solution: methanol 80, eosin B, 0.16, stock blue 24 method: [smear] — + stain, on slide 2 mins. -^ add water, drop by drop, till green scum forms on surface, leave 2 mins. —> water, thorough wash -^ [dry] or —^ neutral mountant, via acetone note: Wright 1910 also recommended (17510, 57:783) the dilution of the working solu- tion with an equal volume of water to be used for 10 minutes. It is nowadays uni- versal practice to substitute a phosphate buffer at pH 6.4 for the water used to dilute the stain on the slide. 13.13 OTHER THIAZIN EOSINATES 13.13 Agulhon and Chavennes 1919 see DS 13.13 Pappenheim 1908 13.13 Bohm and Oppel 1907 methylene blue-thionin-eosin Bohm and Oppel 1907, 114 REAGENTS REQUIRED: A. Water 96, 40% formaldehyde 4, methylene blue 0.3, thionin 0.15; 5. 1% acetic acid; C. 1% eosin B method: sections — > A, some mins. -^ B, tiU no more color comes away —> C, few sees. —f balsam, via usual reagents 13.13 Boye 1940 5310, 33:248 REAGENTS REQUIRED: A. watcr 100, cosin Y 0.1; 5. DS 11.44 Stevenel 1918 method: [methanol-fixed smears] -^ A, 15-20 sees. — > B, flooded on slide, 45 sees. — > A, till differentiated -^ rinse -^ dry 13.13 Endicott 1945 20540b, 20:5^ PREPARATION OF DRY STOCK: To 250 ml. 0.8% toluidine blue add 250 0.4% eosin B. Filter, wash, and dry ppt. WORKING solution: methanol 50, glycerol 50, dry stock 0.3 13.13 Field 1941 see DS 23.33 Field 1941 13.13 Geschickter, Walker, Hjort, and Moulton 1931 20540b, 6:3 REAGENTS REQUIRED: A. watcr 60, glycerol 20, 95% ale. 20, sodium hydroxide 1.47, potassium acid phosphate 0.675; B. ethylene glycol 75, 95% ale. 25, acetic acid 0.2, thionine-eosinate 0.75, barium-eosinate 0.25, azur A 0.25; C. 20% glycerol in 95% ale; D. diethylene glycol monobutyl ether; E. n-butyl phthalate PREPARATION OF B: Dissolve the barium-eosinate with heat; raise solution to boiling, add thionine-eosinate. Filter solution hot. method: [frozen sections of fresh tissue] — > A, till required-^ B, 20-30 sees.—* C, 10 sees. -^ C, fresh solution, 3 sees. — > D, 10-15 sees. -^ E, 20 sees. -^ dammar note: Solution A should be adjusted, if necessary, to pH 7. 348 METHODS AND FORMULAS DS 13.13 13.13 Giemsa 1902 23684,31:429 formula: 0.05% eosin 95, 0.8% azur 5 note: This is the original formula for Giemsa's stain, given in a footnote to the paper cited. From this time on almost any eosin-aznr mixture has been referred to as Giemsa. The formulas of Slider and Downey 1929 and Gatenby and Cowdry 1928 are referred to by their publishers as Giemsa's stain. When reference is made in the present work to DS 13.13 Giemsa 1902 the solution of SHder and Downey 1929 should be em- ployed urdess dry powder is specified. Kopel 1945 (4349, 25 :61) substitutes ethanol for methanol in preparing the working solution. 13.13 Giemsa see also DS 13.13 Langeron 1942, Slider and Downey 1928, Gatenby and Cowdry 1928 13.13 Grosso 1914 see DS 21.3 Grosso 1914 13.13 Haynes 1926a 20540b, 1:68 REAGENTS REQUIRED: A. 1.5% azur II or azur C; B. sat. sol. ethyl eosin in clove oil method: [water] —> a, 5 mins. — > abs. ale. quick rinse ^ B, 30 sees. —> balsam, via xylene 13.13 Haynes 1926b 20540b, 1:107 REAGENTS REQUIRED: A. 2.5% phloxine; B. 0.1% azur I; C. ADS 22.1 Wolbach 1911 method: [sections] -^ water — » A, 15 mins. -^ water, thorough wash -^ B, 30 mins. -^ water, thorough wash — * C, from drop bottle, till differentiated — > abs. ale, minimum possible time — > balsam, via xylene 13.13 Kingsley 1935 20540b, 10:127 STOCK solutions: I. water 50, methanol 10, glycerol 10, buffer pH 6.9 30, methylene blue 0.130, azur A 0.020; II. acetone 70, methanol 20, glycerol 10, methylene violet 0.026, eosin Y 0.090 reagents required: A. stock I 50, stock II 50; B. 0.015% acetic acid; C. acetone 60, acetic acid 0.005, eosin Y 0.0005; D. n-butyl ale. 60, eosin Y 0.001 METHOD FOR SMEARS: [air-dried smear] -^ methanol 3^^ to 1 min. -^ dry -^ A, 5-8 mins. — > water, thorough wash — * dry METHOD FOR PARAFFIN SECTIONS: Water -^ A, flooded on slide, 9-10 mins. -^ water, thorough wash -^ C, rinse -^ D, rinse — > balsam, via xylene TEMPORARY METHOD FOR FROZEN SECTIONS: [blot section to slide] — > A, flooded on slide, 2-3 mins. — » wash — » [examine] - PERMANENT METHOD FOR FROZEN SECTIONS: [blot sectiou to slide] — > A, 4-5 mins. (fresh) or 8-10 mins. (fixed) -^ [thence as for paraffin sections] note: The application of this stain to a blood smear is described under DS 13.10 above. See also DS 21.42 Kingsley 1937. Ritchie 1941 {Tech. Bull, 2 :157) prefers, for smears, to dilute the A stain 8:5 with water. 13.13 Kingsley 1937 see DS 21.42 Kingsley 1937 13.13 Langeron 1942a Giemsa for wet smears — aucl. Langeron 1942, 583 REAGENTS required: A. Water 100, potassium iodide 2, ADS 12.2 Lugol (1905) 3; B. 0.5% sodium thiosulfate; C. water 97, DS 13.13 Giemsa 1902 3; D.\% monosodium phosphate method: [smears fixed in F 3000.0000 Schaudinn 1893 or F 3700.0010 Zenker 1894, 24 hrs.] — > rinse -^ A, 5 to 10 mins. — > rinse — » B, 10 mins. -^ running water 5 mins. -* distilled water — > C, 30 mins. -^ C, fresh solution, 12 hrs. -+ D, if differentiation required — > neutral mountant, via graded acetone-xylene mixtures 13.13 Langeron'l942b Giemsa for sections — auct. Langeron 1942, 583 REAGENTS required: A. 70% ale. 97, ADS 12.2 Lugol (1905) 3; B. 0.5% sodium thio- sulfate; C. water 97, DS 13.13 Giemsa 1902 3 method: [5 M paraffin section of F 3000.0000 Schaudinn 1893 or F 3700.0010 Zenker 1894 material, dewaxed and brought to water] -^ A, 20-30 mins. -^ rinse -^ B, 10 mins. -^ tap water 5 mins. -^ distilled water — > C, 30 mins. — > C, fresh solution, 2 to 12 hrs. — ♦ balsam, via graded acetone-xylene mixtures note: Langeron (loc. cit. p. 751) also refers to this as Wolhach's method; but see DS 13.13 Pappenheim 1908 (note) and DS 23.12 Wolbach 1919. DS 13.13 DYE STAINS OF GENERAL APPLICATION 349 13.13 Groat 1936 11281, 21:978 PREPARATION OF DRY STOCK! Dissolve 1.2 eosin y in 100 water. Dissolve 1 methylene blue, 0.2 methyl violet 2 B, 0.04 thionine in 10 water. Mix solutions, heat to 50°C. and hold at 37°C., 24 hours. Filter. Wash and dry filtrate. FORMULA OF WORKING SOLUTION: methanol 100, dry stock 0.5 method: [blood smears] — > stain, on slide, 5 mins. — > plunge in distilled water till smear rosy-pink — ♦ dry 13.13 Kingsley 1937 11284, 22:524 stock solutions: I. water 80, phosphate buffer pH 6.920, methylene blue 0.070, methyl- ene azur A 0.025, II. acetone 70, methanol 20, glycerol 10, methylene violet 0.018, eosin Y 0.065 WORKING solution: stock I 50, stock II 50 13.13 Kuhn 1933 19764d, 7 :758 PREPARATION OF DRY STOCK: Mix 9 ammonia with 6 2% copper sulfate. Add this to 200 0.5% meth3dene blue and leave 24 hours at 18°-20°C. Then add, in small amounts and with continuous agitation, 40 2% eosin Y. Filter, wash ppt. with 4% ammonia, and dry. stock solution: methanol 60, glycerol 30, dry stock 0.36 w^ORKiXG solution: stock solution 25, methanol 75 13.13 Lillie 1948 azur-eosin Lillie 1948, 82 PREPARATION OF DRY STAIN: Dissolve 1 azur A or C in 60 water. Add 0.8 eosin B or Y dis- solved in 10 water. Mix solutions. Filter. Wash and dry ppt. PREPARATION OF STOCK SOLUTION: glycerol 50, methanol 50, dry stock 1 PREPARATION OF STOCK BUFFERS: I. Water 75, methanol 25, sodium phosphate, dibasic 2.84; II. water 75, methanol 25, citric acid 9.12 PREPARATION OF WORKING SOLUTION: stock stain 0.5, acetone 5, mixed buffers I and II 2, water to make 40 method: [sections] —» water —> stain, 1 hr. —> rinse -^ acetone, tiU dehydrated —» balsam, via xjdene note: The ratio of buffers I and II in the working stain must be adjusted to suit the fi.xative used and the color balance desired. Lillie, loc. cit., recommends from 0.71: 1.3 II to 1:1. This is a slight modification of Lillie 1941 (20540b, 16:1). 13.13 MacNeal 1922 azur I -methylene violet-eosin Y 11006,78:1122 formula: methanol 100, eosin Y 1, azur I 0.6, methylene violet 0.2 method: [air-dried smears] — * flood with stain, 2 mins. — > add water, drop by drop, till green scum forms on surface -^ leave 1 min. -^ water, thorough wash — » neutral mountant, via acetone note: The dry dyes, mixed in the proportion indicated, have appeared in commerce under the name MacNeaVs Tetrachrome. 13.13 Maximow 1909 see DS 12.37 Maximow 1909 13.13 Maximow 1924 azur-eosin 11250,34:549 reagents required: A. water 100, DS 11.122 Delafield 1885 0.1; B. 0.1% eosin Y 10, water 100, 0.1% azur II 10 method: [sections from F 3700.1010 fixed material]—* water -^ A, 24 hrs. -^ water—* B, 24 hrs. — > 95% ale, till differentiated -^ balsam, via xylene 13.13 Medalia, Kahaner, and Singer 1944 see DS 23.33 Medalia et al. 1944 13.13 McNamara 1933 11284, 18:752 REAGENTS REQUIRED: A. water 90, ADS 12.2 Lugol (1905) 10; B. 0.5% sodium thio- sulfate; C. water 100, DS 13.13 Slider and Downey 1929 10, acetone 10, methyl ale. 10, 0.5% sodium carbonate 2; D. ADS 22.1 Wolbach 1911 method: [4 M sections of F 3700.0010 Zenker 1894 material] — » A, 30 mins. — > 95% ale. wash — * water, wash — * B, 10 mins. —^ wash — > C, 15 mins. — » D, till differentiated —* balsam, via acetone and xylene 13.13 Pappenheim 1908 panoptic stain — compl. script. 13172, 4:1244 REAGENTS REQUIRED: A. DS 13.11 May-Grimwald 1902; B. water 97, DS 13.13 Giemsa 1902 3 350 METHODS AND FORMULAS DS 13.13-DS 13.2 method: [dry smear] — * A, flooded on slide, 3 mins. -^ add 10 drops distilled water, mix well, leave 1 min. -^ drain -^ B, 10-15 mins. -^ wash off with jet of distiUed water -^ wash, till differentiated — » drain, dry note: Agulhon and Chavannes 1919 (6630, 82:149) differentiate with 1% boric acid or 1% monosodiura phosphate. Otherwise the technique is identical. Wolbach 1911 (11006, 56:345) uses his resin alcohol (ADS 22.1, Wolbach 1911) for differentiation. Langeron 1942, p. 751 refers to his "method for sections" as Wolbach's technique. 13.13 Pappenheim 1912a 766,42:525 REAGENTS REQUIRED: A. water 75, DS 13.11 May-Griinwald 1902 25; B. water 95, DS 13.13 Giemsa 1902; C. 0.2% acetic acid method: [5 M paraffin sections of F 7000-1000 Orth 1896 or F 3700.1000 Helly 1903 material, dewaxed and brought to water] — + A, 15 mins., 37°C.^^ B, 40 mins., 37°C. — > rinse — > rinse -^ C, till differentiated -^ balsam via acetone 13.13 Pappenheim 1912b 8545, 13:340 REAGENTS REQUIRED: A. Water 75, DS 13.11 May-Griinwald 1902; B. water 97, DS 13.11 Pappenheim 1911 3; C. 0.1% picric acid method: [water] ^ A, 15 mins. at 37°C. ^ B, 30 mins. at 37°C. -^ wash ^ C, till differentiated-^ balsam, via graded acetone-xylene mixtures 13.13 Shortt 1918 9940, 6:124 REAGENTS required: A. DS 11.44 Langeron 1908 10, water 90; B. eosin Y 0.01, water 100 method: [methanol-fixed smears] -^ 1 A mixed with 1, 2, or 3 S (proportion established by trial) -^ quick rinse -^ abs. ale, till differentiated -^ water, to stop differentiation — » dry — > balsam note: The original calls for a 0.1% solution of BorreVs blue. A above is identical in com- position to such a solution. 13.13 Slider and Downey 1929 azur-eosin iVIcClung 1929, 246 formula: glycerol 50, azur II 0.16, azur Il-eosin 0.6, methanol 50 method: [smears, treated with methanol or DS 13.11 Jenner 1899 or DS 13.11 May- Griinwald 1902] -^ equal parts stain and water (or pH 6.4 buffer), 15 mins. — > water, till differentiated — + blot -^ dry note: Gatenby and Cowdry 1928, 490 specify 75 methanol to 25 glycerol. 13.13 Svihla 1924 11006,83:2093 preparation of dry stock: To 50 water add 0.69 methj'lene blue, 0.44 silver oxide, 0.38 sodium bicarbonate. Boil 1 hour and decant supernant liquid which is added to 50 0.62% eosin. Mix thoroughly, filter, wash, and dry ppt. WORKING solittion: methanol 100, sodium phosphate, dibasic 0.125, potassium di- hydrogen phosphate 0.080, potassium hydroxide 0.004 13.13 Ugruimov 1928 see DS 21.3 Ugruimov 1928 13.13 Villain and Comte 1,933 see DS 23.3 Villain and Comte 1933 13.13 Wolbach see DS 13.13 Langeron 1942b (note) 13.13 Wolbach 1911 see DS 13.13 Pappenheim 1908 13.13 Wolbach 1919 see DS 23.12 Wolbach 1919 13.2 Techniques Employing the eosinates themselves. The first and still Thiazins and Their Eosinates the best known formula is that of Mann IN Combination with Other 1894, though in French literature it has Dyes largely been replaced by the method of The combination of the thiazin-eosin- Dominici 1902. The preoccupation of the ates with orange G (DS 13.21) is almost French school with the work of Dominici as old as the utilization of the thiazin- has led to the production of such almost DS 13.21 DYE STAINS OF GENERAL APPLICATION 351 incredibly complex methods as those of Gausen 1929 and Iloucke 1928. Complejv methods of this type have proved of some value in the investigation of pathology, but are not to be recommended to the routine worker. The combination of the tliiazin-eosin- ates with other dyes than orange G (DS 13.22) have not been very successful, the main exception being the technique of KuU 1914 which, though originally de- signed for the demonstration of mitochon- dria, is one r)f the best and surest triple staining methods yet developed. The formula of Rhamy 1930 is of great interest in the staining of blood smears, and yields pictures which are not only of as great diagnostic value but also far more readily preserved than are the standard eosin- methylene blue mixtures. 13.21 IN COMHIXATIUN WITH UKAXGE G 13.21 Arnold 1909 methylene blue-safranin-orange G 1820, 3 :434 REAGENTS REQUIRED: .-1. ADS 12.2 Lugol (1905); B. sat. sol. safranin in 70% ale; C. water 100, methylene blue 7, sodium carbonate 0.5; 1% orange G method: [sections of chromic or dicliromate material] — > A, 5 mins. -^ wash -^ B, 4 hrs. — > wash ^ C, 4 hrs. -^ abs. ale, till dehydrated —>■ D, till differentiated -^ balsam, via usual reagents result: nucleoli, ccntrosomes, red; many cell inclusions, blue; other structures, orange 13.21 Cowdry 1943 phloxine-orange G-azur A Cowdry 1943, 69 REAGENTS REQUIRED: .4. Water 100, phloxine 0.12, orange G 0.3; B. 0.1% azur A method: As Dominici 1902 below note: See also note on Cowdry 1943 modification under Dominici 1902. 13.21 Dominici 1902 eosin Y-orange G-toluidine blue 6630,54:221 REAGENTS REQUIRED: A. water 100, eosin Y 0.5, orange G 0.5; B. 0.5% toluidine blue method: [water] -^^, 5-10 mins. -^ water, quick rinse —> B, 20-30 sees. -^ water. quick rinse -^ 95% ale, till differentiated -^ balsam, via xylene note: Cowdry 1943 p. 69 recommends the substitution of 0.5% acid fuchsin for the eosin Y in .4 al)ove. See also Cowdry 1943 above and Mann 1894 below. 13.21 Gausen 1929 methylene blue-orange G-magenta 66.30,97:1658 FOR.MULA of methylene BLUE SOLUTIONS: 80% alc. 150, lactic acid 3, methylene blue 2.5 FORMULA OF ORANGE G SOLUTION: 80% alc. 100, lactic acid 2, orange G 2 PREPARATION OF COMPLEX: Add bluc to Grange. Heat to 80°C. Cool. Filter. Save both ppt. and filtrate. PURIFICATION OF PRECIPITATE: DLssolvc ppt. from above in 30 80% alc. Heat to 80°C. Cool. Filter. Save filtrate. Reject ppt. PREPARATION OF WORKING SOLUTION: Mix both filtrates with 50 methylene blue solution. Filter. REAGENTS REQUIRED: A. DS 11.43 Zichl 1890; B. working solution method: [sections] — > water —> A, 5 mins. — > water, thorough wash — > B, 4 mins. — > abs. alc, till differentiated -^ neutral mountant, via usual reagents result: nuclei, red; cartilage, blue; muscle, yellow; bone, green. 13.21 Houcke 1928 toluidine blue-orange G-thionin-eosin-azur II 6630, 99 :784 PREPARATION OP STOCK SOLUTIONS: I. Mix 10 1% toluidine blue with 5 1% orange G. Dilute to 100. Leave 24 hours. Decant and leave ppt. dry. Dissolve dried ppt. in 10 methanol. II. Add 17 1% eosin Y to 100 sat. sol. (circ. 25%) thionine. Leave 24 hours. Decant. Dry ppt. Prepare 0.5% solution ppt. in methanol. III. Add 111% eosin B to 10 1% methylene blue. Thence as in II. IV. Add 1.25 1% eosin Y to 40 0.08% azur- eosin. Filter. Dry ppt. on filter paper. Cut in strips and extract with 10 methanol. V. Add 8 1% eosin Y to 10 1% toluidine blue. WORKING solution: water 100, stock I 0.5, stock II 1.5, stock III 1.5, stock IV 1.5, stock V 1.5, 0.1% acetic acid 1.0 352 METHODS AND FORMULAS DS 13.21-DS 13.22 method: [water] ^ stain, 24 hrs. — > abs. ale, minimum possible time —> balsam, via xylene note: Houcke {loc. cit.) recommends varying 'the acidity of the working solution by experiment to adapt it to use after various fixatives. 13.21 Holmes and French 1926 azur C-eosin Y-orange II 20540b, 1:25 REAGENTS REQUIRED: A. 1.5% azur C; B. abs. ale. 99, acetic acid 1, eosin Y 0.025, orange II 0.025 method: [water] -^ A, 5 mins. — > methanol, till color clouds cease, 5-10 sees. -^ B, till no more blue comes away -^ abs. ale, quick rinse — * balsam, via xylene result: nuclei, and some bacteria, blue; cell inclusions and blood, as Giemsa; collagens, bright orange; muscle, pink. 13.21 Kedrovsky 1931 see DS 22.12 Kedrovsky 1931 13.21 Mann 1894 test. 1942 Langeron cit. Masson erythrosin-orange G-toluidine blue Langeron 1942, 613 REAGENTS REQUIRED: A. ADS 12.2 Lugol (1905); B. 5% sodium thiosulfate; C. water 100, orange G 1, erythrosin 0.2; D. 1% toluidine blue; E. 0.2% acetic acid method: [sections] —> water —> a, J^ hr. -^ water, rinse -^ jS, till bleached ^ water, thorough wash —* C, 15 mins. — * water, rinse -^ D, on slide, 1-2 mins. — * water, rinse — > E, till differentiated -^ balsam, via usual reagents note: Langeron {loc. cit.) says "Ce procMe, dit de Dominici, jouit en France d'une assez grande vogue — etc." See, however, DS 13.21 Dominici 1902, above. 13.22 IN COMBINATION WITH OTHER DYES 13.22 Bauer and Leriche 1934 methylene blue-eosin Y-cresyl blue 16550, 42:1385 reagents required: A. water 100, brilliant cresyl blue 0.25; B. DS 13.11 Jenner 1899 (working sol.) method: Mix 4 parts blood with 1 A, as drop on slide, 2 mins. -^ smear — > dry -^ B, 2 mins. — > water, rinse — > dry —* neutral mountant. 13.22 Blank 1942 mercurochrome-azur-eosin 11284,27:1342 STOCK solutions: I. water 82, 40% formaldehyde 9, abs. ale. 5, DS 13.13 Geimsa 1902 2.25, methylene blue 0.25, sodium borate 0.5 reagents: required: A. 0.1% mercurochrome 220 in 25% methanol; B. stock I 5, water 95 method: [sections (nuclei may be hematoxylin stained)] — > water -^ A, \ min. — * wash — > B, 2 mins. ^•90% ale, till no more color comes away 13.22 Geschickter 1930 azur-erie garnet 20540b, 5:81 formula: water 100, azur A 0.8, erie garnet B 0.1 preparation: To the azur dissolved in 80 water add, very rapidly, the garnet dissolved in 20. Filter. method: [frozen sections of fresh tissue] — > stain, 15-20 sees. — > wash — > M 10 mountant 13.22 Houcke 1928a methylene blue-toluidine-thionin-fuchsin 6630, 99 :786 preparation of stock formulas: I. Mix, without agitation, in a small graduate, 14 1 % acid f uchsin with 22 1 % methylene blue. Leave 24 hours. Pour liquid from viscous ppt. which adheres to side of graduate. Dry ppt. and dissolve in 20 methanol. II. Add 0.5 1% acid fuchsin to 10 sat. sol. thionine. Leave 1 hour. Centrifuge. Decant and drain. Dry ppt. in tube. Dissolve in 10 methanol. III. Mix 11 1% toluidine blue with 5 1% acid fuchsin. Then treat as II. working formula: water 100, stock I 2.5, stock II 2.5, stock III 2.5, 1% acetic acid 1 method: [sections] -^ water—* stain }i to 2 hrs. — > abs. ale, shortest possible time -^ balsam, via xylene note: Houcke {loc. cit.) comments that the pH of the'working solution is critical, but the optimum, which varies both with types of tissue and fixatives employed, can only be established empirically. DS 13.22-DS 13.3 DYE STAINS OF GENERAL APPLICATION 353 13.22 Houcke 1928b meOnjlene bhie-rhodamine B 6630, 99 :788 formula: sat. sol. (circ. 5%) methylene blue in 95% ale. 3, sat. sol. aniline 27, 0.5% rhodamine B 70 method: [section] —> water —> stain, 2-3 hrs. — » abs. ale, minimum possible time-* dammar via xylene result: chromatin, blue violet; nucleoli, red; collagens, orange; muscle, deep orange; erythrocytes, bright red. note: For tissues recently fixed Houcke recommends that the aniline solution be diluted 1 : 1 with water. 13.22 Langeron 1942a poli/chrome methylene blue-orcein Langeron 1942, 612 reagents required: A. water 100, polychrome methylene blue 1; B. 70% alcohol 100, orcein 0.25 method: Distilled water — > A, 5 mins. -^ wash — > B, 5 mins. — > abs. ale, least possible time — * balsam, via xylene result: nuclei, blue; connective tissue, red; other structures, polychrome. 13.22 Langeron 1942b polychrome methylene hlue-lannin-oramje Langeron 1942, 612 REAGENTS REQUIRED: A. Water 100, polychrome methylene blue 1; B. DS 11.44. Unna 1892 method: Distilled water -^ A, 5-15 mins. -^ wash -^ B, till no further blue removed — » rinse — > balsam via usual reagents result: nuclei in mitosis, bacteria, some connective tissues, blue; resting nuclei, most other structures, orange. 13.22 Kull 1913 ioluidine blue-aurantia-acid fuchsin 766,45:153 reagents required: A. sat. sol. aniline 100, acid fuchsin 20; B. 0.5% toluidine blue; C. 0.5% aurantia in 70% ale. method: [sections] -^ water -^ A, 1 min. warmed to steaming —> cool—* water, quick rinse — * B, 1-2 mins. — * water, quick rinse -^ C, till sufficient red extracted, 20-30 sees. -^95% ale, till differentiation complete —* balsam, via usual reagents note: Though originally intended for the demonstration of mitochondria, this is an excellent general-purpose stain. For the mitochondria technique see DS 22.2 Kull 1914. 13.22 Masson test. Langeron 1942 thionin-picric acid Langeron 1942, 613 reagents required: A. DS 11.44 NieoUe (1942); B. 0.2% acetic acid; C. sat. sol. picric acid in toluene method: water -^ a, 15 mins. — » wash — > 5, till differentiated -^ abs. ale. till de- hydrated —* toluene — > C, till green —* balsam, via toluene result: nuclei, bacteria blue; other tissues, yellow, blue, or green. 13.22 Rhamy 1930 rnethylene blue-eosin Y-magenta 11284, 15:490 stock, formulas: I. sat. sol. {circ. 6%) magenta in abs. ale; IL sat. sol. {circ. 45%) eosin Y in water; III. sat. sol. {circ. 1.5%) methylene blue in abs. ale working formula: 30% ale 100, stock I 4, stock II 5, stock III 15 method: [sections]—* 70% ale—* stain, 5 mins.—* abs. ale, till differentiated—* bal- sam, via usual reagents 13.22 Unna lest. 1928 Schmorl methylene blue-orcein Schmorl 1928, 76 reagents required: A. DS 11.44 Unna 1892; B. 1% orcein in 95% ale method: [celloidin sections of ale fixed material] -* water—* A, 10 mins. — * thorough wash — * blot —> B, 15 mins. -^ balsam, via bergamot oil 13.3 Techniques Employing methyl green combinations, it has the dis- Methyl Green as the advantage of being very sensitive to alka- NucLEAR Stain lies — so much so that extreme care must Methyl green in combination with py- be taken either to provide a perfectly ronin has been long recognized as an excel- neutral mounting or to use some acid lent stain for smears, but, like all other mountant such as salicylic balsam. The 354 METHODS AND FORMULAS DS 13.30 formula of Pappenheim 1901 has been adapted for use with bacteria by Saathof 1905, and is now widely used for this pur- pose. The combination of methyl green with other dyes is best known from the Ehrhch 1898 (or Heidenhein 1888) "tri- acid" mixtures. These mixtures and their variations continue to occur from time to time in the literature, but it is difficult to justify their employment today. Far better methyl green-acid fuchsin combinations are the two triple stains of Foley, 1930 and 1931, which will give all the staining reactions of the earlier mixtures without their manifest disadvantages. Most of the other formulas in this class are various modifications of the "triacid" mixtures. 13.30 TYPICAL EXAMPLES Staining a section of the suprarenal body in the methyl green-acid fuchsin-orange G stain of Foley 1939 The method of staining here described is the most rational of all the variants of the old Ehrhch's "triacid" stain. These stains are largely designed to differentiate cell types, and should be employed only on organs in which so many cell types are present that many varieties of shade are desired to differentiate between them. The suprarenal bodies fulfill this require- ment and are best obtained from a young mammal. A rabbit is excellent for class demonstration; a two-third-grown male should be selected, killed in the customary manner, eviscerated, and the suprarenal bodies removed from under the peri- toneum before being rinsed in physio- logical sahne solution. There is considerable discussion in the literature as to the best fixative to select for these materials, but a combination of dichromate and osmic acid will provide excellent fixation of the chromaffin mate- rial (which will be retained in paraffin sec- tions) and will thus differentiate at least this cellular material with a reasonable degree of certainty. Numerous fixatives of this general composition have been sug- gested; that of Altmann 1890 (Chapter 18, F 1700.0000 Altmann 1890) is well suited for this particular preparation. No fixative containing osmic acid has much penetrating power, so that the suprarenal body, having been rinsed in physiological saline, should be cut with a sharp scalpel into at least three pieces before being im- mersed in about 50 milliliters of the selected fixative. Fixation is permitted to continue in the dark for about 24 hours before the specimen is removed and washed in running water for at least a further 12 hours. If, after this washing, the outer surface of the specimens are un- duly blackened (they will in any case be a liglit brown color), a few drops of hy- drogen peroxide may be applied in dis- tilled water; but the pieces should not be left in this mixture for longer than it takes the black stain to disappear. They should then be washed in several changes of dis- tilled water before being rewashed in running water for at least three or four hours. The hydrogen peroxide does not destroy the osmic hydroxides precipitated on the surface, but only causes them to revert to the water soluble tetraoxide. The soluble tetraoxide must therefore be removed by prolonged washing unless the blackening is to recur. Dehydrating, embedding, and section- ing should present no difficulty whatever with an object of this type, though it is recommended that sections no thicker than 5 microns should be employed. These sections are attached to a slide in the customary manner, dewaxed, and brought down through the customary dehydrating agents to distilled water, in which the slides may be accumulated until they are required for staining. The only difficulty lies in the prepara- tion of the stain itsejf (DS 13.32 Foley 1930) which, if prepared imperfectly, will contain a precipitate. The addition of the acid fuchsin and the orange G solutions to the glycerol occasions no difficulty; but the greatest care should be taken to have these completely mixed together before the methyl green is added. The methyl green should be added as slowly as possi- ble in small drops and each drop should be thoroughly mixed in before the next is added. The stain should then be allowed to remain for at least 12 hours at room temperature before being filtered im- mediately before each use. DS 13.31 DYE STAINS OF GENERAL APPLICATION 355 Sections are passed directly from dis- essential that the slide be passed from tilled water into the stain, in which they may remain from overnight to about 24 hours. Overstaining does not normally occur. Each slide must be removed separately from the stain and rinsed briefly and rapidly in 95% alcohol. It will be observed that in the alcohol color clouds rise from the sections, and that there is then a brief period when these color clouds cease. If the slide is then left for a longer period in alcohol, further stain will commence to leave it. To secure satisfactory slides, it is alcohol to the carbon-xylene, which is recommended for clearing, at the exact moment when the primary color clouds cease to leave. The purpose of the carbol- xylene is to permit complete dehydration, even though the length of time in alcohol has not been sufficient to remove the whole of the water from the section. The carbol-xylene must be thoroughly re- moved with pure xylene before the slide is mounted in balsam, or the latter will inevitably turn brown. 13.31 METHYL GREEN IN COMBINATION AVITH PYRONIN 13.31 Flinn 1939 see DS 23.219 Flinn 1939 13.31 Grosso 1912 16059, 4:41 formula: water 100, sat. sol. (arc. 9%) pyronin 5, sat. sol. {circ. 5%) methyl green 3.5, sat. sol. {circ. 11%) orange G 3.5 method: water —> stain, 30 sees. — > abs. ale, till differentiated^ n-projiyl ale, if de- hydration insufficient -^ neutral mountant result: nuclei, green; plasma, red, orange, and yellow. 13.31 Langeron 1942 Langeron 1942, 615 STOCK solutions: I. water 100, methyl green 4, phenol 5; II. water 100, pyronin 4, phenol 5 REAGENTS REQUIRED: A. stock I 50, stock II 50; B. ale. 50, acetone 50; C. aniyl alcohol method: distilled water — > A, 15 mins. at 50°C. -^ distilled water, rinse -^ B, till differ- entiated -^ C, till dehydrated -^ balsam, via toluene result: good differential staining of blood cells and glandular cell inclusions. 13.31 Lipp 1940 see DS 23.214 Lipp 1940c 13.31 Pappenheim 1901 1780a, 155:427 STOCK solutions: I. water 100, phenol 0.25, methyl green 1; II. water 100, phenol 0.25, pyronin 1 WORKING formula: stock A 30, stock B 70 method: water ^^ stain, 5-10 mins. -^ water, quick rinse —> neutral mountant, via acetone result: nuclei, violet; lymphocytes and plasma cells, red; other tissues, orange and green. note: For bacteria see DS 23.221 Saathof 1905. 13.31 Sandiford 1937 11431,45:467 formula: water 75, glycerol 20, 95% ale. 5, phenol 1.5, methyl green 0.15. i)yn)iiiii 0.5 13.31 Scott and French 1924 13685, 55:337 formula: water 80, glycerol 16, ahs. ale. 4, phenol 1.6, nietliyl green 0.8, pyronin 0.2 13.31 Unna 1910 test. 1928 Gatenby and Cowdry cit. Gandletz Gatenby and Cowdry 1928, 176 formula: water 100, phenol 0.5, glycerol 20, abs. ale. 2.5, pyronin 0.25, methyl green 0.15 preparation: Griiul the dyes with the ale in a mortar. Heat glycerol to 50°C. and add to mortar in small jxtrtioris while frriiuling. Dissolve phenol in water and use this to wash out mortar with small successive doses. method: water -* stain, 10 mins. 30°C. -> water, rinse — » abs. ale till differentiated — ' balsam, via usual reati;eiits note: For adaptation for bacterial staining, see DS 23.221 Saathof 1905; for staining fungi in skin sections see DS 23.32 Unna 1929. 356 METHODS AND FORMULAS DS 13.31-DS 13.32 13.31 Walton 1939 see DS 23.219 Walton 1939 13.32 METHYL GREEN IN COMBINATION WITH OTHER DYES 13.32 Auerbach test. 1930 Guyer acid fuchsin-methyl green Guyer 1930, 230 STOCK solutions: I. 0.1% acid fuchsin; II. 0.1% methyl green WORKING solution: stock I 40, acetic acid 0.005, stock II 60 method: [3 X sections of mercuric fixed material] -^ water — * stain, 15 mins. — > 95% ale, till green clouds cease — ♦ abs. ale. rinse — > balsam, via xylene 13.32 Biondi test. 1888 Heidenhain orange G-acid fuchsin-methyl green 16155, 63 (suppl.): 40 stock solution: mix 100 sat. sol. (circ. 11%) orange G with 20 sat. sol. {circ. 13%) acid fuchsin. Add slowly and with constant agitation 50 sat. sol. {circ. 5%) methyl green WORKING formula: Water 100, stock 1-2 method: [sections] -^ water —>■ stain, 6-24 hrs. -^ balsam, via usual reagents notes: This stain is variously attributed to Ehrlich, Ehrlich-Biondi, and Ehrlich- Biondi-Heidenhain. Heidenhain (loc. cit.) explains quite clearly " — welche dieselben Ingredientien enthdlt, ivie die von Babes empfohlene Ehrlich'sche Mischung {Berhend's Ztschr f. Mikroskopie, Bd IV, S 232) aber in anderen Verhdltnissen." This would seem to dispose of the "Ehrlich" in the name of the mixture. These new proportions, how- ever, were stated {loc. cit.) as "nach Versuchen von Biondi" and the mixture was later referred to (p. 43) as Biondi'sche Flussigkeit. 13.32 Cooper 1931 see DS 22.11 Cooper 1931 13.32 Ehrlich 1888 Ehrlich's Triacid—compl. script, see DS 13.32 Biondi (1888) (note) 13.32 Ehrlich 1898 test. Lee 1905 orange G-acid fuchsin-methyl green Lee 1905, 212 preparation: Mix 16 sat. sol. {circ. 11%) orange G with 7.5 sat. sol. (circ. 13%) acid fuchsin. Dilute mixture with 40 50% ale. Add slowly and with constant agitation 15 sat. sol. {circ. 5%) methyl green. Then add 10 each 95% ale. and glycerol. 13.32 Foley 1930 rnethyl green-acid fuchsin 763, 45 :340 REAGENTS REQUIRED: A. 2% methyl green 80, 0.1% acid fuchsin 20; B. 0.1% hydro- chloric aeid method: [sections of osmic fixed, or mordanted, material] -^ running water, overnight -^ A, 24 hrs. -^ blot — > B, till differentiated — » balsam, via carbol-xylene result: chromatin, green; muscle, brick red; other tissues, pink. 13.32 Foley 1931 methyl green-acid fuchsin-orange G 763,49:15 preparation: To 10 glycerol and 20 0.1% acid fuchsin and 30 0.1% orange G, add drop by drop, with constant agitation, 30 0.25% methyl green. method: [water] — > stain, 12-24 hrs. -^ blot — * 95% ale, till differentiated, 5-30 sees. — > balsam, via carbol-xylene note: a detailed description of the use of this stain is given under 13.30 above. 13.32 Grosso 1914 see DS 12.211 Grosso 1914 13.32 Guinard 1889 methyl green-acid fuchsin 19076,1:19 preparation: Mix 1% methyl green with 1% acid fuchsin in such proportion as give a violet solution. Add acetic aeid to pH 3. 13.32 Heidenhain 1888 see DS 13.32 Biondi (1888) (note) 13.32 Kahlden and Laurent 1896 see DS 21.3 Kahlden and Laurent 1896 13.32 Kardos 1911 see DS 21.3 Kardos 1911 13.32 Krause 1893 mttliyl green-acid fuchsin-orange G 1780,42:59 formula: sat. sol. {circ. 13%) aeid fuchsin 0.4, sat. sol. {circ. 11%) orange G 0.7, sat. sol. {circ. 5%) methyl green 0.8, water 100 DS 13.32-DS 13.40 DYE STAINS OF GENERAL APPLICATION 3o7 13.32 Krause 1911 test. 1948 Romeis methyl green-acid fuchsin-orange G Romeis 1948, 1G9 formula: water 100, methyl green 3.4, acid fuchsin 4.2, orange G 3 preparation: Grind the (hy dyes together to a line powder. Dissolve in water. method: As Biondi (1888) 13.32 Maresch 1905 see DS 13.43 Maresch 1905 13.32 Mayer 1901 test. 1901 Lee and Mayer methyl green-acid fuchsin-orange G Lee and Mayer 1901, 197 formula: water 60, glycerol 15, 95% ale. 25, orange G 2.6, acid fuchsin 4, methyl green 1.3 . . . • preparation: Grind dyes together and dissolve in mixed solvents. 13.32 Moore 1882 see DS 21.3 Moore 1882 13.32 Morel and Doleris 1902 6630, 54:1255 formula: DS 13.32 Ehrlich 1898 50, 8% formaldehyde 50, acetic acid 0.1 13.32 Oppell test. 1895 Rawitz methyl green-acid fuchsin-picric acid Rawitz 1895, 71 REAGENTS REQUIRED: A. 1% methyl green 60, 1% eosin 1, 1% acid fuchsin 20, abs. ale. 20; B. sat. sol. picric acid 80, abs. ale. 20 method: [sections] — > A, 15 mins. — > B, 30 sees. — > abs. ale, minimum possible time -^ balsam, via usual reagents 13.32 Squire 1892 methyl green-acid fuchsin Squire 1892, 37 preparation: Mix 30 0.5% methyl green with 10 1.5% acid fuchsin. 13.32 Stropeni 1912 methyl green-acridine red 23632, 29 :3()2 reagents required: A. water 100, sodium borate 1; B. water 100, glycerol 20, methanol 30, phenol 2, methyl green 0.05, acridine red 0.25 preparation of B: Grind each dye with 1 phenol and wash out each mortar with 50 water. Mix washings and add other ingredients. method: water -^ A, 10 mins. — > rinse — » B, 30 mins. — > abs. ale. till differentiated —* balsam via xylene 13.32 Thome 1898 methxjl green-acid fuchsin-orange G 1780, 52 :820 formula: sat. sol. {circ. 13%) acid fuchsin 0.15, sat. sol. (circ. 11%) orange G 0.35, sat. sol. (circ. 5%) methyl green 0.6, water 100 13.4 Techniques Employing Acid Fuchsin as the Nucleak Stain Within this subdivision of the complex 20540b, 11:101) prefers phosphotungstic staining techniques he the majority of acid. No one formula in either of these two the methods which are understood today groups can be singled out as better than whenever the term triple stain is used, another, and only one of them (Heiden- Originated by Mallory in 1901, they de- hain 1905) has become sufficiently well pend for the most part upon the fact that known to acquire a popular name. This phosphomolybdic acid will extract acid stain is frequently referred to as Heiden- fuchsin from collagens and leave it in hain's azan because azocarmine is used as muscle and nuclei. Various mixtures are the first solution, then used differentially to stain the decol- orized tissues. The original method of 13.40 typical examples Mallory used methyl blue and orange G and has been widely copied. So numerous p^p^j, nation of a transverse section of have these formulas become that it is Treparation ot a transverse section oi .., ■• , „+„i;;j« Amphioxus using the acid fuchsm- necessary in the present instance to divide •,• i , r^ ^ ■ <• ,, • 1 xu • I, V, 1 u.i;^ anihn blue-orange G stain oi them into those using phosphomolybdic ° acid (DS 13.41), and those using phospho- Maiiory lyui tungstic acid (DS 13.42), though in point Amphioxus is a difficult subject from of fact the results of the stains can scarcely wliich to prepare satisfactory sections, the be distinguished. Mallory himself (1936; more so as it is almost impossible now- 3 58 METHODS AND FORMULAS DS 13.40 adays to secure supplies of living amphi- oxus and to fix them oneself, or to prevent the supplier from whom one secures the fixed material from using Bouin's picro- acetic-formaldehyde fixative (Chapter 18, F 5000.1010 Bouin 1897) in their prepara- tion. If it is possible to secure the hving lancelets, they should be fixed by one of the methods recommended for fixing very heavily muscularized material, for it is otherwise almost impossible to secure un- broken sheets of muscle in the transverse section unless one is prepared to sacrifice histological detail to the interests of mor- phological demonstration. It is also to be regretted that popular demand has forced the biological supply houses to sell only large specimens, because these are too large to be viewed at one time in even the lowest power commonly available on a student microscope. If any selection can be exercised, care should be taken to pick a specimen of not more than 2.5 mm. greatest thickness in order that it may be seen as a whole. If, however, one is forced to use a Bouin-fixed specimen, it may be sectioned without too much difficulty provided that it first be soaked overnight in 1% nitric acid. This treatment destroys much of the fine cytological detail and should not be applied to any specimen in which it is desired to demonstrate, say, the detailed structure of the endostyle. The writer, however, is always prepared to sacrifice such detail as this in a section desired for class demonstration, in order to avoid end- less questioning as to what is tliis and that cavity which will be seen in a section of Bouin-fixed amphioxus handled by routine methods. Apart from this question of fracturing the muscular layers, no difficulty will present itself in sectioning. As many 10- micron sections as are required should be accumulated. If it is desired to place on the same slide a collection of sections from different regions of the animal, reference may be made to the description of this procedure in Chapter 12. When the sections have been mounted on a slide, deparaffinized, and run down to water, it is recommended that they be treated overnight in a saturated solution of mercuric chloride and then washed in running water for at least six hours. This process, though it takes a day, improves the \dvidness of Mallory's stain almost beyond belief when it is applied to a sec- tion of Bouin-fixed material. The actual staining procedure is simphcity itself and, though the original stain of Mallory (DS 13.41 Mallory 1900) has been selected for demonstration, there is no reason why any of the other stains in the same section should not be employed. The solutions present no difficulty in their preparation, though it is recommended that 1 % phos- photungstic acid be substituted for the 1 % phosphomolybic acid specified in the original method. After the sections have been thoroughly washed from the mor- danting in mercuric chloride, they are placed in the 1 % solution of acid fuchsin for a period of about two minutes. Tliis time is not critical, it being desired only to make sure that the entire section is thoroughly stained. On removal the sec- tions are rinsed rapidly in water, to re- move the surplus stain, and are then placed in the 1 % phosphotungstic acid until such time as the red stain has been removed entirely from the connective tissues. This may be judged partly by the cessation of the color clouds which rise from the section or by an examination under the low power of the microscope to make sure that the septa between the myotomes are free from color. The speci- fied time of two minutes is usually suffi- cient, but the sections will not be damaged however long they may be left. On re- moval from this solution they are again quickly rinsed in water, and then placed in the acid-methyl blue-orange G solution where they should remain for at least 15 minutes. The mistake is often made of leaving them for too short a time in tliis stain, for they will have the appearance of being deeply stained after an immersion of only a few moments. It does not matter how long they remain in the solution ; it is the writer's experience that soaking for at least 15 minutes discourages the subse- quent removal of the blue from the tissues. After they are removed from this rather thick staining mixture the slides are thoroughly washed in water. The wash DS 13.40-DS 13.41 DYE STAINS OF GENERAL APPLICATION 359 is designed to remove not only the whole of the adherent stain from the slide, but to permit the oxalic acid also to leach out of the tissues. No differentiation of the stain should take place in water, because such differentiation as is necessary is pro- duced bj' the absolute alcohol used in the next stage for dehydration. It is difficult to take sections stained by this method up through the usual graded series of alcohols, nor will any grave damage be occasioned by the omission of this step. If, however, the preparator is one who insists that his sections should pass through a graded series, mixtures of acetone and water should be substituted for alcohol and water. When the sections reach abso- lute alcohol, they should be watched very closely while being moved continuously up and down in the alcohol. The blue will leave them in great clouds and these clouds will taper off quite rapidly, leaving a terminal point at which no color leaves for a moment or two before a slow stream again starts to appear in the alcohol. As soon as the initial color clouds are seen to cease, the sections should be placed in xylene which stops the differentiation. If the preparator is uncertain of this method, or is trj'ing it for the first time, it is recommended that the slides be thoroughly washed in absolute alcohol Ijut removed to xjdene before the color clouds have ceased to leave. Examination under the low power of the microscope will now show these prejiarations to have a dull purple muscle and an intensely blue con- nective tissue. A few trial sections sliould now be returned to absolute alcohol for a few moments and then back into xjdene and reexamined. It is possible by this means to exercise perfect control over the differentiation, which should be stopi)ed when the muscles and nuclei are clear red, and the connective tissues a clear light blue. No attention should be jxiid, while differentiating, to any of the structures (such as the gonad) which by this method acc}uire a violet coloration. The process should be controlled only by apparent contrast between the pure blues and pure reds in the section. The stains used in this preparation are alkali-sensitive, and it is a customarj' pro- cedure in Europe to mount them in as acid a medium as possible. If one is using one of the synthetic resins, which are neutral, it is strongly recommended that the coversUp, before being applied to the resin, be dipped briefly in a strong solution of salicylic acid in xylene. This salicyhc acid will then dissolve in the resin and pro- vide a permanently acid environment. 13.41 METHODS EMPLOYING THE ACID FUCHSIN-PHOSPHOMOLYBDIC REACTION Waterman 1937 (20540b, 12 :21) recommends that dioxane be substituted for ale. in the dehydration of sections stained by these techniques. Kemohan 1934 (4349, 13 :82) mordants formaldehyde material 4 days in ADS 12.1 Weigert 189(3 followed by 2 days in ADS 12.1 Weigert 1891. 13.41 Bensley test. 1938 Mallory cil. Warren IMallory 1938, 210 REAGENTS REQUIRED: A. DS 22.21 Altiiiann 1890 (sol. .4); B. 1% phosphomolybdic acid; C. water 100, orange G 2, anilin blue 0.5 method: [sections] —> water — > .4, 10 niins. -> rinse -^ B, 10 niins. -^ quick rinse — > C, 1 hr. -^95% ale. till color clouds cease —> balsam, via usual reagents 13.41 Dupres 1935 14425,46:77 REAGENTS HEQiiRED: .4. DS 11.43 Gallcgo 1919; B. water 50, acetic acid 25, 40% formal- dehyde 20; C. 1% phosphomolybdic acid; D. water 100, oxalic acid 4, toluidine blue 0.25, orange G 4 method: [sections of F 3670.0010 Iluffini 1927 material] — > A, 1-10 mins. -^ B, wa.sh — » rinse —' C, 10 mins. -^ wash -> D, 1-2 mins. — > blot — > 95% ale, till differentiated -+ balsam, via usual reagents. note: For toluidine blue in D above there may be substituted cither methylene blue 0.3 or malachite green 0.2 or methyl green 0.3. 360 METHODS AND FORMULAS DS 13.41-DS 13.42 13.41 Kricheski 1931 10540b, 6 :97 REAGENTS REQUIRED: A. 0.25% acid fuchsin; B. 2% methyl blue 30, 1% orange G 30, 1% phosphomolybdic acid 30 METHOD : water — * A, 1-3 mins. -^ water, thorough rinse —> B, 3-5 mins. — > water, quick rinse -^ 70% ale, 2 or 3 dips -^^ 95% ale, 2 or 3 dips -^ abs. ale. till differentiated 1-3 mins. -^ balsam, via xylene result: as Mallory 1901. 13.41 Ladewig 1938 see DS 13.6 Ladewig 1938 13.41 Lee-Brown 1929 see DS 13.41 Mallory 1901 (note) 13.41 Lewis and Miller 1938 see DS 21.421 Lewis and Miller 1938 13.41 Mallory 1901 11189,5:15 REAGENTS REQUIRED: A. 1 % acid fuchsin; B. 1% phosphomolybdic acid; C. water 100, methyl blue 0.5, orange G 2, oxalic acid 2 method: [water] — > .4, 2 mins. — * water, thorough rinse — > B, 2 mins. — > water, quick rinse -^ C, 15 mins. -^ water, thorough wash — > abs. ale, till differentiated -^ balsam, via xylene results: nuclei, red; coUagens, blue; nerves and glands, violet; muscle, red; erythro- cytes and keratin, orange. note: Mallory 1936 (20540b, 11:101) substituted 1% phosphotungstic acid in B. A detailed description of the application of this stain is given under DS 13.40 above. Lee-Brown 1929 (11597, 21:259) differs only in using sol. C before sol. B. Rexed and Wohlfart 1939 (23632, 56:212) recommend that A above be buffered to pH 3.3 with citrate. 13.41 Masson 1912 4956, 14:291 reagents required: A. 4% ferric alum at 50°C.; B. 1% hematoxylin at 50°C.; C. 2% ferric alum; D. 0.1% acid fuchsin; E. 1% phosphomolybdic acid; i^. 1% anilin blue 50, 1 % phosphomolybdic acid 50 method: [water] —> A, 5 mins., 50°C. -^ water, 50°C. rapid rinse-* 5, 10-15 mins., 50°C. — ^ C, till nuclei alone colored — > running water, 15 mins. — > D, 10 mins. -* tap water, if overstaining has occurred — * E, 5-10 mins. — > F, 20 mins. to 1 hr. — » water, quick rinse — > 95% ale, quick rinse — > abs. ale till dehydrated — > balsam, via xylene 13.41 Maxwell 1938 see DS 21.421 Maxwell 1938 13.41 Milligan 1946 Tech. Bull, 7 :57 reagents required: A. water 75, 95% ale 25, potassium dichromate 2.25, hydrochloric acid 2.5; B. 0.1% acid fuchsin; C. 1% phosphomolybdic acid; D.2% orange G in 1% phosphomolybdic acid; E. 1; acetic acid; F. 0.1% either fast green FCF or anilin blue in 0.2% acetic acid method: [sections of formaldehyde material] — * water — * A, 5 mins. rinse —* B, 5 mins. — > rinse — > C, 1-5 mins. — > D, 5-10 mins. —^ rinse —^E,2 mins. — » F, 5-10 mins. — > E, 3 mins. — > 95% ale. — ♦ balsam, via usual reagents 13.41 Rexed and Wohlfart 1939 see DS 13.41 Mallory 1901 (note) 13.41 Schneidau 1937 21400a, 56:260 reagents required: A. 1% acid fuchsin; B. 10% phosphomolybdic acid; C. 0.1% thionine method: [whole objects] — > A, 2-4 mins. — > wash —* B, 1 min. — » rinse —* C, 2-4 mins. —> wash — » balsam, via usual reagents. recommended for: double stained wholemounts. 13.42 METHODS EMPLOYING THE ACID FUCHSIN-PHOSPHOTUNGSTIC REACTION 13.42 Cason 1950 20540b, 25 :225 formula: water 100, phosphotungstic acid 0.5, orange G 1, anilin blue 0.5, acid fuchsin 1.5 method: [Gju sections] —> water -^ stain, 5 mins. —> water, rinse ^ balsam, via usual reagents DS 13.42-DS 13.43 DYE STAINS OF GENERAL APPLICATION 3G1 » 13.42 Heidenhain 1905 Heidenhain's Azan — compl. script. 23G32, 22 :33'J REAGENTS REQUIRED: A. water 100, azocarmine 2, acetic acid 1; B. 0.1% aniline in 95% ale; C. 0.1% hydrochloric acid in abs. ale; D. 5% phosphomolybdic acid; E. water 100, orange G 2, anilin blue 0.5, acetic acid 7.5 method: water— » A, 1 hr., 60°C. -^ water, quick rinse -^ B, till nuclei well marked dip in C, before examining — » C, thorough rinse — > jD, 2 hrs. — > E, 2-3 hrs. — > water, rinse — >• balsam, via usual reagents result: nuclei, scarlet; muscle, orange; coUagens, blue. 13.42 Kohashi 1937 . 8542a, 15:175 reagents required: A. water 100, azocarmine 0.1, acetic acid 1; B. 90% ale. 100, aniline 0.1; C. 1% acetic acid in 95% ale; D. 5% phosphotungstic acid; E. DS 12.31 Pasini (1928) (sol. B) method: [sections] —* water — » A, 12-15 mins. 60°C. — > wash -^ B, till nuclei differenti- ated — » C, li-1 mins. — > rinse — >• D, l-i-l hr. -^ rinse -^ E, 15-20 mins. —+95% ale. till differentiated — > balsam, via c^.rbol-xylene 13.42 Walter 1930 23632, 46 :458 REAGENTS REQUIRED: A. 2.5% ferric alum; B. 2% phosphotungstic acid; C. DS 12.31 Pasini (1928) sol. B method: water -^ A, 2-4 hrs. — > water, quick rinse — » B, 10 mins. — * water, quick rinse — » C, 15-20 mins. — » 95% ale, till color clouds cease — > abs. ale, 1 min. — > balsam, via usual reagents result: nuclei and elastic fibers, purple; coUagens, blue; erythrocytes, orange. 13.43 METHODS USING NEITHER PHOSPHOTUNGSTIC NOR PHOSPHOMOLYBDIC ACID 13.43 Auerbach see DS 13.32 Auerbach (1930) 13.43 Bohm and Oppel 1907 Bohm and Oppel 1907, 115 stock formulas: I. water 100, orange G 0.05, acid fuchsin 0.05, acetic acid 1, 40% formaldehyde 1; II. water 90, methanol 10, 0.25% brilliant cresyl blue 0.25, 40%, formaldehyde 1 WORKING solution: stock I 50, stock II 50 method: water —> stain, 20-30 mins. -^ abs. ale, till differentiated —> balsam, via xylene result: nuclei, red-purple; cartilage, blue; bone, orange; muscle, red. 13.43 Buchholz 1931 see DS 21.41 Buchholz 1931 13.43 Delamare 1905 see DS 13.7 Delamare 1905 13.43 Maresch 1905 23681,16:41 REAGENTS REQUIRED: A. sat. methanol sol. methyl green 50, sat. methanol sol. picric acid 50; B. Q.lb7o acid fuchsin method: [sections] -* abs. ale -^ A, 10 mins. — > water, rinse -» B, 5-10 sees. -» blot -» — > abs. ale dropped on slide, till color changes from dark violet to blue gray -^ bal- sam, via xylene 13.43 Roskin 1946 Roskin 1946, 154 REAGENTS REQUIRED: A. 0.1% indigocarmine in sat. sol. picric acid; B. sat. aq. sol. magenta method: [sections] -> A, 10-30 mins. -^ B, poured on slide, left till greenish scum ap- pears — » abs. ale, till difJerentiated 13.43 Waterman 1937 20540b, 12:21 PREPARATION OF STOCK SOLUTION! water 100, phcnol 5, magenta 3 REAGENTS REQUIRED: A. Water 100, acetic acid 0.6, 40% formaldehyde 0.6, stock 10; B. water 100, picric acid 1, acetic acid 2, indigocarmine 0.25 method: [sections of material fixed in F 4000.0040 Watermann 1937] -^ water -^ A, 5 mins. -^ rinse, 2 sees. -^ B, 90 sees. -+ rinse, 2 sees. -^50% dioxane, 2 sees. -^ bal- sam, via dioxane and xylene result: nuclei, red; collagen, blue; other tissues, yellow and green 362 METHODS AND FORMULAS DS 13.5-DS 13.50 13.5 Techniques Employing Safranin as the Nuclear Stain Safraiiiu is scarcely employed in the English-speaking countries as a histolog- ical nuclear stain, and no current staining technique for animal tissues which re- quires such a procedure is known to the author. Most of the stains placed in this class were developed for plant purposes and there is no doubt that few finer botan- ical stains can be produced. 13.50 TYPICAL example Preparation of a transverse section of a Ranunculus stem using the safranin-crystal violet-fast green-orange II stain of Conant (1940) The inexperienced microtomist must clearly distinguish between those methods of staining plant tissues, such as the pres- ent, which are designed to stain both the cytoplasm and the cell walls, and those such as the example given in the next chapter, which are designed to stain only the cell walls. The latter are much simpler and may be advantageously employed for class-demonstration purposes. The present technique, which is one of the simplest of the many quadruple stains which have been suggested for plant tissues, may be used as a general-purpose stain for all plant material, and the selection, in the present instance, of a Ranunculus stem as a demonstration object is designed solely because of its relative commoness and be- cause of the ease with which it may be prepared. This staining method has the disadvantage that it does not always yield reproducible results, hence it is better for demonstration tlian for research purposes. If it is of primary importance that sections show identical staining when prepared at different times from sliglitly different ma- terial, it is recommended that the tech- nique of Johansen 1940 (DS 13.5 Johansen 1940) be substituted. The species of Ranunculus selected for demonstration purposes is not of great importance, but it must be remembered that the fame which the stem of this plant enjoys for teaching purposes is based upon the structure of Ranunculus acris, the commonest European buttercup, which is, however, so widely dispersed in the northeastern United States that it may be gathered without trouble. So many textbook diagrams are based on the sec- tion of this species that it is well worth the trouble to collect it. The choice of fixative is far less critical in botanical than in zoological micro- techniques, and it will be quite sufficient for the present purpose to use any of those fixatives, known to botanists as AAF fixa- tives, which will be found in Chapter 18 under the heading of F 0000.1010. These alcohol-acetic-formaldehyde mixtures keep indefinitely, and no two botanists have ever agreed as to what are the best con- centrations to be employed. Stems should be collected, so far as pos- sible, toward the middle of the summer, when the structural detail will be found at its best, and the fixative should be taken into the field. The fresh stem should be severed from the plant, and about the lower ^4 of an inch cut off a few moments later to get rid of the air which will have been drawn into the vessels by the tur- gidity of the specimen. The remainder of the stem is then cut into about i-^-inch lengths and placed in a 250 cc. bottle of the fixative. It should remain in this fixa- tive, which may be changed as rapidly as it becomes discolored, until the chloro- phyll has been thoroughly removed, and should then be washed in 70% alcohol until the washings no longer smell of acetic acid. The specimens may then be stored in 70% alcohol, though it has been suggested that slightly better preservation of detail may be secured by the addition of 1 % glycerol to the preserving fluid. This is not, however, a matter of any importance provided less than a year elapses between the time of the collection of the material and the cutting of sections. The stem presents no difficulties in em- bedding either by the dioxane technique or by the more conventional routine of alcohol-xylene. It is suggested that sec- tions be cut at about 15 microns in thick- ness, since these rather thicker than nor- mal sections give a much better idea of general structures when used for teaching purposes. The sections are attached to the slide, deparaffinized in the usual manner, DS 13.50-DS 13.51 DYE STAINS OF GENERAL APPLICATION 3G3 and brought down througli the customary graded alcohols to 70% alcohol. Plant material is far more likely to become dis- torted through insufficient attention being paid to grading alcohols than is animal material. When all the slides have been accumu- lated in 70% alcohol, they may be trans- ferred to the first stain, which is of 1 % safranin in 50 9o alcohol, and may remain in this for any time desired l)y the mounter. The customar}^ limits are from 2 to 24 hours. On removal from the safranin, the slides should be cheeked under the low power of the microscope to make sure that the nuclei of the cells are densely stained. Provided these are stained, all other struc- tures will have acquired the necessary safranin coloration. On removal from the safranin, the slides should be washed in distilled water until no more color comes away. A single slide is then withdrawn, and a saturated solution of crystal violet is poured over the section on the slide and left for about one minute. After being stained with crystal violet, the sections should be rinsed in water until no more color comes away, and then dehydrated as rapidly as possible through two changes of absolute alcohol. This is a rather criti- cal stage in the staining, since the alcohol will remove the violet very readily and one is, in effect, engaged in a race between the minimum time required for dehydra- tion and the minimum time in which the stain will be lost. As soon as the slides are judged to be dehydrated, they are dipped briefly in 1 % fast green dissolved in absolute alcohol in a copliii jar. Now place the slide in a saturated solution of orange II in clove oil. It is essential that the alcohol fiom the dehydration and from the fast green stain be removed as rapidly as possible so that it is desirable to have at least two coplin jars of the orange II- clove oil solution. The first of these is used e.xclusively for rinsing the slide backward and forward until all the adhering alcohol has left it. The slide may then be trans- ferred to the next coplin jar of orange II- clove oil, in which it may be left, while the remaining slides of the batch are brought from 70 '^y alcohf)l through the successive stages of the technique and accumulated in their turn in the orange Il-clove oil. The orange Il-clove oil acts as a differ- entiating agent and the slides may now be observed at intervals, and, if differenti- ation is insufficient, allowed to remain till the required structures can be clearly seen. As soon as difTcrontiation is judged to be complete, the slides are transferred to a fresh jar of clove oil. Here the orange Il- clove oil is thoroughly washed from them and they are then taken to xylene in which the clove oil is removed. The omission of the last clove-oil wash is a cause of fre- quent failure, since any adherent orange- Il-clove-oil solution which is carried over into xjdene will lose the orange II by precipitation, producing a veil over the finer structures. The slides may be left in xj'lene for as long as required before being mounted in balsam in the customary manner. It must again be emphasized that this method is not intended for the class demonstration of skeletal structures, but only to bring out the finer details of the cytoplasmic and nuclear constituents of any plant material. 13.51 OTHER TECHNIQUES 13.51 Conant lest. 1940 Johansen safranin-crystal violel-fast gr((n-(>i(tii(i< II johansen 1040, 87 REAGENTS REQUIRED: A. 1% Safranin in 50% ale; B. sat. sol. {circ. 1%) crystal viukt; C 1% fast green in abs. ale; D. sat. sol. orange II in clove oil method: [sections] — ► 70% ale. -^ A, 2-24 hrs. -^ water, rinse * H, 1 niin. — > water, rinse -^ abs. ale. till dehydrated-^ C, 5-10 dips -^ I), till altohol removed-^ D, fresh solution, till differentiated — > balsam, via xylene note: A detailed description of the use of this stain is given under DS 13.50 above. 13.51 Foley 1929 safmnin-orange G-crystal violet 76:3,43:171 reagents reqcired: A. 1% safranin O; B. N /AO liydrochloric acid; C. 0.3% crystal violet in 70% ale; D. ADS 12.2 Lugol (1905); E. 1% mercuric chloride; F. sat. sol. orange G in clove oil 364 METHODS AND FORMULAS DS 13.51 method: [sections of osmic-fixed, or mordanted, material] — > wash -^ A, overnight — > B, till outline of nuclei distinct — > C, 20 niins. -^ B, till outline of nuclei distinct — » D, till sections deep black, 1-3 mins. — > running water, till iodine removed — > E, till sections bright blue, 1-3 mins. — > water, thorough wash —> blot — > 95%, rinse 5 sees. — * carbol-xylene till differentiated, J£ to 5 mins. —>■ xylene, 2 mins. — > F, 1-2 mins. — > clove oil wash -^ balsam, via xylene 13.51 Henneguy 1898 test. 1907 Bohm and Oppel safranin-methyl violet-orange G Bohni and Oppel 1907, 111 REAGENTS REQUIRED: A. Water 100, ammonium thiocyanate 1, methyl violet 0.1, orange G 0.1; 5. DS 11.42 Zwaademaker 1887 method: [sections] -^ A, 10 mins. — > rinse -^ B, 15 mins. -^ rinse -^ A, 15 mins. — > abs. ale, minimum possible time — ^ balsam, via clove oil result: nuclei, red; cytoplasm, various shades of blue and blue gray. 13.51 Johansen 1940 safranin-methyl violet-fast green-orange G Johansen 1940, 88 REAGENTS REQUIRED: A. DS 11.42 Johanscu 1940; B. 1% methyl violet; C. 95% ale. 30, ethylene glycol monomethyl ether 30, tert. butyl ale. 30; D. clove oil 6, ethylene glycol monomethyl ether 6, fast green FCF q.s. to sat., 95% ale. 36, tert. butyl ale. 36, acetic acid 12; E. 95% ale. 50 tert. butyl ale. 50, acetic acid 0.5; F. sat. sol. orange G in ethylene glycol monomethyl ether 30, ethylene glycol monomethyl ether 30, 95% ale. 30; G. clove oil 30, ethylene glycol monomethyl ether 30, 95% ale. 30; H. clove oil 30, abs. ale. 30, xylene 30 method: [sections] -^ 70% ale. -^ .4, 1-2 days -^ water, rinse ^^ B, 10-15 mins. — > water, rinse —> C, 15 sees. — * D, 10-15 mins. — * E, quick rinse -^ F, 3 mins. — > G, rinse — > H, rinse — > balsam, via xylene result: (plant tissues) dividing chromatin, red; resting nuclei, purple: lignified and suberized tissues, red; cellulose, green-orange; cytoplasm, bright orange; starch grains, purple; fungal mycelia, green. 13.51 Kalter 1943 safranin-crystal violet-fast green-orange II 11284,28:995 REAGENTS REQUIRED: A. Water 50, 95% ale. 50, 40% formaldehyde 4, sodium acetate 0.5, safranin 0.2; B. 0.5% crystal violet; C. clove oil 100, fast green FCF and orange II, each to sat.; D. sat. sol. orange II in clove oil method: [sections] — > water — > A, 24 hrs. -^ rinse -^ B, 1-2 mins. -^ wash — » 95% ale. -^ C, 5 mins. -^ clove oil, till connective tissue green -^ D, 10 mins. —* clove oil -^ xylene, thorough wash — * balsam result: nuclei, red; cytoplasm, pink or light green; muscle, tan; collagen, bright green. 13.51 Laguesse 1901 test. 1907 Bohm and Oppel safranin-crystal violet-orange G Bohm and Oppel 1907, 154 REAGENTS REQUIRED: A. 2% potassium sulfite; B. DS 11.42 Babes 1887; C. sat. aq. sol. crystal violet 50, sat. aq. sol. orange G 2 PREPARATION OF C: Mix the solution. Add just enough water to redissolve ppt. method: [sections] —> A, 12-24 hrs. -^ wash — > B, 6-12 hrs. ^ wash -^ C, 24 hrs. — > blot — ^ abs. ale. minimum possible time — > clove oil, till differentiated —> balsam, via xylene 13.51 Stockwell 1934 safranin-grntial violet-orange G 19938,80:121 REAGENTS REQUIRED: Aa. ADS 12.1 Stockwcll 1934 or Ab. 1% chromic acid; B.l% crys- tal violet 20, 1% safranin O 40, water 40; C. AFS 12.1 LaCour 1931; D. 1% picric acid in 95% ale; E. 95% ale. 100, ammon. hydroxide 0.3; F. 0.2%, orange G in clove oil method: [sections]-^ water —> Aa, if bleaching required (or Ab if not chromic fi.xed) overnight -^ water, thorough wash — » B, 1-6 hrs. — > water, rinse -^ C, 30 sees. -^ 70% ale, quick rinse -^ D, few sees. — » E, few sees. — > abs. ale, few sees. -^ F, few sees. — ^ balsam, via xylene 13.51 Unna 1928 test. 1928 Schmorl safranin-orcein-anilin blue-eosin Schmorl 1928, 343 stock solutions: I. water 50, abs. ale 25, glycerol 10, acetic acid 2.5, orcein 0.5, anilin blue 0.5; II. 80% ale 100, ethyl eosin 1; III. 1%, hydroquinone DS 13.51 DS 13.6 DYE STAINS OF GENERAL APPLICATION 3G5 REAGENTS REQUIRED: A. stock I 50, stock IT 15, stofk TIT 15; B. 1% safranin; C. 0.5% potassium dichroinate method: [sections] — > watei- —* A, 10 mins. —^ wash -^ B, 10 mins. — » thorough wash — ♦ C, 10-30 mins. — > wash — > balsam, via usual reagents result: nuclei, black with red granules; protoplasm violet; collagen, blue; elastic fibers, red recommended for: skin. 13.51 Unna test. 1928 Schmorl safranin-anilin blue Schmorl 1928, 176 reagents required: ^1. 1% safranin; B. water 100, tannin 15, anilin blue 0.5 method: [sections] ^ a, 10 mins. —^ thorough wash — > B, 10-15 mins. —> thorough wash — ^ abs. ale. minimum possible time -^ balsam, via xylene 13.6 Techniques Employing Hematoxylin as the Nuclear Stain Hematoxylin is, of course, the commonest nuclear stain to be employed before any of the plasma techniques (DS 12). The formulas here given are those in which the nuclear staining is an integral portion of a complex technique which cannot be employed in combination with any other nuclear stain. 13.6 Barbrow 1937 11284, 22:1175 STOCK solutions: I. 1% hematoxylin in 95% ale. (well "ripened"); II. water 99, ferric chloride 2, hydrochloric acid 1; III. water 100, picric acid 1, acid fuchsin 1 working solution: stock I 25; stock II 10; stock III 50 method: [frozen sections of unfixed tissues] -^ stain, 1 min. —» wash ^ balsam, via usual reagents. 13.6 Brown and Brenn 1931 see DS 23.221 Brown and Brenn 1931 13.6 Delamare 1905 test. 1907 Bohm and Oppel Bohm and Oppel 1907, 131 stock solutions: I. abs. ale. 100, hydrochloric acid 2, orcein 2; II. sat. aq. sol. picric acid 100, sat. aq. sol. acid fuchsin 0.5, DS 11.123 Ehrlich 1886 1 reagents required: A. stock I 50, stock II 50; B. 0.1% hydrochloric acid method: [sections] — * water —> A, 30 mins. -^ B, rinse -^ tap water, to "blue" hema- toxylin -^ balsam, via usual reagents result: nuclei, violet; muscle and general cytoplasm, yellow; collagens, red; elastic fibers, black. 13.6 Friedlander 1889 Friedlander 1889, 94 formula: abs. ale. 30, hematoxylin 0.5, glycerol 30, sat. sol. potassium alum 30, 1% eosin 10 preparation: Dissolve the hematoxylin in the ale. Add the glycerol and alum. Leave 1 week. Filter. Add the eosin sol. to filtrate. 13.6 Galiano 1928 test. 1928 Findlay 11360, 48:314 reagents required: A. 3% ferric alum; B. water 80, acetic acid 20, hematoxylin 0.2; C. 95% ale. 75 m acetic acid 25, eosin 1.5; D. 0.1% ammonia in 95% ale. method: [sections] —> water -^ a, 15 mins. — > 5, till nuclei darkly stained —>• wash, 15 mins. -^70% ale, 1 min. -^ C, till differentiated — » D, wash— > balsam, via usual reagents 13.6 Kefalas 1926 11360, 46:277 reagents required: A. DS 11.121 Kefalas 1926; B. sat. sol. Biebrich scarlet in acetone method: [sections] —> acetone -^ a, till slightly overstained — > B, till counterstained and differentiated —> acetone —y balsam, via xylene 13.6 Koneff 1936 763, 66:173 reagents required: A. 5% ferric alum; B. DS 11.122 Harris 1900; C. water 100, anilin blue 0.03, phosphomolybdic acid 5, oxalic acid 0.6 13.6 Ladewig 1938 23032,55:215 reagents required: A. DS 11.121 Weigert 1903; B. 1% phosphotungstic acid; C. water 100, methyl blue 0.5, orange G 2, oxalic acid 2, acid fuschin 1 method: [sections of formaldehyde material] -^ water —» .1, 3-5 mins. rinse —> B, 2 mins. — > rinse ^ C, 4 mins. -^ quick rinse — > 95% ale, till dehydrated — > balsam, via xylene 3GG METHODS AND FORMULAS DS 13.6 13.6 Lillie 1945 4349, 25 :33 REAGENTS required: A. sat. 95% ale. sol. picric acid; B. DS 11.121 Weigort 1903; C 1% Biebrich scarlet in 1% acetic acid; D. 3% ferric chloride; E. water 99, acetic acid 1 and either anilin blue 1 or methyl blue 1 or wool green S 1 ; F. 1 % acetic acid method: [sections] — > 95% ale. — > A, 2 mins. -^ thorough wash -> B, 6 mins. — > wash -^ C, 4 mins. — > D, 2 mins. -^ £^, 3-5 mins. -^ rinse -^ F, 2 mins. — > balsam, via acetone and xylene result: nuclei, brown black; muscle and cytoplasm, red; connective tissue, blue or green. 13.6 mile 1948 Lillie 1948, 149 REAGENTS REQUIRED: A. DS 11.121 Weigert 1904; B. 0.02% fast green FCF; C. 1% acetic acid; D. 0.1% Bismarck brown Y in 1% acetic acid method: [sections] -^ water -^ A, Q mins. -^ wash — ♦ 5, 3 mins. — >• C, wash — >■ D, 4-6 mins. -^ balsam, via usual reagents result: nuclei, black; general cytoplasm, gray green; mucus, cartilage, cell granules, brown. note: Magenta, or new magenta, may be substituted for Bismarck brown in D. Eosin Y may be substituted in B, in which case crystal violet or malachite green should be substituted in D. 13.6 Lowenthal 1892 test. 1907 Bohm and Oppel Bohm and Oppel 1907, 119 preparation of stock solutions: I. Dissolve 0.4 carmine in 100 0.05% sodium hydrox- ide. Add 0.25 picric acid; II. to 0.5 hematoxylin dissolved in 50 abs. ale. add 50 1: ammonium alum. WORKING solution: stock I 100, stock II 20, acetic acid 2.5 PREPARATION OF WORKING SOLUTION: Mix the stock solutions slowly and with constant agitation. Leave 24 hours. Filter. Ripen 4 weeks. method: [sections] -^ water — > stain, 24 hrs. -^ wash -^ balsam, via usual reagents 13.6 Mollier 1938 23632,55:472 REAGENTS REQUIRED: A. DS 21.12 Unua-Tanzcr (1896); B. DS 11.121 Weigert 1904; C 1% hydrochloric acid in 70% ale; D. water 100, azocarmine 2, acetic acid 1; E. 5% phosphotungstic acid; F. water 100, naphthol green B 1, acetic acid 1 method: [sections] —> 70% ale. -^ A, 12 hrs. — > wash, till no more color comes away — > B, 1-3 mins. — > rinse -^ C, till nuclei well differentiated — > wash, 15 mins. -^ D, 15-30 mins. —^ rinse -^ E, 3 changes, 2-6 hrs., till collagen decolorized — * rinse -^ F, 15-30 mins. — > 95% ale., 30 sees, with constant agitation -+ balsam, via usual reagents result: nuclei, deep blue; general cytoplasm, purple; elastic fibers, black; collagen, green; erythrocytes, scarlet. 13.6 Mollendorf test. 1946 Roskin Roskin 1946, 158 reagents required: A. DS 11.121 Hansen 1905; B. 1% eosin in 0.3%, acetic acid; C. 2% phosphomolybdic acid; D. 1% methyl blue method: [sections] —> water —> a, 5 mins. ^ distilled water, rinse ^ running water, wash -^ B, 20 mins. —>■ rinse -^ C, 10 sees. -^ rinse -^ D, 1-2 mins. -^ rinse -^95% ale. till color clouds cease — > abs. ale. balsam, via xylene 13.6 Paquin and Goddard 1947 4349, 27:198 REAGENTS REQUIRED: A. DS 11.121 Paquiu and Goddard 1947; B. 0.5% picric acid in 95% ale; C. water 100, phosphotungstic acid 0.1; eosin 0.07, phloxine 0.03, orange G 0.1; D. 0.2% phosphotungstic acid; E. 0.4% acetic acid; F. 0.04% anihn blue in 1% acetic acid method: [sections of F 5600.1000 Masson (1947) material]-^ water-* A, 5 mins. -► wash, 5 mins. -^ B, 15-20 sees. -> wash ->■ C, 5 mins. -> D, 5 mins. -^ E, double rinse -> F, 5 mins. -^ E, double rinse -^ D,b mins. -^ E, 30 mins. -^ 95% ale, 3 dips -^ balsam, via isoamyl ale. and toluene result: nuclei, black; elastic tissue, cherry red; other connective tissues, blue; general cytoplasm, pink. 13.6 Papanicolaou 1941 see DS 23.4 Papanicolaou 1941 DS 13.6-DS 13.7 DYE STAINS OF GENERAL APPLICATION 367 13.6 Petragnani 1928 see DS 23.13 Petragnani 1928 13.6 Renaut k^t. 1889 Friedlander Friedlander 1889, 94 formula: sat. sol. potassium alum in glycerol 65, sat. sol. eosin Y 15, sat. ale. sol. hema- toxylin 20 13.6 Reeve 1948 205401), 23:1:5 REAGENTS REQUIRED: A. watcr 90, DS 11.122 Dolaficid (1885) 10; li. water 40, 95% ale. 60, safranin 0.01, sodium acetate 0.01; ('. xylene 75, abs. ale. 25, sat. sol. fast green FCF in 50:50 clove oil— abs. ale. 2-5 method: [sections] —^ water — * A, 5-15 mins. —' wash -^ B, 5-15 mins. — * rinse — » 95% ale., till no more color comes away -^ C, 1-3 mins. -^ balsam, via xjdene recommended for: general plant histology. 13.6 Reynolds 1936 see DS 23.34 Reynolds 1936 13.6 Romeis 1948 Romeis 1948, 364 REAGENTS REQUIRED: A. DS 21.13 Wcigert 1898 (working sol.); B. DS 11.121 Weigert 1903; C. water 100, azophloxine 0.5, acetic acid 0.2; D. 1% acetic acid; E. water 100, phosphomolybdic acid 4, orange G 2; F. water 100, light green 0.2, acetic acid 0.2 method: [sections] — * 80% ale. —>■ A, 15 mins. -^ wash — » B, 2-3 mins. — > thorough wash — ^ C, 5 mins. — > D, wash — > E, till collagen decolorized -^ D, rinse -^ F, 5 mins. -^ D, 5 mins. — » abs. ale, least possible time -^ balsam, via xylene result: nuclei, black; muscles and general cytoplasm, red; collagen, green; elastic fibers, black. note: The C, D, E, F solutions are from DS 12.31 Goldncr 1938. 13.6 Slater and Dornfeld 1939 20540b, 14:103 REAGENTS REQUIRED: A. DS 11.123 Harris 1900; B. 1% safranin () in sat. aq. sol. aniline; C. 0.5% fast green in 95%, ale. method: [sections by dioxane technique of amphibian embryos from F 5000.1010 Puckett 1937] -^ A, 5 mins. — » C, 2-5 mins., till yolk granules red on green cytoplasm —>■ balsam, via usual reagents 13.7 Other Complex Techniques of General Application This is the final division of complex clear stain and picric-indigo-carmine as staining techniques in which it has been the contrast. This gives magnificent pic- necessary to place all those miscellaneous tures on embryonic material and is so easy techniques which do not fall into the pre- to use that it is safe even in the hands of ceding classifications. Few of them are well beginning classes. The mixture of Tw-ort, known, and the three techniques of Becher of neutral red and liglit green, was at one 1921 should receive wider attention than time even more popular for staining blood they have received. People have become films than the conventional methylene accustomed to regarding the soluble lakes blue-eosonates; it is now rarely heard of, of the oxazines, when they are used at all, and then only for staining of blood para- as nuclear stains, hence no attention ap- sites. It would be an excellent general- pears to have been given to those other purpose tissue stain if it were not for the formulas published by Becher in which extreme sensitivity of both of its ingrcdi- these dyes are used with varying mordants ents to variations in the pll of the final for the purpose of providing a single-solu- mounting media. The methods of Lonn- tion polychrome stain. These stains can berg 1891 and Lynch 1930 are of interest quite safely be used for research purposes, only in that they provide methods of since their results are reproducible and double-staining wholemounts. There is no they are stable to fight and acid. Attention justification for this from a research point should also be drawn to the formula of of view, but they make the most amusing Shumway 1926, which is a rather unusual mounts, which can be used for popular dis- triple stain involving magenta as the nu- play very cfi"ectively. 368 METHODS AND FORMULAS DS 13.7 13.7 Alzheimer 1910 see DS 13.7 Mann 1894a (note) 13.7 Becher 1921a polychrome gallamin blue Becher 1921, 70 formula: water 100, sodium alum 5, gallamin blue 0.5 preparation: BoU 5 minutes. Cool. Filter. method: [sections] —> water -^ stain, 24 hrs. —> water, wash —» balsam, via usual reagents result: nuclei, black; cartilage, violet. 13.7 Becher 1921b polychrome quinalizarine Becher 1921, 55 formula: water 100, chrome alum 5, quinalizarin 0.5 preparation: Boil 5 minutes. Cool. Filter. result: nuclei, blue; muscle and nerves, red. 13.7 Becher 1921c polychrome coeleslin blue Becher 1921, 73 formula: water 100, chrome alum 5, coelestin blue 0.5 preparation: Boil 5 minutes. Cool. Filter. method: [sections] -^ water — > stain, 24 hrs. -^ water, wash —* balsam, via usual reagents result: blue black nuclei, with a violet to red polychrome staining of connective tissues. For coelestin blue as a nuclear stain, see DS 11.41. 13.7 Bensley and Bensley 1938 acid fuchsin-crystal violet Bensley and Bensley 1938, 97 preparation of dry stain: Add a sat. sol. acid fuchsin to a sat. sol. crystal violet till no further ppt. produced. Filter. Wash and drj- ppt. preparation of stock solution: abs. ale. 100, diy stain from above to sat. reagents required: .1. water 72, abs. ale. 18, stock solution 10; B. clove oil 75, abs. ale. 25 method: [sections] -^ water -^.4,5 mins. — > blot — * acetone, till dehydrated —* benzene — » B, till differentiated -^ benzene —> balsam 13.7 Bohm and Oppel 1907 Bismarck brown-dahlia violet-methyl green Bohm and Oppel 1907, 127 REAGENTS REQUIRED: A. Sat. sol. {circ. 1.5%) Bismarck brown: B. 12.212 Roux 1894 method: water — > A, 10 mins. — > water, quick rinse -^ B, 1 min. — > abs. ale. till differ- entiated, few sees. -^ carbol-xylene — * balsam, via xylene result: as 12.212 Roux 1894 but with black nuclei and brown cartilage. 13.7 Bonney 1908 methyl violet-orange G-pyronin 22575,193:547 REAGENTS required: A. Water 100, methyl violet 0.25, pyronin 1; B. acetone 100 2% orange G q.s. PREPARATION Of B: To 100 acetone add 2% orange G till ppt. first formed just redis- solves. method: [sections, mercuric-fixed, or mordanted]—* water —> A, 2 mins. — > B, flooded on drained slide, 1 min. — > balsam, via acetone and xylene result: nuclei, purple; plasma, red and yellow. 13.7 Borrel 1901 see DS 23.33 Borrel 1901 13.7 Buzaglo 1934 quinalizar in-acid alizarin blue-alizarin viridine 4285a, 11 :40 reagents required: A. DS 11.41 Becher 1921b; B. 70% ale. 100, hydrochloric acid 1, orcein 1; C. water 100, aluminum sulfate 10, acid alizarin blue 5; D. 5% phospho- molybdic acid; E. water 100, hydrochloric acid q.s. to make pH 5.8, alizarin viridine 0.2 PREPARATION OF C: Boil 10 minutes. Cool. FUter. method: [sections] —> water -^ A, 2i hrs. -^ rinse -^ B, S changes, 5 mins. in each — » rinse — * C, 7 mins. — » rinse — > D, till muscles differentiated —* wash -^ E, 7 mins. — > blot -^ abs. ale. minimum possible time — » balsam, via carbol-xylene and xylene result: nuclei, dark blue; elastic fibers, brown; muscle, blue; cartilage, green. DS 13.7 DYE STAINS OF GENERAL APPLICATION 3C9 13.7 Calleja 1897 airminr-picro-indiyocannine 23632, 15:323 REAGENTS UKyuiHKD: .1. sat. sol. Uthiuiii carl)onatc 100, canniiic 2; li. {).[% liydro- chloric acid; C. sat. sol. picric acid 100, indigocarmine 0.25; D. 0.2% acetic acid method: [sections] —>■ A, 5-10 mins. — > B. 20-30 sees. -^ wash—* C, 5-10 mins. — » D, few sees. — > balsam, via usual reagents 13.7 Canon 1937 14900,139:549 formula: 70% ale. 100, chlorazol black E 1 method: [sections, plant or animal tissue] — > stain, 15-30 mins. — > wash — > balsam, via usual reagents result: a very pleasing, and well-differentiated series of gray tones with some green. note: This stain is often attributed to Darrow 1940 (20540b, 15:()7) who introduced it into American literature. 13.7 Castroviejo 1932 magenta-picro-indigo carmine 5i)lb, 2:135 reagents required: A. water 100, 40% formaldehyde 0.6, DS 11.43 Ziehl 1882 5; B. DS 12.221 Cajal 1895 100, acetic acid 0.6 method: [sections of formaldehyde material]^ waters A, till nuclei well stained^ wash — > B, till stained -^ wash — » balsam, via usual reagents 13.7 Darrow 1940 see DS 13.4 Canon 1937 13.7 Dobell 1919 methyl bluc-eosin Y-orange G Dobell 1919, 7 REAGENTS REQUIRED: A. DS 13.5 Mann 1894; B. 70% ale. 90, sat. sol (circ. 0.2%) orange G in abs. ale. 10 method: [smears]-^ waters .4, 12 hrs. — > B, applied from drop bottle, till diiTerenti- ated — > abs. ale, minimum possible time — > balsam, via xylene 13.7 Drew-Murray 1919 picro-acid fuchsin-nile blue 1200, 6 :77 REAGENTS REQUIRED: A. DS 12.221 van Gieson 1896; B. 2% nile blue sulfate method: [water] —> a, 1-3 mins. ^ water, thorough rinse ^ 5, 2-24 hrs. -^ water, wash — > A, 1-5 mins. — > wash — > abs. ale, minimum possible time —^ xylene — > clove oil till differentiated — >• xylene -^ balsam result: nuclei and some cell inclusions, blue black; keratin, orange-yellow: collagens, red. 13.7 Ehrlich test. 1905 Lee cit. Grubler indulin-auraniia-eosin Y Lee 1905, 218 formula: glycerol 90, aurantia 6, indulin 6, eosin Y 6 preparation: Digest at 40°C. till completely dissolved. method: [smears] ^ stain, 4-5 hrs., 40°C. ^ water, thorough rinse —> balsam, via acetone and xylene result: nuclei, deep blue; some cytoplasmic inclusions, violet; plasma in general, orange. 13.7 Fraenkel see DS 21.13 Fraenkel (1928) 13.7 Freeborn 1888 picro-nigrosin 645, 9:231 formula: water 100, nigrosin 0.05, picric acid 0.4 method: [sections] — > stains, 5-10 mins. — > wash — > balsam, via usual reagents result: nuclei, black; collagens, blue; other tissues, yellow. 13.7 Gomori 1946 see DS 21.423 Gomori 1946 13.7 Hruby 1933 see DS 22.12 Hruby 1933 13.7 Koneff 1938 see DS 21.421 Koneff 1938 13.7 Kornhauser 1943 Quad Stain— auct. 20540b, 18 :95 reagents required: A. 95% ale. 90, water 10, nitric acid 0.4, orcein 0.4; B. water 100, aluminum sulfate (cryst.) 10, ferric chloride 0.8, acid alizarin blue 2 B 0.35; C. 5% phosphotungstic acid; D. water 100, acetic acid 2, orange G 2, fast green FCF 0.2 preparation of B: Boil the dye in the sulfate solution 10 minutes. Add the ferric chlo- ride dissolved in a little water. 370 METHODS AND FORMULAS DS 13.7 method: [sections of mercuric, or mercuric-chromic, fixed material after removal of mercury by iodine treatment] ^ 85% ale. ^/l, 2-24 hrs. -^85% ale, thorough wash -^ water, via graded ales. -^ B; 5-10 mms. -^ rmse -^ C, till collagen destained -> rmse -^ D, 10 mms. -^ 50% ale, wash -^ balsam, via usual reagents result: elastic fibers, brown; nuclei, blue; cytoplasm and muscle, violet; collagen, green; erthyrocytes, orange. 13.7 Kornhauser 1945 20540b, 20:33 REAGENTS REQUIRED: A. as Kornhauser 1943 above; B. water 100, acetic acid 0.475, sodium acetate 0.023, acid alizarin blue 2 B 0.35, ammonium alum 5; C. water lOo' phosphotungstic acid 4, phosphomolybdic acid 1; £). as Kronhauser 1943 above method: [material as for Kornhauser 1943 above] -» 85% ale, -^ A, 2-24 hrs. -^85% ale, thorough wash -^ water, via graded ales. -^ B, 5-10 mins. —> rinse ^ C, 10-30 mins. -^ quick rinse -^ D, 10 mins. -^ 50% ale wash -^ balsam, via usual reagents result: As Kornhauser 1943 above. 13.7 Krugenberg and Thielman 1917 23632,34:234 REAGENTS REQUIRED: .4.. water 108, anilin blue 0.45, eosin'^. 0.22, phloxine 0.45 PREPARATION OF A: The eosins are each dissolved in 45 water and mixed with the blue dissolved in 18. method: [sections of alcohol-fixed material] -^ A, 2-10 mins. -* rinse ^ abs. ale, minimum possible time -^ balsam, via usual reagents 13.7 Lillie 1945a 4349^ 25 :27 reagents required: A. water 99, acetic acid 1, brilliant purpurin R 0.6, azofuchsin G 0.4; B. 1% acetic acid; C. water 100, picric acid 1, naphthol blue black 1 method: [sections, nuclei stained by DS 11.121 Weigert 1903] ^ water -^ A, 5 mins. -* B, rinse -* C, 5 mins. -^ B, 2 mins. -^ balsam, via usual reagents result: collagen, reticulum, basement membranes, dark green; muscle and glands, brown; erythrocytes, brownish red. 13.7 Lillie 1945b 4349^ 25 :28 REAGENTS REQUIRED: A. either 0.1% fast green FCG in 1% acetic acid or 0.3% wool green FCF in 1% acetic acid; B. 1% acetic acid; C. either 0.2% acid fuchsin or 0.2% violamine R method: [sections, nuclei stained in DS 11.122 Lillie 1948] -^ water -^ A, i mins. -^ B, wash -^ C, 10-15 mins. -^ B, 2 mins. -^ balsam, via usual reagents result: connective tissue, red; erythrocytes, green; muscle and cytoplasm, gray-green. 13.7 Lillie 1945c 4349^ 25 :32 REAGENTS REQUIRED: ^.1% eosiu Y; B. 3% ferric chloride; C. 1% naphthol green B; D. 1% acetic acid method [sections, nuclei stained by DS 11.121 Weigert 1903] -^ water -^ A, 3 mins. -^ rinse -^ .B, 4 mins. -^ rinse ^ C, 5 mins. -^ D, 2 mins. -^ balsam, via acetone and xylene result: general cytoplasm, pink; collagen, green. 13.7 LiUie 1945d 4349, 25 :32 REAGENTS REQUIRED: A. 1% Biebrich scarlet in 1% acetic acid; B. 1% acetic acid; C. 0.5% methyl blue in 0.3% hydrochloric acid method: [sections, nuclei stained by DS 11.121 Weigert 1903]^ waters A, 5 mins. — > S, 2 mins. —* C, 5 mins. -^ balsam, via usual reagents 13.7 Lillie 1945e 4349, 25:41 REAGENTS REQUIRED: A. 1% phloxine B; B. 1% acetic acid; C. water 100, hydrochloric acid 0.25, methyl blue 0.1, orange G 0.6 method: [sections with nuclei stained by DS 11.121 Weigert 1903]^ water —> A, 10 mins. —> B, 2 mins. -^ C, 10 mins. —* B, 5 mins. — > balsam, via acetone and xylene 13.7 Lonnberg 1891 carmine-spirit blue 20796,6:1 REAGENTS REQUIRED: A. Any DS 11.22 formula; B. 0.1% hydrochloric acid in 70% ale; C. sat. 60%, ale sol. spirit blue 100, hydrochloric acid 1 drop; D. 85% ale adjusted with ammonia to pH 8 DS 13.7 DYE STAINS OF GENERAL APPLICATION 371 method: [whole objects] — > A, till stained — > B, till differentiated -^ 70% ale, wash — ♦ C, 15 mius. -~f D, till violet --> balsam, via usual reagents RECOMMENDED FOR: double staining wholemounts. 13.7 Lopez 1946 Tech. Bull., 7 :53 REAGENTS REQUIRED: A. water 100, DS 11.43 Ziehl 1882 10, acetic acid 0.2; B. water 100, 40% formaldehyde 4, acetic acid 0.2; C. water 100, phosphomolybdic acid 1, anilin blue WS 0.5, methyl orange to sat. PREPARATION OF c: Dissolve the acid with gentle heat. Add the anilin blue. After com- plete solution, add an excess of orange. Warm a few minutes. Filter. method: [sections of formaldehyde material] —> water -^ ^, 1 min. — > wash — » B, 3 mins. — + wash -^ C, 3-^-1 min. —> quick rinse —>■ abs. ale, least possil)le time — » bal- sam, via xylene 13.7 Lynch 1930 carviine-indulin 23632, 46 :465 reagents required: A. DS 11.22 Grenacher 1879; B. hydrochloric acid; C. 0.5% hydro- chloric acid in 70% ale; D. sat. sol. indulin in 80% ale. method: [whole objects, preferably mercuric-fixed]^ A, till thoroughly saturated-^ add B, drop by drop, till brick red ppt. formed; leave 12 hrs. -^ C, till object clear pink -^ add D, drop by drop till solution faint blue, leave till blue round edges ^95% ale, 1 hr. -^ balsam, via usual reagents result: thick or dense, structures, red; thin or diffuse structures, blue. recommended for: double-staining wholemounts. 13.7 Mallory 1900 see DS 11.124 Mallory 1900 13.7 Mann 1892 see DS 13.7 Mann 1894a (note) 13.7 Mann 1894a jnethyl hlue-eosin 23632, 11 :490 reagents required: A. water 55, 1% methyl blue 20, 1% eosin 25; B. 0.005% potas- sium hydroxide in abs. ale; C. 1% eosin method: [smears or sections] -^ water -^ A, 24-48 hrs. -^ water, rinse — > abs. ale, till dehydrated -^ B, till red -^ abs. ale, thorough wash — > water — » C, till differentiated -^ balsam, via usual reagents note: Alzheimer 1910 (Nissl and Alzheimer 1910, 409) gives A as water 150 to 35 of each of the dye solutions. Langeron 1942, 598, attributes this formula, without refer- ence, to "1892." Perdrau 1939 (11431, 48:009) recommends mordanting in 2% ammonium molybdate before this method. 13.7 Mann 1894b toluidine blue-erythrosin 23632, 11 :489 reagents required: A. water 100, erythrosin 0.1; B. water 100, toluidine blue 1.0; C. 0.2% acetic acid method: [water] -^ A, 1-2 mins. — > rinse -^ B, on slide, 1-2 rains. -^ C, till differenti- ated -^ balsam, via usual reagents result: nuclei, cartilage, blue; other structures, polychrome red and violet. 13.7 Masson test. 1942 Langeron erythrosin-toluidine blue-orange G Langeron 1942, 613 reagents required: A. ADS 12.2 Lugol (1905); B. 5% sodium thiosulfate; C. water 100, erythrosin 0.2, orange G 1; D. water' 100, toluidine blue 1; E. 0.2% acetic acid method: [sections of F 3700.1000 Helly 1903, F 7000.1000 Orth 1896. or F 3700.0010 Zenker 1894 fixed material] — > water — > A, 30 mins. — > jB, 2 mins. -^ thorough wash -^ C, 1-2 mins. — > rinse — > D, on slide, 1-2 mins. -^ E, till differentiated —> balsam, via usual reagents result: as 13.7 Mann 1894b but with bacteria deep blue. 13.7 Matsura 1925 polychrome neutral red 8542a, 3:107 reagents required: A. 1% congo red in 95% ale; B. 1% phosphomolybdic acid in abs. ale method: [sections] ^^ a, 12-24 hrs. -^ abs. ale, rinse —* B, 5 mins. —> abs. ale, till differentiated — > neutral mountant, via oil of thyrne result: nuclei, red; elastic fibers, red violet; collagen, green; white blood cells, violet; other tissues, brown. 372 METHODS AND FORMULAS DS 13.7 13.7 Merbel 1877 carmine-indigo carmine 645,1:242 STOCK solutions: I. water 100, sodium borate 7, carmine 1.7; II. water 100, .sodium borate 7, indigocarmine 7 PREPARATION OF STOCK SOLUTIONS: Boil 15 mimites. Cool. Filter. REAGENTS REQUIRED: A. stock I 50, stock II 50; B. sat. sol. oxalic acid method: [sections of F 7000.0000 Miiller 1859 material]-^ water-* A, 15-20 mins. -^ B, till differentiated -^ wash — » balsam, via usual reagents 13.7 Norris and Shakespeare lest. 1895 Rawitz carmine-indlgocarmine Rawitz 1895, 67 formula: water 130, sodium borate 8, carmine 1, indigocarmine 4 preparation: Boil the carmine in 65 water with 4 borax for 15 minutes. Cool. Filter. Boil indigocarmine in 65 water with 4 borax 5 minutes. Cool. Filter. Mix filtrates. Filter. 13.7 Petersen 1924 test. 1946 Roskin Roskin 1946, 230 reagents required: A. water 100, aluminum sulfate 10, acid alizarin blue 0.5; B. 5% phosphotungstic acid; C. water 100, acetic acid 8, orange G 2, anilin blue 0.5 method: [sections] —> water —♦ a, 5 mins. —» rinse -h. i?, several minutes —> distilled water, rinse — > C, 2 mins. -^ rinse -^ abs. ale, till dehydrated -+ balsam, via xylene 13.7 Plehn test. 1896 Kahlden and Laurent methyl blue-eosin Kahlden and Laurent 1896, 126 formula: water 20, sat. aq. sol. methyl blue 60, 0.5% eosin in 70% ale. 20, 20% sodium hydroxide 0.5 13.7 Proescher, Zapata and McNaught 1946 Tech. Bull., 7:50 STOCK solutions: I. 0.1% azophloxine; II. DS 11.4 anonymous 1936 working solution: stock I 60, stock II 30 method: [frozen sections of hot-formaldehyde-fixed materials]—* stain, 10-30 sees. — > abs. ale, least possible time for dehydration -^ M 23.1 mountant recommended for: rapid staining for diagnosis. 13.7 Roskin 1946 Roskin 1946, 231 REAGENTS REQUIRED: A. 70% alc. 100, hydrochloric acid 1, orcein 1; B. any DS 11.122 formula; C. DS 12.221 van Gieson 1896 100, acetic acid 0.3; D. water 100, C (preced- ing) 4 method: [sections] —> water —> a, 1 hr. -^ wash, 3 mins. -^ B, till thoroughly over- stained — > distilled water, 3 mins. -^ running water, 3 mins. -^ distilled water, 3 mins. — » C, 5 mins. -^ D, quick rinse — » blot — * abs. ale, minimum possible time — > balsam, via xylene 13.7 Schleicher 1943 Tech. Bull, 4:35 REAGENTS REQUIRED: A. 0.1% azocarmine B in 5% acetic acid; B.b% phosphotungstic acid; C. water 100, acetic acid 0.3, orange G 0.13, anilin blue 0.07 method: [sections of F 3700.1000 Helly 1903 fixed material with mercury removed by iodine treatment] -^ A, 15-30 mins., 56°C. — » rinse — > B, 15-30 mins. till nuclei deep carmine in pink cytoplasm^ rinse—* C, 15-30 mins. — » rinse—* 95% ale, till color clouds cease -^ balsam, via usual reagents 13.7 Schmorl 1928 see DS 21.13 Schmorl 1928 13.7 Shumway 1926 magenta-picro-indigocarmine 20540b, 1 :37 REAGENTS REQUIRED: A. sat. sol. (circ. 1%) magenta; B. sat. sol. (circ. 0.2%) indigo carmine 50, sat. sol. {circ. 1.2%) picric acid 50 method: [sections] -^ water —* a, 20 mins. -^ B, 5 mins. —* 70% alc. till pink, few sees. — * abs. ale, till blue green, several sees. -* balsam, via xylene result: resting nuclei, dark blue; mitotic figures, dark red; cartilage, pink; procartilage, light blue; bone, dark blue; muscle, bright green; nerves, purple. 13.7 Twort test. Minchin 1909 neutral red-light green 17510, 53:755 preparation of dry stock: DUute 50 sat. sol. (circ. 3%) neutral red to 100. Dilute 50 sat. sol. {circ. 20%) light green to 100. Mix at 50°C. Cool. Filter. Wash and dry ppt. DS 13.7 DYE STAINS OF GENERAL APPLICATION 373 WORKING solution: water 70, propanol 30, dry stock 0.5 method: [sections from F 0000.0010 or F 7000. ioOO fixed material] -^ water -^ A, 40°C., 10 mins. — > distilled water, rinse -+ abs. ale, till differentiated — ♦ balsam, via xylene result: nuclei, purple; blood, green; connective tissues, blue green. note: see also DS 23.3 Twort 192-1. Langeron 1942, 565 recommends the addition of 1% phenol to the working solution. 13.7 Volkman and Strauss 1934 azocarmine-naphlhol green-crystal violet 23632, 51 :244 REAGENTS REQUIRED: A. DS 21.13 Volkman and Strauss 1933 {see DS 21.13 Weigert 1898 — note); B. 0.1% azocarmine in 1% acetic acid; C. 0.1% aniline in 95% ale; D. 5% phosphotungstic acid; E. 1% naphthol green B in 1% acetic acid method: [sections] — > water —* A, 1-2 hrs. — > 70% ale, rinse —* wash —* B, 30 mins. — » — > C, quick wash — > D, till collagen decolorized — » wash — + E, 15 mins. -^ wash — » abs. ale, least possible time — > balsam, via xylene result: nuclei and muscles, red; collagen, green; elastic fibers, black. 13.7 Westphal 1880 test. Bohm and Oppel carmine-crystal violet Bohm and Oppel 1907, 193 formula: water 40, abs. ale. 40, glycerol 20, acetic acid 4, ammonium alum 0.4, car- mine 0.4, phenol 0.4, crystal violet 5 preparation: Boil the carmine and alum in the water 15 minutes. Filter. Dissolve the crystal violet in the ale Add to cooled filtrate. Leave overnight. Filter; add the glycerol and acid. 13.7 Williams 1935 11284,20:1185 preparation of stain: Dissolve 1 cresj'l violet and 1 potassium carbonate (anhyd.) in 95 water and 5 40% formaldehyde. Shake for 30 minutes. Add slowly and with con- stant agitation, 3 acetic acid. Shake 30 minutes. Filter and add 5 iospropyl ale method : [frozen sections of unfixed tissues] — + stain, 6 sees. — > wash -^ examine result: nuclei and muscle, blue; fat, yellow; other tissues, pink. 21 Formulas and Techniques for Dye Stains of Special Application Decimal Divisions Used in Chapter DS 20 DYE-STAINING TECHNIQUES OF SPECIAL APPLICATION 21 Selective dye stains for histological elements 21.1 Special stains for skeletal tissues 21.10 Typical examples Preparation of a wholemount of a small salamander with the skeleton stained by the alizarin technique of Gray 1929 Preparation of an embryo salamander to show the cartilagenous skeleton by the method of van Wijhe 1902 Preparation of a transverse section of a root using the acid fuch- sin-iodine green technique of Chamberlain 1915 21.11 Bone and calcified tissues 21.12 Cartilage 21.13 Elastic fiber 21.14 Chitin 21.15 Plant skeletal tissues 21.16 Other skeletal and connective tissues 21.2 Special stains for nervous tissues 21.20 Typical examples Preparation of a transverse section of the brain of a frog using the stain of Bethe 1896 Preparation of a section of spinal cord using the "Weigert- Pal" stain of Anderson 1922 Demonstration of the neuroglial cells of the white matter of the cerebral cortex using the crystal violet stain of Galescu 1908 21.21 Nerve cells and processes 21.211 Methylene blue and toluidinc blue methods 21.212 Hematoxylin methods 21.213 Other methods 21.22 Methods for neuroglia 21.23 Methods for other nervous elements 21.3 Special stains for blood 21.3 Special stains for other tissues 21.41 Designed to differentiate types of tissue not covered under 21.1, 21.2, or 21.3 21.411 Plant tissues 21.412 Animal tissues 21.42 Designed to differentiate types of cells 21.421 In pituitary 21.422 In other glands 21.423 In other structures 374 DYE STAINS OF SPECIAL APPLICATION 375 22 Dye stains for cytological elements (cell inclusions and extrusions not known to be organisms) 22.1 Nuclei 22.10 Typical example Demonstration of mitosis in an onion root tip using the rose bengal-orange G-toluidine blue stain of Kedrovsky 1931 22.11 Other techniques 22.2 Mitochondria and Golgi apparatus 22.20 Typical example Demonstration of mitochondria in the pancreas using the acid fuchsin-toluidine blue-aurantia stain of KuU 1914 22.21 Otlier techniques 22.3 Nissl granules 22.4 Yolk granules and fat granules 22.5 Plastids 22.6 Starch, glycogen, and amyloid granules 22.7 Mucin 22.8 Other cell inclusions and extrusions 23 Selective stains for specific organisms 23.1 Virus, Rickettsia, and Negri bodies 23.10 Typical examples Demonstration of Rickettsia in the scrotum of a guinea pig using the magenta-thionine stain of Macchiavello 1938 Demonstration of Negri bodies in the brain of a guinea pig using the ethyl eosin-methylene blue technique of Stovall and Black 1940 23.11 Methods of staining unidentified "organisms" smaller than bacteria 23.12 Methods for Rickettsia 23.13 Methods for Negri, and other virus-inclusion bodies 23.2 Bacteria 23.20' Typical e.xamples Staining a bacterial film with crystal violet by the technique of Hucker (1929) Demonstration of Gram-positive bacteria in smear preparation by the method of Gram 1884 Demonstration of tubercle baccili in sputum by the technique of Neelson 1883 Demonstration of the flagella of Proteus vulgaris by the method of Tribondeau, Fichet, and Dubreuil 1916 Demonstration of pneumococci in the liver of the rabbit using the phloxine-methylene blue-azur II stain of Mallory 1938 23.21 In smears 23.211 General methods 23.212 Iodine differential methods 23.213 Methods for acid-fast organisms 23.214 Methods for spirochetes 23.215 Flagella stains 23.216 Spore stains 23.217 Capsule stains 23.218 Diphtheria bacilli 23.219 Other methods for bacterial smears 23.22 In sections of tissue 23.221 General methods 23.222 Iodine differential methods 23.223 Methods for acid-fast organisms 23.224 Other methods for bacteria in sections 23.3 Other parasites and commensals 23.30 Typical examples Demonstration of mycelia of Penicillium in orange rind using 37G METHODS AND FORMULAS DS 20-DS 21 the thionin-light green-orange G-erythrosin stain of Margolena 1932 Demonstration of parasitic fungi in tissue scrapings using the technique of Chahners and Marshall 1914 23.31 Plants parasitic in plants 23.32 Plants parasitic in animals 23.33 Animals parasitic in animals 23.34 Animals parasitic in plants 23.4 Other zoological techniques 23.5 Other botanical techniques 24 Miscellaneous dye staining techniques DS 20 Dye-Staining Techniques of Special Application The phrase dye-staining techniques of special application is here used in contra- distinction to the dye-staining techniques of general application which were given in the last chapter. It must not be thought that general techniques are incapable of showing clearly any of the structures or organisms, the special methods for which have been transferred to the present chap- ter. This chapter has only been organized for the inclusion of those techniques which are so specialized that they cannot justi- fiably be employed for any other purpose than those recommended for each. It has been felt that, by the removal of these techniques to a special chapter, it w^ould be possible to avoid concealing what are generally considered to be the techniques of wide utility within a mass of special methods. It will be emphasized again and again, in the remarks under the various headings below, that if one desires, say, to stain chitin or plant skeletal tissues one should look first among the generalized techniques; and only when it has been found that these are unsuitable, should recourse be had to those in the present chapter. These specialized techniques have been divided into three large groups. The first of these (DS 21) includes the techniques for such histological elements as may be required to be stained differentially. This is followed by a section on cytological elements (DS 22), wliich are defined for the purposes of the present work as cell inclusions not known to be organisms. From time to time a confusion has arisen between mitochondria and bacteria, and there is undoubtedly at present some disagreement as to whether some of the small unidentified bodies — smaller than bacteria — which occur in certain inverte- brate cells are actually organisms or cell inclusions of an unidentified function. The third great division (DS 23) comprises selective stains for specific organisms, the majority of which are naturally bacteria and their immediate allies; there are, how- ever, a few other parasites, particularly the fungi, the methods for the selective demonstration of which are too specialized for general use. To preserve continuity of cross reference the dye stains in this chapter are subdivided from DS 20, those in the last chapter having been subdivided from DS 10. 21 SELECTIVE DYE STAINS FOR HISTOLOGICAL ELEMENTS The justification for this section is that it is occasionally required, for purposes of research or class demonstration, that some special structure should be specifically stained. The techniques given are not intended as general-purpose stains and their use should be confined to those cases in which the demonstration of the particu- lar tissue must outweigh all other con- siderations. The three histological ele- ments which it is most commonly desired to differentiate are: skeletal tissues (DS 21.1), both plant and animal, nervous tissues (DS 21.2), commonly studied by techniques inappUcable to other purposes, and, to a lesser extent, certain special stains for specific conditions in blood (DS 21.3), which should be separated DS 21. IDS 21.10 DYE STAINS OF SPECIAL APPLICATION 377 from the more generally employed blood stains. These three divisions have, how- ever, left a necessity for a fourth division, known here as "special stains for other tissues" (DS 21.4), in which are grouped some highly specialized techniques. 21.1 Special Stains for Skeletal Tissues The term skeletal tissue is here taken in its broadest sense to cover the supporting structures of both plants and animals. The supporting structures of vertebrates are covered in the first three sections, those of invertebrates in the fourth sec- tion, and those of plants in the fifth sec- tion. This leaves a small division at the end for certain formulas which do not easily fall into any division. 21.10 TYPICAL EXAMPLES Preparation of a wholemount of a small salamander with the skeleton stained by the alizarin technique of Gray 1929 Though this technique lies on the borderhne l^etween the making of micro- scope slides and the making of museum preparations, the results are sufficiently interesting to warrant inclusion. The tech- nique about to be described deals with the entire skeleton of a small salamander — say Triturus — which is, of course, far too large to mount as a microscope specimen. An exactly similar method of operation, how- ever, applies to the preparation of the skeleton of the hand or arm of a sala- mander, which can be justifiably regarded as an object for microscopic mounting. The method consists essentially in the deposition of a calcium-alizarin lake on the surface of the bones of the specimen, the muscles and skin of which are subse- quently rendered translucent by alkaline hydrolysis, and the replacement of the alkah with glycerol, both to increase the transparency and to render the prepa- ration permanent. Any small salamander may be used. This is an excellent method of turning experimental animals which die into useful class-demonstration specimens. Each sala- mander should be permitted to lie un- touched after death until it is entirely limp. It must then be mounted on a glass shde to hold it in position through subse- quent operations. An ordinary 3" XI" microscope slide serves excellently for Triturus, but the operator will naturally have little difficulty in adapting this tech- nique to any other nmphiliian. The speci- men is placed flat on the slide and the legs and tail maneuvered into a natural posi- tion. Soft silk thread, of the type once used for surgical ligatures, is used to tie the specimen to the glass slide in as many places as possible, so that it will not shift during the hardening operation. These ligatures need not be made too tight, since the object should always be hardened in a horizontal position. The specimen is now hardened in an iodine solution in alcohol, the exact con- centration of which is of Uttle importance. It should be considerably weaker than the solution specified for microscopical prepa- ration, and the suggestion given in the technique below (DS 21.11 Gray 1929) of a 10% dilution of Lugol's iodine in 95% alcohol is approximately correct. The concentration is not critical, and the degree of dilution may be comfortably made b^^ eye rather than by measurement. At least 500 milliliters of this solution should be used for a single salamander, and the author has found it convenient to lay the salamander, or as many sala- manders on slides as will fit the dish, in the bottom of a square glass dish of the type commonly known as a refrigerator jar. This jar is then filled with the iodine- alcohol and placed in a dark spot over- night, or until ne.xt required. The author has invariably found that if the iodine- alcohol is not used in the dark, the speci- men will fall to pieces during the course of subsequent hydrolysis. Though there is no rational basis at present for this ()l)ser- vation, he has made it often enough to recommend that it be followed; and he is sure that objections wliich have been raised to his technique are based on failure to follow this specific instruction, .\fter the specimens have remained in the iodine-alcohol at least overnight they will 378 METHODS AND FORMULAS DS 21.10 be found to be so hardened that they may be stood in a vertical position with- out becoming distorted. They should now (still in the dark) be placed in 95 % alcohol for about 24 hours, the alcohol being changed at least once in this period. It is not necessary to remove all of the iodine from the specimen, but this additional hardening in alcohol makes staining easier, and prevents the break-up of the specimen. Considerable quantities of 5% potas- sium hydroxide should now be prepared, and about 100 milliliters of this taken for each specimen. To each 100 milliliters add 1 millihter of a saturated solution of aUzarin red S in absolute alcohol. Place the specimens directly from alcohol into this potassium hydroxide-dye mixture to allow the rapid diffusion currents to carry the stain into the bones. The time of exposure to the stain is not very impor- tant, though at least 24 hours should be allowed to elapse in the case of Triturus; but the time should be greatly increased if only slightly larger specimens are at- tempted. Rana tigrina, for example, should be left for at least a week in the solution. It must be remembered that the stain will be deposited nowhere save on the bones, but that, unless a thorough impregnation takes place, some of the deeper bones may not become sufficiently pigmented. As the entire process takes some months, it is a pity to find at the conclusion of the period that an addi- tional day in the stain would have avoided wasting the whole lengthy period of preparation. When the specimens are removed from the staining solution, they will be found to be a dull red all over, and, on exami- nation by transmitted light, the small bones of the limbs will be clearly visible as a darker shadow within the reddish flesh. Now place the specimens in fresh 5% potassium hydroxide, changed as often as it becomes colored pink or brown, until the pink color has been removed from the flesh. This is a dual process, for the hydroxide both hydrolizes the skin and muscle, and removes at the same time the absorbed stain. The muscle under this treatment does not become white but remains a yellowish brown. Care must be taken to distinguish between this inevi- table residual yellowish brown color, which is removed in the next treatment, and the pinkish color which results from incom- plete removal of the stain. The time for this removal of the excess stain varies with specimens, and it is only a rough estimate to say that about two months will be required for a Triturus and from three to four months for a frog. No harm will result to the specimen should it re- main as long as six months in this solution and, if a number are being prepared, they may well be forgotten for this time rather than watched closely. The next stage is the removal of the residual brownish color from the muscles. No further translucency is imparted dur- ing this process of decolorization, which is conducted in equal parts of 5% potas- sium hydroxide and 1% ammonia. The solution needs to be changed as often as it becomes discolored, and this changing must be continued until the muscles and skin are bleached to a pure white color. The time for this is again variable, but rarely takes more than about two or three months for a frog or five or six weeks for a Triturus. When the flesh has been bleached, the specimen is placed in 5% potassium hy- droxide, which must be changed daily until the smell of ammonia is no longer apparent, and then transferred to 5% potassium hydroxide containing 5 % glyc- erol. After the animal has become thor- oughly penetrated by this solution, which may be determined by the fact that no further diffusion currents rise from the specimen, it may be placed in 10% glycerol until diffusion currents cease, and so on through increasing concentrations of glycerol, 10% apart from each other, until it is finally in pure glycerol, of which at least two changes should be used. The specimen is now almost glass-clear and shows bright red bones in a glassUke flesh. It is all too frequently discovered at this stage that one or another of the processes has not been continued long enough. If the bones finally disclosed in the thickest portions of the flesh are found to be insufficiently stained, there is nothing to DS 21.10 DYE STAINS OF SPECIAL APPLICATION 379 be done but to throw the specimen away. If, however, nothing is wrong save too much residual brownish color, it is only necessary to wash out the glycerol by immersion in running water overnight and then to replace the specimen in the potas- sium hydroxide-ammonia mixture. The specimens are difficult to mount as microscope slides unless one uses glycerol jelly (see Chapter 5). The glycerol-impreg- nated specimen should be transferred to molten glycerol jelly in a sealed capsule, and kept in it for about a month or until it is completely impregnated. By this time it is probable that the residual alkaUs will, however, have hydrohzed the glycerol jelly to the point at which it will no longer set; but it is possible, by using a fresh batch of glycerol jelly as a mountant, to secure a fairly permanent preparation. If, of course, one is prepared to take the trouble involved in sealing a glycerol mount (see Chapter 3) the specimen may be mounted in a deep cell in glycerol. If the specimen is required as a museum preparation it may be sealed in the customary manner in a museum jar of pure glycerol. Preparation of an embryo salamander to show the cartilagenous skeleton by the method of van Wijhe 1902 Unhke the preparation of a wholemount stained for bone, which has just been de- scribed, the present method lends itself better to microscope-shde preparation than it does to museum jar preparation. A salamander larva has been selected for demonstration purposes for the reason that it is convenient to secure, but the same technique may be used with any embryonic material, and it is surprising that so few of these preparations, by which the development, for example, of a chrondrocranium can be so perfectly shown, find their way into the hands of classes. It does not very much matter what size salamander larva is selected, but a 20-mm. larva of Triturus is not only easy to secure, but is also very generally useful for demonstration purposes. The chief diffi- culty is in the selection of the fixative, and though it is stated that alcohol- hardened specimens arc satisfactory, the author much prefei's to use a mercuric- acetic fixative of the type of Woltereck (Chapter 18, F 3000.0010 Wolterok (1910)). A specimen of the size described sliould be left in fixative for about 24 hours and should then be washed in a dozen changes of 70% alcohol. It is very important that all the mercuric chloride be removed from the specimen; therefore, it should be trans- ferred from the last wash alcohol to a solution of iodine i)rei)ared by adding about 2 millihters of Lugol's iodine (Chap- ter 22, ADS 12.2 Lugol 1905) to 100 milli- liters of 70% alcohol. The specimen re- mains in this iodine wash overnight and is then again washed in 70% alcohol until the last trace of color is removed from it. It cannot be too strongly empha- sized that a specimen which has been fixed in a picric mixture can never under any circumstances be used for a van Wijhe preparation. The staining solution (DS 21.12 van Wijhe 1902) contains 0.1% each of toluidine blue and hydrochloric acid in 70% alcohol. The specimen should remain in this solution until it is completely satu- rated with the color. For the specimen under discussion a period of 24 hours is probably sufficient, but as overstaining cannot occur, a period longer than this may be employed. The subsequent process of differentiation and dehydration is a long one, and it is better to start with a thoroughly stained specimen than to com- plete a lengthy preparation with the dis- covery that an additional day's time in the beginning would have saved wasting the whole period. Differentiation consists merely in washing with 0.1 % hydrochloric acid in 70% alcohol until no more color comes away. Repeated changes of 70% alcohol can be avoided by taking about a liter for a specimen of the size under dis- cussion, and placing this in a tall, narrow, cyhndrical museum jar fitted with a cork from which is suspended a loosely woven cloth bag containing the small embryo. Streams of color will at once start falling from the stained specimen, and when these color streams have ceased, it will be necessary to place the specimen in fresh 380 METHODS AND FORMULAS DS 21.10 acid alcohol, of which two changes of a smaller volume may 1)0 used to remove the last traces of the toluidine bhie. The total time required for differentiation will be from one to two weeks for a specimen of the size indicated, or from three to oijfht months for a ral)l)it embryo two inches long. As soon as differentiation is presumed to be complete, the specimen may be de- hydrated, cleared, and mounted. Nothing is necessary to render these specimens permanent other than the maintenance of an acid environment, and it is therefore recommended that 0.1% of hydrochloric acid be added to all the alcohols used Jor dehydration. The present specimen, which can be prepared as a microscope slide, may then be dehydrated in xylene and mounted in any acid mounting medium, such as an old batch of Canada balsam or one of the synthetic resins to which salicylic acid has been added. Preparation of cleared museum speci- mens is a great deal more difficult. The mounting medium employed is usually benzyl benzoate to which has been added 1 % of methyl sahcylate. The objection to this medium is its sensitivity to water; e\en a small specimen will require de- hydration in many changes of absolute alcohol before it can be mounted. Preparation of a transverse section of a root using the acid fuchsin-iodine green technique of Chamberlain 1915 This is the simplest of all the prepa- rations described in the present section of the work, and can be unhesitatingly recommended to the beginner who has never previously prepared a section of any type. This preparation is designed only to show the skeletal outlines of the cells, the cytological contents of which are removed in the course of the preparation. If cyto- logical detail in a botanical section speci- men is required, reference should be made to the typical preparation of a plant stem described in the last chapter. It does not matter from what source the root is obtained, but the beginner should select some soft root of about >s inch, or rather less, in diameter. If the root is collected from a living plant, it should be thoroughly washed to remove any adherent sand grains, which would spoil the edge of the cutting knife, and then preserved in 95% alcohol until lo- ([uircd. The 95% alcohol should be changed as it becomes discolored, but with this precaution the specimens may be preserved indefinitely. It is even possible to make preparations of this type from dried roots which have been preserved in a herbarium. The best method of swelling and softening these dried preparations is that recommended byLangeron (Langeron 1942, 1263) which requires a 10% solution of phenol in lactic acid. The lactic acid employed is the ordinary commercial solution, in which the phenol should be dissolved immedi- ately before it is required. Pieces of the dried root are then placed in a reasonably large volume of this material and heated over a low flame to a temperature of about 50°C. Within 10 or 15 minutes a com- pletely dried herbarium specimen will have become swollen out to its normal size and softened to the extent that sec- tions may readily be cut from it. The method of sectioning does not par- ticularly matter, but since the sections cannot in any case be subjected to the first process while they are attached to the slide, there is no real advantage in em- bedding in paraffin and cutting in this medium if an ordinary hand microtome is available. Sections can be taken from this microtome by (see Chapter 9) holding them either in pieces of pith or between the cut halves of a carrot. If the sections are cut by hand, they may be transferred immediately after they are cut to a dish of 20% alcohol, and from there to water; if they are cut in paraffin they should be at least 20 microns in tliickness, and the ribbon, as it is removed from the micro- tome, should be dropped directly into a watch glass of xylene in which the paraf- fin will dissolve. The individual sections are then removed from the xylene with a section lifter, passed through absolute al- cohol for the removal of the xylene, and thence downgraded through alcohols until they reach water. By whatever method DS 21.10 DYE STAINS OF SPECIAL APPLICATION 381 the sections are produced, they are ac- cumulated in a small dish of distilled water. These sections will, of course, re- tain the cell contents, which must be re- moved in order that the section may be turned into a true skeleton. The best reagent to use for skeleton- izing a section of plant tissue is either potassium or sodium hypochlorite; the ordinary bleaching solutions sold for household purposes under various trade names are not suitable, since they contain considerable quantities of calcium hypo- chlorite. If, however, the pure salts are not available, the household solution may be employed by adding to it enough of a solution of potassium or sodium carbonate to precipitate the calcareous contents, and then filtering the solution before use. If the pure salts are available, a 1 % solution may conveniently be employed. The sections are removed from the dis- tilled water on a section lifter and trans- ferred to a watch glass of the sodium or potassium hypochlorite solution. If the sections are made from material wliich has been preserved in alcohol, this solution should be used cold, but it must usually be warmed if it is to have the desired effect on materials which have been resur- rected from a dried condition. In either case the operation should be watched very carefully under a low power of the micro- scope, and the action of the hypochlorite should be discontinued as soon as the cells are free of their contents. If the mounter is completely inexperienced in this field, and is unable to determine the point at which the operation should be stopped, it is recommended that a single section should be taken and the skeletonizing followed under a microscope wliile it is timed. When the operation has gone too far, the finer of the cell walls present will be dissolved by the solution. If the period at which the first of the cell walls dissolves is carefully recorded, and one-half of this time taken for the subsequent sections, they will be perfectly cleaned without the shghtest risk of damage to their walls. After they are removed from the hypo- clilorite solution, the sections should be thoroughly washed in several changes of distilled water and then passed into 1% acetic acid in which they are rinsed several times. They are then rewashed in ordinary water until the wash water no longer smells of acetic acid. The skeleton- ized sections from as many roots as it is desired to cut at one time should be ac- cumulated in water until one is ready to stain them, or they may be preserved in- definitely in alcohol. The stain which is recommended in the present case (DS 21.15 Chamberlain 1915a) is freshly prepared when required by mixing equal parts of a 0.2% acid fuchsin solution and a 0.2 '^o iodine green solution. The mixed stains do not remain usable for much longer than one day, but the separate stock solutions may be kept indefinitely. The differentiating solution, which is 1 % acetic acid in absolute alco- hol containing 0.1% iodine, is also stable. The staining solution should be placed in a small capped vial or stoppered bottle, and the sections transferred to it from the water. They should remain in stain for about 2-4 hours, and it is recommended that they should not be left longer than 36 hours or they may suffer from a precipi- tate over the surface. For this reason it is desirable to accumulate as many sections as possible before starting the process. When the staining period is concluded the contents of the vial should be tipped out into a large watch glass. Usually some of the sections will remain stuck to the side of the vial from which the^^ are removed. Under no circumstances should the vial be rinsed with anything except the stain- ing solution, which should be poured back from the watch glass (the sections will have settled to the bottom), swirled around, and returned to the watch glass. A second watch glass, or even a small crystallizing dish, is now filled with the differentiating solution. Each section is re- moved individually with a section Hfter from the stain and placed into the differ- entiating solution, where it may be watched under the low power of a micro- scope as the dish is rocked gently from side to side. Differentiation will usually take place within two or three minutes and is terminated when the lignitied tis- sues are a bright, clear green, leaving a bright red in the nonlignified tissues. This 382 METHODS AND FOEMULAS DS 21.11 Sections prepared in this manner are permanent, and the process is so simple that it can be most warmly recommended as an introduction to plant section-stain- ing techniques for an elementary class. The sections are, however, clearly enough differentiated to be used for instructing a class at any rank, and they will generally be found much better for this purpose than complex quadruple-stained sections in which the cytological detail all too often tends to obscure the clarity of morphological detail, which is the chief requirement in this type of teaching. process of differentiation is also one of dehydration, hence the sections may now be removed with a section lifter from the differentiating solution and placed in a clearing agent. The writer's preference in this method is for terpineol, which has all the advantages of clove oil without the disadvantage of tending to make the sec- tions brittle so that they crack on mount- ing. All the sections may be passed through the differentiating solution and accumulated in terpineol, where they may remain until removed to a slide. Here they are covered with balsam and a coverslip is added. 21.11 BONE AND CALCIFIED TISSUES The techniques in the present section and in the section immediately following, com- prise a rather miscellaneous group, for they include both the methods intended for the staining of skeletons in wholemounts, and methods developed differentiallj^ to stain skeletal structures in serial sections intended for reconstruction. None of these methods need be employed in those cases in which it is desired to pro- vide a good general stain of a section, the other tissues of which should appear in con- trast. Most of the methods in sections DS 12.3, DS 13.2, and DS 13.4 in the last chapter give clear differentiation of bone from cartilage in general sections. The writer's choice is for the technique of Patay 1934 (Chapter 20 — DS 12.32) in which decalcified bone is stained a brilliant green in contrast to the blue of the cartilage. 21.11 Bechtol 1948 20540b, 23:3 REAGENTS REQUIRED: A. 0.01% Biebrich scarlet; B. water 100, citric acid 2.1, methylene blue 0.001; C. 2.1% citric acid method: [formaldehyde-fixed and hydrogen-peroxide-bleached, specimens]-^ water—* A, 24 hrs. — * 95% ale, till no more color comes away — > B, 24 hrs. -^ C, till differenti- ated — > balsam, via usual reagents recommended for: differentiation of bone (red) and cartilage (blue) in wholemounts. 21.11 Bock 1924a 23632, 40:318 reagents required: A. DS 11.121 Hansen 1905; B. glycerol 50, acetic acid 50; C. 1% eosin B in 95% ale. method: [sections of material fixed in F 7000.1000 Bock 1924 and decalcified in AF 21.1 von Ebner (1891)] — > water -> A, 12-18 hrs. -^ thorough wash — > B, till bone alone remains stained, 5-30 mins. -^ running water, 1 hr. -^ C, 32^2 mins. — > balsam, via usual reagents 21.11 Bock 1924b 23632, 40:318 REAGENTS REQUIRED: A. F 7000.1000 Bock 1924; B. AF 21.1 von Ebner 1890; C. 5% potassium alum; D. DS 11.124 Hansen 1905; E. glycerol 50, acetic acid 50; F. 0.4% eosin Y in 95% ale. method: [fresh tissues] —>■ A, 1 wk. to 1 month -* B, till decalcified — > rinse -^ C, 24 hrs. -^ running water, 1-2 days — ♦ [10 fi celloidin sections] — > D, 18 hrs. -^ E, till differen- tiated 5-20 mins. -^ running water 1 hr. -^ F, 5 mins. -^95% of ale, wash-> bal- sam, via usual reagents recommended for: demonstration of osteogenesis in decalcified tissues. 21.11 Cretin 1937 4285a, 14:163 REAGENTS REQUIRED: A. ADS 12.2 Cretin 1937; B. water 100, hematoxylin 1.2, alizarin red S 6, phosphomolybic acid 0.04 method: [sections of decalcified bone fixed in F 5000.1020 Cretin 1937] -^^ water -> A, 36 hrs. -^ wash -» B, 1-24 hrs. -^ wash -> balsam, via usual reagents DS 21.11 DYE STAINS OF SPECIAL APPLICATION 383 21.11 Dawson 1926 20540b, 1:123 REAGENTS REQUIRED: A. 1% potassiuiii hydroxide; B. water 100, potassium hydroxide 1, alizarin red S 0.01; C. P 13.1 Mall 1902 method: [ale. fixed embryos] —> A, till bones clearly visible-^ B, till bones red — > C, till impregnated -^ glycerol, via graded series RECOMMENDED FOR: staining bones in whole mammalian embryos. 21.11 Eros 1928 23681, 42:97 REAGENTS REQUIRED: A. Water 100, acid fuchsin 1, potassium alum to sat.; B.Z% nitric acid in 95% ale. method: [sections] -> water -> a, 10-15 mins. —> rinse —> Zi, till differentiated, 1-12 hrs. — ♦ balsam, via usual reagents recommended for: selective staining of once calcified areas in sections of decalcified material. 21.11 Grandis and Magnini 1900 1852, 34:73 REAGENTS REQUIRED: A. sat. alc. sol. purpurin; B. 0.5% sodium chloride method: [sections]-* 95% ale.-* A, 5-10 mins. -^ B, 3-5 mins. -* 70% ale, till no more color comes away -^ balsam, via usual reagents recommended for: differential staining of decalcified bone. 21.11 Gray 1929a 14706,9:341 formula: sat. sol. sodium borate in 70% alc. 100, sat. sol. {circ. 0.5%) alizarin red S in abs. alc. 1 method: [material, fixed and stored in neutralized formaldehyde] -^ running water, 24 hrs. — > 70% alc, till saturated —> stain, till calcified structures clearly differen- tiated, 32 to 12 hrs. -^ graded ales., each saturated with sodium borate — * benzene — > neutral mountant recommended for: bony skeletons in wholemounts of material too delicate for Gray 1929b. 21.11 Gray 1929b 14706, 9:341 REAGENTS REQUIRED: A. ADS 12.2 Lugol (1905) 10, 95% alc. 90; B. 5% potassium hydroxide 100, potassium hydroxide 50, 1% ammonium hydroxide 50 method: [freshly killed material] — » A, 24 hrs. at least, in dark — > 95% alc, 24 hrs., in dark -^ B, 24 hrs. -^ C, changed as frequently as it becomes colored, till red bones distinctly visible through yellowish translucent muscle (roughly dissected fish verte- bra, 24 hrs. ; whole frog 3 to 4 months) -^ C, till muscle pure white, 6 hrs. to 1 month -^ glycerol mixtures when shrinkage must be avoided result: as gray 1929a. note: Omission of step A is invariably fatal. A detailed description of a preparation by this method is given under DS 21.10 above.j 21.11 Grieves test. circ. 1938 Wellings Wellings circ. 1938, 160 REAGENTS REQUIRED: A. water 99, DS 23.213 Carpano 1916 1; B. acetone method: [ground section of balsam embedded (see Chapter 10) formaldehyde fixed tooth]-* 20% alc, 15 mins. — >A, 12-24 hrs. —> rinse —» JS, till differentiated-* dammar, via oil of bergamot RECOMMENDED FOR: structure of dentine. 21.11 Hanazava 1917 7141, 59:125 REAGENTS REQUIRED: water 10, DS 11.43 Ziehl 1882 10 method: [ground sections of teeth]—* water—* stain, 10 mins. — * 95% alc. till differ- entiated — * balsam, via usual reagents RECOMMENDED FOR: dentine. 21.11 Juge 1940 19288, 47:65 REAGENTS REQUIRED: A. water 30, 95% alc. 70, acetic acid 0.06, methyl green 3; B. abs. alc. 100, acetic acid 0.2, alizarin red S 0.003 method: [formaldehyde-fixed pieces] — > 70% alc, 1 day-* A, 1-3 hrs. — * 70% alc, till cartilage alone stained -* abs. alc, till dehydrated — * B, 12-24 hrs. — ♦ abs. alc, till no more color comes away -^ balsam, via usual reagents RECOMMENDED FOR: differential staining of bone (red) and cartilage (green) in whole- mounts. 384 METHODS AND FORMULAS DS 21.11 21.11 Klaatsch test. 1896 Kahlden and Laurent Kahlden and Laurent 1896, 178 REAGENTS REQUIRED: A. wator 100, hematoxylin 1, picric acid \; B. 1% acetic acid method: [sections] — > A, 1-2 mins. -^ B, 30 sees. -^ balsam, via usual reagents result: cartilage, blue; bone, yellow. 21.11 Kolliker test. 1928 Schmorl Schmorl 1928, 263 reagents required: A. acetic acid; B. sat. aq. sol. indigo-carmine method: [sections of decalcified bone] -^ A, till transparent —> B, 15-60 sees. -^ wash, till differentiated -^ balsam, via usual reagents 21.11 von Korff 1907 1780, 69:515 REAGENTS REQUIRED: A, Water 100, glycerol 7, acid fuchsin 2, orange G 1 method: [sections of chrome, or dichromate, fixed materials]—^ water—* A, 1 min. -^ 95% ale., till differentiated -^ balsam, via usual reagents RECOMMENDED FOR: histogenesis of bone and teeth. result: osteoblasts and odontoblasts, orange; fibrils in degenerating cartilage, red; other tissues, yellow. 21.11 Lundvall 1927a 766,62:353 REAGENTS REQUIRED: A. \% ammonia; B. 4% formaldehyde; C. 0.02% alizarin red S in 90% ale. method: [embryos, or small vertebrates, fixed and bleached in AF 31.1 Lundvall 1927] -^ A, 24 hrs. or till all acid neutralized — > B, 24 hrs. or till all ammonia removed — * 95% ale, till formaldehyde removed —> C, 1-2 days — > 95% ale, till no more color comes away — > [l^enzyl benzoate, via abs. ale. and benzene for museum mounts] or -^ balsam, via usual reagents, for microscope slides recommended for: demonstration of bone and calcified structures in wholemounts. 21.11 Lundvall 1927b 766, 62:353 STOCK solutions: L 0.1%, toluidine blue in 95% ale. IL water 30, 95% ale. 69, acetic acid 1, alizarin red S 0.02 reagents required: A. 1% ammonia; J5. 4% formaldehyde; C. water 15, 95% ale. 35, acetic acid 0.5, stock I 10, stock II 40; D. 0.01% acetic acid method: [embryos, or small vertebrates, fixed and bleached in AF 31.1 Lundvall 1927] -> A, 24 hrs., or till all acid neutralized -^ B, 24 hrs., or tUl all ammonia removed -^ A, 1 day, 40°C. —> D, till no more color comes away -^ 70% ale, till no more color comes away ^^ 95% ale. thorough wash — > [benzyl benzoate, via abs. ale. and ben- zene, for museum specimens] or — » balsam, via usual reagents, for microscope slides recommended for: demonstration of both cartilage (blue) and bone (red) in whole- mounts. 21.11 Morpugo 1908 test. 1928 Schmorl Schmorl 1928, 269 reagents required: A. sat. sol. lithium carbonate; B. 0.025% thionin 100, ammonia 0.1; C. sat. sol. phosphotungstic acid; D. sat. sol. picric acid method: [celloidin sections of F 7000.0000 Mliller 1859 fixed, and nitric acid decalcified, bone] -^ water, thorough wash -^ A, 1 min. -^ B, 3-5 mins. -^ wash -^ C, 5 mins. -^ D, 2-3 mins. — > rinse — » 95% ale. — > balsam, via usual reagents 21.11 Nollister 1934 20540b (abstr. 1935), 10:37 stock solution: water 96.5, glycerol 13, acetic acid 3.5, chloral hydrate 0.8, alizarin red S to sat. reagents required: A. 1 to 4% potassium hydroxide; B. 1-4% potassium hydroxide 100, stock 0.1 method: [whole fish]-* A, till translucent-^ B, till bones stained —> A + increasing quantities of glycerol under UV light till bleached and clear -* pure glycerol 21.11 Rait 1935 11139b, 19:80 formula: 70% ale. 100, 1% acetic acid 1, sat. sol. alizarin in abs. ale. 5 method: [material, fixed and stored in 10% formaldehyde, adjusted with sodium borate to pH 9] -^ running water, 24 hrs. -* 70% ale, till saturated -^ stain, 12-24 hrs. -* 70% ale, wash — ♦ neutral mountant, via usual reagents DS21.il DYE STAINS OF SPECIAL APPLICATION 385 21.11 von Recklinghausen tcit. 1928 Schmorl Sohmorl 1928, 2(;0 REAGENTS REQUIRED: A. 0.1% tliioiiiiie; B. sut. sol. phosphoinolybdic acid in glycerol C. 2% hydroquinone in glycerol; D. 3% phenol in sat. sol. potassium alum.; E. sat. sol. hydroquinone; F. anhydrous glycerol; G. toluene 60, abs. ale. 30 method: [hand or ground sections of nondecalcified bone preserved in P12.3 Kaiserling 1897] — > A, till deep blue -^ rinse — > B, overnight — > C, till no more color comes away -^ D, 1-2 hrs. -^ thorough wash —> E, thorough wash -^ wash — > F, till dehydrated -^ G, till glycerol removed —y balsam, via toluene RECOMMENDED FOR: general bone structure. 21.11 Roehl kd. 1933 Cajal and de Castro Cajal and de Castro 1933, 314 REAGENTS REQUIRED: A. coppcr tetramino solution (see note below); B. DS 11.121 Weigert 190-4; C. ADS 21.1 Weigert 1885 50, water 50 method: [paraffin sections]^.!, 5 mins. -^ wash -^ 7^, 15 uiiiis. —> rinse —> C, till differentiated -^ wash -^ balsam, via usual reagents recommended for: pathological calcareous deposits. note: Copper tetramine is prepared by adding sufficient ammonium hydro.xide to a solution of a copper salt to redissolve the ppt. first formed; neither Cajal and de Castro {loc. cit.) nor Schmorl 1928, 196 specify the strength of the solution. 21.11 Schmorl 1928a Schmorl 1928, 265 reagents required: A. 0.1% thionin; B. sat. sol. phosphotungstic acid; C. 20% formaldehyde method: [paraffin sections of decalcified bone]—* water, thorough wash -^ A, 5 mins. —> wash ^95% ale, 1-2 mins. —> wash ^ 5, till differentiated, few moments—* wash -^ C, 1-2 mins. ^95% ale. -^ balsam, via usual reagents recommended for: general bone structure. 21.11 Schmorl 1928b Schmorl 1928, 266 reagents required: A. 0.1% thionin; B. sat. sol. phosphotungstic acid; C. 5% potas- sium alum method: [paraffin sections of bone fixed in formaldehyde, hardened in F 7000.0000 Muller 1850 and decalcified in AF 21.1 Schmorl] — > water, thorough wash — * A, 10- 30 mins. -^ wash — > 95% ale, 1-3 mins. -^ wash -^ B, 10-25 mins. — * wash, 2 hrs. -^ C, 1-2 hrs. — > running water, 3-12 hrs. — >• balsam, via usual reagents recommended for: demonstration of lamellae. 21.11 Schmorl 1889 23681,10:745 reagents required: A. DS 11.44 NicoUe 1871; B. sat. sol. picric acid method: [celloidin sections] -^ water —» A, 5-10 mins. -^^ wash —> B, J2~l nain. -^ thorough wash— » 70% ale. till no more color comes away —> balsam, via usual reagents result: pro-cartilage, yellow; cartilage, blue; bone, brown black. 21.11 True 1947 20540b, 22:107 REAGENTS REQUIRED: A. Water 100, potassium hydroxide 5, hydrogen peroxide (3%) 0.1; B. 0.01% alizarin red S in 2% hydroxide method: [vertebrate embryos] —* yl, till bones clearly visible —♦ /?, till bones red -^ glycerol, via graded series RECOMMENDED FOR: boncs in wholemounts. 21.11 Weil test. cirr. 1938 Wellings Wellings circ. 1938, 156 REAGENTS REQUIRED: .1. sat. aq. sol. uiercuric chloride; B. 0.1% iodine in 90% ale; C. DS 11.22 Grenacher 1879; D. 1 % hydrochloric acid in 70% ale; E. 40% dried Canada balsam in chloroform method: [1 mm. slices of tooth] —> A, 6-8 hrs. — > wash -^ 70% ale via graded ales. — » B, 12 hrs. — > abs. ale, till decolorized — > water, till rehydrated — > C, 3-7 days — » D, 24 hrs. --> al)S. ale, via graded ales., till ilcliydratcd » chloroform, 24 hrs. — > E, till impregnated — > evaporate till hard — > [grind section (see Chapter 10)] RECOMMENDED FOR: structurc of dentine.' 386 METHODS AND FORMULAS DS 21.11-DS 21.12 note: The method of Must and Rose (Wellings, op. cit. 158) differs only in that whole teeth, ground on each side to expose the pulp, are used. The method of Choquet (Wellings, op. cit., 158) substitutes formaldehyde fixation for mercuric fixation. 21.11 Williams 1941 20540b, 16:23 KEAGENTS requieed: A. 0.1% ammonia; B. water 30, 95% ale. 70, hydrochloric acid 0.05, toluidine blue 0.25; C 2% potassium hydroxide; D.2% potassium hydroxide 100, sat. sol. {circ. 0.5%) alizarin red S in abs. ale. 0.5; E. \% sulfuric acid in 95% ale. method: [formaldehyde-fixed pieces] -^ A, 24 hrs. -^ 5, 1 wk. -^ 95% ale. changed daily, 3 days -^ C, till bones visible — > D, 24 hrs. — > E, till soft tissues colorless -^ balsam, via usual reagents recommended for: demonstration of cartilage (blue) and bone (red) in wholemounts. 21.11 Williams 1946 20540b, 21:55 REAGENTS REQUIRED: A. 3% potassium hydroxide; 5. 2% potassium hydroxide 100, alizarin S q.s to color red; C. 1% sulfuric acid in 95% ale. method: [pieces of formaldehyde-fixed skin] -^ A, Z days ^ B, 24 hrs. — > C, (if differ- entiation required) — » wash — > balsam, via usual reagents RECOMMENDED FOR: placoid, ctenoid, and cycloid scales. 21.12 CARTILAGE 21.12 Bechtol 1948 see DS 21.11 Bechtol 1948 21.12 Becher 1921 see DS 13.5 Becher 1921a 21.12 Curtis 1905 see DS 12.21 Curtis 1905 21.12 Johansen 1932 see DS 11.45 Johansen 1932 21.12 Juge 1940 see DS 21.11 Juge 1940 21.12 Klaatsch (1896) see DS 21.11 Haatsch (1896) 21.12 LaCour 1931 see DS 11.45 LaCour 1931 21.12 Lundvall 1927 766, 62, 353 REAGENTS REQUIRED: A. water 30, 95% ale. 70, hydrochloric acid 1, toluidine blue 0.25; B. 0.25% hydrochloric acid in 70% ale. method: [embryos or small vertebrates fixed and bleached in AF 31.1 Lundvall 1927] — > 95% ale, wash -^ A, \ day, 40°C. -^ B, changed when necessary, 40°C., till no more color comes away -^95% ale, thorough wash -^ [benzyl benzoate, via abs. ale. and benzene for museum specimens] or — > balsam, via usual reagents, for microscope slides RECOMMENDED FOR: demonstration of cartUage in wholemounts. 21.12 Miller 1921 see DS 21.12 van Wijhe 1902 (note) 21.12 Newton 1927 see DS 11.45 Newton 1927 21.12 Semichon test. 1934 Langeron Langeron 1934, 999 formula: water 100, phenol 1, 95% ale. 1, Bismarck brown 0.1 preparation: grind the dye in a mortar with the phenol. Add ale. while grinding. Wash out with 10 successive doses of water. method: [water] -^ stain, 10 mins. — > 95% ale, till differentiated-^ balsam, via cedar oU result: cartilage, dark brown. note: Langeron [lac. cit.) recommends preliminary staining in hemalum and counter- staining in light groon. This defeats the purpose of the technique, which is to render easier the reconstruction from serial sections, of cartilaginous skeletons. 21.12 van Wijhe 1902 16592, 31:47 REAGENTS REQUIRED: A. 70% alc. 100, liydrochloric acid 0.1, toluidine blueO.l; 5. 0.1% hydrochloric acid in 70% ale; C. 0.1% hydrochloric acid in abs. ale; D. benzyl benzoate 99, methyl salicylate 1 DS 21.12-DS 21.13 DYE STAINS OF SPECIAL APPLICATION 387 method: [whole embryos]—* running water, 24 hrs. — » 70% ale, till saturated-^ A, 24 hrs. — > B, till no more color comes away — * C, till dehydrated — > salicylic balsam, via benzene or [(large embryos) -^ D, till cleared] RECOMMENDED FOR: Cartilage in wholemounts. note: Mercuric-fixed embryos, perfectly washed, are best. Picric-fixed material is worthless. A detailed description of the application of this stain is given under DS 21.10 above. Miller 1921 (7()3, 20:415) first bleaches in alcoholic peroxide and u.ses specially purified alcoliol. 21.12 Williams 1941 see DS 21.11 Williams 1941 21.13 ELASTIC FIBERS Much attention lias been paid to the differential staining of elastic elements. Most of the techniques rely on the mordanting power of resorcinol, to which attention was originally drawn by Weigert in 1898. These methods stain even the finest fibers of elas- tic tissue a relatively dense black, and are thus primarily intended for the display of in- dividual elastic fibers running through cartilage and similar structures. The technique of Krajian 1934, on the contrary, is designed to distinguish large masses of elastic tissue from other connective tissues through the differential staining of the elastic fibers in red against blue. The early method of Pfitzner 1887 also deserves retention, since it can be employed after chrome fixatives, when resorcinol techniques do not take well. The method of Pasini (1928) (Chapter 20, DS 12.32) is also used for this purpose, par- ticularly in the sections destined for class demonstration, though the differentiation of fibers is not as good as the methods here given. 21.13 Argaud 1923 6630, 891 :373 formula: 95% ale. 100, orcein to sat, hydrochloric acid 5 method: [sections] -^ 95% ale. -^ stain, few moments — * abs. ale, wash-^ balsam, via xylene 21.13 Delamare 1905 see DS 13.7 Delamare 1905 21.13 Fraenkel lest. 1928 Schmorl Schmorl 1928, 70 STOCK solution: 95 %o ale. 60, water 30, nitric acid 3, orcein 0.75 reagents required: A. 3% nitric acid 90, stock 10; B. DS 12.211 Cajal 1895; C. 3.5% acetic acid method: [sections] -^ water ^80% ale, till differentiated -^ B, 10-15 min. -^ C, rinse — » 95% ale., rinse -^ abs. ale. — > balsam via xylene 21.13 French 1929 20540b, 4:11 formula of dry stock: water 100, crystal violet 0.5, magenta 0.5, dextrin 0.25, resor- cinol 2, 30% ferric chloride 12.5 preparation of dry stock: Boil together everything except the ferric chloride which is added to boiling solution. Continue boiling 2-5 minutes. Cool. Filter. Wash and dry PPt- FORMULA OF WORKING SOLUTION: 95% alcohol 100, dry stock 3, hydrochloric acid 2 preparation of WORKING SOLUTION: Boil the powder 5 minutes in ale. Cool. Filter. Add acid and make up to 100 with ale. method: [sections]^ water -> stain 3-^ to 3 hrs. ^ 95% ale. till differentiated-* bal- sam, via usual reagents result: elastin, dark bluish green. 21.13 French 1940 see DS 22.4 French 1940 21.13 Gallego-Garcia 1936 tcsl. 1936 Findlay 11360, 56:160 REAGENTS REQUIRED: ^4. 0.5% anillii blue; B.\% eosin Y method: [frozen sections of formaldehyde material]-^ A, 10 niins. -^ wash — > B, 10 mins., wash^ M 11.1 Apathy 1892 388 METHODS AND FORMULAS DS 21.13 21.13 Goldmann test. 1896 Kahlden and Laurent Kahlden and Laurent 1896, 144 REAGENTS REQUIRED: A. sat. alc. sol. Crystal violet; B. 0.01% potassium hydroxide in 95% alc. method: [sections]-^ A, 24 hrs. — ^> 95% alc, rinse —> B, till differentiated-^ balsam, via usual reagents 21.13 Gomori 1950 aldchyde-fuchsin—auct. Tech. Bull, 20 iQQS formula: water 30, 95% alc. 70, magenta 0.5, hydrochloric acid 1, paraldehyde 1 method: [paraffin sections] — » 95% alc. — * stain (aged at least 24hrs.) 5-10 mins. — > 95% alc, wash — ^ balsam, via usual reagents note: This stain will also differentiate mast cells, cells of the pancreas (15-20 minutes in stain), certain cells in the pituitary (30 minutcs-2 hours in stain). 21.13 Hart 1908 23681, 19:1 reagents required: A. DS 11.28 Orth (1892); B. water 30, 95% alc. 70, DS 21.13 Weigert 1898 (working sol.) 5, hydrochloric acid 1 method: [sections] -^ water -^ a, 2-5 mins. — > J5, 12-15 hrs. —> wash -* balsam, via usual reagents 21.13 Hart 1908 23681, 19:1 REAGENTS REQUIRED: A. 0.25% potassium permanganate; B. 5% oxalic acid; C. water 75, 95% alc, 25, DS 21.13 Weigert 1898 (working sol.) 5, hydrochloric acid 1 method: [sections] —> A, 10 mins. —* wash — > B, 20 mins. — > wash -^ C, overnight -^ 95% alc, rinse -^ wash — > counterstain, if desired — > balsam, via usual reagents 21.13 Herxheimer 1886 8645, 4:785 REAGENTS REQUIRED: A. abs. alc 50, hematoxylin 2.5, water 50, lithium carbonate 0.04; B. 20% ferric chloride method: [sections] — > water -^ A, 5 mins. to 1 hr. — > rinse -^ B, till fibers differentiated — >• wash -^ balsam, via usual reagents 21.13 Krajian 1934 1789a, 18 :378 reagents required: A. 2% aluminum chloride; B. water 80, sodium citrate 4, neutral red 3, glycerol 20; C. ADS 12.2 Lugol (1905); D. water 100, anilin blue 1.5, orange G 2.5, resorcinol 3, phosphomolybdic acid 1 method: [sections of formaldehyde-fixed material] — > A, 10 mins. — > water, rinse — > B, 30 mins. — > water, rinse — > C, 30 sees. -^ water, rinse — > D, 30 mins. -^ water, rinse — > abs. alc, minimum possible time —^ balsam, via oil of thyme and xylene result: elastic fibers, bright red; other connective tissues, dark blue. 21.13 Kultschitzky 1896 1780, 46 :675 formula: 95% alc 100, water 5, potassium carbonate 0.05, magdala red 2, methylene blue 1 use: elastic fibers in sections of F 7000.0000 Mliller 1859 fixed material note: Conn 1946, p. 112 points out that Kultschitzky {loc. cit.) may have used phloxine rather than magdala red. 21.13 Mallory 1938 see DS 22.8 Mallory 1938 21.13 Manchot test. 1896 Kahlden and Laurent Kahlden and Laurent 1896, 144 reagents required: A. sat. aq. sol magenta; B. water 100, sucrose 100, sulfuric acid 2 method: [sections] -^ water ^^ A, 30 sees. — > rinse -^ B, till differentiated M 11 or 12 mountant 21.13 Pfitzner 1887 23632, 4:82 formula: abs. alc. 100, safranin 10 method: [sections of chrome-fixed material] -^ stain, 48 hrs., 40°C. — > water, wash -^ balsam, via usual reagents result: elastic fibers black on gray. note: This may be u.sed only after chrome fixation. 21.13 Pranter t^st. 1905 Hall and Herxheimer Hall and Herxheimer 1905, 86 formula: water 30, 95% alc. 70, nitric acid 2, orcein 0.1 method: as Unna-Taenzer (1896) DS 21.13 DYE STAINS OF SPECIAL APPLICATION 389 21.13 Romeis 1948 see DS 13.7 Romeis 1938 21.13 Roskin 1946 see DS 13.7 Roskin 1946 21.13 Rubens-Duval lest. 1938 Carleton and Leach Carleton and Leach 1938, 220 formula: water 70, 95% ale. 30, nitric acid 10, orcein 0.1 method: water — > stain, 21 hrs. — > wash — > balsam, via usual reagents 21.13 Schmorl 1928a S(;hinorl 1928, 168 REAGENTS REQUIRED: A. DS 21.13 Wclgcrt 1898 (working sol.); B. DS 11.121 Weigert 1904; C. DS 12.221 Weigert 1904 method: [sections] -^ water —» A, '2-1 l^f- -^ 95%, till differentiated, '^-l Jif. — > water — » B, 5-10 niins. — > wash -^ C, till ditTerentiated — > balsam, via usual reagents 21.13 Schmorl 1928b Schmorl 1928, 168 REAGENTS REQUIRED: A. DS 11.28 Orth (1892); B. DS 21.13 Weigert 1898 (working sol.) CDS 21.41 Weigert 1887 (.4 solution); D. 0.6% hydrochloric acid; ^. ADS 11.1 Gram 1880; F. aniline 60, xylene 30 method: [sections] —> waters A, 2-5 mins. — ♦ 95% ale, wash — » B, 10-30 mins. -^ 95% ale. till differentiated — » wash — >• C, 5-10 mins. -^ D, wash —»■£', 5 mins. — * blot — » F, till differentiated -^ balsam, via xylene recommended for: differentiation of elastic fibers in tissues containing Gram-positive bacteria. note: Bismarck brown may be substituted for magenta in the preparation of B above. 21.13 Schmorl 1928c Schmorl 1928, 168 REAGENTS REQUIRED: A. DS 11.43 Zichl 1882; B. DS 21.13 Weigert 1898 (working sol.); C. 1 % methylene blue method: [sections] -^ water —* A, 1 hr., 37°C. -^ 70% ale, wash -^ B, 20-30 mins. — > abs. ale. till differentiated — > water -^ C, 5-10 mins. — > wash — ♦ balsam, via usual reagents recommended for: differentiation of elastic fibers in tissues without destaining acid- fast bacteria. 21.13 Schmorl 1928d Schmorl 1928, 169 reagents required: A. DS 11.28 Orth (1892); B. 1% hydrochloric acid in 70% ale; C. sat. sol. crystal violet in sat. sol. aniline; 1). Vesuvelin (see DS 21.13 Weigert 1898, note) method: [sections] — > water -^ A, 10-20 mins. — > B, till differentiated, 10-20 mins. — > wash -^ C, 1 hr., 37°C. — > 95% ale, till no more color comes away — » D, 20-30 mins. -^95% ale, wash — > abs. ale, till differentiated —* balsam, via xylene recommended for: differentiation of elastic fibers in tissues without destaining acid- fast bacteria. 21.13 Sheridan 1929 11571b, 12:103 formula of dry stock: water 100, crystal violet 1, resorcinol 2, 30% ferric chloride 9 preparation: as French 1929 working solution: Prepare from ppt. exactly as for French 1929 above. method: [sections] ^90% ale — > stain, 1-2 hrs. —* abs. ale till differentiated—* bal- sam, via xylene result: as French 1929 but much paler. 21.13 Unna test. 1896 Kahlden and Laurent Kahlden and Laurent 1896, 143 reagents required: A. sat. aq. sol. Bismarck brown; B. 50% ale 100, magenta 1, nitric acid 5; C. 25% nitric acid; D. 0.01% acetic acid method: [sections] -^ water — > A, some hours to overnight —* wash — » B, 24 hrs. — > C, quick dip — » D, till differentiated -^ balsam, via usual reagents 21.13 Unna-Taenzer test. 1896 Kahlden and Laurent Kahlden and Laurent 1896, 144 reagents reqitired: ^4. 50% ale 100, orcein 0.8, hydrochloric acid; B. 0.1% hydro- chloric acid in 80% ale. method: sections-^ water—* A, 6-12 hrs. — > B, till differentiated—* balsam, via usual reagents 390 METHODS AND FORMULAS DS 21.13-DS 21.14 21.13 Verhoeff 1908 11006, 50:876 REAGENTS REQUIRED: A. abs. alc. 60, hematoxylin 3, 10% ferric chloride 0.25, ADS 12.2 Lugol (1905) 25; B. 2% ferric chloride PREPARATION OF A: Dissolve hematoxylin in alc. Add ferric chloride. Filter. Add iodine solution to filtrate. method: [sections] -^ water — > A, till black, 10-20 mins. -^ B, till differentiated, 2-10 sees. -^ water, thorough wash — » 95% alc, till no more iodine comes away — » balsam, via usual reagents result: elastic fibers, black. note: Verhoeff (loc. cit.) recommends a 2% solution of eosin Y if counterstaining is required. 21.13 Volkman and Strauss 1934 see DS 21.13 Weigert 1898 (note) and DS 13.7 Volkman and Strauss 1934 21.13 Weigert 1898 23681, 9 :290 FORMULA OF DRY STOCK: Water 100, magenta 1, resorcinol 2, 30% ferric chloride 12.5 preparation OF DRY STOCK: as French 1929 WORKING solution: 95% alc. 100, dry stock 0.75, hydrochloric acid 2 preparation of working solution: as French 1929 method: [sections] -^ 95% alc. -^ stain, 2-24 hrs. —* 95% alc, tUl no more color comes away -^ balsam, via usual reagents result: elastic fibers, blue black. note: Fischer {test. Schmorl 1928, 167) suggested the substitution of safranin or Bis- marck brown for magenta and designated such solution by the suffix -elin. Hence, the names fuchselin (with magenta), vesuvelin (with Bismarck brown) and safranelin which add confusion to the literature. Volkman and Strauss 1934 use crystal violet. 21.13 Unna see DS 12.216 Unna (1928) 21.13 Zieler see DS 23.221 Zieler 1903 21.14 STAINS FOE CHITIN The few methods recorded in this section are not intended for the general staining ot sections of arthropods, for which purpose any of the better-known triple stains can be employed. Bethe 1895 is based on a standard microchemical test for chitin and is designed principally to assist in the reconstruction from serial sections of portions of the endoskeleton which cannot well be made out in cleared specimens. The formulas of Gage, Racovitza, and Smith are intended only for wholemounts and should be used in those cases in which a valu- able specimen has, through carelessness, been left in potash for so long that it has become too transparent. Such specimens are difficult to stain and the formula of Smith is invaluable for this purpose. 21.14 Bethe 1895 23831, 8:544 reagents required: A. water 100, aniline hydrochloride 10, hydrochloric acid 1; B. 7.5% potassium dichromate method: [sections] -^ water —* a, 5 mins. —» water, quick rinse —» B, 1 min. — > tap water, tUl color changes from green to blue — > balsam, via acetone recommended for: demonstration of chitin in sections. 21.14 Chatton 1920 see DS 12.212 Chatton 1920 21.14 Gage 1919 7871,30:142 formula: water 100, hydrochloric acid 1, acid fuchsin 0.2 RECOMMENDED FOR: potash-cleared chitinous skeletons. 21.14 Nuttall 1908 see DS 12.16 Nuttall 1908 21.14 Racovitza test. 1942 Langeron Langeron 1942, 924 REAGENTS REQUIRED: A. 1% pyrogalHc acid; B. 0.5% hydrochloric acid method: [potash-cleared exoskeletons of arthropods] —» water, thorough wash— > A, 3^ to 1 hr. -^70% alc, in strong light, till darkened -> B, if differentiation required —> balsam, via usual reagents note: Vaulx 1920 (5401, 65:214) mordants for 15 minutes in a sat. sol. ferric sulphate before this treatment. DS 21.14-DS 21,15 DYE STAINS OF SPECIAL APPLICATION 391 21.14 Semichon 1920 see DS 12.222 Semichon 1920 21.14 Smith 1926 9940, 14:171 formula: phenol 100, sat. ale. {circ. 1.5%) ethyl eosin 12.5 method: [potash-cleared skeletons of arthropods] — > stain, 15 mins. at 60°C. — > balsam, via clove oil 21.14 Vaulx 1920 see DS 21.14 Racovitza (1942) (note) 21.15 PLANT SKELETAL TISSUES There is a general confusion among beginners unaccustomed to botanical micro- techniques between those destined to show only the skeletal structures and those which are designed to show the nuclei and cytoplasmic constituents of plant material. For the latter purpose any nuclear stain and any counterstain employed for zoological purposes can equally well be used, and many of the triple staining methods common in zoological techniques give admirable results. The staining methods discussed in this section are those which are used to differentiate (usually for teaching purposes) between cellulose, callose, lignified, and suberized tissues. These tissues should be freed from protoplasmic material before being stained, and the customary method of doing this is to immerse them, after sectioning, in a solution of sodium or potassium hypochlorite. They should remain in this solution until the cellular contents have been leeched away, and should then be thoroughly washed in distilled water before staining techniques are applied to them. In general these techniques may be divided into two sections. First, there are those which are destined to distinguish between lignified and cellulose tissues, which is the purpose of almost all the formulas here given ; second, there are the few genuine botanical triple stains in which some fat-staining constituent is employed for the purpose of bringing the suberized tissues into contrast. For the former purpose the only choice between the different methods rests in the color in which it is desired to differentiate the elements. It is interesting to note that the stain universally referred to as "Benda's Stain" in zoology is equally employed in botanical microtechnique as "Land's Stain." The best technique, if it is desired to include the suberized tissues, is that of Bugnon 1919. A good method for class-demonstration purposes in Langeron 1902, in which advantage is taken of the relative densities of the schlerenchyma and parenchyma in order to distinguish between the two. For those who prefer a single solution, there are the methods of Darrow 1940 and Chamberlain 1915a. The stain of Petit 1903, though obsolete, is included for its interest and for the reason that it gives more permanent results than other staining methods for plant skeletal tissues. 21.15 Bugnon 1919 light green-Sudan II I-hemalum 4999,66:919 REAGENTS REQUIRED: A. 70% alc. 100, light green to sat, Sudan III to sat.; B. 11.122 Delafield 1885 method: [sections] -^ 70% alc. -^ A, 10 mins. -♦ water, rinse -> B, 10 mins. -♦ water, wash -^ balsam, via usual reagents result: lignified tissues, green; suberized tissues, red; other tissues, blue. note: Langeron 1942, 1269 suggests the substitution of Petit 1903 (sols. C and D) for B above. 21.15 Chamberlain 1915a acid fuchsin-iodine green Chamberlain 1915, 62 STOCK solutions: I. 0.2%, acid fuchsin, II. 0.2% iodine green WORKING solutions: a. stock I 50, stock II 50; B. abs. alc. 100, acetic acid 1, iodine 0.1 method: [sections] -> water -^ A, 24 hrs. ->■ B, till differentiated -^ balsam, via xylene result: lignified tissues, green; cellulose tissues, red. note: a detailed description of the use of this stain is given under DS 21.10 above. 21.15 Chamberlain 1915b cyanin-erythrosin Chamberlain 1915, 01 ^■^ reagents required: A. 1% cyanin in 50% alc; B. 1% erythrosin method: [sections] ->■ water ->■ A, 5-10 mins. -♦ 50% alc, rinse -^ B, \i to 1 min. -» abs. alc, least possible time -* balsam, via xylene 392 METHODS AND FORMULAS DS 21.15 result: lignified tissue, blue; cellulose tissue, red. note: Emig 1941, 56 states that the less expensive Capri blue may be substituted for cyanin. 21.15 Cole 1903 Cross and Cole 1903, 171 REAGENTS REQUIRED: A. DS 11.26 Cole 1903; B. 20% hydrochloric acid in 95% ale; C. 2% acid green in 95% ale. method: [sections] — > water — > A, 5-10 mins. -^ B, till differentiated, 1-2 mins. — ' 95% ale, till acid free — > C, 30 mins. -^95% ale, rinse — > balsam, via clove oil 21.15 Conant see DS 13.5 Conant 21.15 Conn and Darrow 1946 cit. compl. script, et litt. Sharp. Iron ahun-safranin Conn and Darrow 1946, llA-9 reagents required: A. 3% ferric alum; B. 0.1% hematoxylin; C. 3% safranin O in 90% ale. method: [sections of woody tissues, preferably cut from living material] -^ A, 10-30 mins. -^ water, thorough wash —> B, till stained sufficiently -^ water, rinse -^ C, 1-5 mins. — > water, wash ^90% ale, till dehydrated — > balsam, via benzene result: lignified walls, red; unlignified walls, black. 21.15 Darrow 1940 chlorazol black E 20540b, 15:67 formula: 70% ale 100, chlorazol black E 1 method: [sections] — > 70% ale -^ stain, 5-10 mins. -^ balsam, via usual reagents result: differentiation of cell walls in black and varying shades of brown; cytoplasm, brown, yellow, and green. 21.15 Emig 1941 see DS 21.15 Chamberlain 1915b (note) 21.15 Foster 1934 tannin-safranin 20540b, 9:81 reagents required: A. water 100, tannic acid 1, sod. salicylate 1; B. 3% ferric chlo- ride; C. 1% safranin in 50% ale method: [sections] water—* A, 10 mins. — ^ water, wash -^ B, 5 mins. -^ repeat A —> water—* B, if necessary, until cell walls sufficiently dark -^ 50% ale, 5 mins. — > C, 48 hrs. —♦70% ale, till differentiated — > balsam, via usual reagents 21.15 Jackson 1926 crystal violet-erythrosin 20540b. 1 :33 reagents required: A. 1% crystal violet; B. sat. sol. erythrosin in clove oil; C. 50% xylene in abs. ale method: [sections] -^ water—* A, 15 mins. — * water, rinse — * 95% ale, till dehydrated -^ B, 1-5 mins. — > C, 1-2 mins. —> balsam, via xylene result: lignified tissues, violet; other tissues, red. 21.15 Johansen 1940 safranin-picro-anilin blue see DS 21.15 Smith 1924 (note) 21.15 Johansen 1940 acid fuchsin-iodine green see DS 21.15 Langeron 1942 (note) 21.15 Johansen 1940 acid fuchsin-fast green see DS 21.411 Johansen 1940 21.15 Johansen 1940 see also DS 13.5 Johansen 1940 21.15 Land test. 1915 Chamberlain safranin-light green Chamberlain 1915, 80 reagents required: A. DS 11.42 Babes 1887 or Conn 1915; B. sat. sol. light green in clove oil method: [sections] — * water — > A, 2-24 hrs. -^ 50% ale, till differentiated -^ abs. ale, till dehydrated — > B, 3-30 mins. — > balsam, via xylene result: lignified tissues, red; other tissues, green. note: This method is widely known in zoological literature as "Benda's Stain"; see DS 11.42 Babes 1887 (note). 21.16 Langeron 1942 carmine-iodine green Langeron 1942, 1266 reagents required: A. DS 11.21 Grenadier 1879; B. 0.01% iodine green method: [sections] — * water -^ A, 1-3 hrs. -^ water, rinse -^ B, 5-20 sees. — * balsam, via usual reagents DS 21.15 DYE STAINS OF SPECIAL APPLICATION 393 result: lignified tissues, green; other tissues, red. note: Laugeron {loc. cil.) refers to tliis as la inMhoilc clasxiiiuc Johansen 1940, IKi, uses 1% acid fuchsin as the red counterstain iu tliis tec-huiquc; this modification is essen- tially the method of Chamberlain 1915a. 21.15 Langeron 1902 /rs/. 1942 ips. methylene blue-ruthenium red Langeron 1912, 12G7 REAGENTS RiOQUiUEu: .1. water lUO, i)otassiuni alum 10, methylene blue 1; B. 0.2% ruthenium red method: [sections] -^ water — ♦ A, 5-10 mins. -^ water, wash -^ B, 5-10 mins. — > water, wash — > Ixilsam, via usual reagents result: suberizcd tissues, green; sclerenchyma, violet; lignified tissues, blue; paren- chyma, deep rose. 21.15 Langeron see also DS 21.15 Bugnon 1919 (note) 21.15 Margolena 1934 Bismarck brcnvn-fast green 20540b, 9:71 reagents required: A. 0.5% Bismarck brown; B. 0.3% fast green FCF in clove oil method: [sections of F 5000.1010 Bouin 1897 fixed buds] -^ A, 10 mins. —* rinse — > de- hydrate — » B, till differentiated -^ balsam, via xylene recommended for: Differentiation of walls of devcloi)ing pollen grains. 21.15 Northen 1936 safranin-tannin-crystal violet 20540b, 11 :23 reagents required: A. \% safranin in 50% ale; B. 0.5% tannin in 50% ale; C. 1% ferric chloride in 70% ale; D. 0.5% crystal violet in clove oil method: [sections] — > water — »• .4, 24 hrs. -^ 50% ale, rinse -^ B, 30 sees. -^ 70% ale, two rinses -^ C, 10-20 sees. — > abs. ale, minimum possible time —> D, 30-CO sees. balsam, via xylene result: lignified tissue, red; other tissues, black and purple. 21.15 Petit 1903 alkanet-iodine green-chrome yellow 0630,55:507 reagents required: A. DS 11.3 Guinard 1890; B. 0.01% iodine green in 95% ale; C. sat. sol. lead acetate; D. sat. sol. potassium dichromate method: [sections]-^ waters A, till suberized tissue red ^ 95% ale, rinse —> B, till lignified tissues green —♦70% ale, rinse — > C, 5 mins. — > water, thorough wash -^ D, few moments — > water, thorough wash -^ balsam, via usual reagents 21.15 Popham, Johnson and Chan 1948 20540b, 23:185 reagents required: A. 1% safranin O; B. 2% tannic acid; C. water 60, DS 11.122 Delafield (1885); D. 0.01% hydrochloric acid; E. 0.5% lithium carbonate; F. sat. sol. anilin blue in methyl cellosolve method: [sections] — ♦ water — > A, 24 hrs. — > rinse -^ B,2 mins. — > rinse — > C, 2 mins. — > D, wash -^ E, 5 mins. — ♦ dehydrate -^ F, 5-10 mins. -^ balsam, via usual reagents recommended for: cell walls in stem apex. 21.15 Sharman 1943 20540b, 18:105 reagents required: A. 2% zinc chloride; B. 0.004% safranin O; C. water 100, hydro- chloric acid 0.15, tannic acid 5, orange G 2; Z). 5% tannic acid; E. \% iron alum method: [sections] — ♦ water ^ A, 1 mm. -^ rinse — > J?, 5 mins. — > rinse -^ C, 1 min. -^ rinse -^ D, 5 mins. — * quick rinse —* E, 2 mins. — > balsam, via usual reagents recommended for: cell walls of stem apex. 21.15 Smith test. 1903 Cole acid green-carmine Cross and Cole 1903, 170 reagents required: A. water 75, glycerol 25, acid green 0.1; B. DS 11.26 Smith (1903) method: [sections] -^ water -^ A, 5-10 mins. -> wash — > B, 10-15 mins. — > 96% ale, wash — > balsam, via clove oil 21.15 Smith 1924 safranin-picro-anilin blue 19938,59:557 reagents required: A. DS 11.42 Chamberlain 1915; B. sat. sol. (circ. 1.2%) picric acid 78, sat. sol. {circ. 5%) anilin blue WS 22 method: [sections] —> A, 2-6 hrs. — > water, thorough wash -^ 95% ale, till differenti- ated -^ B, 2 hrs. -^ abs. ale, quick rinse — > balsam, via clove oil and xylene note: Johansen 1940, 82 substitutes his DS 11.42 Johansen 1940 for A above and cites the technique without reference to Smith, as do Conn and Darrow 1946, IIa-11. 394 METHODS AND FORMULAS DS 21.16 21.16 OTHER SKELETAL AND CONNECTIVE TISSUES 21.16 Baird 1935 20540b, 10:35 REAGENTS REQUIRED: A. 1% trj^pan blue; B. 2% neutralized formaldehyde; C. 1% methylene blue; D. \% acid fuchsin method: [inject (for rat) 1 ml. A daily for 4 days] -^ [spread piece of subcutaneous con- nective tissue on slide: dry till edges adhere] -^ B, ^ 2 to \ hr. — > wash — > C, 1 min. — » wash — > D, 30 sees. -^ wash — > balsam, via acetone and xylene RECOMMENDED FOR: Connective tissue spreads for class instruction. 21.16 Bowell test. 1948 Verdcourt 20540b, 23:145 REAGENTS REQUIRED: A. Water 99, acetic acid 1, potassium permanganate 16; B. sat. aq. sol. {circ. 10%) oxalic acid; C. 0.1% Hoffman's violet method: [radulae isolated by potassium hydroxide treatment]-^ wash -^ A, 100 ml., till black -^ B, till decolorized — » wash — ^ C, till stained, 30 mins. -^ balsam, via usual reagents RECOMMENDED FOR: radulae of mollusca. 21.16 Daniell927 11360,47:253 REAGENTS REQUIRED: A. 0.05% quiuone in abs. ale; B. abs. ale. 50, methyl salicylate 50 method: [crustacea in 70% ale] ^ abs. ale, till dehydrated —> A, overnight—^ B, till diffusion currents no longer visible -^ methyl salicylate RECOMMENDED FOR: demonstration of muscles in wholemounts of crustacea. 21.16 Dietrich test. 1928 Schmcrl Schmorl 1928, 273 REAGENTS REQUIRED: A. 1% brilliant black 3B in 0.1% acetic acid; B. 1% safranin; C. methanol method: [3-4 m sections of mercuric chloride fixed material] —> A, 1-5 mins. — + rinse -^ B, 3-10 mins. -^ drain — > abs. ale, till differentiated -^ C ^ balsam, via xylene RECOMMENDED FOR: structure of heart muscle. 21.16 Hueter 1911 test. 1948 Romeis Romeis 1948, 351 REAGENTS REQUIRED: A. 10% phosphotungstic acid; B. DS 11.124 Hueter 1911 method: [sections] -^ water -^ A, 15-20 sees. — > quick wash -^ B, 3-5 mins. — > 50% ale, till differentiated -^ balsam, via usual reagents RECOMMENDED FOR: collagcn fibers. 21.16 Jasswoin 1932 23632, 49:191 REAGENTS REQUIRED: A. 5% fcrric alum 75, 1% hematoxylin 25 method: [sheets of neutralized formaldehyde-fixed material] -^ thorough wash -^ spread on slide —> 95% ale, till dehydrated —> water, tiU rehydrated — >• A, 15-20 sees. — » tap water, wash — ^ [counterstain if desired] —>■ balsam, via usual reagents recommended for: collagen fibers in wholemounts. note: See also DS 11.121 Yasvoin (1946). 21.16 Karlson 1924 6630,90:1122 REAGENTS REQUIRED: A. water 100, eosin 0.7, gallic acid 0.15; B. 3% pyrogallic acid; C 5% methylene blue method: [sections] — > water -^ A, 5-20 mins. -^70% ale, till no more color comes away — > B, 24 hrs. —> wash -^ C, 15 mins. -^70% ale, tiU no more color comes away — * balsam, via usual reagents RECOMMENDED FOR: differentiation of muscle (red) from other connective tissues. 21.16 Miller 1933 11431,37:127 REAGENTS REQUIRED: A. DS 22.21 Altmau 1898 (sol. A); B. 0.5% aurantia in 70% ale; C. 0.25% toluidine blue method: [sections of F 3700.1000 Helly 1903 material] — > water -^ A, on slide, warmed to steaming, 2-4 mins. —> quick wash —^ C, 10-20 sees. — > rinse —> blot — » C, 10 sees. — > methanol, till differentiated — > balsam, via usual reagents recommended for: striae in striated muscle. 21.16 Perdrau 1921 see MS 33.1 Perdrau 1921 DS 21.16-DS 21.20 DYE STAINS OF SPECIAL APPLICATION 395 21.16 Unna 1910 safranin-anilin-blue-tannin Ehrlich, Krause, et al. 1910, 247 REAGENTS REQUIRED: A. 1% safranin; B. water 50, anilin l)lue 0.5, water 50, tannin 15 method: [sections of ale. fixed material] —* A, 10 mins. — > water, thorough wash — > B, 15 mins. — » water, rinse —> abs. ale., till color clouds cease -^ balsam, via xylene RECOMMENDED FOR: demonstration of collagen fibers in alcohol-fixed material. result: collagen, blue. 21.16 Verocay 1908 test. 1948 Romeis Romeis 1948, 351 reagents required: A. \% chromic acid; B. DS 11.122 Delafield 1885 method: [paraffin sections] —> water —* yl, 24 hrs., 46°C. — * wash — > B, wash — > counterstain, if desired -^ balsam, via u.sual reagents recommended for: collagen fibers. 21.2 Special Stains for Nervous Tissues 1-2 hrs. It has become so customary today for all nervous tissues to be stained by one of the metal-staining techniques that it is a surprise to many histologists to learn that excellent methods exist for dye- staining these structures besides those of the classic "Weigert" methods given in division 21.212 l)elow. These stains for nervous tissues are divided into those destined to stain the neuroglia, which, though they cannot, of course, properly be called nervous tissues, are so closely associated with them that the staining technique must be kept in the same place. 21.20 TYPICAL EXAMPLES Preparation of a transverse section of the brain of a frog using the stain of Bethe 1896 The method of Bethe (DS 21.211 Bethe 1896) for the preparation of class-teaching material or demonstration slides from the brains of the lower vertebrates is one of the best arguments against rejecting a technique for the mere reason that it is obsolete. The process is rapid and simple, yet it yields as good images as can be ob- tained by many of the metal-staining techniques, which would involve weeks of work, and which are capricious and un- rehable in the extreme. The technique de- pends essentially upon the fact that solu- tions of methylene blue are differentially absorbed, in a living animal, into the nerve cells and processes of the brain. The dye is fixed in this position with the aid of am- monium picrate, used also as a fixative of the tissues, and finally precipitated in place with the aid of either sodium or ammonium molybdate. A frog is an easy animal on which to experiment, not only because it is readily available, but also because of the simplicity with which the brain may be removed from the cranium. It is necessary to start wdth a saturated solution of methylene blue in'any standard physiological saline solution, but it need not be sterile since the animal is going to be sacrificed shortly after the application. It is simpler and pleasanter to work on an anesthetized frog, and though any anes- thetic may be used for this purpose, the writer's choice for all amphibian anes- thetization is tricaine methanosulfonate, more frequently known under its trade designation of M2.2.2. Tliis should be pre- pared as a 0.2% solution in physiological saline, and the frog placed in about a liter of the solution. The frog will become non- receptive to stimuli after about ten min- utes and is unlikely to die even upon an exposure of some hours. As soon as the frog is thoroughly anes- thetized, fill a 5-milliliter hypodermic syringe with the saturated solution of methylene blue in physiological saline, and inject about 1 milhhter into the ab- dominal cavity. Take care not to damage the internal structures. It is simplest to pick up a fold of the frog's skin with the fingers and to insert the hypodermic syringe into the length of the fold, parallel to the main axis of the body. The point of the syringe should be watched carefully at the beginning of the injection to make sure that it has not merely slipped under the skin, but has actually penetrated through the muscular layers into the coelom. The needle is then withdrawn and the frog returned to its anesthetic saline. After three or four minutes a slight blue 396 METHODS AND FORMULAS DS 21.20 coloration begins to spread over the skin. Leave the frog for about ten minutes and then inject another milhhter of stain into the abdominal cavity. An hour later, check to see if the nervous system has picked up the methylene blue. This can be done by dissecting the hind leg to disclose the sciatic nerve, which is easily found by slipping the handle of a scalpel between the gastrocnemius and the posterior tibi- alis muscles to disclose the nerve lying alongside the bone. If the preparation has been a successful one, the nerve will be stained light blue, and as a final check the nerve may be severed and the cut end ex- amined under a hand lens. If the cut end has a darker stain than does the outer sheath, the impregnation may be con- sidered successful, and one may proceed with preparations to remove and fix the brain. Pin the frog belly down in the bottom of a dissecting pan. Remove the skin from the whole of the head, and scrape the frontoparietals clear of their attached muscles. Break away the pos- terior portion of the cranium so as to leave the way clear for the removal of the upper half to expose the brain. The brain can be removed by inserting the sharp point of a scalpel under the nasal bone, which is then Ufted up and pulled away. This leaves the end of the frontoparietal hanging free so that it may be lifted very carefully T\dth the edge of a scalpel. Do not jiermit it to l)reak away, or the other end will drive down and destroy the cerebellum. As soon as it has been lifted, however, it may be grasped in a pair of blunt-nosed forceps and twisted with a rotary motion toward the center of the skull. Tliis will result in its coming away free without damaging the brain. The same technique may now be emplo3^ed on the other frontoparietal, thus leaving clear the whole anterior region of the brain. The brain will now be exposed save for those portions which are covered by the prootic bones, or by such parts of the frontoparietal as remained attached to the prootic bone when they broke. Both the prootic and the remains of th'e frontoparietal may be lifted off carefully and the upper surface of the brain fully exposed. It is almost impossible to remove the brain from its bed without damaging it. A pair of heavy scissors should now be used to cut through the remains of the nasal and vomer bones, to sever the connection of the parasphenoid bone from the pre- maxillae, and to cut through the prootic bones at the point of attachment of the squamosals and the pterygoids. The en- tire brain may now be lifted out, resting, as it were, on a platter consisting largely of the parasphenoid bone. It will be seen by reference to the tech- nique under discussion (DS 21.211 Bethe 1896) that six alternative fixative solu- tions are suggested. There is no doubt that the solution which contains osmic acid and phosphomolybdic acid is the best, but the very high cost of osmic acid, and the danger of working with this ir- ritating reagent, may lead the operator to choose in preference alternative formula No. 2, which contains ammonium molyb- date and chromic acid. The results are nearly, but not quite, as good with this reagent as with the osmic-sodium-phos- phomolybdate mixture. Ammonium phos- phomolybdate can, of course, be substi- tuted for sodium phosphomolybdate in the solution selected. The first fixation is in a saturated aque- ous solution of ammonium picrate, which is prepared by taking a saturated solution of picric acid in water and adding to it a considerable excess of undissolved picric acid. Ammonia is now added drop by drop, with constant shaking, until the surplus picric acid at the bottom of the container starts to dissolve. Further ammonia should now be added until the solution smells of free ammonia, and it may then be shaken once or twice to complete the saturation. A very emphatic warning must be given at this point that ammonium picrate is a highly explosive compound which can be detonated without diffi- culty by vibration alone. Under no cir- cumstances whatever should dry am- monium picrate ever be allowed in a laboratory, and the fixative solution under discussion should be prepared immediately before use, and then thoroughly washed down the sink as soon as the period of fixation is finished. To leave the vessel containing the ammonium picrate about DS 21.20 DYE STAINS OF SPECIAL APPLICATION 397 until such a time as the dry crust of the salt collects around the lip of the vessel is to invite a serious accident. Like all other picric compounds, however, this material cannot explode in solution, and is there- fore perfectly safe provided that the neces- sary precautions are observed. Now lift the brain on its bed of the parasphenoid bone and place it in a con- siderable quantity of the ammonium picrate solution for about five minutes. It should then be sufficiently hardened on the outside for it to be safe to tip the bone sideways, and to detach the brain from the parasphenoid bone with a sharp scalpel. After another five minutes in the fixative, cut the brain into small pieces (about two or three millimeters long, de- pending upon the region which it is desired to section) and return to the fixative for 15 to 30 minutes. Now transfer the small pieces, without washing, to the niolybdate fixative se- lected and leave for a period of from about four hours to overnight. A more prolonged immersion will not hurt them, but may tend to make the brain tissue very brittle during section cutting. As soon as the small pieces have been placed in the molybdate fixative, the dish containing the ammonium picrate must be tluM- oughly washed and the residual picrate solution washed down the sink. On their removal from the molybdate fixative the pieces are washed thoroughly in running water and then embedded in paraffin. Do not be alarmed that during the course of dehydration in alcohol a certain amount of blue will come from the pieces. The blue which is Uberated during the process of dehydration is that which is not fixed in the nerve cells; this period of dehy- dration therefore serves to differentiate as well as to dehydrate the tissues. Paraffin sections are now cut in the normal manner and mounted on a slide, a process which should present no difficulty with the pres- ent material. It is recommended that sec- tions of about 15 to 20 microns in thick- ness be used if only the nerve cells are desired, or that sections about ten mi- crons thick be used if it is intended to counterstain the sections to show nuclei. If it is only desired to show the brain cells and their processes, the sections may l)P dewnxod, and then mounted rlircctly in balsam. It is usually better, however, to counterstain these sections with some carmine formula, and it is conventional to use for this purpose one of the alum carmines given in Chapter 20 (DS 11.2). The author's preference is for the formula of Mayer (DS 11.21 Mayer 1892) in which the sections are stained in the following manner. After dewaxing, pass the sections down through the customary alcohols to distilled water and place them in the carmine solution. This is a slow acting, but very safe reagent, and the sections may without danger remain in it over- night. They must, however, be watched to make sure that no overstaining takes place, or the process of differentiation may remove the blue from the nerve tis- sues at the same time as it removes the carmine. On removal from the carmine stain the sections should be rinsed brief!}' in a 5% solution of potassium alum to re- move the adherent stain, then washed thoroughly in tap water until they are free from potassium alum. They are then dehydrated, cleared, and mounted in balsam. Preparation of a section of spinal cord using the " Weigert-Pal " stain of Anderson 1922 All of the methods given under section DS 21.212 below may loosely be referred to as "Weigert-Pal" techniques, and that of Anderson 1922 is selected as the e.x- ample for the reason that in the author's hands it has always given the most satis- factory results. The principle difficulty which will be encountered is the prepara- tion of the staining solutions, and this must first be briefly described before pass- ing to a description of the technique itself. First it is necessary to prepare Weigert's primary mordant, the formula for which will be found in Chapter 22 (ADS 12.1 Weigert 1896). Raise 100 milliliters of dis- tilled water to the boil and throw in 5 grams of reagent grade potassium di- chromate, and dissolve it with rapid stir- ring. While the fluid is still boiling vigor- ously, add 2 grams of chromium fluoride, 398 METHODS AND FORMULAS DS 21.20 a little at a time, with brisk stirring be- tween each addition. When the whole two grams have been added, cool the solu- tion, leave overnight, and filter. This primary mordant of Weigert may be kept indefinitely. When required for use in the method of Anderson, convert it to Ander- son's mordant (ADS 12.1 Anderson 1922) by adding 10 milhhters of a 2% solution of calcium hypochlorite to 90 miUihters of primary mordant; this mixed solution is not stable and must be prepared im- mediately before use. Weigert's primary mordant, as such, is also required in the technique. Anderson's hematoxylin stain (DS 21.212 Anderson 1922) is easily pre- pared. Take 10 milhhters of absolute alcohol and dissolve in it 0.5 gram of hema- toxylin. When the hematoxylin is in solu- tion, add three milhhters of 2% calcium hypochlorite and then shake the vessel vigorously for about two minutes. Filter the mixture and make the filtrate up to 100 milhliters before adding three milh- hters of acetic acid; the solution is then ready for use. The technique also calls for the dichromate-sodium sulfate fixative of Muller (Chapter 18, F 7000.0000 Muller 1859). Differentiation requires the differ- entiating solutions of Pal (Chapter 22, ADS 21.1 Pal 1887). These are a freshly prepared 0.25% solution of potassium permanganate, and a solution containing 0.5% each of potassium sulfite and oxahc acid. Both solutions are relatively un- stable and should be prepared immedi- ately before use. With these solutions on hand for the staining process, it is necessary to con- sider the fixative which will be used for the spinal cord. Opinions are widely di- vergent as to the best fixative to employ, the majority of authors preferring merely to use a 5 % solution of potassium dichro- mate. Kultschitsky 1898 (766, 4:223) recommends the cupric-dichromate mix- ture of Erhtzky (F 4700.0000 Erhtzky 1877). The writer does not consider the cupric addition necessary, since sufficient copper mordanting occurs in Weigert's primary mordant. Having selected the fixative, it remains only to secure the spinal cord, which may be taken from any animal available to the investigator. Though the rabbit is widely used, it is somewhat small for demonstra- tion purposes. If access can be had to a slaughterhouse it is very much better to secure a piece of the spinal cord of a pig, though this latter is almost invariably destroyed in the process of pithing with which the commercial killing of pigs is ac- companied. It does not matter in the pres- ent instance that considerable time should elapse between the killing of the animal and the fixation of the cord, since post- mortem changes in the myelin sheaths, which are shown by this process, are gen- erally very slow. Whatever cord is finally selected should be cut into pieces about twice as long as they are broad, and sus- pended in a loosely woven cloth bag in a very large volume of the fixing solution. If, for example, 5 % potassium dichromate has been selected, it is by no means un- reasonable to use a liter of the solution for three or four half-inch lengths of the spinal cord of the pig. The time of fixation does not matter and should be determined by the physical condition of the spinal cord. A simple method of judging fixation in the dichromate (which will not be ap- plicable to the copper-dichromate mix- ture) was suggested by Pal 1887 (23632, 6:92), who took pieces from the fixative from time to time and cut them with a sharp knife in order to view the surface. If they are underfixed, the white matter of the cord will be lighter than the gray matter; fixation and hardening are com- plete when the white matter is a darker brown than the .gray matter. Overfixation is not particularly harmful but underfixa- tion will result in unsuccessful prepara- tions. When fixation is complete the speci- mens are washed in running water until no further dichromate comes away, and may then be stored in 70 % alcohol in the dark. If they are to be used immediately they must be dehydrated and embedded in celloidin (Chapter 18) before being cut into sections of from 15 to 20 microns in thickness by any of the techniques there given. The sections are accumulated in dis- tilled water, and when a sufficient number have been obtained, are transferred to Anderson's mordant (about 50 millihters DS 21.20 DYE STAINS OF SPECIAL APPLICATION 399 for a couple of sections) in a glass-stop- pered bottle, and kept at 37°C. The exact temperature is not critical, 37° being quoted because ovens at that temperature are common in most biological labora- tories. After three or four days at 37°C., the sections are transferred, without wash- ing to Weigert's primary mordant. To avoid curhng, it is recommended that the mordant be heated to about 20°C. and allowed to cool wliile the sections are in it, i.e. for a period of 10 to 30 minutes. Neither of the two mordanting processes are critical as to time, nor is there any method of discovering what is the best time for the particular batch, save by trial and error. After the second mordant bath, the sections are washed in several changes of distilled water. Anderson's hematoxylin is then raised, in a beaker or other con- tainer, to a temperature of about 50°C. The sections are dropped into this stain and allowed to remain for one hour. Each section is then taken from the stain (it should be a deep purple) and transferred without washing to Miiller's fixative for a period of 10 to 20 minutes. This is neces- sary to ensure the presence of dichromate, without which differentiation cannot be controlled. This dichromate bath is pe- culiar to Anderson's technique; other " Weigert-Pal" methods depend on the re- tention of dichromate from the fixing and mordanting solutions. The sections are then transferred from Miiller's solution to distilled water, and washed until no fur- ther color comes away. If the sections are now a deep blue, they may be differenti- ated, but if, through some error of tech- nique, they are more brown than blue, it is desirable to transfer them briefly to a weak (0.5%) solution of sodium bicar- bonate until they acquire a deep blue color. Differentiation is the most critical part of the entire method, and the in- experienced worker should proceed by short steps rather than endeavor to con- duct one operation. The sections are taken one at a time and placed in the potassium permanganate solution for about 30 sec- onds. Each section is then removed, rinsed rapidly in distilled water, placed in the potassium sulfite-oxahc acid solution, and watched under the microscope. After two or three minutes the white matter will be differentiated from the gray matter, but it is presumed that the differentiation will not have proceeded far enough in this brief period. The section is now returned to the potassium permanganate solution, left for a further period of 20 seconds, re- moved, placed in the bleaching solution, and again watched. The process should be stopped just before the tracts are clearly differentiated, for the differentiation will continue while the sections are being finally washed. This is the next stage of the process. Each section, when differ- entiation is complete, should be passed rapidly through two or three changes of water, then dehydrated, cleared, and mounted in balsam in the customary manner. Demonstration of the neuroglial cells of the white matter of the cerebral cortex using the crystal violet stain of Galescu 1908 Many methods for the demonstration of neurogha are described in Chapter 23. The method of Galescu 1908 (DS 21.22 Galescu 1908) is among the more satis- factory of the dye-staining techniques, and has the advantage over the metal-staining techniques that it requires less \agorous attention to detail to secure a passable result. It is not, however, a method which can be recommended for research pur- poses, though it might well prove a useful and interesting demonstration in the hands of a class in microtechnique. It is unimportant what animal is used, though a rabbit is a convenient form from which to obtain the brain. In demonstra- tions of neuroglial structures the animal should not be killed by anesthetics, but by a sharp blow on the head, and then tied face downward on a board while the skin is removed from the top of the cranium. The nasal bones should then be broken out with a hammer and chisel, and the free end of the frontal bone thus exposed gripped in a pair of blunt-nosed phers. A sharp upward jerk, with an inwardly di- rected twist of the hand at the same time, will cause the frontal bone to break away cleanly without damage to the underlying brain structures. The parietal bone is then 400 METHODS AND FORMULAS DS 21.20 removed piecemeal and the process re- peated on the other side of the brain. The membranes are now dissected away from the freshly exposed brain, preferably with forceps rather than with a knife. A sharp scalpel is then used to remove pieces of the cerebral cortex of about three- or four- milUmeter side. As these pieces will in- evitably adhere to the knife, it is not pos- sible to transfer them directly to the 6% mercuric chloride, which is used as a fixa- tive, because this reagent will destroy the surface of the blade. They are, therefore, best washed from the knife into a tube of normal saline, which is poured off as soon as they have sunk to the bottom and re- placed with 6% mercuric chloride. The tube of fixative with its contained piece of brain is then tipped into a large vessel (at least 500 milliliters) of 6% mercuric chlo- ride, which is agitated gently at intervals for the next five hours. When the pieces have thus been fixed, the mercuric chloride is poured off and the brain pieces, without handling, are tipped with the last of the solution into a vial of about 20-milliliter capacity. The last of the mercuric chloride is then poured off and replaced with Galescu's osmic-mercuric-chromic-acetic fixative (Chapter 17, F 1360.0010 Galescu 1918) which serves both to harden them completely and to render insoluble certain of the fatty substances present. This solu- tion is used at a temperature of 37°C., or such approximation of this temperature as can be maintained in an available oven, for about 12 hours. A convenient time schedule is to kill the rabbit in the morn- ing, to commence the fixation in mercuric chloride immediately, to transfer the pieces to the osmic-mercuric-chromic- acetic mixture in the evening, and con- tinue the process the next day. The next morning the osmic-mercuric-chromic-ace- tic mixture is poured off, replaced with a fresh solution, and returned to the oven. In the evening the solution is again re- placed, and this time the specimens are left in the oven for a whole day. The next evening, therefore, the pieces are trans- ferred to running water for an overnight wash. It is an integral part of Galescu's process that alcohol not be used in any stage of the technique, and the pieces are therefore next treated with an acetone-iodine solu- tion to remove the mercury from the tis- sues. This solution is prepared by adding one milhhter of Lugol's iodine (Chapter 19, ADS 12.2 Lugol) to 100 milhhters of acetone. The specimens are treated for 24 hours, and about 100 millihters should be used for half a dozen small pieces of brain. The bottle containing the pieces should be gently tipped from side to side at intervals to prevent the accumulation of depleted solution at the bottom. The specimens are then dehydrated in pure acetone until no further iodine comes away, and embedded in paraffin, prefer- ably by the dioxane techniques. A high- melting-point paraffin should be used, since Galescu's process depends on cutting very tliin sections, 5 microns being about as thick as can be conveniently handled, and three microns being very much better if the skill of the technician permits. The sections are mounted on a slide, de- waxed ^\•ith xylene, and then transferred to acetone for the removal of the xylene. They are next placed in Galescu's crystal violet-oxalic acid stain (DS 21.22 Galescu 1908) for ten minutes, or until examina- tion shows they have acquired a dense violet color. They are then transferred to a beaker of fresh Galescu's stain, and warmed gently on a hot plate, or in a water bath, for about five minutes or until the temperature has reached about 50°C. Each slide is then removed individualh', drained, and flooded from a drop bottle with Lugol's iodine. They should under no circumstances be washed between their removal from the staining solution and their treatment with iodine. The iodine is in its turn drained from the slide and the section blotted gently with a rather stiff grade of filter paper. Each slide is then transferred to a mixture of equal parts of xj^lene and aniline, in which it remains until examination under the high power of a microscope shows that the required neuroglial structures are clearly differ- entiated. This may take from 5 minutes to 2 hours, according to the treatment which the pieces have previously received. As soon as they are found to be differ- entiated, the xylene-aniline mixture is washed off with xylene and the sections mounted in balsam. DS 21.21 DS 21.211 DYE STAINS OF SPECIAL APrLICATION 401 21.21 NERVE CELLS AND PROCESSES Nerve cells and processes are stained more easily by the dye-staining techniques, than they are by metal-staining techniques. It must be admitted that most of the techniques are not so specific, but they are certainly adequate for class demonstrations; and it must not be forgotten that much of the classical research was done by these methods rather than l)y metal stains. These techniques can be divided into three groups: first, the thiazin (methylene blue and toluidine l)lue) methods (DS 21.211) whioli were among the first to be employed; second, the great division of hematoxylin stains (DS 21.212) with which the name Weigert is linked; third, other dye-staining methods (DS 21.213) which have from time to time occurred in the literature. 21.211 Methylene Blue and Toluidine Blue Methods The thiazin nerve-staining techniques, once far more numerous and of wider employ- ment than is today the case, can be broadly divided into two divisions : those which are customarily applied to sections of fixed material, and those which are applied to living tissues for subsequent fixation. In the latter case, some phosphomolybdate or picrate salt is applied to precipitate the blue stain which has been differentially absorbed into the nerve cells. Both methods depend for their success on accurate timing, for if the stain is permitted to act too long, other tissues than nerves become deeply stained ; if too short a time is allowed for their absorption, a most disappointing result will be obtained. The classic technique for injection into the Uving animal is that of Bethe 1896, who published in the journal cited below no less than five separate formulas for materials designed to fix the blue in the Jiving tissues. It is still a^method which should not be ignored, since successful preparations are certainly as good as those to be obtained by the metal-staining techniques. The second type of technique for living materials is that of Cajal 1893, now practically forgotten, in which solid methylene blue is sprinkled upon a freshly cut surface of brain and allowed to absorb for such time as is considered necessary. The stain is very capricious, but the results obtained when it is used suc- cessfully are of great beauty. The technique of Betsa 1910, though slow, is one of the surest methods by which a good demonstration of Purkinje cells may be provided for class-teaching purposes. 21.211 Alzheimer 1910 Nissl and Alzheimer 1910, 409 REAGENTS REQUIRED*. A. ADS 12.1 Weigert 1891; B. sat. aq. sol. phosphomolybdic acid; C. DS 13.7 Alzheimer 1910 method: [10 M sections by freezing technique] -^ water — > .4, 6 hrs. -* wash -^ B, 2-12 hrs. -^ rinse -^ C, till sufficiently stained -^ wash -^ balsam, via usual reagents 21.211 Bacsich 1937 11025, 72:163 REAGENTS REQUIRED: A. ADS 12.1 Bacsich 1937; B. DS 11.124 Bacsich 1937; C. abs. ale. 50, ether 50 method: [celloidin sections of formol material, attached to slide with V 21.1 Mayer 1884 and varnished with celloidin] -* water -^ A, 2 hrs. 36°C. -^ thorough wash -^ B, 2 lirs. 37°C. -^ thorough wash -» 95% ale, till dehydrated -* C, to remove varnish -^ balsam, via usual reagents 21.211 Besta 1910 766, 36:477 REAGENTS REQUIRED: .4. 0.1% thionin; B. 20% creosote in 95% ale; C. creosote method: [sections of F 0000.1200 Besta 1910 fixed material] -> water -> A, 2-3 hrs. -^ B, until differentiated —* C, till clear -» balsam, via xylene note: This method was originally recommended for the demonstration of Purkinje cells but is of wider application. 21.211 Bethe 1896 766, 12:438 REAGENTS REQUIRED: A. sat. sol. {circ. 4.5%) methylene blue; B. sat. sol. (arc. 1.2%); ammonium picrate; C. water 100, sodium phosphomolybdate 5, osmic acid 0.25, hydrochloric acid 0.2 402 METHODS AND FORMULAS DS 21.211 method: [A, by copious injections into the living animal] -^ [small pieces of brain] — > B, 15 mins. — > C, 4-12 hrs. -* wash -^ [paraffin, via usual reagents] — » sections -^ any DS 11.21 formula if counterstain required note: Bethe, loc. cit., gives the following alternative formulas for C: I. water 100, am- monium molybdate 5, hydrochloric acid 0.2; II. water 100, ammonium molybdate 5, chromic acid 1, hydrochloric acid 0.2; III. water 100, ammonium molybdate 5, osmic acid 0.25, hydrochloric acid 0.2; IV. water 100, sodium phosphomolybdate 5, osmic acid 0.25, hydrochloric acid 0.2; V. water 100, sodium phosphomolybdate 5, hydro- chloric acid 0.2. Cole 1933 (20450b, 9, 89) prefers: water 50, glycerol 50, hydrochloric acid 0.5, ammonium molybdate to excess. A detailed description of the use of this technique is given under DS 21.20 above. 21.211 Bethe lest. 1933 Cajal and de Castro Cajal and de Castro 1933, 169 REAGENTS REQUIRED: .4. 5% nitric acid; B. G5% ale. 90, ammonium, hydroxide 10; C. 70% ale. 90, hydrochloric acid 10; D. 4% ammonium molybdate; E. 0.03% toluidine blue method: [small fragments of fresh tissue] -^ A, 24 hrs. — > 95% ale, 24 hrs. — > 5, 1 wk. -^95% ale, thorough wash -^ C, 24 hrs. ^95% ale, thorough wash -^ water, wash -^ D, 24 hrs. -^ [5-8 m paraffin sections] -^ water, 10-12 mins., 60°C. — * E, 1-10 mins. — > water, till differentiated — > abs. ale, till dehydrated — * balsam, via xylene RECOMMENDED FOR: neurofibriUae. 21.211 Bethe and Monckeberg test. 1933 Cajal and de Castro Cajal and de Castro 1933, 335 reagents required: A. 0.25% osmic acid; B. water 100, sodium bisulfite 2, hydro- chloric acid 1; C. 2.5% ammonium molybdate; D. 0.1% toluidine blue method: [pieces of nerve] -> A, 24 hrs. -^ wash, 6 hrs. -^ 96%, ale, 24 hrs. -> wash, 4 hrs. — > B, 18 hrs. -^ wash, 2-4 hrs. -* [2 m-3 m paraffin sections] -^ water — » B, 5-10 mins. 20°-30°C. — > rinse -^ C, 5 mins. 50°-60°C. — > rinse -^ balsam, via usual reagents recommended for: neurofibriUae in medullated fibers. 21.211 Bing and EUerman 1901 1739, 3 :260 REAGENTS REQUIRED: A. sat. sol. (circ. 4.5%) methylene blue; B. sat. sol. (circ. 1.2%,) picric acid method: [hand sections of Bing and Ellerman 1901, F 0000.1300 fixed material] -♦ A, 5-10 mins. -^ B, 1 to 2 mins. -^ acetone, till dehydrated -^ balsam, via xylene 21.211 Cajal 1893 test. Pollack 1900 Pollack 1900, 133 REAGENTS REQUIRED: A. methylene blue (solid powder); B. 0.1% hydrochloric acid; C. water 100, ammonium molybdate 10, hydrochloric acid 0.3; D. F 2000.1000 Cajal 1893; E. 0.3% platinic chloride; F. 0.3%, platinic chloride in abs. ale method: [small pieces of fresh, or living, tissue] -^ A, dusted over surface, 45 mins. -^ B, quick wash ^ C, 2 to 3 hrs. -* rinse -^ D, 3-4 hrs. --' wash briefly -^ E, 15 mins. — > F, till dehydrated -^ [paraffin sections] -> balsam, via usual reagents 21.211 Chang 1936 763, 65:437 formula: water 90, 40% formaldehyde 10, thionin 0.3 method: [fresh brain tissue] — > stain, few days to few months -^ wash -^ [paraffin sec- tions] -^ balsam, via usual reagents result: fiber tracts, red; cell bodies, blue. ■^ 21.211 Cole 1933 see DS 21.211 Bethe 1896 (note) 21.211 Donaggio test. 1933 Cajal and de Castro Cajal and de Castro 1933, 72 REAGENTS REQUIRED: A. pyridine; B. water 100, ammonium molybdate 4, hydrochloric acid 0.2; C. 0.01% thionin 7 method: [small fragments] -^ A, 5-6 days -* wash, 24 hrs. -^ 5, 8 days -^ wash -^ [5 M paraffin sections] -^ C, till sufficiently stained -^ quick rinse -^ B, b mins. -^ balsam, via usual reagents recommended for: neurofibriUae. DS 21.211-DS 21.212 DYE STAINS OF SPECIAL APPLICATION 403 21.211 Feyrter 1936 22575, 296:015 REAGENTS REQUIRED: A. walop 100, tartaric acid 0.5, thionin 1.0 method: [frozen sections of formaldehyde material] — > .1, 5 mius. — ♦ [add coverslipj -> cement with V 12.2 Noyer 1918 recommended for: myelin sheaths. 21.211 Harris 1898 16185a, 1:897 reagents required: A. b% potassium dichroinatc; B. water 100, sodium })orate 1, toluidinc blue 1; C. sat. sol. {circ. 75%) tannic; acid method: a, till white matter dark brown -^ [15 m sections] — » wash — > B, 1-2 hrs. — » rinse — > C, till differentiated -^ balsam, via usual reagents 21.211 Heller, Thomas, and Davenport 1947 205101), 22:111 re.\ge\ts required: A. water 90, M/15 phosphate buffer ])1I 5.0 5, M/2 sodium lactate 5, sodium chloride 0.6, sodium acetate 0.03, dextrose 0.2, methylene blue 0.01; B. water 100, ammonium molybdate 8, potassium dichromate 1 method: [thin, fresh tissues from pentobarbital-killed mammal] -^ A, 37.5°C. with pure oxygen bubbled through solution, till nerves stained, 2-4 hrs. — > B, 1 or more days, — ♦ balsam, via usual reg-gents [or paraffin sections] 21.211 Landau 1934 4285a, 11 :44 reagents required: A. abs. ale. 50, chloroform 50; B. 1% toluidine blue; C. 1% toluidine blue in creosote; D. 1% toluidine blue in chloroform method: [pieces of formaldehyde-fixed material] —>■ wash — > drain — > A, via graded ales. — > 5, 2 days -^ drain and blot -^ C, 2 days — > drain and blot — > chloroform — > [paraffin sections] recommended for: general neurology. 21.211 Nissl test. 1896 Kahlden and Laurent Kahlden and Laurent 1896, 155 REAGENTS REQUIRED: A. 0.5% methylene blue; B. 10% aniline in 90% ale. method: [sections] -^ a, on slide, heated to steaming —> ale, quick rinse ^ B, till differentiated —* balsam, via clove oil 21.211 Schabadasch 1936 4285a, 13:137 formula: water 100, sodium chloride 0.8, sodium pyruvate 0.032, magnesium bromide 0.15, glucose 0.2, methylene blue 0.025 method: perfuse freshly killed animal RECOMMENDED FOR: ucrve endings in fresh tissue. 21.212 Hematoxylm methods The literature of the hematoxylin staining techniques for nerve cells and their processes is almost as confused as the Uterature of the metal-staining techniques for the same purpose. The method is customarily and loosely referred to Weigert, who pubUshed several of the techniques and formulas involved, and who is also responsible for the original mordant used. His name is often coupled with that of Pal, though the latter contributed to the literature only a method of differentiation, and not of staining. What has rendered the situation so ob- scure, however, is the habit of authors of referring to any of these hematoxylin techniques as " Weigert-Pal methods," irrespective of the author to wliom they are assigned. Thus Gatenby and Cowdry 1937, say casually that the techniciue of Wolters 1890 is to be considered "the standard Weigert-Pal technique," even though it differs very appreciably from any of the methods recommended either by Wcigort or Pal. At least, however, Gatenby and Cowdry have given their reference, but the majority of authors in this field use and recommend the most diverse techniques under the name of "Weigert-Pal." If an author says only that he stained a nervous structure by a "Weigert-Pal" technique, it is safe only to assume that he utilized some hematoxylin stain, which was subsequently differentiated by some method involving a bleaching reagent. It is not quite as safe an assumption, but still very probable, that some form of mordant was used before staining. It will have been understood from what has been said that these techniques involve in general a mordant, usually chromic acid, or chromium fluoride, followed by a hematoxylin stain, which is subsequently differentiated either by another mordant (Meyer 1909) or by a bleach, usually the potassium permanganate sodium sulfite mixture of Pal 1887. These methods tend to make sections brittle, but Miller 404 METHODS AND FORMULAS DS 21.212 1926 (20540b, 1 :72) states that this can be avoided by leaving the sections for 12 hours in 80% alcohol between the staining and differentiating processes. In the technique of Olivo- crona 1917, however, the mordant is incorporated with the staining mixture. In spite of the numerous modifications which have been made, the methods of Weigert 1885 and Weigert 1894 are probably the easiest and simplest to employ, though the rationalization of Anderson 1922 is now almost universal in Europe. The numerous techniques which are given below are offered less as suggestions to be followed than in the hope that they may tend to diminish the confusion by assigning to the correct author the technique which he has invented. 21.212 Anderson 1922 11977, 5:65 REAGENTS REQUIRED: A. ADS 12.1 Auderson 1922; B. ADS 12.1 Weigert 1891; C. water to make 100, acetic acid 3, abs. ale. 10, hematoxylin 0.5, 2% calcium hypochlorite 3; D. F 7000.0000 Miiller 1859; E. ADS 21.1 Pal 1887 (A & B sols.) PREPARATION OF c: Dissolve dye in ale. Add hypochlorite. Shake well. Dilute with water and add acid. method: [15-20 m celloidin sections]^ A, 48 to 72 hrs. 38°C. -^ B, 10-30 mins. -^ wash -^ C, 1 hr., 50°C. -^ D, 10-20 mins. — * wash — » E, till differentiated -^ balsam, via usual reagents RECOMMENDED FOR: myelin sheaths. note: a detailed description of the use of this technique is given under DS 21.20 above. 21.212 Anderson 1929 see either DS 11.121 Anderson 1929 or DS 11.123 Anderson 1929. 21.212 Anderson 1942 11431, 54:258 reagents required: A. ADS 21.1 Anderson 1942; B. DS 11.113 Kultschitsky 1889; C. 2.5% potassium dichromate; D. ADS 21.1 Pal 1880 {A and B sols.); E. DS 11.21 Anderson 1926 method: [30 fi frozen sections]-^ A, 1 hr., 50°C. ^ wash -^ B, 30 mins., 50°C -* C, 2-3 mins. -^ wash -^ D (A sol.), 2 mins. -^ D (B sol.), 1 min. -♦ [repeat D (A) -^ D {B) cycle till differentiated] -* wash -^ E, 45-60 mins., 50°C. -* rinse -^ 80% ale. -^ balsam, via usual reagents 21.212 Bolton 1898 11025, 32:245 reagents required: A. 1% osmic acid; B. DS 11.113 Kultschitzky 1889; CADS 21.1 Pal 1887 (A & B sols.) method: [15 fi sections of formaldehyde-fixed material]-^ distilled water -^ A, few mins. -+ rinse — > 5, 3 to 24 hrs. —>■ rinse -^ C, till differentiated -^ balsam, via usual reagents RECOMMENDED FOR: myelin sheaths. 21.212 Clark and Ward 1934 20540b, 9 :34 REAGENTS required: A. 4% ferric alum; B. DS 21.212 Weigert 1885 (sol. C); C. ADS 21.1 Pal 1887 {A and B sols.); D. sat. sol. lithium carbonate method: [sections] -^ A, 2-24 hrs. -^ quick wash -^ B, 1-2 hrs. -^ quick wash -* A, till gray and white matter just distinguishable -^ C (A sol.), till brown -^ rinse -^ C (B sol.) till gray matter bleached-^ wash—* D, 5 mins. -^ wash -^ balsam, via usual reagents 21.212 Donaggio 1939 1820, 22:171 REAGENTS REQUIRED: A. ADS 12.2 Lugol ; B. DS 11.124 Donaggio 1904; C. ADS 21.1 Pal 1880 (A and B solutions) method: [20 fi sections from F 3700.0010 Zenker 1894 material]-^ water -^ A, few moments —> 90%, ale, 1 hr. -^ water, via graded ales. — > B, six hrs. -^ C, (A sol.), 1 min. -^ C (B sol.), 1 min. -> [repeat C (A) --^ C (B) cycle till differentiated] -^ wash — > balsam, via usual reagents recommended for: differentiation of anesthetized nerves (stained) from normal nerves (unstained). 21.212 Fajerstajn 1901 16341, 1:189 REAGENTS REQUIRED: A. 0.5% chromic acid; B. 1% hematoxylin; C. ADS 21.1 Pal 1887 (A and B sols.) DS 21.212 DYE STAINS OF SPECIAL APPLICATION 405 method: [frozen sections of formaldehyde-fixed material] —> water — > A,2i hrs. — > wash -^ B, 24 hrs. -^ wash -^ C (B sol.), till decolorized -> [repeat C (A) -> C (B) till differentiated] — > balsam, via usual reagents RECOMMENDED FOR: myelin sheaths. 21.212 Gudden 1897 15058, 16:24 REAGENTS REQUIRED: A. 0.5% chromic acid; B. water 90, 95% ale. 10, hematoxylin 1, 10% nitric acid 0.5; C. ADS 21.1 Pal 1887 (A & B sols.) METHOD [20 fj. celloidin sections of formalin-hardened material] —> A, 10 hrs. rinse—* 80% ale, till no more color comes away — » B, 6-24 hrs. — > wash — * C, (sol. A), 15-20 sees. -* C, (sol. B) few sees. — » [repeat C (A) ^ C (B) cycle till sufficiently differenti- ated] -* balsam, via usual reagents RECOMMENDED FOR: myelin sheaths. 21.212 Hadjioloff 1928 .see DS 21.212 Pal 1887 (note) 21.212 Howden 1936 see DS 21.212 Bolton 1898 (note) 21.212 Kaiser 1893 15058, 12:364 REAGENTS required: .4. F 7000.0000 Muller 1859; B. F 1700.0000 Marchi 1886; C. water 50, 95% ale. 50, ferric chloride 25; D. DS 21.212 Weigert 1885 (sol. C); E. ADS 21.1 Pal 1887 (sols. A & B) method: [whole brains or pieces] —> .4, till hardened (some methods) -^ 15 mm. slices -* B, 1 wk. -> [25 M sections] -> C, 5 mins. -* wash -» D, 2 to 12 hrs. -^ E, till differ- entiated -^ balsam, via usual reagents recommended for: myelin sheaths. 21.212 Knower 1930 see DS 11.113 Knower 1930 21.212 Kozowsky 1904 see DS 21.212 Pal 1887 (note) 21.212 Kultschitsky 1890 766,6:519 reagents required: A. DS 11.113 Kultschitsky 1889; B. 1.5% lithium carbonate; C. ADS 21.1 Kultschitsky 1889 method: [15-20 n celloidin sections of F 4700.0000 Erlitzky 1877 hardened material] -> A, 1-24 hrs. —>■ B, till no more color comes away -* C, till differentiated — * wash -^ balsam, via usual reagents recommended for: myelin sheaths. 21.212 La Manna 1937 23632, 54:257 reagents required: A. 30% ferric chloride; B. 0.5% ferric chloride; C. DS 11.121 La Manna 1937 method: [paraffin sections of F 7800.1000 La Manna 1937 fixed material] -+ water — > A, 1 hr. — > S, quick rinse — > C, 1 hr. -^ running water, overnight — > B, till differentiated — > thorough wash — > balsam, via usual reagents recommended for: myelin sheaths. 21.212 Landau test. 1924 Spielmeyer Spielmeyer 1924, 97 reagents required: A. 3.5% ferric alum; B. 1% hematoxj-lin; C. "weak" hydrogen peroxide method: [celloidin sections of formaldehyde fixed material]^ water —> A, 12-24 hrs. -» rinse -♦ B, 12-24 hrs. —>■ tap water, 1 hr. -♦ C, till differentiated -> balsam, via usual reagents 21.212 Landau 1938 4285a, 15:181 reagents required: A. ADS 12.1 Landau 1938; B. 10% ferric alum; C. 1%, hema- toxylin D. ADS 21.1 Landau 1938 method: [large pieces of formaldehyde fixed brain tissue] —> thorough wash — > .4, 24 hrs., 25°-30°C. — * thorough wash -^ [15 m paraffin sections] — > water — > B, 3-6 hrs., 25°-30°C. -*■ quick rinse -> C, several hrs., 25°-30° -> thorough wash -^ D, till dif- ferentiated —>■ thorough wash -* balsam, via usual reagents 406 METHODS AND FORMULAS DS 21.212 21.212 Liber 1937 1887a, 24 :230 REAGENTS REQUIRED: A. ADS 12.1 Weigcrt 1896; B. DS 11.113 Kultschitsky 1889; C. 1% lithium carbonate; D. ADS 22.1 Pal 1887 (A and B sols.) method: [paraffin sections of formaldehyde material] — > water — > A, 12 hrs. — > rinse — * B, 1 hr. —>■ rinse —y C, till blue — > thorough wash — > D (A sol.), few seconds — » D, (B sol.), till differentiated — * balsam, via usual reagents 21.212 Lillie 1948a Lillie 1948, 171 REAGENTS REQUIRED: A. 4% ferric alum 50, 1% hematoxylin in 95% ale. 60; B. 0.5% ferric alum; C. ADS 21.1 Lillie 1948; D. 0.1% safranin in 1% acetic acid method: [sections of dichromate fixed or mordanted material] —> 80% ale. — > A, 40 mins., 50°-60°C. — * rinse —> B, 1 hr. -^ C, till blue —* wash — > D, 5 mins. — > balsam, via usual reagents RECOMMENDED FOR: myelin sheatlis. 21.212 Lillie 1948b Lillie 1948, 160 reagents required: A. 2.5% potassium dichromate; B. DS 21.212 Weil 1928 (sol. A) C. 0.5% ferric alum; D. ADS 21.1 Lillie 1948; E. working solution of Sudan I pre- pared as in DS 22.4 Lillie 1948 method: [frozen sections of formaldehyde-fixed material] -+ A, 2-4 days — > wash — > B, 45 mins. 55-60°C. — > wash -^ C, tUl decolorized — > wash — > D, 10 mins. — > wash — > E, 10 mins. — > wash -^ M.ll mountant 21.212 Loyez iest. 1929 Anderson Anderson 1929, 65 reagents required: A. 4% ferric alum; B. abs. ale. 10, hematoxylin 1, water 90, sat. aq. sol. lithium carbonate 2; C. 0.1% hydrochloric acid in 70% ale. preparation of reagent B: Dissolve hematoxylin in water. Add ale. Then add car- bonate solution. method: [15-20 n sections] — > water — * A, 24 hrs. -^ rinse -^ B, 2-4 hrs. 50°C. -^ wash -^ C, till differentiated — > balsam, via usual reagents 21.212 MacConaill test. 1951 Gurr Gurr 1951, 36 stock solutions: I. water 92, acetic acid 8, lead nitrate 2, acid fuchsin 0.5; II. water 96, acetic acid 4, hematoxylin 1 reagents required: A. stock I 50, stock II 50; B. water 100, ammonium molybdate 14, ammonium acetate 0.4 method: [6-12 n sections of formaldehyde-fLxed material] — > 30% ale. — » A, 5 mins. —* wash -^ B, 1-2 mins. -^ thorough wash — ^ balsam, via usual reagents 21.212 Mallory 1936 608b, 12 :569 reagents required: A. sat. aq. sol. (circ. 1%) lead chloride; B. water 90, ADS 12.2 Lugol 10; C. 0.05% hematoxylin method: [3 mm. slices of formaldehyde-fixed material] —>■ A, 6 wks. room temperature or 1 wk. 37°C. -^ wash, 24 hrs. -> 80% ale, till required — > [8 /x sections] — > water —y B, 1 min. ^•95% ale, till color removed — > C, J^ to 1 hr. —* tap water, till blue note: All solutions must be freshly prepared immediately before use. recommended for: general neurological staining. 21.212 Mitrophanov 1896 23632, 13 :470 reagents required: A. ADS 12.1 Mitrophanov 1896; B. DS'll.llS Kultschitsky 1889; C. ADS 21.1 Weigert 1885 method: [15-20 /t celloidin sections] -^ A, 24 hrs. 45°C. -^ rinse — ♦ B, 10 mins. — > rinse — » C, till differentiated recommended for: myelin sheaths. 21.212 Nelson test. 1930 Guyer Guyer 1930, 104 reagents required: A. 1% potassium hydroxide; B. water 75, acetic acid 12, glycerol 12, chloral hydrate 0.75; C. water 75, glycerol 12, DS 11.123 Ehrlich 1886 12, chloral hydrate 0.75 method: [alcohol-hardened small vertebrates]-^ A, till translucent—* B, 3 days — » C, 7 days -^ B, till differentiated -^ glycerol recommended for: staining nerves in wholemounts of small vertebrates. DS 21.212 DYE STAINS OF SPECIAL ArrLICATION 407 21.212 Neumen 1915 4349, 6:71 REAGENTS REQUIRED: A. watcf 100, chroiTiic acid 5, potassium diclironiate 4; B. 3% ferric chloride; C. sat. aq. sol. {circ. 7.5%) cupric acetate; D. sat. ale. sol. {circ. 35%) hematoxylin; E. water 100, potassium ferrocyanide 7, sodium borate 0.5; F. sat. sol. Lithium carbonate method: [celloidin sections] -^ water -^ A, 2-5 mins. —>■ wash -♦ B, 2-5 mins. ->■ wash -^ C, 2-5 mins. -^ wash — > Z), 2-5 mins. -^ wash — > C (fresh sol.), till copious color clouds are produced -> wash -> D, till differentiated -» wash -+ F, 2 mins. -» wash — » balsam, via usual reagents 21.212 Olivecrona 1917 23681,28:521 REAGENTS REQUIRED: A. Water 30, 95% ale. GO, hematoxylin O.G, ferric chloride 0.15, hydrochloric acid 0.3; B. water 100, ferric chloride 0.8, hydrochloric acid 1.0; C. sat. sol. lithium carbonate PREPARATION OF A: Add the dye dissolved in ale. to the iron and acid dissolved in water. method: [sections by freezing technique] -^ 70% ale— >.4, 1 hr. — > wash — > B, till differentiated —^ wash -^ C, till blue -^ balsam, via usual reagents RECOMMENDED FOR: myelin sheaths. 21.212 Pal 1887 Weigert-Pal—compl. script. 23632, 6 :92 REAGENTS REQUIRED: A. 5% potassium dichromate; B. DS 21.212 Weigert 1885 (sol. C) C. ADS 21.1 Pal 1887 (.4 & B sols.) method: [pieces of brain] -+ .1, till white matter dark brown (some wks.) — > wash -* 80% ale, till required — ♦ [section 15 /x to 20 p] -^ water -^ B, 2 hrs. to 1 day — » wash — > C (sol. A), 15-20 sees. -^ rinse —>■ C (sol. B), few sees. -> [repeat C (A) — > C (B) cycle till sufficiently differentiated] —> balsam, via usual reagents recommended for: myelin sheaths. note: Tschernyschew and Karusin 1897 (23632, 13:354) substitute Kultschitsky 1889 DS 11.113 for B above. Kozowsky 1904 (15058,23:1041) substitutes his ADS 21.1 for C above. Hadjioloff 1928 (4285a, 5:431) counterstains in a sat. ale. sol. light green. 21.212 Schroder 1930 23430, 166:588 reagents required: a. ADS 12.1 Schroder 1930; B. DS 11.124 Schroder 1930; C. 0.25% potassium permanganate; D. water 100, oxalic acid 0.5, potassium sulfite 0.5; E. 0.02% lithium carbonate method: [20-30 /x frozen sections of formaldehyde-fixed material) — > A, 1 day, 37°C. — » rinse -^ B, 12 hrs., 37°C. — > wash -^ C, 30 sees. -^ rinse -^ D, 1 min. —>■ thorough wash — » [repeat C ^> D cycle if differentiation insufficient] -^ £", 15 mins. -^ wash — ♦ balsam recommended for: myelin sheaths. note: Schroder 1939 (23430, 166:588) does not differ significantly from the above. 21.212 Schultze test. 1933 Cajal and de Castro Cajal and de Castro 1933, 234 reagents required: A. 1% osmic acid; B.\% potassium dichromate; C. 0.5% hema- toxylin in 70% ale. method: [small blocks of brain tissue] — > A, overnight — ♦ B, in dark, 3 or 4 changes in 24 hrs. — > 50% ale, 24 hrs. in dark -^ C, till stained throughout —> [celloidin or paraffin sections] recommended for: myelin sheaths. 21.212 Seidelin 1911 see DS 11.121 Weigert 1904 (note) 21.212 Sihler lest. 1907 Bohm and Oppel cit. Gad Bohm and Oppel 1907, 268 REAGENTS REQUIRED: A. Water 75, glycerol 12, acetic acid 12, chloral hydrate 0.75; B. water 75, DS 11.123 Ehrlich 1886 12, glycerol 12, chloral hydrate 0.75; C. 0.1% acetic acid in glycerol method: [pieces of fresh muscle] —> A, 18 hrs. -^ B, 3 to 10 days—* C, till differenti- ated — » glycerol 21.212 Smith and Quiegley 1937 608b, 13:491 RE.'VGENTS REQUIRED: A. 4% ferric alum; B. water 97, acetic acid 3, hemato.xylin 1; C. 0.5% lithium carbonate method: [sections] — > water — > A, 15 mins. -^ 70% ale, short wash — » B, 30-60 mins., 55°C. — » rinse — * C, till blue — > wash — > balsam, via usual reagents 408 METHODS AND FORMULAS DS 21.212 21.212 Spielmeyer 1930 test. 1938 Mallory Mallory 1938, 237 REAGEi^Ts required: A. 2.5% ferric alum; B. water 100, 10% ripened hematoxylin in abs. ale. 5 method: [20-30 m frozen sections of formaldehyde-fixed material] ^ ^, 6 hrs. -^ rinse -^70% ale. 10 mins. — > rinse -^ A, till differentiated — > balsam, via usual reagents RECOMMENDED FORt myelin sheaths. 21.212 Tschernyschew and Karusin 1896 see DS 21.212 Pal 1887 (note) 21.212 Vassale 1889 see DS 21.212 Weigert 1885 (note) 21.212 "Weigert-Pal" see almost any method in this section. Gatenby and Cowdry 1937, 526, state Wolters 1890 to be "the standard 'Weigert-Pal' technique." 21.212 Weigert 1885 8645, 3:230 REAGENTS REQUIRED: A. 5% potassium dichromate; B. 3.5% copper acetate; C. water 90, 95% ale. 10, hematoxylin 1, sat. aq. sol. lithium carbonate 1; D. ADS 21.1 Weigert 1885 method: [pieces of brain] — > A, till white matter dark brown (some wks.) -^ [embed in celloidin] -^ B, 55°C., 2 days — > 80% ale, till required — ^ section 15-20 m — » water — > C, 2 hrs. to 1 day — > wash — * D, till differentiated -^ wash -^ balsam, via usual reagents RECOMMENDED FOR: myelin sheaths. note: Vassale 1889 (19460, 15 : 102) uses B after C but it is otherwise identical. Weigert 1886 (7936a, 6:10) substitutes for A his F 6700.0000 fixative. 21.212 Weigert 1904 see DS 11.121 Weigert 1904 21.212 Weil 1928a method for thin sections — auct. 1879,20:392 REAGENTS REQUIRED: A. water 100, ferric alum 2, hematoxylin 0.5; B. 4% ferric alum; C. ADS 21.1 Weil 1928a; D. 0.1% ammonia PREPARATION OF A: Add 50 1% hematoxylin to 50 4% ferric alum. method: [sections less than 30 /x thick] —^70% ale. — > water -* A, 20-30 mins., 55°C. — > B, 5 mins. -^ C, till differentiated -^ wash -^ D, till blue -^ balsam, via usual reagents RECOMMENDED FOR: myelin sheaths. 21.212 Weill928b method for thick sections—auci. 1879,20:32 reagents required: A. 3% potassium dichromate; B. (see Weil 1928a, sol. A, above); C. 4% ferric alum; D. ADS 21.1 Weil 1928a; E. ADS 21.1 Weil 1928b A & B sols.; F. 0.1% ammonia method: [sections more than 30 /x thick] -^ 70% ale. — > water -^ A, overnight—* wash -^ B, 20-30 mins., 55°C. -^ C, 10 mins. -^ D, till differentiated -> E (A), 10 mins. -^ E (B), till bleached — > [repeat E (A) — > E (B) till background sufficiently bleached] -^ wash -^ F, till blue —* balsam, via usual reagents 21.212 Wharton 1937 763, 67 :467 reagents required: A. water 76, acetic acid 12, glycerol 12, chloral hydrate 0.75; B. water 76, DS 11.123 Ehrlich 12, glycerol 12, chloral hydrate method: [thin organs stranded on paper] -^ A, 18 hrs. -^ B, 24 hrs. — > either glycerol or -^ balsam, via carbol-xylene RECOMMENDED FOR: demonstration of nerves in wholemounts. 21.212 Wolters 1890 23632, 7 :466 REAGENTS REQUIRED: A. DS 11.113 Wolters 1890; B. F 7000.0000 Miiller 1859; C. ADS 21.1 Pal 1887 (A & B sols.) method: [15-20 m celloidin sections of Erlitzky 1877 F 4700.0000 hardened material] -^ A, 24 hrs., 45°C. — > B, rinse -+ rinse — > C, till differentiated -* water, wash -^ balsam, via usual reagents RECOMMENDED FOR: myelin sheaths. 21.212 Wolters test. 1896 Kahlden and Laurent Kahlden and Laurent 1896, 172 REAGENTS REQUIRED: A. water 100, vanadium chloride 2, ammonium acetate 6; B. water 100, acetic acid 3, hematoxylin 2; C. 0.1 hydrochloric acid in 80% alcohol DS 21.212-DS 21.213 DYE STAINS OP SPECIAL APPLICATION 409 method: [celloidin sections of F 4700.0010 Wolters (1890) fixed material] —> water — » A, 24 hrs., 50°C. -> wash -^ B, 24 hrs., 50°C. -^ C, till differentiated -* balsam, via usual reagents RECOMMENDED FOR: myelin sheaths. note: Solution A, above, is not Wolters' original mordant for which see ADS 12.1 Wolters 1891. 21.212 Wright test. 1938 Mallory Mallory 1938, 236 RE.vGENTS REQUIRED: A. 10% ferric chloride; B. 0.02% hematoxylin method: [frozen sections varnished to slide with collodion] —> A, 5 mins. — > blot — > B, poured on slide, 30 mins. — > A, till differentiated — * balsam, via usual reagents RECOMMENDED FOR: myelin sheaths. 21.213 Other Methods So deeply have the hematoxylin methods, given in the last section, become embedded in the literature, that it is almost impossible for an author today to suggest the use of any other dye in the staining of nervous elements after mordanting. Particular atten- tion should tlierefore be drawn to the formula of Becher 1921, which is permanent, easy, and simple, and which gives an admirable picture of the nervous elements in a general brain structure. It does not give so sharp a differentiation, either as a silver method, or a well-differentiated hematoxylin one, but it has at least the advantage of providing a permanent, simple, one-solution metliod which can with safety be placed in the hands of a beginner. Attention must also be drawn to the formula of Romeis 1922, which is designed to stain specifically the nervous elements in wholemounts of small inverte- brates. Tlie stain is certain, and completely specific, but it has the unfortunate dis- advantage that no method has yet been found by which it can be rendered permanent. It should, however, be placed in the hands of any class studying small invertebrates, particularly fresh-water oligochaetes, for it is fascinating to a student to watch the development of the nervous structures as they become clearer and clearer. It is also, of course, of immense value in cases in which the morphology of the nervous system plays any part in the taxonomy of an invertebrate. 21.213 Adamkiewicz test. 1896 Kahlden and Laurent Kahlden and Laurent 1896, 165 REAGENTS REQUIRED: A. 0.01% nitric acid; B. sat. aq. sol. safranin O; C. 0.01% nitric acid in abs. ale. method: [sections] -^ A, rinse -^ B, 6-12 hrs. -^ abs. ale, rinse -^ C, rinse — > clove oil, till differentiated — > balsam RECOMMENDED FOR: general neurological staining. 21.213 Alzheimer 1910 Nissl and Alzheimer 1910, 410 REAGENTS REQUIRED: A. 4% formaldehyde; B. F 1600.0010 Flemming 1882; C. sat. aq. sol. acid fuchsin; D. 0.6% picric acid; E. sat. aq. sol. light green method: [fresh tissue] -^ A, 2\ hrs. — * 5, 8 days -^ wash -^ [2-3 ti paraffin sections] -^ 95% ale. -^ C, 1 hr. at 60°C. — > wash till no more color comes away — > D, 15-20 sees. -^ rinse twice -^ E, 20-50 mins. — > balsam via usual reagents RECOMMENDED FOR: general neurological staining. note: See also DS 22.8 Alzheimer 1910. 21.213 Aronson 1890 23730, 28:577 REAGENTS REQUIRED: A. F 7000.0000 Mtlller 1859; B. 2% copper acetate; C. water 85, 95% ale. 15, sodium carbonate 0.03, gallein to sat.; D. ADS 21.1 Weigert 1885 method: [whole brains] —> A, till hardened —> [15 mm. slices]—^ B, 1 wk. — > wash — > [12-25 n sections] — > C, 24 hrs. — * rinse —* D, till differentiated -^ balsam, via usual reagents note: Erlitzky 1877 F 4700.0000 may replace A, with the consequent omission of B. 21.213 Becher 1921 see DS 13.5 Becher 1921c 410 METHODS AND FORMULAS DS 21.213 21.213 Boissezon 1941 4285a, 18:90 REAGENTS REQUIRED: A, acetone; B. sat. 70% ale. sol. Sudan black B method: [frozen sections]^ A, 15 mins. -^ 70% ale, 10 mins. — > B, 5 mins. — > 60% ale, till differentiated -^^ M 12.1 mountant RECOMMENDED FOR: myelin of peripheral nerves. 21.213 Bretschneider 1914 10899, 52:271 REAGENTS REQUIRED: .1. 1% eosin; B. DS 11.122 Delafield (1885); C. 1% phosphomolyb- dic acid; D. DS 13.41 Mallory 1901 (sol. C) method: [sections of formaldehyde-fixed material] — » water —> A, 20 mins. —* wash -^ B, 30 sees. -^ wash -^ C, 2-3 mins. — > wash -^ D, 10 sees. -^ wash — + balsam, via usual reagents RECOMMENDED FOR: insect brains. 21.213 Campbell 1929 .see DS 23.223 Campbell 1929 21.213 Chilesotti test. Bohm and Oppel cit. Schmaus Bohm and Oppel 1907, 289 REAGENTS REQUIRED: A. sodium camiinate 1, uranium nitrate 0.1, water 100, hydro- chloric acid 0.03; B. 0.5% hydrochloric acid; C. F 7000.0000 MiUler 1859 method: [sections of chrome, or dichromate, fixed material] -^ water -^ A, 1 hr. -^ B, till differentiated — » wash — > C, 1 hr. -^ wash — * balsam, via usual reagents RECOMMENDED FOR: axis cylinders. 21.213 Eberspache 1936 .see ADS 22.1 Eberspache 193G 21.213 Gross 1952 Tech. Bull., 22:234 STOCK solutions: I. 4% magenta in 95% ale; II. 6% phenol; III. 1.5% methylene blue in 95%, ale. REAGENTS REQUIRED: A. stock I 25, stock II 75, tergitol 7 0.1; 5. 3% hydrochloric acid in 95% ale.; C. water 100, stock III 30, potassium hydroxide 0.01 method: [smears] -^ A, 5-10 mins. — > rinse—* B, ^ to 1 min. with agitation — > rinse -^ C, 3-5 mins. -^ rinse -^ dry 21.213 Lison and Dagnelle 1935 4285a, 12 :85 reagents required: .1. sat. 70% ale. sol. Sudan black B method: [30 m frozen sections of formaldehyde-fixed material] — > water -^ 70% ale. -^ — > A, 1-12 hrs. -^ 50% ale, till differentiated—* water -^ M 12.1 mountant recommended for: myelin sheaths. 21.213 McManns 1946 see DS 22.21 McManns 1946 21.213 Menner 1935 23822,110:200 stock solution: water 100, oxalic acid 2, potassium antimony tartrate 0.5, azoaeid blue B 1. Boil; cool. Filter. reagents required: A. 0.005% chromic acid; B. water 96, stock .solution 4 method: [pieces of fresh spinal cord] -^ A, 2-8 days -^ rinse — > B, 24 hrs. -^ rinse ^ glycerol, via graded glycerol-water series recommended for: ganglion cells in wholemounts. 21.213 Nikiforoff /< s/. 1896 Kahlden and Laurent Kahlden and Laurent 1896, 166 REAGENTS REQUIRED: A. sat. sol. safrauiu in sat. sol. aniline; B. 0.2% gold chloride method: [.sections of chrome-fixed materials] -^ A, 24 hrs. —> 95% ale, till gray matter begins to differentiate from white -^ B, till violet —>■ thorough wash -^ 95% ale, till gray matter clearly differentiated from white — > balsam, via clove oil recommended for: general neurological staining. 21.213 Nissl test. 1896 Kahlden and Laurent Kahlden and Laurent 1896, 156 reagents required: sat. aq. sol. magenta method: [sections] — > stain, on slide, heated to steaming, few moments -* abs. ale, till color clouds cease — > oil of cloves, till differentiated -^ balsam, via xylene recommended for: nerve cells and processes. DS 21.213-DS 21.22 DYE STAINS OF SPECIAL APPLICATION 411 21.213 Obersteiner Icsl. 1896 Kahlden and Laurent Kahklon and Lament 1896, 175 REAGENTS REyiiKKi): .1. 20% ferric cliloride; U. sat. sol. dinitroresoroinol in 75% ale. method: [pieces of nerve trunks] —* A, 1 to 2 days -^ wash, till wash water iron-free — » B, several wks. — ' [paraffin sections] result: axis cylinders bright green. 21.213 O'Leary test. 1943 Cowdry Cowdry 1943, 144 REAGE.vTs REQUIRED: .1 . 3% potassium dichromatc or F 7000.0000 Miillcr 1859; B. water 100, acetic acid 0.2, abs. ale. 10, brazilin 1; C. 0.25% potassium permanganate; D. ADS 21.1 Weil 1928b FREPAR.\TiON OF b: Dissolve the dye in ale. Allow to "ripen" 1-6 months. Add other ingredients. method: [celloidiii sections of F 7000.0000 Miiller 1859 fixed ti.ssue] — » water -^ A, 12-24 hrs. — » B, till deeply stained —> water, wash — > C, till differentiated, 1-5 mins. — > D, till permanganate decolorized — * water, wash — » balsam, via usual reagents 21.213 Prince 1952 4349, 11:55 formula: sat. aq. magenta 6, 1% erythrosin 17.5, sat. aq. sol. methyl orange 47, sat. aq. sol. anilin blue 29.5 method: [sections of formaldehyde-fixed material] -* water ^ stain, 10-30 sees. — » wash — > abs. ale. — > balsam, via xylene 21.213 Romeis 1922 6628, 175:455 REAGENTS REQUIRED: A. Water 100, benzidine 1, acetic acid 0.1; B. "12 vol." hydrogen peroxide method: [small invertebrates, either living or alcohol-fixed] —> A, Yi to 1 hr. -^ B, dropped directly on animal on slide — * examine recommended for: demonstration of nervous system of small invertebrates. note: The stain is specific but fugitive. 21.213 Schrotter 1902 15058, 21 :338 reagents required: A. water 100, alizarin red S 5, oxalic acid 0.01; B. water 100, sodium carbonate 0.3 method: [sections] -^ A, 2-3 hrs. -^ B, till no more color comes away — > balsam, via usual reagents 21.213 Tress and Tress 1935 20540b, 10:105 reagents required: A. water 100, acetic acid 0.01, cresyl violet 0.5; B. chloroform 75, abs. ale. 12.5, ether 12.5; C. 0.0025 hydrochloric acid in 95% ale; D. 0.01% sodium bicarbonate in 95% ale. method: [celloidin sections of formaldehyde-fixed material] —> water —>• A, 30 mins. 50°C. -^ wash^ 70% ale., till celloidin stain-free^ B, 2-5 mins. C, till differenti- ated -♦ -D, till neutralized ^95% ale, wash -^ balsam, via n-butyl ale. 21.213 Verne 1928 4285a, 5:223 REAGENTS REQUIRED: A. DS 11.44 Schiff 1866; B. water 100, sodium metabisulfite 0.5, hydrochloric acid 5 method: [frozen sections of mercuric or platinie fixed nerves] -* 90% ale, 10 mins. — ♦ A, till deep red -* B, wash in several changes -> rinse -^ M 11.1 Apdthy 1892 recommended for: selective staining of myelin. 21.22 METHODS FOR NEUROGLIA The crystal violet and victoria blue methods for staining the supporting elements of the nervous system are less specific than are the metal stains. They still occur with some fre- quency in the literature, however, and are warmly advocated by many technicians. 21.22 Alzheimer 1910a te&i. 1938 Mallory Mallory 1938, 245 reagents required: A. F 1600.0010 Flemming 1882; B. sat. aq. sol. acid fuehsin; C. 3% picric acid in 30% ale; D. 10% light green method: [thin slices of formaldehyde-fixed material]-* A, 8 days in dark-* wash ^ [2-4 M paraffin sections] -^ B,\ hr., 60°C. -^ wash -^ C, few sees, to 2 mins. -^ wash -^ D, }>2 to 1 hr. — > wash — * 95% ale, till violet — > abs. ale, minimum possible time — > balsam, via xylene 412 METHODS AND FORMULAS DS 21.22 21.22 Alzheimer 1910b test. 1938 Mallory Mallory 1938, 245 REAGENTS REQUIRED: A. ADS 12.1 Wcigert 1891 90; B. sat. sol., {circ. 60%) phospho- molybdic acid, 40% formaldehyde 10; C. DS 13.7 Mann 1892a method: [slices of fresh tissue] — > A, 2 wks. — + wash -^ [10 n frozen sections] -^ B, 2-12 hrs. — > quick wash -^ C, 1-5 hrs. — > water, till color clouds cease -^ 95% ale, till gray matter blue and white matter pink -^^ abs. ale. minimum possible time — > bal- sam, via xylene 21.22 Alzheimer 1910c test. 1938 Mallory Mallory 1938, 245 reagents required: A. ADS 12.1 Weigert 1891; B. 0.5%, acetic acid; G. DS 11.124 Mallory 1891 3, water 97 method: [slices of fresh tissue] — > A, 2 wks. — » wash — » [10-12 ju frozen sections] -^ B, 2 mins. —* C, 2 mins. — > rinse — » balsam, via usual reagents 21.22 Anderson 1923 11431, 26:431 reagents required: A. F 5000.1010 Bouin 1897; B, ADS 12.2 Anderson 1923 stock A 65, stock B 35; C, ADS 21.1 Pal 1887 (A & B sols.); D. 1.5%, Victoria blue ; E. ADS 12.2 Lugol (1905); F. aniline 50, xylene 50 method: [frozen sections of formaldehyde-fixed material]—* A, overnight —> 70% ale, wash — > B, 10-30 mins. — + wash — * C, {A sol.), 5 mins. —* C {B sol.), 5 mins. or until next required -^ [sections attached to slide] -^ D, flooded on slide while boiling, 5 mins. — » drain — > E, dropped on slide 1 min. — > F, 15 sees. -* xylene, till clear — > F, till differentiated -^ balsam, via xylene note: Anderson later (Anderson 1929, 133) recommended equal parts stock A and stock B for mixture B above. 21.22 Anglade and Morel 1901 19219,9:137 reagents required: A. 6% mercuric chloride; B. F 1360.0010 Galescu 1908; C. sat. sol. {circ. 4%o) Victoria blue; D. ADS 12.2 Lugol (1905); E. xylene 35, aniline 65 method: [pieces of tissue]—* A, 5 hrs. -^ B, 12 hrs., 37°C. — > B, (fresh sol.), 12 hrs. 37°C. -^5, (fresh sol.) 24 hrs. 37°C. -^ wash -> [paraffin sections] -^ water -> C, poured on slide and heated to steaming -^ D, used to wash C from slide — * E, till differentiated — + balsam, via xylene 21.22 Bailey 1923 11343, 44:73 formula of dry stock: water 100, orange G 0.5, ethyl violet 1 preparation dry stock: Digest 1 day 40°C. Filter. Wash and dry filtrate. preparation of stock solution: 95% ale. 100, dry powder from above to sat. reagents required: A. ADS 12.2 Gram 1884; B. 3% potassium dichromate; C. water 60, 96% ale. 15, stock solution 25; D. clove oil 75, 95% ale. 25 method: [paraffin sections of F 3700 or F 5000 fixed material] — > A, few mins. — > 95% ale, wash —* B, 3 days — * wash -^ C, 12 hrs. — * acetone, wash — > toluene, few mins. -* clove oil, till toluene removed -^ D, till differentiated -^ balsam, via toluene 21.22 Bauer 1941 1799, 114:71 reagents required: A. 5% ferric alum; B. DS 11.124 Held (1937) 10, water 100; C. ADS 21.1 Weigert 1885; D. 5% sodium phosphate, dibasic method: [paraffin sections of F 3700.0010 Zenker 1894 fixed material] — * water -^ A, 20 mins., 40°C. -^ rinse -^ B, 12 hrs., 40°C. — > rinse -^ C, till differentiated -^ D, wash — > wash — > balsam, via usual reagents 21.22 Benda test. 1933 Cajal and de Castro Cajal and de Castro 1933, 241 reagents required: A. 10% nitric acid, 5. 2% potassium dichromate; C. 1% chromic acid; D. 4% ferric alum; E. "amber yellow" solution of alizarin red S; E. 0.1% tolui- dine blue; F. 1% acetic acid; G. creosote method: [pieces of 5 mm. side] — > 95% ale, 2 days or longer -^ A, 24 hrs. — * wash — > B, 24 hrs. -♦ C, 24 hrs. -^ wash — > [paraffin sections] —* water -^ D, 24 hrs. -+ rinse — > E, 2 hrs. — > rinse — > blot — » E, warmed to steaming, 15 mins. —* F, wash -^ abs. ale, till dehydrated — * G, till differentiated — > balsam, via xylene recommended for: macroglia. DS 21.22 DYE STAINS OF SPECIAL APPLICATION 413 21.22 Beyer 1940 591b, 4:65 REAGENTS REQUIRED : A. F 7000.0000 Miiller 1859; B. DS 11.121 Hansen 1905; C. 2% hydrochloric acid, in 70% ale; D. 0.1% ammonia; E. water 100, acetic acid 0.3, ponceau 2R 0.07, acid fuchsin 0.03; F. water 100, phosphotungstic acid 3, orange G. 2; G 0.5% anilin blue method: [5 M sections of formaldehyde-fixed material] — > A, 3-12 hrs. -^ thorough wash — > fi, 5 mins. — > wash -^ C, till differentiated — > wash — > D, till blue — + wash — » jE, 5 mins. -^ wash —* F, 5 mins. — > quick rinse — > G, 10-15 mins. -^ abs. ale, least possi- ble time —>■ balsam, via xylene RECOMMENDED FOR: astrocytes. 21.22 Brand 1941 see DS 21.22 Holzer 1921 (note) 21.22 Eisath 1911 14370, 20:3 REAGENTS REQUIRED: A. 2% formaldehyde; B. 0.2% mercuric chloride; C. water 70, DS 11.124 Mallory 1891 30; D. ADS 21.1 Eisath 1911 method: [sections by freezing technique of F 7000.1000 Eisath 1911 fixed material] —> A, till required -^ B, 30 sees. — > wash -^ C, on slide, few minutes — > wash — > D, till differentiated — * wash — > balsam, via usual reagents 21.22 Galescu 1908 6630,65:429 reagents required: A. 6% mercuric chloride; B. F 1360.0010 Galescu 1908; C. acetone 100, ADS 12.2 Lugol 1; D. water 80, 95% ale. 20, crystal violet 5, oxalic acid 0.2; E. ADS 12.2 Lugol; F. xylene 50, aniline 50 method: [pieces of brain] -^ A, 5 hrs. -> B, 12 hrs. 37°C. -^ B (fresh sol.), 12 hrs. 37°C. -^ B (fresh sol.), 24 hrs. 37°C. -^ wash -^ C, 24 hrs. ^ [3 m paraffin sections, via ace- tone] — * acetone — » D, 10 mins. -^ D, (warmed till steaming) 5 mins. — > E (poured on slide), few moments — > blot -^ F, till differentiated -^ balsam note: a detailed description of the use of this technique is given under DS 21.20 above. 21.22 Hadjioloff 1929 6630, 102:789 REAGENTS REQUIRED: A. sat. alc. sol. {circ. 0.2%) acid fuchsin; B. sat. aq. sol. {circ. 20%) light green method: [5 M paraffin sections of F 1600.0010 Flemming 1884 fixed material]—* water — » A, 5-15 mins., 60°C. -^ wash -h> B, 10-20 mins. — » wash -^ balsam, via usual reagents 21.22 Held 1909 test. 1933 Cajal and de Castro Cajal and de Castro 1933, 245 reagents required: A. 1% sodium hydroxide in 80% ale; B. 5% ferric alum; C. to 100 1% hematoxylin add an excess of molybdic acid: dilute to an "intense violet" color for use; D. DS 12.221 van Giesen 1896 method: [celloidin sections of material fixed in F 3700.1010 Held 1909] —* A, 5 mins. — > wash —> B, 1 min. — » wash — > C, 24 hrs., 50°C. — > rinse -^ B, till connective tissues decolorized — > wash — > D, 15 sees. — > abs. ale, till no more color comes away — » bal- sam, via usual reagents recommended for: macroglia. 21.22 Holzer 1921 23430, 60 :354 reagents required: A. water 30, 95% alc. 60, phosphomolybdic acid 0.2; B. chloroform 80, abs. alc. 20; C. chloroform 80, abs. alc. 20, crystal violet 5; D. 10% potassium bromide; E. choroform 60, aniline 40, hydrochloric acid 0.3 method: [sections by freezing technique of formaldehyde-fixed material]-^ A, 30-90 sees. — » float to slide, blot with paper soaked in JB — > C, on slide, 3-5 mins. — > D, wash — > blot with filter paper soaked in E -^ E, till differentiated — > balsam, via xylene note: Holzer 1921 (23632, 37:508) differs only in substituting aniline for E above. Brand 1912 (23430, 172:531) recommends counterstaining in his DS 12.221. 21.22 Jakob 1913 test. 1937 Gatenby and Painter Gatenby and Painter 1937, 542 reagents required: A. ADS 12.1 Weigert 1891, 90, 40% formaldehyde 10; B. 0.1% acid fuchsin; C. sat. aq. sol. {cite. 60%) phosphomolybdic acid; D. DS 13.41 Mallory 1901, sol. C 414 METHODS AND FORMULAS DS 21.22 method: [frozen sections of material fixed in A] — > water -^ B, 3-10 mins. — > wash — > C, 1-24 hrs. -^ rinse -* Z), 30 mins. —> 95% ale, till differentiated -* balsam, via usual reagents 21.22 Lehrmitte and Guccione 1909 20080, 19:205 REAGENTS REQUIRED: A. 7% mercuric chloride; B. F 1600.0010 Lehrmitte and Guccione 1909; C. 1.5% Victoria blue; D. ADS 12.2 Lugol; E. aniline 50, xylene 50 method: [sections of formaldehyde-fixed material by freezing technique] -^ A, 2 hrs. — > B, 2 days -^ wash —* strand section on slide -^ C, poured on slide, warmed to steam- ing -^ drain — * Z), 1 min. -^ drain -^ E, till differentiated -^ balsam, via xylene 21.22 Mallory 1938 Mallory 1938, 241 REAGENTS REQUIRED: A. sat. sol. lead chloride; B. 5% oxalic acid; C. DS 11.124 Mallory 1900 method: [2-3 mm. slices of formaldehyde-fixed material] — > ^, 6 wks. -^ wash — > [cel- loidin sections] —* B, 30-60 mins. -^ rinse — > 95% ale. -^ balsam, via usual reagents 21.22 Merzbacher 1909 11392,12:1 reagents required: A. 95% ale. 70, water 30, sodium hydroxide 2; B. sat. aq. sol. Vic- toria blue; C. ADS 12.2 Lugol; (1905); D. aniline 50, xylene 50 method: [10-15 ^ sections] —> A, 2-5 mins. — > rinse ale. ^ water -^ B, 24 hrs.—* C, rinse — > D, till differentiated — > balsam, via x\'lene 21.22 Meyer 1909 15058, 28:353 REAGENTS REQUIRED: A. 5% potassiuiu dichromatc; B. ADS 12.1 Weigert 1891 at 37°C.; C. DS 11.121 Weigert 1904; D. water 70, ADS 21.1 Weigert 1885 30 method: [pieces of brain] — > A, till white matter dark brown -^ [50 m sections by celloi- din technique] -^ water -^ B, 24 hrs., 38°C. -^ wash 70% ale. -> C, 24 hrs. -> D, till differentiated — > balsam, via usual reagents 21.22 Peers 1941 1887a, 32:446 reagents required: A. Q% mercuric chloride; B. AMS 12.2 Lugol; C. 5% sodium sul- fite; D. 0.025% potassium permanganate; E. 5% oxalic acid; F. DS 11.124 Mallory 1900 method: [sections of formaldehyde-fixed material] -* .4, 3 hrs., 57°C. — > rinse -^ B, 5 mins. — > rinse — > C, 5 mins. rinse —* D, 5 mins. —>■ rinse — > £", 5 mins. -^ thorough wash — » F, overnight — > wash — » balsam, via usual reagents 21.22 Potter 1910 23632,27:238 reagents required: A. ADS 12.1 Weigert 1891; B. DS 21.22 Weigert 1903; C. 0.5% potassium permanganate; D. ADS 21.1 Weigert 1885 method: [15 mm. slices of formaldehyde-fixed material] -^ A, 14 days -^ ale. wash — > [15 M celloidin sections] — » water -* B, 2-3 hrs. —> wash -^ C, till differentiation well started —> D, till differentiation completed —> balsam, via usual reagents recommended for: macroglia. 21.22 Rubaschkin 1904 1780,63:577 reagents required: A. sat. aq. sol. (circ. 3%) methyl violet; B. ADS 12.2 Gram 1884; C. aniline method: [celloidin sections of F 4700.0010 Rubaschkin 1904 fixed material]^ A, on slide, 6-12 hrs.—* B, on slide, ^2 to 1 min. — > C, till differentiated —> balsam, via xylene 21.22 Sokolansky 1935 23632, 51 :378 reagents required: A. 6% potassium dichromate; B. DS 11.113 Kultschitskv 1889; C. F 7000.0000 Mliller 1850; D. ADS 21.1 Pal 1887 (A and B sols.) method: [5-8 m frozen sections] -^ A, 24 hrs., 37°C. -^ rinse -^ B, 4-6 hrs., 50-60°C. -> C, 2-3 mins. -^ rinse -^ D {A sol.), few mins. -^ rinse —^D(B sol.), till decolorized — * [repeat D (A) — > D {B) cycle if insufficiently differentiated] —> balsam, via usual reagents DS 21. 22-DS 21.23 DYE STAINS OF SPECIAL APPLICATION 415 21.22 Weigert 1891 see DS 21.22 Weigert 1894 (note) 21.22 Weigert 1894 7276, 17:1184 REAGENTS REQi;iUEu: A. 5% potassiiuu (lichromate; B. ADS 12.1 Weigert 1891; C. water 46, 95% ale. 64, sat. ale. sol. lithium earbonate 4, hematoxylin 1; D. ADS 21.1 Weigert 1885 PREPARATION OF c: Add tlic dye dissolved in ale. to the water and alkali. method: [pieces of brain] — > A, till white matter dark brown (some wks.) — ♦ [embed in celloidin] -^ B, 24 hrs., 55°C. -^> [section, 15-20 n] -^ water -^ C, 4-24 hrs. -* wash, 90% ale. — > wash -^ D, till differentiated -^• balsam, via usual reagents recommended for: neuroglia. note: Weigert 1891 (727(1. 17:1184) omits 1). Weigert 1903 (Khrlich, Krause, etal. 1910, 942) substitutes for C the following: To 50 0.4% ferric chloride add 50 1 % hematoxylin in 95% ale. 21.22 Weigert 1895 lest. 1937 Gatenby and Painter Gatenby and Painter 1937, 337 This method involved the use of a proprietary reagent of secret composition and cannot therefore be further noticed. Any of the Victoria blue or crystal violet methods re- corded in this section may be substituted. 21.22 Weigert 1903 see DS 21.22 Weigert 1894 (note) 21.22 Windle, Rhines, and Rankin 1943 see DS 22.3 Windle, el al. 1943 (note) 21.23 METHODS FOR OTHER NERVOUS ELEMENTS 21.23 Koinikow test. 1933 Cajal and de Castro Cajal and de Castro 1933, 320 reagents required: .4. F 7000.1000 Orth 1896; B. F 7000.0000 Miiller 1859; C. F 1700.- 0000 Marchi 1886; D. sat. aq. sol. phosphomolybdic acid; E. DS 13.7 Mann 1894a {A sol.); F. 0.1% sodium hydroxide in abs. ale; G. 0.01% acetic acid in abs. ale. method: [piece of nerve] —> A, 24 hrs. ^ B, till required—* C, 8-10 days — > [teased preparations or celloidin sections] —* D, 1 hr. -* wash — » E, 24 hrs. — > rinse -* abs. ale, till dehydrated -^ F, till color changes from blue to red -^ wash, abs. ale. — > G, till color changes from red to blue -^ wash, abs. ale. -^ xylene — * white petroleum or "neutral mountant" recommended for: Schwann cells. 21.23 Fieandt test. 1933 Cajal and de Castro Cajal and de Castro 1933, 265 reagents required: A. 10% iodine in 95% ale; B. 0.25% sodium thiosulfate; C. DS 11.124 Mallory 1900; D. 10% ferric chloride in abs. ale. method: [5 M sections of 2 mm. slices fixed in F 3000.0012 Heidenhain 1909] — > water -^ A, few mins. — * 95% ale. till no more color comes away -^ B, 1 hr. —> wash — > C, 10-14 hrs. — > blot -^ D, till differentiated, some hrs. — » blot —> quick rinse — > abs. ale., 24 hrs. — > balsam, via oil of thyme recommended for: gliosomes. 21.23 Nageotte test. 1933 Cajal and de Castro Cajal and de Castro 1933, 325 reagents required: A. 30% ale; B. 0.1% nitric acid; C. DS 11.122 Cajal and de Castro 1933; D. DS 12.211 Cajal 1895 method: [fresh piece of nerve] —* A, 1 day — > B, till fibers dissociated -^ [tease on slide] —y C, till deep blue -^ wash — » D, till differentiated —^ balsam, via usual reagents recommended for: syncytium of Schwann cells. 21.23 Nageotte test. 1933 Cajal and de Castro Cajal and de Castro 1933, 319 reagents required: A. 2.5% ferric alum; B. 1% hematoxylin method: [pieces of nerve, fixed in F 3900.1000 Dominici 1905 and teased on a slide] —y A, some hrs. — > B, some hrs. —> C, till differentiated — > wash —* balsam, via usual reagents recommended for: selective staining of Schwann cells. 21.23 Reich see DS 22.8 Reich (1933) 416 methods and formulas ds 21.3 21.3 Special Stains for Blood Nowhere is the purpose of the present writer in his classification of staining tech- niques more likely to be misunderstood than in tliis division. At least a hundred methods for staining blood liave been advocated, and it is today conventional to utilize only the methods given in Chapter 20 (DS 13.1, 13.2, and to a less extent 13.3). Those techniques are, however, of far wider application, for they may be used for sections, for staining parasites in blood, and for many other purposes. Moreover, blood films themselves may be excellently stained by almost any triple-staining method, though it usually is a surprise to the hematologist who has seen nothing but a methylene blue-eosin prepara- tion to observe the magnificent differentiation which can be secured by many of the DS 12.3 or DS 13.4 techniques. Under these circumstances the writer has included in the present section only those methods of staining blood which are useless for any other purpose. 21.3 Brice 1930 see DS 22.8 Brice 1930 21.3 Bruner and Edwards 1940 see DS 23.219 Bruner and Edwards 1940 21.3 Bacsich 1936 11025, 70:267 REAGENTS REQUIRED: A. water 20, 95% ale. 80, phenol 2.5, Sudan III 0.5; B. DS 11.121 Weigert 1903; C. 0.5% hydrochloric acid in 40% ale. PREPARATION OF A: Boil the dye 5 minutes in 100 95% ale. Add phenol and filter hot. Cool to 4°C., 24 hours. Filter. Add water drop by drop till ale. eoneentration reduced to 80%. method: [air-dried smears] — > A, on slide, 5 mins., 56°C. -^ thorough wash — > B, 30-00 sees. -^ rinse — > C, till differentiated -^ wash — * dry -* M 12.1 Zwemer 1933 21.3 Baillif and Kimbrough 1947 11284, 32:155 REAGENTS REQUIRED: A. sat. sol. Sudan black B in 70% ale.; B. DS 13.11 May and Grunwald 1902; C. DS 13.13 Giemsa 1902 2, water 98 method: [ale. -formaldehyde-fixed smears]—* A, 30-60 mins.-* rinse—* B, on slide, 3 mins. — * add water, 1 min. — * drain -^ C, on slide, 15 mins. -^ wash — > dry 21.3 Buzard 1930 4285a, 7 :264 REAGENTS REQUIRED: A. 1% acid fuchsin; B. 0.8% bromine water; C. 1% hydrochloric acid in 95% ale. methods: [fixed smears] — > A, 30 sees. — > B, 4-5 mins. -^ wash -^ C, till mauve -^ bal- sam, via usual reagents 21.3 Campbell and Alexander test. 1938 Mallory Mallory 1938, 257 REAGENTS REQUIRED: A. acctic acid 0.5, benzidine 0.1, sodium nitroferrieyanide 0.1; B. water 100, 30% hydrogen peroxide 0.1 PREPARATION OF A: Dissolvc benzidine in acetic acid and dilute to 20. Dissolve nitro- ferrieyanide in 20 water and add to benzidine. Dilute mixture to 100. Filter. method: [200 to 300 m frozen sections of formaldehyde-fixed material]^ A, 30 mins. 37°C. with frequent shaking — * rinse -^ B, 30 mins., 37°C. with frequent shaking — > balsam, via usual reagents recommended for: capillaries in brain. result: capillaries black against colorless background. 21.3 Crossmon 1940 20540b, 15:155 REAGENTS REQUIRED: A. water 100, acetic acid 1, chromotrope 2R 0.25; B. 5% phospho- tungstic acid in 95% ale.; C. water 100, aeetie acid 1, methyl blue 0.5; D. 2% acetic acid method: [sections from formaldehyde-fixed material] -^ water — » A, 1-5 mins. -^ rinse -^ B, till erythrocytes alone stained — » C, 2-5 mins. -^ D, till differentiated — > bal- sam, via usual reagents recommended for: differential staining of erythrocytes in sections. DS 21.3 DYE STAINS OF SPECIAL APPLICATION 417 21.3 Cunningham 1920 1845, 26:405 REAGENTS REQUIRED: A. 0.3% brilliant cresyl blue in 95% ale; B. DS 13.12 Wright 1910 method: [dip coverslip in A and dry] — > make smear on clean cover -^ press to A — » separate — ♦ dry — > B, full technique —> dry recommended for: reticulocytes. 21.3 Dekhuyzen 1901 766, 19:536 formula: water 100, osmic acid 1.8, acetic acid 0.6, methylene blue 0.1 METHOD : blood drop on slide -^ stain, mixed with drop — > smear — > dry 21.3 Doherty, Suk and Alexander 1938 1879, 40:158 REAGENTS REQUIRED! .1. Water 50, abs. ale. 50, benzidine 0.5, sodium nitroferricyanide 0.1; B. water 50, abs. ale. 50, acetic acid 2, hydrogen peroxide 0.5, sodium nitroferri- cyanide 0.1 PREPARATION OF REAGENT A: Dissolvc the benzidine in the abs. ale. Dissolve the nitro- ferricyanide in 10 water. Mix and dilute to 100. method; [200-300 m sections of formaldehyde fixed material]—* wash — > A, with fre- quent agitation 10 mins. — > rinse — > 5, with frequent agitation, till capillary net stained — * wash -^ balsam, via usual reagents RECOMMENDED FOR: differential staining of blood in capillaries in sections. 21.3 Dubuscq 1899 1915, 6:481 formula: water 250, copper chloride 0.6, copper acetate 0.6, osmic acid 0.6, acetic acid 0.6, thionin 0.6 method: Mix equal parts stain and arthropod blood. Leave 5 mins. Make smear. 21.3 Ellermaner 1919 23633, 36:56 REAGENTS REQUIRED: A. Water 100, eosin B 1, neutralized formaldehyde 5; B. DS 13.13 Maximow 1924 method: [5 n sections of F 3700.1000 Maximow 1909 material] -* water — > drain — » A, 15 mins. — > wash, 2 mins., 45°C. — > B, 30 mins. -^ wash -^ blot — > abs. ale, till sec- tion blue -^ balsam, via xylene 21.3 Epstein test. 1946 Roskin Roskin 1946, 243 REAGENTS REQUIRED: A. water 100, citric acid 1, toluidine blue 1; B. sat. aq. sol. {circ. 1%) picric acid method: [dried smears] — ^ A, 20-30 mins. -^ rinse -^ B, 1-2 sees. — > wash -^ dry 21.3 Fiessinger and Laur 1930 test. 1942 Langeron Langeron 1942, 1023 formula: sat. ale. sol. cresyl blue 75, sat. ale. sol. (circ. 2.5%) neutral red 25 METHOD : Mix equal parts blood and stain — > smear — ♦ dry note: Recommended for reticulocytes. 21.3 Flinn 1939 see DS 23.219 Flinn 1939 21.3 Freifeld 1931 test. 1950 Jones Jones 1950, 225 formula: water 100, DS 11.43 Ziehl 1882 1, 1% methylene blue 0.7 method: [methanol-fixed smears] —* stain, 1 hr. -^ dry RECOMMENDED FOR: toxic neutrophiles. 21.3 Graham 1916 11343,35:231 REAGENTS REQUIRED: A. 95% alc. 90, 40; formaldehyde 10; B. water 60, 95% ale. 40, a-naphthol 1, hydrogen peroxide 0.2; C. water 60, 95% alc. 40, pyronin 0.1, aniline 4; D. 0.5% methylene blue method: [air-dried smears]-^ A, 1-2 mins. — > wash — > B, 4-5 mins. — > wash — > C, 2 mins. — > wash — * D, 1-2 to 1 min. — » wash — > blot — > dry recommended for: demonstration of oxidase granules. 21.3 Graham 1918 11313, 39:15 REAGENTS REQUIRED: A. 95% all;. 90, 40*;;; fornialdohydc 10; B. water 00, 95 7o alc. 40, hydrogen peroxide 0.2, benzidine q.s. to sat. immediately before use; C. DS 11.44 Loffler 1890 method: [air dried smears] -^ A, 1-2 mins. -♦ wash — ♦ B, 5-10 mins. — » wash — > C, 3 sees. — > wash — > blot -^ dry RECOMMENDED FOR: demonstration of oxidase granules. 418 METHODS AND FORMULAS DS 21.3 21.3 Groat 1936 see DS 13.13 Groat 1936 21.3 Grosso 1914 16059, 6:235 PREPARATION OF DRY STOCK: Add a sat. sol. picric acid to a sat. sol. methylene blue until no further ppt. forms. Filter. Wash and dry ppt. WORKING solution: methanol 100, dry stock 0.5 method: [dry smear] —* 10 drops, placed on slide, 3-4 mins. -^ 10 drops water, added to stain on slide, 5 mins. —>■ wash — » dry 21.3 Hayem 1889 23632, 6 :335 formula: water 100, mercuric chloride 0.25, sodium sulfate 2.5, sodium chloride 0,5. eosin 0.05 method: Mix 1 blood with 100 stain. Make smear, 21.3 Kahlden and Laurent 1896 Kahlden and Laurent 1896, 134 formula: sat. aq. sol. orange G 25, sat. 20% ale. sol. acid fuchsin 25, 95% ale. 15, sat. aq. sol. methyl green 25 preparation: Mix in order given. Leave 24 hours. Decant supernatant stain (do not filter). method: [dry smear] -^ stain 5-10 mins. — > wash —* dry — > balsam 21.3 Kardos 1911 8545, 12 :39 preparation of dry stain: Mix equal parts 2% orange G and sat. sol. methyl blue. Filter. Wash and dry ppt. reagents required: A. DS 13.11 May-Griinwald 1902; B. water 95, DS 13.13 Giemsa 1902 3, sat. sol. above ppt. in methanol 2 method: [smear] —* A, 3 mins. on slide —* add water to A on slide, 1 min. -^ drain -^ B, on slide, 15 mins. — > rinse -^ dry 21.3 Kuhn 1933 see DS 13.13 Kuhn 1933 21.3 Liebmann 1942 20540b, 17:31 REAGENTS REQUIRED: A. DS 13.12 Wright 1910; B. 0.01% acetic acid method: [smears fixed in F 1700.1000 Maximow 1909] -^ A, 24 hrs. —> B, 1-1,' 2 mins. -> balsam, via acetone and xylene recommended for: invertebrate blood. 21.3 Liebmann 1945 20540b, 20 :83 REAGENTS REQUIRED: A. water 100 azur II 0.005, eosin Y 0.0075 method: [fix smears 10 mins. in formaldehyde vapor: flame 5 times] — > A, 6 hrs. -^95% ale, 1-2 mins. — ^ abs. ale, 30-60 sees. -^ balsam, via xylene RECOMMENDED FOR: amphibian blood. 21.3 Lightwood, Hawksley and Bailey 1935 16916, 28:405 formula: abs. ale. 100, nevitral red 0.04, Janus green B 0.3 method: Dip clean slides in stain. Let dry. Make smear on .slide. 21.3 McCullough and Dick 1942 see DS 23.219 McCullough and Dick 1942 21.3 Michaelis 1899 see DS 13.11 Michaelis 1899 21.3 Moore 1882 test. 1884 Cole Cole 1884, vol. 1, 12 REAGENTS REQUIRED: A. water 50, 95% ale. 50, eosin W 1; B. 0.3% methyl green method: [dried smear of amphibian blood] —> A, on slide, 2 mins. -^ rinse -^ B, on slide, 2 mins. — > wash — ♦ dry —* balsam 21.3 Okajima 1917 763, 11 :295 REAGENTS REQUIRED: A. 10% phosphomolybdic acid; B. water 100, alizarin red S 6, phosphomolybdic acid 2.5 method: [sections of material (not picric-fixed)]—* A, 1 niin. -^ water, quick rinse—* stain, 1-24 hrs. — > Ijalsam, via usual reagents result: erythrocytes, orange yellow; other tissvies, colorless. recommended for: specific staining of erythrocytes in sections. DS21.3 DYE STAINS OF SPECIAL APPLICATION 419 21.3 OUver 1934 see DS 22.8 Oliver 1934 21.3 Osgood and Wilhelm 1934 1128-4, 19:1129 REAGENTS REQUIRED: A. 1% brilliant cresyl blue in 0.85% sodium chloride method: Mix 5 ml. each A and oxalated blood. Prepare and dry smear. RECOMMENDED FOR: reticulocytes. 21.3 Pappenheim 1917 8545, 22:15 REAGENTS REQUIRED: A. US 13.11 May and Grlinwald 1902; B. DS 13.13 Slider and Downey (1929) method: [dry smears] —> A,3 mins. on slide — » add equal vol. water to A on slide, 1 min. —y drain — » B, on slide, 15 to 30 mins. — » water till differentiated, al:»out 1 min. — > dry 21.3 Pickworth 1934 11025,69:02 REAGENTS REQUIRED: A. watcr 75, acetic acid 0.5, benzidine 0.125, sodium nitroprusside 0.1; B. water 100, hydrogen peroxide (3%) 0.5 method: [250 m sections of formaldehyde-fixed brain, sectioned by E 11.1 Pickworth 1934] — ♦ thorough wash — > A, 1 hr., 37°C. with continuous shaking — > wash — > balsam, via usual reagents recommended for: blood vessels in thick sections of brain. 21.3 Price-Jones 1933 test. 1937 Gatenby and Painter Gatenby and Painter 1937, 397 formula: water 90, sodium chloride 0.7, 40% formaldehyde 10, gentian violet 0.01 use: Use as blood diluent. Make smear. 21.3 Pryce 1939 11284,49:594 reagents required: A. sat. sol. brilliant cresyl blue in abs. ale; B. DS 13.12 Leishman 1901 method: [dip slides in A and air dry. Make smear on coated slide and prevent evapora- tion by laying another slide not quite in contact with smear. Leave 5 mins. Remove cover. Dry] — ♦ B, on slide, 5 mins. — > dilute stain on slide, leave 5 mins. — > wash till smear changes from blue to red -^ dry recommended for: demonstration of reticulocytes. 21.3 Pugsley 1940 test. 1944 Randall 20540b, 19:150 reagents required: A. water 100, potassium oxalate 0.2; brilliant cresyl blue 1; B. anj^ DS 13.12 mixture method: [mix equal parts A and blood, leave 1 min.] — » make smear — > B, till counter- stained recommended for: reticulocytes. 21.3 Raadt 1912 see DS 13.12 Raadt 1912 21.3 Rossi 1889 23632, 6 :475 formula: water 100, osmic acid 0.5, methyl green 5 method: [blood, drop on slide] -^ stain, mixed with drop -^ make smear —y dry — > glyc- erol, via graded glycerol water mixtures 21.3 Sabin 1923 10910,34:277 formula: abs. ale. 100, neutral red 0.1, Janus green 0.05 method: Dip clean slide in stain. Let dry. Make smear on slide. 21.3 Sato 1928 11284, 13:1058 reagents required: A. 0.5% copper sulfate; B. benzidine 0.1, water 100, hydrogen peroxide 0.2; C. 1% safranin method: [dry smears on coverslips] -^ A, floated on solution, 1 min. — » rinse -^ B, 2 mins. — > wash — > C, 20 sees. -^ wash —* blot -^ dry recommended for: demonstration of peroxidase granules. result: peroxidase granules, blue; basophilic granules and nuclei, red. 21.3 Saye 1943 Tech. Bull., 4:12 reagents required: A. 0.25%, eosin Y in 95% ale, 0.05% thionine in 80% ale. method: [air-dried smear] — » A, 1 min. — > drain briefly —yB,l min. — » wash — + dry 420 METHODS AND FORMULAS DS 21.3 21.3 Schmorl 1928 see DS 22.8 Sclimorl 1928 21.3 Sheehan 1939 11431,49:58 REAGENTS REQUIRED: A. sat. sol. Sudan black B; B. sat. sol. ethyl eosin in 70% ale; C. sat. sol. methylene blue method: [methanol-fixed smear] -^ A, 30 sees. -* wash — » 70% ale, Imin.-^ B, 30 sees. — * wash — >• C, 3 mins. -^ rinse -^ blot — > dry recommended for: lipoid granules in leukocytes. 21.3 Sheehan and Storey 1947 11431, 59:336 stock solutions: I. 0.3%, Sudan black B in abs. ale; II. water 100, abs. ale. 30, phenol 16, sodium phosphate, dibasic (cryst.) 0.3 preparation of b: Add the phenol dissolved in ale. to the phosphate dissolved in water. reagents required: A. stock I 60, stock II 40; B. DS 13.13 Giemsa 1902 1, water 100; C. 0.2% potassium dihydrogen phosphate method: [smear fixed in formaldehyde vapor] -^ A, 10-60 mins. -^ 70% ale., rinse— » wash -^ B, 5 mins. — > C, till differentiated -^ wash dry recommended for: fat granules in leukocytes. 21.3 Simpson 1922 11343, 40:77 formula: 95% ale. 100, neutral red 1, Janus green 0.5 method: Dip clean slide in stain. Leave dry. Make smear on slide. 21.3 Slominski and Cunge test. 1948 Romeis Romeis 1948, 470 preparation of stain: To 100 1% benzidine add 10 of 10% hydrogen peroxide (3%). method: [120 m frozen sections of material fixed in F 8000.1000 Slominski and Cunge (1948)] — > water -^ A, till sufficiently stained — > balsam, via usual reagents recommended for: differential staining of capillaries in sections. 21.3 Steil 1936 20540b, 11 :99 reagents required: A. DS 13.12 Wright 1910 method: [dry smears] —* A, on slide, 1-2 mins. — >■ equal amount water added to stain, 3 mins. — > drain — » flood with water till smear pink -^ drain -^ methanol, 1-2 mins. -^ water, till smear pink -^ abs. ale. —> balsam, via clove oil 21.3 Strumia 1936 11284,21:930 preparation of stock solutions: I. DS 13.13 Giemsa 1902 (dry powder) 0.2, glycerol 12, methanol acetone 44; II. DS 13.11 May-Grtinwald 1902 (dry powder) 0.04, methanol 50, acetone 50. REAGENTS REQUIRED: A. stock I 65, stock II 35; B. 0.2% sodium carbonate method: [air-dried smear] — * 1 ml. A, on slide, 2 mins. -^ add 1 ml. B, leave 3 mins. -^ wash — > dry 21.3 Thompson 1945 1887a, 38:49 REAGENTS REQUIRED: A. 4% forric alum ; B. sat. sol. picric acid 87, 1% acid fuchsin 13 method: [sections or smears, with nuclei prior stained in DS 11.122 Mallory 1938]^ wash-^ A, 1 min. —* rinse -^ B, 15 mins. — > 95% ale., till differentiated -^ balsam, via usual reagents result: structures or pieces containing hemoglobin, green; other structures, yellow, red, brown, and gray. RECOMMENDED FOR: differentiation of erythrocytes, and pieces of erythrocytes, in smears and sections. 21.3 Ugruimow 1928 23632, 45:191 REAGENTS REQUIRED: 0.1% azur II in phosphate buffer pH 6.3 15, 0.1% eosin BA in phosphate buffer pH 6.3 16, water 100 method: [heat-fixed smear] —> stain, 10-12 hrs. ^ acetone, till differentiated —» acid balsam 21.3 Westphal test. 1896 Kahlden and Laurent Kahlden and Laurent 1896, 134 formula: a DS 11.21 Grenacher 1879 30, sat. ale. sol. crystal violet 30, glycerol 30, phenol 25, acetic acid 5 method: [dry smear] -^ stain, 20-30 mins. — » wash — > balsam, via usual reagents DS 21.3-DS 21.411 DYE STAINS OF SPECIAL APPLICATION 421 21.3 Willebrand 1901 see DS 13.11 Willcbrand 1901 21.3 Ziegler 1945 test. 1945 Riley 20504b, 21 :37 REAGENTS REQUiREu: A. Water 50, abs. ale. 50, benzidine 0.5, sodium nitroprusside 0.1; B. water 47, abs. ale. 50, acetic acid 3, hydrogen peroxide (30%) 0.5, sodium nitro- prusside 0.1; C. 2% acetic acid in 70% ale; D. 2% acetic acid in 90% ale. method: [formaldehyde-ti.xed cornea] -^ thorough wash— ♦A, 20 mins. -^ brief wash — > B, 15 mins. -^ B, fresh solution, 45 mins., 37°C. wash — > C, 10 mins. — > D, 10 mins. — > abs. ale. — > M 23.1 mountant RECOMMENDED FOR: demonstration of capillaries in wholemounts of cornea. 21.4 Special Stains for Other Tissues 21.41 designed to differentiate types of tissues not covered under 21.1, 21.2 OR 21.3 21411 Plant Tissues 21.411 Buchholz 1931 20540b, 6:13 REAGENTS REQUIRED: A. 1% acid fuchsin 80, 1%, Hght green in 90% ale. 20; B. 80% lactic acid method: [cortex dissected from pistil killed in formalin-alcohol] —* A, Q hrs. —* rinse -* B, till cleared and differentiated — > [seal in B under coverslip] RECOMMENDED FOR: Staining wholemounts to show pollen tubes. 21.411 Chandler 1931 20540b, 6:25 REAGENTS REQUIRED: A. DS 11.23 Schneider 1880; B. sat. aq. sol. {circ. 0.3%) magenta method: [cortex dissected from pistil killed in formalin-alcohol]—* A, on slide, few sees. — > B, dropped on A on slide, 1 drop, few sees. -^ bolt -^ dehydrate by drop method —>■ balsam, via xylene RECOMMENDED FOR: staining wholemounts to show pollen tubes. 21.411 Gardiner 1888 Arb. hot. inst. Wiirz, 3:52 REAGENTS REQUIRED: A. 50% sulfuric acid; B. water 50, 95% ale. 50, picric acid to sat., anilin blue 1 method: [sections] —>• water —» a, 2-10 sees. -^ wash —> B, 10 mins. -^ wash, till no more color comes away —y glycerol recommended for: demonstration of protoplasmic connections between plant cells. 21.411 Johansen 1939 20540b, 14:125 REAGENTS required: A. DS 11.42 Johansen 1940; B. 1% methyl violet 2B; C. 95% ale. 30, methyl cellosolve 30, tert. butyl ale. 30; D. sat. sol. fast green FCF in equal parts clove oil and methyl cellosolve 15, 95% ale. 40, tert. butyl ale. 40, acetic acid 1.5; E. DS 12.16 Johansen 1940; F. clove oil 30, abs. ale. 30, methyl cellosolve 30 method: [sections] — > 70% ale. -^ A, 1-2 days —>■ rinse -^ B, 10-15 mins. — > rinse -^ C, 5 sees. — » D, 10 mins. —* C, 5 sees. -^ E, 5 mins. — > F, rinse — > balsam, via usual reagents recommended for: plant histology and cytology. 21.411 Johansen 1940 Johansen 1940, 93 REAGENTS REQUIRED: A. 1% acid fuchsin in 70% ale.; B. DS 12.16 Johansen 1940 fast green; C. DS 13.5 Johansen 1940 (solution H) method: [sections] — * 95% ale. — * A, 20 mins. -^ water, rinse — » 95% ale, rinse -^ B, on slide, few sees. -^ C, till clear -^ balsam, via xylene recommended for: differentiation of reproductive structures in marine Phaeophyta. 21.411 Meyer test. 1915 Chamberlain Chamberlain 1915, 130 REAGENTS REQUIRED: A. ADS 12.2 Gram 1884; B. water 75, sulfuric acid 25, iodine to sat. ; C. 3 % methyl violet method: [sections] —» water —» yl, 3-5 mins. — ♦ 4, under coverslip -^ 5, run under coverslip -^ C, run under coverslip, 3 mins. — >• wash — > glycerol recommended for: demonstration of protoplasmic connections between plant cells. 422 METHODS AND FORMULAS DS 21.411-DS 21,412 21.411 Nebell931 20540b, 6:27 REAGENTS REQUIRED: A. water 100, martius yellow 0.05, resorcin blue 0.05, ammonium hydroxide q.s. to give pH 8 method: [sections of styles, or crushed styles, or cortices dissected from styles fixed in F 0000.0010 Carnoy 1887] —+ wash, till acid-free-^ A, 2-5 mins. -^ balsam, via graded ales, containing resorcin blue at pH 8 RECOMMENDED FOR: staining pollen tubes. 21.411 Strasburger test. 1915 Chamberlain Chamberlain 1915, 130 REAGENTS REQUIRED: A. 1% osmic acid; B. ADS 2.2 Gram 1884 60, water 30; C. 25% sulfuric acid; D. 25% sulfuric acid 100, iodine to sat., 3% methyl violet 0.1 method: [sections of fresh material] —> A, 5 mins. -^ wash -^ B, 20-30 mins. -^ C, 24 hrs. —^D,5 mins. — * glycerol RECOMMENDED FOR: demonstration of protoplasmic connections between plant cells. 21.411 Watkin 1925 11211, 15:340 REAGENTS REQUIRED: A. Water 25, glycerol 25, lactic acid 25, phenol 25, methyl blue 0.1 method: Fix, stain, and mount style in A. RECOMMENDED FOR: demonstration of pollen tubes in flowers of Gramineae. 21412 Animal Tissues 21.412 Badertscher 1940 20540b, 15 :29 REAGENTS REQUIRED: A. DS 22.4 Hcrxheimer 1901 method: [thick, freehand sections of formaldehyde-fixed skin] —^70% ale, till formal- dehyde-free -^ A, 12-24 hrs. -* 70% ale. till glands differentiated -> glycerol -> M12 mountant (Chapter 26) recommended for: differential staining of sebaceous glands in wholemounts. 21.412 Behrens 1898 23328, 3 :76 formula: A. ADS 12.2 Merkel (1898); B.\% potassium permanganate; C. 1% sodium sulfite; D. 0.5% formic acid; E. 1% methyl violet method: [sections of formaldehyde-fixed material] -^ A, 4 or 5 days — > B, 10 mins. — > wash -^ C, 5 mins. -^ D, few sees. -^ [repeat C, D cycle till bleached] -^ wash — > E, 2 mins. — > 95% ale, till no more color comes away -^ balsam, via xylene recommended for: bile capillaries. 21.412 Bloom 1925 see DS 21.412 Eppinger 1902 (note) 21.412 Buzz! 1898 14352,8:149 reagents required: A. sat. sol. picric acid; B. \% nigrosin method: [sections of formaldehyde-fixed material] -^ water -^ A, b mins. — ♦ rinse — * B, 1 min. — » rinse — > 95% ale, minimum possible time -^ balsam, via origanum oil recommended for: differentiation of keratin from eleidin in skin sections. result: eleidin, blue-black; keratin, yellow. 21.412 Clara 1933 23639b, 17:698 reagents required: A. ADS 11.1 Clara 1903; B. DS 11.113 Kultschitzky 1889; C. ADS 21.1 Weigert 1885 method: [celloidin sections of formaldehyde-fixed material]—* water—* A, 1 day, 40- 50°C. — > rinse -^ iJ, 1 day, 37°C. -^ wash — > C, till differentiated — > wash — > balsam, via usual reagents recommended for: differential staining of bile capillaries. 21.412 Clara 1934 23507a, 35:1 reagents required: A. water 100, potassium dichromate 3.2, chrome alum 1.6, chromic acid 0.125, ammonium molybdate 0.25; B. DS 11.113 Kultschitzky 1889 method: [sections of alc.-formaldehyde-fixed material]-^ A, 24 hrs., 50°C. — + rinse —> B, till sufficiently stained -^ wash -^ balsam, via usual reagents recommended for: demonstration of bile capillaries. DS 21.412 DYE STAINS OF SPECIAL APPLICATION 423 21.412 Dahlgren 1929 McClung 1929, 306 REAGENTS REQUIRED: A. sat. sol. picric acid 100, nigrosin 2 method: [sections fixed, or mordanted, in picric acid] -^^ waters A, 5 mins. — > 95% ale, till color clouds cease — > balsam, via usual reagents RECOMMENDED FOR: to distinguish muscle from other connective tissues. result: muscle, yellow; other connective tissues, brown. 21.412 Dublin 1944 see MS 32.1 Dublin 1944 21.412 de Galantha test. 1934 Wilbur 1887a, 18:157 reagents required: A. F 3700.0010 Zenker 1894; B. ADS 12.2 Lugol; C. 2% sodium thiosulfate; D. ADS 12.1 Weigert 1896; E. ADS 12.1 Wcigert 1891; /'\ 1% azocarmine in 1% acetic acid; G. 1% aniline in 95% ale; H. 1% hydrochloric acid in 95% ale; /. 5% phosphotungstic acid; /. water 100, acetic acid 8, orange G 2, anilin blue 0.5 method: [paraffin sections of formaldehyde-fixed material] — * water — > A, 1 hr. —> wash -^ B, 5 mins. — > wash — ^ C, till bleached —> wash — > Z), 4 hrs. —y wash —* E, 2 hrs. —>■ running water, 1 hr. — » F, 40 mins. —y G, till nuclei differentiated — » H, wash —^ rinse —> I, 2 hrs. -^ rinse — > /, 5 mins. — > rinse -^ abs. ale, till differentiated — > balsam, via xylene RECOMMENDED FOR: histological differentiation of kidney. 21.412 Eppinger 1902 2526,31:230 RE.'i.GENTs required: A. 4% formaldehyde; B. ADS 12.1 Weigert 1891; C. 1% hema- toxylin; D. 5% cupric acetate; E. ADS 21.1 Weigert 1885; F. 1.5% lithium carbonate method: [small pieces] — + A, 5-10 days -^ B, 10 days -^ wash — > celloidin sections — > C, 24 hrs. -^ rinse — > D, 5 mins. -^ wash — > E, till sections differentiated -^ wash -^ F, till celloidin decolorized — » balsam, via usual reagents recommended for: demonstration of bile capillaries in liver. note: Bloom 1925 (590, 36 :455) uses B 1 week at 37.5°C. and C for only a few moments. 21.412 Forsgren 1928 23639b, 6:647 reagents required: A. 3% barium chloride; B. 4% formaldehyde; C. 0.1% acid fuchsin; D. 1% phosphomolybdic acid; E. DS 13.41 Mallory 1900 (C sol.) method: [3-4 mm. slices of liver] — > A, 6-12 hrs. -^ B, 12-18 hrs. -^^ 95% ale, quick wash — > [5 M paraffin sections] — > water -^ C, 1-3 mins. — > wash — * D, y^-l min. — > rinse -^ E, 3-5 mins. -^ wash — > balsam, via usual reagents recommended for: differential staining of bile duct. 21.412 Hansen 1898 see DS 12.221 Hansen 1898 21.412 Heidenhain 1903 23632,20:179 REAGENTS REQUIRED: .4. 1% thiazin red; B. \% thionin method: [sections of trichloroacetic-fixed material]-^ water —> A, 1-6 hrs., till deeply stained —> rinse— > B, 1-12 hrs. ^^ 95% ale till differentiated—* balsam, via usual reagents recommended for: contraction bands on striped muscle. 21.412 Holmer 1927 test. 1948 Romeis Romeis 1948, 495 reagents required: A. 30% ferric chloride; B. 0.5% hematoxjdin; C. 0.1% ferric chloride; D. sat. sol. lithium carbonate method: [sections of formaldehyde-fixed material] —^ water ^ A, 3-5 mins. —> short wash — > B, 5-10 mins. -^ rinse -^ C, till differentiated — > wash -^ D, till blue -^ balsam, via usual reagents recommended for: differential staining of bile capillaries. 21.412 Kockel 1899 23681, 10:749 reagents required: A. \% chromic acid; B. DS 21.212 Weigert 1885 (sol. C); C. 10% ammonium alum; D. ADS 21.2 Weigert 1885 50, water 50 method: [paraffin sections] —> water -+ A, 5-10 mins. — > quick rinse — > B, 15-20 mins. — > wash — > C, till dark blue — > wash -^ D, till differentiated — > wash — > C, till back- ground decolorized -^ countcrstain with any DS 11.21 formula if required -^ balsam, via usual reagents recommended for: fibrin in sections. 424 METHODS AND FORMULAS DS 21.412 21.412 Kramer 1948 19938, 108:141 REAGENTS REQUIRED: A. 0.5% eosin in 95% ale; B. methyl salicylate method: [Bouin 1897 F 5000.1010 fixed material] -> 50% ale, 10 mins. -^ 70% ale, 1 hr. -^ 95%, ale, 10 mins. -^ A, 6-8 hrs. or until specimen uniform pink color -> 95% ale, 30 mins. -^ B, drops added to ale, at such intervals as wUl prevent collapse of specimen — » B RECOMMENDED FOR: differential staining of muscles in insect wholemounts. result: muscles pink; other tissues greenish. NOTE : Times cited are for 4-day-old housefly larvae. 21.412 Kromayer 1892 1780,39:141 reagents required: A. sat. sol. methyl violet 6 B. 50, sat. sol. aniline 50; B. ADS 11.1 Lugol (1905) 30, water 60; C. xylene 60, aniline 30 method: [5 M sections] -^ water -^ A, 10-15 mins. -+ thorough wash -^ B, 1-30 sees. — > drain -^ blot -^ C, 50°C., till differentiated — > balsam, via xylene recommended for: epithelial fibers in skin. 21.412 Kultschitzky 1875 1780, 46:675 reagents required: A. water 100, acetic acid 3, acid fuchsin 0.5; B.2% acetic acid; C. water 100, acetic acid 3, anilin blue WS 0.5 method: [sections of F 7000.0000 Miiller 1859 material]-^ A, 3-5 mins. -^ B, till no more color comes away -^ C, till differentiated -^ balsam, via usual reagents recommended for: reticulum fibers. 21.412 Lillie 1948 Lillie 1948, 189 REAGENTS REQUIRED: A. water 100, nitric acid 0.5, sodium periodate 1; B. DS 11.44 Schiff 1866; C. 0.5% sodium bisulfite; D. DS 11.121 Weigert 1903; E. 1% orange G method: [sections] — » water —y A, 10 mins. -^ wash -^ B, 3-5 mins. — » C, 3 successive baths, 1>^ mins. each — > wash — > D, 1-2 mins. — > wash — > E, till plasma sufficiently stained — » balsam, via usual reagents recommended for: reticulum fibers. 21.412 Long 1948 see MS 33.44 Long 1948 21.412 Mall test. 1950 Hall and Herxheimer Hall and Herxheimer 1950, 83 REAGENTS REQUIRED: A. water 100, sodium bicarbonate 10, pancreatin 5; B. water 90, 95% ale. 10, picric acid to sat.; C. water 65, 95% ale 35, acid fuchsin, 10 method: [sections by freezing technique] —> A,24: hrs. — > wash, with agitation — * strand sections on slide and dry — * B, dropped on slide and allowed to dry — > C, 30 mins. — » balsam, via usual reagents recommended for: reticulum fibers. 21.412 Neubert 1940 23418(1), 110:709 REAGENTS REQUIRED: A. DS 11.41 Petersen 1926; B. 5% phosphotungstic acid method: [sections] —> water—* A, 5-30 mins. -^ B, till differentiated —» wash -*■ bal- sam, via usual reagents RECOMMENDED FOR: smooth muscle in sections. 21.412 Pinkus 1944 1829, 49 :355 REAGENTS REQUIRED: A. Water 30, 95% ale 70, hydrochloric acid 0.6; orcein 1; B. 0.3% hydrochloric acid in abs. ale; C. DS 13.13 Giemsa 1902 0.6, water 100; D. 0.001% eosin Y in 95% ale method: [sections] —> 70% ale — > A, 1-9-1 hr. —> rinse —>• 95 % ale, few dips -* abs. ale, till decolorized to pale brown — > B, till almost completely decolorized — > wash — * C, 2-12 hrs. -^ blot — > D, tiU dehydrated — > balsam, via usual reagents recommended for: sections of skin. 21.412 Unna 1928 test. 1928 Schmorl Schmorl 1928, 343 reagents required: A. water 100, ammonium alum 10, crystal violet 1.5; B. ADS 12.2 Lugol (1905) 2, water 98, C. aniline 20, xylene 80; D. aniline 50, xylene 50 method: [celloidin sections of ale fixed material] —» water -^ A, 1 hr. — » wash — » B, 30 sees. — > C, 1^-1 min. — > D, dropped on slide, till differentiated — > xylene — » balsam recommended for: fibers in skin. DS 21.412-DS 21.421 DYE STAINS OF SPECIAL APPLICATION 425 21.412 Weigert 1887 8645, 6 :228 STOCK solutions: I. abs. ale. 82, aniline 22, nietliyl violet to sat.; II. sat. aq. sol. methyl violet REAGENTS KEQUiREu: A. stock I 9, stock II 81; B. 0.6% hydrochloric acid; C. ADS 12.2 Gram 1880; D. aniline 60, xylene 30 method: [sections]^ water -^ A, 5-10 mins. — » B, wash -^ C, 5 mins. — * blot — » D, till differentiated — > blot —> xylene — >• balsam RECOMMENDED FOR: fibrin. note: Mallory 1938, 193 substitutes equal parts aniline and xylene for D above; he further suggests prior staining of nuclei with carmine. Both Mallory {loc. cit.) and Schmorl 1928, 158 recommend avoiding chrome or dichromate fixation. Unna {lest. 1928 Schmorl, op. cit., 160) substitutes 1.5% gentian violet in 10% alum for A al)ove. 21.42 DESIGNED TO DIFFERENTIATE TYPES OF CELLS SI. 421 In Pituitary 21.421 Berblinger and Bergdorf 1935 7802, 15:381 REAGENTS REQUIRED: A. 1% cresofuschiu; B. DS 11.21 Grenacher 1879; C. water 100, phosphomolybdic acid 1, orange G 2; D. 5% phosphomolybdic acid; E. 0.2% anilin blue method: [sections of formaldehyde-fixed material] —> A, 12 hrs. — » water, quick rinse -^ C, 5 mins. -^ water, quick rinse -^ D, 2 mins. — * blot, or wipe around sections — > E, 15 mins. — > water, quick rinse -^70% ale, till no more color comes away — » bal- sam, via usual reagents RECOMMENDED FOR: pregnancy cells in hypophysis. result: pregnancy cells, blue with yellow granules; basophil cells, blue with purple granules. 21.421 Biggart 1935 7599, 81 :42 REAGENTS REQUIRED: A. 0.5% eosiu Y 50, 0.3% isamine blue 50; B. 95% ale. 80, 5% sodium carbonate 20 method: [sections of F 3700.1010 fixed material] —^ water -^ A, 50°C., 30 mins. — > rinse — > B, till differentiated — ♦ neutral mountant, via usual reagents recommended for: differentiation in cell types in the pituitary. result: basophil cells, blue; chromophobe cells, light blue; acidophile cells, red. 21.421 Cleveland and Wolfe 1932 763, 51 :410 REAGENTS required: A. DS 11.123 Ehrlich 1886; B. 5% potassium dichromate; C. 5% erythrosin; D. water 100, phosphomolybdic acid 1, orange G 2; E. 1% anilin blue method: [2 to 3 m sections of material fixed in F 7000.1000 Regaud 1910] —> A, 3 mins. — > water, quick rinse — > B, in dark, 3 days, changed daily -^ water, rinse — > C, 20-30 sees. — > water, rinse -^ E, 30-60 sees. — > balsam, via usual reagents 21.421 Colin 1923a 6630, 89:1230 formula: sat. aq. sol. aniline 100, acid fuehsin 12, light green 6 method: [sections of formol material] —» water —> A, 2 mins. at 40°C. —♦ water, till differentiated — * balsam, via usual reagents 21.421 Colin 1923b 6630, 89:1230 formula: water 100, aniline to sat., acid fuehsin 16, methyl blue 2.8 method: as Colin 1923a 21.421 Crook and Russel 1935 11431, 40:256 REAGENTS REQUIRED: A. water 100, potassium dichromate 2.4, acetic acid 4.75; B. ADS 12.2 Lugol (1905); C. 1% acid fuehsin; D. DS 13.41 Mallory 1901, sol. C method: [sections] -* water— > .4, 12-18 hrs. -^ wash —> B, 3 mins. -> 95% ale, till decolorized -^ C, 15 mins. — » wash -^ D, 20 mins. -^ wash ^95% ale., till differenti- ated — > balsam, via usual reagents 21.421 Dawson and Friedgood 1938 20540b, 13:17 REAGENTS REQUIRED: A. 3% potassium dichromate; B. water 99, acetic acid 1, azo- carmine 0.2; C. 1% aniline in 95% ale; Z). 1% acetic acid in 95% ale; E. 5% phospho- tungstic acid; F. water 100, acetic acid 8, anilin blue 0.5, orange G 2 426 METHODS AND FORMULAS DS 21.421 method: [sections from F 3000.1000 Dawson and Friedgood 1938]— > ^1, 12 hrs. -^ rinse -^ B, I hr., 55°C. -^ rinse — > C, till "carmine cells" only remain red — ^ D, rinse —> rinse —> E, 2 lirs. — + rinse -^ F, 12-36 hrs. -^ rinse ^ 95% ale, balsam, via usual reagents RECOMMENDED FORI differentiation of two classes of acidophile cells in the pituitary. 21.421 KonefE 1938 20540b, 13:49 REAGENTS REQUIRED: A. 0.1% aniline in 90% ale; B. 1% acetic acid in 90% ale; C. water 100, acetic acid 1, azocarmine 1; D. 0.06% aniline in 90% ale; E. 5% phospho- tungstic acid; F. water 100, phosphotungstic acid 0.05, oxalic acid 2, orange G 2, anilin blue 0.5; G. \% acetic acid method: [3-4 ix cellulose sections of F 3700.1000 fixed material, attached to slide and cellulose dissolved away] -^ 70% ale -^ A, 45 mins. — * B, 1-2 mins. — ^ C, 2 hrs. 56°C. — * wash -^ D, till nuclei red, cytoplasm pink -^ B, 1-2 mins. — * j^, 4 hrs. — > rinse -^ F, A hrs., till basophils blue — » wash — > E, 3-5 mins. -^ wash -^ G, rinse -^ wash — > neutral mountant, via usual reagents RECOMMENDED FOR: differentiation of basophils (blue), acidophils (orange red) and chromophobes (light gray). 21.421 Kraus 1912 2526, 54 :520 REAGENTS REQUIRED: A. 5% potasslum dichromate; B. DS 11.113 Kultschitzky 1889; C. ADS 21.1 Weigert 1885 method: [5 M paraffin sections of formaldehyde-fixed material] -^ A, overnight, 37.5°C. -» thorough wash^ B, 24 hrs. -> wash-^' C, till /3-cells decolorized -> DS 12.221 counterstain, if desired — > balsam, via usual reagents RECOMMENDED FOR: differential staining of a-cells (black) in hypophysis. 21.421 Lewis and Miller 1938 20540b, 14:111 This is identical with DS 13.41 Kricheski 1931, save that the time in the A and B solu- tions is increased to 30 minutes and 24 hours respectively. The authors make the com- mon error of referring to Kricheski's formula for the B solution as "Mallory's stain." 21.421 Lillie 1948 Lillie 1948, 99 formula: water 90, citric /phosphate buffer, pH 8.5, safranin O 0.05, eriocyanine A 0.05 method: [sections] —* water —> stain, till differentiated —> rinse ^ acetone, till de- hydrated -^ balsam, via xylene result: acidophile cells, l)lue; basophiles, red. 21.421 MacCallum, Futcher, Duff, and Ellsworth 1935 10919, 56:350 reagents required: A. sat. sol. cupric acetate; B. 0.4% ripened hematoxylin; C. 3% potassium dichromate; D. ADS 21.1 Weigert 1885 method: [paraffin sections of F 3700.1000 Helly 1903 material] -^ water -^^ A, 5 mins. — » wash -^ B, \ min. -^ wash — » C, 1 min. -^ wash — > D, till differentiated — > wash — > balsam, via usual reagents recommended for: differentiation of anterior lobe from pars intermedia cells in the pituitary. result: anterior lobe cells with black granules; granules unstained in pars intermedia cells. 21.421 Martins 1933 6630,113:1275 reagents required: A. DS 11.123 Harris 1900; B. 0.1% acid fuchsin; C 1% phospho- molybdic acid; D. 0.5% methyl blue method: [sections of F 3700.1000 Helly 1903] -^ water -^ A, 2-3 mins. -^ wash -> B, 10 sees. — > rinse — + C, 1-2 mins. -^ drain — > D, 2-3 mins. — > balsam, via usual reagents 21.421 Maurer and Lewis 1922 11189, 36:141 reagents required: A. 1.7% acid fuchsin 70, sat. aq. sol. "acid violet" 30; B. clove oil 75, abs. ale 25 method: [sections from F 3600 fixed material] -^ water — > stain, 20-30 sees. — > blot -^ acetone — ^ benzene -^ B, till differentiated — ^ benzene -^ balsam recommended for: differentiation of cell types in pars intermedia of pituitary. DS 21.421 DYE STAINS OF SPECIAL APPLICATION 427 21.421 Maxwell 1938 2054Ub, 13:93 REAGENTS REQUIRED:/!. 1% acid fuchsin; B. 0.02% ammonia; C. 0.1% hydrochloric acid; D. 0.5% phosphomolybdic acid; E. water 100, aniliii blue, orange G 2, phospho- molybdic acid 1 method: [3-5 m sections in E 21.1 Maxwell 1938 of F 3700.1010 Heidenham 1916 fixed material] — > water — > A, 30 mins. — » rinse — > B, till differentiated -^ C, few moments — » D, 3 mins. — > E, 1 hr. — > rinse -^95% ale, till differentiated — * abs. ale. — » bal- sam, via S 41.1 Maxwell 1938 21.421 Perry and Lochead 1939 4349, 19:101 reagents required: A. 3% potassium dichromate; B. 0.1 aniline in 90% ale; C. 1% acetic acid in 90% ale; D. 1 % azocarmine in 1 % acetic acid; E. 90% ale. 30, B (above) GO; F. 5% phosphotungstic acid; G. water 100, oxalic acid 2, phosphotungstic acid 0.05, orange G 2, anilin blue 0.5 method: [4 M sections of F 3700.1010 Heidenhain 1916 fi.xed material] — > water — * A, 12 hrs. — » rinse — > £, 4 hrs. 56°C. followed by 14 hrs. room temperature — > wash -^ C, till nuclei differentiated -^ D, 1-2 mins. -^ E, 4: hrs, — > rinse — * F, 4 hrs. — > wash -^ G, 5-10 mins. -^ wash -^ C, 1-2 mins. — > wash — > abs. ale, till differentiated — > balsam, via xylene recommended for: pituitary of mouse. 21.421 Pearse 1950 20540b, 25 :97 reagents required: A. ADS 11.1 Hotchkiss 1948 (A and B sols.); B. DS 11.43 de Tomasi 1936; C. DS 11.41 Lendrum and McFarlane 1940; D. DS 11.122 Mayer 1896; E. 2% hydrochloric acid in 70% ale; F. water 100, phosphotungstic acid 5, orange G2 preparation of f: Mix ingredients. Stand 48 hours and decant. method: [4-4 n sections F 3700.1000 Helly 1903 fixed material] -> 70% ale -^ A (A sol.), 5 mins. -^ 70% ale, rinse -^ A {B sol.), 1 min. -^ 70% ale, rinse — * B, 10-30 mins. — > thorough wash —* C, J^^-3 mins. -^ rinse -^ D, }-i to 3 mins. -^ E, 10-20 sees. —>■ thorough w^ash —> F, 10 sees. -^ wash, till differentiated, 5-30 sees. -^ balsam, via usual reagents 21.421 Romeis 1940 test. 1948 ips. Romeis 1948, 511 REAGENTS REQUIRED: A. DS 21.13 Weigert 1898; B. 0.1% aniline in 95% ale; C. water 100, acetic acid 1, azocarmine 0.1; D. 1% acetic acid in 95% ale; E. 5% phospho- molybdic acid; F. water 100, acetic acid 3, anilin blue 0.2 method : [5 M sections of formaldehyde, or sublimate-formaldehyde, material] -^ water -^ A, till 6-cells dark blue-black-^ 95% ale, till background decolorized -+ B, 15 mins. — > C, 1 hr. 58°C. — > leave cool, 30 mins. —>■ B, till connective tissues decolorized -^ D, quick wash —* wash -^ E, 4: mins. -^ drain and blot — * F, 40 mins. — > quick rinse — » 95% ale, till no more color comes aw^ay — » balsam, via usual reagents result: a-ceUs pink; /3-cells dark brown violet; 7-cells light violet; 5-cells cobalt blue. 21.421 Severinghaus 1932 763,53:1 REAGENTS REQUIRED: A. F 1670.0000 Severiughaus 1932; B. water 65, pyroligneous acid 35, chromic acid 0.6; C. 3% potassium dichromate; D. DS 22.1 Altmann 1890 sol. A.; E. 1% picric acid in 30% ale; F.\% phosphomolyi)dic acid; G. water 85, 95% ale. 15, methyl green 0.4, "acid violet" 0.3; H.25% ale, 75% clove oil method: [small pieces] -^ A, 24 hrs. -^ wash -^ B, 24 hrs. -^ wash — » C, 3 days — > wash — > [3 M paraffin sections] — > D, on slide heated to steaming 2-3 changes — > wash —f E, till differentiated -^ wash -^ F, 1 min. — > wash -^ G, I min. -^ wash -^ blot — > 95% ale, 5 sees. — > H, till differentiated — > balsam, via xylene 21.421 Spark 1935 11284, 20:508 REAGENTS REQUIRED: A. 0.25% anilin blue; B. DS 11.122 Mayer 1891; C. DS 12.221 van Gieson 1890 method: [sections of F 7000.1000 material] -+ A, \-\\i mins. — > w^ash, 30 sees. — > B, 10 mins.—* wash 2-3 mins. — > C, 1-1^2 mins. — > wash 1 min. — > balsam, via usual reagents result: /3-granules, blue; a-granules, green. RECOMMENDED FOR: differentiation of cell types in pituitary. 428 METHODS AND FORMULAS DS 21,421-DS 21.422 21.421 Wallraff 1939 23507a, 45:631 REAGEXTs REQUIRED: A. ADS 12.2 Salazar 1923; B. 3% ferric alum; C. 0.5% hydro- chloric acid in 70% ale; D. water 100, acetic acid 1, azocarmine 0.1; E. 0.1% aniline in 95% ale; F. 1% acetic acid in 95% ale; G. 0.5% toluidine blue method: [5 M sections of F 5000.1000 Bouin 1897 fixed material, washed free of fixative] — » water —> A, 2-3 mins. -^ wash -^ B, 30 sees. -^ wash -^ C, till /3-cells differenti- ated ^ wash, 70% ale. -^ D, }i-l hr. 58°C. -^ E, till connective tissues decolorized — > F, rinse — > G, 1-3 mins. 95% ale, 24 hrs. -^ balsam, via usual reagents RECOMMENDED FOR: differentiation of /3-cells (black) in hypophysis. 21422 In Other Glands 21.422 Baley 1937a 1U31, 44:272 PREPARATION OF DRY STOCK: Mix equal parts saturated solutions of magenta and orange G. Filter. Wash till pale yellow. Dry. REAGENTS REQUIRED: A. ADS 12.2 Lugol (1905); B. DS 11.123 Ehrlich 1886; C. 0.1% hydrochloric acid in 70% ale; D. water 50, sat. sol. dry stock in abs. ale 50 method: [sections of F 3700.1000 Baley 1937 fixed material] -^ abs. ale — » A, 2 mins. -^ abs. ale, wash — > B, 20 mins. -^ wash -^ C, till pink — > tap water, till blue —> D, on slide, warmed to steaming, 1 min. — > blot — > abs. ale rinse — » A, till red, 10 sees. — > blot -^ abs. ale — * balsam, via xylene recommended for: zymogen granules (red) in pancreas. 21.422 Baley 1937b 11431,44:272 PREPARATION OF DRY STAINS: T. Mix equal parts saturated solutions of magenta and methylene blue. Filter. Wash ppt. till wash-water pale blue. Dry. II. As I, substitut- ing "acid violet" for methylene blue. REAGENTS REQUIRED: A. ADS 12.2 Lugol (1905); B. water 50, sat. sol. either I or II in abs. ale 50 method: [sections of F 3700.1000 Baley 1937 fixed material] — * abs. ale — > A, 2 mins. — > abs. ale, wash — > B, on slide, warmed to steaming, 1 min. —> blot — > abs. ale, rinse -^ A, till red, 10 sees. -^ blot -^ abs. ale -^ balsam, via xylene recommended for: differentiation of ^-cells (red with I, violet with II) from a-cells (blue with I, unstained with II) in islets of Langerhans. 21.422 Baley 1937c 11431,44:272 REAGENTS REQUIRED: A. ADS 12.2 Lugol (1905); B. 1% eosin; C. 1% acetic acid; D. water 100, acetic acid 0.5, anilin blue 0.375, orange G 0.625, resorcin 0.75 method: [sections of F 3700.1000 Baley 1937 fixed material] -^ abs. alc.,-^ A, 2mins. — > abs. ale, wash — > B, 10 mins. — > blot — > ( ', 2 mins. -^ D, 1 min. -^ blot — > abs. ale, rinse -^ A, 10 sees. -^ blot -^ abs. ale — > balsam, via xylene recommended for: differentiation of zymogen granules (bright red); granular cells of acini (dark red) ; )3-cells (red) ; a-cells (blue) in pancreas. 21.422 Bensley 1916 590, 19:37 REAGENTS REQUIRED: A. 1% ammouium chlorostannate; B. DS 11.3 Bensley 1916; C. DS 12.16 Bensley 1916 method: [sections of F 3700.1000 fixed thyroid] — > water —> A, 2 mins. -^ water, quick rinse -^ B, 1-3 hrs. -^ water, wash — ^ C, 1-5 mins. — ^ water, quick rinse — » abs. ale — > balsam, via xylene RECOMMENDED FOR: differentiation of glandular elements of thyroid. 21.422 Bowie 1925 763,29:57 PREPARATION OF DRY STOCK: Mix 60 sat. sol. ethyl violet with 30 sat. sol. Biebrich scarlet. Filter. Wash and dry ppt. REAGENTS REQUIRED: A. water 80, ale 20, dry stock 0.001; B. cloA^e oil 75, abs. ale 25 method: |3 /u sections of F 3700.0010 Bowie 1925 fixed material] — > water -^ A, 24 hrs. -^ acetone, rinse — > B, till differentiated — » acetone — * toluene — » balsam RECOMMENDED FOR: differentiation of cell types in pancreas. result: a-cells, blue; 7-cells, red; ^-cells purple. DS 21.422 DYE STAINS OF SPECIAL APPLICATION 429 21.422 Fujiware 1939 1887a, 27:1030 STOCK solutions: I. 1% ponceau 2R in 1% acetic acid; II. 1% acid fuchsin in 1% acetic acid REAGENTS REQUIRED: A. stock I 00, stock II 30; B. sat. aq. sol. picric acid method: [sections of F 3700.1010 lleidenhain 1916 material] — > A, 5 mins. — > rinse —>■ B, 5 mins. — » balsam, via usual reagents recommended for: androgenic cells in adrenal cortex. 21.422 Gomori 1939 763,74:439 REAGENTS REQUIRED: A. watcr 100, azocamiinc 0.1, acetic acid 2; B. 1% aniline in 90% ale; C. 5% ferric alum; D. water 100, orange G 0.2, anilin blue 0.6 method: [sections of F 5000.1010 Benin 1897 fixed material] -^ water — > A, 45-60 mins., 55°C. -^ rinse — ^ blot — » B, till acinous cells decolorized — > rinse -^ C, 5 mins. — > rinse — » D, till collagen deep blue -^ abs. ale, till differentiated — » balsam, via xylene RECOMMENDED FOR: differentiation cell types in islets of Langerhans. result: a-cells, orange; jS-cells, red; 6-cells, blue. 21.422 Gomori 1941 608b, 17:395 REAGENTS REQUIRED: A. F 5000.1010 Bouiu 1897; B. water 100, sulfuric acid 0.3, potas- sium permanganate 0.3; C. 3% sodium bisulfite; D. DS 11.124 Gomori 1941; E. 1% hydrochloric acid in 70% ale; F. 0.5% phloxine; G. 5% phosphotungstic acid method: [sections of F 5000.1010 Bouin 1897 fixed material]-^ water -^ A, 1 day — > thorough wash -^ B, 1 min. -^ C, till bleached -^ wash — » Z), 10-15 mins., till cells deep blue -^ E, 1 min. —>■ tap water, till blue -^ F, b mins. -* rinse — > G, 1 min. 95% ale, till differentiated -^ balsam, via usual reagents recommended for: differentiation of a, /3 and 6 cells of pancreas. 21.422 Lane test. 1937 Duthie Gatenby and Painter 1937, 420 PREPARATION OF DRY STOCK: Mix 50 1.5% Crystal violet and 50 1.25% orange G. Filter, wash, and dry ppt. PREPARATION OF STOCK SOLUTION: abs. alc. 100, dry powder from above to sat. reagents required: A. water 90, stock solution from above 10; B. clove oil 75, abs. alc. 25 method: [3 M sections of material fixed in either F 3700.0000 Lane (1910) (for a-cells) or F 3700.0000 Lane (1937) (for /3-cells)] -^ water -^ A, 24 hrs. -^ blot -^ acetone, till de- hydrated — ^ toluene -^ B, till differentiated — * balsam, via toluene recommended for: differentiation of a and j3 cells in islets of Langerhans. 21.422 Launoy 1904a test. 1907 Bohm and Oppel Bohm and Oppel 1907, 355 REAGENTS REQUIRED: A. DS 11.122 Launoy 1904; B. 0.1% hydrochloric acid in 70% ale; C. DS 11.43 Ziehl 1882; D. 0.1% light green in 70% alc. method: [sections of osmic, chrome, or platinic fixed material] — > A, 15 mins. -^ wash — > B, till nuclei alone colored -^ wash -^ C, on slide, warmed to steaming, 5-10 mins. -^ wash, till no more color comes away — > D, few moments -^ B, till differentiated — > balsam, via usual reagents RECOMMENDED FOR: cellular differentiation in pancreas. 21.422 Launoy 1904b lest. 1907 Bohm and Oppel Bohm and Oppel 1907, 357 REAGENTS REQUIRED: A. DS 11.122 Lauiioy 1904; B. 0.1% hydrochloric acid in 70% ale; C. DS 11.42 Zwaademaker 1887; D. sat. aq. sol. orange G method: [sections] -^ A, 15-20 mins. -^ B, M to 1 hr. -^ C, 48 hrs. -^ wash till no more color comes away — > D, few moments—* 95% ale, wash — > B, till differentiated-* balsam, via usual reagents RECOMMENDED FOR: cellular differentiation in pancreas. 21.422 Lillie 1948 see DS 21.422 Wiesel 1903 (note) 21.422 Miiller 1950 Mikroskopie, 6:245 REAGENTS REQUIRED: A. watcr 100, potassium permanganate 0.125, sulfuric acid 0.25, B.2% oxalic acid; C. water 100, zinc acetate 0.5, Bismarck brown 0.5; D. 0.6 acetic acid; E. 0.5% light green 430 METHODS AND FORMULAS DS 21.422-DS 21.423 method: [3-5 n sections of F 5000.1010 Bouin 1896 fixed material]^ waters A, 2 mins. -^ wash -^ B, till white — > wash -> C, 2 mins. — * rinse —> D, till differentiated — > wash —> E, 2 mins. -^ wash — > balsam, via usual reagents RECOMMENDED FOR: differentiation of a (green) and 0 (brown) cells in islets of Langer- hans. 21.422 Wiesel 1902 764, 19 :481 REAGENTS REQUIRED: A. 1% toluidine blue; B. 1% safranin method: [sections of F 7000.1000 Wiesel 1902 material] —+ water —» A, 20 mins. — > wash, 5 mins. running water — > B, 20 mins. -^95% ale, till again blue — > balsam, via carbol-xylene RECOMMENDED FOR: demonstration of chromaffin cells in adrenal. result: nuclei, red; chromaffin cells, green; other cells, blue. note: Lillie 1948 (p. 104) cites Schmorl (no reference) in substituting anilin blue for toluidine blue in A above and in specifing "chromate fixed material." 21.422 WiUiamson and Pearse 1923 11025, 57:193 reagents required: A. ADS 12.1 Lugol (1905); B. 0.25% potassium permanganate; C. 5% oxalic acid; D. DS 11.124 Mallory 1900 method: [sections of F 3670.0000 Williamson and Pearse 1923 fixed material]—* water -^ A, 30 mins. —♦95% ale, wash — ♦ B, 10 mins. —♦ rinse —> C, till decolorized—* wash — > D, 24 hrs. —* wash -^ balsam, via usual reagents recommended for: differentiation of cell types in thyroid. 21423 In Other Structures 21.423 Coutelin 1931 899a, 9:188 reagents required: A. 0.5% acid fuchsin; B. 1% phosphomolybdic acid method: [20 n sections from formaldehyde-fixed material] — > water — > A, 5-10 mins. — > water, wash — > B, 10-20 sees. -^ balsam, via usual reagents RECOMMENDED FOR: differentiation of flame cells. result: nuclei, and cilia, of flame cells, red. 21.423 Downey 1913 see DS 21.423 Wright 1910 (note) 21.423 Endicott 1945 20540b, 20 :5 REAGENTS REQUIRED: A. water 60, DS 11.122 Delafield (1885) 30; B. 1% hydrochloric acid in 95% ale; C. 1% sodium phosphate dibasic; D. water 96, citric acid 0.212, sodium phosphate, dibasic (cryst.) 0.241, DS 13.13 Endicott 1945 (working sol.) 4 method: [nitrocellulose sections of F 7000.1000 Orth 1896 fixed, and formic acid decalci- fied, material] — > water —* A, 5 mins. -^ B, rinse 95% ale, quick wash — » C, till blue — > wash -^ D, 1 hr. — >• E, till differentiated — ♦ balsam, via usual reagents RECOMMENDED FOR: differentiation of cell types in bone marrow. 21.423 Fuller 1943 11284, 28:1475 reagents required: A. water 50, DS 11.123 Ehrlich 1886 50; B. water 99, acetic acid 1, ponceau 2 R 0.5, acid fuchsin 0.5; C. water 100, phosphomolybdic acid 1, orange G 2 method: [ale fixed smears] — > water —♦ A, 2 mins. — » wash, 4 mins. -^ B, 2 mins. —> rinse —* C, 2 mins. -^ balsam, via usual reagents RECOMMENDED FOR: vaginal smears. 21.423 Gomoril946 Tech. Bull., 7 -AB REAGENTS REQUIRED: A. 0.05% azocamiinc in 1% acetic acid; B. 1% aniUne in 95% ale; C. 3% phosphotungstic acid; D. water 100, oxalic acid 2, methyl blue 0.5, tartrazine 2 method: [sections of material after other than dichromate fixation] -^ water -^ A, 1-lJ^^ hrs., 60°C. -^ rinse -^ blot -> 95% ale, rinse -> B, till chromaffin cells deep pink -> rinse —> C, 20 mins. -^ quick wash -^ D, 15-40 mins., till collagen deep blue -^ rinse -^ balsam, via usual reagents recommended for: demonstration of chromaffin granules (purple to ruby-red). DS 21.423 DYE STAINS OF SPECIAL APPLICATION 431 21.423 Hamazaki 1935 22575, 295:703 REAGENTS REQUIRED: A. Water 95, abs. ale. 5, phenol 2.8, magenta 0.5; B. 1% hydro- chloric acid; C. ADS 12.2 Lugol (1905); D. 1% sodium thiosulfate; E. 3% hydro- chloric acid method: [6 M sections of F 3700.1010 Hamazaki 1935 fixed material]^ water— > A, 1 hr. -^ wash -^ B, 10 mins. — > C, 30 mins. —> rinse — > D, till iodine-free -^ wash — » E, 30 mins. -^ wash -^ balsam, via usual reagents recommexded for: demonstration of wandering cells (red) in smooth muscle. 21.423 Hoecke and Sebruyns 1952 20540b, 27 :263 REAGENTS REQUIRED: A. sat. sol. Bismarck lirown in 70% ale; B. sat. sol. anilin blue in 2.5% acetic acid; C. 0.1% methyl violet; D. water 30, 95% ale. 70, aluminum chloride 0.1, hematein 0.2; E. 95% ale. 100, saffron 2; F. 0.1% acetic acid in abs. ale. PREPARATION OF E: Digest 6 hours at GO°C. Filter. method: [sections] —> 70% ale. — > A, 3 mins. — > 70% ale., rinse —> water, wash—* B, 2 hrs. -^ C, dropped on slide, 2 mins. — > 70% ale, wash — > D, 30 mins. -^70% ale, rinse —* E, till differentiated -^ F, 30 sees. -^ balsam, via usual reagents recommended for: differential staining of gastric glandular cells. 21.423 Kingsley 1937 8545, 57:87 reagents required: A. DS 13.13 Kingsley 1935 (sol. A); B. 0.008% acetic acid; C. acetone 100, eosin Y 0.001, acetic acid 0.04 method: [sections of F 3500.1000 Kingsley 1937 fixed material] — > water, via butyl ale. — > A, 8-10 mins. -^ rinse -^ B, wash -^ rinse -^ blot -* C, rinse — ♦ "neutral mount- ant," via butyl ale. and xylene recommended for: demonstration of megakaryocytes in sections of bone marrow. 21.423 Levine 1928 11284, 14:172 reagents required: A. 1% thionin; B. sat. sol. orange G in clove oU method: [5 M sections] — ^ water -^ A, 5 mins. -^ water, till no more color comes away — > blot -^ B, few moments -^ clove oil, till differentiated -^ balsam, via xylene RECOMMENDED FOR: mast cells in connective tissue of parathyroid. result: mast cells blue; other structures orange. 21.423 Novak 1910 766,36:217 REAGENTS REQUIRED: A. DS 12.212 Unna 40, 1% eosin in 80% ale. 40, 1% hydro- quinone 12, orcein 0.4; B. 1% safranin; C. 0.5% potassium dichromate method: [sections] -^ water — » A, 5-10 mins. -^ B, 10 mins. — > wash -^ C, 30 mins. —>■ ■wash -^ balsam, via usual reagents RECOMMENDED FOR: demonstration of corpuscles of Herbst. 21.423 Ralston and Wells 1939 591b, 3:72 REAGENTS REQUIRED; A. Water 100, azocarmine 1, acetic acid 1; B. 0.15% aniline in 95% ale.; C. 0.07% acetic acid in 95% ale; D. 0.5% toluidine blue method; [4 M sections of bone decalcified in AF 21.1 Ralston and Wells 1939] -^ water — > A, 20 mins., 56°C. -^ B, till color clouds cease -^ C, till rose red -^ wash — > D, 10-15 sees. — * abs. ale, least possible time for dehydration — >• balsam, via xylene RECOMMENDED FOR: differential staining of bone marrow. 21.423 Shoor 1941 19938,94:545 formula: water 50, 95% ale 50, Biebrich scarlet 0.5, orange G 0.25, fast green FCF 0.075, phosphotungstic acid 0.5, phosphomolybdie acid 0.5, acetic acid 1 method: [alcohol-ether-fixed smears] -+ stain, 1 min. — > 70% ale, rinse -> dammar, via usual reagents recommended for: vaginal smears. 21.423 Papanicolaou 1941 11284,26:1200 reagents required: A. DS 11.123 Ehrlieh 1886; B. water 100, phosphotungstic acid 0.112, phosphomolybdie acid 0.225, anilin blue W.S. 0.06, orange G 0.125, acid fuchsin 0.1, eosin Y 0.21 method; [alcohol-fixed smears] — » water —> A, 2 mins. — > rinse —»• "blue" in tap water —> rinse —^ B, 2-5 mins. -^ rinse — + balsam, via dioxane recommended for: vaginal smears. 432 METHODS AND FORMULAS DS 21.423-DS 22 21.423 Papanicolaou 1942 19938,95:438 REAGENTS required: .4. DS 11.123 Ehrlich 1886; B. 0.5% hydrochloric acid; C. 95% ale. 100, orange G 0.5, phosphotungstic acid 0.015; D. either 95% ale. 100, light green SF 0.225, Bismarck brown 0.05, eosin Y 0.225, phosphotungstic acid 0.2, lithium carbonate 0.0005 or 95% ale. 100, light green SF 0.22, Bismarck brown 0.06, eosin Y 0.22, phosphotungstic acid 0.17, lithium carbonate 0.0005 method: [ether-alcohol-fixed vaginal smears] — > water, via graded ales. —* A, 5-10 mins. -^ B, till differentiated —>■ "blue" — > 95%, ale., via graded ales. -* C, 1 min. -^95% ale, rinse — > D, 2 mins. -^95% ale., rinse -^ balsam, via usual reagents recommended for: vaginal smears. NOTE : The two alternatives under D above were named respectively E A 36 and EA 25 by their inventor. 21.423 Pfaff and Williams 1942 20540b, 17:165 STOCK solutions: I. water 100, acetic acid 2.5, benzedine 0.5, II. 0.5% sodium nitro- ferricyanide REAGENTS REQUIRED: A. stock I 20, stock II 20, water 60; B. water 100, hydrogen per- oxide (30%) 0.2 method: [pieces of formaldehyde-fixed material] ^ A, 30-45 mins., 37°C. — » wash, 37°C. several mins. -^ B, 30 mins., 37°C. -^ wash, 37°C. -^ balsam, via usual reagents recommended for: demonstration of blood vessels in wholemounts. 21.423 Schleicher 1942 20540b, 17:161 reagents required: A. methanol 100, DS 13.12 Wright 1910 (dry powder) 0.17; B. water 100, methanol 5, acetone 0.5 method: [air-dried smear of mixed plasma and myeloid-erythroid layer produced by centrifugation of heparinized bone marrow] -* 0.5 ml. A, on smear, 2 mins. — ♦ add 2 ml. water, 5-10 mins. -^ B, 1-5 sees. -^ rinse -^ drj' recommended for: cells of bone marrow. 21.423 Wright 1910 11373,21:263 reagents required: A. DS 13.12 Wright 1910 {A sol.) 75, 0.2% eosin Y in methanol method: [sections of mercuric-fixed marrow] -^ stain, 10 mins. -^ wash —>• resin in turpentine, via acetone and turpentine recommended for: demonstration of megakaryocytes in sections of bone marrow. note: Downey 1913 (8545, 15:25) specifies fixation in his F 3000.1000 solution. 21.423 Zimmerman 1925 7962, 24:281 reagents required: A. any DS 11.122 formula; B. 1% hydrochloric acid in 70% ale.; C. DS 22.7 Mayer 1896a (working sol.) ; D. sat. sol. aurantia in 50% ale. method: [sections]^ water—* A, till deeply stained—* B, till connective tissues des- tined — > wash — >■ C, 24 hrs. -^ wash —* D, till deeply stained -* 50% ale. tUl differ- entiated — > balsam, via usual reagents result: superficial epithelium, red; parietal cells, red; chief cells, gray blue; other tissues, yellow. recommended for: differentiation of cell types in stomach. 22 DYE STAINS FOR CYTOLOGICAL ELEMENTS It has been very difficult to determine which techniques should lie in the present section and which should have been re- ferred to section DS 23.1. The term cell inclusion is all embracing and might be held to cover the result of almost any technique which has been used to demon- strate the existence of a small particle, the nature of which has never been disclosed or the existence of which has subsequently been doubted. As the term is employed here, the nature of the research for which the technique was designed, rather than the results obtained, has been used as a guide. It is, therefore, very possible that many, if not all, of the techniques designed to show cell inclusions w'ould show these same cell inclusions if the techniques given under DS 23.1 below were to be substi- tuted for them. In the division of cyiological elements there has been followed, in general, the usual classification. The first class, con- taining nuclear techniques, is quite self DS 22 DS 22.10 DYE STAINS OF SPECIAL APPLICATION 433 explanatory, tliough it must be enij)lia- sized that only tliosc teclmiciuos arc in- cluded in this division which are intended for the specific staining of nuclear ele- ments. General nuclear stains are given under the headings DS 11 in Chapter 20. Probably much annoyance will be caused to cytologists by the grouping together, in the second class, of mitochondria and Golgi apparatus. It is not intended, of course, by this grouping to suggest that these are in any way related to each other. They are, however, the chief ])reoccupa- tion of the cytological cytologist, and the techniques intended for their display may therefore be justifiably included in the same place. The ne.xt four sections deal with a variety of cell inclusions too well known to need further explanation at this point; the final class has been erected to contain all those miscellaneous techniques w'hich have been designed to show not only cell inclusions, but also such cell extrusions as cilia, flagella, and the like. 22.1 Nuclei For staining nuclei in cells any of the techniques given in the last chapter (DS 11) may be satisfactorily employed. These nuclear stains are, however, by no means the best when it is desired to bring out for the interest or information of students the various portions of a mitotic figure other than the chromosomes them- selves or when, for one reason or another, it is desired to stain the chromosomes in a manner which leaves them relatively transparent. The majority of these tech- niques have been developed by botanists, but they may be applied equally well to sections of animal material, and are de- signed throughout to demonstrate clearly all the elements, including the spindle fibers, of a mitotic division. Most research workers would prefer, probably, to retain the iron-hematoxylin techniques to which they are accustomed, but it is sincerely to be hoped that the manufacturers of slides for class purposes will learn of the existence of other methods and will cease to sup- ply the now inevitable iron-hematoxylin- stained mitotic figure, which rarely shows more than the chromosomes themselves. 22.10 TYPICAL EXAMPLES Demonstration of mitosis in an onion root tip using the rose bengal-orange G-toluidine blue stain of Kedrovsky 1931 Nobody who has i)repared onion root tip for class demonstration by this meth- od will ever be hkely to revert to the iron hematoxylin metliod formerly cm- ployed, for nothing is more attractive to an elementary class than a polychrome- stained specimen. Though the term o7iio7i root is usually apphed to these preparations, there are several members of the genus Allium which will give better class-demonstration material. The author's preference is for the root tip of the leek {AlUu7n porrum), which can be obtained just as readily as the root tip of an onion. Excellent demon- stration material can be obtained from a leek seedhng about a month old. These can be cultured in the laboratory, for there is no objection to the etiolation which results from the growing of such material in improperly hghted surround- ings. The high temperatures at which most laboratories are maintained is also an ad- vantage, since it causes a more rapid growth and a greater variety of stages to be obtainable in a single section. The writer does not think that there is much choice in fixative for these specimens, as the mercuric-acetic-nitric fixative of Pe- trunkewitsch (Chapter 18, F 3000.0014) gives uniformly successful preparations. The leek seedhng, upon being removed from the soil, should be very, very thor- oughly washed in normal saline solution to remove adherent particles of fine grit which would blunt the knife and the tip of the seedling, with the roots attached, is then dropped into a considerable vol- ume of Petrunkewitsch's fixative. It may remain here overnight before being re- moved and thoroughly washed in several changes of 50% alcohol. This material will present no difficulties in sectioning. It is customary to cut a lon- gitudinal section; and a section thickness of from five to eight microns should be employed rather than the conventional ten microns. The sections are then at- 434 METHODS AND FORMULAS DS 22.10 DS 22.11 tached to the slide in the usual way, dewaxed, and run down tlirough the cus- tomary alcohols to water. An alternative technique, which is not nearly as widely used but is really most successful, is to employ smears rather than sections of the root tip. Though these do not give, of course, a clear picture of the relations of the cells to each other, they at least insure that the majority of mitotic figures will be so oriented on the shde as to occasion no difficulty to the students studying them. These smears may be prepared by taking the tip of the root, placing it on the slide, taking a sharp scalpel, and smearing, quite hterally, the root onto the glass with the pressure of the flat side of the knife and a rotary mo- tion of the hand. These smears are then fixed by being placed face down, resting across two glass rods, in a petri dish of fixative. The depth of the fluid must be such that the lower surface of the shde bearing the smear is in contact with the fixative, which must not, however, be permitted to rise up until the upper sur- face of the shde is wetted by it. After fixation by this method the sUdes are re- moved and washed in several changes of 50% alcohol until the fixative is removed. Two solutions are required for staining. First is DS 21.11 Kedrovsky]l931. To pre- pare this, dissolve 0.3 gram of rose bengal in 50 milhliters of water; add one milliliter of 1% phosphomolybdic acid to the boil- ing solution, one drop at a time, with constant stirring. Cool and add 50 milli- liters of a 1.2% solution of orange G. The only other stain required is a 1 % solution of toluidine blue. Whether sections or smears are being employed, the shdes are transferred from distilled water to the rose bengal-phospho- molybdic acid stain for from 20 to 30 minutes. The time is not particularly critical, and a, pcu'iod of as long as one hour may be employed if it is more convenient. Each slide is then taken separately, rinsed very briefly in distilled water, and trans- ferred to 1 % toluidine blue for 5 minutes. It is transferred directly from toluidine blue to 70% alcohol, in which it is waved Ijackward and forward for about one min- ute to remove the excess stain from the section, and then transferred to 95% alcohol, in which differentiation proceeds relatively slowly. If differentiation in 95% alcohol is too slow, the specimen may be returned to 70% alcohol for a brief period, and then put back in 95% alcohol. The sections must be examined under a high power of the microscope to determine when they are properly differentiated, and it is not safe to do this if they have been removed directly from alcohol. Before being examined, therefore, each section should be dipped in bergamot oil (specified by Kadrovsky) or in clove oil, which is equally safe. The oil should be wiped from the back of the slide and the slide then examined. The differentiation should not be watched with regard to the chromo- somes themselves, but with regard to the spindle fibers, which are far better shown by this than by any other method. In a properly differentiated specimen the spin- dle fibers will be a clear pink against a blue background, and when this condition has been reached the bergamot oil should be washed off thoroughly in xylene, and the specimen mounted in balsam. If the spindle fibers are properly differentiated the chromatin will be a clear dark blue, very readily differentiated from the blue background of the cytoplasm, and the students' attention will clearly be drawn to the nucleoli in the resting nuclei, which are stained a bright red. 22.11 OTHER TECHNIQUES 22.11 Backman 1935 20540b, 10:83 This method, recommended by Gatenby and Painter 1937, 691, calls for a "saturated solution of anthraquinone " ; anthraquinone is not soluble in water and it is not ap- parent which soluble derivative was employed by Backman. 22.11 Balbiani test. 1895 Maggi Maggi 1895, 174 REAGENTS required: A. 1% osmic acid; B. water 100, acetic acid 1, methyl green 1; C. 0.2% ammonia method: [protozoans] —* A, 1 min. B, 3 mins. — > C, till differentiated RECOMMENDED FOR: nuclear detail in ciliates. DS22.il DYE STAINS OF SPECIAL APPLICATION 435 22.11 Barrett 1932 20540b, 7:63 formula: water 25, 95% ale. 25, acctie acid 50, hematoxylin O.OG, ferric alum 0.5 method: [smear preparations]-^ stain, under coverslip, till chromosomes clear —» seal cover RECOMMENDED FOR: poUen mother cells. 22.11 Bradley 1948 20540b, 23:29 REAGENTS REQUIRED: A. F 0000.0010 Bradley 1948; B. 4% ferric alum; C. 50% hydro- chloric acid; D. DS 11.23 BeUing 1921 method: [plant ovaries] —> A, 2 days — > B, 75°C., 3 mins. -^^ water, 75°C. 2 mins. — > fresh water, 75°C., 2 mins. — > cold water, 2-3 mins. —* C, 10 mins. -^ thorough wash —> [scrape ovules from placenta to slide] -^ [apj)ly cover and top to separate cells] — > heat to steaming -^ jar filled witii ale. vapor, 24 hr.s. —> abs. ale, 1 drop run under cover -^ spread M 23.1 mountant round edge of cover, leave in jar with slight ale. vapor content 24 hrs. — > dry 22.11 Bizzozero-Vassale test. 1894 Kahlden and Laurent Kahlden and Laurent 1894, 71 REAGENTS REQUIRED: A. DS 23.211 Ehrlich 1882; B. ADS 12.2 Gram 1884; C. 1% chromic acid method: [sections] — ^ A, 10 mins. — * abs. ale, rinse —> B,2 mins. — > abs. ale, 30 sees. — > C, 30 sees. -^ abs. ale, 30 sees. -^ C, 30 sees. —* abs. ale, 30 sees. — » clove oil, till no more color comes away — » balsam, via usual reagents 22.11 Conn 1943 20540b, 18:189 REAGENTS REQUIRED: A. \% chlorazol blaclc E method: [sections of root tips] — + water —* A, 2 hrs. —> balsam, via usual reagents 22.11 Cooper 1931 591, 18:337 REAGENTS REQUIRED: A. 1% methyl green; B. 1% acid fuchsin; C. 1% erythrosin method: [sections] —* water —> a, 1 hr. -^ water, rinse— *B, 1 min. -^ water, quick rinse -^ C, few sees. — > abs. ale, minimum possible time —>■ balsam, via xylene result: chromatin, green; linin, red. 22.11 Dalton test. 1948 Lillie Lillie 1948, 87 REAGENTS REQUIRED: A. water 55, acetic acid 1, orcein 45; B. 0.15% fast green FCF in abs. ale PREPARATION OF a: Dissolvc dye in hot acid. Cool. Dilute to 100 with water. method: [pieces for chromosomes]—* A, 48 hrs. — > fragments crushed under coverslip on slide coated with V 21.1 Mayer 1884—* ale vapor, 48 hrs. -^ 95% ale, few mo- ments -^ B, few sees. — > balsam, via usual reagents 22.11 Darrow 1944 20540b, 19 :65 REAGENTS REQUIRED: A. 1% safranin O; B.sat. sol. {circ. 1%) anilinblue W Sin 95% ale method: [sections from F 6000.1010 fixed material] — > water — > A, 15 mins. — » rinse —* B, 2 mins. — ^ balsam, via usual reagents recommended for: chromosomes in root tips. 22.11 Geither 1940 test. 1948 Romeis Romeis 1948, 223 reagents required: A. 2% osmic acid; B. F 1600.0010 Benda 1901; C 3% hydrogen peroxide; D. 1% safranin; E. 0.2% light green method: [fresh smears] — » A, 10-20 sees. — > B, 10 mins. —> thorough wash —y C, 10 mins. -^ thorough wash — * D, 10 mins. — > rinse —> E, i'4-l min. — * 95% ale — > balsam, via usual reagents result: chromatin, nucleoli, red; centrosome, spindle fibers, green. 22.11 Hancock 1942 20540b, 17 :79 reagents required: A. 1% chromic acid; B. 0.1% crystal violet; C. 1% iodine and 1% potassium iodide in 80% ale method: [10 IX sections of F 6000.1010 Belling 1928 fixed material]-^ water -^ A, 20 mins. —> 95% ale, rinse —> abs. ale, least possible time —+ clove oil, till clear —> xylene, 2 hrs. — > balsam recommended for: minute plant chromosomes. 436 METHODS AND FORMULAS DS22.il 22.11 Hermann 1893 test. 1905 Bohm and Davidoff Bohm and Davidoff 1905, 76 KEACiKNTs REQUIRED: A. 1% safraiiiii; B. sat. sol. gentian violet in sat. sol. aniline; C. ADS 12.2 Lugol (1905) method: [sections from F 1200.0010 Hermann 1889 fixed material] -^ water -^ A, over- night — * B, 3-5 mins. -h. rinse -^ C, till uniform black -^95% ale. till violet -^ clove oil —> examine —> [repeat 95% ale. if insufficiently differentiated] ^^ balsam, via xylene result: chromosomes, bluish violet; nucleoli, aster, spindle fibers, red. 22.11 Hruby 1933 19938, 77:352 REAGENTS REQUIRED: A. sat. sol. {circ. 1%) magenta; B. sat. sol. {circ. 1.2%) picric acid 50, sat. sol. (circ. 2%) indigo-carmine 50 method: [sections] — » water — > A, 5-20 mins. — > water, thorough wash —> B, 5-10 mins. — ^ 70% ale, rinse —^ abs. ale, till green—* balsam, via usual reagents result: chromosomes, red; nucleoli, blue; spindle fibers, dark blue. 22.11 Johansen 1932 20540b, 7:17 reagents required: A. 1% methyl violet 2B; B. sat. sol. {circ. 1.2%) picric acid in 95% ale; C. 0.1% ammonia in 95% ale; D. abs. ale 50, clove oil 50, erythrosin to sat. method: [sections] -^ water -^ A, 15 to 30 mins. — > water, quick rinse -^ B, till differ- entiated, about 15 sees. -^ C, 15 sees. — > 95% ale, thorough rinse -^ D, 5-10 sees. — > clove oil, 15 sees. — > balsam, via xylene result: chromatin purple, plastin red. 22.11 Kedrovsky 1931 23632,47:433 reagents required: A. water 100, orange G 0.6, rose bengal 0.3, phosphomolybdic acid 0.01; B. 1% toluidine blue PREPARATION OF A: Dissolve the rose bengal in 50 boiling water. Add 11% phospho- molybdic acid slowly and with constant agitation. Cool and add orange G dissolved in 50 water. method: [sections or smears]—* A, 20-30 mins. -^ water, rinses B, 5 mins. -^ 70% ale, 1 min. -^95% ale, till differentiated -^ balsam, via bergamot oil result: chromatin, dark blue; nucleoli, bright red; spindle fibers, pink against blue. note: a detailed description of the use of this stain is given under DS 22.10 above. 22.11 Kurnick and Ris 1948 20540b, 23:17 formula: water 55, 95% ale 9, acetic acid 36, orcein 0.8, fast green 0.1, sodium chloride 0.4 method: [fresh smears, or mounted sections, taken to water] — >• stain, few moments -^ balsam, via usual reagents result: chromatin, red brown; cytoplasm, nucleoli, green. 22.11 Kurnick and Ris 1948 20540b, 23:17 formula: water 55, acetic acid 36, 95% ale 9, orcein 0.8, fast green FCF 0.1 sodium chloride 0.4 method: [smears and squashes]—* A, under cover, few mins. ^ remove cover—* bal- sam, via usual reagents -^ replace cover result: chromatin, brownish red; nucleoli, green. 22.11 La Cour 1941 20540b, 16:169 REAGENTS REQUIRED: A. watsr 55, acetic acid 45, orcein 1; B. 10% acetic acid method: [fresh smear]—* A, under coverslip, 2-3 mins.—* invert slide in B till cover falls off -^ balsam, via usual reagents 22.11 Maneval 1934 19938,80:292 REAGENTS REQUIRED: A. DS 11.43 Maueval 1928; B. 0.01% hydrochloric acid in 95% ale C. clove oil 100, light green 0.075, orange G 0.025 method: [sections] -^ water — * A, 3-5 mins. -+ wash — * B, till differentiated -^ abs. ale, till dehydrated — * C, till stained -^ balsam, via xylene RECOMMENDED FOR: mitosis in plant cells. DS 22,11 DYE STAINS OF SPECIAL APPLICATION 437 22.11 McCUntock 1929 20540b, 4 :53 REAGENTS REQUIRED: A. 25% acetic acid in abs. ale; B. DS 11.23 Belling 1921; C. 10% acetic acid; D. 50% acetic acid in abs. ale; E. 10% acetic acid in abs. ale; F. abs. ale. 50, xylene 50 method: [fresh anthers]^ A, till required-* squeeze contents of anther onto slide, cover with B, crush — > warm to steaming — > cool — + repeat warming 3 or 4 times —* C, in petri -^ E, 2 mins. — > abs. ale, 2 mins. — > F, 2 mins. — > xylene, 2 mins. — > [re- combine slide and cover] -^ balsam recommended for: plant chromosomes. 22.11 Meyer 1886 see DS 11.28 Meyer 1885 22.11 Mitter and Bartha 1948 20540b, 23 :27 reagents required: .1. water 55, acetic acid 45, brilliant cresyl blue 0.75; B. 45% acetic acid method: [whole salivary glands of Drosophila] — > A, in vial, 30-45 mins. -^ transfer glands to slide —>■ drain -^ blot — > cover and tap cover to break nuclei -^ B, under cover, if differentiation necessarj' -^ seal cover recommended for: salivary gland chromosomes of Drosophila. 22.11 Nissl 1894 see DS 11.113 Nissl 1894 22.11 Rawitz see DS 11.42 Rawitz 1895 22.11 van Rosen 1947 14900. 160:121 reagents required: A. 30% acetic acid in 95% ale, B. 30% hydrochloric acid in 95% ale; C. water 50, acetic acid 50, nigrosin, ale soluble 4 method: [fresh tissue]-^ A, 24 hrs. (cooled for roots) -^ A, 1-10 mins. 4°C. -^ wash, 15-30 mins. — + B, on slide, 1-2 mins. -^ blot recommended for: plant chromosomes. note: the dye used was probably indulin, ale sol. 22.11 Sax 1931 20540b, 6:117 reagents required: ^4. 1% crj-stal violet; B. water 20, 95% ale 80, iodine 1, potas- sium iodide 1 method: [smears fixed 1-2 hrs. in F 6000.1010 Navashin 1912]-* 15% ale, thorough wash — » A, 1-5 mins. — » rinse -* B, 30 sees. — » abs. ale, till differentiated —* balsam, via usual reagents recommended for: pollen mother cells. 22.11 Schmorl 1928 SchmorJ 1928, 140 reagents required: A. DS 11.122 Bohmer 1868; B. 1% safranin; C. water 100, picric acid 0.1, tannin 25 method: [celloidin sections of ale fixed material]—* water —> A, 5 mins. — > tap water, till blue -* B, 20 mins. — > wash -^ C, tUl differentiated, 2-5 mins. — * thorough wash -^ balsam, via usual reagents result: smiren Kerne, red; geicohnlichen Kerne, blue. 22.11 Semmens and Bhaduri 1939 20540b, 14:1 reagents required: A. 5% sodium carbonate; B. water 98, 95% ale 12, aniline 0.1, light green 0.5; C. sat. sol. sodium carbonate in 70% ale method: [sections, chromatin stained by DS 11.43 de Tomasi 1936] ^^.1,1 hr. — >• wash, 30 mins. — > B, 10 mins. — > C, rinse —> 95% ale, 10 mins. -^ repeat T^ ale cycle till cytoplasm free of green —> balsam, via usual reagents recommended for: differential staining of nucleoli (green) and chromosomes (purple). 22.11 Smith 1934 20540b, 9:95 reagents required: A. ADS 12.2 Lugol (1905); B. 1% crystal violet; C. sat. sol. {circ. 1.2%) picric acid in abs. ale method: [sections or smears] — > 70% ale, — > .4, 10-20 mins. — > water, rinses B, 15 mins. — » 95% ale, rinse — > C, few sees. —> abs. ale, till yellow removed -^ clove oil, till differentiated -^ xjdene, till clove oil removed — » balsam 438 METHODS AND FORMULAS DS 22.11-DS 22.2 22.11 Togby 1942 20540b, 17:171 REAGExXTs required: .4. 95% ale. 50, hydrogen peroxide (3%) 50; B. 1% chromic acid; C. 1% crystal violet; D.\% iodine and 1% potassium iodide in 50% ale; E. sat. sol. picric acid in 95% ale; F. 0.1% ammonia in 95% ale. method: [sections of F 1670.0010 La Cour 1931a fixed material] -» water -^ A, 30 mins. -^ rinse -> B, 30 mins. -» rinse -> C, 1 hr. -^ rinse -^ D, 30 sees. — > 70% ale, rinse -> .E^, rinse -^ F, rinse -^ abs. ale, till dehydrated -^ clove oil, till differentiated -» xylene, thorough wash — > balsam RECOMMENDED FOR: chromosomes of Crepis. 22.11 Unna test. 1928 Schmorl Schmorl 1928, 139 REAGENTS REQUIRED: A. DS 11.44 Unna 1892; B. 25% tannin method: [sections of F 7000.0000 Miiller 1859 or F 1600.0010 Flemming 1882 fixed material] — > water -^ .l, 2 mins. — > wash — * B, 10-15 mins. -^ wash -^ balsam, via usual reagents 22.11 Wing 1930 23639b, 10:699 REAGENTS REQUIRED: A. ADS 12.2 Wing 1930; B. 1% gentian violet method: [thin sections] —> /4, 5-30 mins. -^ momentary rinse ^ B, 5-15 mins.—* quick rinse — > A, 20-30 sees, differentiation — * abs. ale, least possible time for de- hydration -^ clove oil, till differentiated —>■ balsam, via xylene 22.2 Mitochondria and Golgi tinued by many firms. If it is handled in Apparatus the laboratory, it should be treated with The techniques designed for the demon- t^e respect which is accorded to any high- stration of mitochondria in sections so explosive material. KuU also substituted closely resemble those intended for the ^J'^ ^^^^^^^^ ^^ P^^^^' ^^^^.^°' ^^^ ^''^- demonstration of bacteria under the same I'J^ specified by Altmann m 1890. On conditions (see DS 23.22 below), that it ^^^ somewhat slender basis that Champy is not surprising that for many years there !™ I u^^T ""' '''^''^^\ P'^'P"'^' was confusion in the literature as to the technique has been commonly known whether or not mitochondria existed. The f *^^ Champy-Kull technique, and this now universal acceptance of the existence *^"^, ^.^ ^°^ generally adopted in Enghsh- of mitochondria has, however, unfortu- ^Peak^g countries. In France and Ger- natelv permitted students to become care- 'Z""^^ however, most of what are called less, and it must never be forgotten that Champy-Kull techniques in tins country the bacteria which frequently grow in the f\ ".^P^^^^? . ^^ *^J ^o-M " Parat egg albumen, with which sections are *''^^^"'f ' „^^ ^ "^ .^^^^^^ f -^l^' attached to a slide, will stain in the same f^hromated before being stamed with a colors and be of much the same size and hematoxylin stain The name Pami, how- shape as mitochondria. The original tech- ^^^'' ^^J ^f^f ^^ .*°. ^^ ^PP\^^ *° .^^T'^ nique, for example, of Altmann 1890, any method o staining mitochondria after could be employed very readily for stain- ^eatnient with dichromate, just as the ing acid-fast bacteria in almost any sec- term C/iampy-At/M has come to be trans- tion of tissue terred to any technique m which acid The next development in the staining of ^^^^"' 1^ ^"^ ^' *^! V^n^^rj stain. The mitochondria, after Altmann, was the ^ ^^^^^,^f, ^'^f ^^^^^^^ together in demonstration by Kull in 1914 that the ^^^8 by Volkonsky, who combined the dye, aurantia, could satisfactorily be Pre-dichromating of Parat with the acid- substituted for picric acid, to which it is fuchsin-aurantia techmque of Kull, and chemically closely related, in the differ- followed these wath a double-staining entiation of Altmann's stain. It may be technique using hematoxyUn, orange G, remarked at this point, parenthetically, and aniUne blue by a method closely re- that aurantia is even more explosive than sembling those of the Masson technicjues picric acid, and is so dangerous to manu- described in sections DS 12.31 and DS facture that its supply has been discon- 12.32 above. DS 22.20 DYE STAINS OF SPECIAL APPLICATION 439 22.20 TYPICAL EXAMPLES Demonstration of mitochondria in the pancreas using the acid fuchsin- toluidine blue-aurantia stain of Kull 1914 There are so many modifications of the method of demonstrating mitochondria here described, that it seems desirable to give a fairly detailed description of the original technique, and to leave to the reader the task of determining by subse- quent experimentation which of the nu- merous modifications he would prefer to employ. The method here described, which is most commonly referred to in the hterature as Champy-KuU, has the ad- vantage that it permits considerable ex- perimentation under standardized condi- tions, and jaelds reproducible results, once the timing on any particular batch of material has been estabhshed. The method involves overstaining in acid fuchsin, which is strongly absorbed by the nuclei, the mitochondria, and any bacteria pres- ent. The acid fuchsin is then differenti- ated in a solution of toluidine blue, which removes the acid fuchsin from the nuclei. These remain stained blue, and finally differentiation is conducted in aurantia which removes the unwanted toluidine blue from all parts of the cytoplasm except the mitochondria. A word of warning may be inserted at this point relative to the use of aurantia. This dye is produced from diphenylamine, to the derivatives of which many persons are exceedingly sensitive. Those who have suffered from dermatitis in the handhng of photographic developers should under no circumstances handle solutions of au- rantia, for the dermatitis produced is diffi- cult to get rid of and the sensitivity of the w'orker to this reagent appears to be in- creased by every exposure to it. The dry dye itself is explosive and should always be kept in solution. Pancreas has been selected as a demon- stration object because it is both readily obtainable, and also normally contains very large and clear mitochondria. Baker 1933, 189 recommends the intestinal mucosa of the white mouse for the same reason. The writer prefers the pancreas because it is so much more easily fixed than the mucosa of the mouse; if the in- testine is used it must be split up, and the piece pinned out flat, to enable the fixa- tive to reach the epithehum. It does not matter which particular animal is taken, but if mitochondria are to be well demonstrated, it should be in good health and should be killed by a blow on the head rather than by narcotic drugs. The pancreas is most readily ob- tained in the following manner. Tie or pin out the animal on its back, re- move the whole of the skin from the ab- domen without breaking through the muscular wall of the abdominal cavity, and then remove the wall of the abdominal cavity by an incision made round its periphery. The very considerable loss of blood which results from this may be ig- nored. Now spread the pancreas on a piece of glass by the following method. Hold the glass by the edges in the right hand, and Uft the stomach and upper portion of the small intestine with the left hand, the in- testine being spread away from the stomach with the fingers. This leaves the pancreas stretched in the mesentery. Now lay the sheet of glass flat against the mesentery, with the pancreas spread on the surface, and then pass the stomach and intestine from left to right, while tipping the glass sUde upward, so that the weight of the stomach and of the intes- tine hanging down keep the mesentery stretched over the surface. Run a sharp knife round the edge of the slide so as to cut through the mesentery, lea^dng the pancreas exposed on the glass surface. It is, naturally, necessary to lower the right hand to prevent the weight of the stomach and intestine from dragging the mesentery off the glass when the first cut is made, but a httle practice will enable one to ob- tain the material spread cleanly on the glass slide with httle trouble. Place the slide, with its adherent mesentery flat in a glass dish (naturally with the pancreas on its upper surface) and flood with the fixative. The classical technique of Kull requires the osmic-chromic-dichromate- pyrohgneous fixative of Champy 1913 (Chapter 18, F 1670.0080), which is ap- 440 METHODS AND FORMULAS DS 22.20 plied in three separate stages. The first solution contains 3 grams of potassium dichromate, 1.15 grams of chromic acid, and 1.25 grams of osmic acid in 250 milh- hters of water. This is poured over the pancreas on the sUde and placed in a dark cupboard for about 24 hours. At the con- clusion of this period the fixative is poured off (it may be used many times), the dish filled with distilled water, and rocked gently backward and forward for about half an hour. The pancreas and mesentery will usually become detached from the glass at this stage, and the glass may be withdrawn. The distilled water is then re- placed with the second solution of Champy, which contains 2.5 grams of chromic acid dissolved in a mixture of 35 millihters of pyroligneous acid and 175 milhhters of water. Pyroligneous acid, it may be said, is the product of the dry destructive distillation of wood, and con- tains a mixture of various creosotes and tars in a weak solution of acetic acid. It is unfortunately not at present known which of its various constituents exercise the re- quired effect, but it may be stated quite categorically that weak solutions of acetic acid cannot be substituted for it. This solution should be allowed to act for about 20 hours and the specimen again washed in distilled water for about 30 minutes. At this point the pancreas may be cut into small pieces with a pair of sharp scis- sors. Pieces of about 3-millimeter side should be selected and removed from the distilled water in which the cutting is done to another vial of distilled water. After enough pieces have been selected, the dis- tilled water is poured off and replaced with 3% potassium dichromate for three or four days before being washed in run- ning water for 24 hours. Fixation is now complete. Many of the modifications which have been suggested are for the purpose of diminishing the time in the dichromate bath, by using various di- chromate fixatives at higher temperatures. The method of Champy, however, is reli- able, and though it is slow and cumbrous, it yields far more certain results than many of its modern, more complicated variants. After washing in running water for 24 hours, the specimens are dehydrated and embedded in paraffin in the customary manner before being sectioned. It is recommended that sections about five microns in thickness be employed, and that these be mounted on the shdes in the customary manner. It is now necessary to make the acid fuchsin stain of Altmann 1890 (DS 22.2 Altmann 1890) about which there has been considerable controversy. This solution is prepared by dissolving 20 grams of acid fuchsin in 100 cc. of "aniline water," which is itself a saturated solution of aniline in water. It is better that the solu- tion be made when a little free aniline is present, since the solubihty of acid fuchsin in anihne is far higher than in water. Take, therefore, 100 grams of water, add to it about five milhliters of anihne, and shake vigorously for a few moments. Throw in 20 grams of acid fuchsin, shake vigorously for a few moments, and lay on one side in a warm place. Shake the bottle at inter- vals during the next 24 hours, at the end of wliich time the acid fuchsin (despite statements to the contrary in the Utera- ture) will be found to have dissolved. The failure of some investigators to cause more than a few grams of acid fuchsin to go into solution (see for example the remarks of Gatenby and Painter 1937, 305) is prob- ably due to tlie fact that they first pre- pared a saturated solution of anihne in water, and then endeavored to dissolve the acid fuchsin into it. A mixture of ani- line and water will dissolve, possibly by a process of mutual soluliihty, the required quantity of acid fuchsin. This point must be insisted upon, since tlie entire success of the preparation depends upon the main- tenance of a very high concentration of acid fuchsin in the solution first used for staining. The other staining solutions re- quired in this method are a 0.5% solu- tion of toluidine blue in water and a 0.5% solution of aurantia in 70% alco- hol; neither present any difficulty of preparation. Now take one of the shdes, drain off the water so far as possible, and blot round the section with filter paper before flooding the slide with acid fuchsin. The draining and blotting are necessary, since DS 22.20-DS 22.21 DYE STAINS OF SPECIAL APPLICATION 441 the high concentration of acid fuclisin will not be maintained if it is poured onto a slide over which are gathered films and droplets of water. Lay the slide on a support of some kind and wave a bunsen flame backward and forward underneath it, until steam rises from the surface of the stain; no bubbles must bo i)ermitted to form. Neither the time of staining nor the exact temperature are in any way critical; as soon as the slide has started to steam, the flame is removed and the slide left until it is cool enough to handle. Then use a jet of water from a wash bottle to re- move the excess acid fuchsin. The sections will now be stained a dense purple red, and are transferred to the solution of toluidine blue in a coplin jar for one to two minutes. The purpose of the toluidine blue is to remove the acid fuchsin from the nuclei and to replace it with the blue stain. After one or two minutes pick out the slide, rinse it in distilled water, and place it in the aurantia until the mito- chondria are differentiated through the removal of the acid fuchsin from the cytoplasm around them, and the sulisti- tution of the clear yellow color of the aurantia for this pink color. This process may only with difficulty be controlled under the microscope. It is better to leave a trial slide in the aurantia for two min- utes, to remove it, dehydrate it in a]:)Solute alcohol for the minimum time possible, and then to transfer it to xylene before examining it under the high power of the microscope. If the timing in the toluidine blue and aurantia solutions has been cor- rect, the nuclei will be a vivid, bright blue, the cytoi)lasiu clear yellow, and the mitochondria vivid scarlet. If the nuclei are still purple, and the mitochondria are not clearly differentiated in scarlet, the next trial slide should be left longer in toluidine blue. If, however, the only defect is that the cytoplasm remains a clear pink, the time in aurantia should be increased. By thus juggling the times in the toluidine blue and the aurantia (it is a waste of time to try to control the timing in the acid fuchsin or in the alcohols), it is possible to arrive at a schedule which yields perfect preparations. Once this schedule has been established, it is possible to prepare a long series of slides, in each which there is clear differentiation of nuclei, mito- chondria, and cytoplasm. It must be pointed out that the staining technique of Altmann, which has been used, can also be used to stain bacteria, and that if the sections are mounted on the slide with an excess of either gelatin or egg albumin, and are then ])ermitted to remain damp and warm for 24 hours, bacteria will un- doul)tedly be found, and also that these bacteria have given rise to certain inaccu- rate observations. This can be avoided, however, either by using an adeciuate quantity of a biostatic agent in the adhesive used for the sections, or by avoid- ing leaving them under conditions which will encourage bacterial growth. 22.21 OTHER TECHNIQUES 22.21 Altmann 1890 test. 1928 Gatenby and Cowdry Gatenhy and Cowdry 1928, 3.33 REAGENTS required; A. sat. sol. aniline 100, acid fuchsin 20; B. water 65, sat. sol. picric acid in 95% ale. 35 method: [sections of F 1700.0000 Altmann 1890 fixed material]-^ water A, on slide, heated to steaming, 1 min. — » B, on slide, heated to steaming, till differentiated — > blot ^ abs. ale, minimum possible time — » balsam, via xylene result: mitochondria red on yellow. note: This method is sometimes erroneously referred to Zimmerman. For a slight modi- fication of this procedure see DS 21.421 Severinghaus 1932. Kiyono 1914 (236S1, 25:481) states that formaldehyde-fixed material t-an be ytained by this technique if the sections are treated 2-3 days at 37°C. in AMS 12.2 Kiyono 1914. For an adapta- tion of this technique to blood smears see DS 2.28 Schmorl 1928. Baker 1932 (19400, 30:134) recommends heating tissues intended for this technique with 0.05% to 0.5% parabenzoquinone in isotonic saline for 1 hour prior to fixation. For a detailed de- scription of the preparation of the .4 sol. see under DS 22.20 above. 22.21 Bailey 1920 11343,42:353 REAGENTS REQUIRED: A. DS 22.21 Altnuiuu 1890 (sol. A); B. water 100, acid'violet 1, 10% sulfuric acid q.s. 442 METHODS AND FORMULAS DS 22.21 PREPARATION OF B: Add the acid drop by drop to the dye solution until no further in- crease in intensity of color takes place. method: [sections of F 7000.1000 Regaud 1910 fixed material] -^ A, on slide warmed to steaming, 6 mins. -^ drain -^ water, quick rinse -^ B, on slide, 5 sees. -^ drain — > abs. ale, till differentiated, few sees. —* carbol-xylene — > xylene -^ balsam 22.21 Baker 1933 see DS 22.21 KuU 1914 22.21 Baker 1944 17510, 85:1 REAGENTS REQUIRED: A. water 90, 40% formaldehyde 10, cadmium chloride 1; B. 25% gelatin; C. 2.5% gelatin; D. sat. sol. Sudan black in 70% ale; E. DS 11.21 Mayer 1892 method: [fresh tissues]-^ A, 3 days -^ wash -^ B, 24 hrs., 37°C. ^ [cast as block] -^^ A, 24 hrs. -^ wash —> [15 fi frozen sections, fixed on slide with C] -^ A, till required wash — > 70%, ale. — > D, 5-10 mins. -^50% ale, rinse — » E, till nuclei stained —>■ wash -^ M 11 mountant (Chapter 26) RECOMMENDED FOR: Golgi bodies. 22.21 Baker and Thomas 1933 Baker 1933 REAGENTS REQUIRED: A. DS 22.21 Altmann 1890, sol. A; B. water 65, ale. 35, picric acid 2; C. water 65, ale. 35, picric acid 2 method: [5 M sections of F 1700.0000 Baker and Thomas 1933 fixed material] -^ water -^ A, flooded on slide and heated till steaming, 1 min. — >• cool, 5 mins. -^ B, 30 sees. — > C, till differentiation complete -^ wash — > balsam, via usual reagents 22.21 Benda 1903 7936a, 12 :752 REAGENTS REQUIRED: A. 4% ferric alum; B. water 100, sat. sol. alizarin in abs. ale. 1; C. water 55, sat. sol. aniline 25, hydrochloric acid 0.025, 95% ale. 7.5, sat. sol. crystal violet in 70% ale. 12.5; D. 30% acetic acid method: [sections from material fixed in F 1670.0018 Benda 1903, F 1670.0080 Champy 1913, or F 7000.1000 Regaud 1910] -^ water -* A, 2-5 hrs. -* water, quick rinse ^ B, poured on slide, heated to steaming, 1 min. -^ C, poured on slide, heated to steaming, 1 min. —> water, quick rinse — > D, till nuclei well defined -^ running water, 10 mins. — * blot -^ abs. ale, quick rinse -^ carbol-xylene — > balsam, via xylene result: cell inclusions purple on yellow. 22.21 Bensley 1911 see DS 22.21 Cowdry 1918 (note) 22.21 Benoit 1922 see DS 22.21 Cowdry 1918 (note) 22.21 Cain 1948 17510,89:229 REAGENTS REQUIRED: A. 0.5% iodine in 70% ale; B. 5% sodium thiosulfate; C. DS 22.21 Altmann 1890 (sol. A); D. 0.1% sodium carbonate; E. 1% hydrochloric acid; F. 1 % methyl blue method: [3 M sections of F3700.1000 Helly 1903 fixed material] — > water -^ A. 5 mins. — » rinse — > B, 5 mins. —>■ wash —> C, on slide, heated to steaming, 1 min. -^ E, few sees, [repeat D -^ E cycle if insufficiently differentiated] -^ wash — > F, till counter- stained — > wash -^ E, brief dip — > balsam, via usual reagents 22.21 Cansey 1925 21400a, 44:156 REAGENTS REQUIRED: A. 5% fcrric alum; B. DS 11.111 Regaud 1910 (sol. B) method: [osmic-fixed protozoans] — > A, 30 mins. — > rinse — > B, 45 mins. -^ A, till differ- entiated — > balsam, via usual reagents RECOMMENDED FOR: mitochoudria in protozoans. 22.21 Champy-Kull see DS 22.21 Kull 1914 22.21 Cowdry 1918 6816, 8 REAGENTS REQUIRED: A. DS 22.21 Altmann 1890, A sol.; B. \% methjd green method: [sections of F 7000.1000 Regaud 1910 material] -+ water —> .4, poured on slide, warmed to steaming, G mins. -^ wipe round sections -^ water, quick rinse — ^ B, on slide, 5 sees. -^ drain — ' abs. ale, till differentiated -^ balsam, via toluene result: mitochondria, purple-red on green. DS 22.21 DYE STAINS OF SPECIAL APPLICATION 443 note: The method of Benoit 1922 (6(530, 86 :1 101) differs only in specifying prior fixation in his F 1379.0000 fixcative; that of Bensley 1911 (590, 12:297) in requiring his F 1700.0010. Gough and Fulton 1929 (11431, 32:765) treat sections of formaldehyde- fixed material with their ADS 12.2 mordant before this technique. 22.21 Champy 1911 see DS 22.21 KuU 1913 22.21 Drew 1920 11360, 40:295 REAGENTS required: A. Drew 1920 ADS 12.1; B. 3% ferric alum; C. 0.5% hematoxylin; D. 2% pyridin method: [sections by freezing technique of Drew 1920 F 9000.1000 material] -^ wash — > A, 15 mins. to 1 hr. 50°C. — > rinse — ♦ B, 15 mins. 50°C. — > quick rinse -+ C, 15 mins. 50°C. — » B, till differentiated, room temperature — > i>, 2 mins. — > balsam via usual reagents 22.21 Dufrenoy 1929 see DS 22.11 Milovidov 1928 (note) 22.21 Fain and Wolfe 1939 see DS 11.421 Fain and Wolfe 1939 22.21 Fieandt see DS 21.23 Fieandt (1933) 22.21 Gough and Fulton 1929 see ADS 12.2 Gough and Fulton 1929 22.21 Held test. 1900 Pollack Pollack 1900 reagents required: A. water 100, acetic acid 0.1, erythrosin 0.7; B. water 50 DS 22.3 Nissl 1898 47, acetone 3; C. 0.1% potassium alum method: [sections] -^ water —» a, 2 mins. on slide while warming —» cool —> water, quick rinse — > B, on slide, warmed till no further smell of acetone -^ cool — > C, till sections turn reddish — > water, quick rinse —> blot — » neutral mountant, via propyl ale. result: nuclei, red; nucleoli, nissl granules, mitochondria, blue; plasma, reddish. 22.21 Hirsch and Bretschneider 1938 test. 1948 Romeis Romeis 1948, 233 reagents required: A. 3% ferric alum; B. 0.5% hematoxylin method: [sections of F 7000.1000 R6gaud fixed 1910 material] — * water ^^ A, }i-l hr. 60°C. — > quick wash — * B, 10-20 mins. 60°C. -^ thorough wash — » A, till differentiated 22.21 Hollande 1930 6630, 104:473 reagents required: A. DS 22.21 Altmann 1890, sol. A;B. 0.5% phosphomolybdic acid; C. 1% methylene blue method: [5 M sections of F 3790.1000 Benoit 1922 fixed material, varnished on slide with 5% collodion] —>■ water -^ A, 30 mins. -^ brief wash -^ B, 5 mins. —>■ C, 10-20 mins. -^ rinse -^ abs. ale, 1 min. — * amyl ale. till differentiated —> balsam, via xylene 22.21 KuU 1913 see DS 13.22 KuU 1913 22.21 KuU 1914 766, 45:153 reagents required: A. DS 21.21 Altmann 1890, sol. A; B. 0.5% toluidine blue; C. 0.5% aurantia in 70% ale. method: [sections of F 1670.0090 Champy 1913 fixed material] —> A, on slide, heated to steaming, 1 min. —y water, quick rinse —>■ B, 1-2 mins. — > water, quick rinse -^ C, till mitochondria differentiated — > abs. ale, minimum possible time — ♦ balsam, via xylene note: This technique is often called Champy-Kull. Baker 1933 (p. 189) recommends fixation in F 3700.1000 Helly 1903, followed by "post-chroming" by the method of Parat 1926a below. A detailed description of the application of this technique is given under DS 22.20 above. 22.21 McManns 1946 11431, 58:93 reagents required: A. sat. sol. Sudan black in 70% ale. method: [sections of material fixed 1 month in F 8000.1000 McManns 1946] -^ 70% ale. — > A, 30 mins. -^ M 11.1 mountant note: This also stains myelin and some fat granules. 444 METHODS AND FORMULAS DS 22.21-DS 22.3 22.21 Martinotti 1910 23632, 27 :24 REAGENTS REQUIRED: A. water 75, lithium carbonate 0.5, toluidine blue 1.0, glycerol 20 95% alcohol 5; B. 1% acetic acid preparation: Dissolve in order given and ripen 1 month. method: [distilled water] — » A, overnight —» B, till required inclusions differentiated. 22.21 Milovidov 1928 6630, 98 :555 REAGENTS REQUIRED: A. DS 22.21 Altmann (1920); B. 0.5% aurantia in 70% ale; C. water 100, phosphomolybdic acid 1, sodium hydroxide 0.1; D. DS 11.44 Unna 1892 25, water 75 method: [paraffin sections of F 3700.1000 Milovidov 1928 fixed material] — » abs. ale. — > [varnish in thin collodion] -^ water — ^ A, 80°C., 1-3 mins. -^ wash — > B, few sees. — * wash —* D, few mins. — > rinse — > D, few mins. — > wash -^ balsam, via usual reagents result: mitochondria red; bacteria blue. recommended for: differentiation of mitochondria and bacteria in root nodules of legumes. note: This was reprinted in English by Dufrenoy 1929 (20540b, 4:13) to whom it is often attributed. 22.21 Parat 1926 4285a, 3 :222 REAGENTS REQUIRED: A. DS 11.113 Kultschitzky 1889; B. ADS 21.1 Weigert 1885 method: [sections of F 3700.1000 fixed, and Parat 1926a (below) mordanted material] -^ A, 24 hrs. 37°C. — ♦ B, till mitochondria differentiated 22.21 Parat 1926a 4285a, 3 :220 Any method is commonly referred to as Parat in which small pieces of fixed material are treated with a sat. sol. potassium dichromate for 24-48 hours at 40°C. before being sectioned and stained. 22.21 Volkonsky 1928 4295a, 5 :220 REAGENTS REQUIRED: A. DS 22.21 Altmann 1890, A sol.; B. 0.5% aurantia in 70% ale; C. 1% phosphomolybdic acid; D. DS 11.44 Unna 1892; E. DS 12.16 Unna 1895 method: [sections of F 3700.1000 Helly 1903 fixed and Parat 1926a treated material] -> water — * A, on slide, heated to steaming, 1 min. — > B, till mitochondria differentiated — *■ C, 1 min. — * water, wash — * D, 5-10 mins. -^ water, rinse — > E, till nuclei differ- entiated — » balsam, via usual reagents 22.21 Volkonsky 1933 test. 1942 Langeron Langeron 1942, 1105 REAGENTS REQUIRED: A. Altmann 1890, A sol.; B. water 55, N/1 sodium hydroxide 10, phosphomolybdic acid 1, 95% ale. 35, aurantia 0.25; C. DS 11.44 Volkonsky 1933; D. DS 12.16 Unna 1895 PREPARATION OF B : Dissolve the acid in 30 water. Add the hydroxide. Dissolve the dye in 25 water 35 ale. Add this solution to the first. method: [sections prepared as for Volkonsky 1928 above] -^^ water —» ^, on slide, heated to steaming, 1 min. — » water, rinse — > B, till mitochondria differentiated — > water, rinse— > C, 10-15 mins. -^ water, rinse —> D, till nuclei differentiated-^ abs. ale., minimum possible time -^ balsam, via xylene 22.21 Zimmermann 1896 see DS 22.5 Zimmermann 1896 22.3 NissL Granules Nissl bodies were originally described by Nissl 1894, who used an alkaline solution of methylene blue for their display. His^formula has generally been followed save that toluidine blue has been substituted by most of the recent workers. Deipolli and Pomerri 1938 have substituted magenta for the blue dye in a method which will show mito- chondria and bacteria as well as Nissl bodies. Attention should also be drawn to the methods of Einarson 1932 and 1935 which use two of the oxazine dyes. The best method forjdemonstration is, however, that of Windle, Rhines, and Rankin 1943 who have worked out the satisfactory pH at which staining will take place after a series of given fixatives. DS 22.3 DYE STAINS OF SPECIAL APPLICATION 445 22.3 Addison 1929 cit. compl. script. McClung 1929, 326 foRxMUla: 1% thionin (or toluidine blue or methylene blue or cresyl violet) method: [sections of material fixed in 5% acetic in 95% ale] — + stain, 6-12 hrs. — > water, quick rinse — » abs. ale. till differentiated — » balsam, via xylene note: Spielmeyer 1924, 69, recommends a 0.1% dye solution, used hot. 22.3 Anderson 1929 Anderson 1929, 42 REAGENTS REQUIRED: A. 1% toluidine blue; B. ADS 21.1 Gothard 1898 method: [10 fx sections] — * water — > A, 10-20 mins., 50°C. —y wash -^ B, till differenti- ated — > abs. ale. till dehydrated — > xylene — > [repeat B —y abs. — ♦ xylene, until re- quired picture secured] — > cedar oil 22.3 Bean 1926 20540b, 1 :56 REAGENTS REQUIRED: A. either 1% neutral red or 1% methylene blue; B. either 0.5% methj'l orange or 0.5% eosin in 50% ale. method: [12 jLi sections of F 0000.1010 Bean 1926 fixed material] -^ water — » A, 90°- iOO°C., in water bath, 20 mins. -^ 50% ale, till differentiated —> B, few sees. -^ balsam, via usual reagents 22.3 Clark and Sperry 1945 20540b, 20 :23 reagents required: A. 0.05% lithium carbonate; B. 0.25% thionin in 0.05% lithium carbonate method: [celloidin sections] — * ^4, 5 mins. — > B, till grossly overstained -^ rinse -^80% ale, till differentiated -^ balsam, via butyl ale. and xylene 22.3 Deipolli and Pomerri 1938 14425, 49:123 REAGENTS REQUIRED: A. Water 100, DS 11.43 Ziehl 1882 2.5, acetic acid 0.5; B. water 100, 40% formaldehyde 1, acetic acid 1 method: [sections of alcohol or formaldehyde-fixed material] — > waters A, 3-4 mins. -^ water, quick rinse -^ B, till Nissl bodies differentiated -^ water, wash — > balsam, via usual reagents 22.3 Einarson 1932 608b, 8:295 formula: water 100, chrome alum 5, gallocyanin 0.15 preparation: Boil 20 minutes. Cool. Filter. method: [50 n celloidin sections of F 3700.0010 Zenker 1894 fixed material] —>■ water — » stain, 12-24 hrs. -^ water, wash — > 80% ale, wash -^^ 95% ale, 1 hr. -^ abs. ale, till dehydrated — » abs. ale. and ether, till celloidin removed -^ abs. ale, wash — » — > balsam, via oil of Cretan thyme 22.3 Einarson 1936 11135, 61:105 formula: water 100, gallamin blue 0.2 preparation: Boil 5 minutes. Cool. Filter. method: [sections] —>■ water — » stain 12-24 hrs. —> water, wash -^50% ale, till differ- entiated — > 95% ale, — » balsam, via usual reagents 22.3 Gothard 1898 6630, 5 :530 reagents required: 4. DS 11.44 Unna 1892; B. ADS 21.2 Gothard 1898 method: [celloidin sections of alcohol-fixed material]-^ A, 24 hrs. -^ 80% ale, quick rinse -^ B, till differentiated — * balsam, via usual reagents 22.3 Hansburg 1935 19938,81:364 REAGENTS REQUIRED: A. DS 13.12 Wright 1910 100, DS 13.13 Giemsa 1902 20 method: [10 n paraffin sections] —^ water —»• A, on slide, 2 mins. — » dilute ^4 on slide to 3 times volume, leave 2 mins. — » wash, 1 min. — » 80% ale, 15 sees. — > abs. ale, least possible time -^ balsam, via xylene RECOMMENDED FOR: Nissl granules in spinal cord. 22.3 Held 1900 see DS 22.21 Held 1900 22.3 Huber test. 1943 Cowdry cit. Addison Cowdry 1943, 95 REAGENTS REQUIRED: A. 0.1% toluidiuc bluc; B. 0.05% lithium c;irbonate method: [sections of F 3000.0020 Huber (1943) fixed material]^ A, 15-18 hrs. -» water, wash -^ B, 2 hrs. — * 70% ale, till differentiated — > balsam, via usual reagents 446 METHODS AND FORMULAS DS 22.3 22.3 Johnson 1916 see DS 22.3 Kirkman 1932 (note) 22.3 King 1910 763, 4:213 formula: 1% phenol 100, thionin to sat. method: [sections] -^ stain, 2-3 mins. -^ water, quick rinses 95% ale, till differ- entiated — > balsam, via usual reagents 22.3 Keller 1945 4349,26:77 REAGENTS REQUIRED: A. 0.5% cresyl violet; B. 50% Canada balsam in xylene method: [sections] -^ A, 3-5 mins. —> wash — * xylene, via graded ales. -* B, 2 mins. — > xylene — > [repeat abs. ale. — > J5 — » xylene cycle till differentiation complete] — > balsam 22.3 Kirkman 1932 763, 51 :323 formula: water 100, neutral red 0.2, acetic acid 0.4 method: [sections stained by DS 21.212 Weigert, or similar technique] -♦ water —> stain, 10-20 mins. -^ water, wash 95% ale, till differentiated-^ balsam, via usual reagents note: The technique of Johnson 1916 (763, 11 :297) was essentially the same, with the additional provision that the neutral red be diluted from a 1 % solution which has been "ripened" 1-4 years. 22.3 Lugaro 1905 19219,3:339 reagents required: A. 0.05% toluidine blue; B. 4% ammonium molybdate method: [sections of alcohol, or acetic alcohol, fixed material] — > A, 1-3 hrs. — » water, rinse — » B, 15 mins. abs. ale. till differentiated — > balsam, via benzene 22.3 Nissl 1894 15058, 13 :507 reagents required: A. water 100, castile soap 0.2, methylene 0.4; B. 10% aniline in 95% ale. preparation of a: Dissolve soap in water. Add dye. "Ripen" some months. method: [sections of alcohol-fixed material] -^ stain, in watch glass, heated till bubbles form — > B, in watch glass, till no more color comes away -* transfer to slide and blot -^ oil of cajeput, till clear -^ balsam note: The original calls for venetianische Seife which was presumably the Sapo venetus of the Austrian pharmacopeia of the period. This is identical in composition with the sapo durus of the current pharmacopeia, commonly known as castile soap. Venetian soap, specified by most texts, is a meaningless term in English. 22.3 Roussy and Lehrmitte test. 1938 Carleton and Leach Carleton and Leach 1938, 243 REAGENTS REQUIRED: A. 1% thiouin, B. ADS 21.2 Gothard 1898 method: [sections of formaldehyde-fixed material] -^ water -^ ^, 6 mins. -+ rinse —» 95% ale, 1 min. -^ B, till differentiated -* abs. ale. — > balsam, via xylene 22.3 Sadorsky test. 1900 Pollack Pollack 1900, 100 REAGENTS REQUIRED: A. 1% methylene blue ; B. 1% acetic acid method: [sections of formaldehyde-fixed material]^ A, overnight-^ B, till differenti- ated 22.3 Snider 1943 20540b, 18:53 formula: water 15, 95% ale. 20, dioxane 65, toluidine blue 1 preparation: Dissolve dye in water and ale. Add dioxane. method: [1 cm. thick slabs of fresh brain] -^ stain, 3-5 days -^ [50 n sections by freezing technique, fixed to slide with albumen] -^95% ale., till differentiated-^ balsam, via usual reagents 22.3 Spielmeyer 1924 see DS 22.3 Addison 1929 (note) 22.3 Tsiminakis 1928 23632, 45 :50 reagents required: A. 0.3% potassium permanganate; B. water 100, oxalic acid 0.5, sodium sulfite 0.5; C. 0.5% toluidine blue; D. 30% aniline in 95% ale. method: [sections of chrome-fixed material] -> water -^ A, 5 mins. -> B, 10-15 mins. -» wash -^ C, 4 mins. -^ wash, 10 mins. —^ D, till differentiated -♦ balsam, via terpineol and xylene DS 22.3-DS 22.4 DYE STAINS OF SPECIAL APPLICATION 447 22.3 Windle, Rhines, and Rankin 1943 20540b, 18 :77 STOCK solutions: I. acetate-veronal buffer, pH 3.65. II. 1% thionin WORKING solution: stock I 100, stock II 5 method: [sections from formaldehyde or F 0000.0010 Carnoy 1887 fixed material]-* water-* stain, 10-20 miiis. —> water, till no more color comes away — > 70% ale. till no more color comes away -* balsam, via usual reagents 22.4 Yolk and Fat Geanules Techniques for staining fat granules may be broadly divided into two classes. In the first of these, fat-soluble stains such as Sudan II, or Sudan black B, are applied to sec- tions obtained by the freezing technique, in which the fat is left in its original condition. The other class consists of those in which fixation has taken place with a chromate- or dichromate-formaldehyde mixture, with the result that certain of the unsaturated fatty acids are rendered insoluble in the solvents used for paraffin sectioning, and may thus be stained by almost any method which will bring out small objects. None of the tech- niques given here are intended to be microchemical tests for the various classes of fat. They are strictly histological methods to be used by those who desire to demonstrate the existence of granules rather than to demonstrate the nature of the materials of which the granules are composed. 22.4 Arndt 1925 see DS 22.6 Arndt 1925 22.4 Bacsich 1936 see DS 21.3 Bacsich 1936 22.4 Bell 1914 11431, 19:105 REAGENTS REQUIRED: DS 22.4 Daddi 1896 method: [paraffin sections of F 7000.0010 Tellyesniczky 1898 fixed material] ^70% ale. — > stain 10 mins. — » 50% ale. till differentiated -^ water ^^ Mil mountant (Chapter 26) note: Mulon 1914 (6593, 6:12) differs from above in specifying F 5000.1010 Bouin 1897 fixation and DS 22.4 Herxheimer 1904 stain. 22.4 Benda test. 1911 Mallory and Wright Mallory and Wright 1911, 386 REAGENTS REQUIRED: A. Sat. sol. hematoxylin in 60% ale; B. ADS 21.1 Weigert 1885 method: [frozen sections of material fixed in F 4600.1010 Benda (1911)] -^ water -^ A, 6 hrs. -^ water, wash —>■ B, till differentiated — » [balsam, via usual reagents] or — > water-* Mil mountant. 22.4 Burden, Stokes, and Kimbrough 1942 see DS 23.219 Burdon, et al. 1942 22.4 Clark 1947 11431, 59:337 reagents required: A. 95% ale. 50, acetone 50, Sudan IV to sat.; B. DS 11.123 Ehrlich 1886; C. 0.1% hydrochloric acid in 70% ale. method: [sections by E 11.1 Clark 1947 (Chapter 27)]-* rinse -^ 70% ale. -^ A, 3-5 mins.—* 70% ale, wash-* rinse—* B, 10 mins.—* wash -^ C, till differentiated—* tap water, till blue -^ M 12.2 Moore 1933 22.4 Daddi 1896 1852, 26:143 formula: 70% ale. 100, Sudan III to sat (circ. 0.2%) method: [sections by freezing technique] — > water —> stain, 5-10 mins. — * water, wash — » Mil mountant (Chapter 26) note: Rosenthal 1900 specifies fixation in his F 3000.1000 before this technique. 22.4 Dietrich 1910 22264, 14 :263 reagents required: A. 5% potassium dichromate; B. DS 11.113 Kultschitzky 1889; C. ADS 21.1 Weigert 1885 method: [frozen sections of formaldehyde-fixed material] —* il, 1-3 days, 37°C. — > water, wash -^ C, till differentiated (overnight) —» water, wash—* Mil mountant (Chapter 26) 448 METHODS AND FORMULAS DS 22.4 22.4 Fischler 1904 23681, 16:913 REAGPLVTs rp^quired: A. 12.5% nipric acetate; B. DS 21.212 Weigert 1885 (sol. C); C. ADS 21.1 Weigert 1885 5, water 95 method: [sections by freezing technique of formaldehyde-fixed material] — » water — > A, 2-24 hrs. -* wash -^ B, 20 mins. -^ rinse — ► C, till differentiated -^ Mil or 12 moun- tant (Chapter 26) 22.4 French 1940 1887a, 30:1243 REAGENTS REQUIRED: A. 4% formaldehyde; B. water, 10 DS 21.13 Weigert 1898 50, sat. sol. Sudan III in equal parts acetone and 70% ale. 40; C. any DS 11.122 stain method: [gelatin sections of formaldehyde-fi.xed material] -^ A, 1 day -^ wash -^70% ale, rinse— > A, 15 mins. — > 70% ale, rinse —> water, rinse —> C, 1-2 mins. — > wash -^ M 12.1 mountant RECOMMENDED FOR: simultaneous demonstration of fat and elastic tissue. 22.4 Galesesco and Bratiano 1928 6630, 99:1460 PREPARATION OF STAINING SOLUTION: Digest 50 Gm. fragments of cortical zone of carrots in 100 ml. 95% ale. 2 hours on water bath. Cool. Digest in dark at room temperature 8-10 days. Filter. Dilute to 80% ale. content. REAGENTS REQUIRED: A. Staining solution from above; B. DS 11.122 Bohmer 1868 method: [frozen sections of formaldehyde-fixed material] — > 70% ale. -^ A, 6-24 hrs. —* 70% ale. till no more color comes away ^^ water —> B, till nuclei blue — > wash — > M 12.1 mountant (Chapter 26) 22.4 Gros 1930 23632, 47 :64 reagents required: A. water 50, diacetin 50, scarlet R 0.6 preparation: Heat to 60°C. Cool. Filter. method: [frozen sections] —> water ^ A, 10 mins. 60°C. -^ wash -^ Mil mountant (Chapter 26) 22.4 Herxheimer 1901 23681, 14:891 formula: 80% ale. 100, sodium hydroxide 2, Sudan IV 0.1 method: [sections by freezing technique of formaldehyde-fixed material] — >• 70% ale. —* stain, 2-4 mins. — > DS 11.122 counterstain, if desired — > M12 mountant (Chapter 26) note: Herxheimer 1904 (23632, 21:57) substituted a mixture of equal parts 70% ale. and acetone for the alkaline alcohol; this is the more usually cited formula. In the place cited above he also recommends a sat. sol. indophenol (probably indophenol blue— Conn 1946, 87) in 70% ale. 22.4 Herxheimer 1904 see DS 22.4 Herxheimer 1901 22.4 Kionka 1894 764,1:414 REAGENTS REQUIRED: .4. DS 11.22 Grenacher 1879; B. water 70, 95% ale. 30, orange G 0.05, hydrochloric acid 0.1 method: [paraffin sections of ale. fixed chicken blastoderms] — > A, overnight—* quick rinse -^ B, till differentiated — > balsam, via usual reagents recommended for: yolk granules in embryonic cells. 22.4 Leach 1938 11431, 47:635 REAGENTS REQUIRED: A. 50% diacetin; B. water 50, diacetin 50, Sudan black B to excess PREPARATION OF B: Digest 2 days at 55°C. Cool. Filter. method: [sections by freezing technique of F 1670.0000 Zweibaum (1943) fixed mate- rial] -+ A, 30 sees. -^ B, 2 hrs. -^ A, 30 sees. — > DS 11.21 counterstain, if desired — > Mil or 12 mountant (Chapter 26) 22.4 Lillie 1945 20540b, 20:7 REAGENTS REQUIRED: A. sat. sol. oil blue N in 60% isopropyl ale. 85, water 15; B. 0.1 % Bismarck brown R; C. 5% acetic acid method: [frozen sections of formaldehyde-fixed material] —> A, 5-10 mins. — ♦ water, rinse -^ C, till differentiated, 1 min. -^ water — > M12 mountant NOTE : The dilution of A should be made immediately before use. DS 22.4 DYE STAINS OF SPECIAL APPLICATION 449 22.4 Lillie 1948a Lillie 1948, 159 PREPARATION OF STOCK SOLUTION: Water 40, isopropyl ale. 60, oil blue N to sat. REAGENTS REQUIRED: A. stock 60, water 30; 5. 0.1% Bismarck brown; C. 5% acetic acid method: [frozen sections] -^ A, 5-10 mins. — > wash -^ B, 5 mins. — » rinse — > C, 1 min. — > wash — » Mil mountant 22.4 Lillie 1948b Lillie 1948, 159 PREPARATION OF STOCK SOLUTION: A. isopropyl alc. 100, Sudan brown or oil red 4B or oil red O 1.5 REAGENTS REQUIRED: A. stock 60, Water 40; B. water 80, acetic acid 1.6, DS11.122 Lillie 1941 20; C. 1% sodium phosphate, dibasic method: [frozen sections] — > water— > A, 10 mins. —>■ wash — > 5, 5 mins. -+ rinse — > C, till blue — > wash -^ Mil or Iz mountant (Chapter 26) 22.4 Lillie and Ashburn 1943 1780a, 36:432 formula: sat. sol. Sudan IV in isopropyl alc. 50, water 50 method: [frozen sections of formaldehyde-fixed material] —> water ^ A, 10 mins. — > water, wash -^ DS 11.123 counterstain if desired -^ balsam, via usual reagents 22.4 Martinotti 1914 23543, 91 :425 reagents required: A. 1% chrysoidin; B. 1% potassium dichromate method: [frozen sections of formaldehyde-fixed tissue]—^ water -^ A, some hrs. — > water, rinse — > B, 1 min. — > water, wash — * balsam, via usual reagents 22.4 McManus 1946 see DS 22.21 McManus 1946 22.4 Mulon 1914 see DS 22.4 Bell 1914 (note) 22.4 Peter 1904 see DS 11.25 Peter 1904 22.4 Proescher 1927 20540b, 2 :60 reagents required: A. 50% pyridine; B. water 30, pyridine 70, oil red 0 4; C. 60% pyridine PREPARATION OF B: Add dye to mixed solvents. Leave 1 hour with occasional agitation. FUter. method: [frozen sections of formaldehyde-fixed material]—* A, 3-5 mins. — * B, 3-5 mins. -^ C, till differentiated -^ water —> Mil or 12 mountant (Chapter 26) 22.4 Rosenthal 1900 see DS 22.4 Daddi 1896 (note) 22.4 Schaffer 1926 test. 1933 Cajal and de Castro Cajal and de Castro 1933, 305 reagents required: A. 5% potassium dichromate; B. DS 11.121 Weigert 1904; C. ADS 21.1 Pal 1887 (sol. B); D. sat. aq. sol. lithium carbonate; E. DS 12.221 van Giesen 1896 method: [paraffin sections of dichromate-fixed material] — > water — > /I, 1 wk., 37°C. -^ thorough wash^ B, 1-2 days, 37°C. -» wash -> C, till differentiated -^^ D, 37°C., till dark blue -^ thorough wash — » E, till sufficiently counterstained -^ balsam, via usual reagents 22.4 Sheehan 1939 see DS 21.3 Sheehan 1939 22.4 Sheehan and Storey 1947 see DS 21.3 Sheehan and Storey 1947 22.4 Smith 1907 11431, 12:1 reagents required: A. sat. sol. {circ. 0.2%) Nile blue; B. 1% acetic acid method: [frozen sections of fresh or formaldehj'de-fixed material]—* A, 10-24 hrs. — > water, wash — > Mil or 12 mountant 22.4 Smith and Mair 1911 20214, 25:247 reagents required: A. sat. sol. Nile blue 100, sulfuric acid 0.5; B. 2% acetic acid preparation OF a: Boil under reflux 1-2 hours. Cool. Filter. method: [frozen sections of fresh tissue]—* water—* A, overnight—* B, till differenti- ated -^ water, wash -^ M 1 1 mountant (Chapter 26) result: fatty acid, blue. Neutral fats, red. 450 METHODS AND FORMULAS DS 22.4-DS 22,6 note: The reflux boiling should continued until a xylene extract of a sample shows strong red fluorescence. The chemical validity of this separation has been sharply queried by Kaufmann and Lehmann 1926 (22575, 261:623). For a discussion of the arguments for and against see Gatenby and Cowdry 1937, 280 and Cowdry 1943, 136. The fact remains that fatty cell inclusions are differentiated in red and blue, on a reproducible basis. 22.4 Smith and Rettie 1924 11431, 27:115 BEAGENTS kequired: A. ADS 12.1 Smith and Rettie 1924; B. water 99.5, acetic acid 0.5, hematoxylin 1; C. ADS 21.1 Smith and Rettie 1924 method: [frozen sections of formaldehyde-fixed material] -^ A, 1-2 days, 37°C. — > wash -^ B, 6-18 hrs. -^ C, till differentiated-^ wash— > M 12.1 mountant 22.4 Vrtis 1931 23632, 47 :443 REAGENTS REQUIRED: A. 4% formaldehyde; B. sat. sol. Sudan III in 70% ale. method: [pieces of skin] —> A, overnight -^ wash ^50% ale, J^ to 1 hr. — > B, }i-l hr. — > 50% ale, till differentiated — > glycerol or M/10 mountant. RECOMMENDED FOR: fat glauds in wholemounts of rodent skin. 22.4 Wilson 1950 4349, 31:216 reagents required: A. DS 22.4 Lillie and Ashburn 1943; B. DS 11.124 Gomori 1941; C. 1% acetic acid; D. 0.5% light green method: [5 II frozen sections, mounted on slide] -^ A, 10 mins. -^ thorough wash — > B, 4 mins. —* tap water, till blue -^ C, rinse -^ D, quick dip — > C, rinse — > M 12.1 Kaiser 1880 22.5 Plastics Plastids are commonly demonstrated in sections of plant tissue by a hematoxylin staining technique, or by any other technique which is customarily employed in the histological examination of vegetable structures. The two methods given below are those which dis- tinguish (if distinction is possible) between proplastids and mitochondria. The method of Milovidov 1928 is merely a modification of the standard Altmann acid fuchsin-aurantia mitochondrial staining technique, while the technique of Nemefi 1906 relies on prior mordant- ing in tannin before the use of gentian violet. 22.5 Milovidov 1928 1823, 24:9 reagents required: A. DS 22.2 Altmann 1890 (A. sol.); B. 5% aurantia in 95% ale; C. 2% tannin; D. 1% methyl green method: [sections of F 7000.1000 Regaud 1910 (or any F 6700.1000 fixative) fixed material] —> A, poured on sUde and warmed till steaming, 1 min. — > B, till mito- chondria alone remain red — » 70% ale, rinse -^ C, 20 mins. -^ D, 5-10 mins. -^95% ale, till starch grains sharply differentiated -^ balsam, via usual reagents result: mitochondria and proplastids, red; starch, green. 22.5 Nemec 1906 2626,24:528 reagents required: A. 2% tannin; B. 1.5% antimony potassium tartrate; C. 1% gentian violet method: [paraffin sections of F 5000.0015 Nemec 1899 fixed material]-^ waters A, 1 hr. -^ water, wash -^ B, 5-15 mins. — > water, wash — > C, ^i to 2 hrs. — ^ water, wash -^95% ale, till starch granules well differentiated -^ balsam, via usual reagents note: Schneider 1922, 351 recommends fixation in F 1600.0010 Flemming 1882, with subsequent bleaching in hydrogen peroxide. 22.5 Schneider 1922 see DS 22.5 Nemec 1906 (note) 22.6 Staech, Glycogen, and Amyloid Granules Starch and glycogen are both most commonly stained by the application of iodine, which turns the former blue and the latter brown. These iodine techniques, however, do not leave permanent stains, though endeavor has been made by Reilhes 1941 to provide a degree of permanence in a resin mount. These preparations do not last, at their best, more than a few months. The original method for the differential staining of glycogen DS 22.6 DYE STAINS OF SPECIAL APPLICATION 451 was that of Best 1906, and it has not yet been surpassed. One of the most satisfactory methods for the differentiation of starch granules in plant tissues is that of Milovidov 1928, which is given in section 12.5 above for the reason that it is also intended for the demonstration of the differentiation of the proi)lastids. An endeavor to adapt the Feulgen 1924 technique to the demonstration of starch was made by Cr6tin 1941, and has only the disadvantage that it requires mounting in liquid petrolatum witli the subsequent difficulty of cementing the coverslip in place. In cases in which it is desired to give general differential stain, as well as a stain for glycogen, the technique of Vas- tarini-Cresi 1907 can be confidently recommended. 22.6 Arndt 1925 23681,35:545 REAGENTS REQUIRED: A. sat. sol. glucosc in 4% formaldehyde; B. sat. sol. glucose; C. DS 22.6 Best 1906 (sol. A); D. DS 22.6 Best 1906 (sol. B);E. any DS 11.122 formula, saturated with glucose; F. 70% ale. 50, acetone 50, chlorophyll to sat. method: [pieces of fresh tissue] — » yi, 24 hrs. — > frozen sections — > B, till required — > C, ^i-1 hr. — > D, till differentiated — > E, till nuclei stained — > B, thorough wash — > 70% ale, 1 min. —y F, 15-20 mins. — > 70% ale, 1 min. -^ B, 1 min. —>■ levulose syrup result: glycogen, red; fat, green. 22.6 Bennhold 1922 14674, 2:1537 reagents required: A. 1% Congo red; B. 1% lithium carbonate; C. any DS 11.122 formula method: [paraffin sections]^ water, thorough wash — » A, 15-30 mins. — » B, rinse —» 80% ale, till color clouds cease — > wash — > [C, if counterstain required] — * balsam, via usual reagents recommended for: amyloid. 22.6 Bensley 1939 see DS 22.6 Best 1906 (note) 22.6 Best 1906 1780, 23:520 stock solution: water 75, potassium carbonate 1.2, carmine 2.5, ammonia 25 preparation of stock: Boil dye with salts 15 minutes. Cool. Filter. Add ammonia to filtrate. reagents required: A. stock solution 25, ammonia 35, methanol 35; B. water 50, 95% ale. 40, methanol 20 method: [sections of alcohol-fixed material, stained by any DS 11.12 method] — » water — » A, overnight —> water, rinse — > B, till glycogen granules differentiated -^ balsam, via usual reagents note: Bensley 1939 (20540b, 14:47) recommends pure methanol for B, with subsequent dehydration in acetone. See Neukirch 1909, below, for another way of using these solutions. Zieglwallner 1911 (test. Schmorl 1928, 218) stains fat in his F 1300.0010 mixture before following Best's technique; Schmorl 1928, 218 recommends his F 1600.0010 for the same purpose. 22.6 Birch -Hirschf eld 1887 test. 1928 Schmorl Schmorl 1928, 210 reagents required: A. sat. sol. Bismarck brown; B. 0.5% crystal violet; C. 1% acetic acid method: [frozen sections] -^ A, 5 mins. -^95% ale, wash — > distilled water, wash -^ B, 5 mins. — > C, till differentiated — > thorough wash -^ levulose syrup recommended for: amyloid. 22.6 Cretin 1941 6630, 135 :355 reagents required: A. methanol 100, phenyl hydrazine 1, hydrochloric acid 1; B. DS 11.43 Feulgen 1924; C. water 100, sodium bisulfite 2, hydrochloric acid 0.2 method: [sections of F 5000.0020 Cretin 1941 fixed materials] —* A, overnight — » B, few mins. — > C, several hrs. —> liquid petrolatum, via usual reagents note: Solution A may also be u.sed to mordant the tissues before sectioning. 22.6 Edens see DS 22.6 Schmorl 1928a (note) 452 METHODS AND FORMULAS DS 22.6 22.6 Highman 1946a 1887a, 41 :559 REAGENTS required: A. 0.5% Congo red in 50% ale; B. 0.2% potassium hydroxide in 80% ale. method: [sections of formaldehyde or ale. fixed material]-^ A, 1-5 mins. — » B, 1-3 mins. till differentiated — »• wash — > eounterstain, if desired -^ balsam, via usual reagents recommended for: demonstration of amyloid 22.6 Highman 1946b 1887a, 41 :559 REAGENTS REQUIRED: A. DS 11.121 Weigert 1904; B. 0.1% crystal violet in 2.5% acetic acid method: [sections of formaldehyde or ale. fixed material] — > water -^ A, 5 mins. -^ wash -^ B, 1-30 rains. -^ wash ^ MS 11.1 Highman 1946 note: Lieb 1947 (591b, 17:413) substitutes a 0.3% solution of crystal violet in 0.3% hydrochloric acid for B, above. recommended for: demonstration of amyloid. 22.6 Klein 1906 590, 5 :323 reagents required: A. sat. aq. sol. orange G 50, sat. aq. sol. acid fuchsin 50; B. sat. aq. sol. toluidine blue method: [sections of F 3700.1000 Klein 1906 fixed material] — » water — > A, 1 min. wa.sh —* B, 1 min. — > wash -^ balsam, via usual reagents result: chromatin and prozymogen, blue; zymogen, red. recommended for: granules in Paneth cells. 22.6 Krajian 1952 Krajian 1952, 169 REAGENTS REQUIRED: A. DS 11.123 Harris 1900; B. water 80, glycerol 20, Congo red 3.2; C. 2% sodium cyanide method: [unmounted sections by freezing technique] -^ A, 15 sees. -^ tap water, wash — > B, 15 mins. — » wash -^ C, till differentiated, few sees. — » strand on slide and drain — + M 12.1 mountant recommended for: amyloid. note: Stated by author (loc. cit.) to be modification of DS 22. G Bennhold 1922. 22.6 Langhans 1890 22575, 120:28 reagents required: A. DS 11.22 Mayer 1881; B. ADS 12.2 Gram 1884; C. 2.5% iodine in abs. ale. method: [eelloidin sections of ale. fixed material] — > A, 10-15 mins. — » B, 5-10 mins. — » — > C, till dehydrated -^ oil of origanum recommended for: demonstration of amyloid. 22.6 Lieb 1947 see DS 22.6 Highman 1946b (note) 22.6 Lillie 1948 Lillie 1948, 143 reagents required: A. water 100, nitric acid 0.5, sodium periodate 1; B. DS 11.432 Lillie 1948; C. 0.52% sodium bisulfite; D. DS 11.121 Weigert 1904 method: [sections of formaldehyde-fixed material, varnished on slide with collodion] — » water -^ A, 10 mins. -^ wash —> 5, 15 mins., with occasional agitation —> C, 3 changes, Vi mins. in each — > wash -^ D, 2-5 mins. — > tap water, till blue — > balsam, via usual reagents 22.6 Lubarsch test. 1905 Hall and Herxheimer Hall and Herxheimer 1905, 112 formula: water 20, DS 11.122 Delafield (1885) 40, ADS 12.2 Gram 1884 40 method: [sections to water] — » stain, 5 mins. -^ abs. ale, least possible time for dehydra- tion - > balsam, via xylene recommended for: glycogen. 22.6 Mayer test. 1938 Mallory Mallory 1938, 134 REAGENTS required: .4. 0.5% crystal violet; B. 1% acetic acid; C. 7.5% potassium alum method: [paraffin .sections, uHthoiil removal of paraffin] -"> A, 40°C., 5-10 mins. — > wash -* B, 10-15 mins. -^ wash -> C, few minutes -^ wash -^ [fix to slide] -^ balsam, via xylene recommended for: demonstration of amyloid. DS 22.6-DS 22.7 DYE STAINS OF SPECIAL APPLICATION 453 22.6 Milovidov 1928 sre DS 22.5 Milovidov 1928 22.6 Neukirch 1909 2^568 1 , 20 ::>;} 1 REAGENTS REQUIRED: A. sat. sol. glucose ill sat. sol. mercuric chloride; li. sat. sol. glucose in 80% ale; C. ADS 11.1 Lugol; D. DS 22.6 Best 1906 (sol. A) E. DS 22.6 Best 1906 (sol. B) method: [pieces of fresh tissue] — > .4, 6-12 hrs. — > B, wash -^ (', 6 hrs. — > 95% ale, 24 hrs. or till required --> cclloidiu sections — > water — > [hematoxylin nuclear stain, if required] — » D, overnight -^ rinse -^ B, till differentiated -^ balsam, via usual reagents note: Neukirch {loc. cit.) also recommends glucose-saturated 40% formaldehyde in place of A, above, in which case steps B and C are omitted. Schmorl 1928, 216 finds this better for large pieces. 22.6 Reilhes 1941 6630, 135:554 formul.\: a. abs. ale. 100, iodine 8, ammonium iodide 2 method: [paraffin sections of alcohol-fixed material]—* abs. ale. — > stain, dropped on slide, several minutes — > abs. ale, quick rinse — » dry -^ Venice turpentine, direct 22.6 Schmorl 1928a Schmorl 1928, 210 reagents required: A. 0.5% crystal violet; B.\% acetic acid; C. 7% ammonium alum method: [paraffin sections, or ribbons, not deparaffinized] — > A, floated on surface, 5-10 mins. 40°C. — > wash, floating on surface — » B, floating on surface, till differentiated -^• wash, floating on surface — > C, floating on surface, till required — > wash, floating on surface -^ strand on slide, dry with gentle heat —^ xylene till deparaffinized — » balsam recommended for: amyloid. note: Edens {test. Schmorl loc. cit., 211) uses 0.1% methyl violet in 0.3% hydrochloric acid for 24 hours and omits steps B and C, above. 22.6 Schmorl 1928b Schmorl 1928, 211 reagents required: .4. DS 11.44 Unna 1891; B. 0.5% acetic acid; C. 7.5% ammonium alum. method: [paraffin sections] -^ water — > A, 10-15 mins. -^ rinse — ♦ B, quick rinse — > C, 2-5 mins. — > abs. ale, minimum possible time —> balsam, via xylene recommended for: amyloid. 22.6 Schmorl 1928c see DS 22.6 Best 1906 (note) 22.6 Schmorl 1928d Schmorl 1928, 209 reagents required: A. \% methyl violet; B. 2% acetic acid method: [frozen sections] -^ A, }^-l min. — > B, 2-3 mins. — > thorough wash — » glycerol recommended for: amyloid. 22.6 Vastarini-Cresi 1907 test. 1942 Langeron Langeron 1942, 1173 reagents required: A. DS 21.13 Weigert 1898 (working solution) 150, resorcin 6, cresofuchsin 3, 95% ale 150, hydrochloric acid 6; S 1% orange G in 95% ale method: [paraffin sections] -^95% ale — > A, 2 hrs. 35°C. — > 95% ale, thorough wash —* B, I min. -^ balsam, via usual reagents result: elastic fibers, blue; glycogen, red; plasma, pale orange. 22.6 Zieglwallner 1911 see DS 22.6 Best 1906 (note) 22.7 Mucin 22.7 Highman 1945 20540b, 20 :85 formula: water 75, 95% ale 25, new methylene blue N 0.1, citric acid 0.2 method: [sections] —> water —» stain, 3-10 mins. ^ rinse —> balsam, via acetone and xylene 22.7 Hoyer 1900 1780, 36:310 REAGENTS REQUIRED: A. sat. aq. sol. mercuric chloride; B. 0.005% thionin method: [sections of mercuric chloride-fLxed material] —> water—* .4, 30 sees. — > 95% ale, wash -^ B, 5-15 mins. — > balsam, via S 22.1 Minot (1928) 454 METHODS AND FORMULAS DS 22.7-DS 22.8 22.7 Lillie 1928 23632, 45 :381 REAGENTS REQUIRED: 0.2% toluidine blue method: [sections]—* waters stain, 1 min. — > quick rinse -^ acetone, till dehydrated -^ balsam, via xylene note: This method was republished by Lillie 1929 (4349, 12:120) to which later paper reference is most frequently made. 22.7 Mallory 1938 Mallory 1938, 131 REAGENTS REQUIRED: A. DS 11.122 Mallory 1938, 10, 95% ale. 9, tap water 81; B. 0.1% magenta method: [paraffin sections alcohol-fixed material] -^ water -^ A, 5-15 mins. -^ thorough wash -^ B, 5-10 mins. — > 95% ale, till no more color comes away —> balsam, via xylene 22.7 Masson 1910 test. 1948 Romeis Romeis 1948, 481 REAGENTS REQUIRED: A. Any DS 11.122 formula; B. 5% metanil yellow in 0.2% acetic acid; C. DS 22.7 Mayer 1896a (working sol.) method: [sections of F 5000.1010 Bouin 1896 fixed material]—* water -^ A, till deeply stained — > rinse — > B, till nuclei differentiated -^ wash —>■ C, 5-10 mins. -^ wash — > balsam, via usual reagents 22.7 Mayer 1896a rnucihaematin — compl. script. 14246, 12 :303 REAGENTS REQUIRED: A. water 30, 95% ale, 70, hematin 0.2, aluminum chloride 0.1, nitric acid 0.05 method: [sections of alcohol-fixed material] — > water -^ A, 10 mins. —* 1 hr. — > thorough wash — > balsam, via usual reagents note: Mallory 1938, 129 recommends this for mucin of fibroblastic origin. 22.7 Mayer 1896b mucicarmine — compl. script. 14246, 12 :303 STOCK solution: Heat together for 2 minutes water 2, aluminum chloride 0.5, carmine 1. Add with constant stirring 100 50% ale. working solution: stock 10, 70% ale. 90 method: [sections of alcohol-fi:xed material] —> stain, 10-15 mins. -^ rinse -^ balsam, via usual reagents note: Mallory 1938, 130 recommends this for mucin derived from epithelial cells. 22.7 Merkel test. 1928 Schmorl Schmorl 1928, 164 REAGENTS REQUIRED: A. 5% cresyl violet; B. abs. ale. 50, toluene 50 method: [5 m-7 m sections of alcohol-fixed material]^ A, 10-30 mins. -+ 95% ale, rinse —* B, controlled under microscope, till differentiated — > balsam, via xylene 22.7 Unna 1894 14352, 18 :509 REAGENTS REQUIRED: A. DS 11.44 Unna 1892; B. 0.1% acetic acid; C. 10% potassium dichromate; D. 1% hydrochloric acid in aniline method: [sections of alcohol-fixed material]-^ water—* A, 10 mins. -^ B, rinse—* C, 30 sees. — > blot —>■ D, till differentiated, 15-30 sees. -^ balsam, via usual reagents 22.7 Zimmerman 1925 see DS 21.4 Zimmermann 1925 22.8 Other Cell Inclusions and Extrusions 22.8 Alzheimer 1910 Nissl and Alzheimer 1910, 411 REAGENTS REQUIRED: A. sat. aq. sol. copper acetate; B. DS 11.124 Alzheimer 1910 method: [sections prepared as in steps A, and B (DS 21.213 Alzheimer 1910)] — * water -^ ^, 1 hr. 35°C. — * rinse — * B, 30 mins. — * wash — * balsam, via usual reagents recommended for: demonstration of granules in nerve cell cytoplasm. 22.8 Bensley test. 1915 Chamberlain Chamberlain 1915, 134 reagents required: A. ADS 12.1 Gram 1884; B. sat. aq. sol. copper acetate; C. 0.5% hematoxylin; D. 5% potassium dichromate; E. ADS 21.1 Weigert 1885 method: [sections of F 3700.1000 Bensley (1915) fixed material]-^ water -^ A, some hrs. — > wash -^ B, 5-10 mins. — * wash, 1 min. — * C, 5-10 mins. -^ wash, 1 min. -^ D, 30 sees. — * E, till differentiated — * wash — > balsam, via usual reagents recommended for: canaliculi in plant cells. DS 22.8 DYE STAINS OF SPECIAL APPLICATION 455 22.8 Beyard 1930 4285a, 7 :264 REAGENTS REQUIRED: A. 1% acid fuchsin; B. 0.8% bromine method: [sections] -^ water — > A, 30 sees. — > rinse —>■ B, 4-5 sees, till violet — * 95% ale., till pale mauve —y [DS 11.111 nuclear stain if required] -^ balsam, via usual reagents recommended for: basophilic granules in mast cells. 22.8 Bowie 1935 763, 64:357 PREPARATION OF DRY STAIN: Add 67 0.4% ethyl violet to 33 0.4% Biebrich scarlet. Collect, wash and dry ppt. STOCK SOLUTION: 1% dry stock in 95% ale. REAGENTS REQUIRED: A. 95% alc. 80, Water 20, stock sol. 0.1-0.5; B. 50:50 clove oil: xylene method: [sections of F 7000.1000 Regaud 1910 material] -^ water -> A, 24 hrs. -> blot — » B, till differentiated, 20-30 mins. — > balsam, via benzene RECOMMENDED FOR: pepsinogen granules in gastric mucosa. 22.8 Brice 1930 20540b, 5:101 REAGENTS REQUIRED: A. abs. alc. 100, benzidine 0.3, sodium nitroferricyanide 0.4; B. water 100, hydrogen peroxide 0.5; C. DS 13.12 Wright 1910, working solution method: [air-dried blood smear] — > A, flooded on slide, 1 min. -^ B, equal in quantity to A, flooded on slide, 3 mins. -^ water, thorough wash — * blot — > dry — > C, flooded on slide, 2 mins. — * add water, drop by drop, till green scum forms on surface, leave 2 mins. — > water, thorough wash -^ [dry] or neutral mountant, via acetone recommended for: oxidase granules in erythrocytes. 22.8 Bujard 1930 4285a, 7 :264 reagents required: A. 1% acid fuchsin; B. 0.8% bromine; C. 1% hydrochloric acid in 95% alc. method: [sections] -^ water —> 4, 30 sees. — » j5, 4-5 mins. — > C, till granules alone stained — > DS 11.111 Regaud 1910 if nuclear staining is required — > balsam, via usual reagents recommended for: basophilic granules in mast cells. 22.8 Ehrlich 1891 test. 1938 Mallory Mallory 1938, 174 formula: water 30, 95% alc. 62, acetic acid 8, Hofmann's violet to sat. recommended for: granules in mast cells. 22.8 Hall and Powell 1926 21400a, 45:256 reagents required: A. 0.2% Bordeaux red; B. 4% ferric alum; C. 0.5% hematoxylin method: [flagellates fixed in F 3000.0000 Schaudinn 1893]-^ water—* A, 1-2 days— » B, 1-2 days — > C, 1-2 days — > B, till differentiated —>■ balsam, via usual reagents recommended for: internal structures in euglenoid flagellates. 22.8 Harvey 1907 590, 6 :207 REAGENTS REQUIRED: A. sat. aq. sol. (circ. 7%) cupric acetate; B. 3% potassium di- chromate; C. sat. aq. sol. hematoxylin; D. ADS 21.1 Weigert 1885 method: [sections of F 3700.1000 Harvey 1907 fixed material] -^ A, 1 min. -^ wash — > B, 1 min. — > wash — ♦ C, 1 min. — > wash — > D, till differentiated — » balsam, via usual reagents RECOMMENDED FOR: demonstration of parietal cell granules. 22.8 Hamperl 1926 22575, 259:179 REAGENTS required: A. Any DS 11.21 formula; B. 0.01% methyl violet method: [sections of F 0000.1000 Hamperl 1926 fixed material] —> water -^ A, till nuclei stained —^ wash — > B, till granules stained -^ abs. alc. least possible time — » balsam, via usual reagents recommended for: granules in chief cells of stomach. 22.8 Heidenhain 1894 1780, 42 :665 reagents required: A. 0.01% aniline blue; B. DS 11.111 Heidenhain 1892 (both sol.) method: [sections of F 3000.0010 material] —> A, 24 hrs. — > B, full technique -^ balsam, via usual reagents recommended for: centrosomes in animal tissues. 456 METHODS AND FORMULAS DS 22.8 22.8 Horvath 1931 23632, 47 :463 REAGENTS REQUIRED: A. 1% phosphotungstic acitl; B. 0.1% toluidine blue method: [protozoa, fixed and mordanted 20 mins. in sat. aq. sol. mercuric chloride] — + water, thorough wash -^ A, 10 mins. —>■ water, rinse -^ B, 1 min., 50°-60°C. — > bal- sam, via usual reagents recommended for: demonstration of cilia, cirri, and basal bodies in ciliate protozoa. 22.8 Kraus 1914 22575, 218:107 reagents required: A. DS 11.44 Unna 1892; B. 25% tannin; C. DS 12.16 Fraenkel (1948); D. 2% acid fuchsin; E. 1% phosphomolybdic acid method: [3 iU-5 m sections of formaldehyde-fixed material]-^ waters A, 6 mins.—* wash —> B, till no more color comes away —> C, till nuclei blue — ♦ wash —> D, I min. -^ rinse — * E, 30 sees. — > wash -^ [drain and lalot] — ^ abs. ale. least possible time — > balsam, via xylene recommended for: differentiation of tannic acid fast (red-violet), fuchsinophile (red- dish yellow) and fuchsinophojje (blue) colloid in thyroid. 22.8 Lendrum 1945 11431, 57:267 REAGENTS REQUIRED: .4. DS 11.122 Mayer 1901; B. 1% fast green FCF in 0.5% acetic C. ADS 12.2 Lugol; D. 2% phosphotungstic acid in 95% ale; E. DS 11.46 Lendrum 1945 method: [sections] —>■ A, till thoroughly stained — » wash — > B, 1-2 mins. — * rinse -^ C, 2 mins. -^^ 95% ale, rinse —> D, few moments —> rinse—* E, 2-6 mins. — > wash—* balsam, via usual reagents RECOMMENDED FOR: granules in cystic breast epithelium and basement membrane of renal glomerulus. 22.8 MacCallum et al. see DS 21.42 MacCallum et al, 1935 22.8 Mallory 1938a Mallory 1938, 136 REAGENTS REQUIRED: A. 0.5% magenta in 50% ale. method: [sections with nuclei prior stained by any DS 11.122 method] —> water — ' A. 5-20 mins. -^95% ale, till differentiated -> balsam, via xjdene RECOMMENDED FOR: demonstration of hemafuchsin granules. 22.8 Mallory 1938b Mallory 1938, 151 REAGENTS REQUIRED: A. 1% acid fuchsin; B. 0.1% potassium permanganate method: [sections from F 3700.0010 Zenker 1894 fixed material]—* water—* A, over- night — > drain — * B, 40-60 sees. -^ abs. ale, — * balsam, via xylene result: fibroglia fibrils, bright red; collagen, reddish yellow; elastic fibrils, bright yellow. recommended for: demonstration of fibroglia fibrils. 22.8 Mallory 1898 test. 1938 Mallory Mallory 1938, 138 REAGENTS REQUIRED: A. 95% alc. 75, sat. sol. ammonium sulfide 25; B. water 100, hydrochloric acid 0.5, potassium ferricyanide 10; C. 0.5% magenta in 50% ale method: [sections] -^ water — > A, 1-24 hrs. — * wash — * B, 10-20 mins. — > wash — > C, 5-20 mins. -^ rinse -^95% ale, till differentiated — * balsam, via usual reagents result: nuclei and hemofuchsin, red; hemosiderin, blue. RECOMMENDED FOR: differentiation of hemosiderin and hemofuchsin in sections. 22.8 Mallory 1938 Mallory 1938, 207 REAGENTS REQUIRED: A. DS 11.122 Mallory 1938; B. 0.5% phloxine in 20% ale; C. 0.1% lithium carbonate method: [sections of alc. or formol fixed material] — > A, 1-5 mins. -^ tap water, wash — > B, 10-30 mins. — * tap water, wash -^ C, J^-1 min. -^ tap water, wash — » balsam, via usual reagents RECOMMENDED FOR! hyaliu. 22.8 Mcjunkin 1922 763, 24 :67 REAGENTS REQUIRED: A. watcr 25, 95% alc. 75, benzidine 0.2, hydrogen peroxide 0.2; B. Any DS 11.122 solution; C. 0.1% eosin method: [5 n sections of formaldehyde-fixed material] -^ water — > A, 5 mins. -^ wash -^ B, 2 mins. -^ wash — * C, 20 sees. —> wash — > balsam, via usual reagents RECOMMENDED FOR: peroxidase granules in bone marrow. DS 22.8 DYE STAINS OF SPECIAL APPLICATION 457 22.8 Oliver 1934 11250, 65:266 REAGENTS REQiriREi): A. ADS 12.2 Olivcr 1934; B. DS 11.13 Ziehl 1882 method: [air-dried smear from a 1 : 4 dilution of blood in hirudin saline, incubated 40-50 mins. 37°C.] -^ A, 20 mins. -> wash -^ blot -» B, 20 mins. —>■ wash -> dry RECOMMENDED FOR: " flagella " on erythrocytes. 22.8 Oppler test. 1928 Schmorl Schmorl 1928, 341 REAGENTS REQUIRED: .1. DS 11.24 Oppler (1928); B. 0.5% picric acid in 95% ale. method: [celloidin sections of alcohol-fixed material]-* water-* A, \i-l min. -» blot -^ B, \ min. — + balsam, via usual reagents recommended for: demonstration of eleidin in cells of stratum lucidum of skin. 22.8 Reich test. 1933a Cajal and de Castro Cajal and de Castro 1933, 323 reagents required: A. 1% toluidine blue method: [15 At celloidin sections of F 7000.0000-Muller 1890 fixed nerve] -> water -^ A, 5 mins. -> quick rinse -^ 95% ale. till differentiated-* abs. ale, till no more color comes away — > balsam, via usual reagents recommended for: granules in Schwann cells. 22.8 Reich test. 1933b Cajal and de Castro Cajal and de Castro 1933, 324 REAGENTS REQUIRED: A. water 100, phenol 5, acid fuchsin 1; B. ADS 21.1 Pal 1887 (A and B sols.); C. 2% toluidine blue method: [15 M frozen sections of formaldehyde-fixed nerve] -^ A, 24 hrs. 37°C. — > B {A sol.), 30 sees. -» B, (5 sol.), few moments -* [repeat B (A) -* B (B) cycle till no more color comes away] —> balsam, via xylene results: nuclei, blue; n granules, red; w granules, purple. RECOMMENDED FOR: differentiation of n and tt granules in Schwann cells. 22.8 Russel test. 1894 Kahlden and Laurent Kahlden and Laurent 1894, 73 REAGENTS REQUIRED : A . sat. sol. magenta in 2 % phenol ;B.1% iodine green in 2 % phenol method: [sections of dichromate-fixed material]^ A, 10 mins. ^ thorough wash -> abs. ale, 30 sees. -^ B, 5 mins. -* balsam, via usual reagents RECOMMENDED FOR: miscellaneous cellular inclusions. 22.8 Russell test. 1928 Schmorl cil. Miller Schmorl 1928, 219 REAGENTS REQUIRED: A. DS 11.43 Ziehl 1882; B. water 100, phenolS, iodine green 1 method: [sections]-^ water-* a, 10-30 mins. -^ wash -* abs. ale. Ja-l min. ^ B 5 mins. ~y abs. ale, wash —> balsam, via usual reagents RECOMMENDED FOR: hyalin. 22.8 Russell test. 1948 Lillie cit. Ehrlich Lillie 1948, 105 REAGENTS REQUIRED: A. sat. sol. magenta in 2% phenol; B. 1 % iodine green in 2% phenol method: [sections of F 7000.0000 Miiller 1850]-* waters A, 10-30 mins. -* wash, 5 mins. -^ abs. ale, 30 sees. -* 5, 5 mins. -^ abs. ale, till differentiated -* balsam, via usual reagents results: nuclei, green; Russell's bodies, red. recommended for: demonstration of Russell's bodies. 22.8 Sannomiya 1927 8542a, 5 :202 REAGENTS REQUIRED: A. 10% hydrochloric acid; B. 1% acid fuchsin; C. sat. sol. picric acid; D. 1% phosphomolybdic acid. method: [small pieces fresh pancreas] -* A, 24 hrs. -^ running water, 24 hrs. -^ celloidin sections -* 5, 3 mins. -^ water, thorough wash -* C, 3-4 mins. -^ water, quick rinse -^ D, 1-2 mins. -^ water, rinse -* balsam, via usual reagents results: paranuclei, red. recommended for: demonstration of paranuclear bodies in pancreas. 22.8 Schmorl 1928 Schmorl 1928, 244 REAGENTS REQUIRED: A. DS 22.21 Altmann 1890 (sol. A); B. DS 22.21 Altmann 1890 (sol. B) method: Jblood smears fixed for 1 hr. in 1% osmic acid] ^ quick wash-* A, 5 mins., 55°-60°C. -* wash, till no more color comes away -* Ji, 15 mins. —* rinse —* blot — * abs. ale least possible time — > balsam, via xylene recommended for: Schridde's granules in lymphocytes. 458 METHODS AND FORMULAS DS 22.8-DS 23.10 22.8 Wallace 1931 19938, 74:369 REAGENTS REQUIRED: A. Any DS 11.122 formula; B. 0.5% eosin Y; C. water 45, aniline 45, abs. ale. 10, methyl violet to sat; D. ADS 12.2 Lugol (1905) ; E. aniline 30 xylene 60 method: [5 n sections of F 3700.1010 fixed material] -^ A, till nuclei lightly stained -^ wash -^ B, 30 sees. — > thorough wash — > C, 2 hrs. -^ wash — > D, 10-15 mins. — > wash -^ E, till differentiated —> xylene wash -^ balsam RECOMMENDED FOR: basal bodies of cilia. 23. SELECTIVE STAINS FOR SPECIFIC ORGANISMS There are surprisingly few stains which will selectively stain a specific organism compared to the number of stains which have been proposed for that purpose. Bacteria, for example, may be very well stained by any nuclear staining tech- nique; therefore here are given (DS 23.2) only those methods for staining them which have, by convention and custom, come to be supposed to be "bacterio- logical methods." One is on even less secure ground in dealing with the first class (DS 23.1), in which are given those stains which stain virus, Rickettsiae, and Negri bodies, for it is, of course, impossible that any individ- ual virus molecule could be stained in such a manner as to bring it within the range of an optical microscope. But the methods given in the division DS 23.11 for stain- ing minute unidentified bodies may quite possibly represent aggregates of virus, as they were supposed to do by the inventors of the techniques. The third division here given (DS 23.3) is for the differential staining of parasites in host tissues, to which much attention has been devoted, and for which a number of relatively specific methods have been proposed. The last two divisions (DS 23.4, DS 23.5) contain a few staining techniques of such speciaUzed application that they cannot possibly be given else- where in the present work. 23.1 Virus, Rickettsiae, and Negri Bodies 23.10 typical examples Demonstration of Rickettsiae in the scrotum of a guinea pig using the magenta-thionin stain of Macchiavello 1938^ It is presumed, in the description which follows, that the technician is acquainted with the exceedingly dangerous nature of Rickettsiae and is prepared to take all those aseptic precautions through which he or she may be protected against infec- tion. Even then it must be emphasized that Rickettsiae can only be handled in properly equipped laboratories and under the supervision of a skilled bacteriologist or pathologist. The staining method here described (DS 23.12 Macchiavello 1938) is the basic method which has been adapted by numerous people, who usually refer to their own method as "Maccliiavello." The volume of Macchiavello 1938 is, more- over, not readily available and most work- ers have derived their information as to the method from the pubhcation of Cox 1939 (17302,53:2242) to whom this method is therefore frequently attributed. Rickettsiae are difficult to stain satis- factorily, and though it is suggested that the beginning technician attempt this original technique of Macchiavello for the first preparation, it is very probable that he will have to vary the technique to suit the materials which he is using. The only difficulty likely to be en- countered will be by technicians who are accustomed to staining bacteria by some method in which every effort is made to get these bodies free on the slide without a great deal of cell debris around them. Rickettsiae, on the contrary, cannot be satisfactorily stained and differentiated unless they are within a cell, hence the critical stage of the preparation consists in securing undamaged cells containing Rickettsiae in a layer thin enough to permit their examination with the highest powers of the microscope. Rickettsiae may be recovered either from the walls of the blood vessels or from the subcutaneous tissues of almost any in- fected animal. The guinea pig has been selected for the present demonstration be- DS 23.10 DYE STAINS OF SPECIAL APPLICATION 459 cause it appears to be the most usually employed in laboratories. The guinea pig may be killed by any method; it is then strapped down on a board in the custom- ary manner. Rickettsiae are most readily obtainable to the beginner from the inner surface of the scrotal sac of a male, which has the advantage of being free from hair because it hes entirely within the coelomic cavity and is free from dermal adhesions. The guinea pig is opened by a median in- cision and the skin stretched back. This will disclose perfectly enormous vesicula seminales, and a very large prostate gland. These are pushed to one side to disclose the testes lying within a peritoneal fold. This is washed free of extravasated blood with a jet of normal saline, and a piece about 5 mm. square is cut from the an- terior surface of the sac. This piece is now removed and laid with the inner surface ui)wards in the center of a 3" X 1" glass slide, where it may remain while the body of the guinea pig is being disposed of with the customary precautions. It is now necessary to cut, not to scrape, a few cells from the exposed inner surface of the material. This is best done with a freshly sharpened razor or with a change- able-blade scalpel, using a new blade, cut- ting parallel to the surface of the glass with short, jerky strokes. One should be careful to draw the knife as much as push it. The debris accumulated on the blade of the knife is then spread over the surface of a second chemically clean slide, which is put aside to dry. More sUdes are now prepared in the same manner until a dozen or two smears have been accumulated. As soon as the smears are dry, the slides are placed in a large jar of absolute alcohol, and if care is taken that the entire surface of the slide is covered, the jar may now be lemoved from the operating room and turned over to a technician. If the slides are, moreover, picked one at a time from the jar with sterile forceps and placed in another jar outside the operating room, it is now safe to proceed with the staining without any further aseptic precautions. The preparation of the stain presents few difficulties, for it is a three-step proc- ess involving first, gross overstaining in a magenta solution buffered to a pH of 7.5, followed by differentiation and counter- staining in an acid thionin solution, with a final clearing of the thionin with weak citric acid. Each slide is removed from the absolute alcohol and waved backward and forward in the air until it is dry. The slides are then examined unstained under the low power of the microscope, and those which show more than one or two cells lying on top of each other are rejected. Normally, in a batch of about a dozen slides, one or two will be found in which the smear is so thin that numerous isolated, undamaged cells will be seen in the field of the low power. These slides alone are worth stain- ing, and each must be treated individu- ally. The sUde is placed on a rack, or held in the hand, and flooded with the ma- genta-phosphate buffer stain for three minutes. The magenta is then drained off without washing, and the citric acid-thio- nin solution is flooded onto the surface and off again immediately. The simplest technique is to hold the slide in the left hand, to take a pipetful of the stain in the right hand, allow this to fall over the slide, moving the pipet backward and forward to insure an even distribution, and then to tip the slide immediately onto a thick layer of blotting paper so that the stain runs off. While the stain is being run from the slide in the left hand, the right hand picks up a pipetful of the citric acid solution, and squirts this against the in- clined slide in such a manner as to wash off the blue dye. The slide is then passed into tap water, where it may remain while suc- cessive shdes from the same batch are treated with the stain. The slides should remain in tap water until no further color comes away, and should then be washed in distilled water and air-dried. It is usually not worth while to mount the slides under a coverslip, but to accumulate them dry and to observe them with an oil immersion objective. After each observation the immersion oil may be washed off with xylene. A successful preparation shows deep red Rickettsiae in light blue cells. The usual error is t(^«Riove too much of the blue which, unoerlfc^ very high powers neces- sary, is a desinR(8B background for picking 460 METHODS AND FORMULAS DS 23.10 out the red Rickettsial bodies. The modifi- cation of Zinsser, Fitzpatrick, and Hsi 1938 (11189, 69:179) consists of differ- entiating the magenta stain with citric acid, and in staining in methylene blue for a longer period; this, however, though undoubtedly giving a better background stain, does not give as clear a differentia- tion of the Rickettsial bodies. Demonstration of Negri bodies in the brain of a guinea pig using the ethyl eosin-methylene blue technique of Stovall and Black 1940 Negri bodies are usually diagnosed by the smear technique which is described in Chapter 8. The method here described is intended more for the preparation of class- demonstration material, though it may also, when time is not vital, be used for diagnostic purposes. The guinea pig has been selected as a test animal, since it is so commonly used for diagnostic infection with the virus of rabies, the presence of which in the brain causes Negri bodies to appear in the cells, particularly those of the horn of Amnion. It is presumed that the investigator is acquainted with the aseptic precautions which must be ob- served in handling animals infected with the virus of rabies. The guinea pig is killed, commonly with chloroform, and tied down on its ventral surface on a dissecting board. The head is then skinned and the muscles dissected away from the surface of the cranium. Since only the anterior region of the brain is required, it is advisable to cut with a saw about yi inch anterior to the supra- occipital bone vertically downwards for about % of the distance through the skull and brain. The supraoccipital and pos- terior portions of the cranium are now re- moved with a pair of powerful pliers which will permit the cut end of the parietal bone to be gripped and stripped off with- out damage to the cerebral hemispheres. It does not matter if the cerebral hemis- pheres are shghtly damaged, since ample protection will be afforded •to -the horn of Amnion. The mcmlnanes of the brain should be stripped off with forceps and the back of a large blunt knife inserted in the slit between the two cerebral hemispheres. A twist of the knife to the left will push a hemisphere to one side, so that it can be gripped firmly and drawn back until a cartilage knife, or similar instrument, can be used to sever the hemisphere from the brain stem. This will leave the other cere- bral hemisphere exposed so that it may in its turn be severed. These two hemis- pheres are now removed to a dish of physiological saline, and the carcass of the guinea pig destroyed with the customary aseptic precautions. Each hemisphere is now cut with a horizontal movement of a large knife just about through the middle, and the upper half is thrown away. This will disclose the horn of Ammon at the posterior end. A httle experience may be necessary to determine the exact point at which the cut should be made. Negri bodies are most commonly found in the basal portions of the horn of Ammon, and from this portion a few pieces about ^i centimeter in thickness and a centimeter square should be removed to the selected fixative. Stovall and Black (DS 23.13 Stovall and Black 1940) suggest the use of their method for diagnostic purposes by using acetone as a fixative, which permits rapid dehydration. For class-demonstration pur- poses, however, it is better to use 5% potassium dichromate in which the pieces of brain are suspended in a loosely woven cloth bag for a week or two to harden them. If the sections are to be cut by an inex- perienced technician, it is recommended that every step of the procedure up to this point be conducted by a physician, prefer- ably a pathologist, and the pieces of brain in potassium dichromate turned over to the technician for further treatment. After the pieces have hardened for a week or two, they are washed in running water overnight, embedded in the cus- tomary manner, and cut into sections of about five microns in thickness. These sections are mounted on the slide, de- waxed in xylene, and run down through the alcohols to distilled water. The great advantage of the technique of Stovall and Black is that it involves the DS23.il DYE STAINS OF SPECIAL APPLICATION 461 use of buffers, and thus gives a clearly reproducible result. Stovall and Black rec- ommend phosphate buffers, of which two will be required, one at pH 3, the other at pH 5.5. It is not known to the writer whether or not other buffers than phos- phate can be substituted in this technique. One per cent of ethyl eosin is then dis- solved in the pH 3 buffer, and 0.25% of methylene blue is dissolved in equal jiarts of the pH 5.5 buffer and absolute alcohol. The only other solution required is 0.4% acetic acid. Sections are taken from the distilled water and placed in the buffered eosin solution for two minutes. From this they are passed to distilled water, in which they are rinsed until the whole of the eosin ad- herent to the sUde, but not that in the sections, is removed. They are then dipped up and down in the methjdene blue solu- tion for about 30 seconds. Each slide, without washing, is next placed in acetic acid and watched. When it is taken from the blue stain it will be purple-blue, and it will be sufficiently differentiated when it has changed to brownish red. This color change is relatively sharp and is accom- panied by a cessation of the clouds of blue stain, which will be seen leaving the sec- tion when it is first placed in the acetic acid. The slide is then transferred to running water and thoroughly washed to remove the acid as rapidly as possible. It is recommended that the slides be run through the technique up to this point, one at a time, and accumulated in the wash water. They may then be dehy- drated in the regular manner, either in alcohol, or in any other solvent selected by the preparator, before being mounted in balsam. 23.11 METHODS OF STAINING UNIDENTIFIED ORGANISMS SMALLER THAN BACTERIA Little can be said in favor of the methods given under this section, save that they have appeared in the literature and are certified by their discoverers to place in evidence, in sectioned material, certain particles presumed by the writers of the articles to have been living, and which cannot be, with certainty, assigned to any known group or class of organism. 23.11 Bland and Canti 1935 11431, 40:2,33 formula: DS 13.13 Gatenby and Cowdry 1928 7, phosphate buffer pll 7 100 method: [pieces of infected explant] -^ methanol, 5mins. —* stain, 24hrs. — * acetone, till differentiated — > xylene, till acetone completely removed -^ balsam 23.11 Gutstein 1937 11431,45:313 formula: 1% methyl violet 50, 2% sodium bicarbonate 50 method: [methanol-fixed smears] -^ stain, 20-30 mins., 37°C. — > wash dry 23.11 Hosokowa 1934 test. 1942 Langeron Langeron 1942, 841 stock solution: methanol 50, glycerol 50, DS 13.11 Jennor 1899 (dry powder) 0.8, azur I 0.2, crystal violet 0.01 RsiAGENTS required: A. 1% eosin B; B. stock 100, water 2 method: [smears, fixed and dehydrated in F 0000.0101 Hosokowa 1934] -^ A, poured on slide and warmed to steaming, 1 min. -^ tliorough wash -+ B, 30-40 mins. — » dry 23.11 Laidlaw test. 1936 Pappenheimer and Hawthorne G08b, 12:627 reagents rkquired: A. DS 11.121 Weigert 1903; B. 0.5% hydrochloric acid in 70% ale; C. 1% acid fuchsin; D. 1% phosphomolybdic acid; E. 0.25%, orange G in 70% ale. method: [3 M sections of F 3000.0010 Laidlaw (1936) fixed material]-* waters A, 5 mins. — » B, till nuclei differentiated -^ wash -* C, 5-15 mins. — > rinse — >• D, 30 sees. -^ rinse — » E, till inclusion bodies differentiated -^ balsam, via usual reagents 23.11 Nicolau and Kopciowska 1937 6628,104:1276 reagents required: A. water 65, methanol 35, glycerol 5, oxalic acid 0.15, methyl blue 1.5; B. water 100, oxalic acid 0.06, acid fuchsin 1.5 method: [thin sections of F 5000.1010 Dubsoscq and Brazil 1905 fixed material] — > 95% ale. -^ A, '^ to 1 hr. — > water, rinse -^ abs. ale, rinse — > B, 10-20 mins. -^ abs. ale. — » balsam, via usual reagents 462 METHODS AND FORMULAS DS 23.12 23.12 METHODS FOE RICKETTSIAE The staining of Rickettsiae in sections is relatively easy, provided that an alkaline solution be employed at some stage of the proceedings. The method of Castaneda 1930 and Macchiavello 1938, which involve the use of a phosphate buffer at a pH of 7.5, have now almost replaced the other methods, which are given here largely for their historical value, and to enable the research worker to check, if necessary, the results of the early publishers in this field. Attention must therefore be drawn to the technique of Lupine 1932, in which a definitely acid solution is employed for staining. It must, however, in all fairness, be pointed out that the technique of Lepine would equally well stain bacteria or mitochondria were they present. Indeed, the general warning must be given that the demonstration of Rickettsiae in cells is far more dependent upon the symptoms shown by the animal from which they were taken, than on any demonstration which can be made with an optical microscope. There are numerous methods for staining small par- ticles occurring in cells, which have been given both under the cytological techniques above and in various places in the previous divisions of the work. There is no evidence of any kind that any of the techniques given in this section will stain Rickettsiae to the exclusion of any other small particle or small body. 23.12 Begg, Fulton, and van den Ende 1944 11431, 56:109 REAGENTS REQUIRED: A. M/15 phosphatc buffer pH 7.6 10, 0.1% magenta 90; B. M/50 citrate buffer pH 3.0; C. 1% methylene blue method: [heat-fixed impression smears] — > A, 5 mins. — > wash —* B, }2 to V ^ mins. -^ wash — > C, 30 sees. — » dry 23.12 Bohner test. 1928 Schmorl Schmorl 1928, 428 REAGENTS REQUIRED: A. 1% methyl blue 35, 1% eosin 35, water 100; B. 0.005% sodium hydroxide in abs. ale; C. 0.1% acetic acid method: [sections of ale. or acetone, fixed material] -^ water — * A, J^-5 mins. — > rinse -^50% ale, rinse — » B, 15-20 sees. — > abs. ale, rinse —>■ water, wash — ^ C, 1-2 mins. — ^ abs. ale, rinse — > abs. ale — > balsam, via usual reagents 23.12 Bond test. 1938 Mallory Mallory 1938, 285 REAGENTS REQUIRED: A. Water 100, eosin 0.1, methyl blue 0.1; B. xylene 30, aniline 60 method: [methanol-fixed smears] — * wash —* stain, 4-5 mins. — » wash -^ blot -^ dry -^ B, till differentiated —* balsam, via xylene 23.12 Castaneda 1930 11250, 47:416 reagents required: A. phosphate buffer pH 7.5 100, 40% formaldehyde 5 DS 11.44 Loffler 1890 1; B. 0.1% safranin method: [very thin smears] -^ A, S mins. -^ drain -^ B, on slide, 2-3 sees. -^ water, till no more color comes away -^ dry 23.12 Clancy and Wolfe 1945 19938, 102 :483 reagents required: A. water 100, methylene blue 0.02, magenta 0.02 method: [air-dried smears] -^ xylene, flooded on slide, few mins. -^ dry — > A, 5 mins. -^ wash —)■ dry 23.12 Cox 1939 see DS 23.12 Macchiavello 1938 (note) 23.12 Darzins 1943 23684, 151:18 reagents required: A. ADS 12.2 Lugol 50, water 50; B. 0.1% thionin in 10%, ale method: [air-dried smears] -^ A, 30 sees. -^ rinse — > B, 5 sees. -^ wash -^ dry 23.12 Goodpasture and Burnett 1919 see DS 23.12 Hertig and Wolbach 1924 (note) 23.12 Gracian 1942 lest. 1943 von Brand 20540b, 18:150 REAGENTS REQUIRED: A. 7% potassium dichromate; B. DS 13.13 Giemsa 1902 10, water 90 method: [smear] -» xylene, 3 mins. -^96% ale, 1 min. -> water -^ A, 3 mins. -^ wash -^ B, 10-20 mins. -^ wash —> dry DS 23.12 DYE STAINS OF SPECIAL APPLICATION 463 23.12 Hertig and Wolbach 1924 11343, 44:332 REAGENTS REQUIRED: .4. DS 11.43 Goodpasture and Burnett 1919; B. 40% formalde- hyde; C. sat. sol. picric acid method: [thin sections of F 3700.0000 fixed material] —* A, poured on slide, warmed to steaming, 5 mins. —>■ water, quick rinse -^ B, dropped on slide till no more color comes away — > C, 1 min. -^ balsam, via usual reagents 23.12 Laigret and Auburtin 1938 5310, 31:790 PREPARATION OF DRT "salt": To a sat. sol. thionin add 10% sodium hydroxide until no further ppt. is formed. Filter. Wash and dry ppt. WORKING solution: water 100, phenol 2, ppt. from above to sat. method: [alcohol-fixed smears] — > stain, on slide, 30-50 sees. —* drain — > abs. ale, quick wash —> balsam, via xylene 23.12 Lepine 1932 6630, 109:1162 REAGENTS REQUIRED: A. Water 90, phenol 3, DS 11.43 Ziehl 1890 10; B. sat. aq. sol. {circ. 2.5%) Bismarck brown method: [smears of vaginal scrapings, fixed on slide by F 7000.1000 Rdgaud 1910] — » water, wash -^ B, 2-3 mins. — ♦ water, wash -^ dry result: Rickettsiae red in pale brown cells. 23.12 Lepine 1933 6630, 112:17 stock solutions: I. water 100, phenol 0.5, azur II 1; II. 1% potassium carbonate REAGENTS REQUIRED: A. Water 100, stock I 3.5, stock II 2, 40% neutralized formalde- hyde 5; B. 0.1% safranin method: [very thin smears] — + ^, 3 or 4 mins. -^ water, wash — + B, till differentiated —^ water, wash — > dry 23.12 Macchiavello 1938 Macchiavello 1938, 48 REAGENTS REQUIRED: A. sat. alc. sol. (circ. 4%) magenta 0.4, phosphate buffer pH 7.5 100; B. water 100, thionin 0.01, citric acid 0.13; C. 0.5% citric acid method: [thin smears]—^ A, on slide, 3 mins. -^ B, 2-3 sees. — > C, 2-3 sees. ^ tap water, thorough wash -^ dry note: Zinsser, Fitzpatrick, and Hsi 1938 (11189, 69:179) omit step B and stain for 1 minute in 1% methylene blue after washing off the citric acid. This modification is commonly referred to as " Macchiavello's method." The original method was cited by Cox 1939 (17302, 63 :2242) to whom it is sometimes referenced. A detailed description of the use of this technique is given under DS 23.10 above. 23.12 Nyka 1944 11431,56:264 reagents required: A. 5% magenta in 90% ale; B. 0.01% methyl violet; C. 0.007% acetic acid. method: [3 M sections of F 7000.0000 Miiller 1859 fixed material] -^ 95 %o ale. -^ A, 5-10 mins. — > rinse -^95% ale, 1-2 mins. -^ B, 1-3 mins. -^ rinse — ♦ C, till differentiated -> balsam, via acetone and xylene 23.12 Nyka 1945 11431,52:317 reagents required: A. 0.01% methyl violet; B. 0.05% acetic acid; C. 0.01% metanil yellow method: [sections of formaldehyde-fixed material] —> water— > A, '^ to 1 hr. -^ B, till cytoplasm clear -^ rinse C, few sees. —> balsam, via acetone and xylene 23.12 Roskin 1946 Roskin 1946, 158 reagents required: A. water 80, 95% alc. 20, phenol 1, aniline 1, magenta; B. DS 11.44 Loffler 1890 method: [sections] —> water —» il, 10 mins. —> wash -^ 95% alc. till differentiated—* wash —y B, 15-60 sees, —y wash — > abs. alc., minimum possible time — > balsam, via xylene recommended for: Rickettsiae and Negri bodies. 23.12 Stutzer 1911 23454,69:25 reagents required: A. DS 11.44 Loffler 1890; B.\% tannin method: [thin sections] —» water —+'' A, 5-15 mins. —» rinse —> B, till differentiated-^ wash — » blot -* abs. ale., least possible time — + balsam, via xylene 464 METHODS AND FORMULAS DS 23.12-DS 23.13 23.12 Wolbach 1919 11343, 41:75 REAGENTS REQUIRED! A. 70% alc. 98, ADS 12.2 Lugol (1905) 2; B. 0.5% sodium thio- sulfate; C. distilled water 100, sodium bicarbonate 0.001, methanol 10, DS 13.13 Giemsa 1902 2; D. ADS 21.2 Wolbach 1919 2; E. acetone 70, xylene 30 method: [3 iU-5 m sections of Wolbach F 3700.0000 material, dewaxed and brought to 90% ale] -^ A, 20-30 mins. -^ 70% ale, wash — » water, wash -^ B, b mins. -^ wash -^ C, 30 mins. — > C, fresh solution, 30 mins. — > C, fresh solution, overnight -^ D, flooded on slide, 15-30 sees. — * E, till dehydrated -^ xylene — > cedar oil result: nuclei and Rickettsiae, blue; cytoplasm, red. 23.12 Zinsser and Bayne-Jones 1939 Zinsser and Bayne-Jones 1939, 654 stock solutions: I. phosphate buffer pH 7.5 100, 40% formaldehyde 0.5; II. 1% methylene blue in methanol reagents required: A. stock I 100, stock II 0.75, 40% formaldehyde 5; B. water 100. safranin 0 0.05, acetic acid 0.075 method: as DS 23.12 Casteneda 1930 23.12 Zinnser, Fitzpatrick, and Hsi 1939 see DS 23.12 Macchiavello 1938 (note) 23.13 METHODS FOR NEGRI, AND OTHER VIRUS INCLUSION, BODIES Negri bodies are generally supposed to be aggregations of virus particles, though they may well be the breakdown products of the cell itself. The stains which have been de- veloped for them are far more specific than the stains given for Rickettsiae in the previous section. The method of Schleifstein 1937 is in general employment in the United States today, although a far better demonstration, if speed is not of more importance than accuracy, is to be obtained from the technitiue of Lepine 1935. The "glyceric ether" mentioned in the technique of Manou^lian is not an article of com- merce in the United States, but is so readily available in Europe, where Manoiielian's method is widespread, that it is here included. Particular attention should be given to the formula of Stovall and Black 1940, for it is the only one of the formulas which have been given for the staining of these minute particles in which an acid buffer is emploj-ed. 23.13 Barreto 1944 test. 1945 Conn 20540b, 20:66 reagents required: A. either DS 11.121 Weigert 1903 or DS 11.123 Ehrlich 1886; B. 1% hydrochloric acid in 70% ale; C. water 100, methyl blue 0.12, eosin 0.12; D. 95% alc. 100, picric acid 0.005, acetic acid 0.1 method: [sections] -^ water — > A, till stained — > B, till differentiated — > tap water, till blue — > C, on slide, 15 mins, -^ rinse — > D, till differentiated — » 90% alc. -^ balsam, via usual reagents 23.13 Carpano 1916 test. 1942 Langeron Langeron 1942, 845 reagents required: A. 1% eosin Y; B. DS 12.15 crystal violet 20, water 80; C. ADS 12.2 Lugol (1905) method: [5 fx sections of F 3700.0010 Zenker 1894 fixed material, or smears similarly fixed] —y A, 1 min. — > 95% alc, wash -^ B, warmed to steaming, 5 mins. — > C, used to wash B from slide, 1 min. — > 95% alc, till differentiated -^ balsam, via usual reagents 23.13 Craigie 1933 11431,36:185 reagents required: .4.2% mercurochrome; B. water 100, sodium phosphate, dibasic (12 H2O) 0.35, mercurochrome 0.002, azur I 0.01, methylene blue 0.25 PREPARATION OF B: Mix 1 2% mercurochrome, 57% Na2HP04.12H20, 1 1% azur I. Add 75 water and 25 1 % methj-lene blue. method: [air-dried blood smears or tissue scrapings] — > methanol, 5-10 mins. —* dry -^ rinse — + blot —>■ A, 5-10 mins. —♦rinse — > blot —y B, 5-10 mins. — * quick rinse -^ dry recommended for: Paschen bodies. 23.13 Coles 1935 11360,55:249 reagents required: .4. DS 13.13 Giemsa 1902 (dry .stock) 0.75, glycerol 25, 95% alc. 75; B. water 100, tannic acid 5, orange G I method: [smears] — > A, 24 hrs. -^ wash -^ dry — * B, few moments — * wash -^ dry recommended for: virus inclusions in general. DS 23.13 DYE STAINS OF SPECIAL APPLICATION 465 23.13 Dawson 1934 11284, 20:G59 REAGENTS kequired: . t . 2 '^ pliloxiiic; B. DS 11.44 Loffler 1890 method: [methaiiol-fixed smears] -^ water -» A, 2-5 mins. — > water, wash — » li, 15 sees. -^ 20% ale, till no more color comes away — > balsam, via usual reagents note: a detailed descrii)tiou of the use of this technique is given on p. 75. 23.13 Frotheringham test. 1952 Krajian and Gradwohl Krajian and Gradwohl 1952, 216 kokmula: water 100, sat. aq. sol. magenta 0.9, sat. aq. sol. methylene blue 0.6 method: [smears from hippocampus or cerebellum (see Chapter 8)]— » abs. ale., few moments — + stain, 10-15 sees. -^ blot -^ dry result: Negri bodies red with blue granules. 23.13 Gallego 1923 23454,25:74 REAGExNTs REQUIRED: A. watcr 100, ferric chloride 0.3, nitric acid 0.3; B. water 100, DS 11.43 Ziehl 1882 3, acetic acid 0.3; C. water 100, 40% formaldehyde 0.6, nitric acid 0.3; D. DS 12.211 Cajal 1895 method: [sections by freezing technique of F 0000.1010 fixed material] -^ A, 1-5 mins. — » B, poured on slide, 5 mins. -^ water, wash -+ C, 1-10 mins. -> wash -^ D, I min. — > — > balsam, via usual reagents 23.13 Gerlach 1926 test. 1948 Lillie Lillie 1948, 226 REAGENTS REQUIRED: A. Water 100, DS 11.44 Loffler 1890 12, DS 11.43 Ziehl 1890 6 method: [paraffin sections of alcohol-fixed material]-^ A, poured on slide, heated to steaming, 1 min. -^ repeat 3 further times —> neutral mountant, via usual reagents 23.13 Goodpasture 1925 608b, 1:550 REAGENTS REQUIRED: A. Water 80, alcohol 20, phenol 1, anihne 1, magenta 0.5; B. DS 11.43 Loffler 1890 method: [thin sections of F 3700.0010 Zenker 1894 material] -♦ water -^ A. 10-20 mins, -* wash -^ blot -^ 95% ale, till light pink -> wash — > 8-15 sees, to 1 min. -> abs. ale. till differentiated — > balsam, via xylene result: Negri bodies red on blue. 23.13 Hamilton 1934 11587, 37:139 REAGENTS REQUIRED: A. water 50, 95% ale. 50, ethyl eosin 0.5, eosin Y 0.5; B. sat. sol. potassium alum; C. 0.15% ammonia in 95%, ale.; D. 0.5% methyl blue method: [4 11-6 n sections of F 3700.1000 Helly 1903 fixed material] -^ A, on slide, set ahght and allowed to burn out -* repeat -^ drain -^ B, flooded on slide -^ drain -> B, in jar, 10 mins. -^ wash -^ C, tiU tissue pink —>■ wash -^ D, 10 mins. -^ abs. ale, rinse -^ C, till inclusion bodies (vermilion) clearly differentiated — > wash -♦ balsam, via usual reagents RECOMMENDED FOR: virus inclusion bodies in general. 23.13 Harris 1908 11250, 5:566 reagents required: a. 1%, ethyl eosin in 95% ale; B. water 80, DS 11.44 Unna 1892 20 method: [alcohol-fixed smears] -^ water —> A, 1-3 mins. —» water, quick rinse ^ B, 10 sees. -* water, rinse -^95% ale, tfll differentiated — > balsam, via usual reagents 23.13 Jordan and Heather 1929 20540b, 4:121 reagents required: A. ADS 12.2 Lugol (1905); B. 2% sodium thiosulfate; C. water 100, eosin Y 0.5, phloxine 0.25; D. 0.1% azur B; E. clove oil 30, abs. ale 60 method: [5 M sections by E 22.1 Jordan and Heather 1929 method of F 3800.1000 Dominici 1905 fixed tissues] — > A, 10 mins. — > wash -^ B, till decolorized —>■ thorough wash-* C, 15 mins. -^ wash-* D, 2-5 mins. -» 95% ale, wash -> E, till differenti- ated -^ abs. ale, least possible time — ♦ xylene — > gum elemi 23.13 Kaiser and Gherardini 1934 23684, 131:128 reagents required: A. water 100, phloxine 0.5, aniline 1.5 method: [sections] — * water — > A, 1 min. -^ wash — > balsam, via usual reagents recommended for: Guarnieri bodies 23.13 Krajian 1941 see DS 23.221 Krajian 1942 466 METHODS AND FORMULAS DS 23.13 23.13 Lenz 1907 23684, 44:374 REAGENTS BEQUiRED: A. 0.5% eosin Y in 60% ale; B. water 100, potassium hydroxide 0.1, sat. ale. sol. {circ. 2%) methylene blue 30; C. 0.005%, potassium hydroxide in abs. ale; D. 0.1% acetic acid in abs. ale. method: [paraffin sections of acetone fixed and dehydrated material] -^ 70% ale. -> A, 1 min. -* water, wash —> B, I min. -» blot or drain -* C, till yellowish -^ D, till nerve cells clear blue — > balsam, via usual reagents 23.13 Lepine 1935 6630, 119:804 REAGENTS REQUIRED: A. 0.5% magenta in 50% ale. 50, 0.2% safranin 50; B. abs. ale. 50, acetone 50; C. DS 11.44 Stevenel 1918 method: [paraffin sections of F 3700.0010 fixed material]-^ A, 10 mins. -^ B, quick rinse -^ water, wash -^ C, 1 min. —>■ B, till section turns blue-^ water, wash ^ B, quick dip -^ abs. ale, till section turns lilac -^ balsam, via xylene 23.13 Lillie 1948 Lillie 1948, 225 reagents required: A. abs. ale. 90, water 6, acetic acid 3.25, ethyl eosin 1; B. any DS 11.122 stain; C. 0.25% acetic acid method: [alcohol-fixed material] -* .4, 2 mins. -^ 95% ale, rinse -^ water, wash ^ B, till sufficiently stained -> C, till differentiated -^ balsam, via usual reagents 23.13 McWhorter 1941 20540b, 16:143 reagents required: A. any wetting agent; B. 0.5% phloxine; C. 0.9% sodium chloride; D. 0.5% trypan blue method: [surface shaving from virus infected leaf] -^ A, brief rinse -^ B, 3-8 sees. -» C, brisk wash -^ D, 2-4 mins. — > C, for observation result: viroplasts blue, red, or purple. note: Formaldehyde-fixed material may be used after 1-6 hours in 10%, citric acid. Rich 1948 (20540b, 23:19) recommends pretreatment in water 85, sodium chloride 0.7, 95% ale 10, ether 5. 23.13 ManoueUan 1912 857, 26 :972 reagents required: A. acetone 100, ADS 12.2Lugol (1905) 0.3; B. DS 13.7 Mann 1892 (sol. yl); C. 2% glyceric ether in 95% ale method: [fresh smears] -^ A, 5 mins. -^ acetone, thorough wash -^ B, 1 min. -^ C, till differentiated -* balsam, via usual reagents note: Langeron 1949, 653 states that ADS 22.1 Beauverie and HoUande 1916, diluted 50 : 1 with water may be used in place of glyceric ether. 23.13 Nagle and Pfau 1937 617, 27 :356 reagents required: A. phosphate buffer pH 7.4 90, 8% ale magenta 0.3, sat. sol. methylene blue 0.2 method : [dry impression smear] — » methanol, 2 mins. —> wash -^ B, on slide, heated to steaming, 5 mins. -^ wash -^ dry 23.13 Neri 1909 23684,50:409 REAGENTS REQUIRED: A. watcr 100, iodinc 0.1, potassium iodide 0.2, eosin Y 1; 5. 1% methylene blue PREPARATION OF a: Dissolve the iodine and iodide in a few drops of water. Dilute to 50. Add dye dissolved in 50 water to iodine solution. method: [sections of acetone-fixed, or smears of alcohol-fixed material] -> A, 10-15 mins. -* water, wash -^ B, 5 mins. -* water, rinse -» 95% ale, till differentiated -> balsam, via usual reagents 23.13 Petragnani 1928a test. 1930 Ciferri 20540b, 5:34 REAGENTS REQUIRED: A. ADS 12.2 Petragnani (1928) (working sol.); B. 0.5% eosm B in 50% ale; C. DS 11.122 Mayer 1896; D. 0.1% methylene blue; E. 0.05% sodium hydroxide in abs. ale method: [sections] -^ abs. ale -^ A, 5-10 sees. -^ abs. ale, rinse -^ B, 10-20 sees. -^ wash -^ C, 1 min. -^ rinse -^ D, till violet (not blue) -> blot -^ E, 15-20 sees. -^ 95% ale, till blue — > balsam, via usual reagents result: Negri bodies, eosin-red; capillaries, nuclei, red; nerve cells, blue. DS 23.13-DS 23.2 DYE STAINS OF SPECIAL APPLICATION 467 23.13 Petragnani 1928b test. 1930 Ciferro 20540b, 6:35 REAGENTS REQUIRED: A. ADS 12.2 Petragnaiii (1928) (working sol.); H- 0.5% acid fuchsin in 50% ale. method: [sections] — + abs. ale. -^ A, few sees. -^ abs. ale., rinse ^70% ale, rinse — » B, 30 sees. — > wash — » balsam, via usual reagents 23.13 Parsons 1939 4349, 19:104 REAGENTS REQUIRED: A. water 94, 95% ale. 5.4, acetic acid 0.6, ethyl eosin 0.054; B. DS 11.44 Jadassohn (1928); C. 0.025% acetic acid method: [paraffin sections of formaldehyde-fixed material] —> water—* A, 2 mins. -^ 95% ale., till pink — * B, 2 mins. -^ rinse -+ C, till pale bluish pink — > 95% ale, rinse — > abs. ale, least possible time -^ balsam, via xylene result: Negri bodies, bright orange red with blue central dot; Nissl bodies, blue. 23.13 Rich 1948 see DS 23.13 McWhorter 1941 (note) 23.13 Schleifstein 1937 617, 27:1283 STOCK solution: methanol 50, glycerol 50, magenta 0.9, methylene blue 0.5 WORKING solution: stock 1.25, 0.0025% potassium hydroxide 100 method: [paraffin sections of F 3700.0010 Zenker 1894 fixed material] — * water —* stain, poured on slide, warmed to steaming, 5 mins. — » 90% ale, till pale violet -^ balsam, via usual reagents 23.13 Stovall and Black 1940 591b, 10:1 REAGENTS REQUIRED: A. 1% ethyl eosin in pH 3.0 buffer; B. 0.25% methylene blue in 50% ale at pH 5.5; C. 0.4% acetic acid method: [acetone-fixed material] -^ A, 2 mins. -^ water, rinse -^ B, 30 sees. — > C, till brownish red -^ water, wash — >• balsam, via usual reagents note: a detailed description of the use of this technique is given under DS 23.10 above _ 23.13 Taniguchi, Hosokawa, Kuga, Komora, and Nakamura 1932 test. 1933 Findlay 11360, 53:43 REAGENTS REQUIRED: A. acetone; B. 1% cadmium iodide in 40% formaldehyde; C. water 92.5, 95% ale 5, 40% formaldehyde 2.5, eosin 0.1; D. DS 11.43 Ziehl 1882 method: [air-dried smear] -^ A, 1 min. — > wash -^ B, 2 mins. — * rinse -^ C, 30 sees. — > rinse -^ D, 10 sees. -^ wash -^ dry note: Any turbidity in B is cleared up with a few drops of hydroclilorie acid. 23.13 Turewitsch 23684, 129:381 REAGENTS REQUIRED: A. 2.5% ferric alum; B. 5% tannic acid; C. 0.02% azur I; D. sat. sol. picric acid method: [sections] -^ A, 20-25 mins. — * wash — > B, 10 mins. -^ wash — » D, 10-15 mins. -^ wash -^ balsam, via usual reagents recommended for: Pasehen bodies in cornea. 23.13 Williams 1908 618, 18:10 formula: sat. ale sol. (circ. 6%) magenta 1.25, sat. ale. sol. (circ. 3.5%) methylene blue 25, water 75 method: [smears] -^ cover with stain, heat to steaming — > wash -^ blot — » dry 23.13 Zottner 1934 6630, 115:593 reagents required: A. 30% nitric acid; B. DS 11.43 Ziehl 1882; C. water 100, picric acid 0.5, indigocarmine 0.5 method: [paraffin sections]^' abs. ale — > A, few drops on slide, 2-3 mins. -^ B, on slide, 15 sees. —>■ rinse — > C, 15 sees. -^ abs. ale, till green -^ balsam, via xylene 23.2 Bacteria gether of the techniques given below as Bacteria can be stained by any of the "bacterial stains" is due entirely to the techniques which are customarily applied fact that persons skilled in the staining of to nuclei, as well as the majority of tech- zoological and botanical forms have niques which are used for the demonstra- drifted so far from bacteriological tech- tion of mitochondria. The grouping to- nicians as to tend to throw these bacterio- 468 METHODS AND FORMULAS DS 23.20 logical techniques into a separate branch of the science. This is very unfortunate and has given rise to many misunder- standings in the literature. Bacterial stains can be broadly divided, as they are below, into those which are applied customarily to smears, and those which are applied to the differential staining of bacteria in cells. The former class is very much the larger of the two, for the staining of bacteria in cells is rarely used for diag- nostic purposes, being generally confined to demonstrations in bacteriology and pathology classes. 23.20 TYPICAL EXAMPLES Staining a bacterial film with crystal violet by the technique of Hucker (1929) The technique is so simple that it would be scarcely worth the trouble to describe it, were it not necessary for the benefit of those who have never previously handled bacterial material, and who may wish to attempt this for the first time. The only tools and reagents necessary are a clean glass slide, a wire loop of the type used normally in bacteriology, a drop bottle containing crystal violet stain (DS 23.211 Hucker 1929), and a wash bottle containing distilled water. Take a culture from which it is desired to stain the organisms, and touch the freshly flamed wire loop as lightly as pos- sible either to the surface of the medium in a test tube or to the surface of the col- ony in an agar petri-dish culture. The loop is then touched lightly to the center of the clean slide to transfer the bacteria. The only mistakes likely to be made by the beginner is securing too great a quantity of material, or making too large an area. It must be remembered that the specimen is to be examined under an oil immersion lens so that the smallest possible smear, derived just by touching the slide with the moist platinum loop, will have an ample area for the purpose required. If the micro-organisms have been taken from a test tube containing a liquid cul- ture which has not yet reached a very thick stage of growth, the spot may now be allowed to dry in air; but if the bacteria were taken from a colony on the surface of agar, it is necessary to dilute them with water, taken in the same platinum loop, and touched to the same spot. The spot is then spread with the loop, and the slide is dried. As soon as the shde has dried, which should take only a moment or two if a sufficiently small quantity of fluid has been used, it is taken and "heat-fixed" in the flame of a bunsen burner or a spirit lamp. This is done by taking the slide and passing it twice quite rapidly through the flame of the bunsen. The actual tempera- ture should not exceed about 80°C., and it is customary to hold the slide smear downward as it passes through the flame. Care must be taken that the slide is quite dried before being thus quickly flamed, or the bacteria will burst and be worthless. Now take the flamed and dried smear and place on it a drop of the selected stain (in this case crystal violet), leaving this in place for about 30 seconds. The time is not critical; any time between a half and one minute is satisfactory. It will be noticed that the stain frequently evapo- rates slightly, leaving a greenish film on the surface. It is, therefore, better to wash it off with a jet from a wash bottle than to endeavor to rinse it off. Tliis jet should be directed from the fine orifice of the wash bottle, at an angle of about 30°, to the slide and should be intended to hit the edge of the drop. This will instantly hft off and float away the surface film, and will also wash excess stain out of the preparation. The preparation is now dried, touched with immersion oil, and examined under the oil-immersion lens. Demonstration of Gram-positive bacteria in smear preparations by the method of Gram 1884 The name of Gram is so firmly fixed to these iodine-differential techniques, that it seems ■well to describe the original method without modification, leaving it to the technician to determine for himself which of the numerous modifications, pro- posed in section DS 23.212 below, best fits his particular problem. For the benefit of DS 23.20 DYE STAINS OF SPECIAL APPLICATION 469 those who are not acquainted with bac- teriolog}^ it may be added that it has been customary, since the time of Gram, to utiHze the reactions of bacteria to iodine mordanting as a basis of diagnostic classi- fication. All bacteria, without reference to their nature, may be stained by the method given in the last example, but there are some bacteria from which the stain can be removed by the action of an iodine-potassium iodide solution rein- forced with alcohol. The bacteria from which the stain is not removed are known as Gram-positive; those from which the stain is removed are known as Gram- negative. The solutions required are crys- tal violet (Gram himself used the mixture of dyes known as gentian violet) which is usually prepared as a phenol solution by the method given in Chapter 20 (DS 12.15). There is often a sharp argument between technicians as to whether or not this method of preparation is essential, but it is in customary usage and should be followed b}' the beginner. Gram's iodine solution, the formula for which is given as ADS 12.2 Gram 1884 (Chapter 22) is difficult to prepare unless the technique is exactly followed. Iodine is very soluble in strong solutions of potassium iodide, but is only slightly soluble in weak solu- tions. If the total quantities of iodine and potassium iodide shown are placed in the total quantity of water, a period as long as a week may elapse before a solution is complete; but if the dry iodine and the drj' potassium iodide are mixed together, and a few drops of water are added, a solu- tion will be produced almost instantly. A smear is prepared as described in the last example. The same precautions as to dilution there mentioned must be taken if the material is obtained from a bacterial colony. The smear is then dried, flamed, and a drop of crystal violet poured onto it from a drop bottle, exactly as in the previous example. In this instance, how- ever, it is not desirable to extract too much of the stain with water, hence, after the stain has been acting for two minutes or so, the entire sUde is rinsed rapidly in water, and a drop or two of the iodine solution poured over it. If manj' slides are being stained, it is probably simpler to drop the shde into a coplin jar containing the iodine solution than to pour iodine on it. After the iodine has acted for one min- ute the slide is given a quick rinse to remove the excess iodine, and then placed into absolute alcohol until no more color comes away; unless the film is very thick, this will appear completely to decolorize it. It is then passed from alcohol to water, which instantly stops differentiation, and then dried. Varying types of bacteria re- quire varj'ing periods of differentiation, but it is better for the beginner to use absolute alcohol until no more color comes away, than to endeavor to control the differentiation under the microscope. Though such a preparation is a Gram's preparation by the original technique, it is customary nowadays to pro\'ide a counter- stain of a contrasting color to bring clearly into evidence any Gram-negative organ- isms which may be mixed with Gram- positive. A 1% solution of safranin is widely employed, though Kopelloff and Beerman 1922 (DS 23.212 below) recom- mend a 0.1% solution of magenta for the same purpose. In either case, the second red contrasting stain is allowed to act for five to ten seconds and is then washed off with water. Demonstration of tubercle bacilli in sputum by the technique of Neelsen 1883 When Neelsen pubUshed his original technique for the demonstration of tuber- cle bacilli (DS 23.213 Neelsen 1883), the standard magenta solution used for the staining of bacteria was that proposed in the previous year by Ziehl. Because of this Neelsen's technique was referred to as a modification of Ziehl, and to this day the hyphenated term Ziehl-Xeelsen is applied to almost any method for the demonstra- tion of tubercle bacilh in sputum, irrespec- tive of the author of the technique. It is proposed here to describe the original technique of Neelsen, leaving it to the technician to determine which of the other methods given in this section is more readily applicable to his problem. It may be said in favor of the technique of Neelsen that it gives a better differentia- 470 METHODS AND FORMULAS DS 23.20 tion of tubercle bacilli than do some of the more recent methods. These, though they give good preparation, do tend to cause certain errors of diagnosis through the ability of other bacteria to withstand the lower concentration of acids nowadays employed. It is to be presumed that the sputum collected from the patient will have been placed at the disposal of the technician in the glass vessel in which it was secured. It should be looked over carefully to see if any small yellowish particles exist in it; if they do, one of these particles should be carefully extracted with a sterile bacterio- logical wire loop and utilized in making the preparation. If no such particles are visible to the naked eye, it is, of course, possible that tubercle bacilli will be pres- ent, but due consideration should be given to some method of concentrating these ba- cilli before making the smear. The stand- ard method of concentration is to hy- drolize the sputum to the extent necessary with the aid of a weak solution of potas- sium hypochlorite, which is known to be without action on tubercle bacilli. For a long time, a proprietary compound known as antiformin, which is a strongly alkaline solution of potassium hypochlorite, was also used for the same purpose. About an equal quantity of the selected solution and the sputum are placed in a centrifuge tube, the tube incubated in a serological water bath (37°C.) for about ten minutes, and then centrifuged rapidly. The smear is made from the denser portions which remain at the bottom of the tube. Whichever method is employed, the quantity removed by the sterile loop should be about the size of a large pin- head. That is, a great deal more should be employed than is used for a simple bacterial smear from a known culture. This pinhead of material must be spread over the largest possible area of the shde. This is best done by pressing another shde on it and then drawing the two slides apart with a lateral motion. Both slides are then dried in air and flamed as has been described in the discussion of previ- ous bacteriological preparations. The solutions required for the original Neelsen technique are the phenol-magenta solution of Ziehl (Chapter 20, DS 11.43), a 25% solution of nitric acid, and the poly- chrome methylene blue of Loffler (Chap- ter 20, DS 11.44). The shde is first flooded with the magenta solution and then placed on a metal sheet, where it should be warmed by a bunsen flame to the point at which it is steaming, but at which no bubbles have appeared. If it shows signs of drying, fresh quantities of the magenta solution should be added to it. It may either be left at this temperature for three to five minutes (Neelsen's original recom- mendation) or, as is customary in modern practice, it may be raised to steaming, per- mitted to cool, again raised to steaming, permitted to cool, and so on, until four such cycles have been completed. The slide is then washed in tap water until no further magenta comes away and placed in 25% nitric acid until it is almost com- pletely decolorized. It cannot be decolor- ized too far, but there will usually be, even after prolonged exposure to the acid, a faint pink coloration of the background. The slide is now washed in running water until all the acid is removed, and then treated with a blue stain for about two minutes, to provide a contrasting colora- tion of any other bacteria present. It should then be washed thoroughly, dried, and examined in the customary manner. It must be emphasized that this tech- nique as described is specifically designed to show tubercle bacilU, and is so violent that many bacteria which are acid-fast to less strong acids will be decolorized. Demonstration of the flagella of Proteus vulgaris by the method of Tribondeau, Fichet, and Dubreuil 1916 Examination of the abbreviated form of this technique (DS 23.215 Tribondeau, Fichet, and Dubreuil 1916) below would give one the impression that it was a sim- ple matter to stain the flagella of bacteria. In point of fact it is one of the most diffi- cult preparations known to the science of microtomy. In the first place it is necessary to secure a culture in which the organisms are ac- tively motile and growing; it is useless to DS 23.20 DYE STAINS OF SPECIAL APPLICATION 471 attempt to stain flagella for demonstra- tion purposes in any other than a culture which has been specially prepared for the purpose. It is desirable to take an actively growing culture and to check, under a dark field illuminator, that the organisms are motile. This culture is then subcul- tured, in the evening, into the same me- dium, and checked again the next morn- ing. The temperature of the culture is then raised about ten degrees above that at which it has been maintained overnight, and left at this temperature for about an hour. A final check should be made on the motility of the organisms. The chief difficulty in staining these or- ganisms lies in the fact that the stain is not in any sense of the term a specific stain for flagella. It is only designed to make certain that any minute particle present on the shde will be colored so densely as to become apparent when examined under a microscope. The stain is deposited just as enthusiastically on the body of the bac- teria as it is on the flagella, and, if any- thing, more enthusiastically on every min- ute speck of organic detritus which may remain on the shde. Little or nothing can be done about this organic detritus which may be present in the culture medium, and the reason for subculturing the se- lected culture is to make sure that only young organisms shall be present, and that numerous decomposing bodies of bac- teria shall be absent. The method employed is a mordant process, using an alum-tannic acid solu- tion which must be prepared with some care. Take 100 milhUters of water and add to it at the same time 3.5 grams of potas- sium alum and 3 grams of tannic acid. These should be placed in a 250-milhliter Ehrlenmeyer flask, and warmed over a flame with constant agitation until they are boihng. The flask is now plugged with cotton, autoclaved at 20 pounds pressure for 30 minutes, and allowed to cool until it can be handled. It is filtered, and the filtrate is placed in a refrigerator until chilled. While the filtrate is cooling, pre- pare a 1 % solution of crystal \'iolet. The staining solution, which consists of about 10 parts of the crystal violet to 100 parts to the tannic acid solution, should be pre- pared immediately before use and, after mixing, should be passed through a bac- terial filter to remove from it any frag- ments which might possibly remain and which, adhering to the dried film, would stain just as readily as would the flagella themselves. Having secured an appropriate culture and having made up the staining solution, make a smear in the manner described in the first example of bacterial technique, dry it, flame it, and pour over it a good supply of the mixed solution. Then hold the shde over a very low bunsen flame and heat until bubbles appear. The heating should be continued for about 20 seconds beyond the time when the bubbles first appear. The sUde is then washed off with water, until no more color comes away, and dried before being examined under an oil immersion objective. If everything has gone correctly the bacteria and their flag- ella will be stained a dense black against a background absolutely free from debris. If, however, there is a granular deposit on the background, which has not been de- rived from the culture, a fresh solution should be made, using, say, 7 parts of violet to 100 of the mordant. If the bac- teria, but not the flagella, are stained, take 15 parts of violet to 100 of the mordant, and repeat the })rocess again. No success is possible save by trial and error. Demonstration of diplococci in the Uver of the rabbit using the phloxine- methylene blue-azur II stain of Mallory 1938 This method of Mallory is the best of all the eosin-methylene blue methods which have from time to time been sug- gested for staining bacteria in sections. It has the advantage of giving a first-class histological stain, in addition to differ- entiating bacteria, and it might well be used as a standard procedure in place of the more customary hematoxyUn-eosin, at least in pathological investigations. The liver of a rabbit is so frequently infected with diplococci that it has been selected as a type demonstration, for such infected animals will be found in ordinary labora- 472 METHODS AND FORMULAS DS 23.20 tory investigations, making it unnecessary to go to the trouble of infecting a rabbit for the purpose of securing the necessary demonstration material. If, then, in the course of routine dissec- tions, a sacrificed rabbit is found to have a pneumococcal infection of the liver, which may easily be seen as yellow lesions on the surface, it is only necessary to cut the lesion and some surrounding tissue, and to place it in a suitable fixative. Mal- lory himself recommends fixation in Zenker 1894 (Chapter 18, F 3700.0010) for about 24 hours. As Zenker contains mercuric chloride it is undesirable that the specimen should remain in it for more than three or four days, but the actual time of fixation is not critical. As always, when deahng with dichromate fixatives, a large quantity of fixative should be used, and the object should be suspended in the fixative solution in a loose cloth bag. When fixation is complete the pieces are re- moved from the fixative and washed in running water overnight. They are then embedded in paraffin and sectioned in the ordinary manner. Sections of from five to eight microns are desirable if it is intended to demonstrate the bacteria, though these are quite readily seen in the ten-micron sections customarily employed for histo- logical examinations. When the sections have been fixed on the shde, they may, if desired, be freed from the last traces of mercuric chloride by treating them with iodine and bleaching with sodium thio- sulfate. The writer does not usually do this, but the treatment is insisted upon by Mallory in the description cited. The staining solutions used in this tech- nique are simple to prepare and relatively stable. The first solution is a 2.5 % solution of phloxine in water. Two stock solutions are also required: one of 1% each of methylene blue and borax in distilled water, and the other a 1% solution of azur II in distilled water. Five milUhters of each are added to 90 milUhters of dis- tilled water for use. Differentiation is in Wolbach's resin-alcohol (ADS 21.2 Wol- bach 1911). It is difficult to secure a sufficiently heavy stain in phloxine to withstand the alkaline tliiazine solutions used for couu- terstaining. Mallory recommends that the sections be placed in a coplin jar of the solution in a 55°C. oven and that they remain there for at least an hour. The coplin jar is then removed from the oven and cooled before the sections are re- moved; they should be stained a dense orange. If they have not yet acquired this color, they should be returned to a paraffin oven for a further period. If the sections are satisfactorily stained, the solution may be poured off or the slides removed from it and briefly rinsed in water. The purpose of this rinse is not to differentiate the eosin in the section but to remove it from the glass. The slides bear- ing the sections are then placed in the methylene blue-azur solution in another coplin jar for 5 to 20 minutes; the exact time varies according to the specimen which one is staining and can only be determined by experiment. Mallory rec- ommends that the solution be freshly filtered onto each slide, rather than that the solution be used in a coplin jar; but the writer has not found this nearly as convenient, nor does it appear to be in any way obligatory. After the slides have taken up sufficient methylene blue solution to appear bluish rather than pinkish, they may be accumulated in water, before being differentiated in the resin alcohol one at a time. This differentiation is best conducted in a dish which is large enough to admit the slide in a flat position. The slide is taken in a pair of angle forceps, dipped under the surface of the solution, and waved gently backward and forward for about a minute. As it is being moved backward and forward in the differentiat- ing solution, the blue color will come off in clouds; it is much easier to overdifferenti- ate than to underdifferentiate. The differentiation may be readily con- trolled by inspection under the microscope, though it is not necessary to observe the bacteria, since the nuclei have exactly the same staining reaction. Differentiation should be stopped when the nuclei can be seen, under the low power of the micro- DS 23.211 DYE STAINS OF SPECIAL APPLICATION 473 scope, to be very deep blue, while the general background of the section is pink. After a little practice the required color may be gaged without examination under the microscope. These specimens are not permanent unless the alcohol and resin are removed from tliem. It is desirable, therefore, as soon as differentiation is complete, to dehydrate them in absolute alcohol and then clear them in at least three changes of xylene, so as to make sure that no alcohol can be carried over into the mounting medium. 23.21 BACTERIA IN SMEARS Bacterial smears are rarely, if ever, fixed. Hence, they share with blood smears the ability to be stained by a method depending not only upon the chemical composition of the material, but also upon the isoelectric point of the protein from which it is produced. Smears may be prepared either upon the slide or the coversUp, and are usually from cultures which have been heavily diluted either with the medium upon which they are grown or with an isotonic salt solution. S3.S11 General Methods It would be quite impossible to gather in one place all the stains which have, from time to time, been recommended for use with bacterial smears. Such a listing of techniques would have to include every stain recommended for staining nuclei, and the great majority of syn- thetic stains when adjusted to a suitable pH. Gathered here are only those solutions which are commonly found in laboratories of bacteriology, or those which involve some departure from the normally recognized techniques, such as the early stain of Claudius 1897, or its more recent modification by Spehl 1927. It must be emphasized again that the staining of a particle by any of the methods given is no voucher for the bacterial nature of the particle. Many of the techniques given are almost indistinguishable from the techniques recommended for the display of mitochondria, therefore, some other evidence than staining is required to prove the existence of bacteria. 23.211 Claudius 1897 857, 11 :332 REAGENTS REQUIRED: A. DS 12.15 gentian violet; B. sat. aq. sol. picric acid 50, water 50; C. chloroform method: [dry smears] — > A, flooded on slide, 2 mins. -^ quick rinse — > B, 1 min. — > blot and let dry — > C, till no further color removed — ♦ balsam 23.211 Conn 1928 20540b, 26:257 formula: water 100, phenol 5, calcium chloride 0.01, rose bengal 1 method: [smears of gelatin-suspended soil dried at 100°C.] — » stain, on slide, heated on water, bath, 1 min. — > water, wash — > dry 23.211 Ehrlich 1882 7276:270 formula: abs. ale. 10, gentian violet 5, sat. sol. aniline 90 preparation: Add the aniline to a solution of the dye in ale. method: As Ziehl 1882 see also DS 23.213 Koch 1884 23.211 Goodpasture test. 1938 Mallory Mallory 1938, 274 formula: water 70, 96% ale. 30, aniline 1, phenol 1, magenta 0.6 23.211 Hucker test. 1929 Conn McClung 1929, 93 formula: 95% ale. 20, crystal violet 2, water 80, ammonium oxalate 0.8 preparation: Add the oxalate dissolved in water to the dye dissolved in ale. method: As Ziehl 1882 note: a detailed description of the use of this technique is given under DS 23.20 above. 23.211 Loffler 1890 for formula see DS 11.44 Loffler 1890; for method see DS 23.211 Ziehl 1882 23.211 Maneval 1941 20540b, 16:13 formula: water 95, phenol 3.9, acetic acid 5, ferric chloride; 3, either acid fu<'lisin 0.01 or anilin blue 0.05 or fast green FCF 0.05 or light green 0.05 method: [air-dried or heat-fixed smear] — > stain, 1 min. -^ wash — > dry 474 METHODS AND FORMULAS DS 23.211-DS 23.212 23.211 Manson 1890 for formula see DS 11.44 Manson (1890); for method see DS 23 211, Ziehl 1882 23.211 Pick and Jacobson test. 1942 Langeron cii. Morax Langeron 1942, 1198 formula: water 100, DS 11.43 Ziehl 1882 3, sat. ale. sol. (circ. 2%) methylene blue 1.5 method: [heat-fixed smear] -^ stain, 20-30 sees. -^ wash -^ dry 23.211 Sahli 1885 for formula see DS 11.44 Sahh 1885; for method see DS 23.211 Ziehl 1882 23.211 Spehl 1927 6630, 107 :920 REAGENTS REQUIRED: A. DS 12.15 gentian violet; B. sat. aq. sol. iodine; C. sat. aq. sol. picric acid; D. chloroform; E. DS 12.15 magenta 10, water 90 method: [dry smears] -^ A,2 min. -^ quick rinse -* B, 30 sees. -^ C, 30 sees. -^ dry -^ D, till no more color comes away -^ dry -^ E, 1 min. — » wash -^ dry -^ balsam 23.211 Unna 1892 for formula see DS 11.44 Unna 1892; for method see DS 23.211 Ziehl 23.211 Volkonsky 1933 for formula see DS 11.44 Volkonsky 1933; for method see DS 23.211 Ziehl 23.211 Ziehll882 7276,8:451 REAGENTS REQUIRED: A. DS 11.43 Ziehl 1882, sol. A. method: [heat, or ale., fixed smears] -^ A, poured on slide, 30 sees. -^ water, wash — > dry note: The same technique may be employed with DS 11.44 Loffler 1890, Manson (1929), Martinotti 1910, Sahli 1885, Unna 1892, Volkonsky 1933. See also DS 22.213 Neelsen 1883. SS.212 Iodine Differential Methods The stains given in this section are commonly referred to as "Gram stains," and the organ- isms which retain the color after any of the techniques here given are known as Gram-posi- tive. Certain methods, such as Burke 1922, involve after-staining, usually with safranin, in such a manner as to render those bacteria from which the stain is removed (the Gram-nega- tive bacteria) red, or such color as will contrast well with the Gram-positive bacteria. The methods require, without exception, preliminary heavy overstaining of the smear, the re- moval of the stain with iodine, and usually, the removal of the iodine with acetone before the application of the counterstain. 23.212 Atkins 1920 11056, 5:321 reagents required: A. DS 11.45 Atkins 1920; B. ADS 12.2 Atkins 1920 method: [heat-fixed smear] -^ .4, 1 min. —> wash —> J5, 1 min. — > wash — » 95 % ale, rinse — > dry 23.212 Burke 1922 11056, 7:159 REAGENTS REQUIRED: A. 1% Crystal violet; B. 5% sodium bicarbonate; C. ADS 12.2 Burke 1922; D. ether 25, acetone 75; E. 2% safranin O method: [smears] — ♦ A, flooded on slide — > add 2-3 drops 5 to A on slide, 2-3 mins. — >• C, rinse -^ C, fresh solution, 2 mins. — > water, wash -^ blot —* D, dropped on slide til! no more color comes away -^ dry — * E, 10 sees. -^ water, wash -^ dry result: Gram-positive, blue; Gram-negative, red note: Kopeloff and Cohen 1928 (20540b, 3:64) substitute equal parts acetone-ale. for D above. 23.212 Hucker and Conn 1923 20936, 93:1 REAGENTS REQUIRED: A . BS 23.211 Hucker (1929); B. ADS 12.2 Gram 1884; (7. 95% ale. 10, safranin O 0.25, water 100 method: [smears] —> A, 1 min. — ^ water, wash -^ B, 1 min. — » water, wash — * blot — ♦ 95% ale. 30 sees. -^ blot — > C, 10 sees. — > water, wash — > dry result: Gram-positive bacteria, blue; Gram -negative, red. 23.212 Gram 1884 8645,2:185 REAGENTS REQUIRED: A. DS 12.15 Crystal violet; B. ADS 12.2 Gram 1884; C. absolute ale. DS 23.212 DYE STAINS OF SPECIAL APPLICATION 475 method: [dried smears] -^ A, dropped on slide, 2 mins. — » quick rinse — > B, 1 min. — > quick rinse — > C, till differentiated —^ water, to stop differentiation — > dry — * balsam note: a detailed description of the use of this technique is given under DS 23.210 above. 23.212 Konschegg 1940 14674, 87 :465 REAGENTS REQUIRED: A. watcr 90, 95% ale. 7, aniline 3, gentian violet 2; B. sat. aq. sol. picric acid method: [heat-fixed smears] —* A, 5 sees. -^ B, 5 sees. — > 95% ale, till no more color comes away -^ dry note: This stain, though not using iodine, is intended to demonstrate Gram-positive bacteria. 23.212 Kopeloff and Beerman 1822 11250, 31:480 reagents required: A. water 105, crystal violet 0.75, sodium bicarbonate 1.25; B. ADS 12.1 Atkins 1920; C. ether 25, acetone 75; D. 0.1% magenta method: [smears] — > A, 5-10 mins. — » B, rinse —^ B, fresh solution, 2 mins. -^ blot — > C, dropped on slide, till no more color comes away —> dry — ♦ D, 5-10 sees. — > water, wash -^ dry result: Gram-positive, blue; Gram-negative, red. 23.212 Kopeloff and Cohen 1928 20540b, 3 :64 REAGENTS REQUIRED: A. 1% methyl violet 6B 60, 5% sodium bicarbonate 8; B. ADS 12.2 Kopeloff and Cohen 1928; C. acetone 50, 95% ale. 50; D. 0.1% magenta method: [heat-fixed smear] -^ A, on slide, 5 mins. — > B, on slide, 2 mins. -^ drain -+ C, drop by drop on slide till drippings colorless -^ dry 23.212 Merieux test. 1904 Besson Besson 1904, 247 REAGENTS REQUIRED: A. DS 23.212 NicoUc 1895, sol. A; B. water 100, iodine 0.5, potas- sium iodide 1, eosin Y 2 preparation: Dissolve the iodine and iodide in a few drops of water. Add the eosin dis- solved in 100 water. method: [heat-fixed smear] -^ A, on slide, 10-20 sees. — » rinse -^ B, 5-10 sees. -^ wash dry 23.212 NicoUe 1896 857, 9 :664 reagents required: A. DS 12.15 crystal violet; B. ADS 12.2 Nicolle 1895; C. absolute ale. 85, acetone 15 method: [dried smears] —> A, flooded on slide, 2 mins. -^ quick rinses B, 1 min. — > rinse —^ C, till differentiated — > water, to stop differentiation -^ dry — > balsam 23.212 Schmorl 1928 see DS 21.13 Schmorl 1928b 23.212 Weiss 1940 11284,26:1518 reagents required: A. water 80, ale. 20, gentian violet 3; B. ADS 12.2 Lugol (1905); C. acetone; -D. 2% magenta in 95% ale. method: [heat-dried smears] — > A, 3-5 mins. —>■ warm water, wash -* B, 3-5 mins. -^ warm water — * C, till no more color comes away — > water, wash -^ D, 15 sees. -^ water, wash — > dry 23.212 White and Culbertson 1945 Tech. Bull, 6:53 REAGENTS REQUIRED: A. 1% Crystal violet; B. 5% sodium bicarbonate; C. water 100, mercuric iodide 0.55, potassium iodide 0.45; D. 0.1% safranin 0 method: [heat-dried smears] —> A, 1 ml. on slide -^ B,5 drops, added to'A on slide, leave 1 min. -^ drain — > dry — * C, 1 min. -^ wash —> acetone, till no more color comes away — ♦ dry — > D, 10 sees. — > wash — > dry note: The original calls for "a 1% solution of potassium mercuric iodide"; there is no such salt. The pharmacopeial liquor hydraryyri et potassii iodidi contains mercuric iodide and potassium iodide in the proportions given for C above. The same result could be secured by diluting 55 of the pharmacopeial solution to 100. If the solution is prepared from the dry salts, they must first be dissolved in a minimum of water, and then diluted. 476 METHODS AND FORMULAS DS 23.213 S3.S1S Methods for Acid-fast Organisms "Acid-fast" bacteria were originally so identified because a solution of magenta was not re- moved from them by strong acid. These techniques are commonly all lumped together as " Ziehl-Neelsen techniques" for the reason that the phenol-magenta stain of Ziehl 1882 was in 1883 used for the first demonstration of acid-fast bacteria by Neelsen. It is nowadays, how- ever, most unusual to use so violent an acid solution (25% nitric acid) as did Neelsen, and there is no justification for the blanket retention of the names of these two technicians for every acid differentiated stain which has been proposed. Many, if not indeed most, of the modern techniques do not use acid for differentiation at any stage of the proceedings, and attention should be drawn to the technique of Burke, Dickson, and Phillips 1932 by which acid-fast bacteria may be clearly differentiated from all other bacteria in the section without the use of any acid at all. Similarly, the technique of Fontes 1909 gives a similar differentia- tion using neutral solution. When the term "Ziehl-Neelsen" is loosely used in the literature today, the technique of Giinther 1899 is the one which is most usually intended. 23.213 Alexander 1932 19938, 75:197 REAGENTS REQUIRED: A. DS 11.43 Ziehl 1882; B. 3% nitric acid in 70% ale; C. DS 11.44 Loffler 1890; D. 0.05% sodium hydroxide method: [heat- fixed smears] -^ A, on slide, warmed to steaming, 5 mins. — > rinse — > C, on slide — * D, 6-8 drops, added to C on slide, 2-3 mins. -^ wash -^ dry 23.213 Alexander and Jackson 1944 19938, 99 :307 REAGENTS REQUIRED: A. DS 11.43 Ziehl 1882; B. 3% hydrochloric acid; C. DS 11.44 Loffler 1890; D. 4% sodium hydroxide; E. 0.2% sodium hydrosulfite; F. water 100, light green 0.5, fast yellow 0.5 method: [heat-fixed smear] —> ^, 3 mins. -^ wash —> B, 1-3 mins. —» wash —> C, on slide -^ D, 6 drops added to C on slide, 1 min. -* E, flooded on slide — > thorough wash — > F, flooded on slide — * wash —> dry 23.213 Augusta 1932 6630, 11:719 REAGENTS required: A. DS 11.43 Ziehl 1882 50, DS 12.15 crystal violet (Auguste) 50; B. 95% ale. 60, acetic acid 30, picric acid to sat.; C. 1% methylene blue method: [heat-fixed smears] -^ A, warmed to steaming, 3 mins. — * B, till no more color comes away — » water, wash -^ C, 15 sees. -^ water, wash —* dry 23.213 Blot 1901 see DS 23.213 Neelsen 1883 (note) 23.213 Burke, Dickson, and Phillips 1932 20540b, 7:21 REAGENTS REQUIRED: A. DS 11.43 Ziehl 1882; B. sat. sol. malachite green in acetone method: [smears] — > A, on slide, warmed to steaming, 3-5 mins. -^ water, rinse — + B, flood on slide and replaced as evaporation occurs, 3-5 mins. —^ wash — » dry result: acid-fast bacteria, red; others, green. 23.213 DogUo 1932 20540b (abstr. 1933) 8:76 reagents required: ^4. DS 11.43 Ziehl 1882; B. water 85, 95% ale. 20, sulfuric acid 10, brilliant yellow 0.15 method: [heat-fixed smears] -^ A, warmed to steaming, 2-3 mins. -^ thorough wash — » B, 40-50 sees. — > wash — * dry 23.213 Fontes 1909 13465,1:59 reagents required: A. DS 11.43 Ziehl 1882; B. DS 12.15 crystal violet; C. ADS 12.2 Lugol (1905); D. abs. ale. 60, acetone 30; E. 1% methylene blue method: [heat-fixed smears] — * A, warmed to steaming 2 mins. — > wash — > B, warmed to steaming, 2 mins. —* C, mixed with B on slide, 1 min. -^ D, till no more color comes away -^ water, wash -^ E, 15 sees. -^ water, wash -^ dry 23.213 Fraenkel 1884 2813, 13:1 reagents required: A. DS 23.211 Ehrlich 1882; B. water 50, ale. 30, nitric acid 20 methylene blue to sat. method: [heat-fixed smears] —» A, 12 hrs. -^ water, rinse —> B, dipped until smear changes from blue to red -^ water, wash —>■ dry DS 23,213 DYE STAINS OF SPECIAL APPLICATION 477 23.213 Gabbet 1887 test. 1928 Zinnser and Bayne-Jones Zinnser and Bayne-Jones 1928, 847 REAGENTS REQUIRED: A. DS 11.43 Zichl 1882; B. water 75, sulfuric acid 25, methylene blue 2 method: [heat-fixed smears] — > A, warmed to steaming, 2 mins. —* water, wash —* B, 1 min. — > water, wash —* dry 23.213 Gibbe test. 1903 Cole Cross and Cole 1903, 160 formula: water 45, 95% ale. 45, aniline 9, magenta 6, methyl blue 3 preparation: Grind dyes together and dissolve in aniline and ale. Dilute to 100 with water. method: [heat-dried smears] -^ stain, heated to steaming, 4-5 mins. — * 95% ale, till no more color comes away -^ balsam, via clove oil 23.213 Giinther 1898 Gunther 1898, 347 reagents required: A. DS 11.43 Ziehl 1882; B. 3% nitric acid in 70% ale; C. sat, sol. (circ. 2%) methylene blue method: [coverslip smear] — > A, in watch glass, heated to steaming, 1 min. —* B, 1 min. — » water, wash -^ C, on coverslip, few sees. —* water, wash —* dry 23.213 Herman test. 1904 Besson Besson 1904, 638 reagents required: A. 1% ammonium carbonate 80, 3% crystal violet 20; B. 10% nitric acid; C. 1% eosin in 60% ale. method: [heat-fixed smear] —>■ A, heated to boiling, 1 min. — > B, 4-5 sees. — + abs. ale, till decolorized — > C, 30 sees. -^ abs. ale, quick rinse — > dry 23.213 Koch 1884 1648, 2 :10 reagents required: A. DS 23.211 Ehrlich 1882; B. 25% nitric acid; C. sat. sol. (circ. 1.5%) Bismarck brown method: [smears] —>■ A, 12 hrs. -^ B, momentary dip — > 60% ale, wash —y D, 15 sees. — > water, wash -^ dry note: Much confusion exists in the literature about this technique, which is often at- tributed to Ehrlich (loc. cit., A above) who recommended his solution for general, not diagnostic, staining. The journal reference given above is technically incorrect for, in the two years prior to 1886, the journal was known as the Mitteilung der Kaiserliche Gesundheitsamte, which is, however, not recorded in the World List of Scientific Periodicals. 23.213 MuUer and Chermock 1945 11284, 30:169 reagents required: A. DS 11.43 MuUer and Chermock 1945; 5. 3% hydrochloric acid in 95% ale, C. DS 11.44 MuUer and Chermock 1945 method: [heat-stained smears] -^ A, \ min. -^ B, 30 sees. — > C, 1 min. — » wash -^ dry 23.213 Neelsen 1883 Ziehl-Neelsen — compl. script 23730, 21 :497 reagents required: A. DS 11.43 Ziehl 1882; B. 25% nitric; C. DS 11.44 Loffler 1890 method: [sputum smears, heat-fixed] — > ^, on slide warmed to steaming, 3-5 mins. — ♦ water, rinse -^ B, till faint pink -^ wash -^ C, 2 mins. -^ wash — » dry result: acid-fast bacteria, red; others, blue. note: The B solution is nowadays usually replaced by a 3% solution of hydrochloric acid in 95% ale (Cowdry 1943, 17.) The term Ziehl-Neelsen has come to mean any acid- differentiated magenta stain. Blot 1901 (6539, C. Lyons, 234) recommends 40% formaldehyde in place of C above. A detailed description of the use of this technique is given under DS 23.20 above. 23,213 Pappenheim 1898 2813, 37 :809 reagents required: A. DS 11.43 Ziehl 1882; B. abs. ale 100, glycerol 20, aurin 1, methylene blue to sat. (circ. 2) method: [heat-fixed smear] —> A, heated to steaming, 2 mins. — > B, on slide, drained and renewed 4-5 times -* water, wash —* dry 478 METHODS AND FORMULAS DS 23.213-DS 23.214 23.213 Pottenger 1942 test. 1942 Farber 20540b, 17:183 REAGENTS REQUIRED: A. DS 11.43 Pottenger 1942; B. 3% hydrochloric acid; C. 0.3% picric acid method: [heat-dried, fixed smear] — > A, 15 mins. 65°C. — > B, 30 sees. — > 70% ale, till no more color comes away -^ C, 10 sees. — > rinse — > dry 23.213 Randolf and Mikele 1944 665, 49:109 STOCK solutions: I. 1% magenta in propylene glycol. II. 5% phenol REAGENTS REQUIRED: A. stock I 20, stock II 80; B. 1% hydrochloric acid in 70% ale. C. 1% methylene blue method: [heat-fixed smears] -^ A, 4 mins. —> wash — > B, till decolorized — » C, 1 min. — ' wash -^ dry 23.213 Spehl 1918 see DS 23.213 (note) Spengler 1907 23.213 Schulte-Tigges 1920 7276,46:1225 REAGENTS REQUIRED: A. DS 11.43 Ziehl 1882; B. 10% sodium sulfite; C. sat. aq. sol. picric acid method: [heat-fixed smears]—* A, warmed to steaming, 1 min. — > wash -^ B, till de- colorized —f wash -^ C, few mins. — > wash -* dry 23.213 Spengler 1907 7276, 33 :337 REAGENTS REQUIRED: A. DS 11.43 Ziehl 1882; B. 95% ale. 50, sat. sol. {circ. 1.2%) picric acid 50; C. 15% nitric acid method: [heat-fixed smears] -^ A, warmed to steaming, 1 min. — > B, mixed with A on slide, few sees. -^ 70% ale, wash -* C, 30 sees. — > 70% thorough wash -^ B, 15 sees. -^95% ale, quick rinse — > dry note: Spehl 1918 (6630, 81:248) substitutes a mixture of 3 parts DS 11.43 Ziehl 1882 with 2 parts DS 11.44 NicoUe 1871 for A above. 23.213 Weiss 1942 665, 46:199 REAGENTS REQUIRED: A. ADS 12.1 Weiss 1942; B. 2.5% safranin; C. 15% acetic acid in acetone; D. DS 11.44 Loffler 1890 method: [flame-fixed smears]—* A, on slide, heated to steaming, 5 mins. -^ wash -^ B, on slide, heated to steaming, 5 mins. — * wash -^ C, till decolorized -^ wash -^ D, 1 min. —> wash — * dry 23.213 Ziehl 1882 see DS 23.213 Neelsen 1883 33£14 Methods for Spirochetes Spirochetes are resistant to dye-staining techniques, and are usually better demon- strated either unstained with dark ground illumination, or else with the aid of one of the metal-staining techniques given in Chapter 23. When they are to be stained with dyes, most of the early techniques involve the utilization of Giemsa's 1902 technique (Chapter 20 DS 13.13), usually with the addition of a strongly alkaline differentiating solution, with or without prior mordanting or subsequent differentiation in a solution of tannic acid. More recently, certain techniques have appeared such as that of Noguchi 1921, in which prior mordanting is conducted in a solution buffered to pH of 7.6, with subsequent staining in any stain desirable to the technician. A very unusual and ex- cellent stain, which should possibly have been placed in the section on metal staining, is that of Ono 1938. He utilized the heavy deposition of oxides of manganese from a solution of potassium permanganate, after mordanting with formaldeh_yde. This method tends to cause darkening of the background, because the oxides of manganese will be de- posited on an)^ organic detritus present, but if the culture is relatively free of organisms, or materials other than the spirochetes, the method can be confidently recommended. 23.214 Becker 1920 test. 1928 Schmorl Schmorl 1928, 400 REAGENTS REQUIRED: A. F 0000.1010 Ruge (1942); B. water 100, tannin 10, phenol 1; C. DS 11.43 Ziehl 1890 method: [smears] — > 1 min. — » rinse -^ B, 30 sees. 40°-50°C. — > rinse -* C, warmed to steaming, 30-45 sees. — > wash — > dry —> balsam DS 23.214 DYE STAINS OF SPECIAL APPLICATION 479 23.214 Doutrelepont see DS 23.214 Giacomi (1896) note 23.214 Du 1936 test. 1937 Kennedy 20540b, 12 :37 REAGENTS REQUIRED: A. Water 96, abs. ale. 4, aniline 8, crystal violet 8; B. either 1% sodium hydroxide or 5% potassium carbonate or 1% ammonia method: [air-dried smear] — > 8 drops A, on slide — » 8 drops 5% ale. added — » 8 drops B, added — + leave 2 mins. — > wash —>■ dry Ri-;('OMMENM)i;i) for: Trcpoiicina pallida. 23.214 Duperie 1909 11307, 20:1 formula: water 100, DS 13.13 Giemsa 1902 5, 0.1% sodium carbonate 3 method: [smears from formaldehyde-fixed organs] — + dry — > abs. ale, 5 mins. — > dry -+ stain, 5-30 mins. — > wash -^ dry 23.214 Gelarie 1936 11284,21:1065 reagents required: A. water 100, sodium chloride 10, zirconium oxychloride 2.5; B. 10% citric acid; C. water 100, sodium lactate 1.1, crystal violet 0.25; D. water 100. potassium iodide 0.5, mercuric iodide 0.5; E. water 100, phenol 0.2, methylene blue 0.2 method: [air-dried smears] -^ A, 5 sees. ^ wash —^ B, 10 sees. — > wash -^ C, 30 .sees. — > wash -^ D, 5 sees. — » wash —* E, 5 sees. -^ wash -^ blot — * dry 23.214 Giacomi lest. 1896 Kahlden and Laurent Kahlden and Laurent 1896, 111 reagents required: A. DS 11.43 Ziehl 1882; B. 0.001% ferric chloride; C. 10% ferric chloride method: [smears] -^ A, several mins. 50°C. -^ B, wash — > C, till differentiated — > abs ale. — > balsam, via xylene note: Kahlden and Laurent {loc. cit) state that Doutrelepont substitutes a sat. soL methyl violet for A above. 23.214 Giemsa 1905 7276, 31:1026 REAGENTS REQUIRED: A. Water 100, DS 13.13 Giemsa 1902 1, 0.1% potassium carbon- ate 1 method: [heat-dried smear] — + A, 10-30 mins. — » water, wash — > dry 23.214 Giemsa 1909 7276,35:1751 reagents required: .4. water 100, DS 13.13 Giemsa 1902 1 method: [heat-dried smear] -^ A, on slide, warmed to steaming, 15 sees. — > drain -^ re- peat 4 or 5 times -^ water, wash — * dry 23.214 Goldsworthy and Ward 1942 11431, 54:382 reagents required: A. water 47.5, methanol 50, acetic acid 2.5, Victoria blue 4R 0.2; B. 10% copper sulfate method: [air-dried smears]-^ A, on slide, heated till ale. evaporated —> B, to wash off stain -^ blot -+ dry 23.214 Hoffman 1921 7177,33:1 reagents required: A. water 100, potassium carbonate 0.01, DS 13.13 Giemsa 7.5; B. 25% tannic acid method: [osmic vapor fixed smears]—* water, wash — > A, 1-2 days-* water, wash—* B, 1 min. — > water, wash — > dry 23.214 Harris 1930 19938, 72 :275 reagents required: A. 1% potassium permanganate; B. 2% methyl violet method: [heat-fixed smear] — > A, 8-10 mins. -^ wash —* B, 8-10 mins. —> wash — ♦ dry 23.214 Keil 1929 7176,89:1398 REAGENTS reqttired: A. water 100, glycerol 10, 95% ale. 10, Victoria blue 3, pyronin 0.9, methyl green 0.1 method: [heat-fixed smears] — > A, 3-4 mins. -^ water, wash -^ dry 23.214 Krauss test. 1942 Langeron Langeron 1942, 827 REAGENTS REQUIRED: A. Water 100, potassium carbonate 0.005, DS 13.13 Giemsa 5 method: [heat-fixed smear] —+ A, on slide, heated to steaming, 4 or 5 times—* water, rinse —> B, I min. -^ water, wash — > dry 480 METHODS AND FORMULAS DS 23.214 23.214 Lipp 1940a 7676, 87 :888 REAGENTS REQUIRED: A. 1% potassium h^'droxide; B. 0.5% magenta. method: [methanol-fixed smears] -^ ^, on slide — > B, added to A on slide, 3 mins. — > wash —* dry 23.214 Lipp 1940b 7676,87:888 reagents required: A. 5% potassium permanganate; B. DS 11.43 Ziehl 1882 10, water 90 method: [air-dried smear] — * ^, 3 mins. — > wash —* B, 2 mins. — > wash -^ dry 23.214 Lipp 1940c 7676, 87 :888 formula: water 81, glycerol 10, abs. ale. 9, Victoria blue 4R 3, pyronin 0.9, methyl green 0.1 method : [air-dried smear] — > stain, 3 mins. — > wash -^ dry 23.214 Muhlpfordt 1924 7176, 79:921 reagents required: A. 3% Victoria blue method: [heat-fixed smears] -^ A, 2-3 mins. -^ wash -^ dry note: The method of Keil 1929 is very commonly referred to Muhlpfordt. 23.214 Noguchi 1921 11006, 78:191 reagents required: A. phosphate buffer pH 7.6 90, 40% formaldehyde 10; B. sat. ale. sol. {circ. 8%) magenta method: [smear from equal parts specimen and A, left 5 mins. before smearing] -^ dry —^ B, 1-5 mins. — > water, wash — * dry 23.214 One 1938 test. 1942 Langeron Langeron 1942, 830 reagents required: A. 4% formaldehyde; B. 1% potassium permanganate method: [smears] -^ A, 15 mins. — ^ water, quick wash -^ B, 24 hrs. 60°C. wash -^ dry note: One 1933 (abstract 1935; 20540b, 10 : 112) recommended this process as a mordant before crystal violet. 23.214 Perrin 1943 Tech. Bull., 4:28 reagents required: A. water 60, DS 11.43 Ziehl 1882 25, acetic acid 6, 40% formalde- hyde 6 method: [heat-fixed smears] — > A, 6 mins. — ^ wash -^ dry 23.214 Renaux 1923 6630, 89 :420 reagents required: A. sat. sol. picric acid; B. DS 12.15 crystal violet method: [smears fixed 5 mins. in F 0000.0101 Ruge] — » 95% ale, rinse — » A, 10 mins. -^ water, wash — > B, 10 mins. — > water, wash -^ dry 23.214 Renaux and Wilmaers 1917 6630, 80 :55 reagents required: A. 5% tannin; B. DS 11.43 Ziehl 1882 method: [alcohol-fixed smears]—^ A, warmed to steaming, 30 sees. -^ water, rinse —> B, warmed to steaming, 30 sees. — * water, wash -^ dry 23.214 Sabrazes 1926 6628, 187 :875 reagents required: A. DS 11.43 Ziehl 1882 method: [heat-fixed smears] -^ A, on slide, heated to steaming, 2 or 3 times —> water, wash — > dry 23.214 Sabrazes 1928 6630, 98:239 reagents required: A. water 100, tannin 5, phenol 1; B. DS 11.43 Ziehl 1882 method: [smears fixed 2 mins. in F 0000.1010 Ruge] — » 95% ale, rinse — > A, warmed to steaming, 30 sees. — > water, wash — > B, warmed to steaming on slide 3 or 4 times — * water, wash — > dry 23.214 Sabouraud test. 1904 Besson Besson 1904, 638 reagents required: A. DS 11.43 Ziehl 1890; B. 1.5% potassium permanganate; C. 2% sulfurous acid; D. 1% methylene blue method: [heat-fixed smear] -^ A, 1-2 hrs. — > B, to wash off yl — > C, till decolorized — > wash — ♦ D, 1-3 mins. -^ wash — » dry DS 23.214-DS 23.215 DYE STAINS OF SPECIAL APPLICATION 481 23.214 Tunnicliff 1921 11006, 78:191 REAGENTS REQUIRED: A. DS 12.15 Crystal violet; B. ADS 12.2 Lugol 1905; C 1% safranin O method: [heat-fixed smear] -^ A, 30 sees. -^ water, wash — > B, 30|sccs. — > water, wash -> C, 30 sees. — » water, wash — > dry 23.214 Weiss 1929 11284, 14:1191 REAGENTS REQUIRED: A. acetic acid ; B. ADS 12.2 Weiss 1929; C. sat. aq. sol. crystal violet or safranin or magenta or brilliant green; D. 10% acid green or acid violet or acid fuchsin in 70% ale. method: [drop material and drop A incubated as hanging drop, 37.5°, 15 mins.] -+ air- dried smear — » B, on slide, heated to steaming, 2-5 mins. -+ wash — > C, 2-5 mins. -^ wash — » D, 8-10 mins. -^ wash — > dry note: The selected dyes in C and D above should be of contrasting colors. 23.214 Woolman 1939 test. 1939 Findlay 11360, 59:184 REAGENTS required: A. watcr 97, 40% formaldehyde 2, acetic acid 1; B. water 100 hydrochloric acid 0.7, crystal violet 0.15, copper sulfate 10 method: [air-dried smears]-^ A, 3-5 mins. -+ wash — > abs. ale, 1-2 mins. — > blot—* B, 10-15 mins. — > drain — * dry 23.215 Flagella Stains The term flagella stains is somewhat misleading because there is no known technique which could be employed differentially to stain a flagella protruding from a bacteria without staining every other minute particle of the protein material present. The pur- pose of these stains, however, is to provide so dense a deposition of dye as to assure that these minute particles will become so covered as to be visible in an optical micro- scope. For this reason every possible type of mordant, usually if not always combining tannin with iron, is used before a solution either of one of the thiazins or of magenta. The real difficulty in the staining of flagella is not to deposit the stain, but to differ- entiate the subsequently stained flagella from the background. For this reason the very utmost care must be taken in the cleanliness of the slides employed for the preparation of a smear, and all those precautions proper to metal stains (which are given in Chap- ter 24) can with justice be used in the present instance. If, however, the flagellated organisms are taken from a culture filled with small particles of organic detritus, it is a waste of time to endeavor to stain the flagella on them. A recent method of Fisher and Conn 1942 gives the most minute directions for the selection for the culture as well as for staining the organisms. 23.215 Bailey 1929 16913, 27:111 reagents required: A. ADS 12.2 Bailey 1929; B. ADS 12.2 Bailey 1929 70, DS 11.43 Ziehl 1882 10, hydrochloric acid 10, 40% formaldehyde 10; C. DS 11.43 Ziehl 1882 method: [dry smear] -^ A, 2 mins. — > 5, 7 mins. — > wash — > C, warmed to steaming, 30 sees. — * wash -^ dry 23.215 Bowhill 1898 23684, 23 :667 preparation of stock soiitrriONs: I. water 40, 95% ale. 50, orcein 1; II. water 100, tannin 20 reagents required: A. stock I 50, stock II 50; B. DS 23.211 Ehrlich 1882 method: [heat-fixed smear] — > A, 10 mins. at 50°C. — * wash -^ dry -* B, heat to steam- ing, 30 sees. — * wash -^ dry 23.215 Bunge 1894 8645, 12 :24 formula: water 100, tannic acid 100, ferric chloride 1.4, magenta 0.25, hydrogen per- oxide q.s. preparation: Dissolve the tannic acid and ferric chloride in 90 water. Add the dye dis- solved in 10 water. Add enough hydrogen peroxide just to turn solution brown. method: As DS 23.215 Loffler 1890 482 METHODS AND FORMULAS DS 23.215 23.215 Casares-Gil test. 1946 Conn and Darrow Conn and Darrow, 1946, 111A2-14 REAGENTS REQUIRED: A. water 50, ADS 12.2 Casares-Gil (1946) 50; B. DS 11.43 Ziehl 1890 method: [heat-fixed smears] — > A, on slide, 1 min. -^ water, wash -^ B, on slide, 5 mins. — » wash -^ dry 23.215 Cerrito test. 1904 Besson Besson 1904, 171 REAGENTS REQUIRED: A. 25% tannin 60, 5% ferric alum 30, magenta 0.3; B. water 100, 95% ale. 10, phenol 5, magenta 0.25 PREPARATION OF a: Heat ingredients to 100°C. for 6 hours. Cool. Filter. method: [heat-fixed smear] -^ A, 10 mins. — ^ wash -^ dry — > B, on slide, warmed to steaming, several sees. — > wash -^ dry 23.215 van Ermengen 1894 see MS 31.1 van Ermengen 1894 23.215 Fisher and Conn 1942 20540b, 17:117 STOCK solutions: I. water 100, tannic acid 7.2, ferric chloride 1.5; II 95% ale. 100, magenta 0.5 REAGENTS REQUIRED: A. stock I; B. stock I 54, stock II 8, hydrochloric acid 8, 40% formaldehyde 30; C. DS 11.43 Ziehl 1890 method: [thick smears of washings, incubated 10 mins., from active cultures] —* A, 3}^^ mins. -^ drain -^ B, 7 mins. — » water, wash — > C, warmed to steaming, 1 min. — > water, wash -^ dry 23.215 Gemelli test. 1904 Besson Besson 1904, 172 REAGENTS REQUIRED: A. 0.25% potassiuui permanganate; B. water 100, calcium chloride 0.75, neutral red 0.05 method: [heat-fixed smear] — > A, 10-20 mins. — > wash -^ B, 15-30 mins. -^ wash — » dry 23.215 Gray 1926 11056, 12:273 reagents required: A. ADS 12.2 Gray 1926; B. DS 11.43 Ziehl 1890 method: [heat-fixed smears] —> A, on slide, 10 mins. — > water, wash —* B, 5 mins. -^ water, wash -^ dry 23.215 Inouye 1924 test. 1942 Langeron Langeron 1942, 1219 REAGENTS REQUIRED: A. DS 23.215 Loffler 1890 (sol. ^4); B. water 75, potassium alum 3, sat. ale. sol. (circ. 10%) crj'stal violet 15 method: [heat- fixed smears] — » A, on slide, warmed to steaming, 1 min. — > water, wash -^ B, on slide, warmed to steaming, 1 min. -^ wash -^ dry 23.215 Kendall 1902 see DS 23.215 Pittfield (1910) 23.215 Kulp 1926 20540b, 1 :60 REAGENTS required: A. Water 75, 95% ale. 5 tannic acid 10, ferrous sulfate 4, magenta 0.5; B. 0.6% magenta in sat. sol. aniline preparation of a: Dissolve the tannic acid in 50 water. Add the ferrous sulfate dis- solved in 25 and the dye dissolved in the ale. Leave 18-24 hours. Filter or centrifuge. method: [air-dried smears] — > A, 15 mins. — » thorough wash —> B, 15 mins. — > wash — + dry 23.215 Liefson 1930 11056. 20:203 formula: water 80, 95% ale. 36, potassium alum 2, tannic acid 4, magenta 0.6 preparation: To the alum dissolved in 40 water add the tannic acid dissolved in 20. Add 20 water and 30 ale. To this add the dye dissolved in 6 ale. method: [air-dried smear] -^ stain 10 mins. -^ wash —> [DS 11.44 Loffler 1890, if counterstain desired] -^ dry 23.215 Loffler 1890a 23684, 7:625 reagents required: .4. water 100, tannic acid 12, ferrous sulfate 5, magenta .03; B. water 100, aniline and crystal violet, each to sat., sodium carbonate .001 preparation of a: To the tannic acid dissolved in 60 water, add the ferrous sulfate dis- solved in 30. To this add dye dissolved in 10 water. DS 23.215 DYE STAINS OF SPECIAL APPLICATION 483 method: [heat-fixed smears, still warm] —* A, flooded on slide and warmed to steaming, 1 min. — » 90% ale. till color clouds cease — > B, flooded on slide and warmed to steam- ing, 1 min. -+ water, rinse — > balsam, via usual reagents note: The original method recommended that either 1% sulfuric acid or 1% sodium carbonate be added to A in amounts determined empirically for each organism . Zikes 1930 (23684, 81:161) substitutes his ADS 12.2 for A above. 23.215 Loffler 1890b 23684, 7 :629 REAGENTS REQUIRED: A. Water 80, tannin 20, ferric alum 5, sat. aq. sol. indigocarmine 10, sat. ale. sol. methyl violet 10; B. sat. aq. sol. aniline 100, crystal violet to sat, 10% sodium carbonate 0.1 method: [heat-fixed smears] — > A, flooded on slide and warmed to steaming, 1 min. — ♦ 90% ale, till color clouds cease—* B, adjusted by trial and error to required pH, flooded on slide and warmed to steaming, 1 min. — > wash -^ balsam, via usual reagents note: The formula for A above will come as a shock to those who have not consulted the original reference. Most authors quote "ferrous sulphate" from the fact that the solution of "ferrum sulphitricum oxydulatum ammoniatum" given on p. 629 (loc. cit) is casually referred to as the f err osulph alios ung on p. 630. The formula for the B solution comes from Loffler 1889 (23684, 6:213) and was there recommended for staining after a hematoxylin mordant. 23.215 Maneval 1929 20540b, 4:21 REAGENTS REQUIRED: A. water 100, tannic acid 13, ferric chloride 1, DS 11.43 Ziehl 1882 6.5, hydrogen peroxide (3%) 12; B. water 60, 95% ale. 27.5, aniline 2.5, acetic acid 0.1, magenta 2 method: [smears of distilled water, suspensions of actively motile organisms] —* A, 2-4 mins. — ♦ wash — » B, 2-3 mins. —> wash -^ dry 23.215 Muir test. 1920 Stitt Stitt 1920, 56 REAGENTS REQUIRED: .4. DS 23.217 Muir (1920) sol. B. 80, DS 11.43 Ziehl 1890 20; B. sat. sol. (circ. 4%) potassium alum 100, sat. ale. sol. (circ. 15%) methyl violet 20 method: [heat-fixed smears] -^ A, steamed over bath, 1 min. -^ water, 2 mins. — + dry —* B, on slide, warmed to steaming, 1 min. -^ water, wash -^ dry note: Both A and B solutions are unstable. 23.215 Nicolle and Morax test. 1904 Besson Besson 1904, 169 REAGENTS REQUIRED: A. DS 23.215 Loffler 1890b, (sol. A); B. DS 11.43 Ziehl 1882 method: [heat-fixed smear] -^ A, on slide, heated to steaming, 3 or 4 changes —> B, on slide, heated to steaming, 3 or 4 sees. — ♦ wash -^ dry 23.215 Novel 1939 see MS 33.52 Novel 1939 23.215 Pittfield test. 1910 Heymann Ehrlich, Krause, et al. 1910, 2, 394 STOCK solutions: I. 10% aluminum acetate 10, sat. ale. sol. crystal violet 100, II. 10% tannic acid WORKING solution: stock I 50, stock II 50 method: [heat-fixed smears] — > stain, lightly warmed -^30% ale. till differentiated -* dry notes: Wright 1928 (20540b, 3 :17) cites (with reference to Kendall 1902: 11032, 5 :1936) a method of Pittfield 1902 in which the mordant contains "sat. sol. alum." 23.215 Remy and Sugg test. 1904 Besson Besson 1904, 169 REAGENTS REQUIRED: A. DS 23.215 Loflfler 1890 (sol. /I); B. ADS 12.2 Gram 1884; C. sat. aq. sol. aniline 80, sat. ale. sol. {circ. 10%) crystal violet 0.2, water 20 method: [heat-fixed smear] -^ A, 15-30 mins. -^ B, on slide, few moments -^ wash — > abs. ale, wash — > C, 30 mins. at 37°C. -^ wash — > balsam, via usual reagents 23.215 Rossi test. 1904 Besson Besson 1904, 170 RE.\GENTs required: .4. ADS 12.2 Rossi (1904); B. DS 11.43 Ziehl 1S82 method: [heat-fixed smear] -^^ ^4, 30 mins. — ♦ wash — » dry —* B, heated to steaming, 3-4 mins. — > wash — > dry 484 METHODS AND FORMULAS DS 23.215-DS 23.216 23.215 Ryo 1937 11796, 14:218 STOCK solutions: I. water 100, phenol 2.5, tannic acid 10, potassium alum 4, II. sat. ale. sole. {circ. 10%) crystal violet WORKING solution: stock I 100, stock II 10 method: [heat-fixed smear] -^ stain, 3-5 mins. — » tliorough wash -^ dry 23.215 Sclavo test. 1904 Besson Besson 1904, 172 REAGENTS required: A. 1% tannin in 50% ale; B. 5% phosphotungstic acid; C. DS 23.211 Ehrlich 1882 method: [heat-fixed smears] -^ A, 1 min. —* rinse — > 5, 1 min. — ♦ rinse — > C, heated to steaming, 3-5 mins. 23.215 Shunk 1920 11056, 5:181 reagents required: A. ADS 12.2 Shunk 1920; B. 95% ale. 80, aniline 20; C. DS 11.44 Lofiier 1890 90, 95%, ale. 8, aniline 2 method: [heat-fixed smear] — > A, on slide —> B, added to A on slide, about 10% of quan- tity of A used, 15 sees. — > drain — » C, 15 sees. — > water, wash — > dry 23.215 Trenkmann 1890 23684, 8:385 REAGENTS REQUIRED: A. ADS 12.2 Trenkmann (1904); B. sat. aq. sol. iodine; C. DS 23.211 Ehrlich 1882 method: [heat-fixed smear] — > water — > A, 6-8 hrs. -^ wash —* B, 1 hr. -^ water -+ C, 30 mins. -^ wash — > balsam, via usual reagents 23.215 Tribondeau, Fichet, and Dubreuil 1916 6630, 79:710 STOCK solutions: I. water 100, potassium alum 3.5, tannic acid 3; II. 1% crystal violet PREPARATION OF STOCK I: Mix ingredients. Autoclave, 20 lbs. for 30 minutes. Cool. Filter. WORKING solution: stock I 100, stock II 10 method: [heat-fixed smear] -^ stain, heated to boiling, 30 sees. — > wash —* dry note: If background too granular, decrease proportion of stock II. If flagella not stained, increase proportion of stock II. A detailed description of the use of this tech- nique is given under 23.20 above. 23.215 Wright 1928 see DS 23.215 Pittfield 1910 23.215 Yokata 1924 6630, 90:1303 REAGENTS REQUIRED: A. ADS 12.2 Yokata 1924; B. sat. sol. (circ. 3.5) aniline 100, magenta 0.03 method: [heat-dried smear] -^ A, raised to boiling, 30 sees. — > water, wash — > B, on slide, raised to boiling 2 or 3 times -^ water, wash -^ dry 23.215 Zettnow 1891 see MS 33.1 Zettnow 1891 23.215 Zikes 1930 see DS 23.215 Lofiier 1890 (note) £3 £16 Spore Stains The reagents here given are intended not so much to stain bacterial spores in their free condition as to differentiate a spore within a bacterium, in order that its spore- forming nature may be determined for diagnostic purposes. Tlie majority of these methods depend on differentiating a first stain with an acid solution, and then ap])ly- ing a counterstain to bring into contrast the main body of the bacteria. Other methods depend upon the selective affinity of malachite green or light green for the spore itself, and thus permit the direct coloration of the spore without subsequent differentiation. Probably the surest and simplest method is that of Muzzarelli 1931, though the selec- tion from the methods given is largely a matter of opinion. 23.216 Abbott test. 1920 Stitt Stitt 1920, 55 REAGENTS REQUIRED: A. DS 11.44 Lofflcr 1890; B. 2% nitric acid; C. 1% eosin method: [lieat-fixed smears] — » /I, on slide, raised to boihng 3 or 4 times, 1 min. -^ B, till colorless -^ water, wash — » C, 15 sees. — > water, wash — > dry result: spores blue on yellow. DS 23.216 DYE STAINS OF SPECIAL APPLICATION 485 23.216 Aladar-Anjeszky test. 1904 Besson Besson 1904, 165 REAGENTS REQUiREu: A. 0.5% hydrochloric acid; B. DS 11.43 Ziehl 1890; C. 4% sulfuric acid; D. \% methylene blue method: [air-dried smear] -^ A,Z mins. in beaker at 80°C. — » wash -^ dry -> flame — » B, on slide, heated to steaming, 2 or 3 changes -^ C, till decolorized — > wash — > D, 1 min. — > wash — > dry 23.216 Anjeszky 1909 23G84, 23 REAGENTS REQUIRED: A. 0.5% hydrochloric acid; B. DS 11.43 Ziehl 1890; C. 3%, sulfuric acid; D. 1% malachite green method: [dried, but not flamed, smear] -^ A, heated to steaming, 3-4 mins. -* wash —y dry, -* flame — > B, warmed to steaming, 1-2 mins. -♦ wash -> C, till decolorized -+ thorough wash — > D, 1-2 mins. — * wash — » dry 23.216 Ashby 1938 19938,87:443 REAGENTS REQUIRED: A. 5% malachite green; B. 0.5%, safranin method: [heat-fixed smear] —> A, on slide, heated over steam bath, 1 min. ^ water, wash — > dry 23.216 Besson 1904a Besson 1904, 164 reagents required: A. 5% chromic acid; B. DS 12.15 gentian violet method: [heat-fixed smears] —> A, on slide, 4-5 mins. -^ wash — > B, on slide, 15-20 mins. — > wash — ♦ dry 23.216 Besson 1940b ' Besson 1904, 165 REAGENTS REQUIRED: A. DS 11.43 Ziehl 1882; 25% nitric acid; C. 1% methylene blue method: [heat-fixed smear] —> A, on slide, heated to steaming, 3-4 mins. — > wash —* B, few sees. — >• wash — > C, on slide, 30 sees. — » wash -^ dry result: spores, red; bacteria, blue. 23.216 Bitter 1913 23684, 68:227 REAGENTS REQUIRED: A. 1% formaldehyde; B. DS 11.44 Loffler 1890; C. water 50, sat. ale. sol. {circ. 3%) safranin 50 method: [heat-fixed smears] -^ A, 10-20 mins. — * water, wash — > B, on slide, heated to boiling, 2-3 times, — > water, thorough wash -* C, 30 sees. -^ water, wash -^ dry 23.216 Botelho 1918 6630,81:183 formula: water 50, acetic acid 50, light green 4, acid fuchsin 2 method: [heat-fixed smears]-^ stain, heated to steaming, 3 to 4 times—* water, wash till greenish -^ repeat staining cycle till color sufficiently deep -^ wash — > dry result: spores red on green. 23.216 Bruner and Edwards 1939 11284, 25:543 REAGENTS REQUIRED: A. 5% malachite green; B. 0.5% safranin method: [heat-fixed smears] — > A, 5 mins. -^ wash — >• B, 10 sees. — > wash -^ dry 23.216 Dorner 1926 11988, 6:8 REAGENTS REQUIRED: A. DS 11.43 Ziehl 1890; B. 10% nigrosin method: [suspension of organism in test tube] — » add equal volume A, heat to 100°C., 10 mins. -^ place loopful of mixture on slide, add loopful of B, smear -^ dry note: Snyder 1934 (20540b, 9:71) applies A to smear under saturated blotting-paper cover and counterstains with B. 23.216 Dutton 1928 20540b, 3:140 REAGENTS REQUIRED: A. phosphatc buffer pH 7.6; B. DS 13.12 Wright 1910 (0.15% in methanol) method: Prepare thick suspension of bacteria in A. Add 0.1 B. Seal tube and heat to 100°C. for 10 mins. Cool, unseal tube, and make smear. 23.216 Fraenkell922 23684,89:106 REAGENTS REQUIRED: A. 20% tannin; B. DS 11.43 Ziehl 1890; C. 5% sulfuric acid; D. 0.1% methylene blue. 486 METHODS AND FORMULAS DS 23.216 method: [heat-fixed smears] —>■ A, heated to bubbling -» cool -^ [repeat twice] -* wash -^ blot -* B, warmed to steaming -^ cool -^ repeat twice -* C, till decolorized -* wash -> D, 30 sees. —>■ wash -^ dry 23.216 Gray 1941 14900, 147:329 REAGENTS REQUIRED: A. Water 100 malachite green 0.5, magenta 0.05 method: [heat-fixed smears] -^ A, heated to steaming, 1 min. — * wash —>■ dry 23.216 Kahlden and Laurent 1896 Kahlden and Laurent 1896, 101 REAGENTS REQUIRED: A. DS 11.43 Ziehl 1882; B. 5% nitric acid; C. DS 11.44 Loffler 1890 method: [heat-fixed smear] -^ A, 20-40 mins. -^ B, 1 min. -> wash -^ C, 2 mins. -> wash —y blot — » dry 23.216 Lagerberg 1917 23684, 79:191 REAGENTS REQUIRED: A. sat. sol. {circ. 25%) copper sulfate; B. 20% ammonium hy- droxide; C. DS 11.43 Ziehl 1882; D. 4% sulfuric acid method: [heat-fixed smears]-^ A, flooded on slide -^ B, added drop by drop till ppt. just redissolved -^ warm to steaming — » add several drops A-* B, rinse -^ water, wash -> C, on slide, heated to steaming, 1 min. -^ D, till no more color comes away — > water, wash —* dry 23.216 May 1926 20540b, 1:105 REAGENTS REQUIRED: A. 5% chromic acid; B. ammonia; C. DS 11.43 Ziehl 1882; D. 1% sulfuric acid; E. DS 11.44 Loffler 1890 method: [heat-fixed smear] -> A, on slide, 30 sees. -^ B, added (twice as much) to A on slide; 2 min. -^ rinse -* C, on slide, heated to steaming, 2-3 mins. -^ rinse -^ D, 15-30 sees. -> wash — > E, few drops added to water on slide, 10-30 sees. — » rinse -»■ blot — > dry 23.216 MoUer 1891 23684, 10:9 REAGENTS REQUIRED: A. chloroform ; B. 5% chromic acid; C. DS 11.43 Ziehl 1882; D.1% sulfuric acid; E. 1% methylene blue method: [heat-fixed smears] -^ A, 1-2 mins. -^ abs. ale, rinse -^ water, wash ^- B, 1 min. -^ water, wash -^ B, 1 min. -^ water, wash — > C, on slide, heated to steaming, 5 mins. -♦ water, wash -^ D, till faint pink -^ water, wash —> E, 10 sees. -^ water, wash — » dry 23.216 Muzzarelli 1931 test. 1942 Langeron Langeron 1942, 1216 reagents required: A. water 75, DS 11.44 Manson 25; B. 10% nitric acid; C. 1% eosin method: [heat-fixed smears]—* A, warmed to steaming, 30 sees. — » water, wash -^ B, till no more color comes away — > water, wash — > C, 15 sees. — > water, wash — > dry 23.216 Neisser and Hueppe test. 1928 Schmorl Schmorl 1928, 355 reagents required: A. sat. sol. magenta in sat. sol. aniline; B. 25% sulfuric acid; C. 1 % methylene blue method: [heat-fixed smear] -^ A, 100°C., 1-5 hrs. — > B, 5 sees. -^ abs. ale, till no more color comes away — > water — + C, 3-5 mins. — > wash -^ dry note: Schmorl {loc. cit.) recommends a steam chest for step A. 23.216 Proca 1909 6630,68:307 formula: water 50, DS 11.44 Loffler 1890 50, DS 11.43 Ziehl 1882 4 method: [heat-fixed smears] — > stain, 1 min. -^ water, wash -^ dry result: live bacteria, colorless spores on blue; dead bacteria, blue spores on red. note: See also DS 23.21 Gay and Clark 1924, who have adapted this technique to general use. 23.216 Ruiz 1946 test. 1947 Pemander 20540b, 22:164 reagents required: A.1% methyl violet; B.0.1% eosin Y 100, normal horse serum 30 method: Mix 1 drop A with bacterial suspension, leave 5 mins. Add 1 drop B, leave 3 mins. Make smear and dry. DS 23.216-DS 23.217 DYE STAINS OF SPECIAL APPLICATION 487 23.216 Schaeffer and Fulton 1933 19938, 77:194 REAGENTS REQUIRED: A. 5% malachite green; B. 0.5% safranin O method: [heat-fixed smears] — > A, poured on slide, heated and reheated to steaming, 3-4 times — > water, wash — > B, 30 sees. — » water, wash -^ dry result: spores green on red. 23.216 Shapiro 1944 20540b, 19:65 REAGENTS REQUIRED: A. 5% malachite green; B. 0.5% safranin method: [mix 2-3 drops bacterial suspension with equal amount A. Heat in boiling water bath 15-20 mins. Make smear and heat fix.] -^ wash, 10 sees. -^ B, 1 min. —> wash — » blot — > dry 23.216 Snyder 1934 see DS 23.21G Dorner 1926 (note) 23.216 Tribondeau 1917 6630, 80:880 REAGENTS REQUIRED: A. ADS 12.2 Lugol (1905); B. DS 12.15 crystal violet; C. 0.2% Bismarck brown method: [heat-fixed smears] —> A, heated to steaming, 2-3 times —»• blot — > B, heated to steaming, 2-3 times -^ water, wash result: spores violet on brown. 23.216 Zeeti 1935 20540b (abs. 1930) 11 :87 reagents required: A. water 100, eosin Y 5, phenol 5, iodine 2, potassium iodide 1; B. 0.075% methylene blue in 5%, ale. method: [heat-fixed smears] — > A, heated to boiling, 5 mins. — > wash — > B, 1-2 mins. -* wash — > dry 23.317 Capsule Stains The standard method for staining the capsule of capsulated bacteria is still that of Hiss 1905, though many other methods have been developed. One of the most inter- esting of these is that of Huntoon 1917, in which the capsule is first caused to absorb a solution of nutrose, w^hich is itself subsequent]}' brought into prominence by a chemical reaction specific to it. The other methods largel}^ rely on mordanting with formaldehj'de and subsequently carrying out a standard Gram procedure with counterstaining for the mordanted capsule. 23.217 Anthony 1931 see DS 23.207 Hiss 1905 (note) 23.217 Besson 1904 Besson 1904, 166 reagents required: A. water 100, crystal violet 0.5, acetic acid 1 method: [heat-fixed smear] -^ A, I min. — > rinse — > dry 23.217 Boni test. 1928 Schmorl Schmorl 1928, 359 reagents required: A. V 21.1 Mayer 1884; B. DS 11.43 Ziehl 1890 method: [place a drop of A on slide. Mix in a trace of bacterial culture. Make smear. Dry. Flame] — > B, warmed to steaming, 1 min. — > wash — > dry 23.217 Buerger 1904 test. 1928 Zinnser and Bayne-Jones Zinnser and Bayne-Jones, 1928, 847 reagents required: A. ADS 12.2 Gram 1882; B. DS 23.211 Ehrlich 1882; C. 2% so- dium chloride method: [F 3700.0000 fixed smear] ^ wash ^ 95 % ale. — > wash — > A, 1-3 mins. ^ 95% ale, wash -^ dry — > B, few sees. — > C, wash —* examine in C 23.217 Churchman and Emelianoff 1932 16913, 29:514 reagents required: .4. DS 13.12 Wright 1910; B. phosphate buffer pll 6.4 method: [air-dried smear] —* A, on slide, till evaporation changes color from blue to pink —>■ B, wash —* dry 488 METHODS AND FORMULAS DS 23.217 23.217 Gutstein 1926 23684, 93 :393 REAGENTS REQUIRED; A. sat. sol. ammonium sulfate; B. 5% tannin; C. 1% malachite green method: [air-dried smear] -^ A, 5 mins. —> rinse —* B, 2 mins. — » thorough wash -^ C, 5-60 sees. — » wash -^ dry -^ balsam 23.217 Hiss 1905 11189, 6:317 REAGENTS REQUIRED: A. 0.1% Crystal violet; B. 20% copper sulfate method: [heat-fixed smears] — > A, on slide, warmed to steaming, 3^^ to 1 min. — » B, till no more color comes away — * blot — » dry result: capsule blue. note: Anthony 1931 (19938, 73:319) substitutes 1% crystal violet for A above. Tyler test. 1946 Conn and Darrow cit. Park and Williams 1933 (Conn and Darrow 1946, 111A2-18) adds 0.25% acetic acid to A above. 23.217 Huntoon 1917 11056,2:241 reagents required: A. 3% nutrose; B. water 100, phenol 2, lactic acid 0.5, acetic acid 0.01, magenta 0.1, DS 11.43 Ziehl 1882 1 method: [smear, of culture diluted with A, air-dried]^ B, 30 sees.—* water, wash—* dry note: Solution A must be steam-stertlized (not autoclaved) and preservative added if required to keep. 23.217 Johne test. Kahlden and Laurent 1896 Kahlden and Laurent 1896, 100 reagents required: A. sat. aq. sol. crystal violet; B. 1% acetic acid method: [smears] —> A, 25 to 30 sees. -^ B, 5-10 sees. — » dry 23.217 Kahlden and Laurent 1896 Kalilden and Laurent 1896, 104 reagents required: A. water 60, crystal violet 0.5, acetic acid SO; B. 1% acetic acid method: [smears] —* A, 2i hrs. 37°C. -^ B, wash -^ balsam, via usual reagents 23.217 Klett test. 1928 Schmorl Schmorl 1928, 359 reagents required: A. 1% methylene blue in 10% ale; B. 1% magenta in 10% ale. 7 method: [heat-fixed smears] — » A, warmed to steaming, 3-5 mins. -^ wash -^ B, 5 sees. — » wash and dry 23.217 La wson 1940 11284,25:435 reagents required: A. 5% phosphomolybdic acid; B. DS 13.12 Wright 1910 (working solution) 60, glycerol 30 method: [air-dried smears] — > A, on slide, 30 sees. —* wash —>■ wash, methanol — > 10-20 drops water, 10-20 mins. — > rinse — » dry 23.217 Muir 1916 11431, 20:257 reagents required: A. DS 12.15 gentian violet; B. ADS 12.2 Gram 1884; C. water 100, mercuric chloride 2, potassium alum 1.3, tannic acid 7; D. sat. sol. (circ. 35%) eosin; sat. sol. {circ. 3.5%) potassium alum" method: [mercuric-chloride-fixed, and acetone-washed, smears]^ A, on slide, 2 mins. — > water, rinse -^ B, on slide, 1 min. -^ water, wash -^ methanol 1 min. — ♦ clove oil, heated to fuming, 1 min. -^ methanol, wash —^ water, wash — * C, 5 mins. — > water, rinse -^ D, 30 sees. — * water, wash —* E, 1 min. — > water, rinse —y dry 23.217 Muir test. 1920 Stitt Stitt 1920, 55 reagents required: A. DS 11.43 Ziehl 1890; B. water 100, mercuric chloride 1.5, tan- nic acid 4, potassium alum 3; C. 1% methylene blue method: [heat-fixed smears]-^ A, heated to steaming, 30 sees. -^• 95% ale, rinse—* water, wash — * B, 5-10 sees. —* water, wash — » 95 % ale, 1 min. -^ water, wash — » C, 1 min. —>■ dry 23.217 Raebiger test. 1904 Besson Besson 1904, 166 reagents required: 40% formaldehyde 100, crystal violet 15 preparation: Heat to boiling. Cool. Filter. method: [air-dried smears] — » A, 1 min. —* wash — > dry DS 23.217-DS 23.218 DYE STAINS OF SPECIAL APPLICATION 489 23.217 Schmorl 1928 Schmorl 1928, 359 REAGENTS REQUIRED: A. 1% potassium hydroxide; B. 2% crystal violet method: [heat-fixed smears] -^ A, 3-5 mins. -^ quick rinse —>■ B, 3-4 mins. -^ wash — > dry 23.217 Tyler 1946 see DS 23.217 Hiss 1905 (note) 23.217 Wadsworth 1906 11250,3:610 REAGENTS REQUIRED: A. 40% formaldehyde; B. DS 23.211 Ehrlich 1882; C. ADS 12.2 Gram 1882; D. 0.1% magenta method: [heat-fixed smears] -* A, 2-5 mins. -^ water, rinse -^ B,2 mins. -♦ C, 2 mins. -^95% ale, till no more color comes away -^ D, 30 sees, dry 23.217 Welch 1892 10919,3:81 REAGENTS REQUIRED: A. acetic acid ; B. DS 23.211 Ehrlich 1882 (sol. A); C. 2% sodium chloride method: [dried, not heated, smear] -^ A, few sees. -^ B, drained and renewed on slide till A removed -^ B, fresh solution, 2 mins. -^ C, wash — > examine SS.218 Diphtheria Bacilli Diphtheria bacilli themselves are not susceptible to differential staining. Diagnosis by microscope is dependent upon bringing into prominence the granules w^hich they uniquely have within them. These granules have much the same reactions as any other minute body, hence the methods for their demonstration most strongly resemble those given either for the demonstration of Rickettsiae, or for mitochondria and other cell inclusions. The original method of Neisser 1897 is still the most widely employed and all these techniques are occasionally referred to as Neisser stains even though they may bear no relation at all to the original description. 23.218 Albert 1921 11006, 76:240 REAGENTS REQUIRED: A. Water 100, 95% ale. 2, acetic acid 1, methyl green 0.02, toluidine blue 0.15; B. ADS 12.2 Gram 1882 method: [heat-fixed smears] — > A, 5 mins. -^ blot -^ B, 1 min. -^ water, rinse -> blot -^ dry note: Laybourn 1924 (11006, 83:121) substitutes 0.2 malachite green for 0.02 methyl green in A above. 23.218 Ambrosioni 1940 988, 60 :228 REAGENTS REQUIRED: A. Water 95, acetic acid 5, crystal violet 0.33; B.2% chrysoidin; C. ADS 12.2 Lugol 99, lactic acid 1 method: [heat-fixed smear] -* A, 10-15 sees. -^ B, 20-30 sees. -^ wash — > C, 5 sees. -^ wash —* dry 23.218 Beauverie 1917 6630, 80 :609 REAGENTS REQUIRED: A. DS 11.44 Loffler 1890; B. ADS 12.2 Lugol method: [alcohol-fixed smears] — > A, 2-3 mins. -^ wash — > B, 2-3 mins. -^ wash — > dry note: Mallory 1922 (4349, 8:110) recommends counterstaining in 1% eosin after this technique. 23.218 Cowdry 1943 Cowdry 1943, 62 reagents required: A. DS 23.218 Neisser 1897 (sol. A) 60, 0.3% crystal violet 30; B. 0.3% chrysoidin method: [heat-fixed smear] -^ yl, on slide, H to 1 ^"^- -^ water, wash -> B, on slide, 30 sees. -^ water, wash -^ dry note: See also DS 23.218 Neisser 1897 (note). 23.218 Kinyoun 1915 617, 5:246 formula: water 120, acetic acid 1, 95% ale. 5, toluidine blue 0.01, azur A 0.01, meth- ylene blue 0.01 preparation: Dissolve dyes in ale. Add acidified water. method: [heat-fixed smears] -^ stain, 5 mins. — » water, wash -^ dry result: granules red on blue. 490 METHODS AND FORMULAS DS 23.218-DS 23.219 23.218 Kemp 1931 11284, 16:593 REAGENTS REQUIRED: A. ADS 12.2 Graiu 1884; B. DS 11.44 Loffler 1890; C. 1% safranin method: [heat-fixed smears] ~^ A, 1 min. -> wash -^ B, 20-30 sees. —> wash -^ C, 10-15 sees. -^ wash — * dry 23.218 Laybourn 1924 see DS 23.218 Albert 1921 (note) 23.218 Ljubinsky test. 1946 Conn and Darrow cit. Blumenthal and Lipskerow Conn and Darrow 194G, 111A2-12 REAGENTS REQUIRED: A. Water 95, acetic acid 5, crystal violet 0.25; B. 0.1% Bismarck brown Y method: [heat-fixed smears] -^ A, 12 to 2 mins. -^ water, wash -^ B, 30 sees. -* water, wash -^ dry result: granules, blue-black on yellow. 23.218 Mallory 1922 see DS 23.218 Beauverie 1917 (note) 23.218 Neisser 1897 23454, 24:448 reagents required: A. 95% ale. 2, methylene blue 0.1, water 95, acetic acid 5; B. 0.2% Bismarck brown method: [heat-fixed smears]^ A, 1-3 sees. -^ water, wash -^ B, 3-5 sees. -^ water, wash — > dry note: The timing given is that of the original paper. Modern practice calls for longer staining. Cowdry 1943, 62, for example, recommends l^ to 1 minute in A and 30 seconds in B. 23.218 Ponder lest. 1920 Stitt Stitt 1920, 55 formula: water 100, 95% ale. 2, acetic acid 1, toluidine blue 0.02 method : [heat-fixed smear on coverslip] —* stain, dropped on coverslip -* examine as hanging drop 23.218 Ryn 1940 11796,17:53 REAGENTS REQUIRED: A. sat. aq. sol. potassium aluminate {not alum) 5, sat. ale. sol. methyl violet 1 method: [heat-fixed smear] — * stain, 2-3 mins. -^ wash — > dry 23.218 Stottenberg 1926 7276,20:426 formula: water 100, acetic acid 3, abs. ale. 3, malachite green 0.25, toluidine blue 0.05, hematoxylin 0.01 method: [dry smear] — > stain, 1 min. — * wash — > dry 23.218 Tribondeau and Dubreuil 1917 6630, 80:331 REAGENTS REQUIRED: A. DS 11.44 Nicolle (1942); B. 0.2% Bismarck brown method: [alcohol-fixed smear] — > A, 5 mins. — > water, wash — » 1-2 mins. — > water, wash -^ dry 23.219 Other Methods for Bacterial Smears 23.219 Broadhurst and Paley 1939 11023, 94:525 formula: 95% ale. 62, tetrachlorethane 40, sulfuric acid 0.4, methylene blue 1, magenta 0.08 preparation: Mix 50 ale. with tetrachlorethane and acid. Heat to 55°C. Add methylene blue and shake to solution. Dissolve magenta in 10 ale. and add to blue. method: [thin film of milk dried to slide] — > stain, on slide, 15 sees. — > drain -^ dry — ♦ wash till faint pink -^ dry recommended for: bacteria in milk. 23.219 Bruner and Edwards 1940 11284, 25:543 REAGENTS REQUIRED: A. 5% malachite green; B. 0.5% safranin method: [heat-fixed blood smear] ^ ^4, 5 mins. — ^ rinse — * B, 10 sees. — > wash —> dry recommended for: demonstration of bacteria in leukocytes. DS 23.219 DYE STAINS OF SPECIAL APPLICATION 491 23.219 Burdon, Stokes, and Kimbrough 1942 11056, 43:717 REAGENTS REQUIRED: A. 0.3% Sudan black B; B. 1% safranin method: [emulsify bacteria from slant with .1]-^ make smear—* B, 15 sees. — > wash -^ dry RECOMMENDED FOR: demonstration of fat in bacteria. 23.219 Erb 1929 11284, 14:377 formula: ether 50, methanol 50, methylene blue 0.59 method: [air-dried milk smear] — ♦ stain, 1 min. -^ rinse -^ dry RECOMMENDED FOR: bacteria in milk. 23.219 Flinn 1939 11284,25:316 preparation of stain: Grind 0.15 methyl green with 0.5 pyronin and 15 95% ale. Add while grinding 85 3% phenol. Leave 1 week. method: [fresh blood smears] — » stain, 4 mins. —* wash -^ dry recommended for: demonstration of bacteria (deep red) in leukocytes. 23.219 Gay and Clark 1934 11056, 27:175 REAGENTS REQUIRED: A. DS 11.44 Loffler 1890; B. DS 11.43 Ziehl 1882 method: [heat-fixed smears] — > A, 3-5 mins. — > rinse -^ B, 5-10 sees. — > wash — > dry recommended for: differentiation of living and dead bacteria. note: See also DS 23.216 Proca 1909. 23.219 Kahlden and Laurent 1896 Kahlden and Laurent 1896, 103 REAGENTS REQUIRED: A. 1% eosiu in 96% ale; B. 1% methyl blue method: [dry smears] -^ A, 1-2 mins. 30°C. —>■ blot -^ B, 30 sees. -* wash -^ dry result: cocci, blue; cellular material, red. RECOMMENDED FOR: differentiation of cocci in smears of cellular material. 23.219 McCullough and Dick 1942 20540b, 17:153 formula: water 50, methanol 50, phenol 0.5, sodium chloride 0.5, sodiimi phosphate, dibasic (12 H2O) 0.02, methylene blue 0.02 method: [smears] -^ methanol, 6 mins. -> stain, 10-30 mins. — > wash — » dry RECOMMENDED FOR: demonstration of bacteria in leukocytes. 23.219. Schwitz test. 1896 Kahlden and Laurent Kahlden and Laurent 1896, 103 REAGENTS REQUIRED: A. sat. sol. methyl blue in 5% phenol; B. 0.25% acetic acid; C. 0.2% safranin method: [dried smears] -* A, 5-10 mins. —* B, 2-3 sees. -^ wash -* C, 15-30 mins. -* wash — > dry recommended for: differentiation of cocci in smears of cellular material. 23.219 Shutt 1947 20540b, 22:1 formula: ether 50, methanol 50, methylene blue 0.5, hydrochloric acid 0.35 preparation: Dissolve dye in mixed solvents. Add acid to solution. method: [make smear] -^ stain, 5-10 sees. — > dry recommended for: bacteria in milk. 23.219 Walton 1939 11284, 24:1308 formula: water 100, phenol 2, glycerol 20, methanol 10, methyl green 1, pyronin 0.2 preparation: Dissolve the dyes in the methanol. Add phenol and water. Shake 2 hours per day for 2 days. Filter. Add glycerol to filtrate. method: [heat-fixed smears] -^ stain, on slide, warmed till steaming -^ wash -^ dry result: gonococci, red. recommended for: specific differentiation of gonococcus. 23.219 Weiss 1929 11284, 16:170 reagents required: A. water 85, 95%, ale. 10, acetic acid 5, methylene blue 5; B. 1% safranin method: [heat-fixed smears] -> A, 5 mins. -^ wash -^ B, ^-2 mins. -> wash —> dry recommended for: demonstration of polar bodies (deep blue) in bacterial cells (red). 492 METHODS AND FORMULAS DS 23.219-DS 23.221 23.219 Zaribnecky 1934 2818, 50:224 formula: water 90, naphazarine 0.15, azophloxine 0.1 preparation: Boil the naphazarine and alum chloride in 60 water. Cool. Filter. Add the azophloxine dissolved in 30 water. method: [dried smear of milk] — >• stain, 5-10 mins. —* dry RECOMMENDED FOR: Staining bacteria in milk. 23.22 BACTERIA IN SECTIONS OF TISSUES Relatively few methods are found for staining bacteria in cells for two reasons. First, these methods are rarely, if ever, used for diagnostic purposes but are confined to sec- tions destined for research or teaching; second, the majority of methods which are used for staining bacteria in smears can also be used for staining bacteria in sectioned ma- terial. The methods given below are therefore confined to those which are used exclu- sively for staining sections and which cannot be used for staining smears. For staining sections of specific organisms, for which a technique in smears is recommended, the smear technique should always be tried in preference to one of the techniques here given. 23.221 General Methods 23.221 Foshay 1931 11284, 17:193 formula: water 80, sat. sol. NUe blue sulfate 12, 1% safranin 8 method: [sections] — > stain, overnight —^ rinse — > balsam, via usual reagents 23.221 Fraenkel test. 1928 Schmorl Schmorl 1928, 363 reagents required: A. DS 11.44 Loffler 1890; B. 0.5% acid fuchsin 30, 33% tannin 30, ADS 22.1 Unna (1928) 30 method: [sections]—* A, overnight-^ thorough wash — > B, till differentiated —> wash -^ balsam, via usual reagents result: bacilli, blue-black; nuclei, light blue; other structures, red. 23.221 Guyer 1930 Guyer 1930, 117 REAGENTS REQUIRED: A. DS 11.44 Loffler 1890; B. 0.1% acetic acid method: [sections] — » water — » A, }'2 to 24 hrs. — > B, 10-20 sees. -^ abs. ale, rinse — » balsam, via xylene 23.221 Holmes and French 1926 see DS 13.21 Holmes and French 1926 23.221 Krajian 1941 1887a, 32 :825 REAGENTS REQUIRED: A. DS 11.123 Harris 1900; B. 0.1% hydrochloric acid in 70% ale; C. water 100, zinc sulfate 4, copper sulfate 7; D. 3% brilliant green in C; £^. 5% ammonium nitrate; F. DS 11.43 Ziehl 1882; G. creosote 50, xylene 50 method: [7-10 n frozen sections] —* A, 2 mins. -^ tap water, till blue ^ 5, 5 or 6 dips — > C, 3 mins., on slide — > Z), 5 mins., on slide — > rinse -^ E, 1 min. -^ rinse -^ F, 2 mins., on slide — > rinse — > blot — > dioxane, 2 mins. -^ G, till differentiated -^ dammar, via xylene 23.221 Krajian 1943 11284,28:1602 reagents required: A. DS 11.44 Loffler 1890; B. creosote 25, xylene 75; C. B 100, 95% ale. 5, magenta 1.2 method: [sections] -^ water — > A, 3 mins. — » wash — > isopropyl ale, till dehydrated -^ B, till differentiated — » C, 1 min. — > B, till excess red removed — * xylene, thorough wash -^ balsam 23.221 Kiihne test. 1904 Besson Besson 1904, 260 reagents required: A. DS 11.44 Kiihne (1904)a; B. ADS 12.2 Gram 1884; C. sat. ale. sol. (circ. 2%) fluorescein method: [sections] -^ water — > A, 5-15 mins. -^ wash — > fi, 2 or 3 mins. — * wash —> C, till difl'ercntiated — > balsam, via usual reagents 23.221 Langeron 1942 see DS 13.22 Langeron 1942b DS 23.221-DS 23,222 DYE STAINS OF SPECIAL APPLICATION 493 23.221 Mallory 1938 Mallory 1938, 86 STOCK solutions: I. water ]()(), sodium borate I, mctliylcne blue 1; II. 1% azur TI REAGENTS REQUIRED: A. 2.5% plilo.xiiie; B. stock I 5, stock 11 5, water 90; ('. ADS 21.2 Wolbach 1911 method: a, 1-2 hrs., 50°C. -^ water, wash -> B, 5-20 inins. — > water, wash — > C, till differentiated — > balsam, via xylene note: a detailed description of the use of this technique is given under DS 23.20 above. 23.221 Masson see DS 13.22 Masson (1942) or DS 13.5 Masson (1942) 23.221 Nicolle test. 1904 Besson Besson 1904, 258 REAGENTS REQUIRED: A. J)S 11.44 Kiihnc (1904) a or b; B. 10% tannic acid method: [sections] -^ water — > .1, 2 3 mins. -^ wash — > B, a few sees. —> rinse — ♦ blot — > dehj'drate least possible time — ^ balsam, via usual reagents 23.221 Nicolle see DS 11.423 Nicolle (1942) 23.221 Noniewicz test. 1896 Kahlden and Laurent Kahlden and Laurent 189G, 108 reagents required: A. DS 11.44 Loffler 1890; B. water 99, acetic acid 1, tropeolin 0.1 method: [sections] -^ water -^ A, 2-5 mins. -^ wash — > B, 1-5 sees. — > wash — > dry 23.221 Ollett 1947 11431,69:357 reagents required: A. DS 23.211 Ehrlich 1882; B. ADS 12.2 Gram 1884; C. 2% acetic acid in abs. ale; D. water 75, DS 13.7 Twort (1909) 25 method: [3 fj. sections of formaldehyde-fixed material]—* A, 3-5 mins. -^ rinse—* B, 3 mins. -* rinse —* blot -^ C, till decolorized — > rinse — > D, 5 mins. —> rinse -^ C, till no more color comes away -^ balsam, via xylene 23.221 Pappenheim DS 23.221 see Saathof 1905 23.221 Saathof 1905 Unna-Pappenheim method — compl. script. 7276, 32 :2047 formula: water 75, phenol 1.5, glycerol 20, 95% ale. 5, pyronin 0.15, methyl green 0.5 method: [sections] —* stain, 1-3 mins. — * w^ater, wash — > acetone, till dehydrated -^ bal- sam, via xylene 23.221 Unna see DS 23.221 Saathof 1905 2S.222 Iodine Differential Methods Staining Gram-positive bacteria sections is only another instance in which a standard method has become so embedded in the literature that all other methods are referred to it. In this instance it is the Gram-Weigert technique which dominates the field. Gram, in point of fact, had nothing to do with the technique of Weigert, published in 1887, which depended entirely upon Weigert's contribution of differentiating with aniline the crystal violet stain of Ehrlich, after mordanting with Gram's iodine. So confused, how^ever, has the picture become, that even Weigert's well-known resorcin- magenta method for staining elastic fibers has been recommended for the purpose of differentiating bacteria. Moreover, some authors (see comments under "Gram-Weigert" below) have recommended prior nuclear staining either with carmine or hematoxylin. Most of the techniques recommended here still rely on differentiation with aniline, though the method of Brown and Brenn 1931 substitutes a mixture of acetone and ether for the aniline. 23.222 Brown and Brenn 1931a 10919, 48:69 reagents required: A. DS 11.123 Harris 1900; S. 3% hydrochloric acid in 95% ale; C. 1% ammonia; D. DS 11.45 Brown and Brenn 1931; E. ADS 12.2 Lugol; F. 0.005%, rosanilin hydrochloride; G. 0.1% picric acid method: [sections]-^ water -^ A, 2-5 mins.—* rinses B, till pink—* rinse—* C, till blue — * wash — > Z), 2 mins. — * wash — > ^, 1 min. -^ wash — > blot — * F, 5 mins. — > wash -^ blot -^ acetone, rinse — > G, till yellowish pink -^ balsam, via acetone and xylene result: Gram -positive organisms, red; Gram -negative organisms, blue black. 494 METHODS AND FORMULAS DS 23.222 23.222 Brown and Brenn 1931b 11056, 21:21 STOCK solutions: I. water 100, gentian violet 1; II. water 100, sodium bicarbonate 5, phenol 0.5 REAGENTS REQUIRED: A. stock I 100, stock II 11; 5. ADS 12.2 Lugol (1905); C. acetone 75, ether 25; D. 0.005% magenta; E. 0.1% picric acid in acetone method: [sections, nuclei stained by DS 11.122 technique]—* waters A, on slide, 2 mins. — > rinse — > 5, 1 min. — > wash — > blot — » C, on slide, till no more color comes away -^ blot D, 5 mins. — > wash — » blot E, till color turns to yellowish pink -^ bal- sam, via acetone and xylene 23.222 Glynn 1935 1789a, 20 :896 REAGENTS REQUIRED: A. DS 11.45 Crystal violet; B. ADS 12.2 Gram (1884); C. acetone; D. water 100, magenta 0.05, hydrochloric acid q.s. for pH 2.5; E. sat. sol. picric acid method: [sections] —* water -^ A, 2 mins. — > drain -^ B, on slide, 1 min. — > C, till no more color comes away — > water, wash — > Z), 3 mins. —>■ drain -^ E, on slide, 1 min. -^ C, 10-15 sees. —> balsam, via xylene result: Gram-positive bacteria, violet; Gram-negative bacteria, red. 23.222 "Gram-Weigert" — compl. script. The original method is Weigert 1887 (see below). Almost any technique involving the differentiation of gentian violet with iodine followed by aniline has come to bear this name. Unfortunately Weigert's 1898 (23681, 9:290) resorciii-magenta method (see DS 21.13 Weigert 1898) has also become confused with the 1887 method. Authors who state only that they "stained by the Gram-Weigert method" may have used crystal violet or magenta, with or without prior nuclear staining in carmine (Zinsser, Bayne- Jones, 1939, 860) or hematoxylin (Mallory, 1938, 272), with or without aniline differentiation. 23.222 Haythorn 1929 4349, 12:128 REAGENTS REQUIRED: A. DS 11.122 Mallory 1938; B. 0.1% hydrochloric acid in 70% ale; C. DS 23.211 Ehrlich 1882; D. ADS 12.2 Lugol (1904); E. aniline 60, xylene 30; F. sat. ale. sol. erythrosin; G. aniline 30, xylene 60 method: [sections] — + water —>■ A, 5-10 mins., or till deeply stained — > B, quick rinse — > tap water, wash — > C, 2-5 mins. — > blot -^ D, 2-5 mins. — * E, till differentiated — » F, 30-60 sees. -^ G, till no more color comes away — > balsam, via xylene 23.222 Krajian 1943 11284,28:1602 reagents required: A. DS 11.44 Loffler 1890; B. xylene 65, creosote 35; C. DS 11.43 Krajian 1943 method: [sections]—* water—* A, 3 mins. -^' wash—* abs. ale, least possible time for dehydration -^ B, till differentiated, 2-5 sees. — » C, on slide, 2 changes, 10 sees, each —> blot — * B, on slide till differentiated — > blot — * balsam, via xylene result: nuclei. Gram-negative organisms, Negri bodies, red; Gram-positive organisms, actinomycetes, blue. 23.222 Krajian 1950 Krajian 1950, 196 REAGENTS REQUIRED: A. 5% thorium nitrate; B. DS 11.121 Krajian 1950; C. 1% hydro- chloric acid in 70% ale, D. water 100, potassium iodide 5, zinc sulfate 5; E. DS 11.43 Ziehl 1882; F. 2% sodium sulfite; G. 3% acetic acid; H. 50:50 aniline xylene; I. cresote method: [5 ju to 7 m sections] — > water — * A, 5 mins. -^ rinse -^ B, 3 mins. -^ wash — * C, till no more color comes away — * tap water, till blue -^ D, 5 mins. — > rinse — * E, 7-10 mins. — > wash — * F, 2 mins. -^ rinse — * G, 3 mins. -^ blot — * H, 30 sees. — * /, till pink clouds cease -+ dammar, via xylene result: Gram-positive organisms, blue black; Gram-negative organisms, red. 23.222 Male 1924 11035,42:455 reagents required: A. 0.5% methyl violet; B. ADS 12.2 Gram 1880; C. water 75, 95% ale. 25, light green 0.05, neutral red 0.25 method: [sections] -^ water -^ A, 1-2 mins. -^ B, 1-2 mins. -* 95%, ale, till no more color comes away -^ C, 3-5 mins. -^ wash -> balsam, via usual reagents DS 23.222-DS 23.223 DYE STAINS OF SPECIAL APPLICATION 495 23.222 Mallory 1938 Mallory 1938, 272 REAGENTS REQLriRED: A. 2.5% phloxinc; B. DP 1 1.45 Sterling 1890; C. ADS 12.2 Gram; D. aniline method: [sections of F 3700.0010 Zenker 1894 fixed material, prior stained by DS 1 1.122 method] — > A, 10 mins., 50°C. -^ water, wash — > B, 14 to 1 hr. — »• water, wash -^ C, 1-2 mins. --> blot -^ D, till differentiated — > balsam, via usual reagents 23.222 Verchoeff 1940 11006, 115:1546 REAGENTS REQUIRED: A. DS 11.45 Sterling 1890; B. ADS 12.2 Gram 1880; C. trichlor- ethylene; D. oil of thyme method: [sections] — > water —^.4,2 mins. -^ wash — » /?, 1 min. — > 95% ale, till color clouds appear -^ C, till color clouds cease — » D, 1 min. -^ [examine and repeat 95% ale. — > C — » Z) cycle till differentiation complete] — > balsam, via xylene RECOMMENDED FOR: "Lcptotriches " or Parinaud's conjunctives. 23.222 Weigert 1887 8644, 5 :228 REAGENTS REQUIRED: A. DS 23.211 Ehrlich 1882; B. 0.9% sodium chloride; C. ADS 12.2 Gram, 1882; D. aniline. method: [sections] -^ water —> a, 1 min. -^ fi, wash — > C, 1 min. —» blot -^ Z), till differentiated -^ xylene -^ balsam note: See comments under "Gram- Weigert" above. 23.222 Zeissig 1929 20540b, 4:91 REAGENTS required: .4. DS 23.212 Hucker and Conn 1923; B. ADS 12.2 Gram 1884; C. water 95, ADS 12.2 Gram 1884 5 method: [sections, with nuclei stained] — > A, 2-3 mins. -^ wash — > B, 5 mins. — > wash — > C, till differentiated -^ counterstain, if required -^ balsam, via xylene 23.223 Methods for Acid-fast Organisms Techniques for staining acid-fast organisms in sections essentially follow those for the staining of acid-fast organisms in smears. The most interesting variation is that of Malloiy 1938, in which the acid used is in such a low strength that it permits the retention of hematoxylin stains in the nuclei while still allowing for the differentiation of the bacteria. 23.223 Adams test. 1946 anonymous 4349, 26:13 REAGENTS REQUIRED: A. ADS 11.121 Weigert 1903; B. 0.1% hydrochloric acid in 50% ale, C. DS 11.43 Goodpasture and Burnett 1919; D. 5% acetic acid; E. 0.1% light green in 0.2% acetic acid method: [sections] — > water -^ A,\ min. -^ B, wash — > C, 1 min. —>■ D, till no more color comes away — > repeat C -^ D cycle three times — » wash — > ZJ, 1 min. -^85% ale. — > balsam, via usual reagents result: nuclei, black; bacteria, red; other structures, green. 23.223 Albrecht test. 1943 Cowdry Cowdry 1943, 17 REAGENTS REQUIRED: A. DS 11.43 Albrecht (1943); B. 1% hydrochloric acid in 70% ale; C. DS 11.123 Harris 1900; D. 0.3% hydrochloric acid in 70% ale; E. 0.4%, ammonium hydroxide method: [sections] — ♦ water — ♦ [lay filter paper saturated with A on slide, steam, 3 mins. leave 30 mins.] —>■ B (removing filter paper), till sections deep pink — > wash — > C, 10 mins. -^ wash — > D, till differentiated -^ wash — ♦ E, till nuclei blue -^ balsam, via usual reagents 23.223 Baumgarten test. 1942 Langeron Langeron 1942, 1212 REAGENTS REQUIRED: A. DS 11.43 Zichl 1882; B. 10% nitric acid in 95% ale; C. 1% toluidine blue method: [sections] — * A, ^^-1 hr. — > water, wash — > B, till no more color comes away -^ water, wash — > C, 1-2 mins. — * water, wash — » balsam, via usual reagents RECOMMENDED FOR: Icprosy bacilU in sections. 496 METHODS AND FORMULAS DS 23.223 23.223 Bertrand and Medakovitch 1924 test. Langeron 1942 Langeron 1942, 1204 REAGENTS REQUIRED: A. sat. sol. aniline 90, 95% ale. 10 magenta 5; B. 3% hydrochloric acid; C 1% lithium carbonate; D. 1% malachite green method: [paraffin sections of F 1500.0010 fixed material] -^^ water -^ A, 1 min. 60°C. -^ water, rinse -^ B, till no more color comes away — > water, wash -^ C, 1 min. -^ D, 30 sees. — > balsam, via usual reagents RECOMMENDED FOR: acid-fast bacilli in sections. 23.223 Campbell 1929 4349, 12:129 REAGENTS REQUIRED: A. DS 11.43 Kinyoun (1946); B. 0.5% hydrochloric acid in 35% ale; C. DS 11.122 "Harris"; D. 1% ammonia; E. 1% orange G method: [paraffin sections of F 3700.0010 Zenker 1894 fixed material]^ water -> A, 30 mins. -^ rinse -^ B, 2 changes, till pink -^ C, 2 mins. -^ B, till nuclei differenti- ated-^^ D, till nuclei blue — > wash ^^ E, till plasma orange—* balsam, via acetone and xylene recommended for: demonstration of B. leprae (red) in sections. Myelin sheaths also stain red. 23.223 Czaplewski test. 1896 Kahlden and Laurent Kahlden and Laurent 1896, 117 reagents required: .4. DS 11.43 Ziehl 1882; B. 95% ale. 100, fluorescin to sat., methyl blue to sat. ; C. sat. ale. sol. methyl blue method: [sections] —> water ^ a, 3-5 mins., 37°C. ^ drain -+ B, 12 dips, draining slowly between each — > C, flooded on slide, 1 min. -^ rinse — > dry -^ balsam 23.223 Doubrow 1929 4285a, 6:142 REAGENTS REQUIRED: A. DS 11.43 Ziehl 1882; B. 4.5% picric acid in 95% ale; C. 0.5% light green in 95% ale. method: [sections, nuclei stained by DS 11.111 Regaud 1910] -> A, 45 mins., 50°C. — > B, till only nuclei appear stained -^ water, till yellow stain removed — > C, 1 min. — > balsam, via usual reagents 23.223 Douglas 1932 11284, 17:1131 REAGENTS REQUIRED: A. DS 11.43 Ziehl 1882 100, sodium chloride 0.3; B. 5% nitric acid in 95% ale.; C. 1% azur II 5, DS 11.44 Jadassohn (1928) 5, water 90; D. ADS 22.1 Wolbach 1911 method: [paraffin sections] ^ A, overnight -^ refrigerator, 30 mins. -^ wash —> 5, 1 min. — * wash -^ C, 10 mins. — > wash -^ D, till differentiated -^ balsam, via usual reagents 23.223 Fits 1939 11284,25:743 REAGENTS REQUIRED: A. water 90, methanol 10, phenol 5, new magenta 0.5; B. 20% formaldehyde; C. 2% hydrochloric acid in 95% ale; D. 1% potassium permanganate; E. 2% oxalic acid; F. DS 11.123 Harris 1900; G. DS 12.221 Fite 1939 method: [sections of formaldehyde-alcohol-fixed material] — * water —> A, 12-24 hrs. — + B, 5 mins. — > C, 5 mins. — > rinse -^ D, 2-5 mins. —^E,! min. wash — > F, 2 mins. — » wash —* G, till stained — > 95% ale, — * balsam, via usual reagents 23.223 Flexner test. 1938 Mallory Mallory 1938, 276 REAGENTS REQUIRED: A. DS 11.43 Ziehl 1882; B. ADS 12.2 Gram (1904); C. aniline method: [paraffin sections, nuclei stained by any DS 11.122 technique] —> water —> A, 1 hr. — * water, wash -^ B, ^2 to 1 min. -* water, wash -^ blot — * C, till differentiated —*■ balsam, via xylene 23.223 Fuller 1938 11284,23:416 reagents required: A. 5% ferric alum; B. water 80, glycerol 10, 95% ale. 10, hema- toxyUn 1; C. 5% picric acid in 95% ale; D. 1.5% magenta in sat. aq. sol. aniline; E. 3% nitric acid in 95% ale; F. 0.01% ammonia; G. 1% light green method: [sections] — > water—* A, 5 mins., 50°C. -^ rinse-* B, 5 mins. 50°C. -* C, till nuclei only stained — > D, on slide, heated to steaming 3 mins. — > cool — » reheat, 3 mins. —> E, till sections pale pink —* F, few sees. — > wash, 10 mins. — > G, 5 mins. — > rinse — > balsam, via usual reagents result: bacilli, red; nuclei, blue green; cytoplasm, pale green. DS 22.223 DYE STAINS OF SPECIAL APPLICATION 497 23.223 Haythorne 1929 4349, 12:130 RKAGENTS reqi'iked: .1. 1 )S I 1. 13 Ziclil IS82; li. 10% sulfuric ncid; ('. sat. abs. n\c. sol. orange G method: [sections of F 3700.0010 Zenker 1894 fixed material with nuclei hematoxylin stained] ^ water -> .4, 1 hr., 55°C. -^ wash, 20°C. -> wash, 8° to 10°C. -> B, 8-10°C., till sections pale violet -^ wash, 8°-10°C. —> tap water —+95% ale, from drop bottle, least possible time -^ C, from drop bottle, till sections pale orange -^ blot — > balsam, via xylene 23.223 Krajian 1943 Tech. Bull., 4:45 RE.\GENTS REQriRED: A. DS 11.43 Ziehl 1882; B. water 40, 95% ale. (iO, arsenic trioxide 1; C. DS 11.44 Lofiler 1890; D. creosote 50, isopropyl ale. 50 method: [frozen sections varnished to slide with celloidin, or mounted paraffin ribbons] — » water —> A, heated to steaming, 3 mins. 23.223 Kiihne test. 1904 Besson cit. Borrel Besson 1904, 638 REAGENTS REQUIRED: A. DS 11.122 Bohmer 1868; B. DS 11.43 Ziehl 1890; C. 2% aniline hydrochloride method: [sections] -^ water —* a, 2 mins. —» wash -^ 5, 15 niins. ^ C, few sees. — > abs. ale, till differentiated -^ balsam, via usual reagents 23.223 Langeron 1942 Langeron 1942, 1204 reagents required: A. DS 11.43 Ziehl 1882; B.3% hydrochloric acid; C. 0.4% formal- dehyde; D. water 100, acetic acid 0.6, DS 11.43 Ziehl 1882 0.6; E. water 100, 40% formaldehyde 0.6, acetic acid 0.3; F. DS 12.211 Cajal 1895 method: [sections] —> water —> A, }^ to 1 hr. — > water, wash — > B, till no more color comes away — > C, 5 mins. -^ water, wash -^ D, 3 mins. — > water, rinse — ^ E, till no more color comes away —> water, wash — > F, 1 min. — » 95% ale, till dehydrated — > balsam, via usual reagents 23.223 Langrand 1913 see DS 23.32 Langrand 1913 23.223 LetuUe 1894 test. 1894 Kahlden and Laurent Kahlden and Laurent 1894, 89 reagents required: A. DS 11.122 Bohmer 1868; B. DS 11.43 Ziehl 1890; C. water 100, iodine green 1, phenol 2 method: [sections of F 7000.0000 fixed material]-^ water —> A, 2-3 mins. -^ wash — > B, 15 mins. — > rinse — > C, 5 mins. — » abs. ale, till difTerentiated — » balsam, via clove oil 23.223 Mallory 1938 Mallory 1938, 276 reagents required: A. DS 11.43 Ziehl 1882; B. 0.1% hydroclJoric acid in 70% ale; C. 0.01% ammonia method: [sections, nuclei prior stained, by any DS 11.122 method] —> water -^ A, over- night -^ [or, A, 1 hr., 60°C.] -^ B, 20 sees. -♦ C, wash -^ balsam, via usual reagents note: This is specifically recommended for tubercle bacilli. 23.223 Miiller and Chermock 1945 11284,30:169 reagents reqi:ired: A. DS 11.43 Miiller and Chermock 1945; B. hydrochloric acid in 95% ale; C. water 98, 95% ale 2, fast green FCF 0.005. method: [sections] — » water — > A, 5 mins. -^ rinse — » B, till light pink —* C, 2 mins. — > rinse — > balsam, via usual reagents 23.223 Putt 1951 Tech. Bull, 21:94 REAGENTS REQUIRED: A. water 90, methanol 10, phenol 5, magenta-Ill 1; B. sat. aq. sol. lithium carbonate; C. 5% acetic acid in 95% ale; D. 0.5% methylene blue in abs. ale method: [5 M paraffin sections of formaldehyde-fixed material] — + water —* A, 5 mins. —* B, 1 min. — > C, 3-5 mins., till sections pale pink — > abs. ale, wash — * D, 1 min. — » abs. ale, two changes, 30 sees, each — > neutral mountant, via toluene recommended for: leprosy bacilli. 498 METHODS AND FORMULAS DS 23.223-23.224 23.223 Spoerri 1933 20540b, 23:133 REAGENTS REQUIRED: A. Water 70, 95% ale. 30, crosyl violet 1, toluidine blue 0.5, thionine 0.25; B. 0.2% sulfuric acid method: [4 n paraffin sections attached to slide by V 21.1 Spoerri 1939] -^ water -^ A, 5-10 sees., 90°C. -^ B, 1 sec. -^ rinse 1 sec. — » 95% ale. 2 sees. -^80% ale, 1 sec. — > A, 2 sees.-* [repeat 80% ale. -^ A cycle till stain sufficient] —> balsam, via usual reagents 23.223 Tilden and Tanaka 1945 Tech. Bull, 6 :95 REAGENTS required: A. DS 11.43 Tilden and Tanaka 1945; B. 40%, neutralized form- aldehyde; C. 1% hydrochloric acid in 70% ale.; D. 1% potassium dichromate; E.2% oxalic acid; F. DS 11.123 Harris 1900; G. DS 12.221 Fite 1939 method: [sections of formaldehyde-aleohol-fixed material varnished to slide with nitro- ceUulose] —> water — > .4, 15 mins. — > wash — > 5, 5 mins. — > wash -^ C, 5 mins. — » wash —* D, 2-5 mins. —>■ wash -* E, till brown not quite removed — > wash — * F, 2-5 mins. -^ wash — > G, 3 rains. — > gum damar, via usual reagents 23.22Jf. Other Methods for Bacteria in Sections Within the present class are grouped all those stains intended for sections which, when used for smears, lie within the classes DS 21.214 through DS 21.218. The ma- jority of smear techniques can, in any case, be used for sections. 23.224 Lustgarten test. 1903 Cole Cross and Cole 1903, 162 REAGENTS REQUIRED: A, aniline 3, 95% ale. 20, crystal violet 1, water 100; B. \% potassium permanganate; C. 5% sulfuric acid method: [sections] -^ A, 24 hrs. -^ abs. ak-., till color clouds cease — > B, 10 sees. -+ C, few moments -^ wash — > balsam, via usual reagents RECOMMENDED FOR: Spirochetes in sections. 23.224 Milovidov 1928 see DS 22.21 Milovidov 1928 23.224 Nikiforoff test. 1916 Warthin 4349, 6 :56 REAGENTS REQUIRED: A. Water 65, 95% ale. 35, propaeolin 0.35, methylene blue 1.3, potassium hydroxide 0.0005; B. abs. ale. 50, ether 50 method: [sections of F 3700.0000 Nikiforoff 1916 fixed material] -^ water -^ A, 24 hrs. — * wash — * B, few dips -^ balsam, via usual reagents RECOMMENDED FOR: Spirochetes in sections. 23.224 Schmorl test. 1916 Warthin 4349, 6 :56 REAGENTS REQUIRED: A. DS 13.13 Gicmsa 1902 3, water 97; B. Q% potassium alum method: [thin frozen sections] -^ A, 1 hr. — > A, fresh solution, 24 hrs. -^ B, wash -^ rinse — * [attach to slide, leave tiU nearly dry] -^ cedar oil -^ balsam RECOMMENDED FOR: Spirochetes in sections. 23.224 Smith test. 1924 Mallory and Wright Mallory and Wright 1924, 289 REAGENTS REQUIRED: A. DS 23.211 Ehrlich 1882; B. ADS 12.2 Gram 1882; C. 40% form- aldehyde; D. water 100, eosin Y 2.5, methyl green 0.5 method: [sections of F 3700.0010-5 Zenker 1894 fixed material] — > A, warmed to steam- ing, few sees.—* drain -^ B, wash — > C, wash ^ 95% ale., till no more color comes away — ♦ B, rinse -^ D, on slide, warmed to steaming, few moments — > water, wash — »• balsam, via usual reagents RECOMMENDED FOR: staining capsules in sections. 23.224 Stoughton 1930 1025, 17:162 REAGENTS REQUIRED: A. watcr 100, phcuol 5, thionine 0.1; B. sat. ale. sol. {circ. 0.2%) orange G method: [sections] —> water — > ^4, 1 hr. — > abs. ale, till dehydrated -^ B, 1 min. -^ abs. ale. till no more color comes away — > balsam, via xylene RECOMMENDED FOR: bacteria in plant tissues. DS 23.30 DYE STAINS OF SPECIAL APPLICATION 499 23.3 Other Parasites and Commensals The techniques here given are not only those which are specificall}- intended for the differentiation of parasite from host in sections, but inchide also those techniques which have been developed for the stain- ing of specific parasites, and which cannot reasonably be employed for the staining of any free-living organism. The four classes which have been set up are obviously the only four into which parasites can be di- vided. The techniques which are given in each of the four classes are, without excep- tion, so specific that their individual ap- plication is given under each, 23.30 TYPICAL EXAMPLES Demonstration of myceUa of Peni- cillium in orange rind using the thionin-hght green-orange G-eryth- rosin stain of Margolena 1932 The method here described is one of the easiest and most certain methods of dem- onstrating the penetration of the myceUa of parasitic fungi through the tissues of their plant hosts, though the example se- lected for demonstration is quite possibly an example of a saprophytic, rather than a parasitic, fungus. This material, however, has the advantage that it may be procured without the slightest difficulty, if it is re- quired for class-demonstration purposes, and also that it permits a clear definition of the invading myceha. To secure a growth of Penicilhum on orange rind it is only necessary to take an orange and to remove the wax, with which the marketer has protected and polished it, by rubbing any very fine form of grit gently onto the surface with the fingers. One of the most readily available forms of grit are the scouring powders sold for the cleaning of domestic utensils. If an orange be scrubbed with some of this scouring powder, it will not appreciably damage the surface, but will roughen it sufficienth' to permit the spores of Penicilhum, which are always in the air, to fight directly on the damaged surface. The orange should then be placed under a bell jar on top of moist blotting paper and kept at about 85°F. After a few days, a vigorous growth of Penicilhum will be seen on the surface, and a piece of about j'i-inch side should be cut from the rind and dro])ped into any of the alcohol-acetic-formaklehj^do mix- tures (Chapter IS, F 0000.1010) which may be available. It should remain in this fixative for about three days and then be placed in absolute alcohol in which it should remain, with frequent changes, until such time as the oil has ceased to leave it. The piece should then be em- bedded in paraffin (Chapter 12) and cut into sections of from ten to twelve microns in thickness. These sections are mounted on a slide, deparaffinized, and brought down to water. It is also possible, though not so convenient, to use this same tech- nique on unmounted sections cut on a hand microtome. The staining solutions required are: first, a 0.1% solution of thionin in 5% phenol; second, a 0.5% solution of hght green in 95% alcohol; third, a mixture in the proportion of 1:2 of a saturated al- cohohc solution of orange G with a satu- rated solution of erythrosin in clove oil. The sections are taken from water and placed in the thionin solution for one hour. They are then rinsed in water to remove the excess thionin, or, if many sections are being dealt with, accumulated in water while the next stage is passed through. The sections are then dehydrated before being dipped up and down in the light green until the color changes from blue to green. They may then be returned to water until no more color comes away. All the sections are then taken together and passed to absolute alcohol, in which they remain until they are dehydrated. Several changes of absolute alcohol may be required, but graded alcohols cannot be used because they would extract the stain. The alcohol-clove-oil stain mixture is then dropped, from a drop bottle, on each individual slide and allowed to act for from one to two minutes. The excess stain is then washed off the shde with fresh clove oil, the clove oil removed with xylene, and the section mounted in balsam. 500 METHODS AND FORMULAS DS 23.30 Demonstration of parasitic fungi in tissue scrapings using the technique of Chalmers and Marshall 1914 All the methods for the diagnostic dem- onstration of fungi in tissue scrapings are very much the same, and combine partial hydrolysis (clearing) of the removed epi- dermal tissue in strong alkali 'uath the staining of the fungi by a bacterial stain- ing method, subsequent!}' differentiated in aniline. The present method (DS 23.32 Chalmers and Marshall 1914) is one of the easiest to use and has, therefore, been se- lected for discussion. It is to be presumed that scrapings from the surface of the patient's skin will be removed bj- the phj^sician, and it is recommended that they be brought to the technician in a ■watch glass. The technician should sort over the material presented, and should remove from it with fine forceps those pieces which definitely present the appear- ance of scales. These scales are then placed in a 25 % solution of potassium h}-- droxide at 40°C. for a few hours. The scales must then have the alkali removed, for which purpose it is best to use 15% alcohol rather than water. The washing is most easily done by picking up the scales with a section lifter and passing them through half a dozen watch glasses of 15% alcohol, rather than by pouring off the solution and replacing it with alcohol. It does not matter how long the specimens remain in alcohol. It is often a useful rou- tine procedure to accumulate all of the day's collected scrapings in separate jars of alcohol, and to stain them the first thing the next morning before additional scales are received. When it is desired to stain the material, each separate scale is lifted from the 15% alcohol and placed in the center of a chemically clean shde where it is dried. If the scales have been so softened that they cannot be lifted from the fluid, it is only necessary to pour the contents of the jar into a fingerbowl of 15% alcohol, and then to maneuver the slide under the selected scale. This scale is then held with a needle against the shde, which is hfted from the alcohol, leaving the scale stranded. The objection to the stranding technique is that it is difficult to place more than one scale on a slide, wlicreas if each scale can be lifted and spread out, a dozen tj-pical scales from one patient ma}' be stained on the same shde. It does not matter whether the slides are dried at room temperature or on a warm table, but there is some risk that, if the temperature is elevated too far, the scale will curl off the shde as it dries. A drop of Ehrlich's crystal violet (DS 23.211 Ehrhch 1882) is then placed over the scales, and allowed to remain at room temperature for about 20 minutes. It should be looked at from time to time and, if it shows signs of drying, further quan- tities of crystal violet should be placed on top. At the end of 20 minutes the crystal \dolet is drained carefully from the scales, which are not washed, and replaced with several drops of Gram's iodine (Chapter 22, ADS 12.2) which is allowed to act for a period of about 3 minutes. Differentiation in aniline is very simple, for it is impossible to overdifferentiate. The shde is placed at about a 45° angle in a glass dish, and anihne is allowed to flow over it from a pipet. If the drops are al- lowed to fall from a height the scales may become detached, therefore, the edge of the pipet should be rested just above the scales, and a slow and stead}' stream of anihne allowed to flow over them. This stream of anihne must be continued until no further color comes away, by which time the fungal hyphae Tvdll be perfectly differentiated against a colorless back- ground. They may be mounted in this condition if desired, or, as suggested by Chalmers and Marshall (DS 23.32 below), counterstained for one minute with a 2% alcoholic solution of eosin Y, which is poured over the slide without removal of the aniline. Aniline is then used exactly as before to remove the excess eosin; the flow of anihne is discontinued when no more eosin comes away. The aniline is then washed from the shde with xylene and the specimens are mounted in balsam. Fungal hyphae will be clearly stained bright blue against a yellow background. The prin- cipal purpose of the background is to en- able one to find the scales under a low power of the microscope before using the DS 23.31 DYE STAINS OF SPECIAL APPLICATION 501 high to search for fungal filaments. With- cleared scales in a balsam mount. These out the counterstain it is sometimes very slides may be filed for reference, for they difficult to detect the almost perfectly are quite permanent. 23.31 PLANTS PARASITIC IN PLANTS 23.31 Bostr0m test. 1896 Kahlden and Laurent Kahlden and Laurent 1896, 120 REAGENTS REQUIRED: A. DS 23.211 Ehrlich 1882; B. DS 11.24 Ranvier 1889 method: [sections] — > water — >• A, 10-15 mins. — > B, 5-10 mins. — ► water, wash -^ abs. ale, till differentiated -^ balsam, via xylene RECOMMENDED FOR: Staining actinoniycetes in sections of plant tissues. 23.31 Cartwright 1929 1032,43:412 REAGENTS REQUIRED: .1. 1% safranin; B. water 100, anilin blue 1, picric acid 1 method: [sections of wood] — > water -^ A, few sees. —>■ water, wash — ► B, heated to sim- mering, few mins. -^ water, till no more color comes away — > balsam, via usual reagents RECOMMENDED FOR: hypliae in wood}^ sections. 23.31 Cohen 1935 20540h, 10:25 reagexts required: A. water 97, acetic acid 3, orseillin BB to sat.; B. 1% crystal violet in clove oil method: [sections from F 5G00.1000 Cohen 1935 fixed material] — > water ^ ^4, 30 mins. — > rinse -^ dehj'drate via graded ales. -^ B, 5 mins. -^ xylene, wash — » balsam 23.31 Cornwall 1937 Microscope, 1 -AST reagents required: .4. water 100, picric acid 0.25, methyl blue 0.35; B. 0.25% safranin O in 90% ale. method: [sections of alcohol-fixed wood] — ^ A, 15-20 mins. — » 50% ale, till no more color comes away — > B, 5 mins. -^70% ale, rinse — > abs. ale, till pink recommended for: fungus hyphae (blue) in wood. 23.31 Dickson 1920 19938. 52:63 reagents required : .4 . 2 % phloxine in 85 % ale ; B. clove oil 98, abs. ale 2, light green 2 method: [sections] ^95% ale -^ .4, 5-10 mins. -^95% ale, wash -^ B, 1-3 mins. — » clove oil, till no more color comes away — * balsam, via usual reagents recommended for: staining fungus in plant tissues. note: "Phloxine" is given for solution .4 above on the authority of Conn 1946, 112, who states that Dickson was in error in supposing his stain to be Magdala red. 23.31 Ferraril930 2174,2:81 reagents required: .4. 0.01% ruthenium red; B. 10% potassium hydroxide method: [alcohol-preserved tissues]^ A, till stained (10 mins. to several days) — > B, till differentiated —> glycerol recommended for: staining fungi in plant tissues. 23.31 Garrett 1937 test. 1942 Langeron Langeron 1942, 1285 formula: water 100, sodium hj'droxide 4, brom thymol blue 0.4 method: [fresh root tissue] -^ A, overnight —> glycerol recommended for: staining fungi in roots. 23.31 Hutchins and Lutman 1941 20540b, 16 :63 reagents required: .4. water 88, 95% ale 10, aniline 2, crystal violet 5; B. ADS 12.2 Gram 1884 method: [sections] — » water — > .4, 24 hrs. — > wash -^ B, 24 hrs. — > abs. ale, till color clouds cease —* xylene, till no more color comes away — > balsam - recommended for: actinomyces in potato tuber tissue. 23.31 Israel test. 1940 Johansen Johansen 1940, 226 reagents reqihred: .4. sat. aq. sol. orcein in 0.1% acetic acid method: [sections] ^ water —> A, several hrs. —> abs. ale, till differentiated (several hrs.) —f balsam, via xylene RECOMMENDED FOR: Staining actinoniycetes in sections of plant tissues. 502 METHODS AND FORMULAS DS 23.31-DS 23.32 23.31 Johansen 1940 see DS 13.5 Johansen 1940 23.31 Langeron 1942 Langeron 1942, 1285 REAGENTS REQUIRED: A. Water 100, sodium carbonate 20, benzoazurine 0.2; B. glycerol 90, water 10, cupric sulfate 0.2 method: [pieces or sections, chlorine-bleached if necessary] —» A, 1-2 hrs. — » B, for preservation RECOMMENDED FOR: Staining fungus in plant tissues. 23.31 Lepik 1928 16233, 18:869 REAGENTS REQUIRED: A. 95% alc. 40, phenol 20, lactic acid 40, glycerol 20; B. A (above) 100, anilin blue 0.02, safranin 0.1; C. DS 12.14 safranin method: [sections] — >■ 95% alc. — > A, 1-15 mins. — » B, 2 hrs. — + A, till differentiated -^ abs. alc. till dehydrated — + C, till clear -^ clove oil, till no more color comes away — * balsam, via xylene recommended for: staining of Peronosporales in sections. 23.31 Mangin 1895 5133,8:1 reagents required: A. 10% potassium hydroxide in 90% alc; B. acetic acid 100, orceillin BB q.s.; anilin blue q.s. preparation of B: add enough each of sat. sols, of the two dyes to make a violet mixture. method: [pieces, if necessary chlorine-bleached] -* .4, 1-2 hrs. ^^ 5, till sufficiently stained -^ glycerol recommended for: demonstration of Peronosporales in pieces. 23.31 Margolena 1932 20540b, 7 :25 reagents required: A. DS 11.44 Stoughton 1930; B. 0.5% light green in 95% alc; C. sat. alc. sol. {circ. 0.25%) orange G 30, sat. clove oil sol. erythrosin 60 method: [sections] -^ water -^ ^1, 1 hr. -^ water, rinse — > B, till sections appear green — > water, till no more color comes away -^ abs. alc, till dehydrated — >, C, 1-2 mins. — > balsam, via xylene recommended for: staining fungus hyphae in plant tissues. note: a detailed description of the use of this technique is given under DS 23.30 above. 23.31 Moore 1933 19938,77:23 reagents required: A. 2% ferric alum; B. water 20, glycerol 40, lactic acid 20, phenol 20, phenosafranin 0.5; C. 0.5% ferric alum in 0.5% hydrochloric acid; D. 1% am- monium hydroxide method: [paraffin sections] — » water —* A, 2 hrs. — > water, quick rinse — > B, 5-15 mins. -^ water, rinse — > C, till differentiated — » D, 15-30 sees. -^ balsam, via usual reagents recommended for: staining hyphae in sections. 23.31 Ravn test. 1942 Langeron cit. Strasburger Langeron 1942, 1285 REAGENTS REQUIRED: A. water 100, acetic acid 3, orceillin BB to sat.; B. water 100, acetic acid 3, anilin blue to sat. method: [sections of chrome fixed, or mordanted, material] -^ A, 12-24 hrs. — > water, wash — * B, 1-2 hrs. -^95% alc, till differentiated — > balsam, via usual reagents recommended for: staining fungus in plant tissues. 23.31 Vaughn 1914 1048, 1:241 REAGENTS REQUIRED: A. Water 75, 95% alc. 25, malachite green 0.25, acid fuchsin 0.05, Martins yellow 0.005; B. 0.1% hydrochloric acid in 95% alc. method: [sections] -^ water -^ A, K to 1 hr. -^ water, rinse — » B, till differentiated — > balsam, via carbol-xylene RECOMMENDED FOR: demonstration of hyphae in sections. 23.32 PLANTS PARASITIC IN ANIMALS 23.32 Bachman 1920 1752, 1 :50 formula: water 70, 95% alc 20, acetic acid 3, orange G 0.5, sat. sol. {circ. 1%) crystal violet 7.5 DS 23.32 DYE STAINS OF SPECIAL APPLICATION 503 preparation: Dissolve orange in solvents. Add violet, method: [fresh scrapings]-^ water, on slide -^ dry — > stain, on slide, 2 mins. — > 95% ale, 15 sees. — » water, 15 sees. — > dry RECOMMENDED FOR: staining fungi in tissue scrapings. 23.32 Bardelli and Cille 1928 test. 1942 Langeron Langeron 1942, 1251 REAGENTS REQUIRED: A. acetic acid; B. DS 11.43 Ziehl 1882; C. ADS 12.2 Lugol; D. aniline method: [thin sections] —> A, on slide, warmed gently, 30 sees.—* 96% ale, thorough wash — > dry — > B, 3 mins. — > drain or blot — > C, 5 mins. — > D, till differentiated — » balsam, via carbol-xylene RECOMMENDED FOR: demonstration of Zymonema in sections. 23.32 Berberian 1937 1750, 36:1171 REAGENTS REQUIRED: A. 50% acctic acid; B. ether; C. acetone; D. DS 11.44 Martinotti 1910 75, glycerol 20, 95% ale. 5; E. 0.5% acetic acid method: [scrapings] -+ A, on slide — > dry -^ B, on slide, 20-30 sees. — »• C, 2 changes, 1 min. each -^ abs. ale. — > [series ales, to 70%] — > D, 10-15 mins. -^ E, till differentiated -^ balsam, via acetone and xylene recommended for: fungus hyphae in skin scrapings. 23.32 Besson 1904 Besson 1904, 70 REAGExNTs REQUIRED: A. DS 11.24 Orth (1924); B. DS 23.211 Ehrlich 1882; C. 0.7% sodium chloride; D. ADS 12.2 Gram 1884; E. anUine method: [sections] ^ a, overnight -^ rinse -^ B, 20 mins. -^ C, rinse -^ D, some minutes -^ E, on slide, till differentiated — > balsam, via xylene RECOMMENDED FOR: demonstration of Aspergillus fumigatus in sections of lung. 23.32 Bernhardt 1943 1829,48:533 REAGENTS REQUIRED: A. 10% potassium hydroxide; B. M 13.1 Bernhardt 1943 100, cotton blue 0.5; C. M 13.1 Bernhardt 1943 method: [skin scrapings or nail fragments] — > A, under cover, till clear and soft -^ press cover with blotter — > B, drawn under cover, warmed — > press cover with blotter -^ C, drawn under cover — »• seal 23.32 Bigot 1924 5310, 17:547 REAGENTS REQUIRED: A. DS 12.15 Crystal violet; B. ADS 12.2 Gram 1884; C 1% erythrosin method: [smears, fixed in F 5000.1010 Duboscq and Brazil 1905 and depicrated in lithium carbonate sol.] — > A, 6-12 hrs. — » quick rinse — > B, 1 min. — > water, thorough wash — > C, 30 sees. — » water, wash -* dry RECOMMENDED FOR: demonstration of Zymonema in tissue fragments or exudates. 23.32 Boeck test. 1896 Kahlden and Laurent Kahlden and Laurent 1896, 146 REAGENTS REQUIRED: A. DS 11.44 SahU 1885; B. 0.1% resorcin; C. 3% dilution of 30% hydrogen peroxide method: [scales degreased in ale. and ether] -^ water — > A, 1-2 mins. — > B, J^ to 1 min. —>■ C, till differentiated — > wash — > balsam, via usual reagents recommended for: fungi in skin scrapings. 23.32 Chalmers and Marshall 1914 11587, 17:256 reagents required: A. 40% potassium hydroxide; B. DS 23.211 Ehrlich 1882; C. ADS 12.2 Gram 1884; D. anihne; E. 2% eosin Y in 95% ale. method: [scrapings]—* A, some hrs., 40°C. — > 15% ale. thorough wash-^ strand on slide -^ dry — > B, on slide, 20 mins. — > drain — > C, 3 mins. — > D, till no more color comes away -^ E, 1 min. — ^ D, till no more color comes away — > balsam, via xylene recommended for: staining fungi in tissue scrapings. note: a detailed description of the use of this technique is given under DS 23.30 above. 23.32 Curtis test. 1904 Besson Besson 1904, 699 reagents required: A. DS 11.28 von Orth 1892; B. water 100, potassium hydroxide 0.01, methyl violet 6B 1.5; C. 1% pyrogallic acid 504 METHODS AND FORMULAS DS 23.32 method: [sections] —> a, 10-15 mins. — > wash -* 5, 10 mins. -^ wash -* C, 1 min. wash — ^ balsam, via usual reagents RECOMAiENDED FOR: Saccharomyces in sections. 23.32 Guegen 1905 5293, 21 :42 REAGENTS REQUIRED: A. P 12.2 Amann 1896; B. P 12.2 Amann 1896 100, Sudan III to sat., aniline blue 0.5 PREPARATION OF B: Boil Sudan III in solvent to saturation. Cool. Filter. Dissolve blue in filtrate. method: [scrapings] — > A, till clear —> drain -^ B, added on slide -^ cover -^ seal RECOMMENDED FOR: staining fungi in tissue scrapings. 23.32 Guegen 1906 5293, 22 :224 formula: lactic acid 100, Sudan III 0.1, auilin blue 0.1, ADS 12.2 Lugol 0.1 preparation: Dissolve Sudan III in lactic acid with boiling. Cool. Add other ingredients. method: As Guegen 1905 above. 23.32 Kligman, Mescon, and DeLameter 1951 Tech. Bull, 21 :86 reagents required: A. 1% periodic acid; B. DS 11.43 Feulgen and Rosenbeck 1924; C. 5% thionyl chloride; D. 1% light green method: [sections] — > water -^ A, 5 mins. -^ thorough wash — > B, 10-15 mins. — > C, 5 mins. — * thorough wash —^D,l min. — > balsam, via usual reagents note: This is a synthesis of the methods of Hotchkiss 1948 {Arch. Biochem., 16 :131) and McManns (20540b, 23:99). 23.32 Langrand 1913 9775, 7:128 reagents required: A. DS 11.43 Ziehl 1882; B. 2% aniline hydrochloride; C. DS 12.15 crystal violet; D. ADS 12.2 Gram 1884; E. 0.2% eosin Y method: [sections]—* A, on slide, warmed to steaming, 5 mins.—* water, wash—* B, several dips — * abs. ale, till no more color comes away -^ water, wash — * C, on slide, 3 mins. -^ water, rinse -^ D, on slide, 1 min. —> water, wash — > E, few sees. — * balsam, via usual reagents result: Tubercle bacilli, red; actinomyces, violet. recommended for: demonstration of actinomyces and tubercle bacilli in sections. 23.32 Lemiere and Becue te.st. 1904 Besson Besson 1904, 677 reagents required: A. 30% potassium carbonate; B. 5% eosin; C. 50% sodium acetate method: [dry smear] — * ether, 2-3 mins. -^ dry -^ A, several mins. -^ B, 15 mins. — * C, 5 mins. -^ seal in C recommended for: actinomyces in smears of pus. 23.32 Lignieres 1903 1886, 7 :444 REAGENTS REQUIRED: A. DS 12.15 Crystal violet; B. ADS 12.2 Lugol (1905); C. abs. ale. 80, acetone 20, acetic acid 2, sat. sol. {circ. 12.5%) acid fuchsin 0.3; -D. 1% acetic acid method: [sections, nuclei stained by any DS 11.11 technique] -^ water -^ A, 3 mins. -^ rinse -^ B, 1 min. -^ C, till section turns bright red — * water, till differentiation com- plete -^ D, few dips -^ salicylic balsam, via carbol-xylene RECOMMENDED FOR: demonstration of actinomyces in sections. 23.32 Mahdissan 1935 1798, 85:61 REAGENTS REQUIRED: A. 5% potassium hydroxide; B. 5% acetic acid; C. 1% acid fuchsin; D. 0.01% picric acid in xylene method: [larvae fixed in F 0000.1010 Mahdissan 1935]-^ water -^ A, till skin trans- parent —* B, 5 mins. —> C, 30 mins. — * wash, till no more color comes away — * [graded ales.] -^ D, till clear — * balsam recommended for: demonstration of symbiotic microflora in larvae of scale insects. 23.32 Mallory 1895a lest. 1938 ips. Mallory 1938, 279 reagents required: A. 2.5% phloxine; B. DS 23.211 Ehrlich 1882; C. ADS 12.2 Gram 1884; D. aniline method: [paraffin sections of alcohol- or formaldehyde-fixed material, nuclei stained by any DS 11.122 method] -* water -* A, 15 mins., 50°C.^ water, wash^ B, 5-15 mins. DS 23.32 DYE STAINS OF SPECIAL APPLICATION 505 -^ water, wash — > C, 1 min. — > water, wash -^ blot — > D, till no more color comes away — > balsam, via xylene RECOMMENDED FOR: staining actinomycetes in paraffin sections. 23.32 Mallory 1895b lest. 1938 ips. Mallory 1938, 279 REAGENTS REQUIRED: A. DS 23.211 Ehrlich 1882; B. aniline 100, magenta to sat.; C. aniline method: [celloidin sections of alcohol- or formaldehyde-fixed material attached to slide after nuclear staining by any DS 11.122 method] — > water — » A, 3-10 mins. — > water, wash -^ blot — > B, 1-3 mins. — > C, till differentiated — > balsam, via xylene recommended for: staining actinomycetes in celloidin sections. 23.32 Morel and Dulaus tesl. 1904 Besson Besson 1904, 077 REAGENTS REQUIRED: A. Any DS 11.123 formula; B. 1% Victoria blue in 10% ale; C. ADS 12.2 Gram 1884; D. 1% methyl violet in 10% ale; E. oil of cinnamon 50, abs. ale. 50 method: [sections] ^ ^, till nuclei stained —♦ wash —» B, 3 mins. —> wash —> C, few sees. — > 95% ale, wash ^ D, several minutes —> abs. ale. rinse —> E, till differenti- ated —>■ balsam, via usual reagents recommended for: staining actinomyces in sections. 23.32 Morris test. 1943 Cowdry cit. Mallory and Wright Cowdry 1943, 82 reagents required: A. 95% ale. 50, ether 50; B. 5% gentian violet in 70% ale; C. ADS 12.2 Gram 1882; 1). aniline 100, nitric acid 1 method: [fresh scrapings]—* A, 5-10 mins. — > dry -^ B, on slide, 5-30 mins. -^ C, 1 min. -^ water, rinse —> D, till differentiated —* balsam, via xylene recommended for: staining fimgi in tissue scrapings. 23.32 Priestly 1917 13034, 2:471 reagents required: A. P 12.2 Priestly 1917a orb.; B. chloroform; C. formic acid; D. DS 11.44 Sahli 1885 method: [fresh scrapings] —> A, till clear ^ water, wash -^ B, 5 mins. —> strand on slide -^ dry -^ C, 2-3 mins., 100°C. -^ water, wash -^ D, 10-15 mins. -^ water, rinse -^95% ale, till differentiated -^ balsam, via usual reagents RECOMMENDED FOR: demonstration of hyphae in skin scrapings. 23.32 Schleifif 1940 14674,87:785 REAGENTS REQUIRED: .4. F 0000.0010 Camoy 1887; B. 0.5%, azur I method: [skin scrapings on slide] -^ A, 3-10 mins. — > drain -^ dry — > B, on slide, 2-3 mins. — > wash -^ dry recommended for: demonstration of fungal hyphae in skin. 23.32 Schubert 1937 7176,105:1025 reagents required: A. 2% potassium hydroxide; B. water 50, phenol 25, lactic acid 25, anilin blue 1.25 method: [fresh scrapings] -^ A, }^ to 1 hr. — > water, thorough wash (at least 3 hrs.) —> B, on slide — » examine recommended for: staining fungi in tissue scrapings. 23.32 Swartz and Conant 1936 1752, 33:291 reagents required: A. 10% potassium hydroxide; B. P 12.2 Amann 1890 100, aniliu blue 0.5; C. P 12.2 Amann 1896 method: [skin scraping]—* A, till clear -^ drain -^ wash, on slide -^ B, 5-10 mins. ^ C, till surplus B removed — > M 1 1 movuitant recommended for: demonstration of hyphae in skin scrapings. 23.32 Unna 1929 7176,88:314 formula: water 80, glycerol 10, 95% ale 9, pyronin 0.9, methyl green 0.1, phenol 0.5. method: [10 fx sections of alcohol-fixed material]-^ water -^ stain 5-10 sees. -^ water, rinse —>■ alis. ale, till doliydratcd -^ balsam, via xylene RECOMMENDED FOR: fungi in skin sections. 506 METHODS AND FORMULAS DS 23.33 23.33 ANIMALS PARASITIC IN ANIMALS 23.33 Alii 1944 13685, 96:317 REAGENTS REQUIRED: A. ADS 12.1 All! 1944; B. 4% ferric alum; C. 1% hematein; D. 0.1% hydrochloric acid in 70% ale. method: [fresh smears] — > A, 10 mins. — > wash — » B, 10 sees. — > rinse — > C, 5-10 mins. -^ wash — > D, till differentiated -^ balsam, via usual reagents RECOMMENDED FOR: intestinal protozoans. note: Morrison 1946 substitutes his ADS 12.1 for A and omits B. 23.33 Anonymous 1946 4349, 26:13 REAGENTS REQUIRED: A. water 100, calc. chloride 0.01, phloxine 1; B. DS 11.44 Anony- mous 1946; C. acetone; D. 0.2% acetic acid; E. ADS 22.1 Anonymous 1946 method: [sections] —^ water ^ ^, 5 mins. -^ tap water, 1 min. —^B,l min. — » rinse — > C, 2 mins. — > D, till color clouds cease — > £", 1-2 mins. — > balsam, via acetone and xylene RECOMMENDED FOR: malarial parasites in sections. 23.33 Bidegaray 1926 899a, 4 :385 REAGENTS REQUIRED: ^. ADS 12.2Lugol (1905) ; B. DS 23.212 NicoUe 1895 (dye solution) method: [fresh fecal smear] — > equal parts of A and B, mixed with smear, 2-3 mins. — > examine recommended for: protozoans in fecal smears. 23.33 Bignami test. 1896 Kahlden and Laurent Kahlden and Laurent 1896, 126 formula: 95% ale. 100, magenta, aurantia a.a.q.s. to sat. method: [sections of F 3000.0010 Bignami (1896) fixed material]-^ 95% ale.-* stain overnight -^ abs. ale, till differentiated — > balsam, via xylene recommended for: Plasmodium in sections. 23.33 Borrel 1901 857, 15:57 reagents required: A. sat. aq. sol. magenta; B. DS 12.211 Cajal 1895 method: [sections of F 1260.0030 Besson 1904 fixed material] -^ water ^ ^, 1 hr. -^ B, till differentiated -* abs. ale, minimum possible time — > balsam, via clove oil recommended for: coecidia in tissues. 23.33 Corbin 1946 Tech. Bull, 7:92 reagents required: A. water 80; B. 20; B. water 29, glycerol 19 DS 13.13 Giemsa 1902 (dry powder) 0.29, DS 11.44 Corbin 1946 3.5, 0.5% eosin Y in 25% ale. 5.7, metha- nol 43 method: [air-dried, thick blood smears] -^ A, 2 mins. -^ B, 15 sees. — > wash — * dry recommended for: malarial parasites. 23.33 Chorine 1932 5310,25:561 reagents required: A. A.% formaldehyde; pH 7.5; B. water 100, potassium iodide 2, ADS 12.2 Lugol (1905) 3; C. 5% sodium thiosulfate; D. water 95, DS 13.13 Giemsa 1902 5 method: [thick smears, even when old] ^ A, till bleached—* water, wash ^ B, 3-5 mins. -* water, rinse — > C, till colorless -^ water, wash -^ D, 10-30 mins. — >• water wash — * dry RECOMMENDED FOR: Staining Plasmodium in smears. 23.33 Crough and Becker 1931 19938, 73:212 reagents required: A. acetic acid; B. 0.01% Janus green; C. sat. aq. sol. eosin method: [oocysts, separated by saline flotation, on slide under coverslip] -^ A, drawn under cover, 10 mins. 30-40°C. -^ B, drawn under cover, 10 mins. — » C, drawn under cover, 5 mins. — > wash — » blot — » seal coverslip recommended for: oocysts of coecidia. 23.33 Dammin 1937 11284, 23:192 reagents iiEQi ired: A. water 70, acetic acid 20, 40% formaldehyde 10, picric acid 1; B. 10% sodium hydroxide; C. 5% hydrochloric acid DS 23.33 DYE STAINS OF SPECIAL APPLICATION 507 method: [short lengths compressed between bound slides]-* A, overnight-* remove slides -> running water, 2-3 mins. -^ B, till partial clearing shows reproductive sys- tem orange on yellow -» water, quick rinse -^ C, 1-2 hrs. -^ water, thorough wash -» balsam, via usual reagents RECOMMENDED FOR: diagnostic demonstration of cestode reproductive system. 23.33 Dobell 1919 Dobell 1919, 7 REAGENTS REQUIRED: A. watcr 85, 95% ale. 15, acid fuchsin 0.5; B. \% acetic acid; C. DS 12.211 Cajal 1895; D. 0.2% acetic acid method: [sections] — > water—* A, 10 mins. -» water, thorough wash -> B, on slide, few sees. -^ drain -* C, on slide, 10 mins. -* water, rinse -* D, till differentiated -* abs. ale, till differentiated — * balsam, via xylene recommended for: differential staining of E. histolytica in sections. 23.33 Dobell 1919 see DS 13.7 Dobell 1919 23.33 DobeU 1942a 16035,34:101 reagents required: A. 2% phosphotungstic acid; B. 0.2% hematoxyhn method: [fixed smears] -^ A, 10 mins, — » wash -* B, 10 mins. -^ wash -» balsam, via usual reagents 23.33 Dobelll942b 16035,34:101 reagents required: A. 2% ammonium molybdate; B. 0.2% hematoxylin method: [fixed smears] — * A, 10 mins. — * thorough wash -> B, 10 mins. -^ wash -^ bal- sam, via usual reagents recommended for: intestinal protozoans, particularly Trichomonas. 23.33 Field 1940 21671,34:195 reagents required: A. water 100, sodium phosphate (anhydr.) 1, potassium dihydro- gen phosphate (anhydr.) 1.25, brilliant cresyl blue 1 method: [air-dried smears] -* A, 1 sec. -^ wash, 5 sees. -^ drain — * dry recommended for: Plasmodium in thick smears. 23.33 Giemsa 1935 23684, 134:483 REAGENTS REQUIRED: A. DS 13.13 Giemsa 1902; B. water 100, sodium phosphate, mono- basic 1, eosin Y 0.01 method: [dry smear]—* A, 30 mins.-* water, quick rinse—* B, till differentiated—* water, rinse —^ dry recommended for: staining Plasmodium in smears. 23.33 Ginrich 1941 20540b, 16:159 REAGENTS required: A. DS 13.13 Giemsa 1902 1, water 99; B. DS 13.11 May and Griinwald 1902 method: [thick blood films] —>■ A, 15 mins. -^ wash -^ dry — * B, 30 sees. —* wash — * dry recommended for: permanent slides of Plasmodium. 23.33 Goldman 1952 Cowdry 1952 stock solutions: I. 1 % hematoxylin in 95% ale, II. water 100, sulfuric acid 0.12, acetic acid 1, ferric alum 4 working solution: stock I 50, stock II 50 method: [fixed smears] — > stain, H-3 mins. — > wash -^ balsam, via usual reagents RECOMMENDED FOR: intestinal protozoans. 23.33 Hewitt 1939 11428, 24 (suppl.) :22 REAGENTS REQUIRED: A. 2.5% potassium dichromate; B. water 97, DS 13.13 Giemsa 1902 3 method: [5 fjt sections of F 3700.1000 Helly 1903 fixed material] -^ water -^ A, K to 1 hr. -^ water, quick rinse-* B, 24 hrs.-* water, rinse-* 70% ale, till differentiated —>■ balsam, via graded acetone-xylene mixtures recommended for: staining Plasmodium in sections. 508 METHODS AND FORMULAS DS 23.33 23.33 Hewitt 1939 600a, 29:115 REAGENTS hequired: A. 2.5% potassium dichromato; B. water 100, DS 13.13 Giemsa 2.5, sodium bicarbonate 0.0005 method: [5 M sections of F 3700.0010 Zenker 1894 material] -^ water — > A, 30-60 niins. — > rinse — * B, 24 hrs. — > 70% ale, till differentiated — > balsam, via acetone and xylene recommended for: avian malarial parasites in tissues. 23.33 Hobbs and Thompson 1945 Tech. Bull., 6:29 REAGENTS REQUIRED: A. watcr 100, potassium dihydrogen phosphate (anhyd.) 1.25, sodium phosphate, dibasic (anhyd.) 1, azur B 0.1, methylene blue 0.16; B. 1% eosin in same buffer as A. method: [air-dried smear] -^ ^, 1 sec. — > rinse, 5 sees. -^ B,l sec. — ♦ rinse, 5 sees. —> dry RECOMMENDED FOR: Plasmodium. 23.33 Hollande 1920 1915, 59:75 REAGENTS REQUIRED: .1. 1 % Bosin Y; B.\% phosphomolybdic acid; C. 0.5% light green; D. amyl ale. method: [smears fixed in F 5000.1040 Hollande 1911 or F 4500.1010 Hollande 1918] -> [nuclei stained by any DS 11.111 method and fully differentiated] — ♦ water — > J^^ to 1 min. — > 95% ale, till differentiated -^ D, till dehydrated — > balsam, via xylene recommended for: staining flagellate protozoans in fecal smears. 23.33 Jager test. 1928 Schmorl Schmorl 1928, 432 REAGENTS REQUIRED: A. Any DS 11.122 formula; B. 0.1% eosin Y method: [fecal smears fixed in F 3000.0010 Jager 1928]-^ A, 10 mins. — > wash -^ B, 1-2 mins. — > balsam, via usual reagents RECOMMENDED FOR: parasitic amebas. 23.33 Knowles 1931 9943, 64:271 REAGENTS REQUIRED: A. water 100, acetic acid 1, tartaric acid 0.8; B. methanol; C. buffer pH 7.2; D. DS 13.13 Giemsa 1902 method: [thick smears] —> A, till bleached-^ water, wash ^^ B, 2-3 mins. — > C, wash -^ D, 5-10 mins. — > water, wash -^ dry 23.33 Kofoid test. 1920 Stitt Stitt 1920, 58 stock solutions: A. sat. sol. eosin in normal saline; B. sat. sol. iodine in 5% potassium iodide in normal saline reagents required: A. stock A 50, stock B 50; ^. normal saline method: Mix 1 drop feces with A, another with B. Place on slide and cover both drops with 1 coverslip. recommended for: demonstration of protozoans in fecal smears. 23.33 Langeron 1942 Langeron 1942, 763 REAGENTS REQUIRED: .1. DS 11.22 Grenacher 1879; B. water 100, picric acid 1, anilin blue 0.6 method: [tissues, well washed from fixative]-^ A, 24-48 hrs. -^ 70% ale. thorough wash — > [5 M paraffin sections] -^ water — > B, 12 hrs. -^ water, wash —>■ salicylic bal- sam, via usual reagents recommended for: demonstration of Myxosporid parasites in sections of arthropods. 23.33 Mallory 1897 test. 1938 ips. Mallory 1938, 297 REAGENTS required: A. 0.25% thionin; 5. 2% oxalic acid method: [sections] -^ water — > A, ^ to 1 min. -> B, till differentiated }i to 1 min. -♦ wash — » balsam, via usual reagents RECOMMENDED FOR: differential staining of Entamoeba in sections. 23.33 Markey, Culbertson, and Giordano 1943 Tech. Bull., ^:2 REAGENTS REQUIRED: A. 5% ferric alum; B. water 100, acetic acid 2, hematoxylin 0.1 method: [smears fixed in F 3000.0000 Schaudinn 1893] -* water -> A, 2-3 mins. 56°C. — > rinse — > B, 1-2 mins., 56°C. — > running water, 15-30 mins. -> balsam, via usual reagents RECOMMENDED FOR: protozoans in fecal smears. DS 23.33 DYE STAINS OF SPECIAL APPLICATION 509 23.33 Medalia, Kahaner, and Singer 1944 Tech. Bull., 5 :68 REAGENTS REQUIRED: A. water 100, sodium phosphate, dibasic 0.56, dihydrogen potas- sium phosphate 1.70; B. water 100, sodium phosphate, dibasic 1.70, dihydrogen po- tassium phosphate 0.5G, methylene blue 0.16, azur II 0.10 method: [methanol-fixed smears] — > water — » A, 4 sees. — > rinse — > B, 6 sees. — » rinse — » dry RECOMMENDED FORI Plasmodium in smears. 23.33 Meriwether 1935 4349, 14:64 RE.\GENTS required: .4.. DS 11.26 Meriwether 1935 24, ammonia 36, methanol 36; A. water 45, abs. ale. 36, methanol 18 method: [sections of formaldehyde-fixed material with nuclei hematoxylin stained]—* water —> A, 5 mins. —> B, till differentiated — * balsam, via usual reagents recommended for: differential staining of parasitic amebas in tissue sections. 23.33 Moorthy 1937 11428,23:100 REAGENTS REQUIRED: A. Water 100, sodium chloride 1, mercuric chloride 0.5; B. water 100, methylene blue 0.06, DS 13.13 Giemsa 1902 (solution) 24 method: [larvae of Dracunculus, isolated from Cyclops killed in A]^> ^4, on slide, under coverslip — > B, drawn under coverslip —> C, replacing B, drawn under coverslip — > [seal coverslip] recommended for: larvae of Dracunculus. 23.33 Moschkovsky test. 1946 Roskin Roskin 1946, 287 reagents required: A. water 95, DS 11.44 Moschkovsky (1946) 5; B. 2% tannic acid method: [thick smears] — > A, 10-15 mins. —> rinse -^ B, 1 min. — * rinse -^ dry recommended for: blood parasites in thick smears. 23.33 Noble 1944 19938, 100:37 reagents required: A. water 67.5, 40% form.aldehyde 7.5, acetic acid 25, ferric alum 3; B. 0.5% hematoxylin method: [fresh, moist smear] — > A, on slide, warm to steaming -^> drain — » B, on slide, warm to steaming —* wash -^ blot — > balsam, via dioxane and toluene recommended for: protozoan parasites in fecal smears. 23.33 Reynolds 1936 20540b, 11:167 formula: water 70, DS 11.21 Guyer 1930 30, DS 11.122 Delafield 1885 10 recommended for: nematodes in sections and wholemounts. 23.33 Rukhadze and Blajin 1929 11587, 32:342 formula: lactic acid 30, water 100, carmine 0.3 preparation: Dissolve with boiling method: [living cestodes] — > stain, }^ to 1 hr. — > running water, 3-^ to 3 hrs. — > balsam, via usual reagents recommended for: staining wholemounts of cestodes. 23.33 Schmorl 1928 Schmorl 1928, 421 formula: sat. sol. methylene blue 60, 0.5% ethj'l eosin in 70% ale. 20, water 20, 20% potassium hydroxide 0.4 method: [smears] — > stain, 3-5 mins. — » wash — ► dry recommended for: Plasmodium. 23.33 Schuffner test. 1928 Schmorl Schmorl 1928, 421 reagents required: .4. water 94, glycerol 5, 40% formaldehyde 1; B. any DS 11.122 formula method: [fre.sh blood smear] — ► dry in dark, 6-30 mins. — ♦ A, 5-10 mins. — > wa.sh —* B, 1-10 mins. -^ wash — >• dry — ► balsam recommended for: Plasmodium. 510 METHODS AND FORMULAS DS 23.33 23.33 Shortt 1927 9940, 14:565 fokmula: xylene 75, phenol 25, eosin Y 1 preparation: Dissolve dye in phenol. Dilute with xylene. method: [fixed smears, either dehydrated in ale, or air dried] —> stain, 1-5 mins. -> xylene, thorough wash -^ balsam RECOMMENDED FOR: demonstration of parasitic Sarcodina, and their cysts, in smears. 23.33 Simons 1938 5310, 31:100 REAGENTS REQUIRED: A. Water 100, methylene blue 0.2, sodium chloride 0.6, sodium citrate 1, saponin 0.6, 6% formaldehyde 4; B. 50% glycerol PREPARATION OF A: Dissolvc the dye and sodium salts in water with gentle heat. Cool. Add saponin and shake till dissolved. Add formaldehyde. method: [mix on slide 1 part blood to 5 parts A] — * dry — > B, tiU differentiated -^ seal coverslip RECOMMENDED FOR: staining Plasmodium in fresh blood. 23.33 Sinton and MulUgan 1930 9940, 17 :329 REAGENTS REQUIRED: A. 0.04% sodium hydroxide in 90% ale; 5. 1% Bordeaux red; C. 4% ferric alum; D. DS 11.111 Shortt 1923 (sol. B); E. 0.3% iron alum; F. 0.1% eosin method: [thick smears fixed in F 0000.0010 Gilson, 1897]^ 50% ale. -> A, several hours -^ water, via graded ales. -^ B, 12-24 hrs. — ♦ rinse — > C, 18 hrs. -* rinse -^ D, overnight — > E, till differentiated — * F, 3 mins. — > wash -^ balsam, via usual reagents recommended for: Plasmodium in thick smears. 23.33 Sternberg 1905 23681, 16 :293 reagents required: A. DS 13.13 Giemsa 1902 3, water 97; B. 0.5% 5% acetic acid method: [thin paraffin sections] — > water ^ A, 24 hrs. -^ wash — > B, till section red — > wash — > dry -^ abs. ale, till blue — > dry — * balsam recommended for: Plasmodium in sections. 23.33 Tomlinson and Grocott 1944 591b, 14:36 reagents required: A. water 100, calcium chloride 0.01, phloxine 1; B. water 75, lithium carbonate 0.5, toluidine blue 1, glycerol 20, abs. ale. 5; C. 0.2% acetic acid; D. acetone 75, abs. ale. 25, rosin 3.75, orange G 0.01 method: [sections] — > water -^ A, b mins. -^ rinse -^ B, 45-60 sees. — > rinse -^ acetone, 2 mins. -^ C, till blue clouds cease — » D, 1-2 mins. — » balsam, via acetone and xylene recommended for: Plasmodium in sections. note: Pigmented tissues may be bleached in 5% ammonium sulfite before this stain. 23.33 Tompkins and Miller 1947 591b, 17:755 reagents required: A. 4% ferric alum; B. 0.5% hematoxylin; C 2% phosphotungstic acid. method: [smears fixed in F 3000.0000 Schaudinn 1893 after iodine treatment for re- moval of mercury] -^ water -^ A, 3-5 mins. -^ rinse— > B, 1 min. — * rinse—* C, 2 mins. — > blue in tap water or alkali — * wash — > dammar, via usual reagents recommended for: protozoans in fecal smears. 23.33 Tsuchiuya 1932 11284, 17:1163 reagents required: A. 4% ferric alum; B. DS 13.12 Wright 1910 method: [fecal smears fixed in F 3000.0000 Schaudinn 1893] — > A, 20 mins. — > wash, 3 mins. — > B, on slide, 1 min. — > B, diluted on slide, 5 mins. — > wash — * balsam, via usual reagents recommended for: intestinal protozoans. 23.33 VUlain and Comte 1933 1843, 22 :137 stock solutions: I. 0.08% eosin B; II. 0.08% azur II working solution: stock I 6.5, stock II 6.5, water 87 method: [alcohol-fixed smears] — > stain, }4 to 1 hr. — > wash — » dry note: All solutions must be adjected to pH 7.4 to 7.6. I recommended for: Plasmodium. DS 23.34-DS 23.5 DYE STAINS OF SPECIAL APPLICATION 511 23.34 ANIMALS PARASITIC IN PLANTS 23.34 Strong 1924 624, 4 :345 REAGENTS REQUIRED: A. methanol; B. DS 13.13 Giemsa 1902 method: [smears of latex, partially dried] — > A, 5 mins. — > water, wash -^ B, on slide, 10-15 mins. -^ water, wash — » dry RECOMMENDED FOR: demonstration of Phytomonas (the protozoan not the bacteria) in Euphorbia. 23.4 Other Zoological Techniques 23.5 Other Botanical Techniques The techniques given under this heading are so specific for the purpose and class of plant to which they are applied, that they could not be justifiably given under any other heading. These methods, moreover, are so varied that they cannot be subdivided into sections, hence the specific purpose for which each was intended is given under the individual formula. 23.5 Alcorn and Worley 1936 20540b, 11:119 REAGENTS REQUIRED: A. DS 11.43 Ziehl 1882; B. 2% hydrochloric acid in 70% ale. method: [dried perithecia scraped from leaf] — * A, 48 hrs. 50°C. -^ [place single perithe- cium on slide, open by tapping coverslip] — * A, under coverslip, 15 mins. 95°C. re- placing stain as it evaporates —> B, drawn under cover, till differentiated -* balsam, via usual reagents recommended for: Perithecia of Erysiphaceae. 23.5 Baumgartel 1902 1798, 41 :87 formula: water 99, acetic acid 1, methylene blue 0.5, acid fuchsin q.s. to give violet color method : [alcohol-fixed algae] — » stain, till stained — » water, till no more color comes away -^ balsam, via usual reagents recommended for: staining wholemounts of algae, particularly Cyanophyceae. 23.5 Baumgartel 1917 2626, 64:1138 reagents required: A. water 80, 95% ale. 20, picric acid 0.5, mercuric chloride 1, potassium alum 1, hematin 0.1 preparation: Dissolve all ingredients except the dye in water. Add dye dissolved in ale. recommended for: staining wholemounts of algae. 23.5 Bonnet 1910 6630, 38:103 recommended for: combined fixation and staining of algae. See DS 24 Mennier and Vaney 1910. 23.5 Chamberlain 1915a Chamberlain 1915, 198 reagents required: A. 1% eosin Y; B. 1% acetic acid method: [fresh material] -^ abs. ale, 2 mins. — > A, 2 mins. -^ B, quick rinse -^ water, thorough wash — > glycerol note: This method is also given by Johansen 1940, 245 without reference to source. recommended for: wholemounts of filamentous fungi. 23.5 Chamberlain 1915b Chamberlain 1915, 110 REAGENTS REQUIRED: A. 1% phloxine in 90% ale; B. 1% anilin blue in 90% ale; C. 0.1% hydrochloric acid in 90% ale. method: [algae in 95% ale] -* A, 24 hrs. -> 90% ale, 1 min. — > B, 3-30 mins. —> C, till differentiated — > Venice turpentine, via usual reagents recommended for: wholemounts of filamentous algae. NOTE: A detailed description of the use of this technique is given in Chapter 6. 512 METHODS AND FORMULAS DS 23.5 23.5 Gardiner 1898 test. 1931 Crafts 20540b, 6:127 REAGEN-TS REQUIRED: A. watcr 100, iodiue 0.5, potassium iodide 0.75; B. 10% sulfuric acid; C. water 95, sulfuric acid 5, iodine 1, potassium diodide 1.25; D. 5% sulfuric acid E. water 95, sulfuric acid 5, 0.5% gentian violet q.s. to give green color. method: [sections of living material] ^ ^, 5 mins. -^ B, 5 mins. -^ C, 5 mins. — » D, till iodine starts to fade —>■ E, till dark -^ P 1,3.1 Gardiner 1898 RECOMMENDED FOR: demonstration of protoplasmic connections between plant cells. 23.5 Gutstein 1924 23684, 93 :233 REAGENTS REQUIRED: A. 1% methylene blue; B. 5% tannic acid; C. 1% safranin method: [heat-dried smear] ^^, 2-3 mins. ^ water, wash -^ B, 2 mins. —> water, wash — > C, 2 mins. -^ water, wash -^ dry recommended for: demonstration of nuclei in yeasts. 23.5 Gutstein 1925 23684, 95:1 REAGENTS REQUIRED: A. DS 11.43 Ziehl 1882; B. 5% acetic acid; C. 5% tannic acid; D. 1% safranin method: [heat-dried smears]-^ A, 2 mins.—* B, till no more color comes away-* water, wash -^ C, 2 mins. -^ water, wash -^ D, 2-5 mins. -^ water, wash — » dry recommended for: demonstration of ascospores in yeast. 23.5 Johansen 1940 Johansen 1940, 270 REAGENTS REQUIRED: A. 1% Bismarck brown in 70% ale; B. DS 12.16 Johansen 1940; C. clove oil 50, abs. ale. 25, xylene 25 method: [sections] — > water -^ A, 20 mins. — > 95% ale, till no more color comes away -^ B, 5-8 sees. — * C, till differentiated -^ balsam, via xylene recommended for: staining sections of Thallophyta. 23.5 Kufferath 1929 test. 1942 Langeron Langeron 1942, 1249 reagents required: .4. DS 11.43 Ziehl 1882; B. 2% lactic acid in 95%, ale; C. 1% Nile blue sulfate method: [smear, dried at 100°C.] — > A, on slide, warmed nearly to boiling—* drain —> water, wash -^ B, & few sees. — * water, wash -h> C, 30 sees. -^ water, wash -^ dry RECOMMENDED FOR: staining ascospores in yeasts. 23.5 Langeron 1942 Langeron 1942, 1239 formula: water 100, acetic acid 3, anilin blue 0.5 method: [dried hyphae on slide] ^ stain, 5 mins. -^ water, till no more color comes away -^ balsam, via usual reagents RECOMMENDED FOR: Staining fungus mycelia. 23.5 Maneval 1929 20540b, 4:21 REAGENTS REQUIRED: A. 1% acid fuchsin; B. 5% tannic acid; C. 2% sulfuric acid method: [heat-dried smears] — » A, 1 min. -^ B, 20 sees. — > C, till differentiated — > bal- sam, via usual reagents recommended for: demonstration of nuclei in yeasts. 23.5 Rivalier and Seydel 1932 6630, 110:181 formula: a. P 12.2 Amann 1896 100, anilin blue 1 method: [collodion varnished coverslip preparations] —* stain, 10-50 mins. -^70% ale, rinse—* 90% ale, till differentiated -*• balsam, via graded acetone-xylene series RECOMMENDED FOR: staining fungus mycelium in coverslip cultures. 23.5 Semmens 1937 Microscope, 1 :5 REAGENTS REQUIRED: A . P 12.2 Scmmcns 1937 100, osmic acid 0.04 and either light green 0.5 or anilin blue 0.5 or phloxine 0.5; B. P 12.2 Semmens 1937 method: [fresh algae] -^ .4, 1 hr. — * A, another color, if required, 5-30 mins. — + B, till required — > B, on slide -^ ring cover with V 11.2 Semmens 1937 RECOMMENDED FOR: wholemouiits of algae. DS 23.5-DS 24 DYE STAINS OF SPECIAL APPLICATION 513 23.5 Taylor 1921 21400a, 40:94 REAGENTS REQUIRED: A. 0.05% methylene blue; B. 0.1% picric acid method: [fresh material] -^ A, '2 to 1 min. -^ water, rinse —> B —* examine RECOMMENDED FOR: sheath structure of desmids. 23.5 Yamanouchi test. 1915 Chamberlain Chamberlain 1915, 167 REAGENTS REQUIRED: A. 10% alc; B. 1% safranin in 95% ale; C. 1% crystal violet; D. 1% orange G method: [dried smears] — > A, overnight —* B, i days -^ water, 5 mins. -^ C, 2 days —> — > water, brief rinse — > D, 3 niins. — > 95% ale, brief rinse —> abs. alc. 1 min. —^ clove oil, till differentiated —^ balsam, via xylene RECOMMENDED FOR: wholcmounts of small Chlorophyceae. 24 MISCELLANEOUS DYE-STAINING TECHNIQUES The existence of any techniques in the present miscellaneous class must of necessity be a criticism of the classification adopted by the author. He is, therefore, pleased that few tech- niques have so far evaded the classification which he has developed as to be forced into the refuge of this miscellaneous group. 24 Francotte test. 1942 Langeron Langeron 1942, 1011 formula: water 55, 90% alc. 20, glycerol 20, 40% formaldehyde 5, Bismarck brown 0.05, malachite green 0.10 method: Fresh or preserved plankton is mixed with stain and examined. For perma- nence allow to evaporate under coverslip and replace with P 12.1 Francotte (1942). recommended for: staining mixed plankton collections. 24 Langeron 1942 Langeron 1942, 8GG REAGENTS REQUIRED: A. 5% potassium hydroxide; B. 1% acetic acid; C. any DS 11.24 formula; D. 1% anilin blue in 1% acetic acid method: [roughly dissected radulae] -^ .4, boiling, till clear —> water, rinse —> B, till neutralized — » C, >^ to 1 hr. -^ water, wash — * D, few mins. -^ balsam, via usual reagents recommended for: demonstration of radula of moUusca. 24 Meunier and Vaney 1910 6630, 68:727 reagents required: A. water 100, quinone 0.3 .method: [fresh plankton] -^ A, equal vol., till objects settled out — » 70% alc, 2 changes —> balsam, via usual reagents note: An identical technique was applied to algae by Bonnet 1910 (6630, 38). recommended for: staining mixed plankton collections. 22 Accessory Dye-staining Solutions Decimal Divisions Used in Chapter ADS 10 MORDANTS AND MISCELLANEOUS SOLUTIONS 1 1 Miscellaneous ILl Formulas 12 Mordants 12.1 Formulas for use before hematoxylin 12.2 Formulas for use before other dyes ADS 20 DIFFERENTIATING SOLUTIONS 21 For differentiating hematoxylin 21.1 Formulas 22 For other purposes 22.1 Formulas ADS 10 Mordants and Miscellaneous Solutions These formulas have been separated from the stains with which they are custom- arily associated because many of them are capable of a much wider employment than they usually receive. One might take, for example, the mordant of Casares-Gil (ADS 12.2) which was originally developed for staining the flagella of bacteria, but which can be applied admirably to revive the staining properties of sections of tissues which have been preserved so long in alcohol that they would otherwise be useless. Attention should also be paid to such formulas as those of Drew 1920 (ADS 12.1) which, by combining a chromic-acid and an osmic-acid mordant, enable one to apply stains, usu- ally appHed only to tissues fixed in such fluids, to tissues which have been fixed in picric acid, and which have therefore been rendered incapable of giving a good differentiation with many of the common triple staining methods. 11 MISCELLANEOUS These formulas are of very wide application, and are accordingly placed before the formulas of specific application. 11.1 Formulas 11.1 Anderson 1929 Anderson 1929, 127 formula: water 100, potassium dichromate .375, calcium hypochlorite 1.25 preparation: Dissolve the dichromate and hypochlorite in separate portions of the water. Filter the hypochlorite into the dichromate. use: After osmic stains to prevent removal of alcohol-soluble fats. 11.1 Chura 1925 23632, 42:59 formula: water 70, acetic acid 30, picric acid q.s. to sat. RECOMMENDED FOR: solution of Cytoplasmic inclusions in chrome-fixed material to render chromosomes more evident. note: See also ADS 12.1 Chura 1925. 514 ADS 11.1-ADS 12.1 ACCESSORY DYE-STAINING SOLUTIONS 515 note: The proportions of the above mixture are misquoted by Minouchi 1928 (see ADS 12.1). Gatenby and Painter 1937, 267 assign Minouchi's formula to Chura but give the wrong reference. 11.1 Hotchkiss 1948 Arch. Biochem., 16:131 REAGENTS REQUIRED: A. Water 30, abs. ale. 70, sodium acetate 0.8, periodic acid 0.8; B. water 40, abs. ale. 60, sodium thiosulfate, cryst 2, potassium iodide 2, hydrochloric acid 0.07 method: [sections]— > water-* A, 2-5 mins. -^ 70% ale, rinse—* B, 1 min. -* 70% ale, wash recommended for: originally intended for increasing stainability of polysaccharides. Now recommended for many other purposes. 11.1 Szecsi 1913 7276,39:1584 FORMUL.\: acetone 100, benzyol peroxide 10 recommended for: use as a tissue reviver. 12 MORDANTS 12.1 Formulas for Use before Hematoxylin Stains 12.1 AUi 1944 13685, 95:317 formula: water 25, 95% alcohol 65, tannic acid 10, acetic acid 10, phenol 5 12.1 Anderson 1922 11977, 5:65 formula: ads 12.1 Weigert 1896 90, 2% calcium hypochlorite 10 12.1 Anderson 1942 11431, 54:258 formula: water 100, potassium dichromate 3.75, chromium fluoride 1.875, phospho- molybdic acid 0.64, calcium hypochlorite 0.2 12.1 Bacsich 1937 11025,72:163 formula: water 100, chromic acid 1, ferric chloride 1 12.1 Benda 1893 22246, 7:161 formula: water 40, sulfuric acid 15, ferrous sulfate 80, nitric acid 15 preparation: Dissolve the sulfate in the sulfuric acid and water. Heat to 50°C. Add nitric acid. note: This is roughly equivalent in iron content to a 70% solution of ferric sulfate. This salt, however, is so hygroscopic that it is almost impossible to prepare a solution of known concentration from the solid. The additional acids both stabilize the solution and render it more effective as a mordant. For a solution sometimes known as Benda' s Mordant see Chapter 18, F 4600.1010 Benda (1911). 12.1 Chura 1925 23632, 42 :59 formula: water 100, chromium fluoride 0.5, chrome alum 0.5, potassium dichromate 2.5 recommended for: use after F 5670.1000 Chura 1925 to mordant cytoplasmic inclu- sions. note: See also ADS 11.1 Chura 1925. 12.1 Clara 1933 23639b, 17 :698 formula: water 100, potassium dichromate 3, chrome alum 1.5, chromic acid 1.25, ammonium molybdate 1.25 12.1 Cole 1916 19938,44:452 formula: 95% ale. 50, water 50, ferric chloride 5, acetic acid 10 12.1 Drew 1920 11360,40:295 formula: water 100, chromic acid 2, osmic acid 1 recommended for: tissue reviver before hematoxylin stains. 12.1 Eichhorn 1941 1887a, 31:391 formula: F 3700.0010 Zenker 1894 237.5, nitric acid 12.5 516 METHODS AND FORMULAS ADS 12.1 12.1 Faure 1924 6630, 90 :87 formula: water 100, ferric chloride 0.2, cupric acetate 0.1, hydrochloric acid 2 12.1 Kupperman and Noback 1945 see F 5000.1010 Bouin (note) or F 0000.1010 Lavdowsky 1894 (note) 12.1 Landau 1938 4285a, 15:181 formula: water 100, ferric chloride 0.06, potassium dichromate 5 12.1 Lang 1936 20540b, 11:149 formula: water 100, acetic acid 1, sulfuric acid 0.12, ferric alum 4 RECOMMENDED FOR: use in place of simple ferric alum solutions before hematoxylin stains note: The same solution, diluted with an equal volume of water, may be used for differentiation. 12.1 Merkel test. 1898 Behrens 23328, 3:76 formula: water 100, chrome alum 2.5, copper acetate 5, acetic acid 5 preparation: Add the copper acetate and acetic acid to a boiling solution of the alum. BoU 5 minutes. Cool. Filter. 12.1 Minouchil928 10881,1:231 formula: water 50, picric acid 0.6, acetic acid 50 recommended for: as Chura 1925. note: This formula is attributed by Gatenby and Painter 1937, 267 to Chura 1925 {q.v.). 12.1 Mitrophanow 1896 23632,13:470 formula: water 50, 90% alcohol 50, copper acetate 5 12.1 Morel and Bassal 1909 11024,45:632 formula: water 100, ferric chloride 2, copper acetate 0.04, hydrochloric acid 1 12.1 Morrison 1946 Turtox News, 24:66 formula: water 70, 95% alcohol 30, tannic acid 10, acetic acid 10, phenol 5 12.1 Mullen and McCarter 1941 608b, 17:289 reagents required: A. water 95, acetic acid 5, chromic chloride 5; B. 0.25% potassium permanganate; C. 5% oxalic acid 12.1 Smith and Rettie 1924 11431. 27:115 preparation: Dissolve 2.5 paraldehyde in 2.5 50% hydrochloric acid at 37°C. with continuous shaking. Neutralize with sodium hydroxide and adjust to pH 6 with acetic acid. 12.1 Weiss 1942 665,46:199 formula: water 57, 95% alcohol, 40% formaldehyde 10, acetic acid 10, tannic acid 25 recommended for: treatment of sections of formaldehyde-fixed tissues before staining techniques specifying chromic or dichromate fixation. 12.1 Schroder 1930 23430, 166:588 formula: water 100, potassium dichromate 3.75, chromium fluoride 1.25, sodium sulfate 0.5 note: This can be prepared by mixing equal parts of F 7000.1000 Miiller 1850 and ADS 12.1 Weigert 1896. 12.1 Vastarini-Cresi 1915 see ADS 12.1 Weigert 1896 (note) 12.1 Weigert 1896 Weigert's primary mordant — compl. script. 7936a, 6:14 formula: water 100, potassium dichromate 5, chromium fluoride 2.5 preparation: Dissolve with boiling. recommended for: pretreatment of sections of central nervous system before hema- toxylin stains. See DS 21.212 (Chapter 21). ADS 12.1-ADS 12.2 ACCESSORY DYE-STAINING SOLUTIONS 517 note: Vastarini-Cresi 1915 (10157, 31:38) substitutes ammonium dichromatc for potassium. 12.1 Weigert 1891 test., 1910 ips. Weiyert's secondary mordant — compl. script. Ehrlich, Krause, et al. 1910, 1:231 formula: water 100, chroniiuin fluoride 2.5, acetic acid 2, copper acetate 5 preparation: Add fluoride to boiling water. Boil 5 minutes. Cool to 80°-90°C. and add other ingredients. recommended for: use as a mordant before hemato.xylin staining, particularly of connective tissues in the central nervous system. See DS 21.212 and 21.22 (Chapter 21). note: This was named Gliaheizc by its inventor. Attempts to graft this word onto the English language appear, mercifully, to be failing. For an adaptation of this formula to fixative use see F 4600.1010 Benda (1911) (Chapter 18) and DS 21.22 Jakob 1913 (Chapter 21). 12.1 Williamson and Pearse 1923 see F 3670.0000 Williamson and Pearse 1923 12.1 Wolters 1891 23632, 7:471 formula: water 100, vanadium chloride 2, aluminum acetate 6.5 12.2 Formulas for Use before Other Stains 12.2 Anderson 1923 11431,26:431 formula: a. water 100, sodium sulfite 5, oxalic acid 2.5, potassium iodide 5, iodine 2.5, acetic acid b; B. water 95, ferric chloride 5 preparation of a: Dissolve the oxalic acid and sulfite in 95 water. Dissolve iodine and iodide in 5 water. Mix and add acetic acid. recommended for: mordant before Victoria blue neuroglia stains. note: The proportions of A and B vary according to the technique employed. Equal parts are generally satisfactory. 12.2 Atkins 1920 11056, 5:321 formula: water 100, sodium hydroxide 0.4, iodine 2 preparation: Dissolve the dry ingredients in 10 water. Dilute to 100. 12.2 Bailey 1929 16913,27:11 formula: water 100, ferric chloride 2.5, tannic acid 3.75 12.2 BelUng 1921 651, 54:573 formula: water 55, acetic acid 45, ferric oxide 5 recommended for: use as an ingredient of, or mordant before, aceto-carmine stains. 12.2 Bethe 1896 see DS 21.21 Bethe 1896 12.2 Burke 1922 see ADS 12.2 Lugol (1905) 12.2 Casares-Gil test. Anselmier 16157b, 5 :33 STOCK solution: 70% ale. 75, water 25, tannic acid 25, aluminum chloride 45, zinc chloride 25, rosaniline hydrochloride 3.57 working formula: stock 20, water 80 recommended for: reviving old tissues and as a general mordant. preparation : Dissolve the tannic acid and aluminum chloride in the ale. Dissolve the zinc chloride in the water and add drop by drop, with constant agitation, to first solution. Dissolve rosaniline hydrochloride in mixed solution. 12.2 Cretin 1937 4285a, 14:163 formula: water 100, aluminum chloride 0.7, ferric chloride 0.025, calcium chloride 5 PREPAR.vnoN: Dissolve each salt separately and mix in order given. 12.2 David 1934 23684, 132 :240 formula: water 90, potassium alum 5, mercuric chloride 1.25, tannic acid 4 12.2 van Ermengen see AMS {not ADS) 21.1 van Ermengen 518 METHODS AND FORMULAS ADS 12.2 12.2 Gordon 1939 11284,24:405 formula: water 100, copper sulfate 12, mercuric chloride 2.5, potassium dichromate 1, sodium sulfate 0.5 12.2 Gough and Fulton 1929 11431,32:765 formula: water 100, acetic acid 0.1, mercuric acetate 3 RECOMMENDED FOR: mordanting of fatty tissues prior to staining for mitochondria. 12.2 Gram 1884 see AF 12.1 Gram 1884 12.2 Gray 1926 11056, 12:273 formula: sat. sol. (circ. 6%) potassium alum 50, 20% tannic acid 20, sat. sol {circ. 4%) mercuric chloride 20, sat. ale. sol. (circ. 5%) magenta 4 recommended for: use as Casares-Gil. note: This solution is unstable. It may, however, be prepared from the first three, and the last ingredients, each as a separate solution. Liefson 1930 (11056, 20:203) sub- stitutes 95% ale. for the sat. sol. mercuric chloride. 12.2 Harris 1898 16185, N.V., 47 formula: water 250, potassium ferricyanide to sat. {circ. 85 Gms.), osmic acid 0.1 recommended for: mordanting before DS 11.44 staining. note: Harris recommends cooling to about O'C. before use. 12.2 Kilduffe 1923 11006, 81:2182 formula: water 100, iodine 0.3, potassium iodide 0.6, sodium bicarbonate 1. 12.2 Liefson 1930 see ADS 12.2 Gray 1926 (note) 12.2 Lugo! test. Lee 1905 see AF 12.1 Lugol (1905) 12.2 Marquez 1933 3360, 112 :1056 formula: water 100, chrome alum 10, potassium alum 10, potassium dichromate 10 recommended for: mordanting before staining for mitochondria. 12.2 Michailow 1910 23632,27:19 formula: water 250, sodium molybdate 20, 40% formaldehyde 1.5 recommended for: use before methylene blue nerve stains. See DS 21.211 (Chapter 21). 12.2 Mullen and McCarter 1941 608b, 17 :289 formula: water 95, acetic acid 5, chromic chloride 5. 12.2 Muir see DS 23.217 Muir 12.2 Oliver 1934 11250,55:266 formula: water 100, tannic acid 6.5, ferrous sulfate 11, acid fuchsin 0.7 12.2 Petragnani 1928 test. 1930 Ciferri 20540b, 5 :34 FORMULA OF WORKING SOLUTION: Water 75, methanol 25, acetic acid 0.06 preparation: Stock I. In 100 0.1% acetic acid dissolve with heat 3 potassium alum 2 and 0.5 lead acetate. Stock II. In 100 70% methanol dissolve 14 tannic acid and 4 ferric chloride WORKING solution: Mix 2 stock I with 1 stock II. Add 100 to 300 methanol. 12.2 Ponselle 1913 6630,74:1072 formula: abs. ale. 100, iodine 0.6 12.2 Rossi test. 1904 Besson Besson 1904, 170 formula: water 100, tannic acid 5, potassium carbonate 1 12.2 Salazar 1923 763, 26 :60 formula: water 70, acetic acid 30, tannin 1 12.2 Semmens 1939 Microscope, 3:6 formula: water 100, chromic acid 10, sodium uranate 0.5 use: Before crystal violet nuclear stains. note: For the preparation of the sodium uranate see F 3800.0010 Semmens 1939b. ADS 12.2-ADS 21.1 ACCESSORY DYE-STAINING SOLUTIONS 519 12.2 Shunk 1920 11056,5:181 formula: water 100, tannic acid 200, ferric chloride 1.25 12.2 Stockwell 1934 19938,80:121 formula: water 90, chromic acid 1, potassium dichromate 1, acetic acid 10 RECOMMENDED FOR: As a mordant prior to safranin staining, particularly for plant tissues containing much phlobaphene. 12.2 Trenkmann 1890 23684, 8 :385 formula: water 100, tannic acid 2, hydrochloric acid 0.2 12.2 Weiss 1929 ' 11284,14:1191 formula: 95% ale. 65, tannic acid 65, 40% formaldehyde 32.5, acetic acid 2.5 12.2 Winge 1930 23639b, 10 :699 formula: 96% ale. 80, water 20, iodine 1, potassium iodide 1 12.2 Yokata 1924 6630,90:1303 formula: water 100, tannic acid 5, potassium antimony tartrate 0.5 12.2 Zikes 1930 23684, 81 :161 formula: water 116, tannic acid 15, chrome alum 9, osmic acid 0.112, crystal violet 0.15 preparation: Dissolve the tannic acid in 60 water. Add alum, dissolved in 37.5 and osmic acid dissolved in 11.25. Filter. Add dye dissolved in 7.5. ADS 20 Differentiating Solutions The principal reason for separating these differentiating solutions from the stains with w^hich they are used is that the same formula may be used with thirty or forty stains, and will therefore have either to be given thirty or forty times in the section on dye staining, or, alternatively, be the subject of continual cross references within the section on dye staining. It is hoped that the technician will not hesitate to experiment by applying solutions, designed for differentiating one particular technique, to quite different techniques. As an example, one may quite the formula of Pal 1887 below, the use of which is generally confined to the differentiation of hematoxylin-stained sections of nervous tissue. The author recommends its employment, in another place in this book, for the surface bleaching of parasitic flatworms which have been stained in carmine. Many other examples of this kind of double use could be given and the whole science of microtomy would greatly benefit were persons to experiment more widely with existing solutions, rather than to invent new ones every time they are faced with new problems. Hematoxylin stains are usually differentiated with "acid alcohol" which is either 0.1% (British and American practice) or 1% (German practice) hydrochloric acid in 70% alcohol. The "glyceric ether," used in continental Europe for the differentiation of thiazin stains, is not an article of commerce in the United States. It is prepared by the distil- lation of glycerol over aluminum chloride at room temperature. The distillate, in micro- tomic practice, is diluted with 10% glycerol in 95% alcohol according to the fancy of the supplier or user. 21 FOR DIFFERENTIATING HEMATOXYLIN 21.1 Formulas .21.1 Eisath 1911 14370, 20:3 formula: water 55, 95% ale. 45, tannin 12, pyrogallic acid 6 21.1 Gordon 1936 11284,22:294 formula: water 90, 40% formaldehyde 10, ferric alum 0.25 520 METHODS AND FORMULAS ADS 21.1-ADS 22.1 21.1 Gouillart and Brouardel 1938 Bull. Soc. franc, microsc, 7:140 formula: water 98, acetic acid 2, potassium permanganate 0.01, oxalic acid 0.01 21.1 Kozowsky 1904 15058,23:1041 formulas: a. water 100, potassium permanganate 1; B. water 100, ferric chloride 1 RECOMMENDED FOR: differentiation, by successive immersion in A and B, of hematoxylin stains. 21.1 Kultschitzky 1889 766, 14:223 formula: water 100, potassium ferricyanide 0.1, lithium carbonate 1.0 21.1 Landau 1938 4285a, 15:181 formula: water 100, lithium carbonate 1.5, potassium ferrocyanide 2.5 21.1 Lang 1936 see AMS 12.1 Lang 1936 21.1 Lillie 1948 see ADS 21.1 Weigert 1885 (note) 21.1 Pal 1887 23632, 6:92 formulas: .4. water 100, potassium permanganate 0.25; B. water 100, potassium sulfite 0.5, oxalic acid 0.5 recommended for: use as a differentiator of stains, particularly D.S. 21.22 Weigert by alternate immersion in A and B, and as a general surface bleach. 21.1 Rossolino and Busch 1896 test. Schiefferdecker 1897 23632, 14:55 formula: water 100, oxalic acid 0.05, sodium sulfite 0.05 use: Differentiation of hematoxylin stains. 21.1 Smith and Rettie 11431, 27:115 formula: water 100, potassium ferricyanide 0.5, borax 1 21.1 Weigert 1885 8645, 3:238 formula: water 100, potassium ferricyanide 2.5, sodium borate 2 recommended for: use as a differentiator of hematoxylin stains, particularly those of Weigert. note: Lillie 1948, 161, differs only in containing half the quantity of sodium borate. 21.1 Weil 1928a 1879,20:392 formula: water 100, potassium ferricyanide 1.25, sodium borate 1 21.1 Weil 1928b 1879, 20:392 formulas: a. 0.25% potassium permanganate; B. water 100, sodium bisulfite 0.25, oxalic acid 0.25 22 FOR DIFFERENTIATING OTHER STAINS 22.1 Formulas 22.1 Beauverie and Hollande 1916 6630, 79 :605 formula: ethylene glycol 80, creosote 20 recommended for: differentiating methylene blue stains. 22.1 Dupres 1935 14425,46:77 formula: water 50, 40% formaldehyde 20, acetic acid 25 recommended for: differentiating after nuclear staining with magenta. 22.1 Eberspacher 1936 23684, 138 :92 formula: abs. ale. 90, water 10, urea 4 recommended for: differentiation of smears of acid-fast bacteria in place of acid solu- tions. 22.1 Gothard 1898 6030, 5:530 formula: abs. ale. 50, xylol. 15, beechwood creosote 15, oil of cajeput 20 recommended for: differentiation of methylene blue stains. ADS 22.1 ACCESSORY DYE-STAIXIXG SOLUTIONS 521 22.1 Kiyono 1890 23081,25:181 formula: water 100, potassium dichromate 5, chrome alum 2 UEcdMMENDioi) kor: sce DS 22.21 Altmami 1920. 22.1 Lenoir 1929 0630,101:1203 formula: abs. ale. 50, oil of cloves 50, hydrochloric acid 0.1 RECOMMENDED FOR: differentiation of safranin stains. 22.1 Lewis test. 1900 Pollack Pollack 1900, 86 formula: 2% chloral hydrate 25, oil of cloves 25, abs. ale. q.s. preparation: Shake first two ingredients till emulsified. Add ale. with constant shaking till emulsion clears. RECOMMENDED FOR: differentiation of dyes applied to neural structures. 22.1 Lowit 1891 1780, 38:524 formula: 95% ale. 100, picric acid 1, ADS 12.1 LaCour 1931 1 RECOMMENDED FOR: differentiation of safranin stains in plant tissues. note: Tuan 1930 (205-lOb, 5:103) recommends the addition of picric acid to all alcohols used in differentiation. 22.1 Masson test. 1942 Langeron Langeron 1942, 530 formula: sat. sol. picric acid in 95% ale. 65, 95% ale. 35 RECOMMENDED FOR: differentiation of iron hematoxylin and, particularly, safranin stains. 22.1 Tuan 1930 see ADS 22.1 Lowit 1891 (note) 22.1 Unna test. 1928 Schmorl Schmorl 1928, 154 foRxMULa: glyceric ether 10, water 90 note: See comment imder ADS 20 above. 22.1 Wolbach 1911 11006, 56:345 formula: 95% ale. 100, rosin 0.5 RECOMMENDED FOR: in differentiation of thiazin stains. note: There is no reason to refer to this as colophonium alcohol, even though this phrase occurs in the title of Wolbach's paper. The obsolete term colophoniuyn has now been replaced by rosin or colophony in both the United States and British Pharmacopoeias. Moreover, in spite of his title, Wolbach used "common brown resin" — and says so, in just those words. 22.1 Wolbach 1919 11343,41:75 formula: acetone 90, rosin 15 recommended for: differentiation of thiazin stains, usually after great dilution. 23 Formulas and Techniques for Metal Stains Decimal Divisions Used in Chapter MS 00 GENERAL OBSERVATIONS 01 Classification of methods and formulas MS 10 OSMIC ACID 11 Staining methods 11.0 Typical example Demonstration of Golgi network in the ovary of the earthworm by the Ludford 1925 method 11.1 Staining solutions 11.2 Neurological techniques 11.21 Methods for degenerative changes 11.22 Other neurological methods 11.3 Histological methods 11.4 Methods for cell inclusions MS 20 GOLD 21 Methods using gold alone 21.0 Typical examples Demonstration of termination of the fourth cranial nerve in the superior oblique muscle by the method of Ranvier 1889 21.1 Staining solutions 21.2 Techniques 22 Methods using gold in combination with mercury 22.0 Typical example Demonstration of protoplasmic neuroglia in the cerebral cortex by the method of Cajal 1916 22.1 Staining solutions 22.2 Neurological methods 22.21 Nerve cells and processes 22.22 Neuroglia 23 Methods using gold in other combinations 23.0 Typical example Demonstration of the nervous elements in spinal cord by the method of Gerlach 1872 23.1 Staining solutions 23.2 Neurological methods 23.21 Nerve cells and processes 23.3 Cytological methods 23.4 Other methods MS 30 SILVER 31 Methods using sUver nitrate 31.0 Typical examples Demonstration of the nervous elements of the retina by the method of Balbuena 1922 Demonstration of neuoblasts and axons of the developing spinal cord of the three-day chicken embryo by the method of Cajal 1910b 522 METAL STAINS 523 Demonstration of spirochetes in sections by the method of Dieterle 1927 31.1 Staining solutions 31.2 Neurological methods 31.21 Nerve cells and processes 31.22 Neuroglia 31.3 Cytological methods 31.31 Golgi apparatus 31.32 Other cytological methods 31.4 Histological methods 31.41 Reticular fibers 31.42 Other histological methods 31.5 Bacteriological method 31.51 Methods for spirochetes 31.52 Other bacteriological methods 31.6 Other silver nitrate methods 32 Methods using protein silver 32.0 Typical example Preparation of a transverse section of the sciatic nerve of a cat to demonstrate axis-cylinders by the method of Davenport, Windle, and Rhines 1947 32.1 Neurological methods 32.2 Other protein silver methods 33 Methods using silver diammine 33.0 Typical examples Demonstration of the nerve endings in the taste buds by the method of Bielschowsky 1904 Demonstration of oligodendria and microglia by the method of Pen- field 1938 Demonstration of microglia by the technique of del Rio-Hortega 1921b 33.1 Staining solutions 33.2 Neurological methods 33.21 Nerve cells and processes 33.22 Neuroglia 33.23 Other neurological methods 33.3 Cytological methods 33.4 Histological methods 33.41 Reticulum fibers 33.42 Selective staining of special cells 33.43 Demonstration of calcified and ossified material 33.44 Other histological methods 33.5 Bacteriological methods 33.51 Methods for spirochetes 33.52 Other bacteriological methods 33.6 Other silver diammine methods 34 Methods using silver in combination with other metals 34.0 Typical examples Demonstration of the Purkinje cells in the cerebellar cortex by the method of Golgi 1875 Demonstration of the structure of the superior cervical ganglion by the method of Kolossow 1896 Demonstrations of the neurons and dendrites of the brain of an embryo rabbit by the method of Windle 1926 34.1 Staining solutions 34.2 Neurological methods 34.21 Nerve cells and processes 34.22 Neuroglia 34.3 Histological methods 34.31 Reticulum fibers 34.32 Other histological methods 524 METHODS AND FORMULAS MS 00-MS 01 34.4 Cytological methods 34.5 Bacteriological methods 35 Other silver methods 35.1 Staining solutions 35.2 Neurological methods 35.3 Other methods MS 40 OTHER METALS 41.1 Staining solutions 41.2 Neurological methods 41.3 Histological methods MS 00 General Observations The practice of metal staining, except in neurological techniques, has in late years fallen into some disrepute, largely through the lack of success which has at- tended its use in the hands of the inexperi- enced, or of those not able to observe the very specific precautions which alone can lead to success. The most necessary of these precautions are the utilization of nothing but the purest reagents available, and the maintenance througliout of a con- dition of chemical cleanliness in the glass- ware emi:)loyed. It is difficult to justify the retention of the term metallic iviyregnations for this class of microscopic preparation. Many dyes impregnate materials, and there is certainly no justification for the retention of this term to include only those processes which are supposed to result in the deposi- tion, on the surface of the structure to be observed, of a film of metal or metalfic oxides and hydroxides. It is indeed doubt- ful how far a successful metal stain is ever the result of such a deposition. As will be pointed out later, metallic silver is less frequently found in a successful silver stain than is a silver proteinate, nor is it possible always to draw the fine in gold staining between the absorption of gold salts by the tissues and the deposition of very finely divided colloidal gold through- out their mass. It seems, therefore, prefer- able to retain the old term metal staining for these reactions. Many metal staining techniques should be more widely employed than is at pres- ent the case. This is particularly true of staining with osmium tetroxide or with any of the osmic-chromic fixatives found in Chapter 18. These materials will render the internal organs of a small inverte- brate so sharjjly defined that all after- staining is unnecessary. And no one who has ever examined a jM-operly dehydrated and cleared small crustacean, after fixa- tion in an osmic material, will again be in- clined to try to stain these forms with dyes which yield a more diffuse image. At the present time, however, these techniques are almost confined to the cy- tologist searching for Golgi apparatus, or such other lipid materials as may be dem- onstrated by the surface deposition of osmium hj^droxides, or to the neuroanat- omists, the majority of whose discoveries have been made with the aid of silver impregnations. It is to be hoped, however, that the classification of stains which fol- lows will encourage more biologists to en- deavor to utihze these excellent and rapid techniques more generally than is at pres- ent the case. MS 01 CLASSIFICATION OF METHODS AND FORMLTLAS Two bioad (H)nsidenitions necessitate the employment of a classification of metal stains different from that used for the dye stains in Chapters 20 and 21 . First, none of the metal stains aio of such general appli- cation tliat a division of "stains of general application" is possible. Second, only three metals — in contrast to several hun- dred dyes— are commonly used, so that "osmium," "gold," "silver," and "other metals" form convenient titles for pri- mary divisions. Many metal-staining methods require that the tissues shall have some special MS 10-MS 11 METAL F5TAINS 525 pretreatment in an accelerator or mordant; most metal-staining methods necessitate after-treatments such as development, ton- ing, and the hke. The solutions used in these pre- and after-treatments, however, are very much the same, whatever metal be used for staining. Much space lias therefore been saved by the removal of these accessory ductal staining formulas to Chapter 24, which should be consulted for the composition of an}' solution referenced "AAIS" in the formulas which follow. Each of the four primary divisions of metal stains are secondarily divided, when the variety justifies such division, accord- ing to the particular compound of the metal employed; all are further divided according to the purpose for which the technique is intended. Constant repeti- tion of the formulas for "staining solu- tions," many of which are used in dozens of techniques, has I)een avoided by plac- ing such formulas at the beginning of each of the subdivisions in which they are employed. It is difficult to decide, in the case of osmic acid, which of the staining formulas given should be regarded as fixatives — and more properly should be removed to Chap- ter 18 — and which should be regarded as staining solutions, which are more proj^- erh' retained in the present section. The author has adopted as a criterion the ques- tion of whether or not any after-staining solutions are to be em]:)loyed. When after- staining treatment is an essential part of the technique, the solution has been re- moved to the division on fixatives; when no after-staining is necessary, the formula has been retained in this section as a stain. MS 10 Osmic Acid Osmic acid is a greatly neglected stain, and there is no real justification for its present retention as a reagent which is used almost entirely for the demonstration of the Golgi apparatus. It is not denied that it does this well, but it is a pity that its one-time use as a general histological stain should have sunk into disrepute. Osmic acid is an excellent general-piu'pose stain, either for materials intended for subsequent sectioning with a view to dem- onstrating in class the relative distribu- tion of cells, nuclei, and the like, or for the preparation of wholemounts of small invertebrates. Probably one of the most unfortunate things that has happened to the science of microtomy has been the sub- stitution, for these clear gray and black specimens, of the standard monstrosities which are stained by convention bright red, and the fuzzj^ outlines of which can- not compare for simplicity with those pro- duced by osmic staining. The exact nature of the material laid down when tissues are exposed to osmic acid is not known; it appears fairly cer- tain that it is not metallic osmium. Part- ington and Huntingford (Gatenby and Cowdry, 1928, 29) state that it is a hy- drated form of one of the lower oxides. These oxides are laid down first on unsat- urated fatty acids and, later, on other constituents of the cell and cell wall. Ex- cess blackening may be removed either with hydrogen peroxide or by the stand- ard permanganate-oxalic acid techniques, but in both cases fresh osmium tetroxide is liberated in the tissues. This osmium tetroxide must be thoroughly washed out, or it will redeposit on the places from which it has been oxidized. Osmic acid stains are, in general, permanent. MS 11 STAINING METHODS MS 11.0 Typical Examples Demonstration of Golgi network in the ovary of the earthworm l)y the Ludford 1925 method In this, as in every other metal-staining technique, the first essential is to make certain that all glassware is chemically clean. In this instance two stopi)ered bot- tles of about 25-miUiliter capacity will be required, though if only a single specimen is to be prepared, it will be better to use the small, straight-sided, upright type of stoppered weighing bottles. Both bottles should be soaked overnight in sulfuric-di- 526 METHODS AND FORMULAS MS 11.0 chromate cleaning solution, thoroughly rinsed in tap water, soaked for at least an hour in two changes of large volumes of distilled water, and then dried under such conditions that dust cannot reach them. If either bottle has previously had its stopper greased for any purpose, it is better to reject it outright than to endeavor to clean it. In the first bottle, place 10 to 15 milli- liters of Mann's 1894 osmic-dichromate fixative, whjch will be found under the classification F 1300.0000 in Chapter 18. In the other bottle place a layer of about 3 milhmeters of 2% osmic acid. Before pouring the osmic acid from the bottle in which it is kept, wipe the neck of the bot- tle with a rag soaked in alcohol, and then rinse off the alcohol with distilled water, which is subsequently dried off with a hnt- less cloth. This is necessary, since the os- mic acid will become reduced on the surface of any organic material. It is also necessary to provide six small dishes of about 10- to 15-milhliter capacity, filled with triple-distilled water, for the inter- mediate wash between the two reagents. These dishes should be as clean as pos- sible, but need not be chemically clean, as must those used for the fixative and for the stain. Also required are a chemically clean pipet of the eye-dropper type, a sharp scalpel, and a pair of very fine pointed forceps. If the latter can be of stainless steel, so much the better, but it is not absolutely essential in this technique. Next, remove the ovary from the earth- worm. First, identify the female genital aperture in segment fourteen. This estab- lishes the ventral side of the earthworm. Then wrap the earthworm round the fore- finger, holding its back end between the first and second fingers, so that the genital aperture is towards the tip of the finger. The worm will then be lying on its left side, provided the operator is right-handed and thus has the worm in his left hand. Next, take a sharp scalpel and make an incision covering two segments posterior, and one anterior, to the fourteenth seg- ment about one miUimeter to the left of the genital aperture. Apply considerable pressure and spread the hps of the wound. The ovary will then appear as a small. white, pear-shaped body, which can be removed without difficulty by taking hold of it with fine forceps, just at the point of its insertion, and pulling gently. Now lay the ovary against the inside of the stoppered bottle containing the osmic- mercuric mixture, placing it against the side of the bottle, a good centimeter above the level of the liquid. Under no circum- stances should the tip of the metallic for- ceps be brought into contact with the liquid. The ovary will adhere to the glass surface, thus permitting the withdrawal of the forceps, and it is then only necessary to shake the tube gently, so that the ovary is washed into the fixative. It should re- main in this hquid from a half to one hour (the time is not critical) and should be shaken gently at intervals. After fixation the ovary should be re- moved with the pipet, together with the least possible quantity of fixative, to one of the dishes of wash water. The pipet should then be used to suck water in and out rapidly, so as to mix the contents of the dish. After about five minutes the ovary is removed to the next dish, again being thoroughly rinsed backward and forward, and should remain in each dish for about 30 minutes, with occasional agi- tation. It is better, but not absolutely essential, that it should remain in the final wash water overnight. Remember that if any of the osmic-mercuric mixture is taken over into the osmic stain, there will be a tendency to overfixation, which will make the ovary brittle. When the ovary has been sufficiently washed, transfer it to the osmic acid using the same technique which was used to place it in the original bottle; that is, lay it against the inner wall of the bottle and then remove the forceps. This avoids car- rying over any water and thus diluting the osmic-acid solution. There should be enough osmic acid in the bottle to cover the ovary so that the outline of the tissue shows on the surface. The specimen is now placed in a cupboard in the dark for from ten days to two weeks; but it should be examined daily to make sure that no con- tamination is causing the reduction of the osmic acid anywhere except on the ovary. If, in any of these examinations, the osmic MS U.O-MS 11.1 METAL STAINS 527 acid is seen to be cloudy, or if there is a black precipitate on the bottom of the bottle, the ovary should be removed to another bottle containing a fresh supply of osmic-acid solution. It is a waste of time to add fresh osinic to the existing bottle, wliich has demonstrated, through the reduction of the reagent, that it is al- ready contaminated. When the ovary is sufficiently impreg- nated, the bottle containing it should be filled to the brim with triple-distilled water and tilted backward and forward once or twice to mix the contents. The ovary is then allowed to settle to the bot- tom, and the surplus water is poured off. This should be repeated once or twice, and then only half the water removed, leaving the ovarj^ immersed in about ten milli- liters of water. The bottle with the water in it should then be placed in an oven, or on a water bath, which will maintain a temperature of about 38°C. for one or two days. The ovary is then examined under the surface of water with a binocular mi- croscope. It should present a very dark brown, but not absolutely dead-black, appearance. If it is covered with a dead- black, amorphous deposit, it is evident that it has been too long in osmic acid. If it is light brown, it has not been in osmic long enough. In either circumstance it had better be thrown away. If, however, it has been properly impregnated, it should now be thoroughly washed, either in at least ten changes of triple-distilled water with not less than one hour in each, or over- night in slowly running, triple-distilled water. The ovary is then embedded in paraffin by the methods described in Chapter 12, and sections are cut longitudinally at a thickness of from one to two microns. The sections are dried on a shde, the wax melted, and the slide dropped into xylene to remove the wax. Meanwhile, set up two tubes, or coplin jars, one containing an- hydrous turpentine and the other oil of cedar. As soon as the wax has been dis- solved, pass the sHde into the cedar oil until the xylene has been removed, with- draw the slide, wipe the surplus oil from the bottom, and examine it under an oil- immersion lens. If impregnation has been successful — that is, if the Golgi apparatus and yolk granules are shown as dead-black spots against a pale brown background — return the sUde to xylene until the cedar oil has been removed, and then mount it in balsam under a coverslip. If no signs of Golgi apparatus are present, the shde may be thrown away, and it must be presumed that some mistake has been made in the technique. If, however, as frequently hap- pens, there is some evidence of Golgi apparatus as black dots, but the back- ground is obscured by other scattered black granules, place the sUde in turpen- tine for about two minutes, rinse it rapidly in xylene, return to cedar oil, and re-exam- ine. Repeat these operations until the section has been properly differentiated. Some people recommend that all sections should be treated with turpentine, but this is not usually necessary in the case of so simple an object as the earthworm ovary. If turpentine has been used, wash the slide in a jar of clean xylene in order that all turpentine may be removed from the slide before it is finally mounted. The majority of formulas from this sec- tion have been transferred to the appro- priate section of Chapter 18. Those re- tained were originally suggested as stains and have never been recommended as fix- atives. Simple solutions of osmic acid are not listed separately, but are given with individual techniques. 11.1 van Gehuchten 1927 11.1 Hamilton 1897 11.1 Staining Solutions see F 1700.0000 van Gehuchten 1927 3464, 20:180 STOCK solutions: I. Brain tissue hardened in F 7000.0000 Muller 1859 for 3 weeks 50, F 7000.0000 Muller 1859 100. (Grind to a paste and filter.) II. 1% osmic acid. WORKING solution: stock I 100; stock II 0.5. 11.1 Kolossow 1892 23632,9:39 formula: water 50, 05% ale. 50, nitric acid 2, osmic acid 1 528 METHODS AND FORMULAS MS 11.1-MS 11.21 11.1 Mann 1894 see F 1300.0000 Mann 1894 11.1 Orr 1900 see F 1000.0010 Orr 1900 11.1 Rossolino and Busch 1896 tesl. 1897 Schiefferdecker 23632, 14:55 formula: water 50, 95% ale. 50, 40% formaldehyde 0.2, osmic acid 0.2 11.1 Swank and Davenport 1924 see F 1000.1010 Swank and Davenport 1924 11.1 Swank and Davenport 1935 see F 1000.1010 Swank and Davenport 1935 11.1 Takahashi 1908 see F 1600.0060 Takahashi 1908 11.2 Neurological Techniques 11.21 methods for degenerative changes These methods are commonly known as Marchi methods. For further information see Mettler 1932 (20540b, 7:95), Swank and Davenport 1934 and 1935 (20540b, 9:11 and ibid, 12:45). 11.21 Anderson 1929 Anderson 1929, 68 REAGENTS REQUIRED: A. 2% potassium iodate; B. F 1800.0000 Busch 1898; C. ADS 11.1 Andeison 1929 method: [small pieces of formaldehyde-fixed tissue] — * A, 24 hrs. — > B, 7 daj's —* thor- ough wash — > C, 7 days, 37°C. — » thorough wash -^ [celloidin sections] 11.21 Busch 1898 see MS 11.21 Anderson 1929 for method, and F 1800.0000 Busch 1898 for formula 11.21 van Gehuchten test. 1927 Kingsbury and Johannsen Kingsburj' and Johannsen 1927, 8 :9 REAGENTS REQUIRED: A. 3.7% potassium dichromate; B F 1700.0000 van Gehuchten 1927 method: [fresh tissue]-^ A, 3 wks. — > B, 3 wks. 2 or 3 changes —^ wash —^ [frozen sections] 11.21 Hamilton 1897 3464, 20:180 REAGENTS REQUIRED: A. F 7000.0000 MiUler 1859; B. MS 11.1 Hamilton 1897; C. AMS 21.1 Hamilton 1897; D. 0.25% potassium permanganate; E. 2% potassium sulfite method: [whole brains]-^ A, till hardened, 3-5 months—* [celloidin section enclosed between two coats of celloidin and stripped from the slide] — > B, 24 hrs. 37°C. — > wash — » C, 24 hrs. 37 °C. -^ wash — > D, 24 hrs. — > wash — * E, till decolorized — * wash -^ D ^> E cycle repeated twice — > wash — > C, fresh solution, 24 hrs., 37°C. — * wash — > M 32.1 mountant 11.21 Marchi 1896 19460, 12 :3 reagents required: A. F 7000.0000 Miiller 1859; B. F 1700.0000 Marchi 1886 method: [1 cm. slices fresh tissue] -^ A, 3-5 days -^ wash — » B, 5-14 days — > wash -^ [celloidin sections] 11.21 Mettler 1932 20540b, 7:95 reagents required: .4. 10% neutralized formaldehyde; B. 3% potassium dichromate; C. F 1700.0000 Marchi 1886 method: [pieces] — + A, 24 hrs. -^ [4 mm. slices] — > i5, 2 wks. — > rinse — * C, 5-14 days — > wash -^ [celloidin sections] note: Sol. B should be "aged" 3 months. C should be replaced if it ceases to smell of osmic acid. 11.21 Orr 1900 • 11431, 6:387 reagents required: .4. F 7000.0000 Miiller 1859; B. Orr 1900 F 1000.0010 method: [A, several wks. to months] —> wash -^ [freehand sections]—* B, 1-2 days — > balsam, via usual reagents MS 11.21-MS 11.3 METAL STAINS 529 11.21 Stewart 1936 11431,43:339 REAGENTS Ri-xjiiREi): A. 2% pofassium dichromate; B. 1% osniic acid method: [30 m frozen sections of neutral formal dehyde-fixod material]— > wash — » A, 1 day, 21°C. —> wash, till pale yellow —>■ B, in dark, 16-36 hrs. -^ wash —^ M 30 mountant note: Sections in whicli tlie normal myelin is too deeply stained may be differentiated by ADS 21.1 Pal 1887. 11.21 Swank and Davenport 1935 20540b, 10:88 REAGENTS REQUIRED: A. watcr 100, magnesium sulfate 5, potassium dichromate 3; B. 4% formaldehyde; C. Swank and Davenport 1935 F 1000.1010 method: [animal killed with nembutal] — + A, perfused through aorta — > [central nervous system removed] -^ B, 48 hrs. —* wash — > [celloidin sections] 11.22 OTHEK NEUROLOGICAL METHODS 11.22 Azoulay 1894 766, 10:25 REAGENTS REQUIRED: .4. F 7000.1000 MuUer 1859; B. 0.002% osmic acid; C. 10% tannin method: A, some months -^ [sections] —> 5, 5-15 mins. ^ rinse —> C, warmed till steaming — > wash — > balsam, via usual reagents RECOMMENDED FOR: myelin sheaths. 11.22 Bohm and Oppel 1907 Bohm and Oppel 1907, 261 REAGENTS REQUIRED: A. 1% osmic acid; B. glycerol method: [teased fibers of muscle on slide under coverslip] ^ A, 30 mins. in moist chamber -^ B, substituted for A without moving coverslip -^ [seal coverslip] RECOMMENDED FOR: myelin sheaths. 11.22 Champy, Coujard, and Coujard-Champy 1946 Acta Anatomica, 1 :233 REAGENTS required: A. water 100, osmic acid 0.25, sodium iodide 2.25 method: [2-3 mm. slices of fresh tissues] ^^ A, 24 hrs. -^ running water—* [paraffin sections] RECOMMENDED FOR: Sympathetic nerve endings in gland cells. 11.22 Heller-Robertson Irsl. 1929 Anderson Anderson 1929, 64 REAGENTS REQUIRED: A. ADS 12.2 Weigert 1891; B. 1% osmic acid; C. 5% pyrogallic acid; D. ADS 21.1 Pal 1887, A and B sols. method: [frozen sections] —* A, 24 hrs. — > wash -^ B, in dark, 30 mins. — > wash — > C, 30 mins. —> wash — > D (sol. A), 30 sees. — > wash — > D (sol. B), till colorless — > balsam, via usual reagents RECOMMENDED FOR: myelin sheaths. 11.22 Rossolino and Busch 1896 test. Schiefferdecker 1897 23632, 14:55 REAGENTS REQUIRED: A. 0.5% chromic acid; B. MS 11.1 Rossolino and Busch 1896 method: [sections of formaldehyde-fixed material] —> A, 2-3 hrs. —> rinse -^ .B, 24 hrs. — > wash -^ balsam, via usual reagents RECOMMENDED FOR: granule cells. 11.3 Histological Methods 11.3 Altmann 1878 see V31 Altmann 1878 11.3 Cramer 1919 1200, 6:77 REAGENTS REQUIRED: A. 2% osmic acid at 37°C. method: Expose fragments to vapor from A in stoppered bottle for 13-^ hrs. at 37°C. -^ [sections via usual reagents] RECOMMENDED FOR: Cell differentiation in adrenal gland. 11.3 Hamann 1885 see F 1000.0010 Hamann 1885 11.3 Hermann 1891 1780,37:4 REAGENTS REQUIRED: A. F 1200.0010 Hermann 1889; B. 90% ale; C. crude pyroligneous acid method: a, overnight —* wash B, 1 to 2 wks. — > C, 12-18 wks. — > wash 530 METHODS AND FORMULAS MS 11.3-MS 11.4 11.3 Hirschler 1918 see F 1300.0000 Hirschler 1918 11.3 Kolossow 1892 23632, 9:39 REAGENTS REQUIRED: A. MS 11.1 Kolossow 1892; B. AMS 21.1 Kolossow 1892; C. 0.05% osmic acid method: [small pieces or objects] — » A, 15 mins. -^ wash — > B, 5 mins. -^ C, wash -^ balsam, via usual reagents note: Lee 1891 (23632, 9 :185) claimed this method on the grounds that he had in 1887 (6011, 4:110) published a description of it. Kolossow 1892 (23632, 9:316) pointed out that Lee had given only a general indication of an uncontrolled method of reducing osmic acid with tannin. recommended for: general histological staining. 11.3 Krajian 1940 1887a, 30:766 REAGENTS REQUIRED: A. 1% osmic acid; B. \% phloxine method: [10 M frozen sections of formaldehyde-fixed material]—* A, 60°C., 5 mins.—* thorough wash-^ B, 1 min. — * wash -^ M 12.1 mountant recommended for: fat in frozen sections. 11.3 Lee 1887 6011,4:110 reagents required: A. 1% osmic acid; B. 0.5% pyrogallol method: a, 1-2 days ^ wash -^ B, till green-brown throughout-* [paraffin sections via usual reagents] recommended for: general histological staining. 11.3 Lee 1905 Lee 1905, 255 reagents required: A. F 1200.0010 Hermann 1889 or F 1600.0010 Flemming 1882; B. 0.25% pyrogallol method: a, not more than 30 mins. -* B, 24 hrs. -^ [sections by paraffin technique] recommended for: general histological staining. note: Lee 1905 {loc. cit.) states that this method has been attributed to von Maehren- thal. 11.3 von Maehrenthal see MS 11.3 Lee 1905 (note) 11.3 Sjovall 1905 764,30:261 reagents required: A. 4% formaldehyde; B.2% osmic acid method: [small fragments]-* A, 8 hrs. to 2 days ^ wash-* B, 2 to 15 days -^ 3 n paraffin sections, via usual techniques 11.3 Woronin 1898 test. 1948 Romeis Romeis 1948, 304 reagents required: A. \% osmic acid; B. sat. aq. sol. tannin method: [sections] -^^ waters A, 10 mins.-* B, 10 mins.-* A, 20 mins.-* abs. ale, thorough wash -^ balsam, via xylene recommended for: epithelial tissues. 11.4 Methods for Cell Inclusions These techniques for the demonstration of the Golgi apparatus are commonly referred to as Mann-Kopsch techniques. 11.4 Gatenby 1920a 17510,64:267 reagents required: A. 2% osmic acid at 37°C.; B. F 1700.0000 [AltmanMSOO or F 1670.0000 Champy 1913 method: expose fragments to vapor from A in stoppered bottle at 37 °C. for 112 hrs. — » A, several days — * or B, several days -* wash — * sections via usual reagents 11.4 Gatenby 1920b 17510, 64:267; Langeron 1942, 646 REAGENTS REQUIRED: A. F 1300.0000 Mann 1894; B. 2% osmic acid; C. turpentine method: [small fragments] -* A, 15 mins. to 3 hrs. -^ wash -^ 5, 2 to 3 wks. in dark -^ 3 M sections by paraffin technique — * C, till differentiated NOTE : Langeron 1942, 646 refers to this as the Kopsch-Gatenby technique. MS 11.4 METAL STAINS 531 11.4 Hirschler 1918 1780, 89:271 REAGENTS REQUIRED: A. F irjOO.OOO IlirschlcF 1918; B. 2% osinic acid method: [small fragments] — > A, 1-3 hrs. — > wash — > B, 12-16 days —» wash — » paraffin via chloroform note: This was reprinted, with slight alterations in the timing, in 1924 (6630, 90:83). 11.4 Kopsch 1902 20170, 40:929 reagents required: A. 2% osmic acid method: [small fragments] —> rinse -^ A, in dark, 2 wks. — > wash — » [3 n paraffin sections, via usual reagents] 11.4 Kopsch-Gatenby test. Langeron 1942 see MS 11.4 Gatenby 1920b (note) 11.4 Ludford 1925 113G0, 45:31 reagents required: .4. F 1300.0000 Mann 1894; B. 2% osmic acid; C. turpentine method: [small pieces fresh tissue] —> A, ^^ to 1 hr. — » wash — > B, sufficient to cover piece on bottom of 1 oz. stoppered bottle, 10 days to 2 wks. — > water at 38°C., 1-2 days -^ wash — » [paraffin sections by usual techniques] — > xylene, till wax removed -^ C, if differentiation necessary -^ balsam, via usual reagents 11.4 "Mann-Kopsch" see note under 11.4 above. 11.4 Nassanow 1923 1780,97:136 reagents required: A. F 1670.0000 Nassanow 1923; B. 2% osmic acid at 35°C. method: [small pieces fresh tissue]^ A, 24 hrs. — > wash—* B, 3 to 7 days, 35°C. — > wash -^ [paraffin sections by usual techniques] 11.4 Newcomer 1940 20540b, 15:89 reagents required: A. F 4700.0000 Zirkle 1934; B. 2% osmic acid; C. 1% potassium permanganate; D. 3% oxalic acid method: [growing root tips] -^ A, 4S hrs. -^ wash, overnight, — > B, changed alternate days, 4-6 days -^ wash, overnight — > [5 m paraffin sections] — * water -^ C, 5 mins. — > rinse — > D, 2-3 mins. — > thorough wash — > balsam, via usual reagents recommended for: mitochondria in plant cells. 11.4 Weigert test. McClung 1929, 208 see DS 11.4 Weigl 1912 note 11.4 Weigl 1912 Mann-Kopsch — compl. script. 4346,23:1 reagents required: A. F 1300.0000 Mann 1894; B. 2% osmic acid method: [small fragments] —* A, 30 mins. to 2 hrs. -^ wash —* B, 10 days to 3 wks. — » wash -^ [sections via usual techniques] note : This is almost universally referred to as the Mann-Kopsch technique for the reason that the former invented solution A for another purpose, while the latter recommended solution B for the present one. McClung 1929, 208 refers to the method as WeigerVs Mann-Kopsch. ]VIS 20 Gold Gold is used eitlaer by direct application witli osmium or other metals. Techniques of tlie chloride to partially hydrolized tis- in which gold is used to replace silver- sues, or in combination with mercury, impregnated structures (the toning reac- either from complex solutions or by sue- tion of the photographer) are given under cessive applications, or in combination silver staining in MS 30 below. MS 21 METHODS USING GOLD ALONE These are the most primitive of all the endings in muscle as the classic gold- metal-staining techniques though, when lemon-juice method of Ranvier given be- they are successful, they yield beautiful low. The effect is presumably produced by results. There is still no method as satis- the reducing action exercised by the sheath factory for the demonstration of nerve of the nerve on gold chloride, but there is 532 METHODS AND FORMULAS MS 21.0 no evidence of the exact reaction pro- duced, nor has it been satisfactorily estab- lished that the resultant precipitate is metallic gold. The ver}^ fugitive nature of many of the gold stains would tend to make one beheve that the metal cannot be involved. It cannot be too strongly em- phasized that absolute cleanliness and the rigorous control of the extent of hght ad- mitted during the reaction are two criteria of success. MS 21.0 Typical Example Demonstration of the termination of the fourth cranial nerve in the superior obhque muscle by the method of Ranvier 1889 This method is more than half a century old, but it is one of the simplest and best available, both for training classes in gold techniques and for demonstrating the nerve terminations in muscles. Only two prerequisites are essential to success. The first is that the lemon juice employed shall be from a relatively fresh lemon, shall have been squeezed with as little oil of lemon getting into it as possible, and that it shall then have been filtered through paper; tliis is tedious unless it is done with a filter pump. It is better to prepare the lemon juice immediately before use, and it is not nearly as good, even if it be left overnight. The second essential to success is that the glassware used should be chem- ically clean. In this particular technique four glass-stoppered bottles, or upright glass-stoppered weighing bottles, should be soaked in sulfuric-dichromate cleaning solution, thoroughly washed off in tap water, soaked in distilled water, and then dried under conditions which will leave them as dust-free as possible. In the first bottle place the freshly filtered lemon juice, in the second bottle 1% gold chlo- ride, in the third bottle 0.2% acetic acid, and in the fourth bottle 20% formic acid. There will also be required two glass dishes of triple-distilled water which should be as clean as possible. A rabbit is a good subject for this tech- nique; but if a small shark or dogfish is available, it will be found to be better. In either case, the animal should be killed, the skin removed from around the orbit, and the eye taken out carefully so as to leave all the muscles intact within the or- bit. The fourth nerve, leading to the supe- rior obhque muscle, should then be identi- fied and severed where it leaves the fora- men. The muscle itself should then be cut off, leaving as much as possible in front of the nerve and about a quarter of an inch behind the nerve. Then take a surgical silk or other fine, clean fiber and tie this round the upper end of the nerve. Using this thread as a suspensor, hang the mus- cle by the nerve in the bottle of lemon juice. It will become transparent within a few hours, and may be passed to the next stage at any period between the time when it becomes completely transparent and about the twelfth hour of immersion. The longer period is, in general, better for mammahan tissue, and the shortest pos- sible period for fish tissues. As soon as the muscle has become trans- parent, withdraw it from the lemon juice by the thread, snip off the thread and, hold- ing the muscle by one end in a pair of for- ceps, rinse it thoroughly in one of the jars of triple-distilled water. Then, after having drained off the water by touching the end of the muscle to a clean filter paper, drop the preparation into at least 60 milhliters of 1% gold chloride. Leave it in this solu- tion, with gentle agitation at intervals, for about 20 minutes; it will darken shghtly, from the transparent yellow of the lemon juice to a light brown. One certain way to insure failure of this technique is to use metallic forceps to re- move the muscle from the gold chloride and pass it to the acetic acid. It must be picked up on the end of a glass hook or with a glass spoon. The muscle is then rinsed and placed in the bottle of 0.2% acetic acid. The intensity of hght is critical, for if the object be placed in direct sunhght it will become badly over- stained, while in the dark it will not be- come stained at all. It was recommended by Ranvier originally that this technique should be carried out on days when there were clear, white clouds in the sky from MS 21.0 METAL STAINS 533 which the sunhght was reflected. Though this is possibly ratlier an excessive view, tliere is no doubt that the state of the weather has more effect on the technique than any other single factor. It would perhaps be simplest to suggest that the specimen be exposed to bright daylight, that is, some intermediate condition be- tween the dingy darkness of a winter day and bright, direct sunhght. It should re- main in the acetic acid for from one to two days. The period of time is, of course, de- pendent upon the degree of illumination to which it is exposed. If the technique is successful the entire muscle will change to a dull purple color. The word c?mM should be emphasized. Dai'k purple indicates too great a degree of re- duction, and there will be no differentia- tion of the nervous tissues. A hght mauve color indicates that the material has not been sufficiently reduced, and it is doubt- ful that it is worth exposing it further to dayhght, since prolonged exposure to in- ferior illumination does not have the same effect as a correct length of exposure to the proper illumination. The best way of determining whether or not the impregnation has been success- ful is to examine the muscle under a binocular microscope with the strongest possible illumination from beneath. If the beginnings of the divisions of the nerve witliin the muscle can be seen, the preparation is satisfactory; if, however, the material is so dark that the nerve cannot be followed beyond the point of its entry into the muscle, it is doubtful that it is worth proceeding further. Successful impregnations should be re- moved from the acetic acid and placed in the formic acid for 48 hours in the dark. Then take a straight upright tube of about one inch in diameter by four inches long, and place an inch of pure glycerol in the bottom; with a pipet, very carefully place about a two-inch layer of 20 % formic acid on the top of the glycerol, being careful to mix them as little as possible. The prepa- ration is now transferred to the upper layer of formic acid, and will naturally float at the interphase between the formic acid and the glycerol. After about 24 hours the muscle will have sunk through the glycerol, but it should not be removed until it shows no further streams of formic acid rising from it through the glycerol. The formic acid is then carefully pipetted from the top of the tube, and a fresh portion of glycerol is added. There will probably have been a considerable mixture of formic acid and glycerol by diffusion, thus the muscle will again float at the interphase between this diluted glycerol and the fresh glycerol which has been added. As soon as the muscle has again sunk, thus demonstrating that it is completelj'' impregnated, the partially diluted glycerol should be pipetted from the surface and the muscle transferred to a watch glass or stender dish full of fresh glycerol. This is an excellent time at which to issue the muscle and nerve to a class, and there are manj^ methods which may sub- sequently be followed to secure prepara- tions showing the nerve endings. The sim- plest for class purposes is to divide the muscle into a series of freehand sections taken longitudinally, each section being about J^ o-niillimeter thick. If each stu- dent is provided with one of these sections, he can then examine it under the micro- scope, and will usually without further trouble be able to see the fine terminations of the nerves. To make better and more permanent preparations, it is desirable to take these thin sections and to tease them with needles so as to separate the individ- ual fibers, following the process under a binocular dissecting microscope. Fibers which look as though they might show endings, are then transferred to a slide and mounted permanently in glj'cerol jelly. In place of glycerol jelly one may also employ any of the gum-arabic-glycerol media, or probably, though the author has never tried it, some of the polj^vinyl-alco- hol media. These preparations are not very perma- nent^unless thej'' are preserved in the dark, and no method seems to be known by which permanency can be given to them. Under no circumstances can they be mounted in anything save an aqueous medium. 534 METHODS AND FORMULAS MS 21.1-MS 21.2 MS 21,1 Staining Solutions 21.1 Jabonero 1935 test. 1936 Findlay 11360, 56:160 formula: water 100, glucose 1, gold chloride 0.02 21.1 Kolossow 1888 23632, 5 :52 formula: water 245, hydrochloric acid 25, gold chloride 25 21.1 Ranvier 1880 17510, 80:456 formula: water 200, formic acid 50, gold chloride 2 21.1 Stoehr 1894 test. 1907 Bohm and Oppel Bohm and Oppel 1907, 258 formula: water 200, formic acid 50, gold chloride 2 preparation: Dissolve the chloride in the dilute acid. Bring to boil. Cool. Repeat 3 times. Filter. MS 21.2 Techniques Unless specific recommendations are made, it is to be understood that all the following methods are intended for nerve endings in muscle. 21.2 Apathy 1897 14246, 12:718 reagents required: A. 1% gold chloride; B. \% formic acid method: [fresh tissue] — > A, in dark — * blot — > B, 6 to 8 hrs., evenly illuminated from all sides 21.2 Beckwith test. circ. 1938 Wellings Wellings circ. 1938, 130 reagents required: A. 1% gold chloride; B. 20% sodium hydroxide; C. 10% potas- sium carbonate; D. 10% potassium iodide method: [sections from F 4700.0000 Erlitzky 1877 or F 7000.0000 Muller 1859 fixed material] — > water -^ A, 5-6 hrs. -^ rinse -^ B, 3 mins. -^ drain — > C, 30 mins. — > drain — > D, till differentiated recommended for: nerves in teeth. 21.2 Bensley and Bensley 1936 test. 1952 ips. Cowdry 1952, 8 reagents required: .4. 10% gold chloride; B. 4%, neutralized formaldehyde; C. 1% toluidine blue; D. water 100, ammonium molybdate 2.5, potassium ferrocyanide 0.5 method: [inject mouse intravenously through tail with 1 ml. A] — >• [after death fix lungs in B] —>■ [paraffin sections on slide] -^ water — » C, 10 mins. — >• rinse -^ D, 5 mins. — > balsam, via usual reagents recommended for: alveolar epithelium of lung. 21.2 Boccardi 1886 test. Lee 1905 see MS 21.2 Manfredi 1881 (note) 21.2 Bohm 1907 test. 1907 Bohm and Oppel Bohm and Oppel 1907, 437 reagents required: A. 50%, formic acid; B. 1% gold chloride; C. AMS 21.1 Pritchard 1907 method: [small fragments, fresh tissue] — ^ A, 20 mins. —>■ rinse —>■ B, 20 mins. -^ rinse —>■ C, large volumes, 24-36 hrs. in dark — > paraffin sections via usual reagents note: This technique is .sometimes referred to Carriere 1882 (1780, 21:146) who, how- ever, attributes it without reference to Bohm as do, still without reference, most other authors including those cited. 21.2 Carriere 1882 see MS 21.2 Bohm 1907 (note) 21.2 Cole 1946 20540b, 21 :23 reagents required: A. 10% citric acid in 0.9% sodium chloride; B. 1% gold chloride; C. 20% formic acid method: [muscle pieces] —► .4, 10 mins. — > Zi. 1 hr. -^ C. 10-20 hrs. -> glycerol by evaporation from glycerol-alcohol mixtures 21.2 Cornheim 1867 see MS 21.2 Manfredi 1881 (note) MS 21.2 METAL STAINS 535 21.2 Dependorf 1913 7282, 31 :377 REAGENTS REQUIRED: A. 30% foriiiic acid ; B. 1% gold chloride; C. formic acid method: [small pieces of fresh teeth] — > A, 5-10 mins. -^ B, 2-6 hrs., in dark — > A, 1 day, in dark -^ C, 1 day, in dark -^ [celloidin sections] recommended for: nerves in teeth. 21.2 Drasch 1887 23632, 4:492 REAGENTS REQUIRED: A. 0.5% gold chloride; B. 20% formic acid method: [ pieces of fresh tissue, left without preservative at 4°C. for 48 hrs.] — > A, in dark, >2 to ^i hrs. — > rinse -^ B, till nerves visible -^ glycerol, several changes recommended for: nerve endings in alimentary canal. 21.2 Flechsig 1884 see MS 21.2 Ranvier 1880 (note) 21.2 Freud teat. 1896 Kahlden and Laurent Kahlden and Laurent 1890, 159 reagents required: A. 0.5% gold chloride in 50% ale; B. 5% sodium hydroxide; C. 10% potassium iodide method: [small pieces, or sections, of dichromate-fixed material] -^ water -^ A, 3-5 hrs. — > wash -^ B, 2-3 mins. — > C, 5-15 mins. —> wash -^ balsam, via usual reagents recommended for: axis cylinders. 21.2 Graven 1925 3464, 48 :380 reagents required: A. 25% formic acid; B. 1% gold chloride method: [fresh tissue] —> A, 10-15 mins. -^ blot -^ B, 20 mins., in shade -^ blot -^ A, 10-15 mins, -^ B, 24 hrs., in dark -^ blot -^ A, 10-15 mins. -^ blot -^ B, 24 hrs., in dark -^ wash -^ glycerol 21.2 Hanazawa 1917 7141,59:125 reagents required: A. 1% gold chloride in 95% ale; B. 5% potassium hydroxide; C. 10% potassium iodide method: [embed tooth in resin, grind section to half millimeter (see Chapter 10)] —> A, 2-3 days, in dark -y wash -> B, 15-20 mins. -^ wash —>■ C, 12-24 hrs. -* [grind thin] — > balsam, via usual reagents recommended for: dentine. 21.2 Henocque test. 1895 Rawitz Rawitz 1895, 81 reagents required: A. 1% gold chloride; B. sat. sol. tartaric acid method: [small pieces] — > A, 15 mins. -^ rinse — > B, till reduced 21.2 Kolossow 1888 23032, 5 :52 reagents required: A. MS 21.1 Kolossow 1888; B. 0.02% chromic acid method: [fresh tissue] -^ A,S hrs. — > blot -^ JB, 2 or 3 days in dark 21.2 Jabonero 1935 test. 1936 Findlay 11360, 56:160 reagents required: A. 50% glucose; B. MS 21.1 Jabonero 1935; C. 5% sodium car- bonate method: [frozen sections of fresh tissue] -♦ A, 10 mins., 60°C. -^ B, warmed till sections violet — > balsam, via usual reagents recommended for: myelin sheaths. 21.2 Lowit 1875 20170,71:1 reagents required: A. 50% formic acid; B. 1% gold chloride; C. 25% formic acid; D. formic acid method: [fresh skin] -^ A, until epidermis peels off -^ B, 15 mins. —> C, in dark 24 hrs. — > D, in dark 24 hrs. — > [sections by paraffin technique] — * balsam, via usual reagents recommended for: nerve endings in skin. 21.2 Manfredil881 1946,5:30 REAGENTS REQUIRED: A. 1% gold chlondc; B. 0.5% oxalic acid method: [fresh tissue] -^ A, 30 mins. — » rinse -^ B, until reduced -^ glycerol mounts note: This method is essentially that of Cornheim 1867 (22575, 34:606) save that oxalic acid has been substituted for the acetic acid of the original. Boccardi 1886 (test. Lee 1905, 251) substituted MS 23.1 Boccardi 1886 for B above. 536 METHODS AND FORMULAS MS21.2-MS22.0 21.2 Miller 1923 763, 25 :77 KEAGENTS REQUIRED: A. 4% citric acid; B. 1% gold chloride; C. 30% formic acid method: [fresh tissue] -^ A, 20-30 mins., in dark -» rinse -> B, 20-30 mins., in dark -» C, 2 days -^ wash -^ glycerol 21.2 Nikiforoff see DS 21.213 Nikiforoff 1896 21.2 Ranvier 1880 17510, 80:456 REAGENTS REQUIRED: A. Ranvier 1880 MS 21.1; B. 20% formic acid method: .4, 20 mins. to 2 hrs. -^ B, in dark until reduced note: Flechsig 1884 1739, 463 substitutes 10% sodium hydroxide for B above. 21.2 Ranvier 1889 Ranvier 1889, 813 reagents required: A. fresh filtered lemon juice; B. 1% gold chloride; C. 0.2% acetic acid; D. 20% formic acid method: [fresh tissue] — » A, until transparent -^ rinse -^ B, 20 mins. -^ rinse — > C, in light, 24 to 48 hrs. -^ examine -^ [if successful render permanent by] — > D, 48 hrs. in dark 21.2 Rufini test. 1933 Cajal and de Castro Cajal and de Castro 1933, 348 REAGENTS REQUIRED: A. 20% formic acid; B. 1% gold chloride; C. 1% potassium ferro- cyanide method: [fresh tissue] —* A, till translucent — ♦ wrap in cloth — » B, in dark, 20-30 mins. -^ A, in dark, 24 hrs. — » C, if overstained, till differentiated -^ wash — > glycerol 21.2 Stoehr 1894 test. 1907 Bohm and Oppel Bohm and Oppel 1907, 258 REAGENTS required: A. MS 21.1 stoehr 1894; B. 20% formic acid method: [fresh muscle] — »• A, 45 mins. -^ wash -^ B, in light, 36 hrs. MS 22 METHODS USING GOLD IN COMBINATION WITH MERCURY These methods are mostly used for the demonstration of neuroglia, particularly oligodendroglia and microglia. They ap- pear at first sight to be simple, but are actually more difficult to use than are the silver techniques developed for the same purpose. There appears to be no criterion for success, though this may be rendered the more likely by the use of pure reagents and the most rigorous attention to chem- ical cleanliness of the glassware employed. Nothing appears to be known of the the- ory lying behind these stains. MS 22.0 Typical Example Demonstration of protoplasmic neuroglia in the cerebral cortex by the method of Cajal 1916 This is a deceptively simple technique which is unlikely, in inexperienced hands, to yield as good results as are the silver methods (MS 31.22, 33.22, 34.22) more commonly employed for this purpose. It is essential that the reagents, the distilled water, and the glassware emploj'ed be pre- pared as though one were engaged in a critical analj^sis. For the first step, secure a Uving or freshly killed rabbit and the solution of ammonium bromide fisted in Chapter 24 as AMS 11.1 CajallGlB. This solution must be prepared with reagent quality materials throughout, including the formaldehyde ; and the bottle in which it is placed must be chemically clean. It is desirable to place in the bottom of this bottle a layer of about half an inch, either of fat-free absorbent cotton or of a fine glass fiber. The freshly killed rabbit is tied face down on a board, the upper surface of the head skinned, and the frontal and parietal bones removed with forceps, particular care being taken not to break the blood vessels of the meninges. All extraneous blood should be removed with a gentle washing in either triple-distilled water or with a normal saline made with triple- distilled water. The surface of the brain is then flooded with a relatively large quantity of the ammonium bromide-for- maldehyde solution, which acts as'a hemo- static agent during the removal of the MS 22.0 METAL STAINS 537 meninges. Finally, remove a series of cubes of about one-centimeter side from the cerebral cortex. Stainless steel knives may be used for this purpose, but the author has alwaj's found it more i)ractical to em- ploy the sharp edge of a broken coverslip. If a standard 18-by-22-minimeter cover- slip be broken roughl}'- along a diagonal, it will be found to cut the brain most satis- factorily. This avoids the risk of surface contamination from metals which will in- terfere with subsequent staining. Two or three blocks will be sufficient, and they should be placed in the stoppered bottle containing the fixative, of which at least 250 milhhters should be employed for two or three one-centimeter cubes; four times this volume is not an unreasonable quantity. The blocks should be removed from the fixatives as soon as they are adequately hardened. The exact degree of hardening necessary can best be gaged by pressing gently on the surface of the block with the rounded end of a glass rod. The block should be springy but not hard, and should not be removed until it can be han- dled with forceps without running the risk of crushing any of the internal structures. After removal from the fixative, the blocks are washed in running triple-dis- tilled water for at least a day, or washed in not less than five successive changes of at least 500 milliUters each. After washing, each block is subdivided, preferaljly by the broken edge of a glass coverslip, into five or six smaller blocks, which form a reasonably sized portion for sectioning on the freezing microtome. One of the most fruitful sources of error of this technique is the use of crude commercial mucilages of gum arabic in the course of the section- ing, even after taking stringent precau- tions with the purity of the reagents employed in fixation. It is far better to em- ploy pure sugar solutions than to rely on gum materials which may contain such impurities as will stultify subsequent work. Sections are removed, as made, to a watch glass of triple-distilled water. Again it must be emphasized that the watch glass should be chemically clean. Before commencing to section the material, it is desirable to prepare the staining reagent, the formula for which is given as MS 22.1 Cajal 1916 below. The very greatest care is required in making this solution. All the reagents involved, particularly the mer- curic chloride, should be of the grade sold for analytical analysis; the ordinary com- mercial chloride found in biological lab- oratories is not satisfactory. Cajal does not indicate in his formulas which of the numerous types of gold chloride or mixed gold, potassium, and sodium chlorides he used in the original fornuila. The method of preparing the formula is, however, crit- ical. First, the mercuric chloride is dis- solved in hot triple-distilled water at from 70°C to 80°C. It should give a clear solu- tion entirely free from opalescence, and if the faintest trace of a precipitate is seen, the solution must be rejected and a purer batch of the reagent sought. In a second chemically clean beaker dissolve the gold chloride at room temperature in triple- distilled water. The gold solution is then added to the hot mercuric chloride solu- tion with constant stirring; the utmost care is taken to avoid the production of a precipitate, the presence of which will ren- der the solution valueless. The solution is then brought up to 250 with triple-distilled water. The sections are taken from the triple- distilled water and transferred to the staining solution for from four to six hours in the dark at room temperature. If time is a consideration, it is possible to warm the solution to about 25°C., which reduces the period required for staining to three hours. It is not difficult to determine when staining is sufficient; the sections should, in any case, be examined at hourly inter- vals and be withdrawn from the stain when they have become dark purple. A Hlac color indicates understaining, and they will be spoiled if they are left until they have become brown. The successful application of this technique depends on the ability to judge the exact shade of purple which indicates a satisfactory ter- mination of the staining process. Sections should be removed from the stain and washed for at least three hours in running trit)le-distilled water, or in at least three changes of a considerable volume of triple- distilled water changed at hourly intervals. 538 METHODS AND FORMULAS MS 22.1-MS 22.21 If the preparations are required only for for this purpose. This is no better than temporary examination, they may be, at water from the point of view of removing this point, dehydrated and cleared, but an excess of the fixative, but has the ad- the stain itself is still light-sensitive and vantage of commencing the process of it is necessary to fix the sections if they dehydration. are to be mounted in balsam. Cajal him- After the sections have been adequately self recommends an acid-thiosulfate solu- washed they should be removed one by tion, the formula for which is given in one from the washing solution, placed on a Chapter 24 as AMS 24.1 Cajal 1913. The clean shde, covered with filter paper, and sections should be treated in a large vol- blotted to remove as much of the alcohol ume of this solution for five or ten min- as possible. They may then be dehydrated utes. The time of apphcation is not criti- by dropping alcohol on them from a pipet cal, and it is better to err on the side of too or wash bottle, and cleared in any satis- long apphcation than too short. The solu- factory clearing agent before mounting tion cannot be used twice and should be in balsam. thrown away after each batch of sections These preparations at the best are only have been passed through it. The fixing moderately permanent, and should never solution must, of course, itself be removed be exposed to bright light for long periods by thorough washing and Langeron (Lan- of time, geron 1942, 653) recommends 40% alcohol 22.1 Staining Solutions 22.1 Cajal 1916 21344, 14:155 formula: water 100, mercuric chloride 0.7, gold chloride 0.14 preparation: Dissolve mercuric chloride in 15 water at 70-80°C. Dissolve gold chloride in 15 water at room temperature and add to mercuric chloride. Dilute mixture to 100. 22.1 Raileanu 1930 6630, 104:285 formula: water 100, mercuric chloride 1.6, gold chloride 0.16 preparation: see Cajal 1916 (above) 22.1 Ziehen 1891 15058, 10:65 formula: water 100, mercuric chloride 0.5, gold chloride 0.5 preparation: see Cajal 1916 (above) 22.2 Neurological Methods 22.21 nerve cells and processes 22.21 Apathy 1893 23632, 10:349 reagents required: A. 1% formic acid; B.\% gold chloride method: [paraffin sections of material fixed in F 1300.0000 Apdthy 1893] -^ wash — * A, 1 min. -^ B, 24 hrs. -^ blot -^ A, evenly illuminated from both sides, till reduction complete -^ balsam via usual reagents note: Lee 1905, 254 substitutes 0.1% formaldehyde for the second usage of A above. 22.21 Lee 1905 see MS 22.21 Apathy 1893 (note) 22.21 Nabias 1904 6630, 66 :426 reagents required: A. ADS 12.2 Gram 1884; B. 1% gold chloride; C. sat. sol. aniUne method: [sections of material fixed in mercuric chloride] —> A, till yellow —>■ rinse — ^ B, 5 mins. -^ rinse —>■ C, till differentiated recommended for: ganglia of invertebrates. 22.21 Ogawa 1913 1798, 29 :248 reagents required: A. \% gold chloride; B. \% formic acid method: [smears, fixed in F 3000.0000 or F 3000.0010 mixtures] -^ wash -> A, 24 hrs. -^ wash -^ B, in direct sunlight, till purple -^ balsam, via usual reagents MS 22.21-MS 23.0 METAL STAINS 539 22.21 Ziehen 1891 15058, 10 :65 REAGENTS REQiiREo: A. MS 22.1 Ziclieii 1891; B. ADS 12.2 Lugol 1905 20, water 80 method: [fresh tissues] —> A, 1-0 months, till copper red — + [sections by freezing tech- nique] -^ B, till differentiated — > balsam, via usual reagents RECOMMENDED FOR: axis Cylinders and dendrites. 22.22 NEUROGLIA 22.22 Cajal 1916 21344, 14:155 reagents required: A. AMS 11.1 Cajal 1913; B. MS 22.1 Cajal 1916; C. AMS 24.1 Cajal 1913 method: [fresh tissue] — > ^, 2 to 10 days — > wash -^ [sections by freezing technique] — > rinse -^ 5, 4 to 6 hrs. -^ wash — > C, 6 to 10 mins. -^ wash, 40% ale. -^ balsam, via usual reagents 22.22 Raileanu 1930 6630, 104 :285 reagents required: A. 6% neutralized formaldehyde; B. AMS 11.1 Raileanu 1930; C. MS 22.1 Raileanu 1930; D. AMS 24.1 Raileanu 1930 method: [fresh tissue] -^ A, 24 hrs. at 37°C. — > [sections by freezing technique] —> B, 24 to 48 hrs. -^ wash — > C, in dark, 4 to 7 hrs., till deep violet -^ wash — > D, 15 mins. — » wash, 30 mins., 50% ale. -^ balsam, via usual reagents 23 METHODS USING GOLD IN OTHER COMBINATIONS 23.0 Typical Example Demonstration of the nervous elements in spinal cord by the method of Gerlach 1872 It is a pity that this method should have become obsolete and that it is today cited in so few textbooks. The method is simple and certain, hence it is suitable for class demonstration, and it shares with the method of Ranvier, already described, the distinction of being the only gold tech- nique that may reasonably be so em- ployed. It is not suitable for original research, since the structures which it displays are already well known, but it cannot be surpassed for a method of pre- paring demonstration material. Only four solutions are required, all of which are stable indefinitely. These solu- tions are: first, a 1% solution of ammo- nium dichromate, which must, of course, be prepared from a reagent of analytical grade; second, a 0.01% solution of gold chloride; third, 0.5% hydrochloric acid; and fourth, 0.1% hydrochloric acid in 60% alcohol. These solutions, with the exception of the gold, should be available in relatively large volumes. In the descrip- tion which follows it will be presumed that the method is being utiUzed for the in- struction of a class in an ele mentary tech- nique of gold staining. The instructor should first secure short lengths of spinal cord from a freshly killed mammal. The technique works equally well on the spinal cords of fish and am- phibia, but these are in general too small for convenient handling by a class. The cord should be cut into approximately one-inch lengths and placed in a large volume of the ammonium dichromate so- lution in a stoppered bottle. It is a matter of convenience that this bottle should have about a one-inch layer of fat-free absorbent cotton or of glass fiber on the bottom, to prevent the distortion of the spinal cord through pressure against the glass. The period of fi.xation should be from two to three weeks and is not critical. At the beginning of the week in which the class is to be held, the lengths of spinal cord should be removed from the reagent and washed in running distilled water for at least 24 hours. They may then be left in a bottle of triple-distilled water until required for class purposes. A freezing microtome can be used to cut sections for issue to the class, but the shape and hardness of the spinal cord makes it convenient for freehand section- ing, either between layers of the pith, or by any other method customarily used in the class in question. These freehand sec- 540 METHODS AND FORMULAS MS 23.21 tions should be as thin as possible, though der the low power of the microscope, it is the necessary structures can be seen in time to remove the sections, one at a time, sections as thick as 40 microns. These sec- to 0.1 % hydrochloric acid in 60% alcohol, tions should be washed in several changes where they may remain for a matter of of triple-distilled water, after preparation ten minutes. Immersion in acid of this by the class, and then placed, preferably concentration in alcoholic dilution does in glass-stoppered, chemicaUy clean bot- not cause much further reduction. After ties, in the gold-staining solution and left they have been thoroughly dehydrated to there overnight. If the class does not meet the extent possible in the 60% alcohol, on two successive days, it is probably bet- they should be removed and passed ter for the instructor to handle the whole through other watch glasses where they technique up to this point and to issue to are dehydrated and cleared by the ordi- the class the material in the gold-chloride nary reagents. The sections may then be solution. The sections are taken from the mounted in balsam. gold-chloride solution directly to the 0.5% Beginning students of microtomy are hydrochloric acid, where they are rocked frequently so frightened of the metal- gently backward and forward in a clean staining techniques that they ignore them watch glass for a few minutes. This will completely, and the real value of this reduce the gold and will be a useful lesson method is to provide each student of to the class on the perils of over-reduction, such a class with a gold-stained per- When the outhnes of individual nerve cells manent slide by a method which is very within the ganglia are clearly visible un- nearly foolproof. 23.1 Staining Solutions (Vacant) 23.2 Neurological Methods 23.21 nerve cells and processes 23.21 Ciaccio 1880 13495,10:301 REAGENTS REQUIRED: A. 0.2% acetic acid; B. water 100, gold chloride 0.1, potassium chloride 0.1; C. 0.1% osmic acid method: [fresh amphibian tendons] —> A, tUl transparent —> B, 5 mins. — * C, 1 day in dark and 3 hrs. in sunlight — » C, 24 hrs. -^ M 11 mountant recommended for: nerve endings in amphibian tendons. 23.21 Gerlach 1872 test. Lee 1905 cit. Strieker 1872 Lee 1905, 253 reagents required: A. 1% ammonium dichromate; B. 0.01% gold chloride; C. 0.5% hydrochloric acid; D. 0.1% hydrochloric acid in 60% ale. method: [fresh spinal cord] — > A, 15 to 20 days -^ wash -^ sections freehand, or by freezing technique -^ wash -^ B, 10 to 12 hrs. — > C, wash -^ D, 10 mins. —> balsam, via usual techniques 23.21 Golgi 1880 13497, 32 :382 REAGENTS REQUIRED: A. 2% potassium dichromate; B. 1% arsenic acid; C. 0.5% gold chloride method: [fresh tissue]-^ A, 10 to 20 mins. -^ wash ^^ B, 10 to 20 mins. -^ rinse C, 30 mins. — > wash — > B, in sunlight, until completely reduced -^ wash -^ glycerol 23.21 Kerschner 1908 1780, 71 :522 REAGENTS REQUIRED: A. F 1000.0030 Kerschncr 1908; B. 1% gold chloride; C. 25% formic acid method: [fresh tissue] — > A, until brown -^ wash -^ B, 2-6 hrs. in dark —>■ rinse — > C. 12 hrs. in dark — > C, fresh solution, 24 hrs. in light — > glycerol or M 12 mountant 23.21 Kolossow 1888 see MS 21.2 Kolossow 1888 MS 23.21-MS 30 METAL STAINS 541 23.21 Muschenkoff test. 1907 Bohm and Oppel Bohm and Oppel 1907, 267 REAGENTS REQUIRED: A. 2% potassium dichromate; B. 20% formic acid; C. 0.5% gold chloride; D. 0.1% acetic acid method: [small pieces] -^ A, \ month — > wash — * B, 15-20 mins. -^ rinse — > C, in dark, 30 mins. -* D, in light, till reduced — » M 10 mountant RECOMMENDED FOR: ncrvc endings. 23.21 Upson test. 1896 Kahlden and Laurent Kahlden and Laurent 1896, 160 REAGENTS REQUIRED: A. 1% potasslum dichromatc; B. 1% gold chloride in 2% hydro- chloric acid; C. 10% potassium hydroxide; D. water 75, sulfuric acid 10, ADS 12.2 Lugol (1905) 15, ferric chloride 0.3 method: [fresh spinal cord] -^ A, 4-6 mnths., in dark—* [2-3 mm. thick slabs]—* rinse — * 50% ale, 2-3 days — » 95% ale. tUl green, 2-4 wks. -^ [celloidin sections] — > B, 2 hrs. — * wash -^ C, 30 sees. — * D, till reduction complete -* wash -^ balsam, via usual reagents RECOMMENDED FOR: axis Cylinders. 23.21 Viallane 1883 test. Lee 1905 Lee 1905, 250 REAGENTS REQUIRED: A. 1% osmic acid; B. 25% formic acid; C. 0.02% gold chloride; D. 25% formic acid method: [arthropod tissues] -^ A, until light brown -^ B, 10 mins. -^ C, 24 hrs. in dark -^ 24 hrs. in light 23.3 Cytological Methods 23.3 Beams test. 1930 Guyer Guyer 1930, 150 reagents required: A. water 100, gold chloride 0.2, acetic acid 0.3; B. 5% sodium thiosulfate method: [3 M sections of F 1360.0010 Brouba 1930 material]—* water—* A, till differ- entiated -^ wash — * 5, 2 mins. — * wash — * balsam, via usual reagents recommended for: Golgi bodies in glands. 23.4 Other Methods 23.4 Kupffer 1876 1780, 12 :353 reagents required: A. 0.05% chromic acid; B. 0.01% gold chloride in 0.01% hydro- chloric acid method: [sections by freezing technique of fresh tissue] -^ A, 15 mins. — * rinse — * B, in shade, till violet — > wash -^ glj^cerol recommended for: connective tissue in liver 23.4 Kupffer 1899 1780, 5:219 reagents required: A. 0.01% chromic acid; B. water 100, 40% formaldehyde 0.01, gold chloride method: [frozen sections of fresh liver] — * A, 10 mins. -^ B, 30 hrs. — * wash — * balsam, via usual reagents recommended for: demonstration of astrocytes in liver. MS 30 Silver The techniques of silver staining are commonly compared to those of pho- tography, though the analogy cannot jus- tifiably be maintained, save in the case of the original process by Simarro (Gat- enby and Painter 1937, 477). Photography consists essentially in the reduction to me- tallic silver of particles of silver bromide, maintained in a colloidal environment, and which have been rendered unstable through the absorption of photon energy. Simarro impregnated living forms with solutions of potassium bromide and then sensitized them, as in the photograpliic methods of his day, by immersion in a solution of silver nitrate. Parts of these impregnated animals were then sectioned and exposed to light; such silver bro- 542 METHODS AND FORMULAS MS 30 niide as may have been present was then reduced to the metalHc form by a photograpliic developer. It is doubtful, however, whether or not metallic silver is the end product of any of the reac- tions employed in modern silver-staining techniques. There is no question in photography of any intermediate condition between the bromide and the metal, the various grada- tions of shade being dependent on the total mass of silver present. Many of the silver-staining techniques, on the con- trary, result in varying shades of brown, making it more probable that the end product is some dark colored silver pro- teinate, a hypothesis born out by the fact that some of these stains may be differ- entiated by exposure to distilled water. Thus, in the techniques of del Rio-Hor- tega (MS 33.31 del Rio-Hortega 1925) mitochondria are demonstrated by the process of washing out all impregnated material, other than mitochondria, in water. In the absence of any accurate informa- tion as to the method by which the results are produced, it is best to divide the tech- niques into four great classes according to the reagents employed. In the first class, silver is applied to the tissues as a solu- tion of silver nitrate to which, in some modifications, may be added alcohol or pyridine. Moreover, in many cases, so little pyridine is added that the amount of silver-pyridinium complex present is insignificant. The second class comprises the Bodian techniques in which silver is employed as the proteinate. The third class contains those techniques in which the application of silver nitrate is either followed, or replaced, by immersion in so- lutions containing silver-diammine com- plexes, secured usually by dissolving either silver hydroxide or silver carbonate in ammonia. The fourth class involves prior treatment of the tissues with a solution containing some other metalhc ion, such as a chromate or dichromate, with which the silver subsequently applied is known to react. The first class, designated below as MS 31, is usually referred to as the "Cajal technique," although this name is also associated with the third class. Cajal 1907 (21344, 8:21) distinguished eleven meth- ods by which his results could be pro- duced, and divided the possible reactions into eight classes. In broad outUne it may be said that these techniques involve either the exposure of fresh tissues to silver nitrate, and the subsequent reduction of the absorbed silver to a dark-colored com- plex by exposure to formaldehyde, or al- ternatively, the prior treatment of the tis- sues with a series of "accelerators," the purpose of which is to cause a greater differentiation of types of nervous struc- tures. These techniques are used prin- cipally for the demonstration of nervous structures ; the various modifications which have been proposed have been designated to bring one type of structure more into prominence than another. Much of the classic work in tracing nerve tracts, and in demonstrating fine nerve endings in tissue, was carried out by these methods. This, as are all the other classes, is sub- divided according to the purpose for which the technique is intended. The second class of stains employs a relatively new method, in which silver is adsorbed on the tissues from a silver pro- teinate solution, sometimes in the pres- ence of metallic copper. These techniques have the advantage that no special prepa- ration of the tissues is required and they may therefore be used on ordinary paraffin sections. The next great class, designated MS 33 below, is of the most varied application. Workers in this group, with which the names of Bielschowsky, Cajal, and del Rio-Hortega are associated, were able to adapt silver-staining techniques to the demonstration of the connective tissues of the central nervous system, through the discovery that prior treatment of the tissues with a variety of solutions (vary- ing from uranium nitrate to alcoholic ex- tract of cork crumbs) prevented the stain- ing of nervous elements and brought into contrast their supporting structures. If the silver is dei)osited from a colloidal en- vironment, which may be produced either from solutions of gelatin or in other ways, there is a complete inhibition of the ab- sorption of silver, either by nerves or MS 30 METAL STAINS 543 brain connective tissue, with the result that these techniques are commonly em- ployed for the demonstration of spiro- chetes in tissues and in smears. In general the techniques of class MS 33 are more certain and more accurate than those of the last class, in those cases in whicli it is desired to show a specified structure, but are less useful when it is desired to secure a good general stain of nervous tissues. The final class, here designated as MS 34, is known sometimes as the "dichro- mate-silver method," though other sub- stances than dichromate have been em- ployed. The techniques are experimental, yielding the most brilliant results when a successful impregnation is obtained, but for which it is almost impossible to specify conditions leading to success. Tissues are fixed for varying lengths of time in either potassium dichromate, dichromate-osmic acid, or dichromate-formaldehyde mix- tures. The length of time is entirely crit- ical, but results cannot be predicted or even reproduced with certainty. For this reason, it is usual to start with a large number of pieces in the prehminary fixa- tive, remove groups of these to silver at frequent intervals, and extract from each group a number of pieces after varying times in the silver. Among the numerous pieces thus treated, one may be found which will show tlie required condition. Tliis condition is curious and depends entirely upon the fact that there is not, as one might anticipate, a uniform precipi- tate of silver chromate or dichromate throughout the tissues. In certain cases, under conditions which it must again be emphasized cannot be anticipated, iso- lated nerve cells, with all their dendrites, will become impregnated with a dark- colored precipitate, while neighboring cells and neighboring dendrites will remain entirely unaffected. This is undoubtedly the best method by which the structure of such cells may be demonstrated. Un- successful impregnations show either a uniform granular precipitate throughout the whole tissue or else no staining of any kind whatever. The nature of the material precipitated onto the cell is unknown, though it has the pecuhar property of be- ing unstable unless exposed to oxygen. For this reason such preparations cannot be moimted in the conventional manner un- der a coverslip, but must be placed upon a slide and varnished either with balsam, dammar, or one of the special media recommended. The fugitive nature of the stain pro- duced by this last class, and the moder- ately fugitive nature of those obtained from the previous two classes, may be im- proved in either of two ways. The silver complex may be changed into a gold com- plex, or quite possibly to a colloidal dis- persion of metallic gold, by a process analogous to photographic toning, in which treatment by gold chloride, either alone or in some combination, results in the replacement of silver with gold. This technique may be apphed to any material, but in the formulas which follow it has only been specified if it is indicated by the original author. The second, and more satisfactory, method of securing perma- nency is to change the silver complexes, whatever they may be, to metallic silver itself. There are many methods by which this can be acliieved, the simplest being to expose the sections to a weak (10%) solution of hydrobromic acid until the silver complex has been changed to silver bromide. The sections are then exposed to bright Hght to render the bromide un- stable, and this compound is then reduced to metalUc silver with ordinary photo- graphic developers. Though Globus pro- posed the use of hydrobromic acid for quite another purpose, this technique is often associated with his name. The most important consideration in securing successful results by any silver technique is the absolute purity of all the ingredients used and the absolute cleanli- ness of the glassware employed. Ordinary distilled water is insufficiently pure ; triple- distilled water should be used. In many in- stances the shghtest trace of an impurity at any stage of the proceedings will com- pletely wreck what may be several weeks of subsequent work. The use of com- mercial formaldehyde, for example, in prior fixation should be avoided, and the grade sold as "analytical reagent" should be used. Another important step is to re- move all traces of one solution before plac- 544 METHODS AND FORMULAS MS 31.0 ing the object in the next. Thus, in those techniques in which the term wash is em- ployed, it is understood that all traces of the previous reagent must be re- moved, preferably by running triple-dis- tilled water, before going on to the next step. If running triple-distilled water is not available, it is necessary to make at least ten changes between large volumes of water. Another common cause of failure is the employment of too small volumes of reagents, many of which are weak solu- tions, but which are dependent for their success upon the maintenance of an excess of the solute. Even when working with pieces as small as a few-millimeter cube, it is rarely worth while to employ less than 50 millihters of a solution, in which the object should, moreover, be gently agi- tated from time to time. MS 31 METHODS USING SILVER NITRATE 31.0 Typical Examples Demonstration of the nervous elements of the retina by the method of Balbuena 1922 This technique is included for two rea- sons. First, it is the most complex silver nitrate technique which has yet been developed; second, the excellence of the results obtained in a satisfactory prepara- tion justify this complexity. As the tech- nique involves the utilization of a large number of somewhat unusual reagents, it is best to gather these first. They will be given in the numbered sequence in which they are employed: 1. AMS 11.2 Cajal 1910a. This is the pyridine alcohol of the well-known Cajal techniques, and is made by adding 50 millihters of pyridine to 200 milhliters of absolute alcohol. The solution is stable indefinitely. 2. AMS 11.2 Bal- buena 1922. This is Balbuena's "alcohohc extract of cork crumbs." It is prepared by placing one inch of cork crumbs on the bottom of a 250-miUiliter bottle. The crumbs are best prepared by cutting small pieces (of less than one-millimeter side) from a fresh bottle cork. The bottle is then filled with 70% alcohol and shaken at daily intervals until the solution is a dark yellow-brown color. The reagent is then filtered from the cork crumbs and may be stored indefinitely. 3. MS 31.1 Balbuena 1922 (Balbuena's silver stain). This is pre- pared by dissolving 0.13 grams of silver nitrate in 250 milhUters of triple-distilled water. After solution is complete 2.5 miUi- hters of pyridine are added. This solution will remain stable for a considerable time, if it is preserved in the dark. 4. AMS 21.1 Balbuena 1922. This is Balbuena's devel- oper, which requires the use of two solu- tions. The first of these is an alcoholic extract of amber. This is best prepared by taking 70 milliliters of commercially avail- able oil of amber and adding this to 180 milliliters of 80% alcohol. These are shaken together at daily intervals for seven days, at the end of which time the alcoholic extract is separated in a separa- tory funnel. This remains stable indefi- nitely. The second solution required for Balbuena's developer is a solution of 2.5 grams of hydroquinone in 250 millihters of triple-distilled water. This should be prepared as it is required, for it is very unstable. 5. AMS 22.1 Balbuena 1922, which is Balbuena's toning solution. It is a buffered solution of gold chloride pre- pared by dissolving 2.5 grams of sodium borate in 250 milliliters of triple-distilled water and then adding to this solution of 0.25 grams of gold chloride. This solution also remains stable indefinitely if kept in the dark. 6. A 5% solution of sodium thiosulfate. There will also be required for purposes of embedding; absolute alcohol, a mixture of absolute alcohol and ether in equal pro- portions, and the solutions of celloidin, given above as the standard requirements for celloidin embedding in Chapter 13. If the celloidin block is to be set, as is best in this instance, with the aid of anhydrous chloroform, this reagent will also be re- quired. Having made sure that these re- agents are available, one must next secure the material on which to operate. The retina of a rabbit is an excellent ma- terial on which to start. The eyeUds are removed from a freshly killed rabbit and MS 31.0 METAL STAINS 545 the eyeball removed from the orbit. The eye is then carefully washed in triple- distilled water, and an area about one- millimeter square is cut about three milli- meters to one side of the point of entry of the optic nerve. This usually demonstrates best the nervous elements of the retina. Many of these blocks may, of course, be taken from the same eye. These blocks are placed in the alcoholic pyridine fixing solu- tion in a chemically clean vessel. About 25 miUiliters of the fixative should be used for a piece of retina of the size indicated, and overnight fixation will be ample. The small pieces are removed directly from the fixa- tive to absolute alcohol, in which they should remain at least 12 hours, being gently agitated at intervals to prevent the accumulation of diluted alcohol on the bottom of the chemically clean, stoppered vessel containing them. The absolute alco- hol should then be replaced for a period of at least 24 hours by a mixture of equal parts of absolute alcohol and ether. It can- not be emphasized too frequently that the ether used for dehydration of specimens intended for celloidin embedding should be of the grade sold as "dried over so- dium" and not the water-saturated mate- rial commonly sold for anesthesia. The material is next transferred to the thinnest of the three solutions of celloidin for a period of not less than 12 hours. It is then transferred to the intermediate solution of ceUoidin for a further period of 24 hours, and finally placed in the thickest solution for a further 24 hours. With fragments as small as this, the simplest hardening tech- nique is to remove the piece from the syrupy solution in a clean, dry pipet, and to express the drop containing the piece from the end of the pipet directly into about ten milliliters of anhydrous chloro- form. Again it must be emphasized that this chloroform must be specially dried, preferably over calcium chloride, if the material is to cut well. This small drop of celloidin should remain in the chloroform only long enough to harden, and should under no circumstances be permitted to remain until the chloroform has pene- trated to the contained fragment of retina. A period of about 30 seconds will probably be sufficient before the drop of celloidin is removed to clean, 70% alcohol, where it may remain until it is convenient to con- tinue the process. After not less than one hour in the 70 % alcohol, the drop of hardened celloidin is removed and trimmed until only about one millimeter of celloidin remains on top of the fragment of retina. The trimmed block is now placed in the alcoholic ex- tract of cork where it may remain almost indefinitely. Balbuena, in his original method, suggests between 2 and 20 days. After the fragment has been (to quote Balbuena) "sensitized" in the alcoholic extract of cork, it is again removed to clean, 70% alcohol and washed until the greater part of the brown color has left the superficial layers of celloidin. The fragment is then oriented in thick syrupy celloidin, on the surface of a block of wood adapted to being held in the object holder of a sHding microtome, and the block, with the contained fragment, is then placed under a bell jar with chloroform, or allowed to evaporate in dry air until such time as it is hard enough to cut. Sections are cut with a knife which has been moistened in 70% alcohol and then accumulated in 70% alcohol until a suffi- cient number have been secured. The sections are transferred directly from 70% alcohol to a chemically clean watch glass containing the silver-staining solution. This is best held on a small tripod and is warmed, after the sections have been placed in it, until it begins to steam. The flame is then removed, the reagent allowed to cool down until steam is no longer evident, and then reheated to steaming. This cycle of heating and per- mitting to cool is repeated until the sec- tions have become yellowish. This may take anywhere from three to ten minutes according to the extent to which they have been previously sensitized in the alcoholic extract of cork. As soon as they are yellow-brown, they are allowed to cool in the silver solution. When this has reached room temperature, two or three drops of the alcoholic extract of amber are added to render the solution cloudy. Sufficient extract of amber should be added to render the silver solution defi- nitely milky but not creamy. 546 METHODS AND FORMULAS MS 31.0 As soon as this condition has been reached, add to the mixture as much of the hydroquinone sokition as one has added extract of amber. The watch glass is then gently shaken backward and for- ward until the hydroquinone solution, the milky precipitate of oil of amber, and the original silver solution are thoroughly homogenized. The sections should be watched in this bath as they darken. If, after about five minutes, the sections have not become a definite dark brown, a fresh bath should be prepared by taking more of the original silver stain, rendering it milky with the extract of amber, and then adding to it the required quantity of the hydroquinone solution. The sections should then be removed from the ex- hausted solution and placed in the fresh one. It is rarely necessary to make more than two changes, or to wait more than 10 or 15 minutes, before the sections have become satisfactorily browned. When tliis has occurred they are removed to triple- distilled water where they are very thor- oughly washed in at least five successive changes. The sections are removed from the distilled water directly to the gold toning solution, the dish containing which should be rocked gently backward and forward as though one were developing a photographic plate in a dish. Under this treatment the sections will be seen to change slowly from brown to a dark bluish purple. This change will normally take not more than a minute or two, and if it is not taking place sufficiently rapidly, it in- dicates either an insufficient washing of the section or that the gold has become decomposed through prolonged storage. Additional washing or the substitution of a freshly prepared gold solution will insure satisfactory toning within a few moments. When toning is complete the sections are fixed in the 5% sodium thiosulfate bath, which removes from them the last traces of unwanted silver material. The sections are then washed in at least three changes of triple-distilled water. One of the sections may then be taken, rapidly dehydrated in alcohol, cleared, and ex- amined under a coverslip with the highest power of the microscope. If the nervous elements are not stained at all, the whole batch of sections may be thrown away, and the operation carefully reviewed to decide the point at which the mistake oc- curred. If, alternatively, the entire section is found to be too greatly blackened, the situation may often be improved by pro- longed soaking in thiosulfate solution. If, however, directions have been followed carefully, it is probable that the nervous structures of the retina will be displayed better than they can be by any other technique. If this is the case, the sections may be removed, dehydrated, cleared, and mounted in balsam in the ordinary manner. Demonstrations of neuroblasts and axons of the developing spinal cord of a three-day chicken embryo by the method of Cajal 1910b This technique may be divided into three stages: first, the removal of the chicken embryo from the yolk, and its fixation; second, staining in silver nitrate; third, developing the stain. For the first operation it is necessary to assemble three fingerbowls, two Syracuse watch glasses, a four-ounce, chemically clean, glass- stoppered bottle, scissors, and forceps. It is, of course, also necessarj^ to have an egg which has been incubated for 72 hours. The reagents required are a liter of normal saline heated to around 37°C.; about 4 ounces of chemically pure alcohol, which may be either the ordinary commercial ab- solute alcohol or the latter diluted to 95 % as specified in the original formula; and a solution of 1.5% silver nitrate in triple- distilled water. A chemically clean eye- dropper type pipet is also required. One technique for removal of chicken embryo from the yolk has been described in Chapter 20, but a variation of this tech- nique is required in the. present case. First of all, fill three fingerbowls to within about one inch from tlie top with the normal saline. Break tlie egg into one of the fingerbowls. Breaking an egg, after 72 hours of incubation, into a fingerbowl in such a manner as to avoid also breaking the yolk, is an art to be learned only -with practice. The inexperienced should submerge the egg in saline, break out the air space so as to cause the embryo to drop away from MS 31.0 METAL STAINS 547 the shell, and then remove the shell, piece by piece, from one end of the egg, until a, hole is left of sufficient size for the yolk and attached embryo to be slid from it into the normal saline. Before going fur- ther, the embrj'^o is examined and a piece of filter paper taken of a size which will approximately fit one of the Syracuse watch glasses. A hole is then cut in this filter paper of a size that will leave about a J.^-inch gap around the embryo itself. The next stage is to take a pair of large scissors and make as few cuts as possible around the outside of the extra-embryonic area, so as to isolate the entire blastoderm containing the embryo. The fewer the cuts which are made, and the larger the scissors used, the less will be the leakage of yolk into the surrounding normal saline. It would be ideal if it were possible to re- move the embryo with only four cuts, but this requires a larger pair of scissors than is possessed by most technicians. What- ever method is adopted, the utmost care should be taken to get as little yolk as possible distributed through the normal sahne. A common cause of failure in this metal-staining technique is the carrying over of a considerable quantity of yolk to the final dish of sahne. As soon, there- fore, as the embryo has been separated from the yolk, it is taken with a pair of forceps and pulled gently backward and forward through the saline in the first fingerbowl until it ajDpears to be yolk-free. It is then picked up, preferably in a spoon, transferred to the second fingerbowl of clean saline, and again washed. Transfer- ence to the third fingerbowl, where a con- tinual rinsing of the embryo backward and forward should fail to disclose the slightest milky trace of yolk coming from it, com- j)letes the washing. If, however, milky trails of yolk are still observed coming from the embryo, it must be washed in a fourth batch of saline. Next take a chem- ically clean Syracuse watch glass, and transfer the embryo so that it lies in the watch glass with its ventral surface upper- most. This can most readily be established by observing the entry of the vitelline veins and arteries into the embryo. The saline carried over with the embryo is now removed with an eye dropper. The piece of filter paper with the hole in it is now dipped into clean saline and dropped on top of the embryo in such a manner that the embryo is centered in the hole. Next take the tip of a pair of fine forceps and make, as it were, a series of dots with these forceps on top of the filter paper in the region where the extra- embryonic membranes lie under it. Each time the tip of the forceps is pressed down on the filter paper, it causes the adhesion of the membrane to the paper. Fifty or 60 such dots, evenly spaced around the extra-embryonic membrane, will be none too many to insure proper adhesion. Now place a few drops of alcohol on the filter paper without getting any on the embryo itself. The purpose of this is to insure the adhesion of the embryo to the filter paper for ease in after-handling. Next place a few drops on top of the eml)ryo itself, wait a moment or two, and then very carefully and slowly fill the watch glass with al- cohol. Leave it for three or four minutes and then, pressing downward on the filter paper with the end of the pipet, move the whole filter paper backward and forward to make sure that the embryo is adhering to the filter paper and not to the watch glass. If the filter paper moves without the embryo, thus indicating that the ad- hesion of the embryo is to the watch glass, it is almost certain that the embryo has been placed upside down in the watch glass (that is, with its ventral surface against the glass) and there is nothing which can be done about it save to start with a new embryo. If, however, the embryo is attached to the filter paper, it may be picked up with a pair of forceps and removed to a stoppered, four-ounce jar of alcohol. This jar must be chemically clean. Tip the jar upside down from time to time to make sure that the water coming out of the embryo does not dilute the alcohol on the bottom. The embryo should remain in alcohol for at least 2-1 hours. Make up the solutions which will be re- quired. These are: 1.5% silver nitrate, of which 100 milliliters should be placed in a wide-mouthed, chemically clean, stop- j)ere(l l)ottle; and the developer (AMS21.1 Cajal 1910, Chapter 24) which is pre- 548 METHODS AND FORMULAS MS 31.0 pared by dissolving 2.5 Gm of p3a-ogallic acid in 250 milliliters of water and 15 milliliters of 40% formaldehyde. The hardened embryo is removed from the bottle of alcohol, and drained by touching the corner to a filter paper to re- move as much of the alcohol as possible. It is then dropped into the silver nitrate solution which has been heated to 35°C. It will immediately turn slightly brown, and will float at the top for some time. While it is still floating, place it in an oven at 35°C., in the dark, and leave it for four or five days. It should, however, be examined at daily intervals, in case some material has been carried in which is caus- ing the precipitation of the silver. This will immediately become apparent if the inside of the bottle or the floor of the bottle becomes covered with a brownish or black stain. Should this be observed, immediately remove the embryo to a fresh 1.5% silver nitrate. When the embryo has been sufficiently impregnated, it is washed in triple-dis- tilled water. Damage from shrinkage will be minimized if the wash water be heated to about 25°C., on the assumption that the developing solution which will be used next will be at about 20°C. It is not neces- sary, nor indeed desirable, to remove much of the silver nitrate, the main pur- pose of the rinse being to avoid carrying over the silver nitrate solution to the de- veloping solution. This latter should be in a chemically clean, wide-mouthed, stop- pered bottle, and at least 100 milliliters will be required for the development, which is carried out at room temperature, preferably in the dark, for about 24 hours. The embryo will become black in the developer, and there is no means of finding out whether the impregnation has been successful until it has_been sectioned. Paraffin sections about eight microns thick are now prepared, mounted on slides, dewaxed, and graded down through alcohol into water in which they can be examined under a low-power objective. They should at this point sliow^ neuro- blasts and neurofibrils completely; black- ened, with little or no blackening of other portions of the embryo, save the periphery on which some deposition of black mate- rial is inevitable. If the sections on examination should prove to be uniformly blackened through- out, indicating either overimpregnation or overdevelopment, or far more probably the utilization of impure reagent, nothing can be done about it save to throw the sections away and start with a fresh embryo. If, however, the impregnation is not sufficiently heavy — that is, if the neuroblasts and neurofibrils are perfectly apparent but are only stained a light brown rather than the dense black which should be observed — they may be toned, and at the same time increased in contrast, with Cajal's gold toning solution, which will be found in Chapter 24 as AMS 22.1 Cajal 1910. SHdes are taken directly from distilled water and placed in this solution, and examined at intervals. They will turn from a brown to a purplish shade, and will become darker in so doing. The reaction may be stopped at any point by removing the sections from the toning solution and washing them in distilled water. When the desired degree of intensity has been reached, the sections are thor- oughly washed in distilled water, up- graded through alcohols, and mounted under a coverslip in balsam after the usual reagents. Demonstration of spirochetes in sec- tions by the method of Dieterle 1927 This is a simple method for the demon- stration of spirochetes in post-mortem material. It can be used after many kinds of fixation, though it is usually preferable to take the formaldehyde-fixed material. This is sectioned either by the paraffin or the freezing technique, the latter being so customary in pathological work, that it has given rise to the supposition that this method alone can be used. Better demon- strations for teaching, as distinct from diagnostic, purposes are, however, ob- tained from paraffin sections of about eight to ten microns in thickness. These sections are mounted on slides in the normal way and the slides are accumu- lated in distilled water. Four solutions are required, two of which must be main- MS 31.0 METAL STAINS 549 tained at 55°C. The stain depends for its effectiveness, first, on the inhibition of the staining of nervous elements by exposure to uranium nitrate, and second, on the selective deposition of the silver from a colloidal environment which is, in this case, produced with gum mastic. All four solutions should be prepared before the sections are started. The first solution is 1% uranium (uranyl) nitrate in 70% alcohol. Both the metaUic salt and the alcohol itself should be chemically pure, should be kept in chemically clean bottles, and used in chemically clean dishes. The next solution is a 10% solu- tion of gum mastic in absolute alcohol. This is best prepared by selecting clear, transparent, hght-colored pieces of gum mastic from a large quantity of the gum, grinding the selected pieces of gum mastic of the required weight with dry sand in a mortar, then flooding tliis mixture with absolute alcohol, and shaking until solu- tion is complete. The sand is then re- moved by filtration through glass wool or some other relatively coarse material. The tliird requirement is a 1% solution of silver nitrate in triple-distilled water and must again, of course, be maintained in chemically clean bottles. The fourth solution, which is the developer, is very complicated, but is necessary in the peculiar circumstances under which this technique is employed. The formula will be found under AMS 21.1 Dieterle 1927; but it is so difficult to make up that the brief description there given must be aug- mented. In the first place, mix 150 milli- liters of triple-distilled water with 30 of reagent grade acetone. The technique will be seriously impaired if a low quaUty of acetone is employed. In this mixture, dis- solve first 43»^ grams of hydroquinone, and after the solution is complete, a 9-4-gram of anhydrous sodium sulfite. When the solution of sodium sulfite is complete, add first 30 milliliters of pyridine, and second 30 milhliters of 40 % neutraUzed formalde- hyde. This forms, when it has been brought by triple-distilled water to a total of 270 milhliters, a relatively stable solu- tion. To this then add, drop by drop, ten millihters of a 10 % solution of gum mastic. This emulsion is stable for some days, and can usually be re-emulsified by shaking, should it tend to separate. When these solutions have been pre- pared, and an adequate supply of chem- ically clean coplin jars provided, the sec- tions are immersed for 30 minutes at 55°C. in the uranium nitrate solution. They are then washed in at least three changes of triple-distilled water before being placed in 90% alcohol for two minutes. When they are sufficiently dehydrated they are flooded with 10% gum mastic, or placed in a jar of 10% gum mastic for about 30 seconds. It is less essential to impregnate each individual section than to insure that there is a sohd film of the solution over the surface of the slide. Each slide is then removed individually, drained by one corner to remove surplus gum mastic, and the back of the shde hghtly wiped. They are then rinsed very, very briefly in 90% alcohol to remove the remains of the gum mastic from the back of the shde, and to make sure that drops of gum mastic do not remain between the sections. Immedi- ately after this brief rinse, they are dropped into triple-distilled water where the gum mastic is, of course, precipitated in a colloidal form over the surface and through the material of each section. Each shde is best handled individually in its successive changes through alcohol, mas- tic, and water, all the slides then being accumulated together in triple-distilled water. The utmost care must be taken to pro- vide a chemically clean coplin jar in wliich to place the silver nitrate solution which is next used. This is preheated to 55°C., preferably by leaving it for some hours in the embedding oven. It is then removed briefly from the oven, the slides are dropped into it, and it is returned to the oven for from one to six hours. It is essen- tial that it be kept in the dark during this period. After removal from silver nitrate the sections should be a very pale yellow color. If they appear blackened, it is usu- ally from some impurity in the gum mastic, and there is nothing to do save to take a fresh sample of gum and to experi- ment with it. After removal from silver nitrate, the sections are washed in at least three 550 METHODS AND FORMULAS MS 31.1 changes of triple-distilled water. It is es- microscope, that the gum mastic has been sential at this stage to remove the whole removed. If many slides are being treated of the silver nitrate from the gum mastic in one coplin jar, it is usually better to complex which covers the sections. treat them for about five minutes in a After the sections have been ade- first bath of alcohol and then to pass them quately washed, they are placed in the to a second, clean alcohol bath for at least milky developing solution for from 5 to 15 ten minutes. A very simple test is to lift minutes, or until they become dark brown, the slide from the alcohol and to permit it They should not turn completely black, to drip into some convenient vessel of dis- and it is much better to develop them too tilled water. If the least cloudiness is little than too much. After removal from observed, the slide must be placed in clean the developing solution, they are thor- alcohol and re-treated. Tliis must be re- oughly washed in several changes of peated until such time as the drops of triple-distilled water. This is essential, be- alcohol falling from the surface fail to go cause the developing solution itself will cloudy in distilled water. The sections are tend to darken by oxidation, and, if left in then treated in acetone (to remove any the tissue, will itself become so dark that alcohol-insoluble fraction of the gum it will mask the spirochetes. The sections mastic which may remain), cleared in are then placed in 95% alcohol, in which xylene, and mounted in balsam. Success- they are left until it is apparent, on ex- ful preparations show spirochetes in black amination under a low power on the against a gray background. 31.1 Staining Solutions In all these solutions the silver nitrate is first dissolved in water. The other ingredients are then mixed and added to the silver solution. 31.1 Balbuena 1922 21344,20:31 FORMULA : water 100, silver nitrate 0.05, pyridine 1 31.1 Bauer 1944 608b, 20:297 formula: water 50, 95% ale. 50, acetic acid 0.2, silver nitrate 1.5 31.1 Cajal 1910 21344, 8:5 formula: water 72, silver nitrate 1.4, 95% ale. 28 31.1 Cajal 1921 21344,19:71 formula: water 100, silver nitrate 2, pyridine 2 31.1 Cajall925 21344,23:23 formula: water 60, silver nitrate 1.2, pyridine 2, 90% ale. 30 31.1 Cajal 1925 (est. 1933 /p.s. Cajal and de Castro 1933, 262 formula: water 100, silver nitrate 2, pyridine 3, 40% formaldehyde 5 31.1 Cajal 1929 21344, 26:1 FORMULA : water 100, silver nitrate 2, pyridine 0.3 31.1 Davenport 1930 1879, 24:690 formula: water 10, silver nitrate 10, 90% ale. 90, nitric acid 0.4 31.1 Lauda and Rezek 1928 22575, 269:218 formula: dissolve with heat 0.1 gelatin in 100 water. Cool. Add 3 silver nitrate. 31.1 Levaditi test. 1916 Warthin 4349, 6:56 formula : water 90, pyridine 10, silver nitrate 0.9 31.1 McManns 1943 11431,65:503 formula: water 100, silver nitrate 20, chloral hydrate 1 31.1 Podhradszky 1934 23632, 50 :285 formula: water 10, 95% ale. 90, silver nitrate 10, nitric acid 0.3 MS 31.1-MS31.21 METAL STAINS 551 31.1 del Rio-Hortega 1921 3232, 21:1 formula: water 100, silver nitrate 2, pyridine 1, 95% ale. 5 31.1 del Rio-Hortega 1932 lesl. 1933 Cajal and de Castro Cajal and de Castro 1933, 262 formula: water 100, 95% ale. 1.5, silver nitrate 2, pyridine 1.5 31.1 Shanklin 1951 Cowdry 1952, 270 formula: water 100, silver nitrate 10, pyridine 1 31.2 Neurological Methods 31.21 nerve cells and processes 31.21 Ascoli 1911 3381,25:177 REAGENTS REQUIRED: A. 5% silvcr uitrate in 95% ale; B. 10% silver nitrate; C. AMS 21.1 Ascoli 1911; D. glycerol; E. AMS 22.1 Cajal 1910 method: [leeches, slit open and tied, not pinned, round a cork] — > A, till hard, few mins. — * A, fresh solution after removal from cork, 24-48 hrs. at 40°C. — ^ B, 24-48 hrs. -> rinse -^ C, 5-8 hrs. -^ wash -^ D -^ teased preparations -^ E, 5 mins. —> M 11.1 Apathy 1892 RECOMMENDED FOR: lecches. 31.21 Balbuena 1922 21344, 20:31 REAGENTS REQUIRED: .4. AMS 12.1 Cajal 1910a; B. AMS 12.1 Balbuena 1922; C. MS 31.1 Balbuena 1922; D. AMS 21.1 Balbuena 1922; E. AMS 22.1 Balbuena 1922; F. 5% sodium thiosulfate method: [small pieces] -^^ .4, 12-48 hrs. -^ [sections by celloidin technique] ^ B, 2 to 20 days —>■ rinse, 70% ale. -^ drain —>■ 20 ml. C. heated to steaming, 3-10 mins. -^ cool — > add to sections in 20 ml. C, 2 or 3 drops D {A sol.) — + shake gently —> add 2 or 3 drops D {B sol.) — > shake gently, leave 10-15 mins. —>■ wash —^E,5 mins. —> F, 2 mins. — > balsam, via usual reagents RECOMMENDED FOR: retina. note: a detailed description of this technique is given under MS 31.0 above. 31.21 Bartelmez 1915 11135, 25:87 reagents required: .4. F 0000.0010 Bartelmez 1915; or F 0000.0010 Bartelmez 1915; B. 1.0%, silver nitrate; C. 1.5% silver nitrate; D. 2% silver nitrate; E. AMS 21.1 Cajal 1910 method: [fish larvae] —> A, 60-90 mins. — > 80% ale, wash —>■ wash —>■ B, 24 hrs. 35°C. -^ C, 24 hrs. 35°C. — >• [repeat B -^ C —^ D cycle, till material becomes brown] —> rinse — + E, 24 hrs. — » wash -^ [sections by paraffin technique] recommended for: fish larvae. 31.21 Blair and Davies 1935 590, 69 :303 reagents required: .4. water 81, 40% formaldehyde 9, ammonia 5; B. pyridine; C. 2% silver nitrate; D. DS 21.1 Noguchi 1913 method: [pieces fixed in neutralized formaldehyde] —> A, 2 days -^ wash, 2 days—* B, 2 days —> thorough wash, distilled water — + C, 4 days, 35°C. — > thorough wash — » D, 24 hrs. — > wash — > [sections] recommended for: nerves in heart. 31.21 Boule 1908 15063, 10:15 reagents required: A. F 0000.1010 Boule 1908 or AMS 12.1 Boule 1908; B. 3% silver nitrate in 15% alcohol; C. Boule 1908 AMS 21.1 method: [small pieces] —>■ A, 24-48 hrs. — > drawn or blot — > i5, 7 days, 35°C. -^ rinse, 15% alcohol — > C, 24 hrs. — > [section by paraffin technique] 31.21 Buxton test. 1930 Eltringham Eltringham 1930, 91 REAGENTS REQUIRED: .4.1% silvcr nitrate; B. 1.5% silver nitrate; C 1% gold chloride; D. 2% pyrogallol method: [fresh material] —> .4, 10 days, in dark — » [paraffin sections] — > water — > B, 10 mins., bright sunlight — » wash — > C, 2 mins. — > Z>, 5 mins. — » wash — » balsam, via usual reagents recommended for: insect brains. 552 METHODS AND FORMULAS MS 31.21 31.21 Cajal 1910a methods 1, lA, IB, iC—auct. 21344, 8:3 REAGENTS REQUIRED: A. 1.5% silver nitrate (or see below); B. AMS 21.1 Cajal 1910; C. AMS 22.1 Cajal 1910 METHOD 1 : [fresh tissue] -^ A, 3-4 days, 35°C. -^ rinse -* B, 24 hrs. -* [section by paraffin technique, bring sections to water] -* C (optional), till faint stains sufficiently dark- ened — * balsam via usual reagents RECOMMENDED FOR! nerve cells and processes in embryos in lower mammals. Method lA: Substitute 5% silver nitrate for A above. RECOMMENDED FOR: scnsory nerve endings, invertebrates. Method IB: Substitute 0.75% silver nitrate for A above. RECOMMENDED FOR: Very young embryos. Method IC: Substitute MS 31.1 Cajal 1910 for A above. RECOMMENDED FOR: human tissues and embryos. 31.21 Cajal 1910b methods 2, 2 A, 2B, 2C, 2D—auct. 21344, 8:7 REAGENTS REQUIRED: A. 95% alcohol (or see below); B. 1.5% silvernitrate; C. AMS 21.1 Cajal 1910; D. AMS 22.1 Cajal 1910 METHOD 2: [fresh tissue] — » A, 24 hrs. -^ drain or blot -^ B, 4-5 days, 35°C. -^ rinse — > C, 24 hrs. — > [section by paraffin technique, bring sections to water] — > D (optional), till faint stains darkened — * balsam, via usual reagents RECOMMENDED FOR: general use. Method 2A: Substitute AMS 12.1 Cajal 1910d, for A above. RECOMMENDED FOR: general use when method #2 is unsatisfactory. Method 2B: Substitute AMS 12.1 Cajal 1910a, for A above. RECOMMENDED FOR: peripheral nerve endings. Method 2C: Substitute AMS 12.1 Cajal 1910e, for A above. RECOMMENDED FOR: Spinal cord. Method 2D : Substitute AMS 12.1 Cajal 1910, for A above, and precede A by 24 hrs. in allyl alcohol. RECOMMENDED FOR: human cerebrum and cerebellum. 31.21 Cajal 1910c methods 3, SA, 3B—auct. 21344, 8:9 REAGENTS REQUIRED: A. AMS 12.1 Cajal 1910 (or see below); B. 1.5% sUver nitrate; C. AMS 31.1 Cajal 1910; D. MS 22.1 Cajal 1910 METHOD 3 : [fresh tissue] -^ A, 20 to 48 hrs. -^ drain or blot — >• 5, 4 days at 40°C. or until light gray -^ rinse -^ C, 24 hrs. — > [sections by paraffin technique, bring sections to water] -^ D (optional) till faint stains darkened — > balsam, via usual reagents RECOMMENDED FOR: neurofibrils of large cells. Method 3A: Substitute AMS 12.1 Cajal 1910f for A above. RECOMMENDED FOR: buds of Auerbach. Method 3B: Substitute AMS 12.1 Cajal 1910g for A above. RECOMMENDED FOR: buds of Held. 31.21 Cajal 1910d methods 4, 4A—auct. 21344, 8:11 REAGENTS REQUIRED: A. 4% ueutral formaldehyde; B. AMS 12.1 Cajal 1910; C. 1.5% silver nitrate; D. AMS 21.1 Cajal 1910; E. AMS 22.1 Cajal 1910 METHOD 4: [fresh tissue] — > A, 6-8 hrs. — > running water, 6 hrs. — > B, 24 hrs. — > drain or blot — > C, 4 days at 40°C. — > rinse -^ D, 24 hrs. — > [section by paraffin technique, bring sections to water] — > E (optional), till faint stains darkened -^ balsam, via usual reagents RECOMMENDED FOR: arborizations in adult cerebellum. Method 4A: Substitute fixative of class F 0000.1000 for A above. RECOMMENDED FOR: neurofibrils of large cells. 31.21 Cajall910e method 5— auct. 21344,8:12 REAGENTS REQUIRED: A. 50% pyridine; B. pyridine; C. 90% alcohol; D. 1.5% silver nitrate; E. AMS 21.1 Cajal 1910; F. AMS 22.1 Cajal 1910 METHOD 5: [fresh tissue] — > A, 6 to 8 hrs. — > B, 18-24 hrs. — > running water 6 hrs. -^ C, 24 hrs. —>■ drain or blot -> D, 4-5 days, 35°C. — » rinse — ♦ D, 24 hrs. -^ [section by MS 31.21 METAL STAINS 553 paraffin technique, bring sections to water] — » F (optional), till faint stains darkened — ♦ balsam, via usual reagents RECOMMENDED FOR: neurogenesis and regenerative processes. 31.21 Cajal 1910f methods 6, GA—aud. 21344, 8:14 REAGENTS REQUIRED: A. 10% chloral hydrate) B. AMS 12.1 Cajal 1910 (or see below); C. 1.5% silver nitrate; D. AMS 21.1 Cajal 1910; E. AMS 22.1 Cajal 1910 METHOD 6: [fresh tissue] -^ A, 24 hrs. — > rinse — > B, 24 hrs. -* drain or blot -^ C, 4-5 days, 40°C. -^ rinse -^ D, 24 hrs. — > [section by paraffin technique, bring sections to water] — » E (optional), till faint stains darkened RECOMMENDED FOR: PurkinJG cells, motor end plates. Method 6A: Omit B from above. 31.21 Cajall921 21344,19:71 REAGENTS REQUIRED: A. AMS 31.1 Cajal 1921; 5. 95% alcohol; C. AMS 21.1 Cajal 1921; D. AMS 22.1 Cajal 1921 method: [sections of formaldehyde material by freezing technique] -^ wash -^ A, 12-48 hrs. or till light brown -* B (optional), 30 sees. -^ blot or drain -^ C, 24 hrs. -^ D, till axons of meduUated fibers — > balsam, via usual reagents RECOMMENDED FOR: axons of meduUated fibers. 31.21 Cajal 1925a 21344,23:237 REAGENTS REQUIRED: A. MS 31.1 Cajal 1925; B. AMS 21.1 Cajal 1925; C. 0.2% gold chloride; D. 5% sodium thiosulfate. method: [30-40 m sections, by freezing technique, of formaldehyde material] -> A, few mins. or till hght brown —> rinse, 95% alcohol-* B, 5-15 mins. -* wash — > C (op- tional), tiU required shade -+ D, 5 mins. -^ wash — * balsam, via usual reagents RECOMMENDED FOR: aXOnS. note: This method is frequently confused in the literature with MS 33.21 Cajal 1925. 31.21 Cajal 1925b test. 1933 ips. Cajal and de Castro 1933, 362 REAGENTS REQUIRED: A. AMS 12.1 Cajal 1925; B. 2% silver nitrate; C. AMS 21.1 Cajal 1925; D. 0.2% gold chloride; E. 5% sodium thiosulfate method: [frozen sections of formaldehyde-fixed material] — > A, 1-2 hrs. -^ quick wash -> B, 1-5 mins., 45°-50°C. —> 95% ale, quick rinse -^ C, 1-3 mins. -^ wash -* D, till gray —* E, 5 mins. -^ balsam, via usual reagents recommended for: nerve endings in tongue muscle. 31.21 Cajal 1929 21344, 26:1 REAGENTS REQUIRED: A. AMS 11.1 Cajal 1929; B. AMS 12.1 Cajal 1929; C. MS 31.1 Cajal 1929; D. AMS 21.1 Cajal 1929; E. any AMS 22.1 sol. method: [fragments of fresh tissue] -^ A, 1-2 days — > frozen sections -^ B, till required -^ C, 10 hrs. ^95% ale, 24 hrs. -^ D, 12 hrs. -♦ wash -* E, 10 mins. -^ balsam, via usual reagents RECOMMENDED FOR: sections of cerebellum. 31.21 Cajal 1927 test. 1933 ips. Cajal and de Castro 1933, 188 REAGENTS REQUIRED: A. AMS 12.1 Cajal 1927; B. 1.5% silver nitrate; C. AMS 21.1 Cajal 1910 method: [fragments of fresh tissue] -^ A, 24-48 hrs. -♦ wash, overnight^ B, 3 days, 40°C. — > rinse — >• C, 24 hrs. —* [sections] RECOMMENDED FOR: nerve fibers. 31.21 Cajal 1930 test. 1933 ips. Cajal and de Castro 1933, 190 REAGENTS REQUIRED: A. 5% neutralized formaldehyde; B. 70% pyridine; C. 1.5% silver nitrate; D. AMS 21.1 Cajal 1910 method: [fragments of fresh tissue]-* A, 2-3 days—* wash-* B, 1-2 days-* running water, 24 hrs. -* C, 3 days, 37°C. -* wash -^ D, 24 hrs. -^ celloidin sections recommended for: brains of small mammals. 554 METHODS AND FORMULAS MS 31.21 31.21 de Castro 1926 21344, 23:427 REAGENTS REQUIRED: A. AF 21.1 (le Castro 1926; B. 0.3% ammonia in 95% alcohol; C. 15% silver nitrate; D. AMS 21.1 Cajal 1910; E. 0.2% gold cliloride; F. 5% sodium thiosulfate method: [fresh material] — * A, till decalcified -^ wash, 24 hrs. — » slice, 1 mm. thick -^ B, thorough wash — > wash — > C, 5-7 days, 37°-40°C. -^ wash — > D, 3-4 hrs. — > wash — * [sections] -^ E, 1-5 mins. — > F, 5 mins. — » wash — > balsam, via usual reagents RECOMMENDED FOR: nervcs in teeth. 31.21 Cher 1933 1879, 29:344 REAGENTS required: A. \% ammonia in 95% ale; B. pyridine; C. 2% silver nitrate; D. AMS 21.1 Chor 1933 method: [fresh muscle] -^ A, 24 hrs. -^ wash -^ B, 48 hrs. — > thorough wash — > C, in dark, 72 hrs. — > rinse—* D, 6-8 hrs. ^ rinse —> 95% ale.,-* [paraffin sections by usual techniques] recommended for: motor end plates in primate biceps. 31.21 Cowdry 1912 10157, 29:1 reagents required: A. 1.5% silver nitrate; B. AMS 21.1 Cowdry 1912; C. 0.1% gold chloride; D. 5% sodium thiosulfate method: [pieces fixed in F 0000.0010 Carnoy 1887] -^ water -^ A, 3 days at 40°C. -* rinse — * B, 24 hrs., in dark -^ [paraffin sections] — > water — » C, 2 hrs. — > rinse -^ D, 5 mins. -^ balsam, via usual reagents recommended for: nerve fibrils. 31.21 Davenport 1930 1879,24:690 REAGENTS REQUIRED: A. MS 31.1 Daveuport 1930; B. AMS 21.1 Davenport 1930; C. 0.01% gold chloride; D. 5% sodium thiosulfate method: [celloidin sections mounted on slides and varnished with 2% celloidin] —+80% ale, 5 mins. —* A, overnight or till yellow -^ ale, rinse — > 5, 2 mins. — > ale, wash — > C (optional), till desired color — + D (optional), 2 mins. -^ ether-alcohol to remove celloidin — > balsam, via usual reagents RECOMMENDED FOR: neurofibrils. 31.21 Davenport, Windle, and Beech 1924 20540b, 9:5 REAGENTS REQUIRED: A. 2% ammonia in 95% alcohol; B. 5% pyridin; C. 1.5% silver nitrate; Z). 4% pyrogallic acid method: [fresh embryos]-^ A, 2 days — > B, 24 hrs. — > wash, 1-3 hrs. — > C, 2-3 days, 37''C. — > wash 20 mins. (12 mm. embryos) to 1 hr. (20 mm. embryos) —* D, 4 hrs. —* wash —> section RECOMMENDED FOR: embryos. 31.21 Dogiel test. 1933 Cajal and de Castro Cajal and de Castro 1933, 357 REAGENTS REQUIRED: .4. 1.5% formic acid in 90% ale; B. 2.5% silver nitrate; C. AMS 21.1 Cajal 1910 method: [pieces of fresh tissue] —*• A, 1-2 days—* B, rinse —> B, fresh sol., 5-6 days, 36°C. -* wash -* C, 24 hrs. -^ [sections] RECOMMENDED FOR: corpuscles of Graudry and Herbst. 31.21 da Fano 1920 11360, 40:157 REAGENTS REQUIRED: A. AMS 11.1 da Fano 1920; B. 1.5% silver nitrate; C. AMS 21.1 Cajal 1914; D. 0.2% gold chloride; E. 5% sodium thiosulfate method: [small fragments fresh tissue] — > A, 6-8 hrs. — > rinse — > B, 24-48 hrs. — * rinse — > C, 24-48 hrs. -^ wash — > [section by paraffin technique, bring section to water] — * D, 2 hrs. -^ E, 5 mins. -* balsam, via usual reagents 31.21 Favorsky 1930 766,70:376 REAGENTS REQUIRED: A. 10% ammonia in 95% alcohol; B. pyridine; C. 2% silver nitrate; D. AMS 21.1 Cajal 1910a method: [pieces from F 0000.0010 Favorsky 1930]-^ 50% ale, wash-* A, 2 days -^ wash -^ B, 1-2 days -^ wash — > C, 4-10 days, 37°C. -^ rinse -^ D, 24 hrs. -* wash -^ [paraffin sections] MS 31.21 METAL STAINS 555 31.21 Foley 1938 20540b, 13 :5 REAGENTS REQUIRED: A. pyridine; B. 1% ammonia in 80% alcohol; C. 40% silver nitrate; D. MS 33.1 Davenport 1930; E. AMS 21.1 Davenport 1930; F. 0.2% gold chloride; G. 5% sodium thiosulfate method: [sections of material from F 5000.1010 Boiiin 1897 or F 5000.1080 Foley 1938] —> A, I hr. -^ abs. ale, rinse -^ [varnish on slide with celloidin] — > B, 12-24 hrs. -^ 80% ale, rinse -^ C, 6-8 hrs. 37°C. -> D, 16-24 hrs. -^ 95% ale, rinses E, till fine axons chocolate brown — » 95% ale, rinse —* tap water, wash -^ F, 10 mins. — > wash — > G, 3-5 mins. -^ wash —^ dammar, via alcohol and acetone RECOMMENDED FOR: axons in picric fixed material. 31.21 Foley 1939 763, 73:465 REAGENTS REQUIRED: A. AMS 12.1 Foley 1939; B. a graded series of 50%, 40%, 30%,, 20%, and 10% alcohol each containing 15% pyridine; C. a graded series of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, and 90% aqueous pyridine; D. pyridine; E. 10% silver nitrate; F. AMS 21.1 Ranson 1914 method: [stretched nerves] -^ A, 4°C., 24 hrs. -^ B, 30 mins. in each -^ C, 30 mins. in each (in 50% pyridine remove nerve from stretcher and thread through block of cerebral cortex preserved in 50% pyridine) —^ D, 24 hrs. — > C, in reverse order, 30 mins. in each -^ water, thorough wash — > E, 3-5 days, 37.5°C. —y rinse —* F,2 days — » [paraffin sections] 31.21 Gooding and Stewart 1937 11977, 7:596 REAGENTS required: A. 0.3% ammonia in 90% alcohol; B. 5% nitric acid; C.2% silver nitrate; D. AMS 21.1 Gooding and Stewart 1937 method: [pieces of teeth] — ^ A, 48 hrs. -^ rinse -^ B, 48 hrs. -^ running water, 24 hrs. —>■ C, 4-6 days, 37°C. — » rinse -^ D, 1-2 days, 37°C. -^ wash —> [paraffin or frozen sections] RECOMMENDED FOR: nerves in pulp of teeth. 31.21 Gurdjian 1927 1135,43:1 REAGENTS REQUIRED: A. 1% ammonia in 95% ale; B. AMS 11.2 Cajal 1910d; C. 0.75% silver nitrate; D. AMS 21.1 Ranson 1914 method: [whole brain stems] -^ A, 10-20 days, changing daily -^ drain and blot — > B, 3-5 days, changing daily — > rinse -^ C, 2-4 wks., changing twice weekly —> rinse —> D, 7-10 days — > [sections by paraffin technique] 31.21 Huber and Guild 1913 763, 7:253 REAGENTS REQUIRED: A. 1% ammonia in 95% ale; B.7% nitric acid; C. pyridine; D.2% silver nitrate; E. AMS 21.1 Ranson 1914 method: [entire head, taken from animals injected via the heart with A]—* A, 2-4 days — > wash -^ B, till decalcified — » wash -^^ ^, 3 to 8 days -^ rinse -^ C, 24 hrs. -^ D, 3 to 5 days, 35°C. in dark — > rinse — > E, 24 to 48 hrs. -^ [paraffin sections] 31.21 Jahnel 1917 14370, 42:17 REAGENTS REQUIRED: A. 10% formaldehyde; B. 1% uranium nitrate; C. 95% alcohol; D. 0.5% silver nitrate; E. AMS 21.1 Ranson 1914 method: [fresh tissue] —> A, 15 days or [old formaldehyde-fixed material]—* B, 1 hr., 37°C. — > wash — > C, 1 wk. — > rinse — > I), 1 wk., 37°C., in dark — » wash, in dark — » E, 1-2 days, in dark -^ wash — > [sections by paraffin technique] 31.21 Liesegang 1911 11848, 3:1 reagents required: A. 1% silver nitrate; B. AMS 21.1 Liesegang 1911 method: [sections of formaldehyde-fixed material by freezing technique] — * A, heated if necessary, till yellow —> pour off A, leaving thin layer, add B in excess-* wash — > balsam, via usual reagents 31.21 McManus 1943 11431,65:503 reagents required: A. MS 31.1 McManus 1943; B. AMS 21.1 Bodian 1936; C. 1% gold chloride; D. 2% oxalic acid; E. 5% sodium thiosulfate method: [4-6 n paraffin sections] — ♦ water -^ A, 30 mins., 60°C. — > wash — > B, 10 mins. -^ wash — * C, 10 mins. — > rinse — > D, till fibers distinct — > wash — > E, 5-10 mins. -♦ wash -^ balsam, via usual reagents recommended for: nerve fibers in spinal cord. 556 METHODS AND FORMULAS MS 31.21 31.21 Miller 1944 test. 1944 Randall 20540b, 19:122 REAGENTS REQUIRED: A. 20% silver nitrate; B. MS 33.1 Gros-Schultze (1938); C. 4% formaldehyde; D.2% gold chloride; E. water 100, 40% formaldehyde 1, oxalic acid 2; F. 5% sodium thiosulfate method: [sections of formaldehyde-fixed material] —> A , 1-132 hrs. -^ rinse -^ B, 3 mins.— » rinse — > C, 1 min. -^ wash -^ D, 10 mins. -^ wash -^ E, 20 sees. — > wash -^ F, 2 mins. — ^ wash -^ balsam, via usual reagents recommended for: nerve trunk of oligochaetae. 31.21 de No 1926 test. 1933 Cajal and de Castro Cajal and de Castro 1933, 368 reagents required: A. 50% pyridine; B. 3% nitric acid; C. 0.1% ammonia; D. 15% pyridine in 50% ale; E. 2% silver nitrate; F. AMS 21.1 Cajal 1910 method: [fresh tissue] -^ A, 24 hrs. -^ wash, 24 hrs. -^ B, till decalcified — > wash — » C, 24 hrs. -^ D, overnight -^ E, 6-7 days, 37°C. -* wash -^ F, 24 hrs. -^ wash -> [cel- loidin sections] RECOMMENDED FOR: nerve endings in structures requiring decalcification. 31.21 Okada 1929 8542a, 7:403 REAGENTS REQUIRED: A. 0.02% sodium hydroxide in abs. ale; B. 1.5% silver nitrate; C. AMS 21.1 Okada 1929; D. 5% sodium thiosulfate method: [1 mm. slices of fresh tissue] -^ A, 6-12 hrs. -^ thorough wash — > B, 3-5 days, 37°C. -^ C, 1-2 mins. -> [paraffin sections] -^ D, 15 mins. -^ wash -^ balsam, via usual reagents recommended for: neurofibrils and pericellular net. 31.21 Perez 1931 21344,27:187 reagents required: A. 15% chloral hydrate; B. 0.2% ammonia in abs. ale; C. 1.5% sUver nitrate; D. ADS 21.1 Cajal 1910a method: [2 mm. slices of fresh skin] — > A, 24 hrs. — > rinse -^ B, 24 hrs. — > wash, till rehydrated -* C, 7 days, 37°C. -^ wash -^ D, 24 hrs. -* wash -^ [paraffin sections] recommended for: Meissner's bodies in human skin. 31.21 Podhradszky 1934 23632, 50:285 . reagents required: A. MS 31.1 Podhradszky 1934; B. AMS 21.1 Podhradszky 1934; C. 1% gold chloride in 95% alcohol; D. 5%, pyrogallic acid in 95%, alcohol method: [5 n sections of F 0000.1010 Podhradszky 1934 fixed material, varnished to slide with collodion] -^ 80% ale. -^ A, in dark, 12 hrs., 15°C. -^ 1 hr., 37°C. -^ 95% ale, rinse -^ B, till reduced -^ 96 %o ale, wash -^ C, till gray -^ 95 %o ale, wash -^ D, 3 mins. — > abs. ale, rinse -^ balsam, via xylene 31.21 Rachmanov test. 1946 Roskin Roskin 1946, 257 REAGENTS REQUIRED: A. 10% silver nitrate; B. AMS 21.1 Rachmanov (1946); C. 5% sodium thiosulfate method: [sections of alcohol-fixed material] —> water ^ A, 24 hrs., 37°C. in dark -^^ wash — > B, 1-3 mins. — > wash -^ C, 3-5 mins. — * balsam, via usual reagents 31.21 Ranson 1914 766,46:522 reagents required: A. 1% ammonia in 95% ale; B. pyridine; C. 2% silver nitrate; Z). AMS 21.1 Ranson 1914 method: a, 48 hrs. -^ rinse -^ B, 24 hrs. -» C, 3 days, 35°C., in dark -^ rinse -^ D, 24 hrs. -^ [sections by paraffin technique] 31.21 Rasmussen 1938 7802, 23:263 reagents required: A. 0.2% ammonia in abs. ale; B. 3% nitric acid; C. 0.15% am- monia in 80% ale; D. pyridine; E. 2% silver nitrate; F. 0.75% silver nitrate; G. 4% pyrogallol method: [small pieces fresh hypophysis]—* A, 2-3 days ^ running water, 12 hrs.-* B, 2 hrs. -^ rinse -> 80% ale, several changes, few hrs. -^ C, overnight -> D, 12 hrs -* thorough wash ^^ E, in dark, 24 hrs. -* F, in dark, 24 hrs. -^ E, in dark, 24 hrs. -^ rinse — * G, 24 hrs. — > wash -^ [paraffin sections] recommended for: nerve fibers in hypophysis. MS 31.21 METAL STAINS 557 31.21 Sand 1910 6593, 12:128 REAGENTS REQUIRED: A. 10% iiitric aci(l in acetone; B. 20% silver nitrate; C. AMS 21.1 Sand 1910; D. AMS 23.1 Dand 1910; E. 5% sodium thiosulfate method: [small pieces fresh tissue] — > A, 1 hr. — > A, fresh solution, 24 hrs. — > A, fresh solution, 24 hrs. ^ [sections by paraffin technique, using acetone for dehydration; bring sections to water, via acetone] —* B, S hrs., 37°C. —^ C, 10 mins. — > rinse — > D, 5 mins. — > wash -^ E, 15 sees. — > wash -^ balsam, via usual reagents note: This method was republished later (Sand 1915: 4349, 5:71). 31.21 Schultze test. 1948 Romeis Romeis 1948, 418 REAGENTS REQUIRED: A. 0.5% sodium liydroxidc; 5. 2% silver nitrate; C. water 95, AMS 21.1 Schultze (1948) 5 method: [30-40 ^ frozen sections of formaldehyde-fi.xed material] — » wash -^ A, \ day — > wash, till wash water alkali-free (test with phenolphthalein) — ^ B, lG-24 hrs, — > C, till completely reduced -^ wash -^ balsam, via usual reagents recommended for: nerve bundles in cerebrum. METHOD FOR CELLS IN CEREBRUM: Substitute 0.04% sodium hydroxide for A and 0.5% silver nitrate for B. METHOD FOR MEDULLA, AND CEREBRAL, SPINAL, AND SYMPATHETIC GANGLIA: Substitute 0.8% sodium hydroxide for A and 10% silver nitrate for B. METHOD FOR CEREBELLUM: Substitute 0.16% sodium hydroxide for A and 0.25% silver nitrate for B. METHOD FOR PERIPHERAL NERVES: As for medulla and ganglia, but with a 1% dilution of AMS 21.1 Schultze (1948) substituted for C. note: The methods of Lobe 1937 {lest. Romeis 1948, 420) are essentially the same save that MS 33.1 Lobo 1937 is substituted for B, above, and that A is used at 60°C. for 15 minutes. 31.21 Schultze and Stohr test. 1933 Cajal and de Castro Cajal and de" Castro 1933, 198 reagents required: A. 0.8% sodium hydroxide; B. 2% silver nitrate; C. ADS 21.1 Schultze and Stohr 1933; D. Any AMS 22.1 formula method: [frozen sections of formaldehyde-fixed material] — > A, 1 day -^ wash, 1 hr. — > B, overnight -^ C, till reduced -^ D -^ balsam, via usual reagents recommended for: nerve fibers in sections of nerve tissue. 31.21 Tello 1932 test. 1933 Cajal and de Castro Cajal and de Castro 1933, 369 REAGENTS REQUIRED: A. 50% pyridine; B. AF 21.1 Tello 1932; C. 1% ammonia in 95% ale; D. 1.5% silver nitrate; E. AMS 21.1 Cajal 1910 method: [fresh tissue] -^ A, 24 hrs. — ♦ wash, 24 hrs. -^ B, till decalcified -^ wash, 24 hrs. ^ C, 24 hrs. -^ D, 5-7 days, 37°C. -^ wash -^ E, 24 hrs. -^ wash -^ [celloidin sections] recommended for: nerve endings in structures requiring decalcification. 31.21 Uyama 1926 8542a, 4:389 reagents required: A. 3% silver nitrate; B. AMS 21.1 Uyama 1926 method: [washed, enucleated, eye] ^ A, 3-5 days, 37°C. -+ rinse -> B, 12-36 hrs. -» wash — > [paraffin sections] recommended for: nerve net in retina. 31.21 Walgren 1930 test. 1946 Roskin Roskin 1946, 232 REAGENTS REQUIRED: A. AMS 11.1 Cajal 1933a; B. 1% silver nitrate; C. AMS 21.1 Cajal 1910; D. 0.05% potassium permanganate in 0.1% sulfuric acid; E. 1% oxalic acid method: [fresh fragments]-^ A, 5-9 days—* rinse -^ B, 1-2 days — > wash—* C, 1-2 days —> paraffin sections — > water — > D, few moments — > rinse — » E, few moments — > [repeat D —* E cycle till differentiation complete] — * wash — * balsam, via usual reagents 31.21 Willard 1935 17510,78:475 REAGExNjTS required: A. AMS 13.1 Willard 1935; B. 2.5% silver nitrate; C. AMS 21.1 Willard 1935 558 METHODS AND FORMULAS MS 31.22-MS 31.31 method: [fresh tissue] -^ A, 24 hrs. -^ wash — ♦ 96% ale, 24 hrs. -^ wash -* B, 9-12 days, 37.5°C. -^ rinse -^ C, 12-24 hrs. —> [15 m to 30 m sections by paraffin techniques] RECOMMENDED FOR: innervation of adrenal. 31.22 NEUROGLIA 31.22 Boisi 1927 test. 1933 Cajal and de Castro Cajal and de Castro 1933, 274 REAGENTS REQUIRED: A. AMS 11.1 Cajal 1914; B. AMS 11.1 Noguchi 1913; C. AMS 13.1 Bolsi 1927; D. 2% silver nitrate; E. AMS 24.1 Bolsi 1927; F. 5% sodium thiosulfate in 50% ale. method: [pieces of fresh tissue]—* A, 24-48 hrs. — > B, 1 or more months^ wash—* [frozen sections] — * water -* B, 10 mins. 45°-50°C. —> cool -^ C, 5 mins. — » Z), 5 mins. — > £■, 5 mins. —* F, b mins. -^ balsam, via carbol-xylene RECOMMENDED FOR: microglia. 31.22 Cajal 1925 test. 1933 ips. Cajal and de Castro 1933, 262 REAGENTS REQUIRED: A. AMS 11.1 Cajal 1913; B. 5% formaldehyde; C. MS 31.1 Cajal 1925; D. 0.2% gold chloride; E. 5% sodium thiosulfate method: [pieces of fresh tissue] -^ A, 24 hrs. — > B, till required — > [frozen sections] —>■ C, till dark chestnut, 5-10 mins. -^ wash — » D, till violet —^E,5 mins. -^ wash — > balsam, via usual reagents RECOMMENDED FOR: macroglia and microglia. 31.22 Lobe 1937 test. 1948 Romeis Romeis 1948, 420 REAGENTS REQUIRED: A. AMS 12.1 Lobo 1937; B. 4% formaldehyde; C. 0.04% sodium hydroxide; D. MS 31.1 Lobo 1937; E. AMS 21.1 Lobo 1937; F. 0.2% gold chloride; G. 5% sodium thiosulfate method: [fresh tissue] ^^ A, (see note below for time) -^ B, till required—* [30-40 n frozen sections] -* wash — > C> 5-8 mins., 60°C. — * wash, till wash water alkali-free when'phenolphthalein tested -* D, overnight, 60°C. — * rinse — * E, 3-5 mins. -^ wash —* F, 10-15 mins. -^ G, 5 mins. -^ balsam via usual reagents RECOMMENDED FOR: microglia (3-5 days in ^1), protoplasmic glia (15-30 days in A). 31.22 Merland 1936 4285a, 12 :290 REAGENTS REQUIRED: A. AMS 11.1 Merland 1935; B. 10% silver nitrate; C. AMS 21.1 Merland 1935; D. 10%, sodium thiosulfate method: [frozen section of F 8000.1000 Merland 1935 material]-* A, changed fre- quently, till silver nitrate test shows all bromide eliminated -^ A, fresh solution, 40 mins., 56°C. -^ rinse -* B, 30-60 mins. 5G°C. (till ochre) -* wash -* C, till reduced -^ D, 5 mins. — * balsam, via usual reagents RECOMMENDED FOR: astrocytes. 31.3 Cytological Methods 31.31 golgi apparatus 31.31 Cajal 1912 see MS 31.31 Cajal 1914 (note) 31.31 Cajal 1914 21344, 12:127 REAGENTS REQUIRED: A. AMS 11.1 Cajal 1914; B. 1.5% silver nitrate; C. AMS 21.1 Cajal 1914 method: [Small pieces of fresh tissue]-* A, 10-14 hrs.-* rinse -^ B, 36-48 hrs. -^ rinse -^ C, 8-24 hrs. — » wash —> [paraffin sections by usual techniques] RECOMMENDED FOR: Golgi network. note: This method is assigned, without reference, to Cajal 1912 by Cajal and de Castro 1933, 203. 31.31 Golgi 1908a test. 1933 Cajal and de Castro Cajal and de Castro 1933, 201 reagents required: A. F 8000.1000 Golgi 1908; B. 1% silver nitrate; C. AMS 21.1 Cajal 1914; D. AMS 22.1 Golgi 1908; E. AMS 23.1 Golgi 1908; F. 1% oxalic acid method: [fresh tissue] -* A, 6-8 hrs. — * wash -^ B,3 hrs. to some days -* quick rinse — > C, 12 hrs. -^ wash — > 3 to 5 m sections -^ D, 10-30 mins. -* E, till differentiated -^F, to stop differentiation —> [counterstain, if desired] -^ balsam, via usual reagents RECOMMENDED FOR: Golgi network. MS 31.31-MS 31.41 METAL STAINS 559 31.31 Golgi 1908b /< s^ 1933 Cajal and de Castro Cajal and de Castro 1933, 201 REAGENTS REQUIRED: .4. AMS 11.1 Cajal 1908; B. AMS 12.1 Cajal 1910; C. 2% silver nitrate; D. AMS 21.1 Cajal 1910 method: [pieces of tissue] —> A,24 hrs. -^ wash, 4-6 hrs. —>■ B, 24 hrs. -> quick rinse -> C, 5 days, 37.5°C. -> quick wash -* D, till reduced -^ [sections] RECOMMENDED FOR: Golgi network. 31.31 Rojasl917 21344, 15:30 REAGENTS REQUIRED: A. AMS 11.1 Rojas 1917; B. 1.5% silver nitrate; C. AMS 21.1 Rojas 1917 method: [fresh nerves] —> A, 24 hrs. -^ running water, 24 hrs. -> B, 48 hrs. -^ wash -^ C, 24 hrs. —>■ wash -^ tease -^ glycerol RECOMMENDED FOR: demonstration of Golgi apparatus in wholemounts. 31.31 Weatherford tesl. 1938 Mallory Mallory 1938, 113 REAGENTS REQUIRED: A. F 8000.1000 da Fano 1920; B. 5% trichloracetic acid; C. 1.5% silver nitrate; D. AMS 21.1 Cajal 1910; E. 0.2% gold chloride; F. 25% sodium thiosulfate method: [fresh tissue]^ A, 6-8 hrs. — > wash ^ B, changed daily till ammonium ox- alate calcium test is negative -^80% ale. thorough wash — » A, 1 hr. -^ quick wash — > C, 36-48 hrs. — > rinse -> D, 8-24 hrs. in dark -^ wash — > [4-8 m sections] -^ water -^ E, 5-10 mins. — > F, 10-15 mins. -^ [DS 11.21 counterstain, if required] —^ balsam, via usual reagents RECOMMENDED FOR: Golgi bodies in tissues requiring decalcification. 31.32 OTHER CYTOLOGIC AL METHODS 31.32 Chatton and Lwoff 1930 6630, 104:834 REAGENTS REQUIRED: .4. F 80000.1000 da Fano 1920 100, sodium chloride 1; E. V 22.1 Chatton and Lwoff 1930; C. 3% silver nitrate method: [live protozoa, concentrated on slide] -^ A, on slide, 5 mins. ^^ drains B, 1 drop at 25°C. -^ cool to solidify -^ C, on slide, 5 mins. in dark -^ rinse -^ bright light till sufficiently reduced — ♦ wash — > balsam, via usual reagents RECOMMENDED FOR: impregnation of basal bodies and nerve net in Ciliata. note: Chatton and Lwoff 1940 (6630, 134:229) recommend development in 0.1% hydro- quinone. 31.32 Kingsbury and Johannsen 1927 Kingsbury and Johannsen 1927, 83 REAGENTS REQUIRED: .4. AMS 11.1 Kingsbury and Johannsen 1927; BA% silver nitrate; C. AMS 21.1 Kingsbury and Johannsen 1927 method: [small pieces of fresh tissue] —>■ A, 8-12 hrs. — > rinse -^ B, 24 hrs. —>■ wash — > C, 24 hrs. -^ [wholemount or section] recommended for: demonstration of invertebrate striped muscle. 31.4 Histological IMethods 31.41 reticular fibers 31.41 Cajal 1907 test. 1933 ips. Cajal and de Castro 1933, 290 reagents required: A. AMS 13.1 Cajal 1907; B. water 50, 95% ale. 50, ammonia 0.3; C. 1.5% silver nitrate; D. AMS 21.1 Cajal 1910 method: [pieces of fresh tissue] -> A, 24 hrs. — > running water, 24 hrs. —> B, I day -» rinse -> C, 5 days, 37°C. -^ wash -y D, 24 hrs. -^ wash -> [sections] 31.41 Cajal test. 1933 ips. Cajal and de Castro 1933, 290 reagents required: A. AMS 11.1 Cajal 1933; B. 0.3% ammonia in 95% ale; C. 2% sUver nitrate; D. AMS 21.1 Cajal 1910 method: [pieces of fresh tissue] -+ A, 24 hrs. -^ thorough wash — > B, 24 hrs. -> wash — > C, 3-4 days, 37°C. -^ wash, 1 min. -* D, 24 hrs. — > wash -^ [celloidin sections] 560 METHODS AND FORMULAS MS 31.42-MS 31.51 31.42 OTHER HISTOLOGICAL METHODS 31.42 Lauda and Rezek 1928 22575, 269:218 REAGENTS REQUIRED: A. 0.25% ammonia in 95% alcohol; B. MS 31.1 Lauda and Rezek 1928; C. AMS 21.1 Lauda and Rezek 1928 method: [fresh tissue]-^ A, 24 hrs. — > wash -^ B, 3-4 days — > wash—* C, 24 hrs. -^ wash — > 95% alcohol-^ [paraffin sections] RECOMMEXDED FOR: general histology of kidney. 31.42 Lillie 1928 23632, 45 :380 REAGENTS REQUIRED: A. 2.5% silvcr nitrate; B. AF 21.1 von Ebner (1891b); C. 18% sodium chloride method: [formaldehyde-fixed bones with fixative thoroughly washed out] — > A, 37°C., 4-5 days ^ thorough wash -^ B, till decalcified — > C, till acid-free —* [paraffin sec- tions] recommended for: structure of bone. 31.42 Kossa 1901 2526, 29:163 reagents required: A. 5% silver nitrate; B. 1% pyrogallol; C. 5% sodium thiosulfate method: [frozen sections of undecalcified material] —* water — > A, 10-60 mins., in strong light — » wash —> B, 1-3 mins. — > rinse — * C, 3-5 mins. — > thorough wash — > balsam, via usual reagents 31.42 Kossa test. 1933 Cajal and de Castro Cajal and de Castro 1933, 315 reagents required: A. 0.2% silver nitrate; B. any DS 11.42 solution method: [paraffin sections]—* water -^ A, J2 hr. — * wash — > B, till counterstained — > balsam, via usual reagents 31.5 Bacteriological Methods 31.51 methods for spirochetes 31.51 Armuzzi and Stempel 1924 test. 1928 Schmorl Schmorl 1928, 40b reagents required: A. pyridine: B. 5% uranium sulfate; C. 2% silver nitrate; D. AMS 21.1 Armuzzi and Stempel 1924 method: [frozen sections of formaldehyde-fixed material] -* A, 1^^ hrs. —> wash — > 95% ale, 1 hr. -^ water, 1 min. — > 5, 2 hrs. — > rinse -^ C, 2)^-3 hrs., 37°C. — > rinse -^ D, 10 mins. -* thorough wash -^ balsam, via usual reagents 31.51 Bauer 1944 608b, 20 :297 reagents required: A. MS 31.1 Bauer 1944; B. AMS 21.1 Bauer 1944 method: [blocks of formaldehyde-fixed and nitric acid-decalcified jaws] — * running water wash, 2 daj's —>■ A, changed when cloudy, 18 days, in dark, 37°C. —> wash, 1 day — > B, changed when cloudy, 48 hrs. in dark -* wash — ♦ [paraffin sections] recommended for: spirochetes in tooth buds and jaws. 31.51 Cajal test. 1933 ips. Cajal and de Castro 1933, 192 reagents required: .1. 5% formaldehyde; B. MS 31.1 Cajal 1929; C. AMS 21.1 Cajal 1910 method: [fresh tissues] — > A, 2 days ^95% ale, 24 hrs. — * fi, 2 days, 37°C. — > wash — > C, 24 hrs. — + [section] recommended for: spirochetes in sections. 31.51 Cajal and de Castro 1933 see MS 31.51 Noguchi (1913) (note) 31.51 Dieterle 1927 1879, 18:73 reagents required: A. 1% uranium nitrate in 70% ale; B. 10% gum mastic in abs. ale; C. 1% silver nitrate; D. AMS 21.1 Dieterle 1927 method: [sections, preferably of formaldehyde-fixed material] — >• A, 30 mins., 55°C. — * wash — > 90% alcohol, 2 mins. —* B, 30 sees. -^ drain — > rinse, 90% alcohol —> rinse, water —> C, 1 to 6 hrs., 55°C. in dark —>■ wash -^ D, 5-15 mins. -^ wash — * 96% alco- hol till all traces of B removed — * balsam, via acetone recommended for: spirochetes in sections. MS 31.51 METAL STAINS 5G1 31.51 Eyene and Sternberg test. 1916 Warthin 4349, 6:71 REAGENTS REQUIRED: ,4. 1% silvop iiitiatc; />'. AMS 21.1 EyciH! and Sternberg (1916); C. 10% sodium thiosulfate method: [thin sections of formaldehyde-fixed material] — > wash — > ^4, 30 mins., in dark, 37.5°C. — > B, 1-2 mins. -^ C, 1-2 mins. -^ wash — » balsam, via usual reagents 31.51 Farrier and Warthin 1930 623, 14:394 REAGENTS REQUIRED: .4. 1% silver nitratc at pll 4. 1; B. AMS 21.1 Farrier and Warthin 1930; C. 5% sodium thiosulfate method: [sections of formaldehyde-fixed material] — > A, 30 mins., 37°C., in dark -^ B, till brownish black — > C, 5 mins. — » wash — > balsam, via usual reagents 31.51 Hertzman lest. 1938 Mallory Mallory 1938, 293 REAGENTS REQUIRED: .1. pyridine; B. 1% uranium nitrate; C. 0.25% silver nitrate; D. AMS 21.1 Hertzman 1938 method: [frozen sections of formaldehyde-fixed material] ^ ^, 10 mins. — > wash — > B, 15 mins., 37°C. -^ rinse -^ C, 15-30 mins., 50°-60°C. -^ D, till dark brown, 50°-60°C. — > warm water, wash — » balsam, via usual reagents 31.51 Jahnel test. 1933 Cajal and de Castro Cajal and de Castro 1933, 384 REAGENTS REQUIRED: .4. pyridine; 5. 4% formaldehyde; C. 1% uranium nitrate; D. 1.5% silver nitrate; E. AMS 21.1 Noguchi 1913 method: [2-4 mm. slices of alcohol- or formaldehyde-fixed material] — > A, 1-3 days — ♦ thorough wash -* B, 24 hrs. -^ C, 1 hr., 37°C. -> wash -» D, 5-8 days in dark, 37.5°C. — + wash -^ E, 1-2 days in dark — > [paraffin sections] note: see also MS 31.51 Levaditi 1905 (note). 31.51 Krajian 1935 1829,32:764 REAGENTS REQUIRED: A. 1% sodium cobaltinitrite ; B. AMS 12.1 Krajian 1933; C. 0.75% silver nitrate; D. AMS 21.1 Krajian 1933 method: [frozen sections of formaldehyde material] -^ A, 5 mins. -^ wash ^ 5, 15 mins., 67°C. -^ wash ^ C, 1 hr., 67°C. — > rinse — > C, 15-25 sees. — > wash — > balsam, via usual reagents note: see also MS 33.51 Krajian 1933. 31.51 Krajian 1938 1829,38:427 REAGENTS REQUIRED: A. AMS 21.1 Krajian 1933; B. 0.5% mastic in 95% alcohol; C. 1% silver nitrate; D. AMS 21.1 Krajian 1933 method: [air-dried smears of exudate] -^> A, 5 mins., 37°C. -^ wash ^ B, poured on slide — > drain and breath on surface till cloudy — > rinse — ^ C, on slide warmed to bubbling, 3 mins. -^ C (repeat) — » drain -^ D, 2 mins. — * wash — > dry 31.51 Krantz 1924 14074, 608 REAGENTS REQUIRED: A. 0.1% silvcr nitrate; B. AMS 21.1 Krantz 1924 method: [sections of formaldehyde-fixed material] —> water —> A, 24 hrs., 00°C. — » wash — ♦ B, 30-60 mins. — » wash -^ balsam, via usual reagents 31.51 Knowles, Gupta, and Basu 1932 test. 1938 Hunter 20540b, 13 :46 REAGENTS REQUIRED: A. 0.3% silver nitrate; B. 1% hydroquinone method: [1 mm. slices of formaldehyde-fixed material, washed free of fixative]—* A, 37°C., 24 hrs. —* wash, till free from silver -^ B, 24 hrs. — > [paraffin sections] RECOMMENDED FOR: Spirochetes in avian tissues. 31.51 Levaditi 1905 6630,58:845 REAGENTS REQUIRED: A. 4% formaldehyde; B. 2% silver nitrate; C. AMS 21.1 Levaditi 1905 method: [fresh tissue] —> A, 24 hrs. or till required -^^ 90% ale, 24 hrs. -^ water, till rehydrated — > B, 3-6 days, 37°C. —^ rinse -^ C, 1-3 days in dark -^ wash — > [5 m paraffin sections] note: Schmorl 1928, 403 recommends the substitution of AMS 21.1 Levaditi and Manuelian 1906 for C above; .Jahnel test. 1933 Cajal and de Castro 384 recommend the substitution of AMS 21.1 Jahnel 1933 for C above. 5G2 METHODS AND FORMULAS MS 31.51 31.51 Levaditi lest. 1916 Warthin 4349, 6 :56 REAGENTS REQUIRED: A. MS 31.1 Levaditi (1916); B. AMS 21.1 Levaditi (1916) method: [pieces of formaldehyde-fixed, and alcohol-hardened, tissue] -^ water —> ^ , 2-3 hrs. — » B, 6-9 hrs. —* thorough wash — > [paraffin sections] 31.51 Manouelian 1918 6630, 131 :759 REAGENTS REQUIRED: A. 4% formaldehyde; B. 1% silver nitrate; C. AMS 21.1 van Ermenger 1894 method: [small fragments of tissue] -^ A, 1-2 hrs. -^95% ale. till A removed -^ water, till ale. removed -» B, 1-12 hrs. 50°C. -^ rinse —> C, 1-24 hrs. -> 5 m sections -^ bal- sam, via usual reagents RECOMMENDED FOR: Spirochetes in sections. 31.51 Murray and Fielding test. 1937 Findlay 11360, 57:138 REAGENTS REQUIRED: A. 1% silver nitrate; B. 1% pyrogallol method: [pieces fixed overnight in F 0000.1010 Murray and Fielding 1937] -^70% ale, 30 mins., 50°C. -* A, 30 mins., 50°C. -^ wash -> B, 1 hr., 50°C. -> [paraffin sections] RECOMMENDED FOR: Leptospira iderohaemorrhagica in sections. 31.51 Nakano 1912 test. 1928 Schmorl Schmorl 1928, 404 REAGENTS REQUIRED: A. 4% formaldehyde; B. 1.5% silver nitrate; C. AMS 21.1 Leva- diti 1905 method: [thin slices] -^ A, 10-20 mins., 37°C. —>■ 95% ale, 3-5 hrs. -^ wash -^ B, 4-5 hrs. in dark, 50°C. -> rinse -^ C, 4-10 hrs., 50°C. —> wash —y [paraffin sections] 31.51 Noguchi 1913 test. Schmorl 1928 Schmorl 1928, 407 reagents required: A. AMS 11.1 Noguchi 1913; B. 1.5% silver nitrate; C. AMS 21.1 Noguchi 1913 method: [5-7 mm. slices of formaldehyde-fixed brain tissue] -^ A, 5 days -^ thorough wash -^95% ale, 3 days -^ water, till rehydrated — » B, 3 days, 37°C. -^ wash — > C, 1-2 days — > wash — > [paraffin sections] note: Cajal and de Castro 1933, 383 recommend AMS 21.1 Cajal 1910 in place of C above. 31.51 Para 1946 1789a, 42 :649 reagents required: A. 1% uranium nitrate; B. 1.5% silver nitrate; C. MS 33.1 Steiner 1937 80, 10% rosin in 95% ale. 20; D. AMS 21.1 Levaditi 1905 method: [paraffin sections] — > water ^ A, 30 mins. — > rinse — > 5, 2 hrs., 56°C. — > rinse — > C, 1 hr. -^ rinse -^ D, 10-15 mins. -^ balsam, via usual reagents note: This is the preferred technique. The original offers 3 alternatives to A, 3 alterna- tives to C, and 3 possible substitutes for rosin in C. 31.51 Schmorl 1928 see MS 31.51 Levaditi 1905 (note) 31.51 Steiner 1922 14674, 121 REAGENTS REQUIRED: A. % gum mastic in 95% ale; B. 0.1% silver nitrate; C. A 2.5, 95% ale 25, water 75; D. 5% hydroquinone method: [frozen sections of formaldehyde-fixed material] — » A, 1-2 mins. -^ short wash -^ B, 24 hrs., 37°C. -^ wash, 37°C. -^ C, 10 mins. -^ rinse -^ D, 4-6 hrs. -^ thorough wash — > balsam, via usual reagents 31.51 Steiner 1937 11284, 23:315 reagents required: A. AMS 12.1 Steiner 1937; B. 0.1% silver nitrate; C. 3% gum mastic in abs. ale; D. AMS 21.1 Steiner 1937 method: [frozen sections of formaldehyde-fixed material] —* water —^ abs. ale — > A, 6-8 mins. -^ wash, till gum-free -^ B, heated till bubbles appear and cooled 20-30 mins. — > 95% ale, wash -^ C, 2 mins. ^ wash till gum-free —> D, heat to boiling, cool — » balsam, via usual reagents 31.51 Steiner 1939 11284, 25:204 reagents required: A. AMS 12.1 Steiner 1939; B. 0.1% silver nitrate; C. 12.5% gum mastic in abs. ale; D. AMS 21.1 Steiner 1939 MS 31.51-MS 31.6 METAL STAINS 563 method: [10 M paraffin sections] — » abs. ale. -^ A, l-V/i mins. -^ thorough wash -^ B, 1-13'2 hrs., 100°C. — > abs. ale. via graded alcohols — » C, 5 mius. — > wash -> /), 5 min.s. -^ [counterstain, if desired] — » balsam, via usual reagents 31.51 Steiner and Steiner 1944 11284,29:868 REAGENTS REQUIRED: A. 1% uranium nitrate; B. 1% silver nitrate; C. 2.5% gum mastic in abs. ale; D. AMS 21.1 Steiner and Steiner 1924 method: [paraffin sections] —> water —> A, 3 mins. — > wash—* B, 2 hrs., 56°-58°C. -^ wash -^ dehydrate —* C, 5 mins. — » drain —* D, 12-15 mins. — » wash — > balsam, via usual reagents 31.51 Warthin 1916 4349,6:71 reagents required: A. 2% silver nitrate; B. AMS 21.1 Levaditi 1905 method: [pieces of F 8000.1000 Warthin 1916 fixed, and alcohol-hardened, material) — ♦ wash — > 4, 2 days in dark, 37.5°C. -+ wash, in dark — > B, 48 hrs., in dark — ♦ wash —* [paraffin sections] 31.51 Warthin-Starry 1929 test. Langeron 1942 Langeron 1942, 629 reagents required: A. 1% nitric acid; B. 2% silver nitrate; C. AMS 21.1 Warthin- Starry 1929; D. 5% sodium thiosulfate method: [sections of formaldehyde-fixed material on slides]-^ A, 1-30 mins. — > rinse — » B, 30 mins. to 2 hrs., 55°C., in dark, by dipping section bearing slide into stain, placing another slide on top and laying this sandwich in half its depth of stain — » remove cover slide, lay section bearing slide, sections up, in 3 mm. layer C till brown —> wash, warm water — > Z), 2 mins. -^ wash — > balsam, via usual reagents recommended for: spirochetes in section. 31.51 Yamamoto 1909 23681,20:153 reagents required: ^1. 4% formaldehyde; B. 5% silver nitrate; C. AMS 21.1 Yama- moto 1909 method: [thin slices] — > ^4., 24 hrs. — » 95% ale, 1 hr. — > running water, 24 hrs. — ♦ dist. water, 1 hr. —> B, 47 hrs., 37°C. — > rinse —> C, changed after 1 hr., and whenever turbid, 24 hrs., 37°C. -^ wash — > [celloidin sections] 31.52 OTHER BACTERIOLOGICAL METHODS 31.52 van Ermengen 1894 23684, 15 :969 REAGENTS REQUIRED: .1. 1% silvcr nitrate; B. AMS 21.1 van Ermengen 1894 method: [bacterial smears, fixed 30 mins. in F 1000.0019 van Ermengen 1894] — > water, wash ^^95% ale., wash -^ A, on slide, 15-30 sec. — > B, added to A on slide, 30 sees. — > A, dropped on mixture on slide till ppt. occurs — > water, wash — > dry recommended for: demonstration of bacterial flagella. 31.52 Steiner 1950 591b, 20:489 REAGENTS REQUIRED: A. AMS 12.1 Steiner 1950; B. 0.1% sUver nitrate; C. 2% mastic in abs. ale.; D. 5% pyrocatchol method: [8 n paraffin sections of formaldehyde-fixed tissues] -^ abs. ale. -^ A,5 mins. — »• water, changed till no longer milky -^ B, 14-16 hrs., 60°C. -^ thorough wash — » abs. ale. via graded ales. -^ C, 5 mins. — > D, 1 hr., 60°C. — > wash —> balsam, via usual reagents recommended for: microorganisms in tissues. 31.6 Other Silver Nitrate Methods 31.6 Gomori 1940 16913,44:250 REAGENTS REQUIRED: A. 0.5% silver nitrate; B. 5% sodium acid phosphite; C 2% sodium thiosulfate method: [alcohol-fixed teeth] -^ A, 1 day -^ wash, 1 day — > 5, 1 day — > wash -* C, 12 hrs. -^ [sections] recommended for: carious lesions in teeth. 31.6 Hanazawa 1917 Dent. Cosinos, 59:125 reagents required: A. 2% silver nitrate; B. any AMS 21.1 fornmla; C. 5% sodium thiosulfate 5G4 METHODS AND FORMULAS MS 31.6-MS 32.0 method: [ground sections of teeth] -^ A, 2-5 days — > wash -^ B, till sufficiently reduced — ^ water — > C, 2-3 niins. -^ wash — » balsam, via usual reagents RECOMMENDED FOR: dentine. 31.6 Holmes 1900 11373, 16:371 REAGENTS REQUIRED: A. 0.75% silver nitrate; B. 0.2% sodium thiosulfate; C. sat. aq. sol. picric acid method: [eggs of Planorbis] -^ A, in direct sunlight, till cells clearly demarcated—* rinse —» B, wash ^ wash ^ C, 10 mins. -^ 70% ale, wash ^> balsam, via usual reagents RECOMMENDED FOR: demarcation of cell outlines in invertebrate embryos. 31.6 Roskin 1946 Roskin 1946, 292 REAGENTS REQUIRED: ,4.2% silvcr nitrate; B. 2% hydroquinone method: [specimens fixed in F 0000.1010 Roskin 1946] -^ wash -^ A, 10-18 days, 37°C., in dark — > wash, in dark -^ B, 18-38 hrs., in dark — > wash — * balsam, via usual reagents recommended for: general structure in wholemounts of small fresh-water oligochaetae. MS 32 METHODS USING PROTEIN SILVER These methods, usually referred to as Bodian Techniques (see MS 32.1 Bodian 1936 below) are the only silver methods which give satisfactorj^ staining of nervous structures in paraffin sections mounted on the shde. The original method, and most of the modifications, call for Protargol, a proprietar}^ compound conforming to the specifications for "Protein silver, strong, U. S. P. XL" The restricted availabihty of Protargol has lead the author to specify "silver protein" in the methods which fol- low ; all samples of "silver protein, strong," however, do not give satisfactory results, and some selection may be necessary. Con- fusion is sometimes caused by tlie designa- tions "strong" and "mild" as applied to pharmacopeial preparations since the mild contains about three times as much (19% to 25 So) silver as does the strong (7.5% to 8.5%). The strength, or mildness, from the pharmacopeial point of \dew, depends on the quantity of ionic silver which is found in solutions of the compound. A method of preparation from gelatin is given by Mos- kowitz 1950 (20540b, 25:17). 32.0 Typical Example Preparation of a transverse section of the sciatic nerve of a cat to demon- strate axis-cylinders by the method of Davenport, Windle, and Rhines 1947 This method departs from the classic Bodian technique in that metalhc copper is not used in combination with silver pro- tein to impregnate the nerves. The method is, however, simple and certain, and is recommended in those cases in wliich ab- solute certainty of impregnation of a.xis- cylinders is required. Since the method of fixation is an inte- gral part of this technique, the removal and the fixation of the sciatic nerve will first be described. The fixative recom- mended is a mixture of formamide and paranitrophenol, and is given under the heading F 9000.4000 Davenport, Windle, and Rhines 1947 in Chapter 18. Care must be taken to secure pure formamide, as the commercial grade is worthless for the pur- pose. About 100 milliliters of fixative will be required, and it is prepared by dissolv- ing 5 grams paranitrophenol in 45 milli- Uters of 95% alcohol, and then adding to the mixture 10 grams of formamide. After all these ingredients are mixed, 45 milli- liters of distilled water is added. The cat has been selected, rather than the rabbit recommended in other ex- amples, because of the relatively large size of the sciatic nerve. A freshly killed cat should be secured and the skin removed from the lateral side of the upper part of the leg. This exposes the biceps femoris muscle which may be lifted by slipping the handle of the scalpel under it and running the scalpel down from the origin towards the insertion. The aponeurosis at the knee can then be cut, either with a scalpel or scissors, and the muscle laid MS 32.0 METAL STAINS 565 back to expose the sciatic nerve. If this operation is skilfully clone there will be no bleeding. Before removing the nerve it is desirable to have some form of stretcher which may be used to keep it straight during the process of fixation. The simplest method is to use the nerve as the string of a bow, the bow itself being made either from a fine sliver of bamboo, or from some thin plastic which will apply the required ten- sion. The author prefers the small stiffen- ing devices which are sold for insertion into the collars of men's shirts. These are usually about 2 inches long by Js of an inch wide, and may be cut down the middle with a sharp knife to make two bows from each. The selected piece is laid down flat alongside the nerve, which has been freed from the fascia with some blunt-pointed instrument, and one end of the bow tied firmly to the nerve with a piece of surgical silk. The bow is then bent very slightl.y — from li to Ke of an inch, given sufficient tension — and the other end similarly lashed to the nerve with surgical silk. The required piece of nerve is then severed and lifted out, using the bow as a handle, and transferred to about 100 milliliters of fixative, preferably being suspended in the fixative by a thread tied around the bow. In the fixative recom- mended with this technique, from 24 to 48 hours are sufficient. The usual precautions with regard to the purity of the reagents employed and chemical cleanliness of the glassware must be observed in staining the sections. Staining and washing is most conveniently done in rectangular jars, and it is best to use a single jar for all the staining, fixing, and toning operations, though the usual jars of xylene and alcohols will be required for deparaffinizing and dehydrating. The solutions required in order of their use are: A. 5% silver nitrate. This presents no difficulty in preparation provided that re- agent grades of silver nitrate and triple- distilled water are employed in chemically cleaned glassware. B. 0.2% silver protein. The simplest way to dissolve the silver jM'otein is to sprinkle the dry powder on the surface of the water. The mixture should not be stirred, Init the powder should be allowed to drop through the water of its own weight. When no further silver protein is left on the surface, the material may be stirred rapidly and then placed on one side for use. C. Davenport's developer, the formula for which is given under AIMS 21.1 Daven- port, Windle, and Rhines 1947 in Chapter 24. To prepare the solution .5 grams of sodium sulfite are dissolved in 100 milh- liters of water. When solution is complete, 1 gram of hydroquinone is added and al- lowed to dissolve completely before adding a J^^-gram of potassium metaborate, which is commercially available under the trade name of Kodalk. D. 0.2% gold chloride. E. 0.4% oxalic acid. F. 5% sodium thiosulfate. The technician should now have in front of him two jars of xylene, one jar of absolute alcohol, one jar of 95% alcohol, one jar of 70% alcohol, one jar of 50% al- cohol, one jar of distilled water, and one jar of 5% silver nitrate. He should also have available in beakers a sufficient quantity of the silver protein solution, the developer, the gold chloride, the oxalic acid, and the sodium thiosulfate to fill one of the jars. It is to be presumed that the nerve has been sectioned by the ordinary paraffin technique and the sections, of a thickness of from eight to ten microns, mounted on glass slides. Each slide is then warmed until the wax melts and is dropped into the jar of xylene. When the slides have been in the first jar of xylene long enough for the wax to have been re- moved, they are transferred to the second jar of xylene for a minute and then run down the series to the jar of distilled water. If the slide, on removal from dis- tilled ^^■ater, appears to be greasy, it must be run up the series through the alcohols again into xylene, and then down. If the least trace either of wax or xylene remains in the section, staining cannot be carried out. The slides are transferred one at a time to the jar of 5% silver nitrate, which is then placed in the paraffin embedding oven (presumably at a temperature of about 60°C.) where they remain for ap- 566 METHODS AND FORMULAS MS 32.0-MS 32.1 proximately one hour. While they are in the oven a beaker full of distilled water should be heated to a temperature of 30°C. to 40°C. At the end of the hour, the jar of silver nitrate containing the slides is removed from the oven, and the silver nitrate is either thrown away or poured back into a bottle for further use. The jar is then filled with the warm distilled water and rocked slowly backward and forward for about 30 seconds. This first wash water is thrown away and replaced by a second, which is again used for 30 seconds and, in its turn, thrown away and replaced by a third change for a further period of 30 seconds. Immediately after this third wash, the jar is filled with silver protein solution and left at room tempera- ture for about one hour. The silver protein solution is then thrown away — it is im- possible to use this solution more than once — and the jar is rapidly filled with dis- tilled water, which is instantly poured off again. The purpose of this wash is to re- move the silver protein from the glass sUde and jar without removing any ap- preciable quantity from the sections. The jar is then filled with the developing solu- tion and rocked gently backward and for- ward for one or two minutes. The develop- ing solution is then poured away and the jar containing the sUdes left under a running tap for several minutes; running distilled water is better if it is available. If tap water is used, the jar and sHdes should be rinsed with a couple of changes of distilled water before pouring in the gold chloride toning solution. This is left until the yellow color of the silver stain has been replaced by the gray color of the gold, when the sHdes may again be rinsed in tap water, and the jar filled with the oxalic acid solution. This stage is rather critical and must be watched carefully. The oxalic acid causes great darkening and, if allowed to act too long, will destroy the sharp differentiation of the stain. The slides should be watched carefully, and the oxalic acid should be poured off and re- placed by running tap water as soon as the first signs of darkening are apparent. This may be anywhere from J^ to % of a minute. Darkening will continue for some time after the slides have l)een in tap water; if one waits until they have become dark before starting the washing, the preparation will be spoiled. After the oxahc acid has been thoroughly washed off, the jar should be filled with sodium thiosulfate for one to two minutes, and then placed in running tap water until all traces of the fixative have been removed. Each slide is then individually removed from the jar and run up through the series of the alcohols and xylene. After each is cleared in xylene it maj' be re- moved and mounted in balsam, which gives a permanent preparation. 32.1 Neurological Methods 32.1 Bacsich 1938 test. 1948 Romeis Romeis 1948, 422 REAGENTS REQUIRED: A. 1% protein silver in a jar, on the bottom of which has been placed 2-3 Gms. of clean metallic copper for each 100 ml.; B. AMS 21.1 Bodian 1936; C. AMS 22.1 Bodian 1936; D. AMS 22.1 Bacsich 1938; E. 5% sodium thiosulfate; F. abs. ale. 50, ether 50 method: [celloidin sections of formaldehyde-fixed material, attached to slide with V 21.1 Mayer 1884 and varnished with celloidin] -^ A, 1 hr.. 36°C. -^ wash -^ B, till sec- tions are as dark as possible — ^ wash, 1 min. each in 3 changes of water — > C, 5 mins. — > wash — > D, 5 mins. — » wash —> E, 5 mins. — ► wash -^95% ale. till dehydrated -^ F, to remove celloidin -^ balsam, via usual reagents 32.1 Bodian 1936 763, 65 :89 reagents required: A. 1% protein silver in a jar, on the bottom of which has been placed 2 or 3 Gms. of clean metallic copper per 100 ml.; B. AMS 21.1 Bodian 1936; C. AMS 22.1 Bodian 1936; D. 5% sodium thiosulfate method: [paraffin sections of formaldehyde-fixed material]—* water—* A, 12-48 hrs. at 37°C. — » wash — * B, 10 mins. — » wash — > C, 5-10 mins. — * wash — > D, 5-10 mins. -^ balsam, via usual reagents MS 32.1 METAL STAINS 5G7 32.1 Davenport, McArthur, and Bruesch 1939 20540b, 14:22 REAGENTS REQUIRED: .1. 10% sUvcr nitrate; B. 0.2% protein silver; C. AMK 21.1 Davenport, McArthur, and Bruesch 1939; D. 0.1% }j;ohl chloride; E. 1% diainino- phenol hydrochloride method: [paraffin sections of material fixed 2 to 12 hrs. in F 0000.0023 Davenport, McArthur, and Bruesch 1939] -^ wash -^ A, 1 hr., 58-G2°C. -^ wash -^ B, 1 hr. -^ quick rinse — » C, 1 min. -^ wash —>■ D, till color gray — + wash — > E, dropped on slide, few sees. — > wash — ^ balsam, via usual reagents RECOMMENDED FOR: general nervous histology. 32.1 Davenport, Windle, and Rhines 1947 Conn and Darrow 1947 1C2, 24 REAGENTS REQUIRED: A. 5% silvcr nitrate; B. 0.2% protein silver; C. AMS 21.1 Daven- port, Windle, and Rhines 1947; D. 0.2% gold chloride; E. 0.4% oxalic acid; F. 5% sodium thiosulfate method: [paraffin sections of material fixed in F 9000.4000 Davenport, Windle, and Rhines 1947] — > water -^ A, 1 hr., 58-62°C. — > wash, 3 changes, 30 sees, each — > B, 1 hr. — » quick rinse — > C, 1 min. -^ wash, running water — > D, till gray — > rinse — > E, 15-45 sees, till darkening starts -^ rinse — >• F, 1-2 mins. — > wash — > balsam, via usual reagents RECOMMENDED FOR: axis-cylindcrs. note: a detailed description of the application of this technique is given under MS 32.0 above. The method of Dawson and Barnett 1944 for argentophil granules differs from the above in that the " B —* rinse —* C" cycle is repeated. 32.1 Dublin 1944 1829,50:361 reagents required: A. 1% protein silver; B.\% hydroquinone; C. 0.5% gold chloride; D. 2% oxalic acid; E. 5% sodium thiosulfate; F. water 100, acetic acid 0.1, ponceau 2R 0.75; azophloxine 0.125; G. 0.1% acetic acid; H. water 100, acetic acid 0.1, phos- photungstic acid 1.5, orange G 1; /. water 100, acetic acid 0.1, light green 0.02 method: [6-8 m sections of formaldehyde-fixed material] -^ water -^ yl, 2-4 hrs. ^ rinse -^ B, 5 mins. — > rinse -^ C, till gray —* rinse — * D, 5 mins. —> rinse -^ E, 5 mins. — * wash —* F, 5 mins. — > G, rinse —* H, 5 mins. time -^ balsam, via usual reagents recommended for: skin. 32.1 Foley 1943 20540b, 18:27 reagents required: A. water 50, 95% ale. 50, ammonia 1; B. 1% protein silver in dish containing copper foil varnished with 0.5% celloidin; C water 50, 95% ale. 50, pro- tein silver 0.5, pyridine 1 in dish containing copper foil varnished with 0.5% celloidin; D. AMS 21.1 Foley 1943; E. 0.2%, gold chloride in 0.1% acetic acid; F. 2% oxalic acid; G. 5% sodium bisulfite; H. DS 11.41 Einarson 1932; /. 5% phosphotungstic acid; /. water 97, acetic acid 3.2, anilin blue 0.004, fast green FCF 0.2, orange G 0.8 method: [25 n frozen sections of formaldehyde-fixed material] —* A, 24 hrs. — > B, 6-8 hrs., 37°C. -^ C, 1-2 days, 37°C. -^ 50% ale, rinse -^ D, 10 mins. -^ wash -^ E, 10 mins. — > F, 1-3 mins. — > rinse —> G, 3-5 mins. — > wash -^ H, overnight — » wash — >■ 7, 30 mins. -^ J, 1 hr. — > 95% ale, till differentiated — > balsam, via butyl alcohol and cedar oil 32.1 Humphreys 1939 608b, 15:151 reagents required: A. AMS 12.1 Humphreys 1939; fi. 2% protein silver (in jar con- taining 10 Gm. metallic copper per 100 ml.); C. AMS 21.1 Humphreys 1939; D. \% gold chloride; E. water 99, 40% formaldehyde 1, oxalic acid 2 method: [blood vessels dissected from formaldehyde-fixed material] -^ thorough wash —^ A, 4: hrs. -^ wash, 2 hrs. — > B, 6-24 hrs. — > wash — + C, till completely reduced — » D, till gray -^ wash — > E, till blue —* balsam, via usual reagents recommended for: perivascular nerves in wholemounts of intracerebral vessels. 32.1 MacFarland and Davenport 1941 20540b, 16:53 REAGENTS REQUIRED: A. 1% thallium nitrate; B. 1% protein silver; C 0.1% oxalic acid; D. AMS 21.1 Bodian 1936; E. 0.2% gold chloride; F. AMS 21.1 MacFarland and Davenport 1941; (?. 5% sodium thiosulfate 568 METHODS AND FORMULAS MS 32.1-MS 32.2 method: [15 yu paraffin sections of F 0000.4000 MacFarland and Davenport 1941 mate- rial] —> water —>■ A, 1-2 days, 60°C. — > wash -* B, 1-2 days, 37°C. -^ quick rinse -^ C, till differentiated, 7-15 sees. —>■ wash, 1 min. — > D, 3-5 mins. -^ wash -^ E, 5-10 mins. — + wash, H niin. -^ /^, till sufficiently darkened, 15-30 sees. —* balsam, via usual reagents RECOMMENDED FOR: nerve fibers in adrenal glands of mammals. 32.1 Rogoff 1946 20540b, 21:59 REAGENTS REQUIRED: A. 1% protein silver in a jar on the bottom of which has been placed 4-6 Gm. clean metallic copper per 100 ml.; B. AMS 21.1 Bodian 1936; C. AMS 22.1 Bodian 1936; D. 2% oxahc acid; E. 5% sodium thiosulfate method: [paraffin sections of mosquito larvae fixed in F 4000.0040 Petrunkewitsch 1933] — * water -^ A, 36 hrs., 37°C. — > wash -^ B, 5-10 mins. — > wash -^ C, 2-5 mins. — > quick rinse -^ D, 2-5 mins. -^ wash —>■ E, 5-10 mins. — * wash — * balsam, via usual reagents recommended for: cephalic ganglia of mosquitos. 32.1 Silver 1942 20540b, 17:123 reagents required: A. 0.2% protein silver; B. AMS 22.1 Silver 1942 method: [frozen, celloidin or paraffin sections of formaldehyde-fixed material, loose or on slide, but with embedding material removed] -^ water — » equal parts A and B, 45°C., till stained, 2-3 hrs. —* wash -^ balsam, via usual reagents recommended for: nuclei, fine fibers, nerve terminations. note : this method may be adapted to myelin sheaths by mordanting tissues for 1 week in 3% potassium dichromate 32.1 Stage 1936 20540b, 11:155 reagents required: A. 10% protein silver; B. 0.5% gold chloride; C. AMS 21.1 Stage 1936; D. 5% sodium thiosulfate method: [2 mm. slices fresh brain tissue] —> A, 2-20 days -^ 70% ale, rinse—' [cel- loidin, via graded ales, and ale. -ether] -^^ [section 10-20 ju]^80% ale. -^ B, till gray white, about 1 min. —y wash, 15 sees., or till differentiated — > dissolve celloidin — » balsam note: sections should not be exposed to alcohol lower than 85% between A and B. 32.1 Ungewitter 1943 20540b, 18:183 REAGENTS REQUIRED: A. 25% cliloral hydrate in 50% ale; B. \% protein silver in jar containing 2-3 Gm. metallic copper per 100 ml.; C. AMS 21.1 Ungewitter 1943; D. 1% silver nitrate method: [fresh tissue] — > A, 24 hrs. — * [paraffin sections] -^ water — > B, 24 hrs. -^ rinse — > C, 5-10 mins. — * wash — > D, 10-20 mins. — ^ wash — > [repeat C -^ D cycle till sufficiently stained] — ^ balsam, via usual reagents 32.2 Othek Protein Silver Methods 32.2 Dawson and Barnett 1944 see MS 32.1 Davenport, Windle, and Rhines 1947 32.2 Dublin 1943 Tech. Bull., 4:127 REAGENTS REQUIRED: A. 1% protein silver; B.\% hydroquinone; C 0.5% gold chloride; D. 5% oxalic acid; E. 10% sodium thiosulfate method: [paraffin sections of formaldehyde-fixed material] —* water —» A, overnight, 37.5°C. -^ rinse -^ B, 10 mins. ~> rinse — > C, 5 mins. -* rinse -^ D, 5 mins. — > rinse —* E, 5 mins. — > wash -^ balsam, via usual reagents RECOMMENDED FOR: demonstration of melanin. 32.2 Moskowitz 1950 20540b, 25:17 REAGENTS REQUIRED: A. 0.5% potassium permanganate; B. 5% oxalic acid; C. 1% pro- tein silver; D. water 100, sodium sulfite 5, hydroquinone 1; E. 0.2% gold chloride; F. 2% oxalic acid; G. 5% sodium thiosulfate method: [smears fixed in F 3000.0000 Schaudin 1893 and well washed] -^ A,2 mins. -^ wash -^ B, 2 mins. -^ thorough wash — > C, 36 hrs. at 35°C. — > rinse -^ F, 3 mins. —>■ wash -^ G, 8 mins. — * thorough wash — > balsam, via usual reagents RECOMMENDED FOR: extra-iiuclcar structures in protozoa. MS 33.0 METAL STAINS 569 MS 33 METHODS USING SILVER DIAMMINE 33.0 Typical Examples Demonstration of the nerve endings in the taste buds by the method of Bielschowsky 1904 This method is one of the best of tlic silver diammine techniques for the pur- pose of showing nervous structures, the demonstration of which is the sul)je('t of the next two examples, rather than con- nective tissues. In every type of metal staining so far described, emphasis has been laid on the necessity of securing pure reagents. This warning is the more necessary in the present instance because such common re- agents as sodium hydroxide and formal- dehyde are included among the materials retiuired. The sodium hydroxide employed must be of the analytical grade (purified by alcohol) and if possible free from all traces of chlorides. The formaldehyde em- ployed must be of analytical reagent grade and must be neutralized by the addition of analytical reagent grade borax. The term nexdrality in this instance may be extended to include any pH between 7 and 7.5, but under no circumstances may an acid formaldehyde be employed. The first stage in the preparation is the preservation of small blocks of tissue likely to contain taste buds, in 8 % neutral- ized formaldehyde. These may easily be obtained from a rabbit, in which form the taste buds are concentrated on the lateral surface of the ridges which comprise the foliate papillae. Secure a freshly killed rabbit and disarticulate the lower jaw cf)mpletely, cutting with scissors through the disarticulated joint. A large sharp scalpel or cartilage knife is then passed under the tongue to sever its attachments. The lowei' jaw is removed, and the tongue left in place. The latter can now be re- moved entire, washed free of extravasated blood, and placed with the dorsal surface uppermost on any convenient surface. Ex- amination of the posterior region of the tongue will sliow two oval bulges at the lateral portions of the posterior region. Each of these oval bulges (the foliate papillae) consists of many parallel trans- vorsel}^ arranged ridges with deep grooves between them. Taste buds are closely concentrated on the lateral surfaces of these ridges. Each ridge should be severed from its base by running a cartilage knife or razor roughly parallel to the surface of the tongue. A sufficient number of ridges thus removed may be placed until recjuired in the neutralized 8% formal- dehyde, which is to be changed after six hours. The length of time allowed for hardening in the formaldehyde is im- material, but should not be less than one month. Three or four days before proceeding with the staining technique, the pieces should be removed from formaldehyde and placed directly into pure pyridine for about three da,ys. It is probable that the main function of the pyridine is to cause a differential shrinkage of the nervous elements, which will thus become more apparent on subsequent staining. The success of subsequent operations depends on the removal of every trace of both formaldehj^de and pyridine from the pieces before they are placed in the silver staining solution. Though one could in theory achieve this by washing in running triple-distilled water, the quantities of water required would be so great that the process is not practical. It is therefore recommended that the pieces be washed in running tap water for at least 24 hours (double the time will not hurt them) and then rewashed in triple-distilled water to remove impurities which may have come from the tap water. The second wash should either be in running triple-distilled water, or alternatively in not fewer than five changes of large volumes of triple- distilled water changed daily. Before proceeding further it is necessary to make up the Bielschowsky stock silver solution (MS 33.1 Bielschowsky 1902) used in this technique. First secure a chemically clean 500-milliliter beaker, a chemically clean 250-milliliter graduated flask, a chemically clean glass stirring rod, and a chemically clean l)uret. Place 120 milliliters of triple-distilled water in 570 METHODS AND FORMULAS MS 33.0 the beaker and add to it 2.4 grams of reagent-grade silver nitrate. When the solution is complete add one milliliter of a 40% solution of sodium hydroxide pre- pared from analytical reagent-grade NaOH dissolved in triple-distilled water. Mix thoroughly until the curdlike precipitate is uniformly distributed through the mass. From the buret add reagent-grade am- monia to this curdy material, stirring vigorously after the addition of each drop. As soon as the curdy precipitate is seen to be clearing up, add one drop about every ten seconds, stirring vigorously be- tween additions. It is as well to put the solution in a good light against a black background so as to detect the exact moment at which the last of the precipi- tate has vanished. It is fatal to the tech- nique to add too much ammonia, but it does not particularly interfere with the stain if a slight excess of the precipitated silver hydroxide is present. Some authors have accordingly recommended that after the end point of the reaction has been reached (that is, when the precipitate has been exactly redissolved) one or two drops of a very weak solution of silver nitrate should be added so that the faintest opalescence is produced. The solution is then transferred to a graduated flask and made up to a total volume of 250 milli- liters with triple-distilled water. This is the stock solution which in the present technique is diluted 40 stock to 60 triple- distilled water before use. This staining solution is used after pre- treatment with 3% silver nitrate, immer- sion in which is the next step. This 3% silver nitrate should be in a chemically clean stoppered bottle. To it the well- washed pieces of tongue are removed from triple-distilled water, and there incubated at 36°C. for three days. This reaction is best conducted in the dark, and like reac- tions in all other silver techniques must be watched closely to make sure that no im- purities present in the solution or in the bottle cause a precipitate of metallic silver or of silver salts. If the least cloudiness is seen in the solution, or if a black precipi- tate appears to be accumulating on the bottom, the pieces should immediately be removed to a fresh solution. After three days, each piece of tongue is removed from the silver nitrate solution and rinsed briefly in a large volume of triple-distilled water. The object of this rinse is not so much to remove silver ni- trate from the interior of the material as to prevent carrying over excess silver ni- trate solution into the silver stain. When the pieces have been freed from the sur- face-adherent silver nitrate solution, they are transferred to the diluted staining so- lution, where they remain at room temper- ature for 24 hours. They are then washed in running distilled water for at least 24 hours, or in three changes of large vol- umes of triple-distilled water changed at intervals of about 12 hours, before being placed in a fresh batch of 8% neutralized formaldehyde. The washing which precedes immersion in formaldehyde is one of the most critical steps of the entire technique, for if silver stain or silver nitrate is carried over into the reducing solution, the preparation will be spoiled through a general deposition of silver proteinates. After remaining in the neutral formalde- hyde overnight, the pieces are washed thoroughly in tap water. Sections are then prepared by the ordinary parafhn tech- nique. When deaUng with materials as strongly muscularized as is tongue, how- ever, it is desirable to use benzene as a clearing agent in order to prevent the mus- cle from becoming brittle and thus inter- fering with subsequent sectioning. After the sections have been mounted on a sUde, they are passed down to water, thoroughly washed, and then examined under a high power of the microscope. If the sections clearly display the nerve endings in the taste buds, they may be again upgraded through the successive strengths of alcohol, dehydrated, and mounted in balsam. If, as is frequently the case, the nerve endings are only lightly stained, the sections placed in a very weak solution of gold chloride (0.004%) until the pale brown color of the nervous mate- rial has been replaced by the dark purple of gold. After this treatment the sections are rinsed for a few minutes in 5 % sodium thiosulfate to remove any residual silver which has not been replaced by the gold, MS 33.0 METAL STAINS 571 washed thoroughly in either tap water or distilled water, dehydrated, and mounted in balsam. Demonstration of oligodendria and microglia by the method of Penfield 1938 This is one of the most complex of the silver staining techniques, hut tlie com- plexities are justified by tlie relative cer- tainty with which results may be obtained. A relatively large number of reagents are required and it is best to make sure that all of these are available before starting the technique. These reagents are de- scribed below in the order in which they are required. First is Cajal 1913 formol-bromide solu- tion (AMS 11.1 Cajal 1913— Chapter 24). This is made by adding 38 c. milUhters of neutrahzed 40% formaldehyde to 212 milliUters of triple-distilled water. The formaldehyde must be of reagent grade and should have been neutralized with re- agent-grade borax. The term neutral refers in this instance to any pH between about 7 and 7.6. Five grams of reagent-grade ammonium bromide are then added. This solution is stable indefinitely. The next three reagents required are a 1 % dilution of ammonia, 2% hydrobromic acid, and 5% sodium carbonate. Both the acid and the carbonate must be of the finest grade available; the latter in par- ticular must be chloride-free. The silver stain used is a dilution of del Rlo-Hortega 1921 silver carbonate (MS 33.1 del Rio-Hortega 1921). This is pre- pared from a 10% solution of pure silver nitrate in triple-distilled water, a 5% solu- tion of reagent-grade sodium carbonate (also in triple-distilled water), and am- monia. Place 30 milliUters of 10% silver nitrate in a 250-miUihter beaker and add to this, with constant agitation, 120 milli- liters of sodium carbonate solution. The solution is now allowed to stand until the silver carbonate has fallen to the bottom. Then as much as possible of the supernatant Uquid is poured from the top. The beaker is then filled with a fresh batch of triple- distilled water, thoroughly agitated, and again allowed to settle. The supernatant liquid is poured off and the process re- peated about three times. The precipitate may alternatively be accumulated on a chemically clean filter paper on chemically clean glassware and washed by passing considerable volumes of triple-distilled water through it. Whatever method may be adopted, the silver carbonate is col- lected in approximately 100 milliliters of triple-distilled water; and reagent-grade ammonia is added drop by droj), with agi- tation between drops, until the carbonate is just dissolved. It is essential that the ammonia should not be in excess. At the moment when the precipitate is seen to be clearing up, the rate of addition of am- monia should be reduced to one drop every 10 seconds. The beaker should be observed in a good light against a black background. When the carbonate is dissolved, make up the solution with triple-distilled water to a volume of 240 milliliters. For purposes of the present technique, this stain is diluted 56 of the solution just described to 44 of triple- distilled water. One requires also a 0.4% neutrahzed formaldehyde solution (that is, one milli- Uter of neutralized formaldehyde diluted with 99 milhhters of water), a 0.2% solu- tion of gold chloride, and the usual 5% solution of sodium thiosulfate. All these reagents except the diluted stain are sta- ble. The latter may usually be kept some weeks in a well-stoppered bottle, prefer- ably in the dark. Since the method of Penfield is designed to show both oligodendria and microglia, considerable care must be taken to make sure that the former are present in a nor- mal condition in the brain at the time it is fixed. Ohgodendria (McClung 1929, 362) will be distorted almost beyond recogni- tion if the death of the animal from which they are taken is preceded by coma or deep stupor. Under these circumstances it is best to kill the rabbit used for the prepa- ration by a sharp blow on the occipital region rather than by the more conven- tional method of chloroform or ether. Having killed the rabbit, fasten it face down on a convenient dissecting board, skin the head, remove the parietal and frontal bones with bone forceps, flood the 572 METHODS AND FORMULAS MS 33.0 brain with the formaldehyde-bromide as a liemostatic measure, and tlien remove small pieces from the white matter of the cerebrum or from such other portions of the brain as it is desired to study. The white matter of the cerebrum is recom- mended as a material because of the rela- tive certainty with which the supporting elements witliin it may be demonstrated. The tissue is best removed in blocks of about 3-2 cm. cube, and as tlie author has elsewhere indicated, it is preferable to use for this purpose the broken edge of a coverslip rather than steel instruments. Three or four of these blocks are removed to the formaldehyde-bromide solution in a chemically cleaned stoppered bottle and permitted to remain there until such time as one is ready to proceed with the prepa- ration. It is stated by McClung 1929, 379 that one week in this solution gives excel- lent results; but the original recommenda- tion of Penfield does not place any time limit on the preliminary hardening. When one is ready to prepare and stain sections it is simplest to erect a sort of production line of glass dishes containing the successive solutions and water for the intermediate washings. The size and shape of these dishes is not of the slightest im- portance provided that they are chem- ically cleaned. Dishes in which it is in- tended to leave the reagents exposed for any length of time should be made of pyrex glass and furnished with some kind of lid. In the writer's experience the most readily available and useful dish is a deep pyrex petri dish at least 15 milhmeters in depth, even though these dishes require rather large volumes of solution. Twelve such dishes should be chemically cleaned (that is, soaked in dichromate-sulfuric cleaning mixture), washed in tap water, soaked in distilled water, and dried under dust-free conditions. The first dish should contain triple-dis- tilled water. The blocks of tissue are placed on a freezing microtome and sec- tioned to a thickness of from 15 to 25 microns, and the sections are taken off on a wet knife and accumulated in this dish. When a sufficient number have been ac- cumulated, one may proceed with the staining. In another dish place the 1 % ammonia, and transfer to it the sections from the dish of triple-distilled water. Here they may remain overnight to insure the com- plete washing out of formaldehyde and the neutralization of anj^ residual acid which may be piesent. The next morning fill another dish with 2 % hydrobromic acid and transfer the sec- tions to it one at a time, being careful to pick them up with a glass utensil. It will be found that a 22 X 15 mm. coverslip is excellent for this purpose. As soon as enough sections have been accumulated in the hydrobromic acid, the dish is placed for one hour on the surface of a water bath which is maintained at from 37° to 40°C. While these sections are being treated, fill three more dishes with water and a fourth with the 5% sodium carbonate. The dish containing the sections is now removed from the bath and, again with a glass utensil, the sections are removed to the first dish of wash water. Here they remain for about five minutes while the dish is gently rocked at intervals. All the sections are next removed to the second dish of water for at least 30 minutes of rocking at intervals; and then to the third dish for not less than 15 minutes of similar wash- ing. While the washing in the third dish is being concluded the next dish is filled with 5% sodium carbonate. All the sec- tions are transferred to this and permitted to remain for about one hour, though they may be left two or three times as long without interfering with subsequent stages of the technique. Next take four clean dishes. Fill the first of these with triple-distilled water, the second with the diluted silver stain, the third with triple-distilled water, and the fourth with 0.4% neutrahzed formal- dehyde. Take the first of the sections from the sodium carbonate, rinse it in the first dish of distilled water, and pass it to the dish of diluted silver stain. There it should remain for a period of three minutes. It is then removed, rinsed in the next dish of distilled water, and passed into the dish of formaldehyde. It should turn a dull steel- grey color almost immediately. If the first section does not turn this color, take a second section from the sodium carbonate, MS 33.0 METAL STAINS 573 rinse it, place it in the reduced silver solu- tion for four minutes, rinse it again, and transfer it in its turn to the fornuildehyde. If this section does not then turn grey, repeat the process with another section, leaving it in the reduced silver solution for five minutes. Ry this method one may establish the period of time necessary to secure adequate impregnation of sections in tlie particular batch witli wliicli one is dealing. As soon as this has been estab- lished four or five sections may be taken at a time, placed in the silver, and gently rocked to make sure that some sections do not rest on top of others. Next, the sec- tions are removed all at the same time to the distilled water, in which they are rinsed; and then to the formaldehyde, in which they may be accumulated. The whole batch of sections may thus be passed through stain and accumulated in formaldehyde. Now take four more clean dishes. In the first of these place distilled water; in the second, the gold chloride toning solution; in the third, the 5% solution of sodium thiosulfate; and in the fourth, another bath of distilled water. The entire batch of sections is now taken at one time from the formaldehyde, placed in the distilled water, and there rocked gently to and fro. If there are many sections, change the water so as to insure thorough washing. When they have been sufficiently washed, the entire batch is transferred to the next dish containing the gold chloride. In this solution the dull steel-grey of the sections changes to the purphsh-blue-grey of gold- stained material. They should be left until the change is complete — usually within a few minutes. It does not in the least mat- ter if they are left for several hours. The sections are next taken all at one time from the gold chloride solution and passed without preliminary washing to the sodium thiosulfate solution. This removes from them the silver salts which may not have been completely replaced by the gold. Four or five minutes is sufficient for this change. The sections should certainly not be left here longer than about ten minutes for there is some risk of removing the stain. After the thiosulfate treatment they are passed to the next dish of dis- tilled water, in which they must be rocked back and forth. If anj^ considerable num- ber of sections is included in the batch, this distilled water should be changed once or twice, since it is necessary to re- move all thiosulfate from the sections be- fore mounting them. Now take an entirely new batch of dishes containing the cus- tomary dehydrating and clearing agents, run the sections through, and mount them in halsan:i. This technique may be modified to show astrocytes, and thus present a very com- plete picture of the supporting structures of the cortex of the cerebrum. Simply leave the sections too long in the silver stain. This will result in a less clear pic- ture of the oligodendria and microglia, but may be desirable for class demonstration purposes. To achieve this result leave them in the silver staining solution until they have changed to a definite dark straw color. This will usuall}' require from five to ten minutes, and may be judged by eye without difficulty. The rest of the process is in every way similar. If the reader is trying this technique for the first time, it might be of interest for him to leave two or three sections in the silver stain while he is taking the remainder through the test of the technique. Demonstration of microglia by the technique of del Rio-Hortega 1921b The last example demonstrated the use of del Rfo-Hortega's silver-carbonate tech- nique for the demonstration of both oligo- dendroglia and microdendroglia. The pres- ent method demonstrates the use of del RIo-Hortegu's silver-hydroxide technique for a differential demonstration of micro- glia. The technique is much quicker and shorter than that of Penfield and is to be recommended where the demonstration of oligodendroglia is not required. The procedure of securing the blocks of tissue and fixing them in Cajal's formal- dehyde ammonium-bromide mixture is identical with that of the last example, to which reference should be made. Pieces of tissue, however, are fixed in a slightly dif- ferent manner. Thev are allowed to remain 574 METHODS AND FORMULAS MS 33.0 in a large volume of the formaldehyde bromide for from two to three days at room temperature before being removed to a fresh batch of bromide in a chemically clean beaker which is then raised to a temperature of 55°C. for about ten min- utes. The bromide is then cooled to room temperature and sections taken from it by the freezing technique described in Chap- ter 15. Sections should be about 25 mi- crons in thickness, and cut with a knife moistened in distilled water. Sections are removed from this knife to a weak solution of ammonia, where they may remain until required ; certainly at least overnight. Before proceeding further it is neces- sary to make up the customary solutions. The only one presenting any difficulty is the silver complex of del Rio-Hortega. This is given below as MS 33.1 del RIo- Hortega 1916. All that has already been said about the necessity of the absolute chemical cleanliness of the glassware and the purity of reagents can be repeated at this point. Triple-distilled water, reagent- grade sodium hydroxide, and reagent- grade ammonia are essential. Place 50 milhliters of 10% silver nitrate in a chemically clean 500-milliliter beaker and add to it, with constant stirring, 3.5 milhUters of 40% sodium hydroxide. Stir until the white curd of silver hydroxide is uniformly distributed. Then while still stirring add about 250 milliliters of triple- distilled water. Allow the precipitate to settle, pour off the supernant liquid, and add a further 250 milliliters of triple-dis- tilled water. This washing by decantation should be repeated at least three times, and the precipitate allowed to settle after the last washing until all but 50 milliliters of the wash water can be discarded. The wet precipitate is then removed to a chem- ically cleaned graduated cylinder and the volume brought up to 75 milliliters before being transferred back to the beaker. Ammonia is added drop by drop until the precipitate is just redissolved. As has been recommended in previous examples, the ammonia should be added slowly. When there are signs that the precipitate is clear- ing up, the (hops shouUl be added at inter- vals of from 10 to 15 seconds, with con- stant stirring between additions. A strong light and a dark background assist in de- termining the end point of the reaction. The mixture is now placed in a graduated flask and brought up to a volume of 250 milhliters with triple-distilled water. In the present method this staining solution is used at full strength. Other solutions also required are : a 4 % solution of neutralized formaldehyde (re- member that reagent-grade formaldehyde neutralized to a pH between 7 and 7.5 with reagent-grade borax must of neces- sity be employed), a 2% solution of gold chloride, and the regular fixing solution of sodium thiosulfate. As in the last ex- ample, it is recommended that staining be carried out in chemically cleaned petri dishes. In the present instance not more than half a dozen such dishes will be re- quired. In the first of these place triple- distilled water; in the second, the stain; in the third, triple-distilled water; in the fourth, the neutralized formaldehyde; in the fifth, triple-distilled water; and in the sixth and seventh, the gold chloride and the fixative. Now take sections from the weak ammonia, place them in the first dish of triple-distilled water, and rock them back and forth until they are alkali- free. A trial section is placed in the silver staining solution for approximately three minutes, then removed from the stain, rinsed rapidly in the next dish of distilled water, and placed in the neutralized form- aldehyde. There, in from three to five minutes, it should have assumed a clear grey color, which will not become appreci- ably darker for another five minutes. This reaction time indicates that three minutes in the stain is satisfactory. If the darken- ing is too great or appears too quickly, the time that the section is left in the stain must be reduced. On the other hand, if the section fails to darken, the time must be increased. When the correct time has been established, all the sections may be passed successively through the stain, the dis-- tilled water, and the neutralized formal- dehyde. After a maximum time of approx- imately ten minutes in the formaldehyde, the sections are removed to the next dish of triple-distilled water and thoroughly washed before being toned for ten to twenty minutes and fixed for five minutes. MS 33.1 METAL STAINS 575 They may then be mounted in balsam in the ordinary manner. It will be immediately apparent that this method is both (juicker and simpler than the method of Penfield; but it does not give such certain results, nor does it stain oligodendria except by an occasional accident which cannot be forecast. In the author's opinion it is less generally satis- factory than Penfield's technique even for the demonstration of microglia. 33.1 Staining Solutions The solutions of diammino silver used in these techniques are usually prepared by precipitating silver nitrate with sodium hydroxide and then adding ammonia drop by drop until the precipitate is just redis- solved. It is much better to have a faint opalescence remain than to use too much ammonia. Some authors (Fontana 1912, Masson 1928, Gomori 1937) ' recommend a second addition of a few drops of silver nitrate solution to insure an excess of this reagent; but see Gros-Schultze 1938 below, in which an excess of ammonia is used. Sodium hydroxide as the initial precipi- tant is sometimes replaced by potassium hydroxide (Foot 1927a, Gomori 1937), sodium carbonate (Cajal 1925, del Rio- Hortega 1917, 1921, 1923, and 1927), lithium carbonate (Foot 1927b, Laidlaw 1929, del Rio-Hortega 1919), or potassium oxalate (Herrera 1932). Ammonia is used to produce as well as to dissolve the original precipitate by Fontana 1912, Gros-Schultze 1938, Lillie 1948, Masson 1928, and Weil and Davenport 1933. Tri- ethanolamine is used both as precipitant and solvent by Zettnow 1891. Gomori 1936 used methenamine, and Herrera 1912 used ethylamine, for the same purpose. Most of the solutions contain radicals derived from the original silver nitrate as well as from the precipitant and solvent. On the contrary, the precijjitate is washed after either filtration or decantation in the formulas of Foot 1927b, Jalowy 1937, Landlaw 1929, and del Rio-Hortega 1916 and 1921. The result of all this is of course to pro- vide solutions of diammino silver of various strengths and with various im- purities. The table below summarizes the solutions recommended, hsting (1) grams of silver per liter, (2) reagent used to pro- duce the original precipitate, and (3) re- agent used to dissolve this precipitate. Many of these solutions may appear to be identical, but most authors are insistent that the staining solution must be pre- pared by the method and in the concentra- tion which they recommend. The original specifications are therefore given immedi- ately following this table. For the sake of uniformity each formula has been ad- justed to a final volume of 100. SUMMARY OF DIAMMINO SILVER STAINING SOLUTIONS Author Gms Ag/L Precipitant Solvent Agduhr 1917 3.2 NaOH NH4OH Arcadi 1948 6.3 NazCOs NH4OH Belezky 1931 57 NaoCOa CsHsN Bensley and Bensley 1938a 0.6 NaOH NH4OH Bensley and Bensley 1938b 12.7 NaOH NH4OH Bertrand and Guillain 1934 4.4 NaaCOs NH4OH Bielschowsky 1902 6.1 NaOH NH4OH Cajal 1920a 7.6 NaOH NH4OH Cajal 1920b 1.9 NaOH NH4OH + CsHsN Cajal 1925 3.8 NasCOa NH4OH + CsHsN Cajal (1933) 6.3 NH4OH NH4OH del Carpio 1930 0.6 NaOH NH4OH Cone and Penfield 1929 3.2 NasCOa NH4OH Fajersztajn 1901 12.7 NH4OH NH4OH da Fano 1914 12.7 NaOH NH4OH da Fano 1919 15.9 NaOH NH4OH Fontana 1912 1.6 NH4OH NH4OH > Literature references to the authors cited in this and the next two paragraphs and in the table which follows are given in the descriptions of the preparation of the solutions. 570 METHODS AND FORMULAS MS 33.1 SUMMARY OP DIAMMINO SILVER STAINING SOLUTIONS — (Continued) Author Foot 1924 Foot 1927a Foot 1927b Gatenby and Stern 1937 Glees, Meyer, and Meyer 1946 Gluck 1938 Gluckman 1943 Gomori 1937 Gomori 194G Gordon 1930 Gordon and Sweets 1936 Gros-Schultze (1938) Herrera 1932 Holmes 1942 Jalowy 1937 Kalwaryjski 1938 King 1937 Krajian 1933 Laidlaw 1929 Lawrentjew (1933) Levi 1907 Lillie 1948 Lobo 1937 Long 1948 Maresch 1905 Masson 1928 Patton 1907 Penfield 1935 del Rio-Hortega 1916 del Rio-Hortega 1917a del Rio-Hortega 19J7b del Rio-Hortega 1919 del Rio-Hortega 1921 del Rio-Hortega 1923 del Rio-Hortega 1927 del Rio-Hortega 1932 Robb-Smith 1937 Rogers 1931 Romanes 1946 Wallart 1935 Weber 1944 Weil and Davenport 1933 Wolff 1905 Zettnov 1891 Gnis Ag/L Precipitant Solvent 15.9 NaOH NH4OH 0.6 KOH NH4OH 6.3 LiCOs NH4OH 25.4 NaOH NH4OH 63 NH4OH NH4OH 6.3 NH4OH NH4OH -t- CsHoN 95 NH4OH NH4OH 25.4 KOH NH4OH 1.9 C6H12N4 C6H:2N4 3.2 NH;OH + NaOH NH4OH 6.3 NH4OH + NaOH NH4OH 127 NH4OH NH4OH 3.2 K2C2O4 C2H5NH2 0.06 NH4OH NH4OH 12.7 NaOH NH4OH 31.7 NH4OH NH4OH 12.7 NaOH NH4OH 18.4 NaOH NH4OH 30.5 LiCOa NH4OH 127 NH4OH NH4OH 21.5 NaOH NH4OH 31.8 NH4OH NH4OH 31.7 C5H5N C5H5N 79 L1CO3 NH4OH 63.5 NaOH NH4OH 15.9 NH4OH NH4OH 6.3 NaOH NH4OH 127 NH4OH NH4OH 12.7 NaOH NH4OH 15.9 NaaCOs NH4OH 15.9 NaoCOs NH4OH + CfiHsN 7.9 LiCOs NH4OH 7.9 NaoCOs NH4OH 7.9 NaoCOa NH4OH 12.7 Na.COa NH4OH 7.9 UCOz NH4OH + CsHsN 18 NaOH NH4OH 61 NaOH NH4OH 0.45 NH4OH NH4OH 58 NH4OH NH4OH 15.9 NH4OH NH4OH 15.9 NH4OH NH4OH 63.5 NaOH NH4OH 2.5 N(CHoCH20H)3 N(CHoCH20H)3 33.1 Agduhr 1917 23632, 34:1 preparation: Mix 5 10% silver nitrate witii 1 25% sodium liydroxide. Dilute to 100 and add just enough ammonia to redissolve ppt. 33.1 Amprino 1936 see MS 34.1 Amprino 1936 33.1 Arcadi 1948 20540b, 23:77 PREPAHATiox: To 10 10% silver nitrate add 30 5% sodium carbonate. Add just enough ammonia to dissolve ppt. and dilute to 100. 33.1 Belezky 1931 22575, 282:214 PREPARATION: To 50 cach of 17% silver nitrate and 10% sodium carbonate add just enough pyridine to dissolve ppt. MS 33.1 METAL STAINS 577 33.1 Bensley and Bensley 1938a Bcnsley and Bensley 1938, 109 preparation: Add 0.1 40% sodium hydroxide to 10 1% silver nitrate. Add just enough ammonia to redissolve ppt. Dihite to 100 nd. 33.1 Bensley and Bensley 1938b Bensley and Bensley 1938, 109 preparation: Add 0.75 40% sodium hydroxide to 100 2% silver nitrate. Add just enough ammonia to redissolve ppt. 33.1 Bertrand and Guillain 1934 6630, 115:706 preparation: To 7 10% silver nitrate add 27 5% sodium carbonate and then just enough ammonia to dissolve ppt. Dilute to 100. 33.1 Bielschowsky 1902 15058, 21 :579 preparation: Add 0.4 40% sodium hydroxide to 48 2% silver nitrate. Add just enough ammonia to dissolve the ppt. Dilute to 100 ml. 33.1 Cajal 1920a hst. 1933 ips. Cajal nnd de Castro 1933, 253 preparation: To 12 10% silver nitrate add 0.5 40% sodium hydroxide. Wash ppt. by decantation. Suspend ppt. in 100 water. Add just enough ammonia to dissolve ppt. 33.1 Cajal 1920b (ed. 1933 ips. Cajal and de Castro 1933, 253 preparation: Dilute 25 MS 33.1 Cajal 1920a to 100. Add 1 pyridine. 33.1 Cajal 1925 21344, 2:157 preparation: Prepare 50 MS 33.1 del Rio-Hortega 1921 (below). Add 1.5 pyridine. Dilute to 100. 33.1 Cajal test. 1933 ips. Cajal and de Castro 1933, 319 preparation: To 100 1% silver nitrate add just enough ammonia to redissolve the ppt. first formed. 33.1 del Carpio 1930 test. 1932 Findlay 11360, 52:155 preparation: To 1 10% silver nitrate add 0.3 40% sodium hydroxide. Add just enough 20% ammonia to dissolve ppt. and dilute to 100. 33.1 Cone and Penfield /t.s/. 1929 Anderson Anderson 1929, 90 preparation: Add 5 10% silver nitrate to 20 5% sodium carbonate. Add just enough ammonia to dissolve ppt. Dilute to 100. 33.1 Craigie 1928 see MS 33.1 Zettnov 1891 (note) 33.1 Davenport, Windle, and Beech 1934 20540b, 9:5 preparation: Add 5 ammonia to 40 2% sodium hydroxide. Add just enough (about 40) 8.5% silver nitrate to give a slight permanent opalescence. 33.1 Debauche 1939 see MS 33.1 Rogers 1931 (note) 33.1 Fajersztajn 1901 test. 1933 Cajal and de Castro Cajal and de Castro 1933, 223 preparation: To 100 2% silver nitrate add just enough ammonia to redissolve the i)i)t. first formed. 33.1 da Fano 1914 2190, 3:14 preparation: Add 0.2 40% sodium hydroxide to 10 20% silver nitrate. Add just enough ammonia to dissolve ppt. Dilute to 100. 33.1 da Fano 1919 - 11454,52:1919 preparation: As da Fano 1914 above but diluted to 80 instead of 100. 33.1 Fontana 1912 7170, 55:1003 preparation: To 100 0.25% silver nitrate add ammonia until ppt. first formed is just redissolved. Then add 0.25% silver nitrate drop by drop until a faint permanent opalescence is produced. 33.1 Foot 1924 11284, 9:778 preparation: Add 1 40% sodium hydroxide to 25 10% silver nitrate. Add just enough ammonia to dissolve ppt. Dilute to 100. 578 METHODS AND FORMULAS MS 33.1 33.1 Foot 1927a 1887a, 4:42 preparation: Add 0.1 40% potassium hydroxide to 10 1% silver nitrate. Add just enough ammonia to redissolve ppt. Dilute to 100 ml. 33.1 Foot 1927b 1887a, 4:212 preparation: To 10 10% silver nitrate add 10 sat. aq. sol. (circ. 1.5%) lithium car- bonate. Allow ppt. to settle. Decant. Wash ppt. by decantation several times. Sus- pend washed ppt. in 25 water. Add enough ammonia to not quite dissolve ppt. Dilute to 100. Filter. 33.1 Gatenby and Stern 1937 Gatenby and Painter 1937, 487 preparation: Add 0.6 ml. 40% sodium hydroxide to 20 20% silver nitrate. Add just enough ammonia to dissolve ppt. Dilute to 100. Filter. 33.1 Glees, Meyer, and Meyer 1946 11025, 80:101 preparation: To 100 10% silver nitrate in 50% ale, add just enough ammonia to redis- solve the ppt. first formed. 33.1 Gluck 1938 test. 1939 Foot 4349, 19:169 preparation: To 10 10% silver nitrate add 0.5 40% sodium hydroxide. Wash ppt. by decantation and add just enough ammonia to dissolve ppt. Dilute to 100, and add 2 pyridine. 33.1 Gluckman 1943 4285a, 20:132 preparation: To 100 15% silver nitrate add just enough ammonia to redissolve the ppt. first formed. 33.1 Gomori 1937 608b, 13:993 preparation: Mix 10 10% potassium hydroxide with 40 10% silver nitrate. Add am- monia till ppt. dissolved. Add 10% silver nitrate drop by drop until ppt. just redis- solves on shaking. Dilute to 100 ml. 33.1 Gomori 1946 591b, 10:177 preparation: Add 5 5% silver nitrate to 100 3% methenamine. Shake until ppt. is dissolved. 33.1 Gordon 1936 11284,22:294 preparation: To 5 10% silver nitrate add just enough ammonia to redissolve ppt. first formed. Add 5 3% sodium hydroxide. Again add just enough ammonia to dissolve ppt. Dilute to 100. 33.1 Gordon and Sweets 1936 608b, 12:545 preparation: To 10 10% silver nitrate add just enough ammonia to redissolve ppt. first formed. Add 10 3% sodium hydroxide. Again add just enough ammonia to dis- solve ppt. Dilute to 100 ml. 33.1 Gros-Schultze test. 1938 Mallory Mallory 1938, 227 preparation: Prepare a stock solution by adding just enough ammonia to 100 20% silver nitrate to redissolve the ppt. first formed. Prepare working solution by adding about 2 (more or less according to experience) ammonia to 100 stock. note: This solution is given as "Gros" (without reference) by Cajal and de Castro 1933, 178. 33.1 Gros 1933 see MS 33.1 Gros-Schultze 1938 (note) 33.1 Herrera 1932 test. 1933 Cajal and de Castro Cajal and de Castro 1933, 275 preparation: To 20 5% potassium oxalate add 5 10% silver nitrate and 0.3 abs. ale. Add just enough 33 % ethylamine to dissolve ppt. Dilute to 100. 33.1 Holmes 1942 11431, 64:132 preparation: To 100 0.01% silver nitrate add 1 drop ammonia. 33.1 Jalowy 1937 23639b, 27 :667 preparation: Add 1 40% sodium hydroxide to 20 10% silver nitrate. Mix thoroughly and filter. Wash ppt. thoroughly and suspend in 20 water. Add just enough ammonia to dissolve ppt. Dilute to 100 ml. MS 33.1 METAL STAINS 579 33.1 Kalwaryjski 1938 test. 1939 Findlay 11360, 59:36 preparation: To 50 10% silver nitrate add 50 ammonia. 33.1 King 1937 1879, 38:362 preparation: Add 20 10% silver nitrate to 80 5% sodium carbonate. Add just enough ammonia to dissolve ppt. 33.1 Krajian 1933 1789a, 16:376 preparation: Add 1 10% sodium hydroxide to 29 10% silver nitrate. Add just enough ammonia to dissolve ppt. Dilute to 100. 33.1 Laidlaw 1929 608b, 5:239 preparation: To 16 60% silver nitrate add 185 sat. aq. sol. {circ. 1.5%) lithium car- bonate. Wash ppt. by decantation three or four times and suspend washed ppt. in 60 water. Add just enough ammonia to dissolve ppt. and dilute to 100. 33.1 Lawrentjew test. 1933 Cajal and de Castro Cajal and de Castro 1933, 361 preparation: To 100 20% silver nitrate add not quite enough ammonia to redissolve ppt. first formed. Immediately before use add from 3% to 8% (according to experi- ence) ammonia. 33.1 Levi 1907 14225, 18:292 prep.^ration: Mix 17 20% silver nitrate with 17 40% sodium hydroxide. Add just enough ammonia to dissolve ppt. Dilute to 100 ml. 33.1 Lillie 1948 see MS 33.1 Weil and Davenport 1933 (note) preparation: To 20 ammonia add 10% silver nitrate, drop by drop, until a faint permanent-opalescence is produced. Dilute to twice the volume. 33.1 Lobo 1937 test. 1948 Romeis Romeis 1948, 418 formula: water 62.5, 95% ale. 37.5, silver nitrate 1.25, pyridine 2.5 33.1 Long 1948 20540b, 23:69 preparation: To 10 50% silver nitrate add, drop by drop with constant agitation, 100 sat. sol. lithium carbonate. Wash ppt. 5 times by decantation. Add not quite enough ammonia to dissolve ppt. Dilute to 100, filter, heat to 50°C. for 30 minutes in open vessel, cool, filter. 33.1 Maresch 1905 23681,17:641 prep.\ration: To 100 10% silver nitrate add 0.5 40% sodium hydroxide. Add just enough ammonia to dissolve ppt. 33.1 Martinez 1931 see MS 34.1 Martinez 1931 33.1 Masson 1928 608b, 4:181 preparation: Add to 12.5 20% silver nitrate enough ammonia to redissolve ppt. first formed. Then add 20% silver nitrate drop by drop until a slight permanent turbidity is produced. Dilute to 100. 33.1 Paton 1907 1424b, 18:567 preparation: Add 14% sodium hydroxide to 100 1% silver nitrate. Add just enough ammonia to dissolve ppt. 33.1 Penfield 1935 608b, 11:1007 preparation: To 100 20% silver nitrate add just enough ammonia to redissolve ppt. first formed, then add 3 drops excess ammonia. 33.1 del Rio-Hortega 1916 21344, 14:181 preparation: To 20 10% silver nitrate add 1.4 40% sodium hydroxide. Add just enough ammonia to dissolve ppt. Dilute to 100 ml. note: Cajal and de Castro 1933, 211 wash the ppt. by decantation before re-solution. 33.1 del Rio-Hortega 1917a 21344, 15:367 preparation: To 25 10% silver nitrate add 75 5% sodium carbonate. Add just enough ammonia to dissolve ppt. 580 METHODS AND FORMULAS MS 33.1 33.1 del Rio-Hortega 1917b 21344, 16:367 preparation: To 100 MS 33.1 del Rio-Hortega 1917a add 1 pyridine. 33.1 del Rio-Hortega 1919 3231, 9:68 preparation: To 50 ml. sat. aq. sol. {circ. 1.5%) lithium carbonate add 12 10% silver nitrate. Allow ppt. to settle and wash by decantation. Add just enough ammonia to dissolve ppt. Dilute to 100. 33.1 del Rio-Hortega 1921 3232, 21:14 preparation: To 50 5% sodium carbonate add 12 10% silver nitrate. Leave ppt. settle and wash by decantation. Add just enough ammonia to dissolve ppt. Dilute to 100. 33.1 del Rio-Hortega 1923 21344, 21 :95 preparation: To 50 5% sodium carbonate add 12.5 10% silver nitrate. Add just enough ammonia to dissolve ppt. Dilute to 100 ml. 33.1 del Rio-Hortega 1927 3232, 27:199 preparation: To 80 5% sodium carbonate add 20 10% silver nitrate. Add just enough ammonia to dissolve ppt. 33.1 del Rio-Hortega 1932 test. 1933 Cajal and de Castro Cajal and de Castro 1933, 263 preparation: To 100 MS 33.1 del Rio-Hortega 1917 add 1 pyridine. 33.1 Robb-Smith 1937 11431,45:312 preparation: To 28.5 10% silver nitrate add 0.6 10% sodium hydroxide. Add just enough ammonia to redissolve precipitate and dilute to 100. 33.1 Rogers 1931 763, 49:81 preparation: To 48 20% silver nitrate add just enough ammonia to redissolve the ppt. first formed. Then add 0.5 ammonia and 48 water, note: Debauche 1939 (20540b, 14:121) differs only in using twice as much 10% silver nitrate. 33.1 Romanes 1946 11025,80:205 preparation: To 2 5% silver nitrate add just enough ammonia to redissolve the ppt. first formed. Dilute to 100. To this add 33 1% gelatin, 2.5 0.5% tannic acid, and 1 pyridine. 33.1 Steiner 1937 11284, 23:293 preparation of stock solutions: I. Dissolve 1 silver nitrate in 20 water. Add am- monia until ppt. first formed is not quite dissolved. Dilute to 100. II. Dissolve 0.2 silver nitrate in 100 boiling water. Add 0.165 sodium potassium tartrate and boil till ppt. turns grey. Filter hot. III. 25% gum arable. WORKING solution: stock I 40, stock II 60, stock III 4 33.1 Wallert 1936 4285a, 12 :254 preparation: To 100 10% silver nitrate add 1.5 40% sodium hydroxide. Add just enough ammonia to dissolve ppt. 33.1 Weber 1944 4285a, 21 :45 formula: To 25 10% silver nitrate add not quite enough ammonia to redissolve ppt. first formed. Dilute to 100. 33.1 Weil and Davenport 1933 21458, 14:95 preparation: To 40 ammonia add just enough 10%, silver nitrate (about 60) to produce a permanent opalescence. 33.1 Wolff 1905 766, 26:135 preparation: To 100 10% silver nitrate add 40% sodium hydroxide until no further ppt. is produced. Then add just enough ammonia to dissolve ppt. 33.1 Zettnov 1891 23684,11:689 preparation: To 100 0.4% silver nitrate add triethanolamine until ppt. first formed is just redissolved. note: Craigie 1928 (3566, 9:55) is identical. MS 33.21 METAL STAINS 581 33.2 Neurological Methods 33.21 nerve cells and processes 33.21 Agduhr 1917 23632,34:1 REAGENTS REQUIRED: A. 20% neutralized formaldehyde; B. 3% silver nitrate; C. MS 33.1 Agduhr 1917; D. 0.5% acetic acid; E. 0.004% gold chloride; /''. 5% sodium thiosulfate method: Fix .1, 5 days -^ wash -^ B, G days in dark — ♦ wash —> C, 20 hrs. -+ D, 1 hr. -> wash, 1 hr. -^ .4, 1 to 4 days —> [section by paraffin technique, bring sections to water] -^ E, 1 hr. -^ F, 2 mins. -^ balsam, via usual reagents note: Agduhr emphasized the need for thorough washing, particularly between A and B and for the use of large volumes (100 ml. for 5 nun. cubes) of A. recommended for: neurofibrils. 33.21 Bielschowsky 1904 method for pieces — auct. 11478,3:109 reagents required: A. 8% neutralized formaldehyde; B. pyridine; C. 3% silver nitrate; D. MS 33.1 Bielschowsky 1902 40 ml., water 60 ml.; E. 0.004% gold chloride; F. 5% sodium thiosulfate method: [Fix A, 1 month or till required] -^ B, 3 days -* running water, 24 hrs. — > dis- tilled water, wash -» C, 3 days, 36°C. -> rinse -^ D, 24 hrs. -^ wash -^ A, 10 hrs. -^ [sections by paraffin technique, bring sections to water] —> E, 1 hr. -^ F, 2 mins. -* balsam, via usual reagents 33.21 Bielschowsky 1905 see MS 33.21 Bielschowsky 1910 33.21 Bielschowsky 1908 test. 1933 Cajal and de Castro Cajal and de Castro 1933, 365 reagents required: A. 8% formaldehyde; B. 5% nitric acid; C. 4% silver nitrate; D. MS 33.1 Bielschowsky 1902; E. 0.2% gold chloride; F. 5% sodium thiosulfate method: [fresh tissue] — > A, till hardened -+ B, till decalcified — > wash, till acid-free — > A, 24 hrs. — > wash — > [frozen sections]-^ water ^^ C, 24 hrs. in dark -^ rinse -^ D, till brown, 20 mins. -> wash -> A, 10-15 mins. -^ rinse -^ E, till grey -^ F,b mins. -♦ balsam, via usual reagents recommended for: nerve endings in structures requiring decalcification. 33.21 Bielschowsky 1910 method for sections — auct. 11478,3:169 REAGENTS REQUIRED: A. 2% silver nitrate; B. MS 33.1 Bielschowsky 1904; C. 8% neutral formaldehyde; D. 0.004% gold chloride; E. 5% sodium thiosulfate method: [sections by freezing technique, of formaldehyde-fixed material, from which the formaldehyde must be completely washed out before sectioning] -^ A, 24 hrs. in dark —>■ rinse — > B, 15 to 20 mins. — > wash — » C, 15 to 30 mins. —> wash — > D, 1 hr. — » E, 2 mins. — > balsam, via neutral reagents notes: Bielschowsky 1905 (11478, 4:227) varies from above in substituting a few seconds in 2% acetic acid for the wash between B and C above. Favorsky 1906 (11478, 6:260) substitutes 10% silver nitrate for A above. Boeke 1910 (766, 35:193) substi- tutes his F 0000. 1000 for plain formaldehyde in fixation ; otherwise his method is es- sentially that above RECOMMENDED FOR: peripheral nerve endings. 33.21 Boecke 1910 see MS 33.21 Bielschowsky 1910 (note) 33.21 Cajal 1925 21344,23:157 reagents required: A. AMS 11.1 Cajal 1913; B. MS 33.1 Cajal 1925 33.1; C. 4% neu- tralized formaldehyde; D. 0.2% gold chloride; E. 5% sodium thiosulfate method: [sections by freezing technique of materials hardened 5-30 days in A] — > A, 4 hrs. -^ wash -^ B, warming if necessary, till dark amber -> wash — » C, 1 min. -^ D, till required color —* E, 2 mins. — > balsam, via usual reagents note: This technique is frequently confused with MS 31.21 Cajal 1925. RECOMMENDED FOR: Cerebellum. 582 METHODS AND FORMULAS MS 33.21 33.21 Davenport, Windle, and Beech 1934 20540b, 9:5 REAGENTS REQUIRED: A. 50% pyridine; B. 1.5% silver nitrate; C. MS 33.1 Davenport, Windle, and Beech 1934; D. 0.4% formaldehyde method: [embryos fixed 2 days in F 0000.1020 Davenport, Windle, and Beech 1934] -^ wash, 1 hr. ->■ A, 1-2 days -» wash, 2-4 hrs. -> i?, 3 days, 37°C. -^ wash, 20 mins. (12 mm. embryos) to 1 hr. (20 mm. embryos) -^ C, 6-24 hrs. -^ wash, 15 mins. -» D, same time as C — » wash — > [section] RECOMMENDED FOR: embryos. 33.21 Debauche 1939a 20540b, 14:121 REAGENTS REQUIRED: A. \% ammonia; JS. 2% ammonia; C. 20% silver nitrate; D. am- monia; E. 1.2%, formaldehyde; F. 1% gold chloride; G. 5% sodium thiosulfate method: [tissue fragments fixed in F 5000.1010 Debaissieux 1935]-* A, 2-6 hrs. — > [frozen 25 n sections] -^ B, 15 mins. -^ C, 45°C. till light brown -» D, added drop by drop to C containing sections until ppt. first formed is not quite redissolved -^ rinse — > E, till rich brown -^ F, till deep blue ^ G, 10 mins. — > wash — > balsam, via usual reagents RECOMMENDED FOR: invertebrate neurology in free sections. note: This method was republished by the same author (1939: 966, 59:23). It may be applied to sections cut by the paraffin technique, if the wax is removed and the impregnation conducted before the section is attached to a slide. 33.21 Debauche 1939b 20540b, 14:21 REAGENTS required: A. 2% ammonia; B. 10% silver nitrate; C. MS 33.1 Debauche 1939; D. 1.2% formaldehyde; E. 1% gold chloride; F. 5% sodium thiosulfate method: [serial sections from E 21.1 Debauche 1939 blocks of F 5000.1010 Debaissieux 1935 fixed material] -^ water — > A, 10 mins. — » quick rinse — > B, 10 mins. 45°C. — * C, 1-5 mins. -^ quick rinse -^ D, till rich brown — * E, till blue — > F, 5 mins. —* wash -^ balsam, via usual reagents recommended for: invertebrate neurology in serial sections. 33.21 Doinikow test. 1933 Cajal and de Castro Cajal and de Castro 1933, 341 reagents required: A. 8% formaldehyde; B. pyridine; C. 2% silver nitrate; D. MS 33.1 Bielschowsky 1902 method: [blocks of fresh tissue] — > A, some months — > wash — > B, 24-48 hrs. —> running water, 24 hrs. — * distilled water, some hrs. — > C, 4-5 days, 35°C. -^ rinse —y D, 48 hrs. — + thorough wash -^ A, 48 hrs. — > wash — > [celioidin sections] recommended for: regenerating nerve fibers. 33.21 da Fano 1914 2190, 3:14 REAGENTS REQUIRED: A. 2% sUver nitrate; B. MS 33.1 da Fano 1914; C. 8% neutralized formaldehyde; D. 0.004% gold chloride; E. 5% sodium thiosulfate method: [small pieces, fixed in formalin or F 7000.1000 Orth 1896] -^> wash^ [sections by freezing technique] — > wash -^ A, in dark, 6 hrs. to 3 days -^ B, 20 to 30 mins. —*■ wash — > C, 15-20 mins. -^ wash — ^D, 1 hr. -^ E,2 mins. — * balsam, via usual reagents 33.21 da Fano 1919 11454, 52:1919 REAGENTS REQUIRED: A. pyridine; B. 2% silver nitrate; C. MS 33.1 da Fano 1919 33.1; D. 8% neutralized formaldehyde; E. 0.004% gold chloride; F. 5% sodium thiosulfate method: [pieces, fixed 8% formaldehyde, 1-2 months] — > wash — + [sections by freezing technique] — ♦ wash — > A, 6-12 hrs. -^ wash -^ B, 24-48 hrs. -^ rinse -^ C, 15 to 20 mins. — »• rinse —> D, 2-3 hrs. -^ wash -^ E, \ hr. -^ F, 2 mins. — * balsam, via usual reagents 33.21 da Fano 1920 11454, 53:1919 REAGENTS REQUIRED: A. pyridine; B. 9>% formaldehyde; C. 2% silver nitrate; D. MS 33.1 da Fano 1919; E. 8% neutral formaldehyde; F. 0.004% gold chloride; G. 5% sodium thiosulfate method: [pieces, fixed 8% formaldehyde, 1-2 months]^ wash—* [sections by freezing technique] — * wash —> 4, 4 to 12 hrs. -^ wash -^ B, 24 hrs. 37°C. — > C, 1-2 days —> rinse -^ D, 30 mins. -^ rinse — * E, 2-3 hrs. -^ wash — > F, 1 hr. —> G, 2 mins. -^ balsam, via usual reagents MS 33.21 METAL STAINS 583 note: The method above is the most complex of eight pubhshed in the same paper; the others: (a) reverse steps .4 and B above; (b) substitute 60% methyl alcohol for A above; (c) as b but with order of A and B reversed as in (a); (d) reverse steps A and B, substituting AMS 11.1 da Fano 1920 for A; (e) substitute AMS 11.1 da Fano 1920b for A; omit B; (f) substitute 50; pyridine for A; omit B; (g) omit A and B. RECOMMENDED FOR: neurofibrils (original and a) in adult (f) and embryonic (g) material, general neurological staining (e), and pericellular baskets (b, c, d). 33.21 Fajersztajn 1901 test. 1933 Cajal and de Castro Cajal and de Castro 1933, 223 REAGENTS REQUIRED: A. MS 33.1 Fajersztajn 1901; B.2% formaldehyde; C. 0.2% gold chloride; D. 5% sodium thiosulfate method: [frozen sections of formaldehyde material] -^ water -^ A, 5-10 mins. -^ B, few moments — > C, till violet —>^D,5 mins. — * wash — > balsam, via usual reagents recommended for: axis cyUnders in ganglia, cerebellum, spinal cord. 33.21 Favorsky 1906 see MS 33.21 Bielschowsky 1910 (note) 33.21 Final 1947 6630, 141:198 reagents required: A. 20% silver nitrate; B. 40% formaldehyde; C. MS 33.1 Gros (1938); D. 1% ammonia; E. 0.1% acetic acid method: [paraffin sections of F 5000.1010 Bouin 1897] -> water -^ blot -^ A, 24 hrs., 37°C. — * blot — > B, on slide, 1-2 sees. — > blot -^ C, till sufficiently differentiated about 30 sees. -^ D, wash — > E, rinse -^ balsam, via usual reagents 33.21 Glees, Meyer, and Meyer 1946 11025, 80:101 reagents required: A. 0.1% ammonia in 50% alcohol; B. 10% silver nitrate; C. 4% formaldehyde; D. MS 33.1 Glees, Meyer, and Meyer 1946; E. 5% sodium thiosulfate method: [15 ju frozen sections of formaldehyde material] —* A, 24 hrs. —>■ wash -^ B, till brown, 1-5 days -^ C, 1 hr. — > wash — > D, 30-60 sees. —* C, 5 mins. — > wash -^ E, 10 mins. — > wash — >• balsam, via usual reagents 33.21 Gros-Schultze test. 1938 Mallory Mallory 1938, 227 reagents required: A. 20% silver nitrate; B. 20% formaldehyde; C. MS 33.1 Gros- Schultze 1938; D. 20% ammonia; E. 0.5% acetic acid; F. 1% gold chloride; G. 5% sodium thiosulfate method: [frozen sections of formaldehyde-fixed material] -+ A, in dark, 5-20 mins. — > B, 4 washes in 4 successive portions — > C, till axis cylinders differentiated — » D, rinse — > E, rinse -^ F, 1 hr. —> G, 1 min. -^ wash -^ balsam, via usual reagents recommended for: nerve endings. note: Cajal and de Castro 1933, 178 give this technique as "Gros." Szatmari 1936 (23639b, 24 :239) precedes A, above, with 24 hours soaking in pyridine which is then thoroughly washed out. 33.21 Holmes 1942 11431, 54:132 REAGENTS REQUIRED: A. Xylene 30, acetic acid 70; B. MS 33.1 Holmes 1942; C. AMS 21.1 Holmes 1942; D. 0.2% gold chloride; E.2% oxalic acid; F. 5% sodium thiosulfate method: [15 n paraffin sections of formaldehyde material]—* A, 1 hr. -^ water, via graded ales. -^ B, 5-24 hrs. 37°C. —> rinse —y C, 30 sees. — > thorough wash -^ D, 3-5 mins. -^ rinse — > E, 5-10 mins. -> F, 5 mins. -^ wash -^ balsam, via usual reagents 33.21 Kernohan 1930 21400a, 49:58 reagents required: A. 20% silver nitrate; B. MS 33.1 Bielschowsky 1902 20, water 80; C. 4% formaldehyde; D. 0.2% gold chloride; E. 2% sodium thiosulfate method: [sections either by paraffin or collodion techniques]—* wash—* A, 1 hr. ^ wash ^ B, 1 to 4 mins. —> wash — > C, 1 min. -^ D, 2 mins. —* E, 2 mins. -^ balsam via usual reagents 33.21 Landau 1940 4285a, 17 :65 REAGENTS required: A. 10% neutralized formaldehyde; B. 20% silver nitrate; C. MS 33.1 Gros-Schultze (1938); D. 0.04% neutrahzed formaldehyde; E. 0.2% gold chloride; F. 5% sodium thiosulfate; G. 10% potassium iodide 584 METHODS AND FORMULAS MS 33.21 method: [paraffin sections of formaldehyde material]—* A, 24 hrs. in dark — > B, 1-2 hrs. 35°-40°C., in dark -^ rinse — > C, 5 mins. -^ add few drops D to C —> repeat addi- tions of C till sections sufficiently stained — * thorovigh wash -^ E, till blue grey — >• F, 5 mins. -^ G, till differentiated —> wash —* balsam, via usual reagents, or M 11.1 Landau 1940 33.21 Lobo 1937 see MS 33.21 Schultze (1948) (note) 33.21 Miskolczy test. 1933 Cajal and de Castro Cajal and de Castro 1933, 341 KEAGENTs requieed: A. 10% silver nitrate; B. MS 33.1 Cajal 1925; C. 0.5% acetic acid; D. 8% formaldehyde; E. 0.2% gold chloride method: [blocks of formaldehyde fixed material] —> [graded ales, till dehydrated]-^ xylene, 2-3 hrs. -^ [graded ales., till rehydrated] -^ water — > A, 2-7 days -^ wash —>■ B, 2-5 hrs. -^ wash — > C, 10-30 mins. — > wash — > D, 24 hrs. — * wash —>■ [paraffin sec- tions on slide] — > water —* E, 5-10 mins. — » wash -^ balsam, via usual reagents recommended for: axis cylinders. 33.21 Paton 1907 14246, 18:576 REAGEXT-s REQUIRED: A. 4% neutralized formaldehyde; B. 1% silver nitrate; C. MS 33.1 Bielschowsky 1902; D. 2% acetic acid; E. AMS 21.1 Paton 1907; F. 0.004% gold chloride; G. 5% sodium thiosulfate method: [whole fish embryos] —'■A, 1 to 5 days —>■ wash — > B, 4 to 7 days — > rinse — > C, 24 hrs. -^ wash — > Z), 5 to 15 mins. — > wash —>■ E, 12 hrs. — > [sections by paraffin technique, bring sections to water] —> F, I hr. — > G, few minutes -^ balsam, via usual reagents 33.21 Penfield 1935 608b, 11:1007 REAGENTS REQUIRED: A. watcr 50, citric acid 10.5, 40% formaldehyde 50; B. 20% silver nitrate; C. 20% formaldehyde; D. MS 33.1 Penfield 1935; E. 20% ammonia; F. 5% acetic acid; G. any AMS 22.1 formula; H. 5% sodivmi thiosulfate method: [small blood vessels dissected from material perfused for 3 days with A]~^ wash -^ B, 2 hrs. -^ C, 4 changes, each 100 ml. -^ D, till stained — > E, 1-2 mins. -^ F, till neutral — > G, till toned — > H, 10 mins. —> wash -^ l^alsam, via usual reagents RECOMMENDED FOR: perivascular nerves of pia mater. 33.21 Pullinger 1943 11431, 56:97 REAGENTS REQUIRED: A. 0.1% ammouia; B. MS 33.1 del Rio-Hortega 1917a; C. 4% formaldehyde method: [cornea fixed in situ by injection of formaldehyde, 1 day, then cut from eye and fixed 3 more days] — > wash — > A, overnight—* wash—* B, 37°C., 4 hrs. — > wash—* C, 15 mins. — ^ [sections] 33.21 Rogers 1931 763,49:81 REAGENTS REQUIRED: A. 10% formaldehyde; B.3% ammonia in 90% ale; C. 40% silver nitrate; D. MS 33.1 Rogers 1931; E. 4% acetic acid: F. 0.3% gold chloride in 1% acetic acid; G. i% oxalic acid; H. 5% sodium thiosulfate method: [fresh tissue] — ♦ ^4, 1 wk. —>■ wash —^B,! day — * [paraffin sections] — * C, 13 hrs. — * 80% ale, wash — * D, 20 mins. -^ E, wash —>■ A, 2-5 mins. -^ F, 10-15 mins. — * wash — * G, if stain not sufficiently intense -^ H, 5 mins. — > wash — > balsam, via usual reagents RECOMMENDED FOR: axis Cylinders and nerve endings. 33.21 Romanes 1946 11025, 80:205 REAGENTS REQUIRED: A. MS 33.1 Romanes 1946; B. AMS 21.1 Romanes 1946; C. 0.3% gold chloride in 2% acetic acid; D. 2% oxalic acid in 0.04% formaldehyde; E. 5% sodium thiosulfate method: [paraffin sections] —* water —* A, 4-24 hrs. 58°C. —* rinse ^ B, 5 mins. -^ [repeat A — * B cycle if insufficientl.y stained] -^ wash — * C, 5 mins. -^ rinse — * D, 10-15 mius. —> wash —* E, 5 mins. — > wash — > balsam, via usual reagents MS 33.21-MS 33.22 METAL STAINS 585 33.21 Schutz 1908 15058, 27:909 UKAGKNTS uioQi ihkd: .1. 2% silvcp iiitrato; H. MS 33.1 Binlschowsky 1901; C. 8% neu- tralized i'onaahlehyde; D. 1% acetic acid; E. 0.02% gold chloride; F. 5% sodium thiosulfate method: [sections by freezing technique of formaldehyde fixed material] —* wash — > A, 24 hrs. in dark — > wash -^ D, 10 mins. — » E, 30-49 mins. (till dark grey) -^ rinse -+ F, 2 mins. — * balsam, via usual reagents 33.21 Szatmari 1936 see MS 33.21 Gros-Schultze 1938 (note) 33.21 Szepsenwol 1938 GG30, 120:089 KKAOKNTs RixjiiRKi) I .4. F 0000.1030 Szcpseuwol 1935; B. 1% silver nitrate; C. 3% silver nitrate; 1). MS 33.1 Bielschowsky 1902 10, water 90 method: a. 10 to 20 days, 55°C. -^ running water, 24 to 48 hrs. — > J5, 1 to 3 days, 35°C., in dark — » C, 7 to 10 days, 35°C., in dark — > wash -^ D, several hrs. -^ wash —^ E, 12 to 15 hrs. — > paraffin sections via cedar oil note: This method is a slight modification of Szepsenwol 1937 (4285a, 14:168). 33.21 Wallart 1935 4285a, 12:254 reagents required: A. AMS 12.1 Wallart 1935; B. 2% silver nitrate; C. MS 33.1 Wallart 1935; D. 4% neutralized formaldehyde method: [3-5 mm. slices of formaldehyde material]-^ 4, 6 hrs. — > thorough wash — ► B. 5 days, 37°C. -^ rinse -^ C, 1-2 days -^ wash — > D, 12-18 hrs. —* wash — » [par- affin sections] -^ AMS 22.1 treatment — > balsam, via usual reagents recommended for: nerves in organs rich in lipids. 33.21 Weber 1944 4285a, 21 :45 reagents required: A. AMS 12.1 Weber 1944; B. water 50, isopropanol 25, dioxane 25; C. 1% pyridine; D. 3% silver nitrate; E. MS 33.1 Weber 1944; F. AMS 21.1 Weber 1944 method: [small fragments]—* A, 0°C., allow to rise to room temperature —>• 55°C., 1 month -+ B, wash, 30 mins. — > C, wash 12 hrs. -^ wash, till wash-water no longer gives chloride test for silver -^ E, 24 hrs. — > F, 24 hrs. —* paraffin, via usual reagents —y [5 fj. sections] — > balsam 33.22 NEUROGLIA 33.22 Achucarro 1911 3231, 1:139 reagents required: A. sat. aq. sol. tannic acid; B. MS 33.1 Bielschowsky 1902 4, water 96; C. 4% formaldehyde method: [sections, by freezing technique, of formaldehyde-fixed material]—*/!-^ warm till steaming -^ rinse — » B, 10 mins. — > C, 10 mins. -^ wash — * balsam, via usual reagents recommended for: macroglia. 33.22 Arcadi 1948 20540b, 23 :77 reagents required: A. ammonia; B. 2% hydrobromic acid; C. MS 33.1 Arcadi 1948; D. 0.4% formaldehyde; E. 5% sodium thiosulfate method: [15 /x frozen sections] — > A, 100 ml. jar — > displace A with slow stream of water over 24 hr. period — * A, fresh solution — » wash as before -^ A, fresh solution, 7 mins. -* B, I hr., 38°C. -^ wash -» C, 30-45 mins. -* wash -^ D, 30 sees. -* wash -* E, 2 mins. -^ wash — > balsam, via usual reagents recommended for: oligodendria in material stored for many years in formaldehyde. 33.22 Barker 1934 11977, 7:293 reagents required: A. AMS 11.1 Cajal 1913; B. 0.5% ammonia; C. MS 33.1 del Rio-Hortega 1917a; D. 1% formaldehyde; E. 0.2% gold chloride; F. 5% sodium thiosulfate method: [3 mm. slices of fresh tissue] — > A, 3 days -^ [25 m frozen sections] — * water — > A, 10 mins., 55°C. — > wash — + B, 2 mins. — > wash ^ C, 3 mins. — > rinse -^ D, I min. — > wash —> E, 5 mins. ^ i^, 1 min. — * wash — * balsam, via usual reagents recommended for: microglia. 586 METHODS AND FORMULAS MS 33.22 33.22 Belezky 1931 22575, 282:214 REAGENTS REQUIRED: A. MS 33.1 Bclezky 1931; B. 4% neutralized formaldehyde method: [sections by E 11.1 Belezky 1931] -* water, wash — > A, few sees. -^ wash — » B, till blackened — > balsam, via usual reagents note: a may be diluted and used for a longer period. 33.22 Bertrand and Guillain 1934 6630, 115:706 REAGENTS REQUIRED: A. AMS 11.1 Bertrand and Guillain 1934; B. pyridine; C. 0.2% hydrofluoric acid; D. 5% sodium carbonate; E. MS 33.1 Bertrand and Guillain 1934; F. 2% formaldehyde; G. 0.2% gold chloride; H. 5% sodium thiosulfate method: [fresh ganglia] -^ A, 4-7 days, renewed daily, 4°-5°C. — > wash — » 12 m frozen sections -^ B, 12-24 hrs. -> C, 1 hr., 37°C. -> wash -^ D, 4 hrs. -> E, till yellow -^ rinse —>■ F, till brown — * wash -^ G, till grey -^ H, 10 mins. — > wash ^ balsam, via usual reagents recommended for: oligoglia in spinal and sympathetic ganglia. 33.22 Bolsi test. 1933 Cajal and de Castro Cajal and de Castro 1933, 261 REAGENTS REQUIRED: A. AMS 13.1 Bolsi 1933; B. MS 33.1 Cajal 1920; C. 2% formal- dehyde; D. 0.2% gold chloride; E. 5% sodium thiosulfate method: [frozen sections of formaldehyde fi.\ed material] — > A, 30 mins., 50°C. — > wash -^ B, 5-10 mins. — > wash -^ C, till reduced — > D, till violet — > £, 5 mins. — > balsam, via usual reagents recommended for: macroglia. note: Cajal and de Castro {loc. cit.) suggest substitution of their AMS 13.1 for A above. 33.22 Cajal 1920 test. 1933 ips. Cajal and de Castro 1933, 253 reagents required: A. AMS 11.1 Cajal 1913; B. AMS 11.1 Cajal 1920; C. MS 33.1 Cajal 1920b; Z). 6% neutralized formaldehyde; E. 0.2% gold chloride; F. 6% sodium thiosulfate method: [small pieces] -^ A, 4 days -^ frozen sections — ^ B, 4 hrs., 37°C. -^ wash -^ C, 40°-45°C., tQl deep brown -^ rinse — > D, few moments — > thorough wash — > E, 10-20 mins. — > rinse -^ F, 5 mins. — > wash — » balsam, via clove oil and xylene recommended for: macroglia. 33.22 Cajal and de Castro 1933 see MS 33.22 Bolsi (1933) (note) 33.22 Cone and Penfield test. 1929 Anderson Anderson 1929, 90 REAGENTS REQUIRED: A. 0.1% ammonia; B. 5% hydrobromic acid; C. 5% sodium carbonate; D. MS 33.1 Cone and Penfield (1929); E. 0.5% formaldehyde; F. 0.2% gold chloride; G. 5% sodium thiosulfate method: [20 n sections by freezing technique] -^ A, overnight -^ wash -^ B, I hr., 37°C. — > wash -^ C, 1 hr. — * D, 3-5 mins., or till brown —* E, 1 min. with agitation — ^ wash -^ F, till grey -^ G, 5 mins. — > balsam, via usual reagents recommended for: microgUa. 33.22 Gans test. 1933 Cajal and de Castro Cajal and de Castro 1933, 277 reagents required: A. 2.5% ammonium bromide; B. 0.1% ammonia; C. MS 33.1 del Rio-Hortega 1921; D. 0.4% formaldehyde; E. 0.2% gold chloride; F. 5% thiosulfate method: [25 m frozen sections of formaldehyde fixed material] — > A, 2 hrs., 37°C. — * B, quick rinse -^ C, 2 hrs. — > rinse — > D, 5 mins. —> E, 15 mins. -^ F, 5 mins. -^ balsam, via usual reagents recommended for: microglia. 33.22 Herrera 1932 test. 1933 Cajal and de Castro Cajal and de Castro 1933, 275 reagents required: A. AMS 11.1 Herrera 1932; B. MS 33.1 Herrera 1932; C. 4% formaldehyde method: [pieces of fresh tissue] —> A, 2 to 20 days — » 25 ^ frozen sections —> water -^ B, 1 min. — > rinse — » C, 5 mins. — > wash — > balsam, via usual reagents recommended for: microglia. 33.22 Ingleby 1929 4349, 12:91 reagents required: A. AMS 11.1 Cajal 1913; B. 10% tannic acid; C. 1% ammonia; D. MS 33.1 da Fano 1919; E. 8% formaldehyde; F. 0.2% gold chloride; G. 5% sodium thiosulfate MS 33.22 METAL STAINS 587 method: [slices of brain tissue] -> A, 2-4 days — » wash ^ [15 /x frozen sections] -* B, 5- 7 mins., 50°C., then cool 10 mins. —>■ C, till pliable -> D, till yellow-brown — > rinse —>■ E, 1 min. -^ wash — > F, till groy — > G, 5 mins. — > wash — > balsam, via usual reagents note: F 5000.1010 Ingleby 1925 may be substituted for A if the time be reduced to 1 day. 33.22 King 1937 1879,38:362 REAGENTS REQUIRED: A. \% ammouia; B. 5% ammonium bromide; C. AMS 11.3 King 1937; D. 3% sodium sulfite; E. MS 33.1 King 1937; F. 4% formaldehyde method: [frozen sections of formaldehyde-fixed material]—* A, till required-^ B, 10 mins., 50°C. -> C, 2 mins. -^ rinse -^^ D, 2-3 mins. -^ E, Z changes, 1 min. each -♦ rinse -^ F, till brown — > wash -^ balsam, via usual reagents recommended for: microglia and oligodendroglia. 33.22 McCarter 1940 608b, 16 :233 REAGENTS REQUIRED: A. 1% ammouia; B. 4% hydrobromic acid; C. 5% sodium carbon- ate; D. 5% ammonium alum; E. MS 33.1 del Rio-Hortega 1917a; F. 0.4% formal- dehyde; G. 0.2% gold chloride; H. 5% sodium thiosulfate method: [20 m frozen sections of formaldehyde material] -> A, few mins. to overnight, according to age ^ B, 37°C., 1 hr. ^ wash ^ C -^ equal volume D, added to C (ignore precipitate), leave 1 hr.-3 days -^ wash -^ E, 2-5 mins. — » F, 10 mins. -♦ wash -^ G, till blue-grey -^ H, 5 mins. -^ balsam, via usual reagents recommended for: oligodendria and microglia. 33.22 Penfield 1928 608b, 4:153 REAGENTS REQUIRED: A. AMS 11.1 Cajal 1913; B. 1% ammonia; C. 2% hydrobromic acid; D. 5% sodium carbonate; E. MS 33.1 del Rio-Hortega 1921 56, water 44; F. 1% formaldehyde; G. 0.2% gold chloride; H. 5% sodium thiosulfate method: [fresh tissue] —> A, until wanted-* [sections, by freezing technique]-^ A, 5 mins. -^ B, overnight -^ C, I hr., 38°C. -♦ wash -^ D, 1 hr. ^ rinse -^ E, 3-5 mins. or till light brown -^ rinse -^ F, I min. with agitation -^ wash G, till blue grey -^ H, 5 mins. — * balsam, via usual reagents recommended for: microglia. 33.22 Penfield 1924 3464, 47 :430 REAGENTS required: A. AMS 11.1 Cajal 1913; B. MS 33.1 del Rio-Hortega 1921; C. 1% formaldehyde; D. 0.2% gold chloride; E. 5% sodium thiosulfate method: [blocks of fre.sh tissue] -^ A, 4S hrs. -^ 95%) alcohol, 36-48 hrs. -> wash -^ sec- tions, by freezing technique —>■ wash -* B, 30 mins. to 2 hrs. or until light brown -* C, with agitation, 1 min. -^ D, till preferred color ^ ^, 1 min. -^ balsam, via usual reagents recommended for: oligodendroglia. 33.22 Polak 1947 18794, 61 :508 REAGENTS REQUIRED: AA % urauium acetate; B. MS 33.1 del Rio-Hortega 1917a; C. 1% formaldehyde; D. 0.2% gold chloride; E. 5% sodium thiosulfate method: [5-10 m sections of formaldehyde material]—* water—* A, 24 hrs.—* B, till transparent, 15-60 sees. -^ rinse -* C, till brown -^ rinse ->■ D, till grey -* rinse -^ E, 5 mins. — > balsam, via usual reagents 33.22 del Rio-Hortega 1916 21344, 14:181 REAGENTS REQUIRED: A. 8% formaldehyde adjusted with ammonia to pH 8; B. 10% tannic acid; C. 0.1%, ammonia; D. MS 33.1 Bielschowsky 1902 2, water 98 method: Fix A, 2 to 3 days —>■ [sections by freezing technique] -^ B, heated till steam- ing, 10 mins. -^ cool -^ C, till again flexible -^ D, till dark yellow -* rinse -^ A, 10 mins. — * balsam, via usual reagents note: del Rio-Hortega for A specifies the pH as that which gives a blue reaction with litmus. recommended for: protoplasmic astrocytes. 588 METHODS AND FORMULAS MS 33.22 33.22 del Rio-Hortega 1917 21344, 15:367 REAGENTS REQUIRED: A. MS 33.1 del RIo-Hortega 1917b; B. 8% neutralized formalde- hyde; C. 0.2% gold chloride; D. 5% sodium thiosulfate method: [sections by freezing technique of AMS 11.1 Cajal 1913 fixed material] -^ wash — > A, 45°C., till black -^ rinse -^ B, 30 sees. -^ wash -^ C, till bluish -^ D, 5 mins. — > wash — > balsam, via usual techniques RECOMMENDED FOR: astrocytes. note: Cajal and de Castro 1925 p. 255 cite this (without journal reference) as "Rio- Hortega 1918" and recommend it for macroglia. 33.22 del Rio-Hortega 1918 21344, 15:105 reagents required: A. AMS 13.1 del Rio-Hortega 1910; B. 0.1% ammonia; C. MS 33.1 del Rio-Hortega 1910 10, water 90; D. 8% neutralized formaldehyde method: [sections, by freezing technique, of formol material] -^ A, 5 mins. 45°C. — > B, till transparent — > C, 1st dish, 1 min. -^ C, 2nd dish, 1 min. -^ C, 3rd dish, till brown -^ quick rinse -^ D, 30 sees. —^ wash -^ balsam, via usual techniques recommended for: protoplasmic neuroglia. 33.22 del Rio-Hortega 1919 3231, 9:68 reagents required: A. 4% formaldehyde; B. MS 33.1 del Rio-Hortega 1917 26, water 74; C. 0.4% neutralized formaldehyde; D. 0.2% gold chloride; E. 5% sodium thiosulfate method: [smears, or small pieces] — > A, 24 hrs. — > [section pieces by freezing technique] —^ wash —> B, 5 to 10 mins. -^ C, till reduced -^ D, 30 sees, to 1 min. -^ E, 1 min. -^ wash—* balsam, via usual reagents 33.22 del RIo-Hortega 1921a 3232, 22:1 reagents required: A. 4% formaldehyde adjusted to pH 8 with ammonia; B. MS 31.1 del Rio-Hortega 1921; C. MS 33.1 del Rio-Hortega 1917a 29, water 71, pyridine 1; D. 0.4% formaldehyde method: [sections by freezing technique of formaldehyde material] -^ ^4, 1 min. 50°C. -^ B, 50°C. till brown —^ wash — » C, 50°C. till dark brown -^ wash -^ D, b mins. — > balsam, via usual reagents recommended for: oligodendria. 33.22 del Rfo-Hortega 1921b 3232, 22:1 reagents required: A. AMS 11.1 Cajal 1913; B. 0.1% ammonia; C. MS 33.1 del Rio- Hortega 1921; D.4% neutralized formaldehyde; E. 0.2% gold chloride; /^. 5% sodium thiosulfate method: Fix small pieces A, 2-3 days room temperature, then raise to 55°C. 10 mins. — > [25 M sections by freezing technique] — > B, wash — * C, 3 mins. -^ D, 10 mins. -^ wash — > E, 10 to 20 mins. -^ F, 5 mins. -^ balsam, via S 41.1 del Rio-Hortega (1938) recommended for: microglia. 33.22 del Rio-Hortega 1923 21344, 21:95 reagents required: A. 2% silver nitrate 100, pyridine 1.5; 5. 1% pyridine; C. MS 33.1 del RIo-Hortega 1923 100, pyridine 1.5; D. 4% formaldehyde; E. 0.2% gold chloride; F. 5% sodium thiosulfate method: [frozen sections of formaldehyde-fixed material] ^ wash —> A, 50°C. till brown some hrs. -^ B, wash -^ C, 50°C. till sepia -^ wash -^ D, 10 mins. -^ E, till violet —y F, 30 sees. —> blot on slide -^95% ale, dropped on slide -^ balsam, via S 41.1 del Rio-Hortega (1938) recommended for: neuroglia in pineal. 33.22 del Rio-Hortega 1927 3232, 27:199 reagents required: A. AMS 11.1 Cajal 1913; B. 5% sodium sulfite or B. AMS 13.1 del Rio-Hortega 1927; C. MS 33.1 del Rio-Hortega 1927; D. 0.4% neutralized formaldehyde; E. 0.2% gold chloride; F. 0.5% sodium thiosulfate method: [fix small pieces A, 2-3 days room temperature then raise to 55°C. 10 mins.] — » [25 n sections by freezing technique] — > B, either solution, several hours — > C, 1st dish, 1 min. -^ C, 2nd dish, 2 mins. — > 1), 10 mins. -^ E, 10-20 mins. -^ F, 5 mins. — » balsam, via S 41.1 del Rio-Hortega (1938) RECOMMENDED FOR: microglia. MS 33.22 METAL STAINS 580 33.22 del Rfo-Hortega 1928 lest. 1933 Cajal and de Castro Cajal iuul B, 10 mins. — > wash — > C, 5-15 niins. —> D, 5 mins. —>■ E, 15 mins. -^ F, 5 mins. — > balsam, via usual reagents RECOMMENDED FOR: oligodendria. 33.22 del Rio-Hortega 1932 lest. 1933 Cajal and de Castro Cajal aiul de Castro 1933, 2(13 REAGENTS REQUIRED: A. AMS 11.1 Cajal 1913: B. 0.1% ammonia; C. AMS 13.1 del Rio-Hortega 1932; D. MS 31.1 del liio-Hortega 1932; E. MS 33.1 del Rio-Hortega 1932; F. 0.1% formaldehyde; G. 0.2% gold chloride; //. 5% sodium thiosulfate method: [pieces of fresh tissue] -^ A, till required — > [frozen sections] — » B, wash — » C, 10 mins.-* D, 10-15 mins., 45°-50°C. -> E, till deep brown -^ 80% ale, wash ^ 95% ale, wash -^ F, 2-3 mins. — > C, till slate gray — > //^, 5 mins. -^ balsam, via usual reagents recommended for: gliob lasts. 33.22 del Rio-Hortega test. 1933 Cajal and de Castro Cajal and de Castro 1933, 268 reagents required: A. AMS 11.1 Cajal 1914; B. AMS 11.1 del Rio-Hortega 1923a; C. 0.1% ammonia; D. MS 33.1 del Rio-Hortega 1933; E. 4% formaldehyde; F. 0.16% gold chloride; G. 5% sodium thiosulfate method: [2-3 mm. slices fresh material]^ A, 2-3 days -^ B, 2-3 days, 25°-35°C. ^ [frozen sections] — » C, thorough wash -^ wash — > D, 45°-50°C., till deep straw colored — > wash — > E, few mins. — > F, 15 mins. — > G, 5 mins. — > balsam, via usual reagents recommended for: gliosomes. 33.22 del Rio-Hortega 1933 see MS 33.23 del Rio-Hortega 1933 33.22 del Rio-Hortega 1929 3232, 30:1 reagents required: A. 0.1 % anunonia; B. MS 33.1 del Rio-Hortega 1917 90, pyridine 10; C. 0.2% neutralized formaldehyde; D. 1% gold chloride; E. 5% sodium thiosulfate method: [sections by freezing techni(iue of material fixed in F 7000.1000 del Rio- Hortega 1929] —>■ A, quick rinse —> wiish -^ B, at 55°C. till light brown -^ wash, 50% ale. -^ C, 5 mins. -* D, 30 sees, to 1 min. —>^E,l min. — » wash -^ balsam via usual, reagents 33.22 Rodriguez test. 1933 Cajal and de Castro Cajal and de Castro 1933, 285 reagents required: .4. AMS 11.1 Rodriguez 1933; B. equal parts A and 3% am- monium oxalate; C. MS 33.1 Herrera 1932; D. O.A% formaldehyde method: [pieces of fresh tissue] -^ A, 1-3 days -* [25 ^-30 m frozen sections] —* B, 24 hrs. —> quick wash -^ C, 1 min. — > D, 5 mins. -* balsam, via usual reagents recommended for: oligodendria. 33.22 Weil and Davenport 1933 21458, 14:95 reagents required: A. MS 33.1 Weil and Davenport 1933; B. 6% formaldehyde method: [celloidin sections] —» water —> A, 15-20 sees. —+ rinse —> 5, until brown —> wash — > balsam, via usual reagents recommended for: microglia. 33.22 Winkler 1935 23430, 153:160 reagents required: A. 2% hydrobromic acid; B. 2.5%, sodium carbonate; C. MS 33.1 del Rio-Hortega 1916 20, water 80; D. 8% formaldehyde method: [frozen sections of formaldehyde material] —* wash -* 0.3% alcohol, 2-8 hrs. • -^ wash -^ A, 14-24 hrs. -^ wa.sh -> B, 6-24 hrs. -* C, '2-2 mins. -* rinse -v D, till reduced —> DS 13.11 May and Griinwald 1902, 24 hrs., if plasma cell staining re- quired — > balsam, via usual reagents recommended for: microglia and plasma cells. 590 METHODS AND FORMULAS MS 33.23-MS 33.3 33.23 OTHER NEUROLOGICAL METHODS 33.23 Cajal test. 1933 ips. Cajal and de Castro 1933, 319 REAGENTS REQUIRED: A. AMS 11.1 Cajal 1933a; B. MS 33.1 Cajal 19.33b; C. AMS21.1 Cajal 1933 method: [pieces of fresh nerve] -> A, 24 hrs. -> thorough wash — » B, 48 hrs. — ♦ wash — » C, 6-12 hrs. — * teased preparations or celloidin sections RECOMMENDED FOR: Schwann cells. 33.23 Cajal test. 1933 ips. Cajal and de Castro 1933, 343 REAGENTS REQUIRED: A. AMS 11.1 Cajal 1933b; B. water 100, MS 33.1 Bielschowsky 1902 1; C. AMS 21.1 Cajal (1933) method: [pieces of fresh nerve] -^ A, 24 hrs. -^ wash -^ B, several hrs. — * rinse — » C, several hrs. -^ teased preparations RECOMMENDED FOR: peritubular connective sheath. 33.3 Cytological Methods 33.3 Fieandt and Sazen 1936 23632, 53:125 REAGENTS REQUIRED; A. 4% neutralized formaldehyde; B. 3% silver nitrate; C. MS 33.1 del Rio-Hortega 1916; D. 0.5% acetic acid; E. 0.02% gold chloride; F. 5% sodium thiosulfate method: [20-30 m celloidin sections of material fixed in Y 7000.1010 Wittmaak (1910) and decalcified by AF 21.1 Fieandt and Sazen 1936] — > [4-6 m paraffin sections neither deparaffined nor attached to slide] -^ wash, 2 days ^ A, 3 days 37°C. — > wash, 24 hrs. -^ B, 31 2 days, 37°C. -^ rinse -* C, 6 hrs. —>■ D, 15 mins. -^ A, 10 mins. — * wash, 15 mins. -^ E, 2 hrs., 37°C. -* F, 2 mins. -^ wash, 12 hrs. — > [attach to slide] -^ balsam, via usual reagents recommended for: Golgi apparatus in cells of inner ear. 33.3 Pritchard 1951 test. 1952 Cowdry Cowdry 1952, 212 REAGENTS REQUIRED: A. MS 33.1 Foot 1927a 50, water 50, ammonia 0.1; B. 0.04% formaldehyde; C. 2% potassium ferricyanide method: [sections of tissue fixed in F 7000.1000 Regaud 1910 or F 3700.1000 Helly 1903 and post-chromed 3 days in 3% potassium dichromate] -^ water —> A, 20 sees., with constant agitation — ^ B, large volume, 20 sees. —* wash -^ C, till differentiated — > wash -^ [counterstain, if desired] — > balsam, via usual reagents note: Regaud fixation for mitochondria: Helly for Golgi. 33.3 del Rio-Hortega 1916 21344, 14:181 reagents required: A. 4% formaldehyde; B.3% tannic acid; C. 1% ammonia; D. MS 33.1 del Rio-Hortega 1916 10, water 90 (3 dishes required); E. 1% gold chloride (2 dishes required); F. 5% sodium thiosulfate method: a, 10 days—* [sections by freezing technique] —> B, heated till steaming, 5 mins. -^ C, till flexibility restored -^ D, 1st dish, 1 min. —»■ D, 2nd dish, 1 min. -» D, 3rd dish, 2 mins. -^ wash -^ E, 1st dish, 5 mins. -> E, 2nd dish, 55°C., 20 mins. -> F, 2 mins. — > wash — > balsam, via usual techniques recommended for: mitochondria. 33.3 del Rio-Hortega 1916 test. 1933 Cajal and de Castro Cajal and de Castro 1933, 211 reagents required: A. 3% tannin; B. 0.75% ammonia; C. MS 33.1 del Rio-Hortega 1916 10, water 100; D. 0.2% gold chloride; E. 5% sodium thiosulfate method: [5 M sections by freezing technique of formaldehyde-fLxed material] —> A, 5 mins., 50°-55°C. —>■ B, till flexibility and transparency regained -^ C, 1 min. in 3 successive baths -^ thorough wash -* D, 10 mins. 40°-45°C. -* E, 5 mins. -^ thor- ough wash —> balsam, via usual reagents recommended for: centrosomes in nerve cells. MS 33.S-MS 33.41 METAL STAINS 591 33.3 del Rio-Hortega test. 1933 Cajal and de Castro Cajal and de Castro 1933, 267 REAGENTS REQUIRED: .4. AMS 11.1 Cajal 1914; B. AMS 11.1 del Rfo-Hortega 1933; C. 0.1% ammonia; D. MS 33.1 del Rio-Hortega 1917; E. 0.2% formaldehyde; F. 0.16% gold chloride; G. 5% sodium thiosulfate method: [2-3 mm. slices of fresh material] -^ A, 2-3 days — > B, 2-3 days, 25°-35°C. — > [frozen sections] — > C, till flexible — » wash — > D, 1-5 mins. — > wash — > E, 5 mins. — » F, 15 mins. -^ G, 5 mins. —* balsam, via usual reagents recommended for: mitochondria. 33.3 del Rio-Hortega 1925 3231, 25:34 reagents required: A. AMS 11.1 Strong 1903; B. 1% ammonia; C. AMS 33.1 del Rio-Hortega 1921 56, water 44; D. 1% formaldehyde; E. 0.2% gold chloride; F. 5% sodium tliiosulfate method; [small blocks]-* A, 2-8 days -^ [10 m sections by freezing technique]-* B, very brief wash -* wash -^ C, 5 mins. — » rinse, 15-20 sees, with very gentle agitation —* D, till grey — > wash -^ E, 15 mins. -^ F, 5 mins. — > balsam, via usual techniques note: This formula can be modified to stain gliosomes differentially by (1) adding 1% pyridine to C and staining at 50% C till sections turn brown and (2) increasing to 10% the concentration of D. All other steps are the same. 33.3 del Rio-Hortega test. 1933 Cajal and de Castro Cajal and de Castro 1933, 269 reagents required: A. AMS 11.1 del Rio-Hortega 1933b; B. 0.1% ammonia; C 2% silver nitrate; D. MS 33.1 del Rio-Hortega 1917; E. 0.4% formaldehyde; F. 0.16%, gold chloride; G. 5% sodium thiosulfate method: [2-3 mm. slices of fresh tissue] ^ ^4, 3 or 4 days, on ice -^ [frozen sections] -* B, thorough wash — » wash — ♦ C, 15 mins. — > rinse — * Z), 1 min. — > rinse -^ E, 1 min. -^ F, 15 mins. -* (7, 5 mins. -* balsam, via usual reagents recommended for: mitochondria in nerve cells. 33.4 Histological Methods 33.41 reticulum fibers 33.41 Amprino 1936 see MS 34.31 Amprino 1936 33.41 Bensley and Bensley 1938a Bensley and Bensley 1938, 109 REAGENTS REQUIRED: A. 1% potassiuiii permanganate; B. 5% oxalic acid; C. ADS 12.2 Lugol (1905) D. 5% sodium thiosulfate; E. sat. sol. tannic acid in 95% ale; F. 0.1% ammonia; G. MS 33.1 Bensley and Bensley 1938a; H. 8% formaldehyde; /. 0.2%, gold chloride method: [sections] -^ water -^ A, 1-5 mins. -^ B, till bleached — * wash -^ C, 3-5 mins. -^ wash -^ D, till bleached -* wash -^ E, 5 mins., 56°C. -^ F, rinse -^ G, 5-10 mins., 56°C. — > wash -^ H, 5 mins. -^ wash -^ I, till violet -* D, 5 mins. — * wash — > balsam, via usual reagents 33.41 Bensley and Bensley 1938b Bensley and Bensley 1938, 109 REAGENTS REQUIRED: A. 2% silver nitrate; B. MS 33.1 Bensley and Bensley 1938b; C. 8% neutralized formaldehyde method: [sections] — > water — > A, overnight — + B, 15-30 mins. — > rinse -* C, 3 mins. — »• balsam, via usual reagents 33.41 del Carpio 1930 test. 1932 Findlay 11360, 62:155 REAGENTS REQUIRED: A. 0.25% potassium permanganate; B. 5% oxalic acid; C. 2% silver nitrate; D. MS 33.1 del Carpio 1930; E. 4% neutralized formaldehyde; F. 0.2% gold chloride; G. 5% sodium thiosulfate method: [sections, already dye-stained]-* water—* A, 15-20 mins. — > rinse -^ B, till bleached — > wash — > C, 24 hrs. — > rinse -^ D, 30 mins. —* double rinse — » E, 15 sees. — * wash -^ F, till gray — > wash — > G, 5-10 mins. -* wash — > balsam, via usual reagents recommended for: reticular fibers in sections previously dye-stained. 592 METHODS AND FORMULAS MS 33.41 33.41 Foot 1924 11284, 9:778 REAGENTS REQUIRED: A. ADS 12.2 Lugol (1905); B. 1% sodium thiosulfate; C. 0.25% potassium permanganate; D. 5% oxalic acid; E. 2% silver nitrate; F. MS 33.1 Foot 1924; G. 2% formaldehyde; H. 1% gold chloride; /. DS 11.121 Weigert 1903; J. DS 12.221 Weigert 1904 method: [G m sections of F 3700.0010 Zenker 1894 material] -^ water -^ A, 5 mins. -^ B. 3-^ min. —> C, 5 mins. -^ D, 15-30 mins. -> wash — > E, 48 hrs. — > rinse -^ F, 30 mins, -^ rinse -^ G, 30 mins. ^ i/. 1 hr. -^ wash ^ /, 1 min. — > tap water, till blue — > /, 30 sees. -^ balsam, via usual reagents 33.41 Foot 1927a 1887a, 4:136 reagents required: A, B, C, D, as MS 33.41 Foot 1924; E. AMS 11.1 Foot 1927; F. 0.1% ammonia; G. MS 33.1 Foot 1927a; H. 8% neutral formaldehyde; /. AMS 22.1 Foot 1927; /. 5% sodium thiosulfate method: [material as for MS 33.41 Foot 1924] -^ A -> B --^ C --^ D, as MS 33.41 Foot 1924^^ water— > E, 15 mins. at 37°C. -^ F, 30 sees. — * rinse —+ G, 5 mins. in each of 2 changes —* wash —> H, 3 mins. — > 7, 3 mins. — > rinse — > J", 3 mins. — > wash -^ bal- sam, via usual reagents 33.41 Foot 1927b 1887a, 4:212 reagents required: A, B, C, D as MS 33.41 Foot 1924; E. Foot MS 33.1 1927b; F. 8% neutral formaldehyde; G. 0.2% gold chloride; H. 5%, sodium thiosulfate; I. DS 11.123 Harris 1900; J. DS 12.221 van Gieson 1890 method: [material as for AIMS 33.41 Foot 1924]-^ A ^ /? -^ C -> D, as AMS 33.41 Foot 1924 -^ water -^ E, 15 mins. at 45°C. -^ wash -* F, 2 mins. — » rinse -^ (?, 2 mins. -^ H, 2 mins. — » wash — ^ 7, 3-5 mins. -^ tap water, till blue — > J, 45 sees. — > balsam, via usual reagents 33.41 Gluck 1938 te.^f. 1939 Foot 4349, 19:169 reagents required: A. ADS 12.2 Gram 1880; 5. 5% sodium thiosulfate; C. AMS 11.1 Cajal 1913; D. MS 33.1 Gluck 1938; E. 2% formaldehyde; F. 0.2% gold chloride method: [sections of F 3700.0000 Zenker 1894]^ water-* A, 4-8 hrs. -^^ 70% ale, wash — > water -^ B, 5 mins. -^ thorough wash -^ C, 24 hrs., 37°C. -^ wash -^ 7), 5 mins. -^ rinse -^ E, 5 mins. — > wash — > F, till gray —^B,5 mins. — * wash — > balsam, via usual reagents 33.41 Gridley 1951 Tech. Bull., 21:71 reagents required: .4. 0.5% periodic acid; B. 2% silver nitrate; C. MS 33.1 da Fano 1919; D. 1.2% formaldehyde; E. 0.5% gold chloride; F. 5% sodium thiosulfate method: [6 n sections]—* water-* A, 15 mins. — > rinse -^ 7?, 30 mins. — » wash—* C, 15 mins. — * rinse — * 7), 3 mins. —>■ wash — » F, 5 mins. -^ wash -^ F, 5 mins. 33.41 Gomori 1937 608b, 13 :993 reagents required: A. 1% potassium permanganate; B.2% potassium metabisulfite; C. 2% ferric alum; D. MS 33.1 Gomori 1937; E. 4% formaldehyde; F. 0.2% gold chloride ; (?. 2 % sodium thiosulfate method: [paraffin sections, brought to water, of formaldehyde-fixed material] —> A, 1-2 mins. — * rinse —> B, I min. — > wash -^ C, 2 mins. — > wash — > 7), 1 min. — * quick rinse -* E, till reduced -^ wash -^ F, 10 mins. -^ rinse -^ B, 1 min. -^ wash ^ G, 1 min. — > wash -^ balsam, via usual reagents 33.41 Gordon and Sweets 1936a 608b, 12:545 reagents required: A. 0.5% potassium permanganate 95, 3% sulfuric acid 5; B. 1% oxalic acid; C. 2.5% ferric alum; D. MS 33.1 Gordon and Sweets 1936; E. 4% form- aldehyde; 7'. 0.2% gold chloride; G. b% sodium thiosulfate method: [sections, on slide, from fornuildehyde or F 3000.1010 material] -^ water -* A, 5 mins. — ► wash — * B, till white — * wash — * C, 15-20 mins. — * wash — * 7), few sees. -^ rinse — > E, till reduction complete — > wash -^ F, 1-3 mins. — > wash — * G, 5 mins. —> balsam, via usual reagents recommended for: reticulum of spleen. MS 33.41 METAL STAINS 593 33.41 Gordon and Sweets 1936b 608b, 12:545 ri<;agp:nts RKgriREi): .1. 0.5';;, |)()jassium prniKuifiaiiatc 05, .'}% sulfuric Mcid 5; /?. 1% oxalic acid; C. 2.5% ferric alum; D. MS 'SAA Foot l'J27a; E. 4% formaldehyde; F. 0.2% gold chloride; G. 5% sodium thiosulfate method: [sections from formaldehyde or F 5000.1010 Bouin 1S97 material] -^ water —* A, 1-5 mins. -^ wash — > B, till white -^ wash — * C, 15 to 30 mins. —* wash — * D, few sees. — > E, till brown — * wash — > F, l-,i mins. — > wash — + (r, 5 mins. — > wash — > bal- sam, via usual reagents RECOMMENDED FORI reticulum of spleen. 33.41 Jalowy 1937 23G.3nb, 27:6(57 UEAGENTs REQUIRED: .4. MS 33.1 Jalowy 1937; B. 1% ammonia; ('. 10% neutralized formaldehyde method: [sections of formaldehyde fixed materials] —>■ water — > A, 5-30 mins. 30°C. — » rinse — > B, rinse —^ C, 2-10 mins. — » balsam, via usual reagents recommended for: collagen and reticular fibers in skin 33.41 Krajian 1933 1789a, 16:376 reagents required: A. 10% ammonia; B. 0.3% potassium permanganate; C. 1.5% oxalic acid; D. 5% silver nitrate; E. 33.1 Krajian 1933; F. 12% formaldehyde method: [5-10 m sections by freezing technique of formaldehyde-fixed material] -^ water — > ^, 15 mins. 60°C. -^ wash —> B, 5 mins. -^ rinse -^ C, till decolorized — > wash — > D, 1 hr. 60°C. -^ j5;, 15 mins. 60°C. -^ wash ^ /^, 1-3 mins. 60°C. -* wash -» balsam, via aniline and xylene recommended for: reticular and collagen fibers in frozen sections. 33.41 Laidlaw 1929 608b, 5 :239 REAGENTS REQUIRED: A. 1% iodine in 95% ale; B. 5% sodium thiosulfate; C. 0.5% potassium permanganate; D. 5% oxalic acid; E. MS 33.1 Laidlaw 1929; F. 0.4% form- aldehyde; G. 0.2% gold chloride method: [sections of F 5000.1010 Bouin 1897 fixed material] -^ water -^ A, 3 mins. -^ rinse — » .B, 3 mins. -^ rinse — > C, 3 mins. — > wash — > D, 5 mins. — * wash — > E, 10 mins. —> wash -^ F, 10 mins. —> rinse —^ (7, 10 mins. wash —> balsam, via usual reagents recommended for: reticulum fibers in skin. 33.41 Levi 1907 14425, 18 :292 REAGENTS REQUIRED: A. 2% silver nitrate; B. MS 33.1 Levi 1907; C. 5% formaldehyde; D. 0.5% gold chloride; E. 5% sodium thiosulfate method: [paraffin sections of F 3700.0010 Zenker 1897 fixed material]—* water—* A, 24 hrs. —* B, 20-40 mins., till brown — * rinse — > C, 5-10 mins. — * wash -^ D, 2 hrs. — » wash -^ E, 10-15 mins. -^ wash — > balsam, via usual reagents recommended for: reticulum fibers in lymphatic glands. 33.41 LUlie 1948 Lillie 1948, 187 REAGENTS REQUIRED: A. 0.5% potassium permanganate; B. 5% oxalic acid; C. 1% uranium nitrate; D. MS 33.1 Lillie 1948; E. 4% formaldehyde; F. 0.2% gold chloride; G. 5% sodium thiosulfate method: [paraffin in sections varnished on slide with colloidin] -^ A, 2 mins. — > wash — » B, 2 mins. — > wash —* C, 5-10 sees. — * D, on slide 3 mins. -^ rinse, E, 2 mins. -^ wash — > F, 2 mins. — > wash — > G, 2 mins. — » wash — » balsam, via usual reagents 33.41 Long 1948 see MS 33.44 Long 1948 33.41 Maresch 1905 23681, 17:641 REAGENTS REQUIRED: ^.2% silvcr nitrate; B. MS 33.1 Maresch 1905; C. 8% formalde- hyde; D. 0.2% gold chloride in 0.2% acetic acid; E. 5% sodium thiosulfate method: [sections of formaldehyde fixed material] — > A, 12-24 hrs. — > rinse—* B, 2-30 mins. -^ wash -* C, 1 hr. — > D, 1 hr. -^ E, 30 mins. —>■ balsam, via carbol-xylene recommended for: reticulum fibers in liver. 594 METHODS AND FORMULAS MS 33.41 33.41 Negrin 1941 1887a, 31:108 REAGENTS REQUIRED: A. 10% sodium Cyanide; B. MS 33.1 del Rio-Hortega 1921; C. 1% pyridine; D. 0.4% neutralized formaldehyde; E. 10% potassium thiocyanate; F. 1% gold chloride; G. 5% sodium thiosulfate method: [sections] —» water —> a, 10-20 mins., 50°C. —» thorough wash -^ B, 10-20 mins., 50°C. — » C, wash — > D, 1-3 mins. — » E, till differentiated —^ thorough wash —> F, 5 mins. — > wash — > G, 2 mins. -^ wash — > balsam, via usual reagents 33.41 Perdrau 1921 11431,24:117 REAGENTS REQUIRED: A. 0.25% potassium permanganate; B. DS 21.212 Weigert 1885; C. ADS 21.1 Pal 1887sols. A and 5; I>. 2% silver nitrate ;£•. MS33.1daFano 1919; F- 8% neutralized formaldehyde; G. 5% sodium thiosulfate method: [sections of formaldehyde fixed material] — > A, 10 mins. — > wash -^ B, 2 hrs. -^ wash -^ C, {A) 15-20 sees. -^ C, {B) few sees. — > wash — + D, in dark, 12 hrs. -^ ^, 20-30 mins. -^ wash — » jP, 15 mins. -^ wash — » G, 5 mins. — * wash —> balsam, via usual reagents 33.41 del Rio-Hortega 1916a 21344, 14:181 REAGENTS REQUIRED: A. 1% taunic acid in 95% alcohol; B. MS 33.1 del Rio-Hortega 1916 10, water 90 (3 dishes required); C. 8% neutralized formaldehyde method: [sections (see note below)] -^ A, 5 mins. at 55°C. — ♦ quick rinse —> B, 1st dish, 1 min. — ^ B, 2nd dish, 1 min. —* B, 3rd dish, 2 mins. -+ wash -^ C, 30 sees. -^ wash — > balsam, via usual reagents note: del Rio-Hortega {loc. cit.) specifies formaldehyde material. Gatenby and Stern (Gatenby and Painter 1937, 549) state that alcohol or "Bouin" (presumably F 5000.1010 Bouin 1897) material may be used provided that it be "re-formalized" for a few days; Cajal and de Castro 1933, 291, suggest that material from this fixative be stored in 4% formaldehyde. 33.41 del Rio-Hortega 1916b 21344, 14:181 reagents required: A. 1% tannic acid in 95% ale; B. MS 33.1 del Rio-Hortega 1916 10, water 90 (3 dishes required); C. 1% gold chloride; D. 5% sodium thiosulfate method: [sections by freezing technique of formaldehyde material] —> A, 5 mins., 55°C. — * rinse rapidly -^ B, 1st dish, 1 min. — > B, 2nd dish, 1 min. — > B, 3rd dish, till dark brown — * thorough wash — > C, 30 to 40 mins., 40°C. — > Z), 5 mins. -^ wash -^ balsam, via usual reagents 33.41 Robb-Smith 1937 11431,45:312 REAGENTS REQUIRED: A. 10% ammoiiia; B. 0.25% potassium permanganate; C. 1.5% oxalic acid; Z). 5% silver nitrate ; .B. AMS33.1 Robb-Smith 1937;F. 15% formaldehyde; G. 0.2% gold cUoride; H. 5% sodium thiosulfate methods: [paraffin ribbons dried on slide, wax not removed] — > A, 15 mins. -^ wash -^ B, 5 mins. — » wash -^ C, till bleached -^ wash -^ D, 1 hr. — ♦ rinse -^ E, 15 mins. — > wash -^ F, Z mins. -^ wash G, 3 mins. -^ H, 1 min. — » wash —>■ dehydrate — > xy- lene, till wax removed — » balsam 33.41 Snessarew 1910 766,36:40 REAGENTS REQUIRED: A. 5% ferric alum; B. 10% silver nitrate; C. MS 33.1 Bielschowsky 1902; D. 8% formaldehyde method: [sections by freezing technique of formaldehyde fixed material] — > A, 4 days changed daily -* wash -^ B, 48 hrs. — > rinse — > C, 24 hrs. — > wash -^ D, till black- ened -^ balsam, via usual reagents 33.41 Studricka 1906 23632, 23:416 REAGENTS REQUIRED: A. 3% silver nitrate; B. MS 33.1 Wolff 1905; C. 4% formaldehyde; D. 0.5% gold chloride; E. 5% sodium thiosulfate method: [sections of fixed and decalcified material] -^ A, 4 days — > wash -* 5, till yellow-brown — > rinse — * C, till dark brown -* D, till gray-black -* wash —* E, 1 hr. — > wash — ^ balsam, via usual reagents recommended for: reticulum fibers in cartilage, bone, and dentine. note: Zimmermann 1908 (23632, 25:8) differs only in reducing A to 2 days and in diluting B fourfold. MS 33.41-MS 33.42 METAL STAINS 595 33.41 Urechia and Nagu 1931 6630, 106:498 REAGENTS HioQi'iRED: .1. AMS 11.1 Cajal 1913; B. sat. sol. Miinnonimii sulfide; C. MS 33.1 del Rio-llortega 1917a; D. 50% ale; E. 0.4% foniK.l.lciiy.l.'; /'. 0.2% gold chloride; G. 5% sodium thiosulfate method: [fresh tissue]-^ .4, 3-4 days —> sections by freezing technique -► B, 15-30 mins. -^ wash — > C, 2-4 hrs. — > D, quick wash, 2 changes-^ E, 5-10 niins. —^ F, 15 mins. -^ wash — > G, 5 mins. -^ wash — * balsam, via usual reagents RECOMMENDED kor: rcliculuiii fibers in nervous tissues. 33.41 Wilder 1935a fi08b, 11:817 RE.\GENT8 heqiired: ,4. 10% phosphouiolybdic acid; B. 1% uranium nitrate; C. MS 33.1 Foot 1927a; D. AMS 21.1 Wilder 1935; E. 0.2% gold chloride; F. 5% sodium thiosulfate method: [sections of F 3700.1000 or .1010 matcrial[ —> waters .4, 1 min. -^ wash -^ B, 5 sees. — » rinse — > C, 1 min. — > 95% ale. quick rinse -^ D, 1 min. — > wash — > E, 1 min. -^ rinse -> F, 1-2 mins. -^ [dye stains as in MS 33.41 Foot 1924 to 1927b, if required] —* balsam, via usual reagents RECOMMENDED FOR: reticuluui of spleen. 33.41 Wilder 1935b 608b, 11:817 REAGENTS REQUIRED: A. 1% potassium permanganate; B. 5% oxalic acid; C. 1% ura- nium nitrate; D. Bielschowsky 1902 MS 33.1; E. AMS 21.1 Wilder 1935; F. 0.2% gold chloride; G. 5% sodium thiosulfate method: [sections] — > water — > ^, 1 min. —y rinse -^ B, 1 min. — + wash — > C, 5-10 sees. -^ rinse D, 1 min. -^ 90% ale, rinse -^ ^, 1 min. -^ rinse -^ F, 1 min. — > rinse —> G, 1 min. -^ wash — > balsam, via usual reagents recommended for: reticulum fibers. 33.41 Zhookin 1937 11284,22:1284 re.\gents required: .4. 0.001% potassium permanganate; B. MS 33.1 Lawrentjew (1933); C. 0.04% formaldehyde, pH 7; D. 0.2% gold chloride; E. 5% sodium thio- sulfate method: [frozen sections of formaldehyde material] — > water ^ .4, 20 sees. -^ rinse — » B, 2-3 mins. -^ rinse -+ C, till reduced -^ wash — > D, 1 min. —* E, 5 mins. -^ wash — > balsam, via usual reagents 33.41 Zimmermann 1908 see MS 33.41 Studnicka 1906 (note) 33.42 SELECTIVE STAINING OF SPECIAL CELLS 33.42 Gluckman 1943 4285a, 20 :63 REAGENTS REQUIRED: A. MS 33.1 Gluckmau 1943; B. 5% sodium thiosulfate method: [parafhn sections of F 5000.1010 Bouin 1897 material] -^ water -^ A, \0 mins., 60°C. —* B, 30 sees. — > wash — » balsam, via usual reagents RECOMMENDED FOR: argcntiphil cells of intestine. 33.42 Gomori 1946 519b, 10:177 REAGENTS REQUIRED: A. 5% chrouiic acid ; B. 1% sodium bisulfite; C. MS 33.1 Gomori 1946 50, water 50, sodium borate 0.2; D. 0.1 % gold chloride; .B. 2% sodium thiosulfate method: [sections] -^ water —> ^, 1-1 J^^ hrs. —» wash -^ B, 1 min. -^ wash —> C, 1-3 hrs. till glycogen granules dark brown -^ wash -^ D, 5 mins. —* E,b mins. — * wash — > [counterstain if desired] — * balsam, via usual reagents recommended for: glycogen. 33.42 Jacobson see MS 33.42 Masson 1923b (note) 33.42 Masson 1923a test. 1937 Duthie Gatenby and Painter 1937, 414 reagents required: A. 0.003% ammonia; B. MS 33.1 Fontanna 1912; C. AMS 22.1 Cajal 1910; D. 2% sodium thiosulfate method: [frozen sections formaldehyde fbced materials] -^^ .4, 2-3 hrs. -^ B, in dark, 36 hrs. -^ wash — > C, 10 mins. — > wash — ♦ Z), 1 min. -^ wash — ► balsam, via usual reagents recommended for: argentophil cells. 59G METHODS AND FORMULAS MS 33.42-MS 33.44 33.42 Masson 1923b test. 1948 Lillie cit. Lee Lillie 1948, 102 REAGENTS requiked: A. MS 33.1 Foatana 1912; B. 0.1% gold chloride; C. 5% sodium thiosulfate method: [sections of formaldehyde material] -^ water -^ A, 12-48 hrs. in dark — >• wash — > B, 4-6 mins. — > wash — > C, 1 min. — > wash — > [counterstain if required] -^ balsam, via usual reagents recommended for: argentaffin cells. note: Lillie {loc. cit.) states that Lison (no ref.) "used the sequence given above" and that Jacobson (no ref.) omitted the gold chloride. 33.42 Masson 1928 608b, 4:181 REAGENTS REQUIRED: A. 0.1% ammonia; B. MS 33.1 Masson 1928; C. AMS 22.1 Cajal 1948 method: [2-3 mm. slices of F 5000.1010 Bouin 1897 fixed material]-^ wash-* A, 24 hrs. — > rinse — > B, 24 hrs. -^ rinse — » C, 24 hrs. — » [sections] RECOMMENDED FOR: argentaffin cells. 33.42 Ogata-Ogata 1923 2526, 71 :376 REAGENTS REQUIRED: A. 1% ainmonia; B. MS 33.1 Maresch 1905 50, water 50; C. 3% sodium thiosulfate; D. 4% formaldehyde method: [small blocks of fresh tissue] -^ A, 1-2 hrs. in dark -^ B, 3-5 hrs. in dark — > A, 30 mins. changed several times, in dark -^ C, I hr. in dark — > wash — > D, 1-2 days —* [frozen sections] RECOMMENDED FOR: chromaffin cells. 33.42 Shanklin 1951 Cowdry 1952, 270 REAGENTS REQUIRED: A. MS 31.1 Shanklin 1951; B. 5% sodium sulfite; C. 0.6%, pyri- dine; D. MS 33.1 del RIo-Hortega 1927; E. AMS 21.1 Shanklin 1951; F. 0.2% gold chloride; G. 5% sodium thiosulfate method: [7-10 m sections of formaldehyde material]—* water—* A, 24°C. 24 hrs.—* wash -^ B, 1 hr. -^ C, 1-2 mins. -^ wash -^ D, 2-5 mins. -^ rinse -^ E, 1 min. -* rinse -^ i^, 1 min. -* G, 1-2 mins. -* wash — > [counterstain if desired] — * balsam, via usual reagents recommended for: parenchyma of pineal. 33.43 DEMONSTRATION OF CALCIFIED AND OSSIFIED MATERIAL 33.43 Gomori 1933 608b, 9 :253 REAGENTS REQUIRED: A. 1.5% silvcr nitrate; B. water 100, sodium hypophosphite 5, 0.5% sodium hydroxide 0.2; C. 5% sodium thiosulfate; D. 7% sulfosalicylic acid method: [blocks of tissue or embryos not more than 2 mm. thick] -* 95% ale. 2-4 days — * wash -^ A, 6-10 days, changed once or twice -^ thorough wash -* B, 4-8 days -^ wash — * C, 2 days — > wash -^ D, till decalcified, 1-3 days -^ sections -^ [counterstain if desired] recommended for: demonstration of calcium in tissues. 33.43 Orban test. circ. 1938 Wellings Wellings circ. 1938, 190 REAGENTS REQUIRED: A. 0.25 %o potassium permanganate; B. 5% oxalic acid; C. 2% silver nitrate; D. MS 33.1 Bielschowsky 1902; E. 2% formaldehyde; F. 1% gold chloride; G. 5% sodium thiosulfate method: [celloidin sections of decalcified, F 3700.1010 Heidenhain 1916 fixed, teeth] -* water -* A, 5 mins. -* wash -* B, 15 mins. —>■ wash -^ C, 24 hrs., in dark -^ wash -^ D, 30 mins. -^ wash -^ E, 30 mins. -^ wash -^ F,l hr., wash -> G, 2 mins. -♦ wash -» balsam, via usual reagents recommended for: general structure of tooth pulp, and particularly development of dentine from it. 33.44 OTHER HISTOLOGICAL METHODS 33.44 Jacobson 1939 11431, 49:1 REAGENTS REQUIRED: A. MS 33.1 Foutaua 1912; B. 5% sodium thiosulfate method: [sections of formaldehyde material] -^ water — * A, 12-24 hrs. -^ wash, 1 min. — > i?, 1 min. — > wash — * balsam, via usual reagents recommended for: differentiation of argentophil cells in gastrointestinal tract. MS 33.44-MS 33.51 METAL STAINS 597 33.44 Long 1948 20540b, 23 :69 REAGENTS REQUIRED! .1. 0.25% potassiuni permanganate; B. 5% oxalic acid; C. 10% silver nitrate; D. MS 33.1 Long 1948; E. 0.05% ammonia; F. 0.4% neutralized form- aldehyde;(r. 0.2% gold chloride; H. 5% sodium thiosulfate; /. 0.1%, azocarmine in 1% acetic acid; J. 5% phosphotungstic acid; K. water 99, acetic acid 1, either light green SFY (adult tissues) or fast green FCP' (embryonic tissues); L. 1% acetic acid method: [sections of F 3700.1010 lieidenhain 1916 after removal of mercury with iodine] -* water — > .A, 2 mins. -^ wash, 5 mins. -^ B, 2 mins. -> thorough wash -» C, 12 hrs., in dark -> wash -^ D, 25-30 mins., 40-45°C. -^ E, one dip -* F, 10-20 mins. -^ wash -^ G, 15-30 mins. -^ rinse -^ H, 2 mins. -^ wash -^ I, 15-30 mins. -> rinse -^ /, 3-6 hrs. —^ rinse -^ A', 3 mins. — > rinse -> L, rinse (or longer if differentiation necessary) -^ dammar, via isopropyl alcohol and xylene RECOMMENDED FOR: differentiation of reticular fibers (dense purple), collagen (green), and myofibrils (red). 33.44 Mclndoo 1928 1789a, 6:598 REAGENTS REQUIRED: A. 10% formaldehyde; B. MS 33.1 del Rio-Hortega 1919 50, water 50, pyridine 1; C. 20% formaldehyde buffered to pH 7; D. 2% sodium thiosulfate method: [small pieces] -^ A, 20 days -^ [frozen sections] — > B, in watch glass heated to steaming, till brown -^ rinse -^ C, 1 min. -^ rinse -^ D, I min. -^ wash, 2-3 days — > balsam, via usual reagents RECOMMENDED FOR: demonstration of bile capillaries. 33.44 del Rio-Hortega 1926 3231, 26:107 REAGENTS REQUIRED: A. MS 33.1 del Rio-Hortega (1933); B. 0.4% formaldehyde; C. 5% sodium thiosulfate method: [frozen sections of formaldehyde material] —>■ water — » A, till brown, 50°-55°C. — » wash — > B, 5-10 mins. — » C, 5 mins. -^ wash — * balsam, via usual reagents recommended for: epithelial fibrils. 33.5 Bacteriological Methods 33.51 methods for spirochetes 33.51 Ferguson test. 1916 Warthin 4349, 6:56 reagents required: A. 2% silver nitrate; B. MS 33.1 Bielschowsky 1902; C. 8% form- aldehyde; D. 0.01% gold chloride in 0.01% acetic acid; E. 5% sodium thicsulfate method: [sections] — > water -* A, 12-24 hrs. -^ B, 15-30 mins. -^ rinse — > C, 3 mins. — > wash — > D, 2 mins. — > wash -^ E, 1 min. -^ balsam, via usual reagents 33.51 Fontana 1912 7176,55:1003 reagents required: A. F 0000.1010 Fontana 1912; B. AMS 13.1 Fontana 1912; C. MS 33.1 Fontana 1912 method: [fresh smears] -> .4, 2 mins. -^• wash -^ B,5 sees., 40°C. -^ wash -♦ C, dropped on slide and warmed, 2-30 sees. — > repeat, until sufficiently darkened — > wash — > dry — > balsam, if permanent preparation is required note: Langeron 1942, 636 recommends F 0000.1010 Ruge 1942 in place of A above. 33.51 Gordon 1936 see 33.6 Gordon 1936 33.51 Lancelin, Seguy, and Debreuil 1926 6630, 94:557 reagents required: A. F 0000.1010 Fontana 1912; B. AMS 13.1 Fontana 1912; C. MS 33.1 Fontana 1912; D. AMS 22.1 Lancelin, Seguy, Debreuil 1926 method: [fresh smears] -* A,2 mins. -^ wash -^ B,5 sees., 40°C. -^ wash — » C, dropped on slide and warmed, 20-30 sees. -^ rinse -^ D, till sufficiently darkened -^ dry -^ balsam, if permanent preparation required 33.51 Krajian 1933 test. 1935 abstract 20540b, 10:1935 REAGENTS REQUIRED: A. 2% sodium cobaltinitrite; B. AMS 12.1 Krajian 1933; C. MS 33.1 Krajian 1933; D. AMS 21.1 Krajian 1933 method: [5 M frozen sections of formaldehyde material]-^ A, 5 mins. -^ wash -^ B, 15 mins. -> rinse — > C, 15-25 sees. — > wash — > D, 5 mins. — ♦ wash -^ balsam, via usual reagents note: See also Krajian MS 31.51 Krajian 1935. 598 METHODS AND FORMULAS MS 33.51-MS 33.6 33.51 Langeron 1942 see MS 33.51 Fontana 1912 (note) 33.51 Seguin 1939 829, 10:838 REAGENTS REQUIRED: A. AMS 21.1 van Ermengen 1940; B. 33.1 Fontana 1912; C. 0.5% gold chloride; D. 0.25% platinic chloride method: [osmic fixed smears] -^ dry — > abs. ale. 10 mins. — > dry -^ A, on slide, 10 mins. -^ thorough wash — > B, 10 mins. — > wash — > C, few mins. — > rinse -^ D, few mins. — » dry 33.51 Steiner 1937 11284,23:293 REAGENTS REQUIRED: A. 1.5% ammonia; B. MS 33.1 Steiner 1937 method: [air-dried films] -^ A, till dehemoglobinized -^ wash — * 5, on slide, 40-90 mins. -^ drain —> wash — > dry 33.52 OTHER BACTERIOLOGICAL METHODS 33.52 Craigie 1928 3566,9:55 reagents required: A. AMS 13.1 Zettnow 1891; B. MS 33.1 Craigie 1928; C. 0.03% gold chloride method: [smears from formolized suspensions] — > dry, 37°C. -^ heat, 100°C., 5 mins. -^ water, 5 mins. -^ rinse -> dry, 100°C., 5 mins. — > A, 5-10 mins., 100°C. -^ wash -^ fi, on slide, warmed to steaming and held warm till smear brown -^ wash —>■ C, 30 mins. — » wash -^ dry recommended for: bacterial smears. 33.52 Novel 1939 857, 63:302 reagents required: A. water 100, tannic acid 10, ferrous sulfate 16, magenta 0.4; B. MS 33.1 Fontana 1912 method: [air dry smears] — > A, 1-1,^2 niin. -^ rinse -^ B, till chocolate, }i to 13^^ min. — > rinse — * dry recommended for: bacterial fiagella. 33.52 Zettnow 1891 23684, 11:689 REAGENTS REQUIRED: A. AMS 13.1 Zettuow 1891; B. MS 33.1 Zettnow 1891 method : [Smear preparations on coverslip] — > A, in dish heated on water bath, 5-7 mins. — > water, wash -^ B, poured on slip, heated to steaming till margin blackens — > wash -^ dry recommended for: bacterial fiagella. 33.6 Other Silver Diammine Methods 33.6 Gordon 1936 11284, 22:294 REAGENTS REQUIRED: A. 2.5% ferric alum; B. water 100, gelatin 1, 2% sodium car- bonate 0.1; C. MS 33.1 Gordon 1936; Z>. AMS 21.1 Gordon 19.36 method: [formaldehyde fixed smears] -^ A, 10 mins. -^ wash -^ B, dip, drain —>■ quick rinse -^ C, 5-15 mins. -^ wash at 60°C. -^ D, till reduced -* wash -^ balsam, via usual reagents recommended for: blood smears containing parasites. 33.6 Haymaker and Sanchez-Perez 1935 19938, 82 :355 REAGENTS REQUIRED: A. watcr 90, 40% neutralized formaldehyde 10, sodium chloride 0.45; B. 0.5%, ammonia; C. MS 33.1 Haymaker and Sanchez-Perez 1935; D. MS 33.1 del Rio-Hortega 1923 50, water 50; E. 0.4% formaldehyde; F. 0.2% gold chloride; G. 1% sodium thiosulfate method: [clots on coverslip] -* A, 24 hrs. -+ B, 5 mins. -^ wash -> C, 40°C. till clot yellows (about 10 mins.) -^ wash -^ D, 40°C. till clot brown (about 9 mins.) -♦ wash -->■ E,5 mins. -» F, 40°C. till violet (about 10 mins.) -^ G, 5 mins. -^ wash -^ balsam, via usual reagents RECOMMENDED FOR: cells in tissue culture. 33.6 Kalwaryjski 1938 test. 1939 Findlay 11360, 59:36 REAGENTS REQUIRED: A. ADS 12.2 Lugol ; B. 2.5% sodium thiosulfate; C. MS 33.1 Kal- waryjski 1938; D. 5% sodium thiosulfate MS 34.0 METAL STAINS 599 method: [pieces of fresh muscle infested with Trichiiicna] -^ A, 10 mins. — > wash — » B, till muscle {not worms) decolorized -^ wash — » C, till worms tiro iodine free -^ wash — * D, till muscle colorless—* wash—* balsam, via usual reagents RECOMMENDED FOR: Trichlnella in muscles. MS 34 METHODS USING SILVER IN COMBINATION WITH OTHER METALS 34.0 Typical Examples Demonstration of the Purkinjc cells in the cerebellar cortex by the method of Golgi 1875 This method is not one which can be recommended to anybody who desires hurriedly to produce a slide. It is an ex- perimental method given here for those who have a genuine interest in the pro- duction of beautiful slides. The two other Golgi processes given under MS 34.21 be- low are considerably quicker but they are less certain and give less opportunity for experiment. By the method about to be described it is possible, with patience, to secure a better slide than can be obtained by any other method. The first step is to make up at least 100 milliliters of Miiller's 1859 dichromate- sulfate (Chapter 18 F 7000.0000 Miiller 1859) and weigh out, into each of three small tubes or capsules, one gram of finely powdered potassium dichromate. Next prepare about 250 milliliters of a 0.75% solution of silver nitrate and secure two chemically cleaned lOO-milhUter stop- pered bottles and a chemically clean crys- talhzing dish of about 30 milliliter capacity. Kill a rabbit, tie it face down on a board, skin the head, and remove the parietal and frontal bones with bone for- ceps. The cerebellum should then be washed free of extravasated blood with normal saline or triple-distilled water, and blocks of the cerebellar cortex removed with the broken edge of a coverslip or with a stainless steel knife. (Many failures in using this technique are due to cutting blocks of material with an ordinary steel scalpel.) The blocks should measure about }i mm. to % mm. cube. At least 30 are required for experimental purposes. These blocks are now placed in the stoppered bottle of Miiller's fluid (F 7000.0000 Mul- ler 1859). It is desirable to place a half- inch layer of fat-free cotton or fine glass fiber on the bottom of the bottle to pre- vent the pieces of material from pressing against the glass. The pieces should be left in Miiller's fluid for five days, and the bottle gently shaken daily to avoid ex- hausting the fixative in the vicinity of the pieces. On the fifth day add one of the one- gram bottles or capsules of dried potas- sium dichromate, tip the bottle up and down until the chemical is thoroughly dis- solved, and then let it stand five days longer. On the tenth day add another gram of potassium dichromate and let it stand five days longer. On the fifteenth day remove two or three of the blocks and place them without washing in about 25 milliliters of 0.75 silver nitrate in the crystalizing dish. Add one gram of potas- sium dichromate to the fluid which holds the remaining blocks, dissolve as before, and leave for two days more. Returning now to the two blocks which, for experimental purposes, have been put in the silver solution: these should be agitated rapidly to keep the precipitate from adhering to the surface as it forms. The silver nitrate after a few minutes should be poured off and replaced with fresh solution. This process of pouring off the contaminated solution and adding fresh is continued until the pieces rest at the bottom and no longer exude streams of a silver chromate precipitate. After they have remained in this satisfactory condition for about ten minutes they are removed to a stoppered bottle containing 100 milliliters of fresh 0.75 % silver nitrate. There they may remain indefinitely. They should be marked in some manner, pref- erably by notching the edge, so that they can be identified. Two more blocks should be removed from the dichromate solution at intervals 600 METHODS AND FORMULAS MS 34.0 of a day, washed, as has been described above, in successive portions of silver ni- trate solution, notched by a different sys- tem, and transferred to the large bottle of silver nitrate at intervals of a day until all the blocks have been transferred. There will finally be 30 blocks covering 15 peri- ods of immersion in the dichromate solu- tion. It must be emphasized that the suc- cess of the process depends on the length of time the blocks stay in the dichromate. As for the length of time in silver nitrate, it should not be less than 48 hours, but it may be extended for several weeks with the assurance that the impregnation will not be affected. Forty-eight hours after the last of the blocks has been placed in silver nitrate, pour off the solution, add about 50 milliliters of triple-distilled wa- ter, tip the bottle up and down once, pour off the distilled water, and fill the bottle with 90% alcohol. After 24 hours the alco- hol is replaced, and the process repeated as often as the reagent becomes discolored. Though it is perfectly possible to obtain adequate sections by freezing techniques, it is usually better to use the celloidin method. An objection has always been that prolonged exposure to strong alcohol tends to remove the stains; but this has not been the author's experience. To avoid wasting time preparing sec- tions from unsatisfactory blocks, reject the hopeless cases by an examination of a freshly cut surface under a binocular mi- croscope. For this purpose arrange the blocks in order of their removal from the dichromate. Examine first the block which has been hardened for the longest time. Slice one of the faces of this block with a razor-sharp scalpel and examine it under the surface of 95% alcohol with a binocu- lar microscope. It will almost invariably appear as a uniform sheet of chrome yel- low, at best only slightly granular. This indicates, as anticipated, that the block has been in the dichromate too long. Next take the block which has remained for the least time in the dichromate and slice its edge. It is almost certain to have a pale, transparent, cheesy appearance without any opaque yellow speckling. This indi- cates, as anticipated, that the block has been for too short a time in the dichro- mate. Work through the series, taking blocks alternately from groups impreg- nated for a long and for a short time. It soon becomes apparent that the plain opacity of the long-impregnated blocks begins to break up into speckles, whereas golden yellow opaque spots begin to ap- pear in the cheesy, transparent, under- impregnated blocks. Somewhere between these two lies the perfect specimen, prefer- ably one lying intermediate between the two which have been reached as the end points of unsatisfactory material. As has been stated, it is customary to section blocks of Golgi-impregnated mate- rial by the freezing technique, on the ground that the prolonged immersion in alcohol necessary for celloidin sectioning tends to degrade the finer details of the preparation. This has not been true in the writer's experience, unless dehydration has been unnecessarily prolonged. It is usually sufficient to put the selected block in about 25 miUiUters of absolute alcohol for about two hours. Next, replace the absolute alcohol with a mixture of equal parts of absolute alcohol and ether and let stand two hours longer. Then place the block for about three hours in the thin, or first, solution of celloidin customarily employed for embedding. Since it will probably now be toward the end of the day, it is the author's custom to transfer the block directly from the thin syrupy solution of celloidin to the thickest solu- tion of celloidin and to leave the material in this overnight. It may be removed from this thick syrup next morning and hard- ened by whatever technique is customary. In this instance immersion in 70 % alcohol is probably simplest. When the celloidin is hardened, sections about 25 microns thick are cut with a knife moistened with 70% alcohol and accumulated in 70% al- cohol. Each section is then separately re- moved to a slide and examined under me- dium power of an ordinary microscope. One or two sections will soon be found which will demonstrate to perfection the aborizations of Purkinje cells. These sec- tions should be transferred to a separate watch glass of 70% alcohol. When suffi- cient sections showing the required struc- ture have been accumulated, they may be MS 34.0 METAL STAINS 601 mounted in cedar oil. This type of mount- ing is necessary, since it is a peculiarity of this technique that sections prepared by it may not be mounted under the surface of a coverslip or they will bleach within an interval varying from a few months to a year; whereas if they are merely var- nished to a shde they may be preserved indefinitely. In the present instance they may be dehydrated in absolute alcohol, as it docs not matter whether or not the celloidin is dissolved from the section. If one is deahng with brittle material or with material for which the celloidin forms the cliief support, it is essential to com- ])lete the dehydration in some alcohol in which celloidin is not soluble. Golgi him- self recommended guaiacol. Whatever me- dium is used for dehydration, clearing sliould always be carried out in cedar oil. When it is required to make a more permanent preparation of an individual mount, it is simplest to take it from the thin cedar oil, lay it on a slide, blot the cedar oil from it with the finest filter paper available, and place over the surface a layer of the kind of thickened cedar oil once sold for use with oil immersion objec- tives and still obtainable from some sup- pliers as "cedar oil (special for micro- scopy)." This oil hardens in about 48 hours. Two layers will give enough pro- tection, and the only objection to this method of mounting is the tendency of dust to accumulate on the surface. When this has happened to the extent that ex- amination of the specimens is becoming difficult, it is only necessary to wash off the cedar oil with absolute alcohol an'd replace it with fresh cedar oil. Such drastic treatment may not even be necessary, and it is often possible to remove the dust with a cloth moistened in cedar oil. Those who are not prepared to indulge in the many experimental processes men- tioned in this technique are recommended to the technique of Golgi 1880 which is universally known as "Golgi's quick process." This involves osmic-dichromate fixation and the entire operation may be concluded in as little as three or four days. It does not, liowever, j^ield results so beautiful, nor is it always i)()ssil)le to se- cure results at all. The advantage of the present slow method is that, since numer- ous blocks are removed at intervals, one is certain to find at least one which has the correct degree of impregnation. Demonstration of the structure of the superior cervical ganglion by the method of Kolossow 1896 This is an interesting mixed silver-osmic method which is ideally suited for the demonstration of nerve fibers in cells within sympathetic ganglia, a subject most difficult to impregnate by other silver techniques. Only two solutions are re- quired. The first is Kolossow's 1897 osmic- dichromate fixative (see in Chapter 18, F 1700.0000 Kolossow 1897) and the second, the silver-osmic stain of Kolossow (see MS 34.1 Kolossow 1897), which is prepared by adding to 250 milliliters of triple-distilled water, first five grams of silver nitrate and then, when this has been completely dissolved, one gram of osmic acid. The easiest material on which to become acquainted with this technique is the an- terior (or superior) sympathetic cervical ganghon of a rabbit. Before commencing the dissection of the rabbit it is as well to prepare an adequate supply of the fixative solution and to store this in a chemically clean glass-stoppered bottle. A rabbit is then killed and tied, ventral side upper- most, on a dissecting board. A loop of cord is passed around the anterior region of the head, which is drawn over the edge of the board so as to stretch the neck as far as possible. The neck is then skinned and the superficial fascia removed, the muscles being pulled laterally away from the tra- chea. If this is done in about the central region of the neck, two nerves will im- mediately become apparent. The smaller of these is the descending ramus of the hypoglossal and the larger is the fused tenth nerve and sympathetic nerve from the ganglion sought. This larger nerve is then followed forward by parting the fascia of the muscle to a region just behind the tympanic bulla. Here it will be found to split into two, each of the two smaller tributaries swelling, after a distance of about one half inch, into a ganglion. The 602 METHODS AND FORMULAS MS 34.0 larger of these two ganglia, which is also the one closer to the surface of the neck, is the ganglion nodosum of the vagus nerve ; while the smaller is the anterior (or su- perior) cervical sympathetic ganglion which is the one sought. This ganglion is then cut out from the surrounding tissue and dropped directly into the fixative, where it should remain for ai)proximately three days. It is difficult to gauge the exact time and it might be well to remove the ganglion from the other side and place it in the same solution. One ganglion may then be removed from solution at the end of two days and the other at the end of four days, with reasonable certainty that one or the other will be in a condition suitable for staining. When the ganglion is removed from the fixing solution it should be rinsed very l:)riefly in distilled water, drained of distilled water on the surface of filter paper, and then transferred directly to the staining solution which is of course kept in a chemically clean stoppered bottle. It remains in the staining solution for from two to three days and is then placed in 90% alcohol. Sections are pre- pared either in celloidin or by the freezing technique and are mounted in exactly the manner described for the method of Golgi given in the last example. Demonstration of the neurons and dendrites of the brain of a rabbit em- bryo by the method of Windle 1926 This is a mixed dichromate-osmic-silver method, of the same general type as Golgi's mixed process (MS 34.21 Golgi 1900) but better and more certain for em- bryonic material. Only three solutions are required. The first of these is a 3.5% solu- tion of reagent-grade potassium dichro- mate in triple-distilled water. The second is Windle's 1926 osmic-dichromate fixa- tive (see, in Chaiiter IS, F 1700.0000 Windle 1926). Tiie third is 0.75% silver nitrate. The brain of a 14-day rabbit em- bryo is used in the following example. At least a liter of the first solution and half a liter of the second will be required. Secure a rabbit on the fourteenth day of gestation and kill it, j^referably by a blow on the head. Lay the rabbit, ventral side uppermost, on a dissecting board, tying its four legs outward so as to leave the abdomen stretched. Remove the skin with extreme care, washing away the milk with a stream of water, and remove as many as possible of the milk glands from the ab- dominal surface so as to leave the mus- cular layer exposed. Remove the greater part of the abdominal wall, disclosing the two uteri, one on each side. These will con- tain a series of globular expansions, each about the size of the egg of a bantam hen. Two ligatures should be tied between each ])air of these globular expansions and the uterus cut between the Ugatures, the resultant pieces being removed separately to a clean dish. It will be found on exam- ination that the uterus is not uniformly swollen but is extended to one side more than the other. Each piece is pinned down with the largest expansion u])permost. With a sharp knife, cut horizontally across the top of the glol)ular expansion. With a little i)ractice it is by this means possible to remove a large area without liberating much blood. Each embryo will then be seen clearly and should be removed with a spoon to a dish of normal saline, where it is rinsed to remove as much as possible of the extravasated blood before being trans- ferred to a clean dish of saline for the re- moval of the membranes. The embryo is now placed in a bottle which contains a large volume of 3.5% potassium dichro- mate. On the bottom of the bottle there should be a layer of fat-free cotton or fine glass fiber. The bottle should be gently agi- tated at intervals to prevent the accumu- lation of exhausted fixative on the bottom. After about two days in potassium dichro- mate, the embryo will be hard enough to handle with safety. It should be removed to a clean dish of potassium dichromate, in which the skin is carefully dissected away from the upper portion of the head, the rudiments of the skull removed, and the entii'e brain passetl, with extreme care and without any washing at all, into the osmic- dichromate solution. Here it should re- main three daj^s more. It is then removed from the osmic-dichromate solution and ])assed to another large volume of 3.5% ])otassium dichromate. The purpose of this is to wash out the osmic acid without MS 34.1-MS 34.21 METAL STAINS 603 removing any of the dichromate. It is safer silver nitrate solution. The brain is then to leave the embiyo at least a week in this removed to a fresh hatch of at least 100 solution and desirable to change tlie di- milliliters of silver nitrate and stored in a chromate at least once during this period, dark cupboard for from seven to ten days. The brain may now be removed, placed on The bottle should be examined at daily a pad of chemically pure filter paper, and intervals and the solution changed at any left there until as much as possible of the time, whenever either a yellow precipitate surface-adherent dichromate has l)een re- is accumulating at the bottom of the moved from it. It is then placed in a clean bottle or a brownisli stain is appearing on crystaUizing dish containing from 25 to 50 the sides. At the end of ten days the brains milliliters of the silver nitrate solution, are removed to a large bottle of 95% al- and rocked back and forth to prevent the cohol after having been cut in half by a precipitate of silver chromate from set- sagittal section. They may remain in al- tling on the surface of the brain. After cohol until required or, as soon as de- about ten minutes of agitation, the now hydration is complete, may be sectioned cloudy silver nitrate should be removed by the celloidin technique. These sections and replaced with a fresh batch of silver are then cleared and mounted under cedar nitrate; and the process repeated until no oil just as though they were Golgi further precipitate washes off into the preparations. 34.1 Staining Solutions In most cases, the two metals are supplied from separate simple solutions recorded under the individual techniques. 34.1 Amprino 1936 4285a, 13:223 preparation: To 20 20% chromic acid add sHghtly more 10% potassium hydroxide than is necessary to change color from red to yellow. Add 25 this solution to 75 10% silver nitrate. Wash ppt., suspend in 100 water, and add just enough ammonia to secure complete solution. 34.1 Berkely 1897 10920, 6:1 preparation: To 100 1% silver nitrate add 10 0.25% phosphomolybdic acid. 34.1 Hill 1896 3464, 9:1 formula: water 100, silver nitrate 0.8 gm., formic acid 0.1 34.1 Juschtschenko test. 1933 Cajal and de Castro Cajal and de Castro 1933, 124 formula: water 100, silver nitrate 2.5, osmic acid 0.5 34.1 Kolossow 1897 test, da Fano 1928 Gatenby and Cowdry 1928, 608 formula: water 100, silver nitrate 2, osmic acid 0.4 34.1 Martinez 1931 te.<^t. 1932 Findlay 11360, 52:152 preparation: To 50 10% silver nitrate add 50 2% sodium tungstate and then just enough ammonia to redissolve the ppt. 34.1 Oliveira 1936 22575,298:523 prepar.\tion: To 5 10% silver nitrate add 10 5% potassium dichromate. Collect ppt. and wash till washings are color-free. Suspend ppt. in 40 water and add just enough ammonia to dissolve ppt. Dilute to 85. 34.1 Renaut test. 1907 Bohm and Oppel cit. Regaud Bohm and Oppel 1907, 387 formula: water 100, osmic acid 0.15, picric acid 0.6, silver nitrate 0.25 34.2 Neurological Methods 34.21 nerve cells and processes 34.21 Andriesen 1894 see MS 34.21 Golgi 1880 (note) 34.21 Berkely 1897 10920, 6:1 REAGENTS REQUIRED: A. F 7000.0000 Muller 1859; B. F 1700.0000 Berkely 1897; C. 0.25% silver nitrate; D. MS 34.1 Berkely 1897 G04 METHODS AND FORMULAS MS 34.21 method: [large pieces] -^ A,2 wks. or until firm -^ [3 mm. slices from A]-^ B,3 days — > drain or blot -^ 25 ml. C, agitate gently -^ repeat successive washings in 25 ml. C, till no further ppt. appears in solution — »• D, 2-3 days at 26°C. -^90% alcohol, rinse — > [frozen, celloidin, or freehand sections] -^ dammar or cedar oil ivilhout cover 34.21 Bolton 1898 see MS 34.21 Gerota 1896 (note) 34.21 Brookover 1910 see MS 34.21 Golgi 1880 (note) 34.21 Bubenaite 1929 23632, 46 :359 REAGENTS REQUIRED: A. 2.5% potassium dichromate; 5. 2% silver nitrate method: [pieces of formaldehyde-fixed material]-^ A, 2 days, 34°C. -^ B, rinse —> B, fresh solution, 1-2 days, 34°C. -^ wash -^ [paraffin sections] RECOMMENDED FOR: ganglion cells. 34.21 Cajal 1890 see MS 34.21 Golgi 1880 (note) 34.21 Cajal 1891 Cajal' s double impregnation — auct. 6011,8:130 REAGENTS REQUIRED: A. F 1700.000 Golgi 1880; B. 0.75% silver nitrate; C. F 1700.0000 Cajal 1891 method: [small blocks, fresh tissue] -^ 100 ml. A, 2-3 days -^ drain -^ 25 ml. B, agitate gently -^ repeat successive washings in 25 ml. B, until no further ppt. appears in solu- tion -^ 100 ml. B, 24 to 48 hrs. -^ rinse -* A or C, 1-2 days -^ drain -^ wash in B as above -^ 100 ml. B, 36-48 hrs. ^^ 90% ale. quick wash -^ sections, either frozen, free- hand or collodion -^ dammar or cedar oil without cover note: Durig 1895 (766, 10:659) substitutes Durig 1895 F 7000.1000 for A and C above. 34.21 Durig 1895 see MS 34.21 Cajal 1891 (note) 34.21 Fish 1895 see MS 34.21 Golgi 1880 (note) 34.21 Gerota 1896 10157, 13:108 reagents required: A. 4% neutralized formaldehyde; B. 4% potassium dichromate; C. 0.5% silver nitrate method : [whole brains] -^ A, very large volumes, 1-2 wks. — * [small pieces from A] —^ B, 3-5 days — > drain, or blot -^ 25 ml. C, agitate gently -^ repeat successive washings to 25 ml. C, until no further ppt. appears in solution -^ 100 ml. C, 10-20 days -^95% alcohol quick wash — » [sections, either freehand, frozen or collodion] — * dammar, or cedar oil, without cover note: Bolton 1898 (11360, 20:244) differs from above in substituting 1% ammonium dichromate for B, and 1 % silver nitrate for C. 34.21 Golgi 1875 test. 1903 ips. Gohji's slow process — auct. Golgi 1903, 1:128 reagents required: A. F 7000.0000 MuUer 1859; B. potassium dichromate; C. 0.75% silver nitrate method: [small blocks, fresh tissue] — > 100 ml. A, 5 days — > add 1 gm. B, leave 5 days — > add 1 Gm. B, leave 5 days — > add 1 Gm. B, leave until sufficiently hardened — ♦ drain or blot — > 25 ml. C, agitate gently -^ repeat successive washings until no further ppt. appears in solution — ♦ 100 ml. C, 24 to 48 hrs. — > 90% ale, changed when discolored, till required -^ [sections, either frozen, freehand, or collodion] -^ dammar, or cedar oil without cover note: a detailed description of this technique is given under MS 34.0 above. 34.21 Golgi 1880 test. 1903 ips. Golgi' s quick process — auct. Golgi 1903, 1:162 reagents required: A. F 1700.0000 Golgi 1880; B. 0.75% silver nitrate method: [small blocks, fresh tissue] -^ 100 ml. A, 2 to 3 days -^ drain -^ 25 ml. B, agi- tate gently — > repeat successive washings in 25 ml. B until no further ppt. appears in solution -^ 100 ml. B, 24 to 48 hrs. or until required -^90% ale. quick wash — + [sec- tions, either frozen, freehand, or collodion] —* dammar or cedar oil without cover note: Cajal 1890 substitutes F 1700.0000 Cajal 1890 for A above. Lachi 1895 (14425, 5:15) substitutes F 7000.1000 Lachi 1895 for 48 hours for A above, dell' Isola 1895 MS 34.21 METAL STAINS 605 (3248, 2) substitutes F 1700.1000 dell' Isola 1895 for A above. Strong 1895 (266, 10:494) substitutes F 7000.1000 Strong 1895 for A above. Fish 1895 (21400a, 17:319) substitutes either F 1700.1000 Fish 1895 or F 7000.1000 Fish 1895 for A above using 3 days in both A and B. Brookover 1910 (11135, 20 :49) precedes A by fixation in 4% neutralized formaldehyde. Vassale and Donaggio 1895 (14425, 6:82) substitute their F 7000.2000 for A above. Andreizen 1894 (3579, 1:909) substitutes his method (F 1700.0000) of fixation for A above. Hill 1896 (34G4, 9:1) substitutes MS 34.1 Hill 1896 for B above. Sehrwald 1889 (23632, 6:456) and Mann 1902 {test, da Fano 1936 in Gatenby and Painter 1937, 509) coat the object with 10% gelatin before trans- ferring to silver, 34.21 Golgi 1900 test. 1903 ips. Golgi's mixed process — auct. Golgi 1903, 2 :685 REAGENTS REQUIRED: A. 2% potassium dichromate; B. F 1700.0000 Golgi 1900; C. 0.75% silver nitrate method: [small blocks of fresh tissue] — > yl, 2 to 30 days -^ 5, 3 to 19 days — » drain or blot -^ 25 ml. C, agitate gently — > repeat successive washings in 25 ml. C until no further ppt. appears in solution —>■ 100 ml. C, 24 to 48 hrs. or until required ^90% ale, quick wash -^ [sections, either frozen, freehand, or collodion] — > dammar, or cedar oil, without cover 34.21 Golgi test. 1933 Cajal and de Castro Cajal and de Castro 1933, 357 REAGENTS REQUIRED: A. F 8000.1000 Lawrcntjcw (1933); B. 2% silver nitrate; C. AMS 21.1 Cajal 1914 method: [pieces of tissue] — > A, 8-10 days — > B, rinse — > B, fresh sol., 5-7 days -^ wash -^ C, 24 hrs. -^ sections RECOMMENDED FOR: nervc endings. 34.21 Hill 1896 see MS 34.21 Golgi 1880 (note) 34.21 deir Isola 1895 see MS 34.21 Golgi 1880 (note) 34.21 Juschtschenko test. 1933 Cajal and de Castro Cajal and de Castro 1933, 124 REAGENTS REQUIRED: A. F 1700.0000 Cajal 1890; B. MS 34.1 Juschtschenko (1933) method: [sympathetic ganglia] — > A, 1-5 days — + quick wash —^ blot dry — * B, 2-3 days -^ [section] — > dammar, or cedar oil, without cover RECOMMENDED FOR: Sympathetic ganglia. 34.21 Kolossow 1897 test, da Fano Gatenby and Cowdry 1928, 608 REAGENTS REQUIRED: A. F 1700.0000 Kolossow 1897; B. MS 34.1 Kolossow 1897 method: [whole ganglia] —> A, 1-7 days -^ rinse and blot -^ B, 2-3 days -^ 90% ale, quick wash —y [sections either frozen, freehand, or collodion] — > dammar or cedar oil without cover recommended for: sympathetic ganglia. note: a detailed description of this technique is given under MS 34.0 above. 34.21 Kopsch 1896 766, 11 :727 REAGENTS REQUIRED: A. F 7000.1000 Kopsch 1896; B. 3.5% potassium dichromate; C. 0.75% silver nitrate method: [small pieces, fresh tissue] — > 100 ml. A, 24 hrs. in dark —>■ 100 ml. B, 48 hrs. —y drain -^ 25 ml. C, agitate gently — > repeat successive washings in 25 ml. C until no further ppt. appears in solution — ♦ 100 ml. C, 2 to 6 days ^95% ale, quick wash — ► [sections, either freehand, frozen, or collodion] -^ dammar, or cedar oil, without cover note: Schreiber 1898 substitutes his F 7000.1000 for A above and his F 7000.1000 for B above. 34.21 Lachi 1895 see MS 34.21 Golgi 1880 (note) 34.21 Lawrentjew test. 1933 Cajal and de Castro Cajal and de Castro 1933, 359 REAGENTS required: A. F 8000.1000 Lawrentjew 1933; B. S% neutralized formalde- hyde; C. 20% silver nitrate; D. MS 33.1 Lawrentjew 1933; E. 30% ammonia; F. 0.3% gold chloride; G. 5% sodium thiosulfate 606 METHODS AND FORMULAS MS 34.21-MS 34.22 method: [fresh pieces] ^ ^, 1 hr. — > B, 4-6 days — * wash -^ [frozen sections] -^ C, 1-5 mins. -^ B, 1-2 mins. -^ D, till nerve endings clearly shown —* E, I min. —^ wash —^ F, till gray — >• G, 5 mins. -^ balsam, via usual reagents RECOMMENDED FOB: peripheral nerve endings. 34.21 Mann 1902 see MS 34.21 Golgi 1880 (note) 34.21 Sala 1891 23632, 9 :389 REAGENTS required: .4. F 1700.0000 Golgi 1880; B. 0.75% silver nitrate method: [entire ganglion] -^^ .1, 3 days — > rinse — * B, 2-3 days — * rinse — > A, 4 days — > wash — > sections by freezing technique — » dammar or cedar oil without cover 34.21 Sanchez test. 1933 Cajal and Castro Cajal and Castro 1933, 128 REAGENTS REQUIRED: A. F 7000.1000 Sdnchez 1933; B. 0.5% silver nitrate; C. 0.75% silver nitrate method: [invertebrates] — » A, changed daily, 3-5 days — > B, with agitation, 1-2 mins. -^ C, 2-3 days—* A, with agitation, 1-2 mins. — > A, changed daily, 1-3 days ^ B, with agitation, 1-2 mins. — > C, 1-3 days -^ [celloidin sections] recommended for: nerve fibers in invertebrates. 34.21 Schreiber 1898 see MS 34.21 Kopsch 1896 (note) 34.21 Sehrwald 1889 see MS 34.21 Golgi 1880 (note) 34.21 Smith 1930 Turtox News, 8:91 RE.AGENTS REQUIRED: A. 4% potassium dichromate; B. 0.75% silver nitrate method: [2 mm. slices of formaldehyde material]-^ rinse—* A, 3-5 days — > wash — > blot — * B, 15 mins. with constant agitation —^ B, fresh solution, 24 hrs. — > section recommended for: general neurology. 34.21 Smirnow 1895 1780, 52:201 REAGENTS REQUIRED: A. F 7000.1000 Smirnow 1895; B. 3.5% potassium dichromate; C. F 1700.1000 Smirnow 1895; D. 0.5% silver nitrate; E. 1.0% silver nitrate method: [fresh whole cerebellum] -^ A, 1-8 wks. — > [split halves from A] —* B, 2-5 wks. -^ [1 cm. slices from B] — > C, 7 to 10 days — > drain or blot -^ 100 ml. D, 24 hrs. -^ E, 36-48 hrs. -^ [frozen, celloidin, or freehand sections] -^ dammar, or cedar oil, without cover 34.21 Strong 1895 see MS 34.21 Golgi 1880 (note) 34.21 Timofecheff test. 1933 Cajal and de Castro Cajal and de Castro 1933, 125 REAGENTS REQUIRED: A. F 1700.0000 Tiiiiofecheff 1933; B. water 100, silver nitrate 1, formic acid 0.01, sodium sulfate 0.001 method: [pieces of fresh tissue of 1 cm. side] -^ A, 6-7 days -^ quick wash blot — > B, 2-3 days -^ [section] recommended for: nerve endings in male sex organs. 34.21 Vassale and Donaggio 1895 see MS 34.21 Golgi 1880 (note) 34.21 Windle 1926 11135,40:229 REAGENTS REQUIRED: A. 3.5% potassium dichromate; B. F 1700.0000 Windle 1926; C. 0.75% silver nitrate method: [fetal brains] —> A, 48 hrs. — * B, 72 hrs. -^ .4. 5-6 days — > drain or blot — > 25 ml. C, agitate gently —^ repeat successive washings in 25 ml. C, till no more ppt. produced in solution — ► 100 ml. C, 7-10 days in dark — » [half brains from C]-* 95% alcohol, till dehydrated —>■ [sections by celloidin technique] -> dammar, or cedar oil without cover note: a detailed description of this technique is given under MS 34.0 above. 34.22 NEUROGLIA 34.22 Cajal test. 1933 ips. Cajal and de Castro 1933, 282 REAGENTS REQUIRED: A. 6% formaldehyde; B. F 7000.1000 Cajal 1933; C. 1% silver nitrate; D. 1% silver nitrate in 1% chloral hydrate MS 34.22-MS 34.32 METAL STAINS G07 method: [small pieces of fresh tissue]—* A, 2-5 days — > B, 3-5 days — > C, with rapid agitation -^ C, fresh sol. — > repeat till clouds of ppt. cease — > Z), 2 days -^90% ale. wash —^ [paraffin sections] -> dammar or cedar oil ivithoul cover RECOMMENDED FOR: oligodendria. 34.22 Martinez 1931 3232, 31 :653 REAGENTS REQUIRED: A. AMS 11.1 Cajal 1913; B. 0.5% ammonia; C. MS 34.1 Martinez 1931; D. 4% formaldehyde; E. 0.2% gold chloride; F. 5% sodium thiosulfate method: [fresh pieces] —> .1, 1 hr., 55°C. -^ wash ^ [frozen sections] —> B, 5 mins. -^ rinse —>■ C, 15 mins. -^ wash -^ D, till pale yellow -^ wash — + E, 5 mins. — > E (fresh solution), 15 mins., 50°C. — > F, till purple -^ wash — > balsam, via usual reagents 34.22 del Rio-Hortega 1928 list. 1933 Cajal and de Castro Cajal and de Castro 1933, 281 reagents required: A. F 7000. lOOO del Rio-Hortega 1928; B. 1.5% silver nitrate method: [fresh blocks of tissue] — > .4, renewed daily, 2-3 days —^ rinse —* B, 2ot 3 days — > 90% ale, wash —> [paraffin sections] — » dammar or cedar oil without cover recommended for: oligodendria. 34.3 Histological Methods 34.31 reticulum fibers 34.31 Amprino 1936 4285a, 13 :223 reagents required: .1. 2% tannin in 95% ale; B. MS 34.1 Amprino 1936 30, water 100; C. 15% formaldehyde method: [sections of formaldehyde-fixed material] —♦ water —+ A, 30-40 mins., 55- 58°C. -^ water — > B, few mins. — > B (fresh solution), few mins. — > B (fresh solution), till light yellow — > rinse -^ C, till reduced — > balsam, via usual reagents 34,31 Oliveira 1936 22575, 298 :523 reagents required: A. 10% phosphomolybdic acid; B. 1% uranium nitrate; C. MS 33.1 Foot 1924; D. AMS 21.1 Oliveira 1936a; E. MS 34.1 Oliveira 1936; F. AMS 21.1 Oliveira 1936b; G. 0.2% gold chloride; H. 5% sodium thiosulfate method: [paraffin sections] -^ A, I min. — » rinse —fB,5 sees. — + quick rinse -^ C, 1 min, -^95% ale, rinse — > D, 1 min. -^ wash -^ E, 15-20 mins. 56°C. — > wash -^ F, \ min. -^ wash—* G, 5-10 mins. 56°C. -^ H, 6-10 mins. —> thorough wash — > balsam, via usual reagents 34.31 Oppel 1890 766, 4:144 reagents required: A. 5% potassium chromate; B. 0.75% silver nitrate method: [small pieces ale. fixed material] — > water — » A, 24 hrs. —>■ wash -^ B, 24 hrs. — > wash — > [frozen tangential sections] recommended for: lattice fibrils in liver. 34.32 other histological methods 34.32 Kupffer 1889 20188, 5:219 REAGENTS REQUIRED: A. F 1700.0000 Cajal 1890; B. 0.75% silver nitrate method: [small pieces of liver] -^ A, 24 hrs. —> wash —>■ B, 48 hrs. — > [sections] 34.32 Martinotti 1888 test. 1889 Behrens, Kossel, and Schiefferdecker Behrens, Kossel, and Schieffer- decker 1889, 211 REAGENTS REQUIRED: A. 2% arseuic trioxide; B. F 7000.0000 Mtiller 1859; C. water 12, silver nitrate 8, glycerol 80; D. 0.75 sodium chloride method: [small pieces] -^ A, till decalcified -^ wash — » B, 15 mins. -^ rinse —>■ C, 24-48 hrs. — > rinse — > sections — » 95% ale. — + D, few minutes -^ balsam, via usual reagents recommended for: elastic fibers, 34.32 Regaud and Dubreuil 1903 trst. 1907 Bohm and Oppel Bohm and Oppel 1907, 175 reagents required: A. 1% protein silver 50, 1% osmic acid 50; B. any DS 11.2 stain method: [fix flat sheet of epithelium in A]—> wash — > B, till nuclei are stained recommended for: delineation of cell outlines in epithelium. ^^^ METHODS AND FORMULAS MS 34.32-MS 35.1 34.32 Renaut test. 1907 Bohm and Oppel cit. Regaud Bohm and Oppel 1907, 387 REAGENTS REQUIRED: A. MS 34.1 Renaut (1907) method: " a " is copiously injected into the testes, which are then hardened in alcohol. Frozen sections are then mounted in balsam and exposed to light. RECOMMENDED FOR: demonstration of lymph vessels in testes. 34.32 Schultze 1907 1780, 69:544 REAGENTS REQUIRED: A. Water 100, sUver nitrate 2, osmic acid 0.1; B. 1% hydroquinone method: [pieces of tissue] -> A, 30 mins. -^ rinse -» B, 24 hrs. -^ wash -* balsam, via usual reagents recommended for: demonstrating outlines of epithelial cells. 34.4 Cytological Methods 34.4 Aoyama 1930 23632, 46:490 REAGENTS REQUIRED: A. F 8000.1000 Aoyama 1930; B. 1.5% silver nitrate; C. AMS 21.1 Aoyama 1930 method: [small pieces] ^ A, 3-4 hrs. -^ rinse -^ B, 10-15 hrs., 25°C. -^ rinse -> C, 5-10 hrs. -^ [paraffin sections] RECOMMENDED FOR: Golgi network. 34.4 Bubenaite 1937 test. 1937 Gatenby and Painter Gatenby and Painter 1937, 313 REAGENTS REQUIRED: A. 4% formaldehyde; B. 25% potassium dichromate; C. 2% silver nitrate method: a, 1-2 days -» rinse -> 5, 2 days 35°C. -^ rinse -> C, wash, renewing solution when necessary, till no further ppt. occurs in solution -^ C, fresh solution, 1 to 2 days 35°C. -^ [sections] RECOMMENDED FOR: Golgi apparatus. 34.4 Golgi-Verratti test. Cajal and de Castro Cajal and de Castro 1933, 200 REAGENTS REQUIRED: A. F 1270.0000 Veratti 1890; B. water 100, potassium dichromate 2, copper sulfate 1; C. 1% silver nitrate method: [fragments of fresh tissue]^ A, 3-4 wks. ^ wash^ B, overnight-^ C, 4-8 hrs. — > [sections] RECOMMENDED FOR: Golgi apparatus. 34.4 Veratti see MS 34.4 Golgi- Veratti 1933 34.5 Bacteriological IVIethods 34.5 Fontana 1923 9170, 64:234 REAGENTS REQUIRED: A. F 0000.1010 Rugc 1942; B. 0.75% neoarsphenamine or AMS 13.1 Iron 1924; C. Fontana 1912 MS 33.1 method: [fresh smears] -^ A,2 mins. -» wash -^ B, dropped on slide and warmed, 20-30 sees. -^ wash -^ C, dropped on slide and warmed, 20-30 sees. -^• repeat until suffi- ciently darkened -^ wash -^ dry — > balsam recommended for: spirochetes in smears. 34.5 Safford and Fleisher 1931 20540b, 6:43 REAGENTS REQUIRED: A. AMS 13.1 Safford and Fleisher 1931; B. MS 33.1 Fontana 1912 method: [moist smears] -^ A, heated to steaming, 2 mins. -^ wash -^ dry -^ B, heated to steaming, 1-2 mins. -^ wash -^ dry recommended for: bacterial flagella. 34.5 Williams test. 1924 Mallory and Wright Mallory and Wright 1924, 279 note: This method involves a proprietary developer of undisclosed formula and cannot be further noticed. 35 OTHER SILVER METHODS 35.1 Staining Solutions 35.1 David 1934 23684,132:240 preparation: Dissolve 1 silver nitrate in 4 water. Add to 1.2 sodium sulfate in 2 hot water. Wash ppt. by decantation and suspend in 100 water. MS 35.1-MS 41.2 METAL STAINS 609 35.1 Lugaro 1932 tesl. 1933 Cajal and de Castro Cajal and de Castro 1933, 260 Stock I. 9% sodium thiosulfate; Stock II. 2% silver bromide in 9% sodium thiosulfate; Stock III. 0.3% silver iodide in 9% sodium thiosulfate II III Working solutions A 4 16 1 B 5 15 2 C 6 14 4 D 7 13 5 E 8 12 6 F 9 11 7 G 10 10 8 H 11 9 12 35.2 Neurological Methods 35.2 Lugaro 1932 test. 1933 Cajal and de Castro Cajal and de Castro 1933, 260 REAGENTS REQUIRED: .4. MS 35.1 Lugaro 1932; B. 8% formaldehyde method: [pieces fixed 3-8 days in 7% formaldehyde on ice] — ♦ 10 ml. A in flask in dark, 3 hrs. -^ 7 ml. B added to flask, 5-8 days -^ wash -^ frozen sections -^ balsam, via usual reagents recommended for: neuroglia. note: Any one of the eight working solutions given under 33.1 Lugaro 1932, or an as- sortment of them, may be selected. 35.3 Other Methods 35.3 David 1934 23684, 132 :240 REAGENTS REQUIRED: A. ADS 12.2 David 1934; B. MS 35.1 David 1934 method: [heat-fixed smear] -^ A, IQ mins. -^ wash — » heat dry -^ B, heated to steam- ing, till brown — > wash — > dry recommended for: bacterial flagella. MS 40 Other Metals 41.1 Staining Solutions 41.1 del Rio-Hortega 1927 test. 1933 Cajal and de Castro Cajal and de Castro 1933, 279 formula: water 94.5, hydrochloric acid 5.5, potassium ferrocyanide 2.25 41.2 Neurological Methods 41.2 Azoulay 1894 4956, 49 :924 REAGENTS REQUIRED: A. 0.5% ammonium vanadate; B. 2.5% tannin method: [thin celloidin sections of dichromate fixed material] — > A, poured over section on slide, 2-30 sees. — » rinse -> B, poured over section on slide, 20-30 sees. — > [repeat A — > rinse — ♦ B, till staining sufficient] -^ balsam, via usual reagents recommended for: cerebellum. 41.2 Cox test. 1933 Cajal and de Castro Cajal and de Castro 1933, 130 REAGENTS REQUIRED: A. F 3700.0000 Cox 1895; B. 5% sodium bisulfite method: [large pieces] — » A, 2-3 months -^ 90% ale, till mercuric chloride extracted — > [celloidin sections] -^ B, till dark brown -^ M 23.1 Cox 1891 . recommended for: general neurology. 41.2 Flater 1895 1780,45:158 REAGENTS REQUIRED: A. 4% potassium dichromate; B. 0.1% mercuric chloride method: [whole brains]-^ A, 2-3 months-^ 0.5 mm. slices-^ B, changed when dis- colored, 9-12 months — » wash — > [celloidin sections] -^ balsam, via carbol-xylene recommended for: brains of small vertebrates. 610 METHODS AND FORMULAS MS 41.2-MS 41.3 41.2 Golgi 1878 Golgi 1903, 1:143 REAGENTS REQUIRED: A. 1% potassium dichromate; B. 1.5% potassium dichromate ; C. 2% potassium dichromate; D. 1% mercuric chloride method: [large pieces] -^ ^, 1 month —> B, 1 month — * C, 1 month or till required —* 1-2 cm. cube blocks^ D, changed daily, till block is decolorized-^ wash—* [cel- loidin sections] -^ wash -> AMS 24.1 Golgi (1937) -^ M 23.1 mountant recommended for: axis cylinder and dendrites in brain. 41.2 Krohntal 1899 23632, 16 :235 REAGENTS REQUIRED: A. F 8000.1000 Krohntal 1899; B. water 90, 40% formaldehyde 10, hydrogen disulfide to sat. method: [fresh tissue]—* A, 4-5 days — > B, with agitation, few mins. — * B, changed daily, 5 days —* [celloidin sections] recommended for: general neurology. 41.2 Pollaillon test. circ. 1938 Wellings Wellings circ. 1938, 132 reagents required: A. 10% ferric chloride; B. 30% tannic acid method: [sections to water]—* A, 24 hrs. -^ wash — > B, till dark enough ^^ wash — > M 11.1 mountant recommended for: nerve fibers in teeth. 41.2 vom Rath 1895 see F 1250.0010 vom Rath 1895 41.2 del Rio-Hortega 1927 test. 1933 Cajal and de Castro Cajal and de Castro 1933, 278 reagents required: A. AMS 11.1 Cajal 1913; B. 0.1% ammonia; C. 10% hydrochloric acid; D. MS 41.1 del Rio-Hortega 1927; E. 1% sodium carbonate; F. 0.1% hydro- chloric acid method: [blocks of fresh tissue]-^ A, some days -^ [20-25 m frozen sections]—* B, thorough wash -^ thorough wash — * C, 1 min. — > D, 50°-60°C., with gentle rocking, until solution begins to cloud -* E, until sections transparent — * F, few sees. — > [re- peat D —* E —^ F cycle until stain satisfactory]^ [counterstain in any DS 11.21 formula if desired] -* balsam via usual reagents recommended for: pathological microglia. 41.2 van der Stricht 1895 see F 1200.0000 van der Stricht 1895 41.2 Welters 1891 see DS 21.212 Wolters 1891 41.3 Histological Methods 41.3 Foster 1934 20540b, 9:91 reagents required: A. water 1, tannic acid 1, sodium salicylate 1; B. 3% ferric chloride method: [sections] -* water —> ^ , 10 mins. — > wash — * iS, several m ins. —» wash —> counterstain, if required — > balsam, via usual reagents recommended for: cell walls of apical meristem. 41.3 Hogan test. 1883 Cole Cole 1883, 5 reagents required: A. 10% ferric chloride in 90% ale; B. 2% pyrogallol in 90% ale. method: [sections]^ 90% ale. -^ A, 2 mins. -^ 90% ale, rinse —> B, till sufficiently stained — » 90% ale, wash -^ water —<■ glycerol -^ M 10 mountant recommended for: general histology. 41.3 Hogan le.'■ ale, rinse -^ B,2 mins. -* ale wash — * balsam, via usual reagents recommended for: cartilage. 41.3 Kenney 1928 1887a, 5:283 reagents reqi ired: A. water 90, 40% formaldehyde 10, sodium sulfantimonate 1 method: [pieces] -^ A, 24 hrs. —>■ wash -^ [sections] recommended for: reticulum fibers. MS 41.3-MS 41.4 METAL STAINS 611 41.3 Leber 1860 1789a, 14:3 REAGENTS REQiiREu: A. \% ferric chloride; B. 5% potassium ferrocyanide method: [small fragments] — > A, 5 mins. — > rinse -> B, 2-3 mins. RECOMMENUEU FORI delineation of cell outlines. 41.4 Other Methods 41.4 Fol 1883 23635, 38:491 REAGENTS REQUIRED: .1. 1% ferric chloride; B. 1% gallic acid in 95% ale; C. 0.1% hydrochloric acid in 70% ale. method: [living Tintiunopsidae] -^ A, till dead -^ wash, 70% ale. — > B, 1 day —y C, till differentiated — > balsam, via usual reagents recommended for: marine Tintiunopsidae. 41.4 Sander 1935 23454, 116:335 REAGENTS REQUIRED: A. Water 99, sulfuric acid 1, potassium permanganate to sat. method: [heat- fixed smear] -^ A, 2 mins. -> wash -* dry RECOMMENDED FOR: spores in bacteria. 24 Accessory Metal Staining Solutions Decimal Divisions Used in Chapter AMS 10 SOLUTIONS USED BEFORE STAINING ("ACCELERATORS" AND "MORDANTS") 100 General observations 11 Formaldehyde mixtures 11.1 Formulas 12 Alcohol mixtures 12.1 Formulas 13 Other mixtures 13.1 Formulas AMS 20 SOLUTIONS USED AFTER STAINING 200 General observations 21 Developers 21.1 Formulas 22 Toning solutions 22.1 Formulas 23 Differentiating solutions 23.1 Formulas 24 Fixing solutions 24.1 Formulas AMS 10 Solutions Used Before Staining AMS 10.0 GENERAL OBSERVATIONS As Chapter 18 has been devoted to fixative formulas, it appears necessary to justify the retention in this place of formulas used to fix materials before metal staining tech- niques. Formulas are retained in this section only when they are not considered by the author to be adapted to any other purpose. Fixatives developed for use prior to silver staining which would appear to have applications outside this specific field are given in Chapter 18. It is impossible, moreover, to distinguish in many of the formulas given in this chapter among those intended to fix the tissues, those designed to serve as mor- dants, and those supjiosed to exercise some physical effect so as to render more apparent some structure in the material after it has been metal-stained. Solutions of uranium, cobalt, and the like, might be considered chemical mordants which would render ma- terials subsequently stained more clearly apparent: the reverse is the case. These ma- terials are used mostly as inhibitors which, by preventing the absorption of various metal-stains upon the nervous elements of nervous structures, permit the demonstration of their supi)orting or connective tissues. The exact role jilayed by pyridine, so fre- quently an ingredient of this group of solutions, is again doubtful; it has been suggested that it may cause no more tlian the differential shrinking of certain structures, particu- larly neurofibrillae, with the result that they become mechanically differentiated. It is recognized that the division into aqueous and alcoholic solutions is untenable on scientific grounds, but it is retained on grounds of convenience. 612 ACCESSORY METAL STAINING SOLUTIONS 013 AMS 11 Formaldehyde Mixtures AMS 11.1 FORMULAS 11.1 Bertrand and Guillain 1934 6G30, 115:706 formula: water 86, 40% formaldehyde 14, ammonium bromide 2, pyridine 0.3 11.1 Cajal 1904 test. 1933 Cajal and de Castro Cajal and de Castro 1933, 189 formula: water 100, 40% formaldehyde 25, ammonia 1 11.1 Cajal 1908 test. 1933 ips. Cajal and de Castro 1933, 201 formula: 40% formaldehyde 50, acetone 50, ammonia 0.2 11.1 Cajal 1913 21344, 11:255 formul.\: water 85, neutralized 40% formaldehyde 15, ammonium bromide 2 11.1 Cajal 1914 21344, 12:127 formula: water 64, 40% neutral formaldehyde 12, 95% ale, uranium nitrate 0.8 11.1 Cajal 1920 test. 1933 ips. Cajal and de Castro 1933, 254 formula: water 100, 40% formaldehyde 12, ammonium bromide 6 11.1 Cajall929 21344,26:1 formula: water 100, 40% formaldehyde 15, chloral hydrate 5 11.1 Cajal test. 1933 ips. Cajal and de Castro 1933, 290 formula: water 37.5, 40% formaldehyde 37.5, pyridine 25 11.1 Cajal test. 1933a ips. Cajal and de Castro 1933, 319 formula: water 100, 40% formaldehyde 15, uranium nitrate 1 11.1 Cajal test. 1933b ips. Cajal and de Castro 1933. 343 formula: water 65, pyridine 25, 40% formaldehyde 12 11.1 da Fano 1920a 11360, 40:157 formula: water 100, neutralized 40% formaldehyde 15, cobalt nitrate 1 11.1 da Fano 1920b 11454, 53:1919 formula: water 40, 40% formaldehyde 10, pyridine 50 11.1 Foot 1927 1887a, 4:42 formula: water 100, tannic acid, 0.15, ammonium bromide 3.5, 40% formaldehyde 5 11.1 Herrara 1932 test. 1933 Cajal and de Castro Cajal and de Castro 1933, 276 formula: water 75, 40% formaldehyde 15, pyridine 2.5, acetone 2.5, ammonium oxalate 3 11.1 Kingsbury and Johannsen 1927 Kingsbury and Johannsen 1927, 83 formula: water 15, 40% formaldehyde 100, uranium nitrate 0.15 11.1 Klatzo 1952 Lah. Invest., 1:346 formula: water 90, 40% formaldehyde 10, potassium dichromate 5, chloral hydrate 3 11.1 Lobo 1937 test. 1948 Romeis Romeis 1948, 420 formula: water 83, 40% formaldehyde 20, ammonium bromide 2.7 11.1 Merland 1935 4285a, 12 :290 formula: water 100, 40% neutralized formaldehyde 10, cobalt nitrate 2 11.1 Noguchi 1913 test. Schmorl 1928 Schmorl 1928, 407 formula: water 75, 40% formaldehyde 15, acetone 5, pyridine 5, ammonium bromide 3 11.1 Raileanu 1939 6630, 104:285 formula: water 90, 40% neutralized formaldehyde 10, anunonium bromide 6 614 METHODS AND FORMULAS AMS 11.1-AMS 12.1 11.1 del Rio-Hortega 1925 see AMS 11.1 Strong 1903 11.1 del Rio-Hortega test. 1933a Cajal and de Castro Cajal and de Castro 1933, 267 formula: water 90, 40% formaldehyde 10, ferric alum 7 11.1 del Rio-Hortega test. 1933b Cajal and de Castro Cajal and de Castro 1933, 269 formula: water 90, 40% formaldehyde 10, ammonium bromide 2, ferric alum 7 11.1 Rodriguez test. 1933 Cajal and de Castro Cajal and de Castro 1933, 285 formula: water 75, 40% formaldehyde 15, acetone 2.5, pyridine 2.5, potassium oxalate 3 11.1 Rojas 1917 21344, 15:30 formula: water 80, 40% formaldehyde 12, pyridine 20, manganese nitrate 1 11.1 Strong 1903 11135,13:296 formula: water 90, 40% formaldehyde 10, ferric alum 6 note: The formula of del Rio-Hortega 1925 (3231, 25:34) does not differ significantly. AMS 12 Alcohol Mixtures 12.1 FORMULAS 12.1 Balbuena 1922 21344, 20:31 preparation: Macerate cork crumbs in 70% ale. till a dark yellow-brown solution is obtained 12.1 Boule 1908 15063, 10:15 formula: 95% ale. 80, 40% formaldehyde 20, acetic acid 0.4, ammonia 0.04 12.1 Cajal 1910a 21344, 8:1 formula: abs. ale. 90, pyridine 10 12.1 Cajal 1910d 21344, 8:1 formula: 95% ale., 100, chloral hydrate 2 12.1 Cajal 1910e 21344, 8:1 formula: 95% ale. 100, nicotine 1 12.1 Cajal 1910f 21344, 8:10 formula: 95% ale. 80, glycerol 16, ammonia 0.8 12.1 Cajal 1910g 21344, 8:11 formula: 95% ale. 100, ethylamine 1 12.1 Cajal 1925 test. 1933 ips. Cajal and de Castro 1933, 363 formula: water 50, 95% ale. 50, ammonia 1 12.1 Cajal 1927 test. 1933 Cajal and de Castro Cajal and de Castro 1933, 188 formula: 95% ale. 100, pyridine 40, chloral hydrate 4 12.1 Cajal 1929 21344, 26:1 formula: 70% ale. 100, ammonia 0.2 12.1 Foley 1939 763, 73:465 formula: 95% ale. 50, water 50, pyridine 15, ammonia 1, potassium dichromate 1.5 12.1 Humphreys 1939 608b, 15:151 formula: 95% ale. 90, 40% formaldehyde 5, acetic acid 5 12.1 Krajian 1933 623, 17:127 formula: 95% ale. 40, acetone 40, glycerol 20, formic acid 12, uranium nitrate 4 12.1 Steiner 1937 11284,23:315 formula: abs. ale. 100, uranium nitrate 0.8, gum mastic 3 12.1 Steiner 1939 11284, 25:204 formula: abs. ale. 100, gum mastic 12, uranium nitrate 0.8 AMS 12.1-AMS 13.1 ACCESSORY METAL STAINING SOLUTIONS 615 12.1 Steiner 1950 591b Tech. Bull, 20:489 formula: abs. ale. 100, uranium nitrate 1, mastic 1 12.1 Wallart 1935 428oa, 12:254 formula: abs. ale. 30, pyridine 30, ether 30, ammonia 0.03 12.1 Weber 1944 SWlG—auct. 4285a, 21 :4o formula: isopropanol 45, dioxane 45, water 10, 40% formaldehyde 20, acetic acid 2, formic acid 2, chloral hj-drate 5, cobalt nitrate 0.3 note: The acids are added immediately before use. 13 Other Mixtures 13.1 formulas 13.1 Bolsi 1927 test. 1933 Cajal and de Castro Cajal and de Castro 1933, 274 formula: water 80, glycerol 20, ammonia 0.3 13.1 Bolsi test. 1933 Cajal and de Castro Cajal and de Castro 1933, 261 formula : water 60, acetone 20, pyridine 20, ammonia 4 13.1 Cajal 1907 test. 1933 ips. Cajal and de Castro 1933, 290 formula: 33% acrolein 10, water 90 13.1 Cajal and de Castro 1933 Cajal and de Castro 1933. 261 formula: hydrogen peroxide (3%) 100, oxalic acid to sat. 13.1 David 1934 see ADS 12.1 David 1934 13.1 Fontana 1912 7176,55:1003 formula: water 100, tannic acid 5, phenol 1 13.1 King 1937 1879, 38:362 formula: water 30, ammonia 30, pyridine 30 13.1 Loffler 1890 see DS 23.215 Loffler 1890 (sol. A) 13.1 del Rio-Hortega 1918 21344, 15:165 formula: water 100, ammonium bromide 1, tannic acid 3 13.1 del Rio-Hortega 1932 test. 1933 Cajal and de Castro Cajal and de Castro 1933, 263 formula: water 30, ammonia 30, pyridine 30 13.1 Saflford and Fleisher 1931 20540b, 6:43 formula: water 100, picric acid 0.25, tannic acid 5, ferrous sulfate 7.5 13.1 Tron 1924 2190, 13 formula: water 100, neoarsphenamine 0.7, acetic acid 0.4 13.1 Willard 1935 17510,78:475 formula: water 40, 95% ale. 40, pyridine 20, chloral hydrate 2.5 13.1 Zettnow 1891 23684, 11:689 formula: water 115, tannic acid 5, antimony potassium tartrate 0.75 preparation: Dissolve the acid in 100 water at 60°C.; add the tartrate dissolved in 15 water. AMS 20 Solutions Used after Staining 20.0 GENERAL OBSERVATIONS It is extraordinary how far the science of microtomy as it is seen in these formulas has lagged behind the photographic methods w-hich it is supposed to resemble. (Whether this resemblance is justified has already lieen (juestioned.) Photographic developers are designed to reduce to metallic silver, particles of silver bromide which have been ren- dered unstable through the absorption of photon energy. Neither silver bromide nor 616 METHODS AND FORMULAS AMS 21.1 light enters into the majority of metal staining reactions. The best that can be said of these organic reducing agents, therefore, is that they will under certain empirically estabhshed conditions reduce either to the metalUc form, or in most cases to metallic oxides or proteinates, some unstable metallic complex which has formed on the surface of the cell which it is desired to demonstrate. None of the more recent photographic developing techniques appears to have been tried, and the majority of the formulas given below appear entirely barbaric to a practical photographer. No formula has been included in this section unless it is specifically recommended by the original author of one of the metal stains given above. Most of the more successful metal stains are re- duced with the aid of formaldehyde, and it remains yet to be demonstrated clearly that any of these pseudophotographic developers are indeed an improvement over this simple reagent. 21 Developing Solutions 21.1 foemulas 21.1 Armuzzi and Stempel test. 1928 Schmorl Schmorl 1928, 406 formula: water 100, gum arable 12, hydroquinone 0.25 21.1 Ascoli 1911 3381,25:177 formula: water 90, sodium sulfite, cryst. 10, amidol 0.5 21.1 Balbuena 1922 21344,20:31 formula: a. digest 30 oil of amber with 70 of 70% alcohol 1 week, separate; B. 1% hydroquinone note: The original calls for Tinctura succini for solution A. This tincture is not in the Spanish Pharmacopaeia, the formula given above being from the Portuguese {test. Squire 1899 Companion to the Pharmacopaeia, p. 612). Langeron 1942, 627 uses teinture alcoolique du succin {du Codex) which is prepared {test. Squire, loc. cit.) by macerating 1 part powdered amber in 10 parts 80% alcohol. This would have a very much lower oil content. 21.1 Bauer 1944 608b, 29:297 formula: water 100, gallic acid 1.43, tannic acid 0.86, sodium acetate 2.86 21.1 Boccardi 1886 test. Lee 1905 Lee 1905, 252 formula: water 80, formic acid 20, oxalic acid 0,3 21.1 Bodian 1936 763,65:89 formula: water 100, sodium sulfite 5, hydroquinone 1 21.1 Boule 1908 15063, 10:15 formula: water 100, 40% formaldehyde 6, 95% ale. 15, hydroquinone 1 21.1 Cajal 1910a 21344, 8:3 formula: water 250, 40% formaldehyde 15, pyrogallol 2.5 21.1 Cajal 1910b 21344, 8:3 formula: water 250, 40% formaldehyde 15, hydroquinone 2.5 note: Cajal {loc. cit.) sometimes substituted 5 ml. pyridine, and sometimes 1.25 Gms. sodium sulfite for the formaldehyde. 21.1 Cajal 1914 21344, 12:127 formula : water 100, hydroquinone 2, sodium sulfite, anhydr. 0.75, 40% formaldehyde 4 note: Cajal and de Castro 1933, 202, refer this formula, without reference, to Golgi 1908. 21.1 Cajal 1921 21344, 19:71 formula: water 70, 40% formaldehyde 30, hydroquinone 0.3 21.1 Cajal 1925 21344,23:237 formula: water 70, 40% formaldehyde 20, hydroquinone 0.3, acetone 15 AMS2I.1 ACCESSORY METAL STAINING SOLUTIONS G17 21.1 Cajal 1929 21344,26:1 formula: water G5, acetone 15, 40% formaldehyde 20, hydroquinoue 0.3 21.1 Cajal lest. 1933 ips. Cajal and de Castro 1933, 320 formula: water 100, 40% formaldehyde 7.5, sodium sulfite, anhydrous 0.25, hydro- quinoue 1.5 21.1 Chor 1933 1879, 29 :344 formula: water 95, 40% formaldehyde 5, pyrogallol 4 21.1 Cowdry 1912 10157,29:1 formula: water 100, pyrogallol 1, 40% formaldehyde 5 21.1 Davenport 1930 1879,24:690 formula: 95% ale. 100, 40% neutralized formaldehyde 2, pyrogallol 2, 50% dextrin 0.4 21.1 Davenport, McArthur, and Bruesch 1939 21540b, 14:23 formula: stock I. water 90, sodium sulfite 10; stock II. water 95, sodium bisulfite 5, diamminophenol hydrochloride 1 WORKING solution: stock I 100, stock II 20 21.1 Davenport, Windle, and Rhines 1947 Conn and Darrow 1947, I-C2 :24 formula: water 100, sodium sulfite dessic. 5, hydroquinone 1, potassium metaborate 0.5 21.1 Dieterle 1927 1879, 18:73 formula: water 60, acetone 12, hydroquinone 1.8, sodium sulfite 0.3, pyridine 12, 40% neutral formaldehyde 12, water, q.s. to bring volume to 25; 10% sol. gum mastic in abs. ale. add 10 just before use 21.1 van Ermengen test. 1942 Langeron Langeron 1942, 831 formula: water 100, gallic acid 2, tannic acid 1, sodium acetate 4 21.1 van Ermengen 1894 23684, 15:969 formula: water 100, gallic acid 1.5, tannic acid 0.8, sodium acetate 2.9 21.1 Eyene and Sternberg test. 1916 Warthin 4349, 6:71 formula: water 100, gelatin 3, gum arable 15, silver nitrate 0.8, hydroquinone 0.75 preparation: Dissolve each ingredient in part of the water. Mix in order given. 21.1 Farrier and Warthin 1930 623, 14 :394 STOCK solutions: I. 5% gelatin, II. 2% silver nitrate, III. 5% hydroquinone working solution: melt 75 stock I at 45°C., add 15 stock II and then 5 stock III 21.1 Faulkner and Lillie 1945 20540b, 20:81 formula: water 96, acetate buffer (pH 3.6) 4, silver nitrate 0.3, hydroquinone 0.15, gelatin 4 preparation: Dissolve ingredients separately in buffered water. Mix immediately before use. 21.1 Foley 1943 20540b, 18 :27 formula: water 85, acetone 15, sodium sulfite, anhydrous 2, boric acid 1.4, hydro- quinone 0.3 21.1 Golgi 1908 see AMS 21.1 Cajal 1914 (note) 21.1 Gomori 1933 see MS 33.1 Gomori 1933, sol. B 21.1 Gooding and Stewart 1937 11977, 7:596 formula: water 95, 40% formaldehyde 5, pyrogallol 2 21.1 Gurdjian 1927 see AMS 21.1 Ranson 1914 (note) 21.1 Holmes 1942 11431, 64:132 formula: water 100, sodium sulfite, anhydr. 5, sodium bisulfite 2.5, p-diammino benzene hydrochloride 0.5 618 METHODS AND FORMULAS AMS 21.1 21.1 Heitzman test. 1938 Mallory Mallory 1938, 293 formula: 5% gelatin 75, 2% silver nitrate 15, 1% hydroquinonc 2.5 21.1 Hewer 1933 see AMS 21.1 Ranson 1914 (note) 21.1 Huber and Guild 1913 see AMS 21.1 Ranson 1914 (note) 21.1 Humphreys 1939 608b, 15:151 formula: water 100, hydroquinone 5, sodium sulfite 10 21.1 Jahnel 1917 see AMS 21.1 Hanson 1914 (note) 21.1 Jahnel test. 1933 Cajal and de Castro Cajal and de Castro 1933, 384 formula: water 100, acetone 10, pyridine 10, pyrogallol 4 21.1 Kallius 1893 764, 2:271 formula: water 66, 95% ale. 34, hydroquinone 0.08, sodium sulfite 0.8, potassium carbonate 1.6 note: Curreri 1908 (766, 32:432) recommends gold toning after this. 21.1 Kingsbury and Johannsen 1927 Kingsbury and Johannsen 1927, 83 formula: water 100, 40% formaldehyde 6, hydroquinone 2, magnesium sulfate "a minute quantity" 21.1 Kolossow 1892 23632, 9:38 preparation: Mix 20 25% tannin with 20 25% pyrogallol. Filter. Add to filtrate 35 water, 15 95% ale. and 10 glycerol. 21.1 Krajian 1933 623, 17:127 formula: water 60, acetone 10, pyridine 10, 40% formaldehyde 10, hydroquinone 1.2, sodium sulfite 0.25, sat. sol. gum mastic in 95% alcohol 10, V 21.1 Mayer 1884 0.05 note: Krajian 1935 (1829, 32:764) is identical. Krajian 1938 (1829, 38:427) omits the Mayer's albumen. 21.1 Kranz 1924 14674, 608 formula: water 100, gum arable 12.5, pyrogallol, 1 21.1 Krautz 1924 test. 1928 Schmorl Schmorl 1928, 405 formula: water 100, gum arable 8, pyrogallol 0.2 21.1 Lauda and Rezek 1928 22575, 269:218 formula: dissolve with heat 0.1 gelatin in 100 water. Cool. Add 3 hydroquinone 21.1 Levaditi 1905 6630, 8 :845 formula: water 100, 40% formaldehyde 5, pyrogallol 3 21.1 Levaditi and Manouelian 1906 6630, 60:134 formula: water 90, acetone 10, pyrogallol 3.6 21.1 Levaditi test. 1916 Warthin 4349, 6 :56 formula: water 77, acetone 8, pyridine 15, pyrogallol 3 21.1 Liesegang 1911 11848, 3:1 preparation: To 50 of a 50% solution of gum arable add 50 20% potassium hydro- sulfide. note: The polassium hydrosulfide referred to is most easily prepared by passing H2S into 20% KOH to saturation. The solution is very unstable. 21.1 Lobo 1937 test. 1948 Romeis Romeis 1948, 420 formula: water 70, 40% formaldehyde 19, acetone 11, hydroquinone 0.3 21.1 MacFarland and Davenport 1941 20540b, 16 :53 formula: water 100, oxaHc acid 2, 40% formaldehyde 1 21.1 Noguchi 1913 test. Schmorl 1928 Schmorl 1928, 407 formula: water 95, 40% formaldehyde 5, pyrogallol 4 AMS 21.1 ACCESSORY METAL STAINING SOLUTIONS 619 21.1 Okada 1929 8542a, 7 :403 formula: water 100, 40% formaldehyde 5, pyrogallol 2 21.1 Oliveira 1936a 22575, 298:523 formula: water 100, 40% neutralized formaldehyde 3, 0.01 uranium nitrate 21.1 Oliveira 1936b 22575,298:523 formula: water 70, 40% formaldehj^de 30, hydroquinone 0.3 21.1 Paton 1907 14246, 18:576 formula: water 80, hydroquinone 0.8, 40% formaldehyde 8 21.1 Podhradszky 1934 23632, 50:285 formula: water 95, 40% formaldehyde 5, pyrogallol 5 21.1 Pritchard hst. Bohm and Oppel 1907 Bohm and Oppel 1907, 438 formula: water 98, formic acid 1, amyl ale. 1 21.1 Ranson 1914 766,46:522 formula: water 95, 40% formaldehyde 5, pyrogallol 4 note: The formulas of Gurdjian 1927 (11135, 43:1), Hewer 1933 (11025, 67:350) Huber and Guild 1913 (703, 7:253) and Jahnel 1917 (14370, 42) are essentially the same. 21.1 Rachmanov test. 1946 Roskin Roskin 1946, 257 STOCK solutions: I. water 100, borax 0.8, sodium sulfite 4, II. water 100, p-aminophenol sulfate 0.4, hydroquinone 1, sodium sulfate (crystal) 0.8 WORKING solution: stock I 50, stock II 50. note: It might be presumed that the "sulfate" in II was a misprint for "sulfite," but the Russian words are quite distinct. Roskin does not cite the original source. 21.1 Rojas 1917 21344, 15:30 formula: water 80, 40% formaldehyde 5, sodium sulfite, anhydrous 0.25, hydro- quinone 1 note: Romeis 1948, 456 gives the third ingredient as "sulphal." 21.1 Romanes 1916 11025, 80:205 formula: water 100, sodium sulfite, crystal 10, pyrogallol 1, hydroquinone 1 21.1 Schultze test. 1948 Romeis Romeis 1948, 418 formula: water 100, hydroquinone 2.5, 40% formaldehyde 5 21.1 Schultze and Stohr test. 1933 Cajal Cajal 1933, 363 stock solution: water 100, hydroquinone 2.5, 40% formaldehyde 5 WORKING solution: stock 1, water 19 21.1 Shanklin 1951 Cowdry 1952, 270 formula: water 90, 40% formaldehyde 10, pyridine 1.3 21.1 Stage 1936 20540b, 11:155 formula: water 70, neutralized formaldehyde 30, hydroquinone 0.3 21.1 Steiner 1937 11284,23:315 formula: water 100, hydroquinone 5, 6% gum mastic in abs. ale. 0.2 21.1 Steiner 1939 11284, 25:204 formula: water 100, hydroquinone 5, 12.5% gum mastic in abs. ale. 0.5 21.1 Steiner and Steiner 1944 11284,29:868 stock solutions: I. 0.6% hydroquinone, II. 2.5% gum mastic in abs. ale. III. Dissolve, with boiling, 0.2 silver nitrate and 0.165 sodium potassium tartrate in 100 water. Cool, filter. WORKING solution: stock I 60, stock II 20, stock III 20 21.1 Ungewitter 1943 20540b, 18:183 formula: water 100, p-mothylaminoplicnol svilfate 0.2, sodium sulfite (dessic.) 10, hydroquinone 0.5, sodium borate 0.1 620 METHODS AND FORMULAS AMS 21.1-AMS 22,1 21.1 Uyama 1926 8542a, 4:389 formula: water 100, 40% formaldehyde 12.5, pyrogallol 1.75 21.1 Warthin -Starry 1929 test. 1942 Langeron Langeron 1942, 630 STOCK solutions: I. 10% gelatin; II. 10% starch; III. 2.5% hydroquinone in 40% acetone; IV. 2% silver nitrate preparation: Prepare 50 ml. each I and II, dissolving the first with warm and the second with boiling. Mix. Take 6 ml. Ill and add to mixture. working solution: immediately before use mix 5 parts of the above mixture with 1 part stock IV 21.1 Weber 1944 4285a, 21 :45 formula: water 100, 40% formaldehyde 5, hydroquinone 2, sodium citrate 1 21.1 Wilder 1936 608b, 11:817 formula: water 100, 40% neutralized formaldehyde 0.5, uranium nitrate 0.015 21.1 Willard 1935 17510,87:475 formula: water 90, 40% neutralized formaldehyde 10, hydroquinone 1 21.1 Yamanoto 1909 23681,20:153 formula: water 100, pyrogallol 2, tannic acid 1 22 Toning or Metal Exchange Solutions Toning depends for its value on tlie fact that a solution of gold will replace metallic silver, or many silver compounds, in the solid state. The principle use is either to render more ap- parent weakly stained materials, or to improve the keeping qualities of the preparations on the ground that gold is less subject to natural deterioration than is silver. The simple solu- tions of gold chloride usually employed are listed with the staining techniques in Chapter 22. 22.1 formulas 22.1 Balbuena 1922 21344,20:31 formula: water 100, sodium borate 1, gold chloride 0.1 22.1 Bodian 1936 763, 65:89 formula: water 100, gold chloride 1, acetic acid 0.1 22.1 Cajal 1910 21344, 8:1 formula: water 100, ammonium thiocyanate 3, sodium thiosulfate 3, 1% gold chloride 0.2 22.1 Cajal 1921 21344, 19:71 formula: water 100, ferric alum 4, oxalic acid 1 22.1 Cajal test. 1948 Lillie Lillie 1948, 101 formula: water 100, ammonium thiocyanate 3, sodium thiosulfate 3, gold chloride 0.01 22.1 Cajal and de Castro 1933 see AMS 24.1 Cajal and de Castro 1933 22.1 Foot 1927 1887a, 4:43 formula: water 100, gold chloride 0.2, mercuric chloride 0.5 22.1 Golgi 1908 tod. 1933 Cajal and de Castro Cajal and de Castro 1933, 202 STOCK solutions: I. water 100, sodivim thiosulfate 3, ammonium thiocyanate 3; II. 1% gold chloride WORKING solution: stock I. 100; stock II. 1.5 22.1 Lancelin, Seguy, and Dubreuil 1926 6630, 94:557 formula: 1% sodium thiosulfate 30, 1% ammonium thiocyanate 30, 1% gold chloride 30 22.1 Sand 1910 6593,12:128 formula: water 85, 2% solution ammonium thiocyanate 15, 2% gold chloride 3 AMS 22.1- AMS 24.1 ACCESSORY METAL STAINING SOLUTIONS 621 22.1 Simard and Campenhout 763, 63:143 STOCK solutions: I. 6% ammonium thiocyanate; II. 6% sodium thiosulfate; III. 2% gold chloride WORKING solution: stock I. 50; stock II. 50; stock III. just enough to produce ppt. 23 Differentiating Solutions 23.1 formulas 23.1 Golgi 1908 test. 1933 Cajal and de Castro Cajal and de Castro 1933, 203 formula: water 100, sulfuric acid 0.1, potassium permanganate 0.05 24 Fixing Solutions Solutions of sodium thiosulfate are employed to "fix" p]iotoj;rapliic images because both silver chloride and silver bromide are readily dissolved by them, whereas metallic silver is soluble only with difficulty. Wliile it is doubtful how far silver bromide enters into any metal staining reaction, it is inevitable that when tissues are exposed to solu- tions of silver salts a certain amount of silver chloride is formed. As this substance is unstable in the presence of light, it seems reasonable that it should be removed either b}' simple thiosulfate or by one of the solutions given below. Care must be exercised, however, not to remove the silver from fine structures by prolonged exposure to sodium thiosulfate. Most of the formulas given are combined toning and fixing solutions. 24.1 FORMULAS 24.1 Bolsi 1927 test. 1933 Cajal and de Castro Cajal and de Castro 1933, 274 formula: water 95, 40% formaldehyde 5, gum arable 0.05 24.1 Cajal 1913 21344, 11:255 formula: water 70, sodium thiosulfate 5, 95% ale. 30, sodium metabisulfite 10 note: The original formula calls for "liquid sodium bisulfite." The quantity of dry salt above presumes that Cajal was employing the common technical solution of 38° Be which contains approximately 50% by weight of the dry salt. To restore the original formula 20 Gms. of solution should be substituted. 24.1 Cajal 1925 see MS 31.1 Cajal 1925b 24.1 Cajal and de Castro 1933 Cajal and de Castro 1933, 130 formula: water 100, sodium thiosulfate 25, ammonium thiocyanate 2, potassium alum 1, lead acetate 1, gold chloride 0.2 24.1 Golgi test. 1937 Gatenby and Painter Gatenby and Painter 1937, 513 STOCK solution: water 100, sodium thiosulfate 15.5, potassium alum 2, ammonium thiocyanate 1, sodium chloride 4. Let stand 1 week. Filter. WORKING solution: "used working solution" 40, stock 50, 1% gold chloride 7 24.1 Raileanu 1930 6630,104:285 formula: water 62, 95% ale. 38, sodium thiosulfate 1.2 25 Solvents and Oils Decimal Divisions Used in Chapter S 00 GENERAL OBSERVATIONS S 10 DEHYDRATING AGENTS (e.g. solvents miscible with water, but not with wax, embedding media, or resinous mounting media) S 11 Table of physical properties of dehydrating agents S 20 "CLEARING" AGENTS (e.g. solvents not miscible with water, but miscible with either wax, embedding media, or resinous mounting media) S 21 Table of physical properties of essential oils S 22 Table of physical properties of synthetic reagents S 30 "UNIVERSAL" SOLVENTS (e.g. solvents miscible both with water and with either wax, embedding media, or resinous mounting media) S 31 Table of physical properties of "universal" solvents S 40 RECOMMENDED MIXTURES S 41.1 Formulas S 00 General Observations Solvents and oils are used in micro- scopical technique principally for the pur- pose of preparing objects for mounting whole in resinous media (Chapter 6) or for section cutting after embedding in one of the ways discussed in Chapters 12 through 15. At one time these reagents and mixtures of them could be clearly divided into two groups: those intended for the removal of water (dehydrating agents) and those intended for the removal of alcohol (clearing agents). These two groups are recorded under S 10 and S 20 below. For the past decade, however, a number of reagents have been recom- mended which may be used either for dehydration or clearing. These, which are given under S 30 below, are referred to as universal solvents for the reason that they are miscible with water and with resinous mounting media and with wax embedding media. S 10 Dehydrating Agents The function of a dehydrating agent as the name indicates is to extract water from the tissues in order that some other solvent not miscible with water may be substituted. Ethanol is the solvent most commonly used in making microscope slides and is referred to throughout the present work either as abs. ale. or 95% ale., as the case may be. Where ale. or alcohol is referred to without qualification the ordinary 95% alcohol (190 proof al- cohol) of commerce is intended. Most good dehydrating agents are naturally hygroscopic and should there- fore always be stored over some dehydrat- ing agent which will remove from them the water that they absorb from the air. Weight for weight there is no dehydrating agent as efficient as anhydrous sodium sulfate, but the solubility of this in some 622 S 11-S 21 SOLVENTS AND OILS 623 dehydrating reagents renders it dangerous to use. The only all-round dehydrating agent which can be recommended is calcium sulfate, commercially available in suitable form as Drierite. It is not usual to include glycerol as a dehydrating agent even though it will, in point of fact, readily extract water from tissues. The advantage of glycerol is that it permits the transfer of material from water to absolute alcohol without the danger of collapse. Objects may be placed in dilute gljTerol, the glycerol concen- trated by evaporation, and the most delicate material then transferred to ab- solute alcohol. This reverses the direction of osmotic pressure so that the pressure tends to keep the object expanded. S 11 PHYSICAL PROPERTIES OF DEHYDRATING AGENTS Evaporation I Suitability Solvent Solvent Boiling rate for use with action on action on Other point (ethanol = stained Canada nitro- Name names °C 100) material balsam cellulose Acetone , , 56 340 + + + + + + + + Ally! ale. , , 97.1 ? + + ? 0 Diethylene glycol . . 245 <1 ? + + + + + Dipropylene glycol , . 231.8 <1 + + + + + + Ethanol 95% ale. abs. ale. 78.3 100 + + + + + + * Ethylene glycol methyl monomethyl ether cellosolve 124.5 14 + + + + + + Glycerol , . , , + 0 0 Isopropanol Isopropyl ale, pro- panol 2 82.3 88 + + + ? 0 Methanol methyl ale, wood ale 64.5 207 + + + + + + + + Propylene glycol , , 188.2 <1 + + + 0 Triethylene glycol . . 287 <1 + + + + + * Alcohol is not a solvent for nitro -cellulose bu t is an excellent cosolvent wi ith ether. A 50-50 mixture usually employed. S 20 Clearing Agents The phrase clearing agent, as an alter- native to the more correct de-alcoholiza- tion agent, is used because the high refrac- tive index usually renders the object more or less transparent. Clearing agents are required either before resinous mounting media or before wax embedding media. It is recommended in general that essen- tial oils be used for preparing whole- mounts and that the sjmthetic clearing agents be used before embedding. S 21 ESSENTIAL OILS Essential oils are the natural oils col- lected from the leaves, stems, flowers, and fruits of plants. The table below presents the important characteristics of those commonly employed in microscopical technique. The column showing the strength of alcohol from which specimens may be transferred to the oil in question supposes the oil to be anhj'drous. Most essential oils, as they occur in commerce, are water-saturated and this water must be removed with some dehydrating agent before they are used. It must be remem- bered that "miscibihty with Canada bal- sam," shown in the seventh column, does not of necessity mean that these oils are miscible with any other resin used for mounting; and, particularly, when syn- thetic mounting media are employed, pre- liminary tests should always be made. The column showing the "solubility of nitro- cellulose in" each oil should be consulted before clearing nitrocellulose-sections, to make sure that the support will not be dis- solved while the specimen is cleared. 624 METHODS AND FORMULAS S 21-S 22 21 PHYSICAL PROPERTIES ESSENTIAL OILS Lowest- strength alcohol from which Suitabil- objects ity for Miscibil- Miscibil- Solubility Refrac- may be use with ity with ity with of nitro- Other tive trans- stained molten Canada cellulose Name names index ferred materials paraffin balsam in Oil of bay Oil of myrcia 1.51 80% ? ? CO 0 Oil of bergamot 1.46 90% + + 00 00 + Oil of cajeput 1.47 80% + + + CO 00 0 Oil of caraway 1.50 90% + CO 00 Oil of cedar wood 1.50 95% + + + 00 00 0 Oil of cheno- Oil of ' podium American wormseed 1.47 80% + CO 00 0 Oil of cii\namon Oil of cassia 1.6 95% + CO 00 0 Oil of citronella . . 1.47 90% + + CO 00 0 Oil of clove 1.53 75% + + + OO 00 ++ Oil of Cretan Oil of Cre- origanum tan thyme ? 90% + + 00 00 0 Oil of eucalyp- tus 1.46 80% + + 00 00 0 Oil of juniper 1.48 100% + + + 00 0 Oil of lavender 1.46 80% + + 00 00 0 Oil of lemon 1.47 95% + CO 00 0 Oil of lilac 1.48 90% + + + ? + + + 0 Oil of marjo- Oil of" ' ram origanum (in error) ? 90% + + M 00 0 Oil of origanum Oil of wild marjoram ? 90% + + 00 00 0 Oil of thyme Oil of ■ origanum (in error) 1.50 90% + + + 00 00 0 Oil of tur- Turpentine, pentine Gum turpentine 1.47 95% 0 CO 00 0 Oil of white Oil of cedar thuja ? 75% + -f- 00 00 0 Oil of winter- Methyl green salicylate 1.54 100% + + + 00 00 0 S 22 SYNTHETIC CLEARING AGENTS These materials are more usually em- ployed before embedding either in wax or in nitrocellulose, and data is, therefore, not available as to the low^est strength of alcohol from \\liich the transfer can be made, since it is customary to dehA-drate completely before either of these proc- esses. A column has been included, how- ever, which shows the solubility of water in these reagents (not the solubility of these reagents in water) which may serve as an indication of their sensitivity to im- S22 SOLVENTS AND OILS G25 perfect dehydration. If they are to be used solvents with Canada l^alsam is not of to clear objects before making whole- necessity the same as their miscibdity mounts in resinous media, it must be with any other resin used for the remembered that the miscibihty of these mountmg. 22 PHYSICAL PROPERTIES OF SYNTHETIC CLEARING AGENTS Suit- Evapo- ability Misci- Misci- Sohibil- Re- ration Solubil- for use bility bility ity of frac- Boil- rate ity of with with with nitro- Othor tive lag (ethanol water stained molten Canada celhilose Name names index point = lUO) in materials parafHn balsam in ^"^cetate 141 ? 2% 0 ++ « « Aniline aniline oil 1.59 184 ? 1^% 0 '^ ++ !? Benzene benzol 1.50 80 185 0.06% + + + - + + + 0 ^""chloride .. 1.40 78.6 .. 0-08% ? + + + - 0 Carbon „ , , n disulfide .. 1.63 46.3 ? ? + + oo co 0 Carbon chloride .. 1.46 76.7 ? >0.01% + + + cc co 0 Chloro- „ , , , n form .. 1.45 61.5 ? ? + + + ^ ^ 0 Creosote, Beechwood wood creosote ? 200+ ? .. + oo « 0 Dichlor- ethyl ether Ethyl acetate Ethyl benzo- ate Ethylene dichlo- , , - ride .. ? 85.6 ? 0.15% + + Ethylene cellosolve glycol acetate mono- ethyl ether acetate Ethyl ether, ether sulfuric ether 1.30 34.6 970 1.3 + + + 2 -Hep- methyl tanol amyl ^ carbinol 1.42 162 ? 5.2% + « « 0 Hexanol amyl „ carbinol 1.47 157.2 ? 7.2% + co =o 0 ? 178.5 ? 0.28% ++ + + + + + + * 1.44 77.1 180 3.3% .. 0 + + + + + + 1.51 212 ? >0.01% + + + « CO 0 156.4 6 6.5 ? ? + + + + + + 00 Isopropyl (. --*- ? 88.4 150 1.8% 0 « 00 0 1.52 199 ? >0.01% + + + " CO 0 benzo- niobe ate 626 METHODS AND FORMULAS S 22-S 31 22 PHYSICAL PROPERTIES OF SYNTHETIC CLEARING AGENTS— {Continued) Suit- Evapo- ability Misci- Misci- Solubil- Re- ration Solubil- for use bility bility ity of frac- Boil- rate ity of with with with nitro- Other tive ing (cthanol water stained molten Canada cellulose Name names index point = 100) in materials paraffin balsam in Methyl oil of salicyl- winter- ate green 1.54 222 >0.01% + + + 00 00 0 Pheynl Salol salicyl- ate 173 , , >0.1% + + 00 00 0 Pinene IA7 155 >1% + 00 00 0 Terpineol . . 1.48 219.8 >0.1 5% + + + ? + + + 0 Toluene toluol 1.50 110.6 70 0.05% + + + 00 00 0 Xylene xylol 1.50 138 19 >0.01% + + 00 00 0 * These materials are not ethanol. A 50-50 mixture of in themselves solvents of nitrocellulose but are excellent cosolvents when mixed with the solvent with ethanol is usually employed. S 30 Universal Solvents These materials are coming more and more into favor as a means of avoiding the use of two solutions, either before mount- ing in resinous media or embedding in wax. Though they are undoubtedly suit- able for routine procedures, the writer prefers not to employ them for deUcate objects, particularly those containing cavities which may become distorted through heavy diffusion currents and osmotic differences. The danger in the use of these materials hes both in the original transfer from water into them and in the transfer from them to paraffin. Unless time is the essence of the technique em- ployed, it is recommended that dehydra- tion be conducted with mixtures of these solvents with water and that the transfer to wax be through graded mixtures of solvent with wax. 31 PHYSICAL PROPERTIES OF UNIVERSAL SOLVENTS Name Acetic acid Butanol Cresol Diacetone alcohol Diethylene dioxide Diethylene glycol mono- butyl ether Other names n-butyl ale, pro- pyl carbi- nol diace- tone dioxane butyl carbitol Re- frac- tive index BoU- ing point 1.37 118 Evapo- ration rate (ethanol = 100) ? Solubil- ity of water in Suit- ability Misci- • for use bility with with stained molten materials paraffin 0 0 Misci- bility with Canada balsam + + + Solubil- ity of nitro- cellu- lose in 1.34 117.7 180- 200 20% 50% + + 0 00 00 00 00 * 0 1.30 169 1.42 101.5 + + + + + 230.4 <0.1 + + + + S 31-S 40 SOLVENTS AND OILS 627 31 PHYSICAL PROPERTIES OF UNIVERSAL SOLVENTS— (Continued) Name Diethylene glycol mono- ethyl ether Diethylene glycol mono- ethyl ether acetate Dimeth- oxytetra ethylene glycol Diethylene glycol mono- methyl ether Ethylene glycol mono- butyl ether Ethylene glycol mono- ethyl ether Ethylene glycol mono- methyl ether acetate Phenol Other names carbitol carbitol acetate methyl carbitol butyl cello- solve cello- solve methyl cello- solve acetate Evapo- Re- Boil- ration frac- ing rate tive point (ethanol index °C. = 100) carbolic acid Triethylene meth- glycol oxytri- methyl glycol ether acetate acetate Triethyl phosphate Solubil- ity of water ? 201.9 <0.l 194.2 <0.1 ? 171.2 0.2 135.1 1.54 144.5 182 11 244 >0.1 21.5 >0.1 Suit- ability for use with stained Misci- bUity with molten Alisci- Solubil- bility ity of with nitro- Canada cellu- m materials paraffin balsam lose in + + 217.7 <0.1 « 276 + + 00 + + 00 + + 00 +++ 00 00 0 00 00 00 + + + =1 + + + + + + 00 00 00 + + + +++ + + +]. + + + + + 0 + + + + + 0 * These materials are not in themselves solvents of nitrocellulose but are excellent cosolvents when mixed with ethanol. A 50-50 mixture of the solvent with ethanol is usually employed. S 40 Recommended Mixtures The majority of the mixtures given below were designed to produce materials having properties not available in solvents which could have been secured in the days when the ^28 METHODS AND FORMULAS S 41.1 mixtures were developed. Thus many of them consist of mixtures of phenol with some clearing agent notoriously sensitive to water. Phenol is an excellent coujiler and permits xylene to be used with, say, 70% alcohol. These mixtures are included in the present place only because they are widely recommended in the literature. The modern worker would be well advised to seek in the tables given above some pure solvent having characteristics of the mixtures given below, and to substitute this pure solvent for the mixture which he had intended to use. 41.1 Formulas 41.1 Amann 1899a 23632,16:38 formula: chloral hydrate 50, p-chlorphenol 50 41.1 Amann 1899b 23632,16:38 formula: chloral hydrate 60, phenol 30 41.1 Apathy test. Guyer 1930 cit. Kornhauser Guyer 1930 65 formula: chloroform 30, origanum oil 30, cedar oil 30, abs. ale. 7.5 phenol 7 5 recommended for: clearing celloidin blocks prior to double embedding. 41.1 Apathy test. 1942 Langeron Langeron 1942, 438 formula: chloroform 32, oil of thyme 16, oil of cedar 32, alcohol 8, phenol 8 41.1 Cole 1903 Cross and Cole 1903, 193 formula: phenol 100, glycerol 3 RECOMMENDED FOR: clearing arthropod material before mounting in glycerol. 41.1 Cole 1947 20540b, 22 :103 formula: xylene 60, toluene 12, beechwood 12, aniline 12 41.1 Dunham test. 1937 Gatenby and Cowdry Gatenby and Cowdrv 1937 108 formula: white oil of thyme 75, clove oil 25 ' RECOMMENDED FOR: clearing celloidin sections. 41.1 Eycleshymer test. 1915 Chamberlain Chamberlain 1915 36 formula: cedar oil 30, bergamot 30, phenol 30 41.1 Gage 1890 21400a, 12:120 formula: turpentine 60, phenol 40, 95% ale. q. s. to complete solution 41.1 Gage 1896a Gage 1896, 176 formula: xylene 75, castor oil 25 RECOMMENDED FOR: clearing and hardening nitrocellulose blocks. 41.1 Gage 1896b Gage 1896, 176 formula: turpentine 70, phenol 30 41.1 Gatenby and Painter 1937 Gatenby and Cowdry 1937, 108 formula: creosote 40, bergamot oil 30, origanum oil 10, xylene 20 RECOMMENDED FOR: clearing celloidin sections. 41.1 Gothard test. 1929 Anderson Anderson 1929 128 formula: creosote 50, cajuput 40, xylene 50, abs. ale. 160 41.1 Maxwell 1938 20540b, 13:93 formula: oil of cedar 30, oil of thyme 40, xylene 15, abs. ale. 15 41.1 Minot test. 1928 Schmorl Schmorl 1928 162 formula: oil of thyme 80, oil of cloves 16 41.1 del Rio-Hortega test. 1938 Mallcry Mallory 1938 249 formula: xylene 80, phenol 10, creosote 10 ' 41.1 Weigert 1891 7276, 17:1184 formula: xylene 35, aniline 65 S41.1 SOLVENTS AND OILS 629 41.1 Weieert test. 1938 Mallory carbol-xylol — compl. script ^ Mallory 1938, 98 formula: xylene 75, phenol 25 41 1 Zirkle 1930 19938, 71:103 REAGENTS required: A. 5% ale; i?. 11% ale; C. 18% ale; D. 30% ale; E. 45% ale 90, n-butanol 10; F. 62% ale 80, n-butanol 20; G. 77% ale 65, n-butanol 35; H. 90% ale 45, n-butanol 55; /. abs. ale 25, n-butanol 75; J. n-butanol method: [water] -^ ^, 1-5 hrs. -^ B, 1-5 hrs. -^ C, 1-5 hrs. -^ D, 1-5 hrs - E, 1-5 hrs -^ F 12 hrs. -^ G, 1 hr. -> H, 1 hr. -^ /, 1 hr. -> J, several changes if necessary, tiU dehydration complete -^ [paraffin] ^ note: This is the well-known " Zirkle's butyl alcohol schedule for plant tissues. It is equally applicable to animal tissues. 26 Mounting Media Decimal Divisions Used in Chapter M 00 GENERALITIES M 10 MOUNTANTS MISCIBLE WITH WATER 11 Gum arable media 11.1 Formulas 12 Gelatin media 12.1 Formulas 13 Other media, including mixtures of 11 and 12 13.1 Formulas M 20 MOUNTANTS MISCIBLE WITH ALCOHOL 21 Gum mastic media 21.1 Formulas 22 Venice turpentine media 22.1 Formulas 23 Gum sandarac media 23.1 Formulas 24 Other media, including mixtures of 21, 22, 23 24.1 Formulas M 30 MOUNTANTS NOT MISCIBLE WITH EITHER ALCOHOL OR WATER 31 Canada balsam media 31.1 Formulas 32 Gum damar media 32.1 Formulas 33 Other natural resin media, including mixtures of 31 and 32 33.1 Formulas 34 Synthetic resins and plastics 34.1 Formulas M 00 Generalities A mountant is a material in which an object may be permanently preserved for microscopical examination and which has inherent in it the property of holding the coversUp in place. There is a tendency to confuse niountants with preservative media, wliich are dealt with in Chapter 17. A preservative is a material in which an object may'^be permanently preserved but which does not have in it the inherent property of holding the coverslip in place, and which must, therefore, be sealed with cement or by some other method. Mountants may be divided into three groups according to the treatment which the object must receive before mounting. The first group {M 10 below) contains those mountants which are miscible with water, and to which, therefore, the object may be directly transferred either from water or from glycerol. The second class (M 20 below) comprises those mountants which are miscible with alcohol and to which objects maj' therefore be trans- ferred after dehydration but without having passed through a clearing agent. The third class of mountants (M 30 below) are the conventional resins and 630 M 11,1 MOUNTING MEDIA ^'31 balsams to which objects can be trans- drated and cleared in the usual ferred only after they have been dehy- manner. M 10 Mountants Miscible with Water This section may be divided into three ration of Crustacea These media have the groups. M 11 contains conventional gum disadvantage tliat they must be melted arable media (of which Farrants' is the l)efore use and are therefore, not nearly tvoe) to which objects may be trans- as simple to use as the gum arable media, er'red eUher directly or from water. These The third group, M 13, which will prob- media should be far more widely used ably become more numerous as time goes Than is commonly the case, for a great on, employs water-tluckemng agents other deal of time is wasted in dehydrating and than gum arable or gelatin. Any one o intransferringtobalsamobjectswhichwere these three groups may be "^^^^d with better mounted in gum arable. The second stains for special purposes and the best group (M 12) contains glycerol jellies known media of this type are those of which are widely used by botanists and to Zirkle, some of which will be found in each a lesser extent by zoologists for the prepa- of the three sections. 11 GUM ARABIC MEDIA 11.1 Formulas 11 1 Allen test. 1937 Gatenby and Cowdry Gatenby and Cowdry 1937, 221 ' FORMULA- water 45, glycerol 11, 40% formaldehyde 4.5, gum arable 45 pheparation: Dissolve gum arable in water. Mix formaldehyde m the glycerin and add slowly, with constant stirring, to gum. 11.1 Andre test. 1942 Langeron ^^^TT 9nn^' ^^^ formula: water 50, glycerol 20, gum arabic 30, chloral hydrate 200 preparation: Dissolve gum arabic in water. Mix glycerol in gum and add chloral hydrate. 11.1 Apathy 1892 23632,89:1065 formula: water 30, gum arabic 30, levulose 30 preparation- Dissolve gum arabic in water. Add levulose to solution. NOTE - Much grief in the prevention of bubbles may be avoided by reducing the water to 20 and using commercial levulose syrup in place of the dry sugar. ■ "•' rr.: iriJ.T^ic acid 3, dextrose syrup 5, .urarawifcMora, hydrate 75 pheparation: Dissolve the acid in the water with the syrup and gum arabic. Add chloral hvdrate to the solution. NOTE- Swan 1936 (4184, 27:389) states that Berlese first disclosed the formula to Davidson in 1919, who communicated it to Lee by whom it was published m 1921. Doetschman 1944 (21400a, 63:175) uses three times as much water in his formula lor "15erlese." . • 11 1 Brun 1889 see P 12.3 Brun 1889 11.1 Chevalier 1882 Chevalier 1882, 319 formula: water 60, gum arabic 20, glycerol 20 11 1 Davies circ 1865 Davies, 82 formula: water 30, gum arabic 30, glycerol 30, arsenic trioxide 0.1 ''•' F^RM^LATatef 35, glycerol 20, dextrose syrup 20, gum Irabiclo! 'chloral hydrate 20, sat. aq. sol. magenta 0.3 11.1 Doetschman 1944 see also M 11.1 Berlese (note) 11 1 Ewie test. 1944 Doetschman 21400a, 63 :175 , , , . on formula: water 35, glycerol 12, dextrose syrup 3, gum arabic 20, chloral hydrate 30 032 METHODS AND FORMULAS M 11.1 11.1 Fabre-Domergue 1889 see P 12.3 Fabre-Domergue 1889 11.1 Farrants IcM. 1880 Beale Beale 1880, 68 FORMULA : water -10, glycerol 20, gum arabic 40 note: This mixture requires a preservative. Arsenic, camphor, and phenol have all been recommended. If only dirty gum arabic is available the mixture may be filtered through glass wool. The author's name is almost always misspelled "Farrant." 11.1 Faure 1910 979,8:25 FORMUL.A.: water 50, chloral hydrate 50, gum arabic 30, glycerol 20 11.1 Gater 1929 4184, 19:367 formula: water 10, acetic acid 2.7, gum arabic 8, chloral hydrate 74, glucose syrup 5, cocaine hydrochloride 0.3 11.1 Gerlach 1885 see P 12.3 Gerlach 1885 11.1 Highman 1946 1789a, 41 :559 formula: water 50, gum arabic 25, sucrose 25, potassium acetate 25 11.1 Hogg 1883 Hogg 1883, 237 formula: water 75, gum arabic 25, phenol 5 preparation: Dissolve gum arabic in 25 water. Dissolve phenol in 50 water and mix with gum. 11.1 Hoyer 1882 2981,2:23 formula: water 50, gum arabic 50, chloral hydrate 2 preparation: Use water and chloral hydrate to dissolve gum. 11.1 Landau 1940 4285a, 17:65 formula: water 30, gum arabic 30, dextrose 30, glucose 5 11.1 Langerhaus 1879 23833,2:575 formula: water 20, glycerol 25, gum arabic 60, phenol 1 preparation: Dissolve gum in 20 water and filter. Add glycerol and phenol to filtrate. 11.1 Lieb 1947 Abopon mountant — auct. 591b, 17:413 This involves a proprietary product of secret composition and cannot, therefore, be further noticed. 11.1 Lillie and Ashburn 1943 1789a, 36:432 formula: water 100, gum arabic 50, sucrose 50, thymol 0.1 11.1 Marshall 1937 11977, 7:565 formula: water 50, gum arabic 0.5, gum tragacanth 1.5, P 12.2 Archibald and Marshall 1931 50 preparation: Dissolve gum arabic in water with gum tragacanth with boiling. Cool. Mix P 12.2 Archibald and Marshall 1931 with gums. Filter. 11.1 Martin 1872 Martin 1872, 169 formt'la: water 50, gum arabic 50, glycerol 25, camphor 0.2 11.1 Morrison 1942 Turtox News, 20:157 formula: water 50, glycerol 20, acetic acid 3, gum arabic 40, chloral hydrate 50 11.1 Robin 1871a Robin 1871, 372 formula: water 45, gum arabic 15, glycerol 30 11.1 Robin 1871b Robin 1871, 372 formula: water 100, gum arabic 50, glycerol 50 11.1 Robin 1871c Robin 1871, 372 formula: water 60, gum arabic 20, glycerol 20 11.1 Schweitzer 1942 test. 1946 Roskin Roskin 1946, 200 formula: water 65, gum arabic 20, chloral hydrate 8, glycerol 7 M 11.1-M 12.1 MOUNTING MEDIA 633 11.1 Semmens 1938a CS13 — auct. Microscope, 2:120 formula: water 40, gum arabic 20, DS 11.23 Belling 1921 11.1 Semmens 1938b CSlS—and. Microficope, 2 -.120 formula: water 45, gum arabic 10, chloral hydrate 25, acetic acid 37.5, carmine 0.5 preparation: Dissolve the chloral hydrate in 25 acetic acid with 25 water, raise to boiling, stir in carmine, cool, and filter. Dissolve the gum in 20 water and 12.5 acetic acid. Mix the solution.s. 11.1 Swan 1936 4184, 27 :38<) formitla: water 20, gum arabic 15, chloral hydrate GO, glucose syrup 10, acetic acid 5 11.1 Womersley 1943 21054, 67:181 formula: water 100, 95% ale. 50, gum araliic (powder) 40, phenol 50, chloral hydrate 50, glucose syrup 10, lactic acid 20 preparation: Mix ale. and powdered gum to a smooth paste. Flood 100 water onto paste. Stir rapidity. Leave 2 hours, then filter. Evaporate till volume 100. Grind phenol and chloral hydrate in a mortar till solution complete. Add to solution. Add syrup and lactic acid to mixture. 11.1 Zirkle 1940 20540b, 16:144 formula: water 65, formic acid 41, gum arabic 10, sorbitol 10, ferric nitrate 0.5, carmine 0.5 preparation: Dissolve the gum in the solvents. Incorporate the iron and then the dye. note: See also M 12.1, M 13.1 Zirkle 1940. 12 GELATIN MEDIA It is presumed, in all the formulas that follow, that a gelatin is employed which will give a crystal-clear solution in water. Such purified gelatins are today available on the market for bacteriological use. If commercial gelatin is being used, it is necessary that it should first be clarified, and directions for doing this are given in all the older for- mulas. Soak the gelatin overnight, drain it carefulh', and then melt it on a water bath at about 40°C. Then add, for each 100 milliliters of the fluid so produced, the wdiites of tw'o fresh eggs. These are mixed thoroughlj^ with the molten gelatin and the temperature of the water bath is then raised to boiling and left until the whole of the egg white is coagulated. The medium must not be stirred during this time. The egg white coagulates in large lumps, which may readily be strained out through cheesecloth, and which retain, attached to them, all the fine particles which cause cloudiness of the gelatin. 12.1 Formulas 12.1 Baker 1944 Baker 1944, 173 formula: water 65, gelatin 5, glycerol 35, cresol 0.25 preparation : Soak gelatin in 25 water for 1 hour, then melt at 60°C. Mix glycerol in 40 water with cresol, then heat to 60°C. and mix with gelatin. 12.1 Beale 1880 Beale 1880, 07 formula: clarified gelatin 50, glycerol 50 12.1 Brandt 1880 23632,2:69 formula: gelatin 40, glycerol 60, phenol 0.5 PREPARATION : Soak gelatin in water for 24 hours. Drain and melt. Mix glycerol and phenol with molten gelatin. Clarify s.a. 12.1 Bruere and Kaufmann 1907 4349, 2:11 formula: gelatin about 25, glycerol about 50, water q.s., 40% formaldehyde 0.1 preparation: Soak the gelatin overnight. Drain, melt, and add an equal volume of glycerol. Clarify s.a., filter, and add formaldehyde. 12.1 Carleton and Leach 1938 Carleton and Leach 1938, 115 formula: water 60, gelatin 10, glycerol 70, phenol 0.25 preparation: Melt gelatin in water at 80°C. Raise glycerol and phenol to 80°C. and add. 634 METHODS AND FORMULAS M 12.1 12.1 Chevalier 1882 Chevalier 1882, 297 formula: water q.s., gelatin 25, pyroligneous acid 10 preparation: Soak the gelatin in water, drain, and melt. Add pyroligneous acid to molten gelatin. 12.1 Deane test. 1877 Fray Frey 1877, 135 formula: water 30, gelatin 15, glycerol 55 preparation: Dissolve gelatin in water with heat and add glycerol. 12.1 Dean test. 1880 Beale Beale 1880, 67 formula: gelatin 30, honey 120, 95% ale. 15, creosote 0.2 preparation: Soak gelatin overnight. Drain. Melt on water bath. Heat honey on water bath. Mix with gelatin. Mix creosote in ale. and add to mixture when cooled to about 35°C. Filter. 12.1 Delepine 1915 4349,5:71 formula: gelatin 5.2, sat. sol. arsenic trioxide 19, glycerol 71 preparation: Dissolve gelatin in hot arsenic solution, add glycerol. 12.1 Fischer 1912 23632,29:65 formula: water 100, sodium borate 2, gelatin 10, glycerol 17 preparation: Dissolve ingredients with heat and maintain at 40°C. until the medium remains liquid on cooling to room temperature. 12.1 Forbes 1943 21400a, 62:325 formula: water 40, gelatin 9, glycerol 50, phenol 0.6 12.1 Geoffrey 1893 11074,7:55 formula: water 100, chloral hydrate 10, gelatin 4 12.1 Gerlach 1885 see P 12.3 Gerlach 1885 12.1 Gilson test. 1905 Lee Lee 1905, 273 preparation: Soak gelatin in water overnight, drain, and melt. To 50 of this add 50 glycerol and enough chloral hydrate to bring the total volume to 50. 12.1 Guyer 1930 Guyer 1930, 96 formula: water 50, gelatin 8, glycerol 50, egg white (fresh) about 10, phenol 0.25 preparation: Soak gelatin in water overnight. Dissolve with gentle heat. Add egg white to warm gelatin. Autoclave 15 minutes at 15 lbs. and filter. Add glycerol and phenol to filtrate. 12.1 Heidenhain 1905 23632, 20:328 formula: water 60, gelatin 13, glycerol 10, 95% ale. 20 preparation: Dissolve gelatin in water with glycerol. Add ale. drop by drop to solution. 12.1 Kaiser 1880 3445, 1 :25 formula: water 40, gelatin 7, glycerol 50, phenol 1 12.1 Kisser 1935 23032, 51 :372 formula: water 60, glycerol 50, gelatin 16, phenol 1 12.1 Klebs /cs/. 1877 Frey Frey 1877, 135 preparation: Soak isinglass in water, drain, and melt. Add enough glycerol to increase volume by one half. 12.1 Legros test. 1871a Robin Robin 1871, 371 formula: water 20, gelatin 10, glycerol 30, sat. aq. sol. arsenic trioxide 30 preparation: Melt gelatin in water. IVIix glycerol and arsenic with molten gelatin. 12.1 Legros test. 1871b Robin Robin 1871, 372 formula: water 50, gelatin 10, arsenic trioxide 0.3, glycerol 30, phenol 0.1 preparation: Soak gelatin in 20 water some hours and melt. Dissolve arsenic in 30 water and add to molten gelatin. Add glycerol and phenol to mixture. M 12.1 MOUNTING MEDIA G35 12.1 Martindale test. 1884 Cole Cole 1884b, 49 formula: water 50, gelatin 5, 95% ale. 3, egg white 3, glycerol 50, saUcyhc acid 0.25 preparation: Soak gelatin in water and melt. Add ale. to molten gelatin. Add remam- ing ingredients to mixture at 30°C. Mix well and heat to 100°C. for 5 minutes and filter. 12.1 Moreau 1918 5293,34:164 formula: water 42, gelatin 7, glycerol 50, phenol 1 preparation: Soak gelatin in water and melt. Add glycerol and phenol to molten gelatin. 12.1 Muir test. circ. 1938 Wellings Wellings circ. 1938, 146 formula: sat. aq. sol. thymol 100, glycerol 5, gelatin 10, potassium acetate 0.5 12.1 Nieuwenhuyse 1912 test. 1916 Kappers 4349, 5:116 REAGENTS required: A. 30% gelatin; fi. 4% formaldehyde method: Sections on slide are covered with a fairly thick layer of A and, after chilling, placed in B for 30 mins. Air dry at 30°C. until transparent. 12.1 Nordstedt 1876 test. 1883 Behrens Behrens 1883, 180 formula: water 42, gelatin 14, glycerol 56 12.1 Roskin 1946 Roskin 1946, 123 formula: water 42, gelatin 7, glycerol 50, phenol 0.5 preparation: Soak gelatin in water 2 hours. Melt. Incorporate glycerol. Filter. 12.1 Roudanowski 1865 11024, 2:227 formula: water q.s., gelatin 20, glycerol 50 preparation: Soak gelatin in water overnight. Drain and melt. Mix glycerol with molten gelatin. 12.1 Schact test. 1883 Behrens Behrens 1883, 181 formula: water 36, gelatin 12, glycerol 48 12.1 Squire 1892 Squire 1892, 84 formula: water 25, gelatin 6.5, glycerol 50, chloroform 0.6, egg white 5 preparation: Soak gelatin in water 24 hours, drain. Mix glycerol in 25 water with 0.1 chloroform, heat, and add to soaked gelatin. Heat to solution. Add egg white and clarify s.a. Filter. Add enough water to make filtrate 100. Add 0.5 chloroform and stir well. 12.1 Wood 1897 3430,24:208 formula: water 100, clarified gelatin 20, glycerol 10, 40%, formaldehyde 1 preparation: Dissolve gelatin in water with glycerol on water bath. Add formaldehyde immediately before use. 12.1 Wotton and Zwemer 1935 see M 12.1 Zwemer 1933 (note) 12.1 Yetwin 1944 11428,30:201 formula: water 83, gelatin 5, glycerol 17, chrome alum 0.3, phenol 0.3 12.1 Zirkle 1937a 19938,85:528 formula: water 50, acetic acid 50, glycerol 1, gelatin 10, dextrose 4, ferric chloride 0.05, carmine to sat. 12.1 Zirkle 1937b see DS 11.23 Zirkle 1937 12.1 Zirkle 1940a 20540b, 15:143 formula: water 60, acetic acid 50, gelatin 10, sorbitol 10, ferric nitrate 0.5, carmine 0.5 preparation: Mix all ingredients except dye. Bring to boil and add dye. Boil 5 minutes. Do not filter. note: See also M 11.1 and M 13.1 Zirkle 1940. 12.1 Zirkle 1940b 20540b, 15:143 formula: water 55, acetic acid 45, gelatin 10, gluconic acid 15, ferric nitrate 0.5, carmine 0.5 preparation: As Zirkle 1940a. 036 METHODS AND FORMULAS M 12.1-M 13.1 12.1 Zirkle 1947 20540b, 22:87 formula: water 45, acetic acid 30, lactic acid 15, Rolatin 10, orcein to sat. PREPARATION : Dissolve the gelatin in water. Add other ingredients, boil 2-3 minutes. 12.1 Zwemer 1933 Gluchrodd—auci. 763,57:41 formula: water 80, gelatin 3, glycerol 20, chrome alum 0.2, camphor 0.1 preparation: Dissolve gelatin in 50 water with heat. Add glycerol to hot solution. Dis- solve chrome alum in 30 water and add to liot mixture. Filter, then add camphor. note: This formula was republished by Wotton and Zwemer 1935 (20540b, 10:21). 13 OTHER WATER-MISCIBLE MOUNTANTS 13.1 Formulas 13.1 Archibald and Marshall 1931 16035, 23:272 formula: water 60, gum tragacanth 0.5, ale. q.s., gum acacia 1.5, lactic acid 12, glycerol 12, phenol 12 preparation: Add enough alcohol to gum tragacanth to make thin paste. Flood 10 water on paste with constant stirring. Dissolve gum acacia in 50 water. Mix with tragacanth mucilage. Add acid, glycerol, and phenol to mixed gums. Filter. RECOMMENDED FOR: wholcmounts of invertebrate larvae. 13.1 Bernhardt 1943 1752,48:533 formula: water 1, glycerol 2, phenol 1, lactic acid 1 13.1 Downs 1943 19938, 97 :639 STOCK solution: dissolve 15 polyvinyl ale. in 100 water at 80°C. working formula: .stock 56, lactic acid 22, phenol 22 note: The same formula was republished, with proper acknowledgment, by Huber and Caplin 1947 (1829, 56:763) to whom the medium is often attributed. 13.1 Huber and Caplin 1947 see M 13.1 Downs 1943 (note) 13.1 Jones 1946 16730a, 21:85 formula: water 35, polyvinyl ale. 6.3, sat. sol. picric in abs. ale. 18, lactophenol 45 formula: mix polyvinyl ale. to a paste with the picric solution. Add water to paste and stir to jelly. Add lactophenol to jelly and heat to transparency on water bath. 13.1 Gray and Wess 1950 11360, 70:290 formula: water 30, lactic acid 15, glycerol 15, 70% acetone 20, polyvinyl ale. 6 preparation: Add the acetone slowly and with constant stirring to the dry resin. Mix half the water with the lactic acid and glycerol and add to resin mixture. Add remain- ing water slowly and with constant stirring. Heat on a water bath until clear. 13.1 Monk 1938 19938,88:174 formula: levulose syrup 35, pectin gel 35, water 20, thymol 0.1 13.1 Monk 1941 21400a, 60:75 formula: dextrose syrup 30, pectin gel 30, water 30 note: Karo brand dextrose and Certo brand pectin are specified in the original of both of Monk's formulas. 13.1 Roudanowski 1865 11024,2:227 formula: water q.s., isinglass 5, glycerol 8 preparation: Leave isinglass in water and soak overnight. Drain and melt. Add glycerol to molten material. 13.1 Watkin 1925 see DS 21.41 Watkin 1925 13.1 Zirkle 1937 19938,85:528 formula: DS 11.23 Belling 1921 80, levulose syrup 10, pectin jeUy 10 note: The original specifies Karo brand levulose syrup and Certo brand pectin jelly. M 13.1-M 22.1 MOUNTING MEDIA 637 13.1 Zirkle 1940a 20540b, 15:142 formula: water 60, acetic acid 50, dextrin 10, sorbitol 10, ferric nitrate 0.5, carmine 0.5 preparation: Dissolve dextrin in water. Add other ingredients in order given. Boil, cool, and filter. note: See also M 11.1 and 12.1 Zirkle 1940. 13.1 Zirkle 1940b 20540b, 15:144 formula: water 55, acetic acid 55, sorbitol 5, pectin gel 10, levulose syrup 10, ferric nitrate 0.5, carmine 0.5 preparation: Mix all ingredients except pectin. Leave some days. Filter. Incorporate pectin. note: The original calls for Certo brand pectin and Karo brand levulose syrup. M 20 Mountants Miscible with Alcohol Media of this tyjDe, into which objects may l^e mounted directly from alcohol with- out the necessitj^ of clearing, fall into three classes. In the first class (M 21) are the media based on gum mastic; in the second (M 22) are the media based on Venice turpentine. Both these media are regularly used for objects which are considered too delicate to withstand the action of a clearing agent. The third class (M 23), containing the gum sandarac media, comprise those formulas which are usually referred to as neutral mountants. These are widely used both for substances wliich are considered too delicate to preserve in Canada balsam, which involves prior clearing, or for sections which have been stained in mateiial which fades rapidly under the influence of acid balsam. Many are derived from the original euparal of Gilson, the formula for which has never been disclosed and which is a preparatory substance of secret composition. These media have a refractive index much lower than the gum mastic or gum turpentine media so that the}^ cannot satisfactorih- be used for thick objects. It may be pointed out that "dry" Canada balsam is soluble in absolute alcohol and has from time to time been recommended. It is the writer's experience that this solution is not satisfactory, for mounts made with it darken more rapidh' than those made from balsam which has been dissolved in hydrocarbons. 21 GUM MASTIC MEDIA 21.1 Formulas 21.1 Artigas 1935 13461, 10:71 formula: 95% ale. 100, gum mastic 30, beechwood creosote 100 preparation: Dissolve gum in ale. Centrifuge or filter. Add creosote and evaporate till no ale. remains. recommended for: nematode worms after clearing in creosote. 21.1 Hoyer 1921 6630,84:814 preparation: Suspend mastic in a cloth bag in a considerable volume of 95% ale. Withdraw bag in which remain gross impurities. Shake solution thoroughly, allow to settle, and decant clear solution. Evaporate to required consistency. 22 VENICE TURPENTINE MEDIA 22.1 Formulas 22.1 Langeron 1942 Venice turpentine-alcohol Langeron 1942, 654 preparation: Dilute crude Venice turpentine with an equal volume of 95% ale. Mix well and allow impurities to settle. Decant and re-evaporate to convenient consistency, 22.1 Vosseler 1889 23632, 6:292 formula: 95% ale. 50, Venice turpentine 50 preparation: Mix ingredients. Allow to settle. Decant. 638 METHODS AND FORMULAS M 22.1-M 24.1 22.1 Wilson 1945 20540b, 20:133 formula: Venice turpentine 25, phenol 50, proprionic acid 35, acetic acid 10, water 20 preparation: As M 22.1 Zirkle 1940. 22.1 Zirkle 1940 20540b, 15:147 formula: water 25, acetic acid 15, proprionic acid 35, phenol 55, Venice turpentine 20, ferric nitrate 0.5, carmine 0.5 preparation: Mix the Venice turpentine with the proprionic acid. Add the phenol and acetic acid. Then add the water, in which the ferric nitrate has been dissolved, slowly and with constant stirring. Incorporate the dye in this mixture. note: See also M 31.1 Zirkle 1940. M 23 GUM SANDARAC MEDIA 23.1 Formulas 23.1 Armitage 1939 Microscope, Z:215 preparation : To 100 of a syrupy filtered solution of gum sandarac in dioxane add the fluid produced by the mutual solution of 3 salol and 2 camphor. 23.1 Buchholz 1938 20540b, 13:53 formula: oil of eucalyptus 60, paraldehyde 30, gum sandarac to give required consist- ency 23.1 Cox 1891 1780,37:16 formula: alcohol 150, sandarac 150, turpentine 60, camphor 30, lavender oil 45, castor oil 0.5 preparation: Dissolve sandarac in ale. Dissolve camphor in turpentine and then mix in ale. solution. Add oils to mixed solutions. 23.1 Denham 1923 Camphoral — auct. 11360,43:190 stock i: chloral hydrate 50, camphor 50 preparation of stock i: Grind ingredients in a mortar till solution complete. stock II : gum sandarac q.s., isobutyl ale. q.s. PREPARATION OF STOCK II : Make a thin solution of the ingredients. Shake with activated charcoal. Filter. Evaporate to a thick syrup. WORKING medium: stock I 60, stock II 30 23.1 Gilson 1906 Euparal — compl. script. 6011,23:427 note: This is a proprietary mixture of secret composition and cannot be further noticed. The reference cited does not disclose the composition. 23.1 Mohr and Wehrle 1942 20540b, 17:157 formula: camsal 10, gum sandarac 40, eucalyptol 20, dioxane 20, paraldehyde 10 note: Camsal is produced by the mutual solution of equal quantities of camphor and phenyl salicylate (salol). This medium may be diluted with dioxane. It may be colored green (in imitation of green euparal) by adding a solution of copper oleate in eucalyptol. 23.1 Shepherd 1918 21400a, 37:131 formula: sandarac 30, eucalyptol 20, paraldehyde 10, camsal 10 note: Camsal is a mixture of equal parts camphor and phenyl salicylate. Shepherd (loc. cit.) recommends dissolving the sandarac in 150 abs. ale, filtering under an- hydrous condition and re-evaporating to dryness. 24 OTHER ALCOHOL-MISCIBLE MEDIA 24.1 Formulas 24.1 Hanna 1949 11360,69:25 formula: sulfur 40, phenol 100, sodium sulfide 2 24.1 SeUer 1881 21400a, 3 :60 formula: Canada balsam 40, abs. ale. 60 M 30-M 31.1 MOUNTING MEDIA G39 M 30 Mountants Not Miscible with either Alcohol or Water The great majority of all mounts are prepared in these media of which Canada balsam (mixtures containing which form the first class, M 31), is the best known. Gum damar (M 32) is a substance which is so variable in composition that it is difficult to recom- mend it. It has less tendency to become either yellow or acid with age than has balsam, provided that one secures a good specimen. But there are numerous accounts in the hterature of mounts which have become granular within a few years of having been made. It would appear probable that these mounts were made from an impure sample of the gum, and the worker who wishes to use damar media is recommended to be very particular as to his source of supply. The next class (M 33) covers the few otlier natural resins which have from time to time been proposed for mounting media as well as mix- tures of these resins with Canada balsam and with gum damar. The last class (M 34 below) is Hkely to increase very rapidly with time. It includes numerous synthetic resins which have been proposed as a substitute for the natural resins usually employed. No methcrylate mixtures are included since there is abundant evidence in the literature (Richards and Smith 1938: 19938, 87:374) that they are worthless. Unfortunately, many authors have proposed media based on resins of which only a trade name is quoted. These have not been included since they are almost impossible to duplicate. Formulas using trade names have, however, been included if a reasonable chemical identification of the resin is given in the original description. 31 CANADA BALSAM MEDIA Canada balsam is the natural exudate of Abies balsamea. It consists of a resin {Canada resin in the formulas given below) dissolved in a variety of hydrocarbons. The material sold on the market as dried balsam has had the lower boiUng-point natural hydrocarbons driven off with heat but the higher boiling-point fractions, which act as natural plasti- cizers, remain. This dried balsam is commonly used as a 40% solution in xylene or ben- zene. If true Canada resin is used, a plasticizer must be added. A method of purifying Canada balsam is given by Bensley and Bensley 1938, 38. Neutral balsams are a delusion and some M 34 formula should be used in their place. 31.1 Formulas 31.1 Apathy 1909 8338,22:18 formula: Canada balsain 50, cedarwood oil 25, chloroform 25 31.1 Becher and DemoU 1913 Becher and DemoU 1913, 107 formula: Canada resin 40, abs. ale. 40, terpineol 20 31.1 Curtis 1905 salicylic balsam — co^npl. script. 1863, 17 :603 formula: dried Canada balsam 30, sat. sol. salicylic acid in xylene, 70 31.1 Hays 1865 21400a, 1:16 formula: Canada balsam 50, chloroform 50 preparation: Mix ingredients and allow to stand 1 month. Decant. Evaporate to required consistency. 31.1 Sahli 1885 23632,2:5 note: This paper is often quoted as recommending a solution of balsam in cedar oil. Sahli recommends only that sufficient of the oil used for clearing be left to soften the balsam. 31.1 Semmens 1938 CS15a—auct. Microscope, 2 'AQQ formula: Dried Canada balsam 40, xylene 20, DS 12.16 McLean 1934b 20 note: The substitution [of DS 12.16 McLean 1934a (eosin) gives CS15b and McLean 1934a (erythrosin) gives CS15c. 640 METHODS AND FORMULAS M31.1-M34,l 31.1 Zirkle 1940 20540b, 15:149 formula: water 20, acetic acid 15, proprionic acid 40, phenol 65, oleic acid 10, dried Canada balsam 10, ferric nitrate 0.5, carmine 0.5 preparation: Mix the balsam with the acetic and proprionic acids. Add the oleic acid and then the phenol. Incorporate the water in which the ferric nitrate has been dis- solved. Then mix in the dye. 32 GUM DAMAR MEDIA Gum (himar is the natural e.xudate of Shorea iviesneri, but it is almost always adul- terated and usually contains solid impurities. The raw gum should be dissolved in chloroform, filtered, and evaporated until the chloroform is driven off. The purified gum is then dissolved in lienzene or toluene to a suitable consistency. M 32.1 Formulas 32.1 Cooke circ. 1920 Cooke circ. 1920, 49 preparation: Warm together till dissolved equal parts of gum damar, benzene, and turpentine. Filter. Evaporate to desired consistency. 32.1 Vogt and Jung test. circ. 1890 Francotte Francotte, 248 formula: Canada balsam 30, benzene 100, gum damar 30 preparation: Dissolve ingredients separately, then mix solutions. 33 OTHER RESINS AND MIXED RESINS 33.1 Formulas 33.1 Artigas 1935 see M 21.1 Artigas 1935 33.1 Chevalier 1882 Chevalier 1882, 327 formula: chloroform 90, rubber 3, gum mastic q.s. to give required consistency 33.1 Fremineau tcit. 1883 Chevalier Chevalier 1882, 297 formula: Canada balsam 60, gum mastic 20, chloroform q.s. to give required fluidity 33.1 Lacoste and de Lachand 1943 4285a, 20:159 formula: toluene 100, rosin 120 33.1 Noyer 1921 6630, 84:814 preparation: Take decanted solution of mastic from M 21.1 Noyer 1921 and evaporate to dryness. Redissolve in xylene. This solution was once known in commerce as erenol. 33.1 Rehm 1893 23632, 9:387 formula: benzene 100, rosin 10 33.1 Seller 1881 Seller 1881, 90 formula: naphtha 17, turpentine 15, Canada balsam 45, damar 23 note: Seller {loc. cit.) recommended this either as a mountant or as a ringing cement, 33.1 Southgate 1923 3506,4:44 preparation: Digest 200 crude Yucatan elemi in the cold with 200 95% ale. Filter and evaporate filtrate to dryness and redissolve in benzene to a suitable consistency. note: The solution of amyrin-free gum elemi thus obtained is stated to preserve Giemsa or other DS 13.1 stains indefinitely. 34 SYNTHETIC RESINS 34.1 Formulas 34.1 Deflandre 1933 Bui. soc. franc, microsc, 2 :07 formula: coumarone resin 20, xylene 80, monobromonaphthalene 1 M 34.1 MOUNTING MEDIA 641 34.1 Deflandre 1947 Kumadex—aud. Doflandre 1947, 96 preparation: Mix 3 parts of a syrupy solution of a couinarone resin in xylene with 1 part of Canada balsam. 34.1 Fleming 1943 11360, 63:34 formula: Naphrax 40, xylene 59, dibutyl phthallate 1 notk: The resin mentioned is a high refractive index (1.7-1.8) naphthalene derivative, the synthesis of which is fully described in the reference cited. 34.1 Gray and Wess 1951a 11300, 71:197 formula: ethyl cellosolve 68, isoamyl phthalate 12, polyvinyl acetate 20 RECOMMENDED FOR: sections. 34.1 Gray and Wess 1951b 11300, 71:197 formula: ethyl cellosolve 50, isoamyl phthalate 10, polyvinyl acetate 40 RECOMMENDED FOR: wholemounts. 34.1 Groat 1939 763, 74:1 formula: toluene 40, "nevillite 1" or "V" 60 note: The nevillites are mostly hydrogenated coumarones of which clarite (nevillite 1) is the best known. Nevillite V is a naphthaline polymer. 34.1 Kirkpatrick and Lendrum 1939 DPX—aud. 11431, 49:592 4 formula: xylene 80, tricresyl phosphate 15, " Distrene-80 " 10 note: Distrene-80 is a polystyrene with a molecular weight of about 80,000. 34.1 Skiles and Georgi 1937 19938, 85:367 formula: vinylite 20, xylene 80 method: used to varnish bacterial films 34.1 Wicks, Carruthers, and Ritchey 1946 20540b, 21:121 formula: "Piccolyte" 60, xylene 40 note: There are a whole series of beta-pinene polymers marketed under the general name "piccolyte." The authors cited found "WW-85," "WW-100," "S-85," and "S-100" the most suitable. 27 Embedding Media Decimal Divisions Used in Chapter E 00 GENERAL OBSERVATIONS E 10 MEDIA MISCIBLE WITH WATER 11.1 Formulas E 20 MEDIA NOT MISCIBLE WITH WATER 21 Wax media 21.1 Formulas 22 Nitrocellulose media 22.1 Formulas 23 Resinous media 23.1 Formulas 24 Other media 24.1 Formulas E 00 General Observations The term embedding media covers all those materials which are used to surround, im- pregnate, and support specimens which are being sectioned. The technique of section cutting is discussed in Chapters 10 through 15. Embedding media may be divided into those which are miscible with water (E 10) and to which, therefore, objects may be transferred without special preparation, and those which are not miscible with water (E 20) and thus require preliminary treatment of the specimen. E 10 Media Miscible with Water Water-miscible embedding media are in some cases intended for surrounding an ob- ject which is to be frozen before it is cut, and in other cases intended to be hardened by other means and cut without freezing. No general division between these two groups is possible because many media are suitable for both purposes. When, however, no specific method is given, it is to be presumed that the media are intended for objects to be cut on a freezing microtome in the manner described in Chapter 15. E 11.1 Formulas 11.1 Anderson 1929 Anderson 1929, 129 formula: syrupus simplex' (B.P.) 45, dextrin 14, 80% ale. 45 preparation: Boil the dextrin in the syrupus simplex till solution complete. Add ale. little by little with constant shaking. 11.1 Apathy test. 1948 Romeis Romeis 1948, 108 formula: water 87.5, glycerol 12.5, gelatin 25 method: [object from water] -^ 50% glycerol-* 30% embedding medium, 40°C., 24 hrs. -^ embedding medium, 40°C. 24 hrs. — > dessicator, 50°C. till volume reduced to half -^ cast as block -* cool -^95% ale, 24 hrs. — > terpineol, 24 hrs. -^ [section] 642 E 11. 1 EMBEDDING MEDIA 643 11.1 Belezky 1931 22575, 282:214 REAGENTS REQUIRED: ^.. Water 75, phenol 7.5, gelatin 25; B. 4% neutralized formalde- hyde method: [small pieces] —> A, 2-3 hrs., 37°C. —» cool —> cut small blocks containing pieces from mass -* B, 2-5 days — > [frozen sections] 11.1 Blank and McCarthy 1950 11284, 36:776 formula: Carbowax 4000 90, Carbowax 1500 10 method: [fixed and washed tissues] —> water ^ "wax," 56°C. till impregnated —> [block] note: The Carbowaxes (products of the Carbide and Chemical Corporation) are solid polyethylene glycols. "1500" has about the consistency of petroleum jelly, "4000" about that of 58° paraffin; other grades are available. All grades are water soluble, which makes embedding easy, but it is very difficult to flatten ribbons before they disintegrate. 11.1 Brunottil892 11074,6:194 reagents required: A. water 100, gelatin 10, acetic acid 15, mercuric chloride 0.5; jB. 5% potassium dichromate method: [water] -^ 1 part A, 3 parts water, till impregnated —» A, till impregnated —> B, dropped in with as much A as possible adhering 11.1 Bunge test. 1877 Frey Frey 1877, 71 formula: egg white 72, 10% sodium carbonate 7.5, tallow 27 preparation: Whip egg white and sodium carbonate together. Melt tallow and in- corporate with egg white. method: embed from water. Harden in alcohol 11.1 Chatton 1923 6630,88:199 formula: water 100, agar 1.3, 40% formaldehyde 2.5 preparation: Dissolve agar in boiling water. Add formaldehyde to hot solution. method: [object from water] —> surface of thin slab cast from medium —> pour just liquid medium on top, allow to set — * 95% ale. note: This is primarily intended for setting and orienting small objects, the block being re-embedded in paraffin. 11.1 Clark 1947 11431, 59:337 reagents required: A. \2.b% gelatin; B. 2b% gelatin; C. 2% formaldehyde; D. 0.5%, gelatin; E. 40% formaldehyde method: [pieces of formaldehyde fixed material] — > thorough wash —y A,24 hrs. 37.5°C. — > B, 24 hrs. —^ [make block and refrigerate — trim block] -^ C, 24 hrs. — * [frozen sec- tions] -^ 50% ale. -^^ D, few moments -^ place on clean slide -^ drain — > vapor from E, 1 hr., 27°C. -^ C, till required recommended for: fatty tissues. note: a detailed description of this technique is given in Chapter 15. 11.1 Cole 1884 Cole 1884, 39 formula: water 24, gum arable 36, sugar 18, water 16 recommended for: use on freezing microtomes. 11.1 Cutler 1935 1887a, 20:445 reagents required: A. 95% ale. 50, glycol stearate 50; B. glycol stearate method: [fixed tissues] -♦ 95% ale, via graded series -^ A, 56°C., 12-24 hrs. -^ B, 24-48 hrs. -^ [make block] note: Ribbons may be attached to slide and processed exactly as paraffin ribbons. 11.1 Flemming test. 1877 Frey Frey 1877, 71 formula: hard soap 75, 95% ale. 25 11.1 Frey 1877 Frey 1877, 71 formula: isinglass 30, water q.s., glycerol 30 preparation: Soak isinglass in water. Drain. Melt. Mix glycerol with molten isinglass. method: embed from 50% glycerol. Harden in alcohol 644 METHODS AND FORMULAS E 11.1 11.1 Gaskell 1912 see E 11.1 Romeis 1948 11.1 Gerlach 1885 see M 12,1 Gerlach 1885 11.1 Godfrin test. 1942 Langeron cit. Brunotte 1889 Langeron 1942, 440 formula: water 25, glycerol 20, gelatin 2.5, 90% ale. 150, castor oil soap 50 preparation: Dissolve gelatin in water with glycerol on water bath. Mix oil in ale. and add to hot solution. method: [woody tissues]-^ 95% ale. in vacuo to remove air — > water, some hrs. — > medium, freshly prepared — * continue heating till skin forms on surface — » withdraw object 11.1 Graff 1916 see E 11.1 Romeis 1948 11.1 Hartley 1938 Microscope, 2 :46 formula: water 20, glycerol 40, gum arable 20, thymol 0.1 method: [bundles of textile fibers] — * medium, till saturated -^ dry — > section RECOMMENDED FOR: transverse sections of textile fibers. 11.1 Hermga and ten Berge 1923 23632, 40:166 method: [object washed free of fixative] -> 10% gelatin, 2-5 hrs., 37°C. -^ 20%, gelatin, 37°C., 10 mins. — > [make block, cool] -^ [frozen sections] -^ sat. sol. thymol — > attach to slides with V 21.2 Heringa and ten Berge 1923 -^ stain 11.1 Kaiser 1880 see M 12.1 Kaiser 1880 11.1 Langeron 1942 Langeron 1942, 439 formula: water 60, gum arable 20, glycerol 20 RECOMMENDED FOR: use On freezing microtome. 11.1 Lebowich 1936 1887a, 22 :782 formula: 56°C. paraffin 80, stearic acid 20, diethylene glycol 1.5, ethanolamine 1.5 preparation: Mix completely the molten waxes, preferably with high-speed electric mixer. Add other reagents and continue mixing 30 minutes at 85°C. REAGENTS REQUIRED: A. acctone; B. soap from above method: [fixed material]—* A, till dehydrated^ B, 56°-62°C. (in vacuo for large ob- jects), till impregnated-^ [cast in block and section] —> [mount sections by V 21.3 Lebowich 1936] 11.1 Lubkin and Carsten 1942 19938, 97:168 formula: water 80, polyvinyl ale. 16, glycerol 20 preparation: Dissolve the ale. with continuous stirring at 75°-85°C. Incorporate glycerol while cooling. method: Place well-washed tissue blocks in medium in covered dishes. Heat to 56°C. for 2 hrs. daily. When solidified (8-9 days), trim and cut as though the block were nitrocellulose. 11.1 Nicolas 1896 2844, 3 :274 reagents required: A. 3% gelatin; B. 10% gelatin; C. water 90, gelatin 20, glycerol 10; D. 5% formaldehyde method: [water] -> A, 1-2 days, 25°C. -^ B, 1-2 days, 25°C. -^ C, 2-3 days 35°C. -» [transfer to paper box, cool] -^ D, till hard enough to cut 11.1 Pickworth 1934 11025, 69:62 formula: water 100, phenol 1, gum arable 25 11.1 Polzam test. circ. 1890 Francotte Francotte, circ. 1890, 287 formula: dried hard soap 15, glycerol 35, 95% ale. 50 preparation: Boil ingredients with reflux to a syrupy mass. method: [objects too delicate for paraffin embedding]-* 95% ale. — > medium at 30- 40°C. — » [cool as block] — > [section] E11.1-E21 EMBEDDING MEDIA 045 11.1 Romeis 1948 Romeis 1948, 109 formula: water 75, phenol 0.75, golatin 25 method: [objtMit washed free of fixative) -* 50% enibeddinf; jiiediuin, 21 hrs., 37°C. —> embedding ni(>diurn, 30 mins. -^ [set as blo(;k; evaporate till surface tacky]—* 10% formaldehyde, 1-2 days —* [frozen sections] note: This is a synthesis by Romeis {loc. cit.) of the methods of Gaskell 1912 (11431, 20:17), and Graff 1916 (14674, 63:1482). 11.1 Salkind 1916 6630,79:16 REAGENTS REQUIRED: A. cherry gum 25, water 50, lead subacetate solution (N.F. VI) 25, acetic acid 1.5; B. ammonia preparation: Dissolve gum in water. Mix lead subacetate solution and acetic acid and add to filtrate. method: [water] — > A, till impregnated — > [paper boat] — > vapor of B, till hard enough to cut note: The original method, and also the citation in Gatenby and Cowdry 1937, 108 call for "Extract of Saturne" (sic). Gray 1816 {Treatise on Pharmacology, 318) refers to a solutionj'obtained by boiling litharge with distilled vinegar as "Goulard's Extract of Saturn." The Merck Index, 1940, 315 refers to the NF solution given above as "Goulard's Extract." Whether or not the strength of Salkind's solution was that of the NF does not appear to be ascertainable. 11.1 Samuel 1944 11025,78:173 reagents required: A. 0.65% agar in 2% formaldehyde; B. 1.3% agar in 2% formalde- hyde method: [small objects] -^ film of B cast on slide -^ A, around but not over object — > film of B over object -* paraffin 11.1 Tobias 1936 Tobias 1936, 74 formula: water 60, gum arabic 40, phenol 0.5 11.1 Webb 1890 11360, 113 formula: water 100, phenol 2.5, dextrin q.s. to give a thick syrup 11.1 Zwemer 1933 763, 57:41 reagents required: A. b% gelatin; B. 10% gelatin; C. 4% formaldehyde method: [formaldehyde fixed tissues]-^ A, 24 hrs., 37.5°C. ^ B, 12 hrs. 37.5°C. -> [cast block] -^ C, till required — > [sections by freezing technique] E 20 Embedding Media Not Miscible with Water Before material can l)e embedded in these media for cutting, it must l^e subjected to whatever treatment is specified in Chapters 12 through 14. In general, liowever, this treatment consists of the removal of water from the specimen with a dehydrating agent and the substitution for the dehj'drating agent of a solvent whicli is itself miscible with the embedding material. Three main types of media are distinguished in the present chapter. First (E 21.1) come the numerous wax media which have been suggested to re- place straight paraffin in the production of sections; second, nitrocellulose media, more commonly referied to as " ceUoidin," in which there are few media but a considerable number of methods, j^articularly for double embedding; third (E 23) resinous materials which are intended for the inclusion of matter from which sections are to be ground, as indicated in Chapter 10, rather than cut. 21 WAX MEDIA The original method of cutting sections eml)ed(led in wax, which is described in detail in Chapter 12, was to dehydrate the specimen with alcoliol, to remove the alcoliol either with an essential oil or a hydrocarbon, and then to embed the material in straight paraf- fin. This technique has been modified both tlirough the introduction of substitutes for, or adjuvants to, paraffin, and by the introduction of other solvents. In the section which 646 METHODS AND FORMULAS E 21.1 follows, therefore, the formulas which may be used to replace paraffin are given and also some techniques by which any material may be embedded in any selected formula. 21.1 Formulas 21.1 Altmann test. 1942 Langeron Langeron 1942, 422 formula: 60°C. paraffin 85, tristearin 10, beeswax 5 21.1 Beyer 1938 591b, 2:173 formula: paraffin 100, rubber 2, beeswax 0.5 21.1 Bragg 1938 23639b, 28:154 REAGENTS REQUIRED: A. water 20, 95% ale. 40, aniline 30; B. 95% ale. 30, aniline 60; C. aniline; D. aniline 50, toluene 50; E. toluene; F. sat. sol. 53° paraffin in toluene; G. 53° paraffin method: [embryos in 70% ale] -^ A, 2-6 hrs. -^ B, 2-6 hrs. -^ C, \ hr. or till clear — > D, 1-6 hrs. -^ E, 1-3 hrs. -» F, 1-4 hrs. -^ G, 3-4 hrs., 56°C. -* [cast in block] note: This is recommended for heavily yolked embryos. 21.1 Debauche 1939 20540b, 14:121 FORMUL.\: paraffin 80, beeswax 20 21.1 Dufrenoy 1935 6630, 119:375 method: [water] -^50% methylal, 30 mins. -> methylal, 30 mins. — > methylal 2, Uquid petrolatum 3, 30 mins. -^ methylal 1, liquid petrolatum 3, 30 mins. — > liquid petro- latum, 30°C., 30 mins. — >• [paraffin] 21.1 Gray 1941 U.S. Patent 2,267,151 formula: paraffin (MP 58°C.) JO, rubber 5, beeswax 5, spermaceti 5, nevillite "5" ("clarite") 15 note: This composition melts at about 50°C. but will cut 5 m ribbons at a room tem- perature of 85°F. By increasing the resin to 30 it is possible to cut 1 ju ribbons. 21.1 Gudden test. 1895 Rawitz Rawitz 1895, 31 formula: stearic acid 48, lard 48, beeswax 2 21.1 Hance 1933 19938,77:353 stock: Dissolve about 20 crude rubber, in small pieces, in 100 paraffin heated to smoking. WORKING formula: paraffin 100, stock 4-5, beeswax 1 21.1 Hetherington 1922 11428,9:102 method: [nematodes] —> F 0000.0010 Hetherington 1922-^ methyl salicylate —> wax 21.1 Johnston 1903 11032,6:2662 formula: paraffin 99, asphalt 0.1, para rubber 1 preparation: Heat ingredients to 100°C. with occasional stirring for 48 hours. Decant. 21.1 Hsii and Tang 1939a 20540b, 14:151 formula: Japan wax 90, beeswax 10 21.1 Hsii and Tang 1939b 20540b, 14:151 formula: Japan wax 30, paraffin 70 21.1 Larbaud 1921 see E 21.1 Peeters 1921 21.1 Langeron 1942 Langeron 1942, 415 method: [water] -^95% ale. -^ amyl ale. 24 hrs. -^ liquid petrolatum, 2-3 changes in 24 hrs. — > [paraffin] 21.1 Maxwell 1938 20540b, 13:93 formula: paraffin 56-58° 100, rubber paraffin (see E 21.1 Hance 1933) 4-5, bayberry wax 5-10, beeswax 1 E 21.1-E 22.1 EMBEDDING MEDIA 647 21.1 Peeters 1921 6630,85:15 method: [95% ale] -^ amyl ale. — * amyl ale, fresh portion, 52°C. -^ equal parts paraffin and amyl ale. 52°C. — ♦ paraffin note: Larbaud 1921 (6628, 172:1317) uses butyl ale. by the same technique. 21.1 Pohlman test. 1930 Guyer Guyer 1930, 43 FORMULA : 52° paraffin 10, bayberry wax 1 21.1 Ruffini 1927 Ruffini 1927, 28 formula: paraffin (MP 52°-54°C.) 100, beeswax 10, lard 15 21.1 Seller 1881 Seller 1881, 48 formula: paraffin 65, tallow 35 21.1 Sherman and Smith 1938 591b (tech suppl.), 2:171 preparation of stock: To 100 paraffin (MP 55°C.) at 78°C. add 12.5 crepe rubber at the rate of about 3 every 12 hours; continue heating till homogeneous. WORKING medium: paraffin (MP 55°C.) 100, stock 2.5, beeswax 5 21.1 Spec 1885 23632, 2 :7 method: Heat paraffin of 50^0. melting point until it turns yellow. 21.1 Steedman 1947 17510,88:123 formula: ethylene glycol monostearate 10, ethylene glycol distearate 73, stearic acid 5, ethyl cellulose 4, castor oil 8 21.1 Steedman 1949 14900,164:1084 formula : diethylene glycol distearate 80, ethyl cellulose (low viscosity) 4, stearic acid 5, castor oil 4, diethylene glycol monostearate 5 note: This composition melts at 53°C. but cuts well at 80''-90°F. 21.1 van Walsem 1892 22238, 1 :32 formula: paraffin MP 55°C. 95, beeswax 5 recommended for: very large sections. 21.1 Waterman 1939 20540b, 14:55 formula: paraffin 80, stearic acid 16, spermaceti 3, bayberry wax 1 note: This mixture melts about 3°C. below the melting point of the paraffin base, but it is sufficiently hard to cut good sections at room temperatures. 22 NITROCELLULOSE MEDIA Nitrocellulose is a very loose term covering a large number of mixtures of chemical compounds. These mixtures are usually differentiated according to the viscosity of a standard solution which is tested by timing the rate of fall of a steel ball. A half-second nitrocellulose would, therefore, be of low viscosity and a thirty-second nitrocellulose of very high viscosity. Only the very low viscosity nitrocelluloses are suitable for embed- ding, and the term celloidin (a registered trademark), is now used for any nitrocellulose suitable for the purpose. In the section which follows, are given mostly methods rather than formulas. 22.1 Formulas 22.1 Bauer 1941 23632, 58:44 method: [object, washed free of fixative]—* equal parts pyridine and 4% celloidin, 24 hrs. — > 9% celloidin, 24 hrs. — > [make block] 22.1 Brown 1948 20540b, 23 :83 reagents required: A. 2% celloidin; B. 4% celloidin; C. 6% celloidin; D. chloroform; E. benzene; F. sat. sol. paraffin in benzene; G. paraffin method: [perfectly dehydrated specimens] — » A, 12-24 hrs. — > B, 12-24 hrs. — >• C, 2-4 days — ♦ D, vapor, till firm — * E, 6-12 hrs. in each of three changes — » F, 37°C., 1 day — ♦ G, 12-24 hrs. in each of three changes 648 METHODS AND FORMULAS E 22.1 22.1 Espinasse test. 1937 Gatenby and Painter Gatenby and Painter 1937, 96 REAGENTS REQUIRED: A. methyl benzoate 100, celloidin 1 method: abs. ale. -^ A, till permeated -^ benzene — > paraffin 22.1 Field and Martin 1894 23632, 11:6 method: Dissolve celloidin moistened with toluene in a mixture of equal parts toluene and abs. ale. to give a thick solution. Warm to 25°C. and add, little by little, as much paraffin as the solution will take up. Pass object to mass from equal parts toluene and abs. ale. When impregnated transfer to a sat. sol. paraffin in chloroform. Evaporate solvents at 56°C. and treat block as paraffin block. 22.1 Heinz 1923 14674,70:913 REAGENTS REQUIRED: A. abs. alc. 50, ether 50; B. methj'l salicylate 50, ether 25, abs. ale. 25, celloidin 1; C. chloroform; D. sat. sol. paraffin in chloroform method: [pieces from abs. ale] -^ A,\2 hrs. — > B, 24 hrs. -^ C, 12 hrs. -^ D, 12 hrs. — > [paraffin] 22.1 Jordan and Heather 1929 20540b, 4:121 formula: abs. alc. 25, ether 25, methyl salicylate 25, celloidin 1 method: [fixed and dehydrated pieces] — > 50:50 ether abs. alc, 8 hrs. -^ celloidin mix- ture 24 hrs. -^ sat. sol. paraffin in chloroform 2-3 hrs. — > paraffin, 2 changes, till permeated -^ [make block] 22.1 Peterfi 1921 23632, 38:342 FORMUL.\: methyl benzoate 100, collodion 1 method: [abs. alc] — > methyl benzoate —> medium, till perfectly clear —> cedar oil — > paraffin 22.1 Reichardt and Wetzel 1928 23632, 45 :476 reagents required: A. methyl benzoate; B. 1% celloidin in methyl benzoate; C. 10% paraffin in methyl benzoate; D. paraffin (50°C.) method: [objects to be embedded] — > abs. alc. till perfectly dehydrated — > A, till clear -^ B, 3-5 days -^ C, 1-2 days, 40°C. -^ D, 1 day -^ [make block] 22.1 Richardson 1934 11571b, 13:81 formula: celloidin 110, abs. alc. 25, ether 110 PREPAR.\TiON : Leave celloidin in alc. overnight. Mix ether in alc. solution and leave several daj^s. method: [formol fixed material] —> acetone 2 hrs. — > clove oil, till clear -^ celloidin, 6 hrs. — » [prepare block in usual manner] 22.1 Seki 1937a 23639b, 27:278 method: [dehydrated object] — > methanol, 24 hrs. — > equal parts methanol and ether, 12 hrs. —* 1% celloidin in methanol 12 hrs. — > [evaporate to ?^rds volume] -^ chloro- form, 24 hrs. -^ anhydrous butyl alc, 12-24 hrs. -^ benzene, 3-5 hrs. — > sat. sol. paraffin in benzene, 35°C., ^ 2~1 hr. —>■ paraffin 22.1 Seki 1937b 23639b, 27:282 method: [dehydrated object] —» 2% celloidin in methanol, 1 day — >• 4% celloidin in methanol, 2 days -^8% celloidin in methanol, 4 days -^ [set in block] -^3% chloro- form in 70% alc, 1 day -^ section 22.1 Stepanow 1900 23632, 17:185 REAGENTS REQUIRED: A. clovo oil; B. ether 80, abs. alc 4, clove oil 20, celloidin 6 method: [dehydrated tissues] —> A, 3-6 hrs. — > B, 3-24 hrs. -^^ [cast block, allow to evaporate until transparent] — > chloroform 22.1 Tschernyachinsky 1930 23632,47:200 method: [objects impregnated with 8% celloidin in abs. alc.-ether] —* equal parts 8% celloidin and clove oil, 2-24 hrs. — » chloroform, 30 mins. — ^ sat. sol. paraffin in chloro- form 56°C. 1 hr. -^ paraffin E 22.1-E 24.1 EMBEDDING MEDIA G49 22.1 Wolf 1939 23632, 56:57 method: [ol\iect, washed free of fixative] -^ laboratory glycerol, G hrs. —> anhydrous glycerol, 6 hrs. — > wipe free frt)in glycerol — ^ suspend in 8% cclloidin, 2 days — > [make block] 23 RESINOUS MEDIA Resinous media, as has been explained in Chapter 10, are used for supporting mate- rials from which sections are to be ground rather tlian cut. No specific mixtures have been recommended for this puri)osc, though Canada balsam, damar, and ordinary resin have all been employed. The only formula at present occupying this section is designed to permit cutting thin sections in a polymerized resin, instead of a material hardened by evaporation, as are the nitrocelluloses of the last section. 23.1 Formulas 23.1 Bourdon 1943 4285a, 20:40 preparation: Catalyze vinyl acetate with 3% benzoyl peroxide and warm till syrupy. Embed in this material impregnated, after dioxane dehydration, with vinyl acetate. Polymerize at 37°C. for 48 hours followed by 48 hours at 56°C. Cut as though the block were made of nitrocellulose. Sections of 2 /i-3 fj. may be obtained. 24 OTHER MEDIA Three media, which cannot justifiably be included in either of the previous divisions, are given under this heading. 24.1 Formulas 24.1 Barlow 1938 test. 1938 "A.B." Microscope, 2:150 formula: paraffin (43°C.) 45, ethyl cellulose (low viscosity) 10, stearic acid 15 24.1 Brain 1950 11360, 70:313 stock solutions: I. 1% celloidin in 98% ale; II. 1% celloidin in abs. ale; III. 2.5% cel- loidin in methyl salicylate note: Stock solutions I and II are prepared by dilution from a 30% solution of celloidin in the usual 50:50 alc.-ether mixture. reagents required: A. 2% gelatin; B. 10% gelatin; C. AF 21.1 Brain 1950; D. stock I; E. stock II; F. 60 stock II, 30 stock III; G. 50 stock II, 50 stock III; H. 30 stock II, 60 stock III; /. stock III; /. benzene; K. 60 benzene, 30 paraffin; L. 50 benzene, 50 paraffin; M. 30 benzene, 60 paraffin; TV. paraffin 56°MP method: [well-washed, formaldehyde-fixed material] -^ A, 4 days, 37°C. -* B, few hrs. 37°C. —f [cast in block of B] — ^ C, changed daily, until decalcification complete (45 days for mouse jaws) -^ running water, 24 hrs. -^50% ale, 30 mins., 37°C. -^70% ale, 30 mins., 37°C. -^ 90% ale, 30 mins., 37°C. -* D, Ui hrs., 37°C. -^ E,3 changes, each 30 mins., 37°C. -^ F, 1 hr., 37°C. -^ G, 1 hr., 37°C. -> H, 1 hr., 37°C. -^ /, 4 days, 37°C. ^ J,2 changes each 2^ hrs., 37°C. -^ K, 1 hr., 45''C. -^ L, 1 hr., 45°C. -> M, 1 hr., 45°C. -* A'', 3 changes each 8 hrs., 58°C. -^ [cast block in N] recommended for: teeth, particularly for showing relation of enamel to dentine. 24.1 Cutler 1935 1887a, 20:445 method : glycol stearate used as paraffin but embedding from 95 % ale. 28 Various Formulas Decimal Divisions Used in Chapter V 00 GENERAL OBSERVATIONS AND ARRANGEMENT OF FORMULAS V 10 CEMENTS, LUTES, AND VARNISHES 11 Fluid at room temperatures (applied from a brush) 11.1 Aqueous base 11.2 Non-aqueous base 12 Solid at room temperatures (applied molten^ 12.1 Aqueous base 12.2 Non-aqueous base 13 Other cements and lutes 13.1 Formulas V 20 ADHESIVES 21 For attaching sections to slides 21.1 For attaching paraffin ribbons 21.2 For attaching sections in nitrocellulose 21.3 For attaching individual sections requiring staining 22 For attaching whole objects to slides 22.1 For objects requiring further manipulation 22.2 For objects not requiring further manipulation 22.3 Label adhesives 23 Other purposes 23.1 Formulas V 30 INJECTION MEDIA 31 For injection at room temperatures 31.1 Formulas 32 For injection warm 32.1 Formulas V 40 CLEANING METHODS AND FORMULAS 41.1 Methods and Formulas V 50 MISCELLANEOUS FORMULAS 51.1 Formulas V 00 General Observations This chapter includes those formulas now the custom, greatly to the detriment which cannot reasonably be placed in any of the art, to use for mounting only those other section. It is divided into three main materials which themselves support and and two subsidiary divisions. The for- cement the coverslip in position. The mulas in the first division (the cements, writer has elsewhere in the present work lutes, and varnishes— V 10) were at one expressed his opinion of the undesirability time of more interest to microtomists than of this custom, and it is to be hoped that is now generally the case. This indicates the inclusion of many of these older less a reduction in the value of the for- formulas for cements and varnishes, de- mulas than it does the changing fashion in signed to attach the coverslip to the slide the preparation of microscope slides. It is and to preserve within it a fluid medium, 650 VIO VARIOUS FORMULAS 651 may encourage mounters to revert to fluid mounts. The next section (on ad- hesives — V 20) is self-explanatory, though few people today seem to think it neces- sary^ to cause to adhere to a slide any ob- ject except a paraffin ribl)on, or, even in that case, to realize that materials other than egg albumen may be used with ad- vantage. The next section (on media for injection — V 30) contains many formulas which cannot be ignored by any preparer of microscope slides whether for teaching or research purposes. The two last sections (on cleaning methods and formulas — V 40, and miscellaneous formulas — V 50) are of little importance but provide a final rest- ing-place for formulas which the writer considers worth publishing but which can- not be placed elsewhere. V 10 Cements, Lutes, and Varnishes These materials are used for two pur- poses. The first is the attachment of objects, either supporting structures such as cells (see Chapters 1-3) or objects for examination, to sHdes; or second, they are used to seal edges of covershps in order to retain under the cover some mounting medium. The formulas are here divided into those which are in fluid form and those which must be melted before application. It must be remembered, how- ever, that the majority of the soHd cements may be dissolved in a suitable solvent and used as though they were fluid. If this is done, it is well to heat the dried cement to get rid of the last trace of solvent. Care must be taken, in following old directions as to the sealing of micro- scopical preparations, that one uses actu- alh^ the material intended by the writer and not that which at the present time passes under the same name. Thus the modern marine glue bears no relation at all to the solution of shellac in gutta percha which was the marine glue used by the early microtomists. The formula given below (V 12.2 Harting 1880) is very close to the original marine glue, but there is no means of finding out whether or not it is identical with the material ("G 4") specified by the early writers and blindly copied by many later ones. Another source of constant confusion is gold size. This was originally partially polymerized and oxidized linseed oil diluted with turpentine, and was specified for the use of gilders because it remained tacky for a long time, thus enabhng the leaf gold to be applied to a complex ornament over a long period. Modern varnishes having the same property of remaining tacky are available on the market under the name of gold size, but are entirely unsuitable for the preparation of microscope slides. Another misunderstood term is sealing wax. In the earlier days sealing wax contained con- siderable quantities of Venice turpentine and of beeswax, as well as the shellac of which it is now almost completely com- posed. In the following pages will be found formulas giving so far as is possible the original ingredients. Shellac itself is a term capable of mis- interpretation. Orange shellac is a natural exudate, caused by insect damage, from the bark of many resinous trees. It is a mixture of alcohol-soluble resins and naphtha-soluble waxes. White shellac is produced by the action of chlorine on alka- line-aqueous solutions (in part "soaps") of orange shellac. The alcohol-soluble fraction of this chlorinated shellac poly- merizes very rapidly, at temperatures just below its melting point, into a material which is insoluble in alcohol or any other common solvent. Hence a "cell" turned from V 11.2 Gage 1901 (below) can be used for an alcohol mount if the dewaxed shellac called for in the formula is prepared from white shellac and the ring-baked. BeUido 1927 (11360, 47 :27) specifies heat treatment for shellac cells used in the mounting of diatoms in bromonaphthalene. The only confusion which is likely to arise among solvents is in the use of the term benzine, a petroleum fraction now- adays sold as hgroin. It has none of the properties of benzene nor should it be con- fused with the low-boihng-point petroleum ether which is sometimes specified in its place. 652 METHODS AND FORMULAS V 11.1-V 11.2 11 FLUID AT ROOM TEMPERATURES 11.1 Aqueous Base 11.1 Bellido 1897 lest. 1927 ips. 113G0, 47:9 formula: water 48, acetic acid 50, abs. ale. 2, gelatin 6 preparation: Soak gelatin in water overnight. Melt at 90°C. and add other ingredients. note: Don Ernesto Caballero Bellido 's Spanish writings fall naturally under the name "Caballero." The present writer uses "Bellido" because that is the last name under the title of the English paper cited. The Royal Microscopical Society avoids the dilemma neatly bj omitting any reference to this paper in the author index of the volume in question. 11.1 Caballero 1897 see V 11.1 Bellido 1897 (note) 11.1 Semmens 1938 CSll — auct. Microscope, 2 :\2Q formula: water 65, acetic acid 25, gum arable 20 11.1 Spence 1938 see V 11.1 Gage 1901 (note) 11.2 Nonaqueous Base 11.2 Beale 1880a Gold size Beale 1880, 54 formula: linseed oil 100, red lead 4, umber 1.5, white lead 5, yellow ochre 5 preparation: Mix oil, red lead, and umber thoroughly. Boil 3 hours. Allow to settle. Decant clear fluid. Grind white lead and yellow ochre together. Mix with boiled oil. Reboil 3 hours. Allow to stand. Decant. 11.2 Beale 1880b Brunswick black Beale 1880, 55 formula: Trinidad asphalt 50, boiled linseed oil 50, turpentine 100 preparation: Boil asphalt in linseed oil for some time. Cool. Mix turpentine with cooled mass. note: Beale directs that 4' 2 oz. of linseed oil be boiled with half an ounce of litharge "until quite stringy." 11.2 Beale 1880c Sealing wax varnish Beale 1880, 55 formula: 95% ale. 100, sealing wax 40 note: This cement should be made with a sealing wax of the period, not with the modern variety which is mostly rosin. A typical contemporary recipe is: 5 parts shellac, 9 parts Venice turpentine, 7 parts rosin, 6 parts pigment. (Spon, Workshop Receipts, 2nd series. London 1883). 11.2 Beale 1880d Damar cement Beale 1880, 55 formula: benzene 60, gum damar 40 11.2 Beale 1880e Cement for glass Beale 1880, 60 formula: chloroform 65, rubber 1, gum mastic 35 preparation: Dissolve chloroform in rubber. Add gum mastic to solution. Digest at 50°C. 1 week. Allow to settle. Decant. 11.2 Behrens 1883 Behrens 1883, 192 formula: abs. ale. 50, ether 10, turpentine 50, copal in fine powder 50 preparation: Dissolve ingredients with prolonged but very gentle heat. 11.2 Bell's cement This was a proprietary product of secret composition; the formula appears to have been lost. 11.2 Bellido see V 11.2 Hitchcock 1884 (note) 11.2 Benoit-Bazille lest. 1942 Langeron Langeron 1942, 666 formula: tiu'pentine 30, asphalt 20, gold size 50 preparation: Dissolve asphalt in turpentine. Add gold size to solution. V 11.2 VAEIOUS FORMULAS G53 11.2 Brooke test. 1880 Beale flexible Brunswick black Beale 1880, 55 formula: Brunswick black 90, rubber solution 10 note: Beale does not specify proportions. Those given have served the author. 11.2 Carney test. 1937 Gatenby and Cowdry Gatenby and Cowdry 1937, 232 formula: tolu balsam U), (Janada balsam 20, chloroform 30, shellac 20 preparation: Mix tolu balsam and Canada balsam with heat. Cool. Dissolve chloro- form in shellac. Add to cooled mixture. note: The cement is to be diluted with chloroform until it flows freely from the brush. 11.2 Chevalier 1882 Chevalier 1882, 305 formula: asphalt 15, turpentine 15, gold size 60 preparation: Dissolve asphalt in turpentine. Add gold size to solution. 11.2 Davies circ. 1865 Davies, 24 formula: rubber 0.5, ligroin 70, asphalt 30 preparation: Dissolve rubber in ligroin. Dissolve asphalt in solution. 11.2 Eulenstein test. 1880 Beale Brunswick black Beale 1880, 55 formula: Brunswick black 50, gold size 50, Canada balsam 5 11.2 Frey 1877 Frey 1877, 133 formula: chloroform 80, rubber 1.25, gum mastic 20 preparation: Dissolve chloroform in rubber 1.25. Add gum mastic in fine powder to solution. 11.2 Gage 1901 Gage 1901, 204 formula: V 11.2 Hitchcock 1884 100, Venice turpentine 2, castor oil 2 note: Spence 1938 {Microscope, 2:127) omits the Venice turpentine. 11.2 Gram-Riitzon test. 1883 Behrens cit. Poulsen Behrens 1883, 191 formula: abs. ale. 25, ether 50, Canada balsam 25, shellac 25 11.2 Groves test. 1883 Hogg Hogg 1883, 246 formula: chloroform 50, gum mastic 50, bismuth nitrate q.s. preparation: Dissolve chloroform in gum mastic. Add enough bismuth nitrate to form a thick cream with gum solution. 11.2 Hitchcock 1884 test. Gage 1901 Gage 1901, 204 preparation: Half fill a bottle with shellac. Fill with 90% ale. and leave to complete solution. Place in a separatory funnel with one-half its volume of naphtha. Shake well, allow to separate, retain the ale. fraction, and evaporate to suitable consistency. note: This method of dewaxing shellac is said by Spence 1938 {Microscope, 2 :127) to be due to Pelz, about 1876. It is certainly German in origin, since benzol (an obvious mistranslation of benzin) is frequently specified. Bellido 1897 (11360, 47:9) merely decants the clear supernant fluid from an alcoholic solution of shellac which has stood for some weeks. 11.2 Hogg 1883a Hogg 1883, 221 formula: gum damar 20, turpentine 20, gum mastic 20, chloroform 40 preparation: Dissolve gum damar 20 and turpentine with heat. Cool. P'ilter. Dissolve gum mastic and chloroform in cold. Filter. Mix with filtered damar. 11.2 Hogg 1883b Hogg 1883, 222 formula: acetic acid 90, gum ammoniac 2, gelatin 20 preparation: Dissolve gum ammoniac in acetic acid. Dissolve gelatin in gum .solution. note: This formula wets gla.ss readily and was originally designed for sealing glycerol mounts. 11.2 James 1885 11360, 1101 formula: gum damar 40, ligroin 100, zinc oxide q.s. prepar.\tion: Dissolve gum damar in ligroin. Heat zinc oxide to dryness. Cool. Moisten with benzine. Add damar solution with constant stirring. Leave 12 hours. Decant from any gross particles. 654 METHODS AND FORMULAS V 11.2-V 12.1 11.2 Kitton test. 1883 Hogg Hogg 1883, 247 formula: a. white lead 30, red lead 30, litharge 30, turpentine q.s. to make a paste; B. gold size note: For use mix about 3 parts B with loi A. This mixture sets very rapidly and forms a very hard cement which may be used for the preparation of cells. 11.2 Mohr and Wehrle 1940 Cellohalm—auct. 20540b, 15:173 formula: toluene 77, ethyl cellulose 7.7, Canada balsam 15 11.2 Pelz see V 11.2 Hitchcock 1884 (note) 11.2 Perruche 1939 Bull. soc. franc, microsc, 8:147 formula: benzene 100, aluminum stearate 1.5 USE : For making water-repellent barriers for the temporary restraint of aqueous fluids on slides. 11.2 Robin 1871 flexible asphalt cement Robin 1871, 379 formula: carbon disulfide 60, asphalt 30, Venice turpentine 10 11.2 Rousselet 1898 11479,7:93 formula: benzene 40, gum damar 25, gold size 30 preparation: Dissolve benzene in gum damar. Mix gold size with solution. 11.2 Seller 1881 see M 33.1 Seller 1881 11.2 Semmens 1937 Microscope, 1 :5 preparation: Dissolve 25 gum damar in 250 each of chloroform and xylene. Filter and evaporate to 100. RECOMMENDED FOR: ringing lactophenol mounts. 11.2 Spence 1938 see V 11.2 Gage 1901 (note) 11.2 Thiersch test. 1871 Robin blue varnish Robin 1871, 381 formula: 95% ale. 100, shellac 60, spirit blue 1, castor oil 0.4 11.2 Woohead test. 1884 Cole Cole 1884b, 50 formula: benzene 50, gum damar 50, zinc oxide 6 preparation: Dissolve gum damar in benzene. Grind zinc oxide with solution. 11.2 Zimmermann test. 1883 Behrens Behrens 1883, 191 formula: shellac 50, 95% ale. 100, anilin green 2 preparation: Dissolve ingredients. Filter. Evaporate to required consistency. 12 SOLID AT ROOM TEMPERATURES (APPLIED MOLTEN) 12.1 Aqueous Base 12.1 de Greet 1904 23833, 28:406 formula: zinc oxide 25, water 90, gelatin 20 preparation: Mix zinc oxide with a little water to make a paste. Add rest of water gradually to secure smooth suspension. Heat to 80°C. on water bath. Dissolve gelatin in hot suspension. note: This cement is excellent for making alcohol-resistant glass-to-glass seals. 12.1 Marsh 1878 Marsh 1878, 45 formula: gelatin 100, water q.s., creosote 0.6 preparation: Mix gelatin and water and soak for 12 hours. Drain. Melt. Add creosote to molten gelatin. 12.1 Renter test. 1938 Carleton and Leach Carleton and Leach 1938, 116 formula: water 100, gelatin 20, potassium dichromate 0.5 preparation: Dissolve gelatin in 90 water at 80°C. Add potassium dichromate dis- solved in 10 water. V 12.1-V 12.2 VARIOUS FORMULAS G55 12.1 Riiyter 1934 4285a, 11 :410 preparation: Saturate 100 water with oil of thyme. Soak 20 gelatin overnight. Melt. Add thyme water to make 100. Add 10 5% potassium dichromate. Keep in dark. note: This formula is identical with Riiyter 1935 (23632, 51 :374). 12.1 Seller 1881 Seller 1881, 93 formula: acetic acid 100, gelatin 27, gum ammoniac 2.2 preparation: Dissolve the gum in the acid. Filter. Warm and dissolve gelatin. note: Seiler {loc. cit.) recommends that the cooled ring should be painted with 2% potas- sium dichromate and exposed to light. 12.2 Nonaqueous Base 12.2 Apathy 1889 23632, 6 :164 formula: paraffin 60°C. 50, Canada balsam 50 preparation: Heat ingredients till golden brown. note: The identical composition can be achieved by dissolving 45 parts Canada resin in 55 parts molten wax. 12.2 Beale 1880a marine glue Beale 1880, 55 formula: ligroin 100, Para rubber 50, shellac 50 preparation: Dissolve Para rubber in half ligroin with gentle heat. Dissolve shellac in half ligroin with gentle heat. Cool. Mix thoroughly. note: The mixture may either be evaporated until solvent free, in which case it is used as a thermoplastic cement, or diluted with more ligroin for use as a varnish. 12.2 Beale 1880b French cement Beale 1880, 59 formula: rubber 100, lime q.s. preparation: Melt the rubber over an oil bath. Add the lime, little by little, until the mixture thickens. Scrape into a mortar and heat with a pestle until cool enough to knead with the hands. note: The cement, when cool, should be of the consistency of stiff modelling clay. It is remarkably waterproof and adheres perfectly to glass. 12.2 Belling 1926 2975, 50:160 formula: paraffin 50, gum mastic 50 12.2 Cigalas test. 1942 Langeron Langeron 1942, 677 formula: beeswax 70, rosin 30, lard 10 preparation: Dissolve ingredients together. note: This is an excellent waterproof cement for temporary aquaria or for mending leaks. 12.2 Coburn 1915 4349,5:71 formula: white lead ground in oil 50, raw linseed oil 7, rosin 29, 95% ale. 7, shellac 7 preparation: Mix the oil with the white lead and raise to the melting point of the rosin which is then incorporated. While cool but still liquid, stir in the shellac dissolved in the ale. 12.2 Fant 1932 in verb. formula: anhydrous lanolin 30, rosin 55, "dried" Canada balsam 10 preparation: Melt ingredients together. note: This formula was verbally communicated to the author by Fant in 1932 and by the author communicated to others. This lead to an unsigned note in Watson's Microscope Record 1934, attributing the composition to Gray; the error has been perpetuated by Gatenby and Cowdry 1937, 230, and others. 12.2 Gage 1896 Gage 1896, 179 formula: V 11.2 Hitchcock 1884 100, castor oil 10, Venice turpentine 10 12.2 Gray 1934 see V 12.2 Fant 1932 (note) 12.2 Griffiths test circ. 1865 Davies Davies, 22 formula: rosin 50, beeswax 10, red ochre 10, Canada balsam 20 656 METHODS AND FORMULAS V 12.2-V 13.1 12.2 Harting test. 1880 Beale gutta percha cement Beale 1880, 56 formula: turpentine 90, gutta percha 6, shellac 6 preparation: Dissolve turpentine in gutta percha with stirring at 50°C. Strain. Dis- solve shellac in strained fluid at 60°C. Continue heating until a test drop placed on a cool surface becomes reasonably hard. note: This is to be used as a thermoplastic cement, particularly for attaching hard rubber cells. 12.2 Hood and Neill 1948 20540b, 23 :217 formula: asphalt -±2, Canada balsam 15, paraffin (48°C.) 28, pitch 15 12.2 Kroenig 1886 1780, 27:657 formula: beeswax 20, rosin 80 12.2 Lataste test. 1942 Langeron Langeron 1942, 677 formula: paraffin 60, rubber scrap 30 preparation: Heat paraffin and rubber scrap till solution takes place, taking equal pre- cautions against fire and suffocation of the technician. 12.2 Martin 1872 Martin 1872, 169 formula: rosin 80, beeswax 20, tallow 10, oil color as desired 12.2 Mendeleef test. 1942 Langeron Langeron 1942, 677 formula: beeswax 12.5, rosin 50, ochre 20, linseed oil 0.5 preparation: Melt wax. Add rosin when hot. Stir to perfect mixture. Grind ochre and oil to a paste. Add to hot mixture. note: Any other appropriate pigment may be used in place of the ochre. 12.2 Muir and Judah 1915 4349, 5:71 formula: Trinidad asphalt 85, boiled linseed oil 12, oil of amber 3 preparation: Raise the oils to Iwiling and add 25 asphalt, boil 30 minutes and cool. Re- melt and add remaining asphalt in 4 portions, cooling and remelting between each addition. Boil 45 minutes after last addition. 12.2 Noyer 1918 6630, 81:741 formula: rosin 80, lanolin 20 preparation: Melt together. 12.2 Oschatz 1842 test. 1847 Cooper Cooper 1847, 207 formula: sealing wax 50, white lead ground in oil 50 preparation: Melt ingredients together. 13 OTHER CEMENTS, LUTES, AND VARNISHES 13.1 Formulas 13.1 Langeron 1942 Langeron 1942, 327 formula: white beeswax 65, Venice turpentine 35 recommended for: rolling into pellets to support coverslip. 13.1 Martin 1872 dead black varnish Martin 1872, 172 preparation: Mix lamp black and gold size in a mortar to a thick cream. V 20 Adhesives The adhesives given in the present section may be distinguished from the cements, lutes, and \arnishes of the last section by the use to which they are put. A cement or varnish is required to have properties other than that of causing one object to adhere to another. The adhesives most commonly employed in microtomy are those used to attach sections in paraffin ribbons to a slide. It is not absolutel\- necessary to use an adhesive for this purpose, because a section of animal material, flattened with distilled water on a chemically clean slide, will remain attached through most subsequent manipulations. When an adhesive for paraffin ribbons is used, it may be applied in either of two ways V 21.1 VARIOUS FORMULAS G57 without regani to the specifications given by the inventor: the material may be first smeared on the shde, or it may be (hhited considcr;d)ly witli water and tliis (hluted ad- hesive used to flatten the section. It may be tliought curious that formulas for label ad- hesives should be included, but there is certainly no commoner reason for the loss of a valuable preparation than the detachment of the label. It is warmly recommended that one of those formulas be employed which contain a small quantity of glycerol for the purpose of preventing the absolute liardening of the adhesive. The entire label and not only the adhesive upon it should be moistened to avoid a differential contraction in dry- ing, which invariably strips the adhesive from the glass. 21 FOR ATTACHING SECTIONS TO SLIDES 21.1 For Attaching Paraffin Ribbons 21.1 Artschwager 1919 see V 21.1 Szombathy 1918 (note) 21.1 Bohm and Davidoff 1905 Japanese method — compl. script. Bohni and Davidoff 1905, 30 REAGENTS REQUIRED: A. V 21.1 Mayer 1884; B. 0.5% gum arabic method: slides are very thinly coated with A and then dried at 70°-80°C. to coagulate albumen. Sections are flattened on the slide with warm B. note: In European literature, other than English, this method is fairly universally known as the "Japanese method" (cf. Cajal and de Castro 1933, 65; Spielmeyer 1924, 48) 21.1 Claoue 1920 ted. 1942 Langeron Langeron 1942, 481 formula: alcohol 50, ether 50, pyroxylin 0.4, castor oil 3, camphor 2 preparation: Dissolve alcohol, ether, and pyroxylin. Add castor oil and camphor to solution. method: applied at any time from a drop bottle. The slide should be blotted free of excess reagent before the adhesive is applied. recommended for: cementing in place sections which have loosened 21.1 Cobe and Schoenfle 1946 Tech. Bull., 7:31 preparation: In 100 boiling 0.2% potassium dichromate dissolve 0.02 gelatin. Boil 5 minutes, cool, filter. method: flatten ribbons on bath of warm solution, strand on slide, and dry. 21.1 Crabb 1935 19938,80:530 reagents required: A. colloidin USP 40, abs. ale. 20, ether 20, amyl acetate 20; B. collodion USP 50, abs. ale. 25, ether 25 method: [ribbons, spread and flattened on slide and thoroughly dried] -^ A, flooded on slide, 30 sees. -> blot -^ B, flooded on slide —>■ drain -^ dry -^70% ale, 5 mins. -^ 95% ale, quick wash — * xylene 21.1 David 1935 19938, 82:179 formula: water 100, "waterglass" 1, ammonia 1 21.1 Gravis 1889 4992, 15:72 formula: water 100, agar 0.1, camphor 0.1 preparation: Dissolve agar in water with boiling. FUter. Add camphor to filtrate. 21.1 Haupt 1930 see V 21.1 Szombathy 1918 (note) 21.1 Heidenhain 1905 23632,22:331 formula: water 75, albumen 2, 95% ale. 25 preparation: Dissolve albumen in 50 water. Filter. Mix 95% ale. with 25 water. Add to filtered solution. 21.1 Hollande 1911 1823,13:171 formula: garlic 50, water 80, chloroform 1 preparation: Crush ingredients. Triturate. Leave 24 hours. Filter. note: For some reason this appears to work better than a pectin solution of comparable strength. G58 METHODS AND FORMULAS V 21.1 21.1 Land test. 1915 Chamberlain Chamberlain 1915, 114 REAGENTS REQUIRED: A. 1% gum arabic; B. 0.5% potassium dichromate METHOD : Smear A on slide. Flood with B. Warm to flatten sections. Drain, dry. 21.1 LUlie 1945 20540b, 20 :99 formula: water 100 dried egg albumen 5, sodium chloride 0.5, glycerol q.s., 0.01% merthiolate 0.5 preparation: Shake the water, albumen, and sodium chloride gently together till dis- solved. Philter. Add glycerol to make double volume of filtrate. Add merthiolate to mixture. 21.1 Masson 1928 608b, 4:181 reagents required: A. 0.25% gelatin; B. 40% formaldehyde method: flatten sections on A. Drain. Expose to vapor of B at 50°C., 20 mins. to overnight. 21.1 Masson test. 1942 Langeron Langeron 1942, 476 reagents required: A. 1% gelatin; B. 95% ale. 80, 40% formaldehyde 20 method : [paraffin ribbons] — > float on A and warm to flatten — * drain -^ dry heat to melt paraffin -^ xylene, till wax removed — * abs. ale. -^ B, 5 mins. — > 50% ale. 21.1 Mayer 1884 11360,4:317 formula: fresh egg white 50, glycerol 50, sodium salicylate 1 preparation: Agitate ingredients at intervals for some days. Filter. recommended for: spreading in thin layer on slide; or, diluted 20: 1 with water, flattening sections. 21.1 McDowell and Vassos 1940 1789a, 29:432 formula: water 90, starch 3, 10% hydrochloric acid 0.5, thymol 0.1 preparation: Mix starch in 30 water to make a smooth paste. Raise 60 water to boiling. Add to starch paste. Add hydrochloric acid to starch. Boil 5 minutes. Cool. Add thymol to cooled starch. 21.1 Moreau 1918 5293, 34:164 REAGENTS REQUIRED: A. 0.01% gelatin, freshly prepared; B. 90% ale. 80, 40% formal- dehyde 20 method: sections are flattened on warm A and dried. After passage through benzene and abs. ale, B is applied from a drop bottle. 21.1 Regaud test. 1942 Langeron Langeron 1942, 481 REAGENTS REQUIRED: A. coUodiou USP 20, ether 40, abs. ale. 40; B. 70% ale. method: [sections attached to slide but apparently loose] -^ abs. ale. —* A, 2 mins. — » drain —>£,—* stain, etc. recommended for: cementing in place paraffin sections which appear loose after dewaxing. 21.1 Reinke test. 1928 Schmorl Schmorl 1928, 77 formula: egg albumen 50, glycerol 50 preparation: Beat egg white until stiff. Leave till the mass has reverted to a clear fluid. Filter and add an equal volume of glycerol. 21.1 Riiyter 1931a 23632, 47 :226 formula: water 80, V 21.1 Mayer 1884 1, acetone 20, methyl benzoate 0.3 preparation: Mix 21.1 Mayer 1884 with water. Mix acetone and methyl benzoate and add to first mixture. method: use to float and flatten ribbon recommended for: attaching ribbons from double-embedded (nitrocellulose-paraffin) blocks. 21.1 Riiyter 1931b 23632, 48 :226 formula: water 80, acetone 20, methyl benzoate 0.6 method: flood under ribbons on slide. Warm. Dry. use: as Riiyter 1931a V21.1-V21.3 VARIOUS FORMULAS 050 21.1 Schneidau 1937 21559,56:258 formula: water 100, e^;g all)uin(Mi 0.0(1, ulycorol l.G, sodium salicylate O.OG KECOMMENDKi) FOR: iiattriiiiig ami fixing i)arallin rihhoii.s. 21.1 Spoerri 1939 199;i8, 90:200 formula: water 100, starch 3, hydrochloric acid 0.2 preparation: Suspend the starcli in ;30 water; add 70 boiling water and stir. Add acid and boil 5 minutes. 21.1 Szombathy 1918 230:52,34:3:34 REAGENTS REQUIRED: A. Water 100, gelatin 1, glycerol 15, .sodium salicylate 0.2; B. water 98, 40% formaldehyde 2 method: coat slide thinly with .4. Flood with B on which sections are flattened in tlie usual manner. Dry. note: This method is usually known to botanists from a paper ])y Artschwager 1919 (3430, 67:373). Haupt 1930 (205401), 5:97j substitutes 2 pheuortor the salicylate in A above. 21.2 For Attaching Sections in Nitrocellulose 21.2 Heringa and ten Berge 1923 23632, 40:166 REAGENTS REQUIRED: A. 3% gelatin; B. 5% sodium sulfate method: [coat slides with A] — >• dry —> soak in B, 2 hrs. —> wash --> dry 21.2 Langeron 1942a Langcron 1942, 482 rea(;ents required: A. V 21.1 Mayer 1884; B. oil of cloves; C. abs. ale; D. abs. ale. 50, ether 50 method: [section from 70% ale] — > press to slide coated with A —* blot with consider- able pressure — > B, from drop bottle, few mins. -^ C, in jar, 15 mins. —> D, till nitro- cellulose dissolved — > abs. ale. — > stain, etc. 21.2 Langeron 1942b Langeron 1942, 483 reagents required: A. water 100, gelatin 10, phenol 0.5; B. 4% formaldehyde; C. abs. ale; D. abs. ale. 50, ether 50 method: [section from 70% ale] — > press to slide coated with A —> blot thoroughly -^ B, 5 mins. —> C, 2-3 mins. —^ D, till nitrocellulose dissolved — > abs. ale — > [stain, etc.] 21.2 Linstaedt 1912 763, 6:445 reagents required: .1. water 100, dextrin 3, sucrose 3; B. 4% celluloid in acetone; C. 1% celloidin in ether-ale method: coat glass plates with A and dry; spray or dip coat with B; store till required. Arrange sections on water film on coated plate; blot; spray with C. When partially dry dip in 70% ale, then in water till celluloid sheet bearing sections can be detached. recommended for: preparation of sheets of sections which may be cut up with scissors, after staining, for issue to classes. 21.2 Riiyter 1931 see V 21.1 Rliyter 1931 21.3 For Attaching Individual Sections Requiring Staining 21.3 Altmann 1894 tcsl. 1905 Bohm and Davidoff Bohm and Davidof!" 1905, 78 reagents required: A. 3% gutta percha in chloroform; B. collodion USP method: Slide is coated with A, dried and stored. When warmed, coated slides liecome tacky. Sections are pressed to tacky surface, cooled, and varnished with B. 21.3 Chiovenda 1936 test. 1937 Foot 4349, 17:173 formula: water 84, V 21.1 Reinke (1928) 12, glycerol 4, merthiolate 9.5 method: soak sections in adhesive; strand on clean slide; drain; dry at 30-40°C.; place in abs. ale 21.3 Fol test. 1937 Gatenby and Cowdry Gatenby and Cowdry 1937, 116 formula: water 72, 95% ale 30, acetic acid 7, gelatin 1.5, chrome alum 0.1 preparation: Dissolve acetic acid in gelatin with heat. Mix 95% ale in 70 water and add to solution. Dissolve chrome alum in 2 water and add to mixture. method: clean slides are dipped and dried. When again moistened, the surface becomes sticky without dissolving. GGO METHODS AND FORMULAS V 21.3-V 22,1 21.3 Heringa 1924 6630, 91:931 REAGENTS REQUIRED: A. 3% gelatin; B. 2.5% sodium sulfate method: [clean slides] -^ A, dip -^ dry -^ B, I hr. —^ water, thorough wash -^ dry note: Slides so treated are soaked for a few moments in water before having sections pressed to them. 21.3 Langeron 1942 Langeron 1942, 678 formula: water 81, gum arable 30, aluminum sulfate 0.6 preparation: Dissolve gum arable in 75 water. Dissolve aluminum sulfate in 6 water and add to gum solution. 21.3 Lebowich 1936 1887a, 22 :782 reagents required: A. 0.5% gelatin in 1% (of "3%") hydrogen peroxide; B. 2% neutraUzed formaldehyde; C. 2.5% gum arable method: Dip chemically clean slide in A. Drain and dry. Dip in B. Drain and store. Use C to float and flatten sections. Drain, dry section in place. note: This technique was originally intended for use with E 11.1 Lebowich 1936. Moritz 1939 (20540b, 14:17) recommends 0.25% gelatin in A. 21.3 Masson test. 1942 Langeron Langeron 1942, 457 REAGENTS REQUIRED: A. 70% alc; B. 70% ale. 50, ether 50; C. abs. ale. 50, ether 50, pyroxylin 0.25; D. 80% alc. method: [sections floating in A] -^ strand on slide -^ B, from drop bottle-^ C, in jar, cautious dip — > drain — * D 21.3 Moritz 1939 see V 21.3 Lebowich 1936 (note) 21.3 Obregia 1890 15058,9:295 REAGENTS REQUIRED: A. dcxtrose syrup 60, dextrin syrup 20, 95% alc. 40; B. 2% celloidin method: Coat slides with A, very thinly. Dry slowly. Flatten section on sticky surface. Heat to 60°C. Remove paraffin with solvent. Remove solvent with abs. alc. Pour on B. Dry. Strip film with sections attached. note: The strength of syrup is not important. The consistency should be about that of the simple syrup of the pharmacopeia. 21.3 Schallibaum 1883 1780, 22 :689 formula: 2% pyroxylin 20, oil of cloves 80 method: [make thin layer on slide] -^ press on section -^ dry 10 mins. -^ heat to 50°C. 21.3 Windeholz 1923 7276, 70:877 formula: "waterglass" 30, water 70 method: spread a thin film on slide; leave till just dry; strand section on film. Blot and leave 15 mins. note: "Waterglass" is a commercial solution of sodium silicate of very variable com- position. 21.3 Zimmermann 1896 Zimmermann 1896, 40 REAGENTS REQUIRED: A. V 21.1 Gravls 1889; B. collodion USP 5, abs. alc. 25, ether 75 METHOD : sections -^ flattened on slide using A -* drain -^ dry -^ varnish in place with B 22 FOR ATTACHING WHOLE OBJECTS TO SLIDES 22.1 For Objects Requiring Further Manipulation 22.1 Chatton and Lwoff 1930 6630, 104 :834 formula: water 100, gelatin 10, sodium chloride 0.05 RECOMMENDED FOR: attaching fixed protozoans on slide before silver staining. Use at 25°C. 22.1 Giesbrecht 1881 23833,4:255 REAGENTS REQUIRED: A. 1% shellac to 95% alc; B. clove oil method: Coat slides thinly with A. Dry. Moisten with B. Arrange objects on oil film and then evaporate oil at 60°C. V 22.2-V 30 VARIOUS FORMULAS 06 1 22.2 For Objects Not Requiring Further Manipulation 22.2 Gage 1896 Gage 1896, 180 formula: V 22.3 Gage 1896 50, acetic acid 25, water 25 RECOMMENDED FOR: a thin layer is dried on a slide or coverslip. Objects are arranged on this and then caused to adhere by breathing on them. 22.2 Martin 1872a Martin 1872, 169 formula: c'liloroform 80, gutta percha 20, tallow 5 22.2 Martin 1872b Martin 1872, 169 formula : rosin 75, beeswax 15, Canada balsam 5 22.2 Meakin 1939 Microscope, 3:17 formula: water 12.5, dextrin 25, glycerol 75, phenol .3 preparation: Dissolve dextrin in water with heat. Add other ingredients. use: attaching minute objects, particularly butterfly scales. 22.3 Label Adhesives 22.3 Gage 1896 Gage 1896, 179 formula: water 30, 95% ale. 30 acetic acid 30, gelatin 25, glycerol 10 preparation: Dissolve acetic acid in gelatin with occasional shaking at about 30°C. Add water, 95% ale, and glycerol in order given after solution of gelatin is complete. 22.3 Marpmann 1886 23328, 2:151 formula: water 70, 95% ale. 5, gum arable 18, gum tragacanth 5, glycerol 23, oil of thj^me 0.4 preparation: Dissolve gum arable in 35 water. Mix gum tragacanth and 95% ale. to make a smooth cream. Flood 35 water on tragacanth cream. Leave 3 hours. Combine with gum arable solution. Mix glycerol with the oil of thyme and combine with mixed gums. 22.3 Martin 1872 Martin 1872, 171 formula: water 30, gum arable 30, gelatin 15, glycerol 1.5, camphor 0.1 preparation: Dissolve gum arable in warm water. Let gelatin soak overnight. Drain. Melt. Mix with warm gum. Add glycerol and camphor to mixture. 23 OTHER PURPOSES 23.1 Formulas 23.1 Apathy 1912 23632, 29 :449 formula: abs. ale. 50, ether, 50, celloidin 16, clove oil 33 preparation: Dissolve ether and celloidin in abs. ale. Add clove oil to solution. recommended for: attaching celloidin blocks to wood blocks. 23.1 Rossi-Regaud test. 1927 Ruffini Ruffini 1927, 39 formula: collodion US? 25, ether 35, abs. ale. 40 note: The formula here given has been adjusted from that cited by Ruffini to com- pensate for the difference between US and Italian pharmacopeial collodion. 30 Injection Media There is a widespread delusion that the only materials which may be used for injec- tion are those suspended in a colloid. Actually any material of a particle size too great to pass througli the walls of the vessel may be used and will be held in place either in a wholemount or in the course of sectioning, just as are the blood corpuscles. Injection media should always be strained before use, since the entire injection may be destroyed through one large particle choking off the major vessel through which the injection is being inserted. The WTiter's preference is always for those injection methods, whereby one inserts first one material in solution, and then, after it, another which will cau.se the injection mass to precipitate in place. A solution of lead acetate, followed by one of po- 662 METHODS AND FORMULAS V 31.1 tassium dichromate, may be used to fill the very finest capillaries with an opaque yellow pigment. This is one of the oldest known methods of injection and cannot be equaled by any of the more modern substitutes. 31 FOR INJECTION AT ROOM TEMPERATURE 31.1 Formulas 31.1 Altmann 1878 test. 1893 Schaffer 23632, 10:191 formula: olive oil 50, 95% ale. 25, ether 25 method: small pieces of decalcified bone are impregnated in vacuo, washed in water, stained in 1% osmic acid for 24 hrs., and then sectioned. 31.1 Beale 1880a Beale 1880, 111 formula: water 30, 95% ale. 15, acetic acid 0.5, glycerol 60, ammonium hydroxide 0.3, carmine 0.6 preparation: Dissolve the carmine in 5 water with the ammonia. Mix the acetic acid with 30 glycerol. Add this, with constant stirring, to the carmine solution. Mix 30 glycerol, 15 ale, and 25 water. Add this to carmine-acid mixture. 31.1 Beale 1880b Beale 1880, 109 formula: water 123, 95% ale. 30, glycerol 12, potassium ferrocyanide 0.8, ferric chloride 0.6 preparation: Dissolve the potassium ferrocyanide in 24 water with 6 glycerol. Dissolve ferric chloride in 24 water with 6 glycerol. Add slowly, with constant agitation, to the ferrocyanide solution. Mix ale. in 75 water. Add to blue solution slowly and with constant agitation. 31.1 Beale 1880c Beale 1880, 111 formula: water 32, 95% ale. 10, glycerol 45, carmine 0.4, ammonium hydroxide 0.2, acetic acid 0.3 preparation: Mix carmine with water to make paste. Add ammonia to dissolve paste. Add 15 glycerol to carmine solution. Mix 15 glycerol with acetic acid. Add slowly, with constant stirring, to carmine. Add 15 glycerol, ale, and 32 water to mixture in order given. 31.1 Beale 1880d Beale 1880, 363 formula: water 30, glycerol 60, potassium ferrocyanide 0.2, ferric chloride 0.13, hydro- chloric acid 0.1 preparation: Dissolve potassium ferrocyanide in 30 glycerol. Dissolve ferric chloride in 30 glycerol and add slowly, with constant agitation, to the ferrocyanide. Mix hydro- chloric acid with water. Add slowly, with constant agitation, to the blue mixture. 31.1 Bensley 1929 590,40:146 preparation of stock solution: Dissolve 0.4 silver nitrate in 10 water. Add 5% sodium phosphate drop by drop until no further ppt. forms. Wash by deeantation. Accumulate ppt. in 3 parts water. Add 2.8 citric acid to wet ppt. Dilute with water to 100. preparation of working solution: stock 25, 1% sodium citrate 75 method: inject after washing out blood with sodium citrate. Fix injected pieces in 4%, formaldehyde. Cut frozen sections and develop with any AMS 21.1 formula. 31.1 Brucke 1865 1780, 1 :87 formula: 2.17% potassium ferrocyanide 20, 12% sodium sulfate 80, 10% ferric chloride 20 preparation: Mix 40 sodium sulfate with potassium ferrocyanide. Mix 40 sodium sulfate with ferric chloride. Add to above solution slowly and with constant stirring. 31.1 Doyere 1841 test. Cooper 1847 Cooper 1847, 156 REAGENTS REQUIRED: A. solutiou of potassium chromate; B. solution of lead acetate method: inject A through an artery until it runs from a vein. Then inject B. V31.1 VARIOUS FORMULAS GGS 31.1 Frey 1877a Frcy 1877, 111 formula: water 100, load acetate 30, potassium chromatc 14 preparation: Dissolve lead acetate in 50 water. Dissolve potassium chromatc in 50 water. Add to acetate solution slowly and with constant stirring. 31.1 Frey 1877b Frey 1877, 118 formula: water 100, barium chloride 40, sulfuric acid q.s., glycerol 10, ale. 10 prepar.\tion: Dissolve barium chloride in water. Add sulfuric acid drop by drop till no further ppt. forms. Leave 24 hours, then pour off one-half of supernatant fluid. Add glycerol and ale. to residue. 31.1 Mayer 1888 14246,8:307 formula: water 105, potassium ferrocyanide 2, ferric chloride 1 preparation: Dissolve potassium ferrocyanide in 5 water. Add ferric; chloride anil 100 water to ferrocyanide solution slowly and with constant agitation. 31.1 Mayer 1910 Lee and Mayer 1910, 250 formula: water 100, gelatin 10, chloral hydrate 10 preparation: Melt gelatin in water. Add chloral hydrate to hot solution. 31.1 Mozejko 1910 23632,27:374 formula: water 100, gelatin 7.5, sodium salicylate 12.5 preparation: Melt gelatin in water. Add sodium salicylate to hot mixture. 31.1 Pearl 1902 see V 31.1 Tandler 1901 (note) 31.1 Ranvier 1875 Ranvier 1875, 120 formula: V 31.1 Beale 1880b 80, glycerol 20 31.1 Richardson test. 1880 Beale Beale 1880, 110 formula: water 30, 95% ale. 4, glycerol 60, potassium ferrocyanide 0.65, ferrous sulfate 0.33 preparation: Dissolve the potassium ferrocyanide in 15 water with 30 glycerol. Dis- solve the ferrous sulfate in 15 water with 30 glycerol. Add slowly and with constant stirring to ferrocyanide solution. Add 95% ale. to mixture. 31.1 Richardson tcsl. 1877 Frey Frey 1877, 117 formula: water 100, potassium ferrocyanide 3.3, ferrous sulfate 1 preparation: Dissolve potassium ferrocyanide in 50 water. Dissolve ferrous sulfate in 50 water. Add slowly, with constant stirring, to ferrocyanide. note: Frey (loc. cit. p. 118) states that if the quantities of salts be doubled, glycerol may be substituted for half the water. 31.1 Robin 1871a Robin 1871, 33 formula : glycerol 100, acetic acid 5, carmine 3 preparation: Grind carmine in 50 glycerol to a smooth mass. Mix acetic acid in 50 glycerine and add drop by drop, with constant stirring, to carmine until pH about 4. 31.1 Robin 1871b Robin 1871, 34 formi'la: sat. sol. {circ. 30%) potassium ferrocyanide 13, glycerol 64, sat. sol. {circ. 32%,) copper sulfate 23 preparation: Mix ferrocyanide in 32 glycerol. Mix the copper sulfate in 32 glycerol. Add to ferrocyanide drop by drop with constant agitation. 31.1 Robin 1871c Robin 1871, 35 formula: sat. sol. (circ. 30%) potassium ferrocyanide 45, glycerol 50, 27% ferric chloride 1.5 preparation: Mix the ferrocyanide in 25 glycerol. Mix ferric chloride in 25 glycerol. Add drop by drop, with constant stirring, to ferrocyanide solution. 31.1 Robin 1871d Robin 1871, 36 formula: water 70, glycerol 100, cadmium sulfate 30, sodium sulfide 20 preparation: Dissolve cadium sulfate in 40 water with 50 glycerol. Dissolve sodium sulfide in 30 water with 50 glycerol. Mix slowly and with constant stirring into cadmium solution. 664 METHODS AND FORMULAS V 31.1-V 32,1 31.1 Seller 1881 Seller 1881, 78 formula: water 100, gelatin 4.5, silver nitrate 0.2 preparation: Add the silver dissolved in 35 water to the gelatin dissolved in 65. method: drain blood vessels and fill with medium. Develop in any AMS 21.1 formula. RECOMMENDED FOR: demonstration of endothelium of blood vessels. 31.1 Tandler 1901 23632,18:22 formula: water 100, gelatin 5, potassium iodide 6 preparation: Melt gelatin in water. Add potassium iodide to hot mass. Cool. note: Pearl 1902 (11032, 5:1736) is essentially the same. 31.1 Thoma 1899 1739,74:270 formula: water 60, glycerol 40, indigo-carmine 0.15, sodium chloride 1 PREPARATION : Dissolve indigo-carmine in 50 water. Add glycerol to solution. Dissolve sodium chloride in 10 water. Add slowly to mixture with constant agitation. 31.1 Hagmann 1940 20540b, 15:115 REAGENTS REQUIRED: A. Water 90, acetic acid 10, Santomerse no. 3 1, trypan blue 2; B. water 75, 40% formaldehyde 10, acetic acid 15, barium chloride 40 method : chloroformed insects are placed under vacuum in a device which permits their being dropped into A in vacuo. After 15 mins. the pressure is released and the insects transferred to B for not less than 3 hrs. Specimens may be stored in 70% ale, mounted in balsam, and sectioned by celloidin or paraffin techniques. Santomerse is a wetting agent. recommended for: injection of insect trachea. 32 FOR INJECTION WARM 32.1 Formulas 32.1 Bensley test. 1929 Moore cit. Knouff 4349, 12 :55 PREPARATION OF COLLOIDAL CARMINE: Dissolvc 40 cariuine in 100 water with 40 am- monia. Leave mixture 12-24 hours. Filter. Boil filtrate until ammonia-free. Add 95% ale. in excess to filtrate to precipitate carmine. Filter. Dry ppt. WORKING mass: Dissolve carmine from above in 50 water. Soak 25 gelatin in 25 water. Melt and mix with carmine. 32.1 Carter 1862 Arch. Med., 3 :2S7 formula: water 90, acetic acid 5, gelatin 12, ammonia 5, carmine 6 preparation: Dissolve carmine in ammonia. Melt 9 gelatin in 60 water. Mix with carmine. Dissolve 3 gelatin in 30 water with acetic acid. Add slowly with constant agitation to hot carmine-gelatin mixture. 32.1 Fol 1883 23635,38:492 STOCK i: Soak 100 gelatin in water. Drain. Melt. stock ii: Warm carmine in 75 water with 25 ammonia. Leave overnight. Filter. Add acetic acid drop by drop until color changes to bright red. preparation of stock mass: Stock I 100; stock II 100. Add the carmine solution to the molten gelatin. Stir well. Chill and shred the chilled mass. Wash the shreds in running water overnight. Drain, remelt, and cast in sheets. Dry sheets. preparation of working mass: Soak stock mass in enough water to cover. Soak 10 minutes. Drain. Melt. 32.1 Fol 1884 Fol 1884, 13 preparation of stock mass: Soak 50 gelatin in sheets in V 32.1 Fol 1883, stock II for 48 hours. Drain. Rinse. Transfer rinsed sheets to 100 0.1% acetic acid solution. Leave overnight. Wash 3 hours in running water. Dry. preparation of working mass: As Fol 1883 32.1 Frey 1877 Frey 1877, 112 formula: water ^..s., gelatin 25, sat. aq. sol. {circ. 40%) barium chloride 50, sulfuric acid o s preparation: Add acid to barium chloride until no further ppt. produced. Soak gelatin in water. Drain. Melt and incorporate with barium sulfate. V 32.1 VARIOUS FORMULAS 665 32.1 Harting (est. 1877a Frey Frey 1877, 111 formula: water 60, gelatin 20, lead acetate 5, potassium dichromate 2.7 preparation: Dissolve lead acetate in 20 water. Dis.solve dichromate in 40 water. Mix slowly and with constant agitation with acetate solution. Heat to 35°C. Soak gelatin in water. Drain. Melt and incorporate with warm lead chromate mass. 32.1 Harting test. 1877b Frey Frey 1877, 112 formula: water 11.5, gelatin 25, lead acetate 25, sodium carbonate 25 preparation: Dissolve lead acetate in 6.5 water. Dissolve sodium carbonate in 5 water. Mix slowly and with constant agitation with acetate solution. Heat to 35°C. Soak gelatin in water. Drain. Melt and mix with load carbonate. 32.1 Hoyer 1882 2981, 2:19 formula: water 80, gelatin 20, glycerol 10, silver nitrate 2, 1% pyrogallic acid 2, chloral hydrate 2 preparation: Soak gelatin in 30 water a few hours. Melt. Dissolve silver nitrate in 50 water. Mix with gelatin. Add 1 % pyrogallic acid to mixture. Agitate few minutes. Add glycerol and chloral hydrate to mixture. 32.1 Krause 1909 23632, 26:1 formula: water 2000, sodium borate 100, carmine 1.5, gelatin 100, 2% HCl q.s., camphor 0.3 preparation: Dissolve carmine in water with sodium borate with boiling. Cool. Chill. Soak gelatin in chilled carmine solution 2-3 days. Drain. Rinse. Transfer rinsed sheets, with added hydrochloric acid, to solution, 6 hours. Drain. Wash. Drain. Melt. Add camphor to molten mass. Cool. 32.1 MacCallum 1926 590,38:153 formula: water 30, gelatin 7, carmine 4, ammonia 4 preparation: Dissolve carmine in ammonia. Add 20 water. Leave 24 hours. Filter. Boil filtrate to volume 10. Add 10 water to reduced filtrate. Again boil to volume 10. Soak gelatin in water. Melt. Mix with carmine solution. 32.1 Moore 1929 4349, 12:55 formula: water 90, gelatin 25, carmine 6, ammonia q.s., potassium iodide 5, acetic acid q.s. preparation: Melt gelatin in 60 water. Soak. Mix carmine in 30 water. Add enough ammonia to dissolve carmine. Add potassium iodide to carmine solution. Mix with gelatin at 25°C. Add enough acetic acid to give pH 7.2. 32.1 Robin 1871a Robin 1871, 23 formula: lard 40, spermaceti 40, beeswax 10, turpentine 15 32.1 Robin 1871b Robin 1871, 32 formula: water 60, glycerol 30, gelatin 10, arsenic trioxide 0.5, phenol 0.1 preparation: Melt the gelatin in water with arsenic trioxide. Add glycerol and phenol to the solution. note: This mass may be mixed with any pigment. 32.1 Robin 1871c Robin 1871, 40 formula: water 130, gelatin 15, silver nitrate 30 preparation: Dissolve gelatin in 100 water with heat. Cool to 30°C. Dissolve silver nitrate in 30 water. Warm to 30°C. Mix with gelatin. 32.1 Stirling test. Cole 1884 Cole 1884b, 29 formula: water 50, gelatin 25, acetic acid q.s., carmine 4, ammonia 4 preparation: Soak gelatin in water. Drain. Melt. Make to 50. Mix ammonia in 50 water and carmine. Leave overnight. Filter. Add acetic acid to filtrate drop by drop until color changes. Add to molten gelatin. 32.1 Thiersch 1865 1780, 1:148 formxtla: water 65, gelatin 15, carmine 10, ammonia 10 preparation: Mix carmine in 30 water with ammonia. Leave overnight. Agitate. Filter. Dissolve gelatin in 35 water with heat. Add to filtrate. 666 METHODS AND FORMULAS V 32.1-V 41.1 32.1 Thiersch test. 1871 Robin Robin 1871, 36 formula: water 140, gelatin 60, potassium dichromate 7.5, lead nitrate 10 preparation: Dissolve 30 gelatin in 70 water with potassium dichromate. Cool to 30°C. Dissolve 30 gelatin in 70 water with lead nitrate. Cool to 30°C. Mix slowly and with constant agitation into dichromate solution. Raise, while constantly stirring, to 100°C. Strain. 32.1 Thiersch test. 1877 Fray Frey, 1877, 113 formula: stock I sat. aq. sol. {circ. 16%) ferrous sulfate; stock II sat. aq. sol. (circ. 33%) potassium ferrocyanide; stock III sat. aq. sol. {circ. 10%) oxalic acid; stock IV gelatin, soaked, strained, and melted WORKING injection mass: Mix stock I 6 and stock IV 15 at about 60°C. Cool to 30°C. Mix stock II 12, stock IV 30. stock III 12 at about 60°C. in order given. Cool to 30°C. Add previous mixture to this slowly and continuously with constant stirring. Without ceasing to stir, raise temperature slowly to 100°C. Strain for use. note: This really excellent blue injection mass fell into disrepute through Robin's un- fortunate mistranslation (Robin 1871, p. 35) of Thiersch's original directions. Thiersch took "eine halt gesalligte Losung von schwefelsaurem Eisenoxydul," which was rendered by Robin as "une solution froide satur^e de protoxyde de fer." The wide circulation by Robin of these very obviously impossible directions led to the dropping of the method from the literature. 32.1 Thiersch (est. 1877 Frey Frey 1877, 115 formula: water 22, gelatin 40, potassium chromate 1, lead nitrate 5 preparation: Dissolve potassium chromate in 12 water. Soak 20 gelatin in water. Drain. Melt and incorporate with chromate solution. Keep at 35°C. Dissolve lead nitrate in 10 water. Soak 20 gelatin in water. Drain. Molt and incorporate at 35°C. with lead solution. Then add to lead gelatin mixture at 35°C. slowly and with constant stirring. Raise to 100°C., stirring at intervals for 1-2 hours. Strain. 32.1 Woodhead test. Cole 1884 Cole 1884b, 29 formula: water 100, gelatin 10, carmine 4, ammonia 8, acetic acid q.s. preparation: Dissolve carmine in ammonia. Soak gelatin in water. Melt at 40°C. Add to carmine. Add acetic acid to mixture, drop by drop, until color changes. V 40 Cleaning Methods and Formulas In addition to the five standard methods given below, many people clean slides by flaming them with a bunsen burner. It is difficult to find a satisfactory cloth with which to dry slides after they have been washed, and unless a piece of old linen is available it is usually bettei' to rinse in alcohol and air dry. It was at one time customary to store all clean slides and covers in 95% alcohol, which was alleged to prevent deterioration of the surface. There is no known method of removing surface cloudiness from an old slide made of bad quality glass. 41.1 Methods and Formulas 41.1 acid alcohol — ronipl. script. metuod: dip slides in acid alcohol and dry without rinsing. Masson 1929 (4341), 12:81) recommends 10%, nitric acid in 95% ale; Gray (1952, 113) prefers 1% acetic acid in 70% ale. 41.1 Lysol method: heat Lysol lo :il)()ut 150°C. Dip slide and rinse off in water. note: This method will even remove resin, laccjuers, varnishes, and parafFm. 41.1 scouring powder method: make a thin cream of any kitchen scouring powder and water. Dip slides, air dry, and repack in boxes. Polish with cloth before use. 41.1 sulfuric-dichromate conipl. script. formula: to 40 sat. a(|. sol. potassium dichromate add, witli due precautions, 60 sulfuric acid V 41.1-V 51.1 VARIOUS FORMULAS 667 method: soak slides and covers for about a day; wash thoroughly in running water note: The cleaning solution may be used as long as any crystals remain at the bottom. 41.1 trisodium phosphate method; soak slides overnight in a 15% solution of trisodium phosphate. Rinse and dry. V 50 Miscellaneous Formulas 51.1 Formulas 51.1 Andre text. 1942 Langeron Langeron 1942, 930 formi'la: water 'M), acetic acid ;30, chloral hydrate 40 RECOMMENDED FOR: rcswelling dried arthropods, 51.1 Baker 1941 11300,61:75 formula: water 36, 95% ale. 54, glycerol 10 RECOMMENDED FOR: softening materials already embedded in paraffin which have proved too tough to cut. The block should be soaked until experiment shows that the material will cut. 51.1 Foster and Gifford 1947 20540b, 22:129 formula: 95% ale. 80, glycerol 10, hydrofluoric acid 10 RECOMMENDED FOR: softcning refractory plant materials before sectioning. 51.1 Gifford 1950a 20540b, 25:161 formula: water 36, 95% ale. 54, acetic acid 10 RECOMMENDED FOR: Softening paraffin-embedded plant tissues after the block has been trimmed to expose a surface of the material. 51.1 Gifford 1950b 20540b, 25:161 formula: water 32, 95% ale. 48, acetic acid 20 RECOMMENDED FOR: as above, but with very refractory material. 51.1 Guyer 1930 Guyer 1930, 30 formula: abs. ale. 55, ether 45, pyroxylin 7, camphor 0.3 preparation: Dissolve ether in abs. ale. with pyroxylin. Add camphor to solution. RECOMMENDED FOR: Scaling, by dipping, stoppers, or corks of vials. 51.1 Hetherington 1922 11428,9:102 formula: water 25, lactic acid 50, 95% ale. 25 RECOMMENDED FOR: rcswelling of dried plant specimens. 51.1 Langeron 1942 Langeron 1942, 8.52 formula: paraffin 65, rubber 35 RECOMMENDED FOR: bottom of Small dishes for dissection. 51.1 Lendrum 1944 20540b, 19:143 formula: glycerol 90, aniline 10 RECOMMENDED FOR: as V 51.1 Baker 1941. 51.1 Schmorl 1928 Schmorl 1928, 90 formula: anhydrous chloroform 32, oil of thyme 16, oil of cedarwood 32, abs, ale. 8, phenol 8 RECOMMENDED FOR: storage of celloidin blocks 51.1 Vesseler 1891 23632, 7:461 formula: beeswax 60, Venice turpentine 30 RECOMMENDED FOR: wax for Supporting specimens. 61.1 Wilson 1946 Tech. Bull, 7 :57 formula: abs. ale. 100, celloidin 4, gum mastic 0.625 preparation: Dissolve celloidin in 75 ale, 25 ether. Evaporate ether and add gum dis- solved in 25 ale. RECOMMENDED FOR: vamishing edge of microtome knife to prevent striations in sections caused by minute nicks in edge of knife. Abbreviations Used The abbreviations used in this work have been drawn from three sources. The first group, invented by the author for bibliographic purposes, is explained fully, with examples, in the introduction. The second group consists of standard pharmaceutical abbreviations, such as a. a. and s.a. used to abbreviate the directions given for the preparation of solutions. The third group contains the common abbreviations such as q.v. and loc. cit. employed in written English. The list which follows contains the meaning of all abbreviations used in the book. ale. a.a. abs. auct. BP cf. circ. cit. compl. script. Gm. in litt. in verb. loc. cit. ml. n.d. N.F. op. cit. ppt. q.s. q.v. s.a. sat. sat. ale. sol. sat. aq. sol. sic sol. test. test. ips. USP of each the amount indicated absolute alcohol author British Pharmacopoeia compare approximately quoting numerous writings gram received as an unpublished written communication received verbally the place [already] quoted milliliter no date National Formulary the work [already] quoted precipitate enough which see in the customary manner saturated saturated alcoholic solution saturated aqueous solution exactly as shown solution according to according to himself United States Pharmacopoij' 669 Books and Periodicals Cited A. Books The following list of books is not intended to be a complete bibliography but only a list of books cited in the preceding pages. A complete bibliography of books on microtechnique has been prepared under the joint authorship of the present writer and his wife (Gray and Gray. Bibliograph}/ of Books on Microtechnique. Dubuque, Iowa, Wm. C. Brown Co., 195-4). Cited as Alzheimer 1910 Anderson 1929 Apdthy 1896-1901 Baker 1945 Beale 1880 Beccari 194G Becher 1921 Becher and Demoll 1913 Behrens 1883 Behrens, Kossel, and Schiefferdecker 1899 Belling 1930 Bensley and Bensley 1938 Besson 1904 Bohm and Oppel 1907 Boitard 1921 Title see Nissl and Alzheimer 1910 Anderson, J. How to stain the nervous system. Edin- burgh, E. Livingstone, 1929. Apathy, Stefan. Die Mikrotechnik der thierischen Mor- phologie, Zwei Abtheilung. Braunschweig, Harald Bruhn, 1896-1901. Baker, John R. Cytological technique, 2nd ed. London, Methuen, 1945. Beale, Lionel S. How to work with the microscope, 5th ed. London, Harrison, 1880. Beccari, Nello. Elementi di technica microscopica, 4th ed. riveduta di \J. Ignesti. Milan, Societa Editrice Libraria, 1946. Becher, Siegfried. Untersuchungen liber Echtfarbung der Zellkerne niit kimstlichen Beizenfarbstoffen. Berlin, Borntraeger, 1921. Becher, S., and R. Demoll. Einfiihrung in die mikro- skopische Technik. Leipzig, Quelle und Meyer, 1913. Behrens, Wilhelm. Hilfsbuch zur Ausflihrung mikro- skopischer Untersuchungen im botanischen Laborator- ium. Braunschweig, Harald Bruhn, 1883. Behrens, W., A. Kossel, and P. Schiefferdecker. Die Gewebe des menschlichen Korpers und ihre mikrosko- pische Untersuchung, erste Band: Das Mikroscop und die Methoden der mikroskopischen Untersuchung. Braunsch- weig, Harald Bruhn, 1889. Belling, John. The use of the microscope, 1st ed. New York, McGraw-Hill, 1930. Bensley, R. R., and S. H. Bensley. Handbook of histo- logical and cytological technique. Chicago, University of Chicago Press, 1938. Besson, Albert. Technique microbiologique et sero- therapique. Paris, Bailliere, 1904. Bohm, Alexander, and Albert Oppel. Manuel^ de tech- nique microscopique, traduit de Tallemand par Etienne de Rouville, 4th ed. Paris, Vigot, 1907. Boitard. Nouveau manuel complet du naturaliste prepara- teur, Nouvelle ed. par Maigne. Paris, Mulo, 1921. 670 BOOKS AND PERIODICALS CITED G71 Cited as Boneval 1890 Cajal and de Castro 1933 Carleton and Leach 1938 Carnoy 1884 Carpenter 1891 Chamberlain 1915 Chamberlain 1932 Chevalier 1882 Clayden 1948 Cole 1883 Cole 1884 Conn 1946 Cooper 1847 Cowdry 1943 Cowdry 1952 Cross and Cole 1903 Davies circ. 1865 Davies 1880 Deflandre 1947 Dobell 1919 Ehrlich, Krause, et al. 1910 Eltringham 1930 \ Fischer 1899 Title lioncval, Rem'. Nonvcau n^xudc i)rati(iuc de technique microscopiciue appliqucc a Thistologie et a rcuil)ryogenic. Paris, Maloine, 1890. Ramon y Cajal, S., and F. de Castro. Elemcntos do tecnica micrografica del sistema nerviosa. Madrid, Tipo- grafia Artistica, 1933. Carleton, H. M., and E. 11. Leach. Histological Tech- nique. 2nd ed. London and New York, Oxford University Press, 1938. Carnoy, Jean Baptiste. La biologic celhdaire. Paris, Doin, 1884. Carpenter, William B. The microscope and its revelations, 7th ed. edited by W. H. Dallenger. Philadelphia, Blakis- ton, 1891. Chamberlain, Charles J. Methods in plant histology, 3rd ed. Chicago, University of Chicago Press, 1915. Idem. 5th ed. 1932. Chevalier, Arthur. L'ctudiant micrographe, 3rd ed. Paris, Chevalier, 1882. Clayden, E. C. Practical section cutting and staining. Brooklyn, N. Y., Chemical Publishing Co., 1948. Cole, Arthur C. Studies in microscopical science, 2 vol. London, Bailliere, Tindall and Cox, 1883-1884. The methods of microscopical research, n. d., bound with Cole 1883, vol. 2. Conn, H. J. Biological stains. Geneva, N. Y., Biotech Publications, 1946. Cooper, Daniel. The microscopic miscellany ; being selec- tions from the Microscopic Journal. London, privately printed, 1847. Cowdry, E. V. Microscopic technique in biology and medicine. Baltimore, Williams and Wilkins, 1943. Laboratory technique in biology and medicine, 3rd ed. Baltimore, Williams and Wilkins, 1952. Cross, M. I., and Martin J. Cole. Modern microscopy, 3rd ed. London, Bailliere, Tindall and Cox, 1903. Davies, Thomas. The preparation and mounting of micro- scopic objects. New York, William Wood, circ. 1865. The preparation and mounting of microscopic objects, edited by John Mathews. London, David Bogue, 1880. Deflandre, G. Microscopie pratique, 2nd ed. Paris, Lech- evalier, 1947. Dobell, Clifford. The amoebae living in man. London, John Bale, Sons and Danielsson, 1919. Ehrlich, Paul, Rudolf Krause, Max Mosse, Heinrich Rosin, and Karl Weigert. Enzyklopadie der mikro- skopischen Tochnik, 2nd ed., 2 vol. BerUn, Urban und Schwarzenberg, 1910. Eltringham, H. Histological and illustrative methods for entomologists. Oxford, Clarendon, Press, 1930. Fischer, Alfred. Fixirung, Farbung und Bau des Proto- plasmas. Jena, Fischer, 1899. G72 METHODS AND FORMULAS USED IN MAKING MICROSCOPE SLIDES Cited as Flemming 1882 Francotte circ. 1890 Frei-Sulzer 1946a Frei-Sulzer 1946b Frey 1877 Friedlaender 1885 Friedlaender 1889 Gage 1896 Gage 1901 Gatenby and Cowdry 1928 Gatenby and Painter 1937 Gerard 1887 Golgi 1903 Graupner 1934 Gray 1952 Griffith and Henfrey 1875 Gunther 1898 Gurr 1951 Guyer 1917 Guyer 1930 Hager 1886 Hall and Herscheimer 1905 Heidenhain 1892 Hogg 1883 Title Flemming, Walther. Zellsubstanz, Kern und ZelltheUung. Leipzig, Vogel, 1882. Francotte, P. Manuel de technique microscopique. Paris, Lebegue, circ. 1890. Frei-Sulzer, M. Mikroskopische Untersuchungsmethoden. Zurich, Andre Schlege], 1946. Lohnende Objekte fur mikroskopische Unter- suchungen und ihre Preparation. Zurich, Andre Schlegel, 1946. Frey, Heinrich. Das Mikroskop und die mikroskopische Technik, 6th ed. Leipzig, Wilhelm Engelmann, 1877. Friedlaender, Carl. The use of the microscope in clinical and pathological examinations, 2nd ed., translated by Henry C. Coe. New York, Appleton, 1885. Mikroskopische Technik zum Gebrauch bei medicinischen und pathologisch-anatomischen Untersuchungen, 4th ed. by C. J. Eberth. Berlin, Fischer, 1889. Gage, Simon Henry. The microscope and microscopical methods, 6th ed. Ithaca, N. Y., Comstock, 1896. The microscope and an introduction to micro- scopic methods and to histology, 8th ed. Ithaca, N. Y., Comstock, 1901. Gatenby, J. Bronte, and E. V. Cowdry. BoUes Lee's microt- omist's vade-mecum. Philadelphia, Blakiston, 1928. Gatenby, J. Bronte, and Theophilus S. Painter. Themicrot- omist's vade-mecum (Bolles Lee), 10th ed. Philadelphia, Blakiston, 1937. Gerard, R. Traite pratique de micrographie. Paris, Doin, 1887. Golgi, Camillo. Opera omnia, 3 vol. Milan, Hoepli, 1903. Graupner, Heinz. Mikroskopische Technik. Leipzig, Aka- demische Verlagsgesellschaft, 1934, Gray, Peter. Handbook of basic microtechnique. Phila- delphia, Blakiston, 1952. Griffith, J. W., and Arthur Henfrey. The Micrographie Dictionary, 3rd ed., 2 vol. London, John van Voorst, 1875. Gunther, Carl. Einflihrung in das Studium der Bakteriol- ogie mit besonderer Beriicksichtigung der mikrosko- pischen Technik, 5th ed. Leipzig, Georg Thieme, 1898. Gurr, Edward. Microscopic staining techniques No. 3. London, Gurr, 1951. Guyer, Michael F. Animal micrology, 2nd ed. Chicago, University of Chicago Press, 1917. Idem. 3rd ed. 1930. Hager, Hermann. Das Mikroskop und seine Aniwendung. Berlin, Julius Springer, 1886. Hall, Walker, and G. Herscheimer. Methods of morbid histology and clinical pathology. Philadelphia, Lippin- cott, 1905. Heidenhain, Martin. Festschrift Herrn A. von KoUiker zur Feier seines fiinfzigjahrigen medicinischen Doktor- jubiljiums. Leipzig, Wilhelm Engellmans, 1892. Hogg, Jabez. The microscope. London, Routledge, 1883. BOOKS AND PERIODICALS CITED 673 Cited as Johansen 1940 Jones 1950 Kahlden 1894 Kahlden and Laurent 189G Kingsbury and Johannsen 1927 Kisser 1926 Langeron 1916 Langcron 1934 Langeron 1942 Langeron 1949 Laporte 1946 Ledermann 1903 Lee 1885 Lee 1890 Lee 1901 Lee 1905 Lee and Mayer 1910 Lillie 1948 Maggi 1895 Mallory 1938 Mallory and Wright 1897 Mallory and Wright 1911 Mallory and Weight 1924 Marsh 1878 Martin 1872 Mayer 1920 McClung 1929 Meakin and Swatman 1949 Meyer 1915 Title Johansen, Donald Alexander. Plant microtechnique. New York, McGraw-Hill, 1940. Jones, Ruth McClung. McClung's handbook of micro- scopical technique, 3rd ed. New York, Hoeber, 1950. Kahlden, C. von. Methods of pathological histology* translated and edited by H. Morley Fletcher. London, Macmillan, 1894. Kahlden, C. von, and O. Laurent. Technique micro- scopique. Paris, Carr6, 1896. Kingsbury, B. F., and 0. A. Johannsen. Histological tech- nique. New York, Wiley, 1927. Kisser, Josef. Leitfaden der botanischen Mikrotechnik. Jena, Fischer, 1926. Langeron, M. Precis de microscopic, 2nd ed. Paris, Masson, 1916. Idem. 5th ed. 1934. Idem. 6th ed. 1942. Idem. 7th ed. 1949. Laporte, L. J. Ce qu'il faut savoir sur le monde micro- scopique. Paris, Lechevalier, 1946. Ledermann, R. Die mikroskopische Technik. Vienna, Alfred Holder, 1903. Lee, Arthur BoUes. The microtomist's vade-mecum. London, Churchill, 1885. Idem. 2nd ed. 1890. Idem. 5th ed. 1901. Idem. 8th ed. 1905. Lee, A. Bolles, and P. Mayer. Grundziige der mikrosko- pischen Technik fiir Zoologen und Anatomen, 4th ed. Berlin, Friedlander, 1910. Lillie, R. D. Histopathologic technic. Philadelphia! Blakiston, 1948. Maggi, L. Tecnica protistologica. Milan, Hoepli, 1895. Mallory, Frank Burr. Pathological technique. Phila- delphia, Saunders, 1938. Mallory, Frank Burr, and James Homer Wright. Patho- logical technique. Philadelphia, Saunders, 1897. Idem. 5th ed. 1911. Idem. 8th ed. 1924. Marsh, Sylvester. Section cutting. London, Churchill, 1878. Martin, John H. A manual of microscopic mounting. London, Churchill, 1872. Mayer, Paul. Zoomikrotechnik. Berlin, Borntrager, 1920. McClung, C. E. Handbook of microscopical technique. New York, Hoeber, 1929. Meakin, S. H., and C. C. Swatman. Mounting diatoms. London, Arthur Barron, 1949. Meyer, Arthur. Erstes mikroskopisches Praktikum, 3rd cd. Jena, Fischer, 1915. 674 METHODS AND FORMULAS USED IN MAKING MICROSCOPE SLIDES Cited as Nissl and Alzheimer 1910 Patzelt 1948 Pollack 1900 Pritchard 1851 Prowazek 1922 Queckett 1855 Ranvier 1875 Rawitz 1895 Richards 1949 Romeis 1948 Roskin 1946 Ruffini 1927 Sass 1940 Schmorl 1928 Schneider 1922 Seiler 1881 Spielmeyer 1924 Squire 1892 Stitt 1916 Stitt 1920 Stohr 1901 Tobias 1936 Wellings circ. 1938 Wethered 1898 Title Nissl, Franz von, and Alois Alzheimer. Histologische und histopathologische Arbeiten liber die Grosshirnrinde. Jena, Fischer, 1910. Patzelt, Viktor. Anleitung zu mikroskopischen Unter- suchungen. Vienna, Urban und Schwarzenberg, 1948. Pollack, Bernard. Les methodes de preparation et de coloration du systeme nerveux, traduit de I'allemand par Jean Nicolaide. Paris, Carre et Naud, 1900. Pritchard, Andrew. A general history of animalcules. London, Whittaker, 1851. Prowazek, S. von. Taschenbuch der mikroskopischen Technik der Protistenuntersuchung, 3rd ed., bearbeitet von V. Jollos. Leipzig, Barth, 1922. Queckett, John. A practical treatise on the use of the microscope, 3rd ed. London, Bailliere, 1855. Ranvier, L. Traite technique d'histologie. Paris, Savy, 1875. Rawitz, Bernhard. Leitfaden fiir histologische Untersuch- ungen. Jena, Fischer, 1895. Richards, Oscar W. The effective use and proper care of the microtome. Buffalo, American Optical Co., 1949. Romeis, Benno. Mikroskopische Technik, 15th ed. Munich, Leibniz, 1948. Roskin, G. E. Mikroskopecheskaya technika. Moscow, Sovetskaya Nauka, 1946. Ruffini, Angelo. Procossi di tecnica embriologica ed istologica. Bologna, Licinio Cappelli, 1927. Sass, John E. Elements of botanical microtechnique. New York, McGraw-Hill, 1940. Schmorl, G. Die pathologisch-histologischen Untersuch- ungs-methoden, 15th ed. Leipzig, Vogel, 1928. Schneider, Hans. Die botanische Mikrotechnik. Jena, Fischer, 1922. Seiler, Carl. Compendium of microscopical technology. Philadelphia, Brinton, 1881. Spielmeyer, W. Technik der mikroskopischen Untersuch- ungen des Nervensy stems. Berlin, Springer, 1924. Squire, Peter Wyatt. Methods and formulae used in the preparation of animal and vegetable tissues for micro- scopical examination. London, Churchill, 1892. Stitt, E. R. Practical bacteriology, blood work and parasitology. 4th ed. Philadelphia, Blakiston, 1916. Idetn. 6th ed. 1920. Stohr, Philipp. Textbook of histology including micro- scopical technique, 4th American ed. translated by Emma L. Bilstein and edited by .\lfred Schafer. Philadelphia, Blakiston, 1901. Tobias, J. Carroll. The student's manual of microscopic technique. London, Chapman and Hall, 1936. Wellings, A. W. Practical microscopy of the teeth and associated parts. New York, Staples, n.d. {circ. 1938). Wethered, Frank J. Medical microscopy. Philadelphia, Blakiston, 1898. BOOKS AND PERIODICALS CITED 675 Cited as Whitman 1885 Wythes 1851 Zimmerman 1893 Zimmerman 189G Zinnscr and Bayne-Jones 1939 Title Whitman, Charles Otis. Methods of research in micro- scopical anatomy and embryology. Boston, Cassino, 1885. Wythes, Joseph H. The microscopist. Philadelphia, Lindsay and Blakiston, 1851. Zimmerman, A. Botanical microtechnique, translated by James Ellis Humphrey. New York, Henry Holt, 1893. Botanical microtechnique, transhited by James Ellis Humphrey. Westminster, Archibald Constable, 189G. Zinnser, Hans, and Stanhope Bayne-Jones. A textbook of bacteriology, 8th ed. New York, Appleton-Century, 1939. Cited as Amer. J. Med. Tech. Arb. hot. inst. Wurz. Arch. Biochem. Arch. Med. Bull. Amer. Soc. A. Microsc. Bull. Soc. Franc. Microsc. J. Pal. Lab. Invest. Micrologist Microscope Microsc. Rec. Mikroskopie Rep. Mass. Gen. Hosp. Tech. Bull. Turtox News. Ward's Bui. B. Periodicals Not Cited in World List Title The American Journal of Medical Technology. Houston, Texas. Arbeiten der botanischer Institut der Universitat Wiirz- burg. Archives of Biochemistry. New York. Archives of Medicine. New York. Bulletin of the American Society of Amateur Microsco- pists. Pittsburgh. Bulletin de la societc francaise de microscopic. Paris. Journal of Paleontology. Tulsa, Oklahoma. Laboratory Investigation. New York. The Micrologist. Manchester, England. The Microscope and Entomological Monthly. London. Watson's Microscope Record. London. Mikroskopie. Zentralblatt flu- mikroskopische Forschung und Methodik. Vienna. Annual Reports of the Massachusetts General Hospital. Boston. Technical Bulletin of the Registry of Medical Tech- nologists. Muncie, Indiana. Turtox News. Chicago. Ward's Natural Science Establishment Bulletin. Roch- ester, New York. C. Periodicals Listed in World List of Scientific Periodicals 2nd Edition 1934 Cited as Title G5 Abhandlungen der Koniglichen Siichsischen Gesellschaft (Akademie) der Wisscn- schaften, Mathematisch-Physische Klasse. Leipzig. 466 Allgemeine Zeitschrift fiir Psychiatrie und psychisch-gerichtlichc Medizin. Berlin. 590 American Journal of Anatomy. Baltimore. 591 American Journal of Botany. Lancaster, Pennsylvania. 591b American Journal of Clinical Pathology. Baltimore. 600a American Journal of Hygiene. Baltimore. 608b American Journal of Pathology. Boston. 617 American Journal of Public Health. New York. 618 American Journal of Public Hygiene and Journal of the Massachusetts Association of Boards of Health. Boston. 623 American Journal of Syphilis. St. Louis, Missouri. 626 American Journal of Tropical iViedicine. Baltimore. 676 METHODS AND FORMULAS USED IN MAKING MICROSCOPE SLIDES Cited as Title 645 American Monthly Microscopical Journal. Washington, D. C. 651 American Naturalist. Boston. 665 American Review of Tuberculosis. New York. 763 Anatomical Record. Philadelphia. 764 Anatomische Hefte: Abteilung 1. Wiesbaden. 766 Anatomischer Anzeiger. Jena. 825 Annales de chimie (et de physique). Paris. 829 Annales de dermatologie et de syphiligraphie. Paris. 857 Annales de I'lnstitut Pasteur. Paris. 899a Annales de parasitologie humaine et comparee. Paris. 915 Annales des sciences naturelles: (a) Botanique, (b) Zoologie. Paris. 966 Annales de la Socicte scientifique de Bruxelles. Louvain. 979 Annali di botanica. Rome. 988 Annali d'igiene (sperimentale). Turin. 1006 Annali di nevrologia. Naples. 1025 Annals of Applied Biology. Cambridge. 1032 Annals of Botany. London. 1048 Annals of Missouri Botanical Garden. St. Louis, Missouri. 1200 Annual Report of the Cancer Research Fund. London. 1683 Arbeiten aus den Zoologischen Instituten der Universitat Wien und der Zoo- logischen Station in Triest. Vienna. 1739 Archiv ftir Anatomie und Physiologic. Leipzig. 1752 Archiv fiir Dermatologie und Syphilis. Vienna and Leipzig. 1756 Archiv fiir Entwicklungsmechanik der Organismen. Leipzig. 1780 Archiv fiir mikroskopische Anatomie (und Entwicklungsmechanik). Bonn. 1789a Archiv fiir pathologische Anatomie. Bratislava. 1798 Archiv fiir Protistenkunde. Jena. 1799 Archiv fiir Psychiatric und Nervenkrankheiten. Berlin. 1820 Archiv fiir Zellforschung. Leipzig. 1823 Archives d'anatomie microscopique. Paris. 1825 Archives de biologic. Paris. 1829 Archives of Dermatology and Syphilology. New York. 1843 Archives de I'lnstitut Pasteur de Tunis. Tunis. 1845 Archives of Internal Medicine. Chicago. 1852 Archives italiennes de biologic. Pisa. 1863 Archives de medecine e.xperimentale et d'anatomie pathologique. Paris. 1878a Archives of Neurology and Psychiatry. Chicago. 1879 Archives of Neurology and Psychiatry. London. 1883 Archives of Ophthalmology. New York. 1886 Archives de parasitologie. Paris. 1887a Archives of Pathology and Laboratory Medicine. Chicago. 1915 Archives de zoologie e.xperimentale et generale. Paris. 1946 Archivio per le scienze mediche. Turin. 1949 Archivio zoologico (italiano). Napoli. 2174 Atti deU'Istituto botanico dclla Universita di Pavia. Milano. 2190 Atti della Societa lombarda di scienze mediche e biologiche. Milano. 2526 Beitrjige zur pathologischen Anatomie und zur allgemeinen Pathologic. Jena. 2626 Bericht der Deutschen Botanischen Gesellschaft. Berlin. 2627 Bericht der Deutschen Chemischen Gesellschaft. Berlin. 2701 Bericht der Naturforschenden Gesellschaft zu Freiburg im Breisegau. 2813 Berliner klinische Wochenschrift. Berlin. 2818 Berliner tierarztliche Wochenschrift. Berlin. 2842 Bibliographia zoologica. Leipzig. 2844 Bibliographic anatomique. Revue des travaux en langue frangaise. Paris and Nancy. 2975 Biological Bulletin of the Marine Biological Laboratory. Wood's Hole, Massa- chusetts. 2981 Biologisches Zentralblatt. Leipzig. 3231 Boletln de la Sociedad espafiola de biologla. Madrid. BOOKS AND TERIODICALS CITED 677 Cited as Title 3232 Boletln de la Real Sociedad espafiola de historia natural. Madrid. 3248 BoUettino della Reale Accademia medica. Genoa. 3381 BoUettino della Societa medico-chirurgica. Pavia. 3389 BoUettino della Societa zoologica italiana. Roma. 3430 Botanical Gazette. Chicago. 3432 Botanical Magazine. Tokyo. 3445 Botanisches Zentralblatt. Jena and Dresden. 3464 Brain: a Journal of Neurology. London. 3566 British Journal of Experimental Pathology. London. 3579 British Medical Journal. London. 3678 Bulletin de I'Academie Royale de Belgique, Classe des sciences. Brussels. 3919 Bulletin biologique de la France et de la Belgique. Paris. 4184 Bulletin of Entomological Research. London. 4285a Bulletin d'histologie appliquee a la physiologic et a la pathologic et de technique microscopique. Paris. 4346 Bulletin international de I'Academie des sciences de Cracovie (de I'Academie polonaise des sciences). Cracow. 4349 Bulletin of the International Association of Medical Museums and Journal of Technical Methods. Montreal and Washington, D. C. 4604 Bulletin of the Museum of Comparative Zoology at Harvard College. Cambridge, Massachusetts. 4956 Bulletin et memoires de la Societe anatomique de Paris. 4992 Bulletin de la Societe beige de microscopie. Brussels. 4999 Bulletin. Societe botanique de France. Paris. 5133 Bulletin de la Societe d'histoire naturelle d'Autun. 5293 Bulletin de la Societe mycologique de France. Paris. 5310 Bulletin de la Societe de pathologic exotique. Paris. 5392 Bulletin de la Societe vaudoise des sciences naturelles. Lausanne. 5401 Bulletin de la Societe zoologique de France. Paris 6011 Cellule. Lierre. 6593 Compte rendu de I'Association des anatomistes. Paris and Nancy. 6628 Compte rendu hebdomadaire des seances de I'Academie des sciences. Paris. 6630 Compte rendu hebdomadaire des seances et memoires de la Societe de biologic, Paris. 6816 Contributions to Embryology (Publications of the Carnegie Institution). Washing- ton, D. C. 7033a Cytologia. Tokyo. 7137 Denkschriften der Medizinisch-naturwissenschaftlichcn Gesellschaft zu Jena. 7141 Dental Cosmos. Philadelphia. 7175 Dermatologische Studien. Hamburg. 7176 Dermatologische Wochenschrift. Leipzig. 7177 Dermatologische Zeitschrift. Berlin. 7276 Deutsche medizinische Wochenschrift. Leipzig. 7282 Deutsche Monatsschrift fiir Zahnheilkunde. Berlin. 7599 Edinburgh Medical Journal. Edinburgh. 7802 Endocrinology. Glendale, California. 7871 Entomological News. Academy of Natural Sciences, Philadelphia. 7936a Ergebnisse der Anatomic und Entwicklungsgeschichte. Wiesbaden. 7962 Ergebnisse der Physiologic. Wiesbaden. 8338 Fauna und Flora des Golfes von Neapel und der Angrenzen Meeresabschnittc. Berlin. 8542a Folia anatomica Japonica. Tokyo. 8545 Folia haematologica. Leipzig. 8645 Fortschritte der Medizin. Halle. 9170 Giornalc italiano dellc malattic venerec c della pelle. Milan. 9775 Hygiene dc la viandc et du lait. Paris. 9940 Indian Journal of Medical Research. Calcutta. 9943 Indian Medical Gazette. Calcutta. 10157 Internationale Monatsschrift flir Anatomic und Physiologic. Leipzig. 678 METHODS AND FORMULAS USED IN MAKING MICROSCOPE SLIDES Cited as Title 10606 Jahrbucli fiir wissenschaftlichc Botanik. Berlin. 10881q Japanese Journal of Zoology. Tokyo. 10899 Jenaische Zeitschrift fiir Natm-wissenschaft. Jena. 10919 Johns Hopkins Hospital Bulletin. Baltimore. 10920 Johns Hopkins Hospital Reports. Baltimore. 10996 Journal of the American Chemical Society. Easton, Pennsylvania. 11006 Journal of the American Medical Association. Chicago. 11022 Journal of the American Veterinary Medical Association. Ithaca, New York. 11024 Journal de I'anatomie et de la physiologic normales et pathologiques de I'homme et des animaux. Paris. 11025 Journal of Anatomy (and Physiology). London. 11032 Journal of Applied Microscopy (and Laboratory Methods). Rochester, New York. 11035 Journal of the Royal Army Medical Corps. London. 11056 Journal of Bacteriology. Baltimore. 11074 Journal de botanique. Paris. 11075 Journal of Botany, British and Foreign. London. 11130 Journal of the College of Science, Imperial University of Tokyo. 11135 Journal of Comparative Neurology (and Psychology). Philadelphia. 11139b Journal du Conseil permanent international pour I'exploration de la mer. Copen- hagen. 11147 Journal of Dental Research. Baltimore. 11189 Journal of Experimental Medicine. New York. 11211 Journal of Genetics. Cambridge. 11250 Journal of Infectious Diseases. Chicago. 11284 Journal of Laboratory and Clinical Medicine. St. Louis, Missouri. 11295 Journal of the Linnean Society (Botany). London. 11307 Journal des maladies cutanees et syphilitiques. Paris. 11343 Journal of Medical Research. Boston. 11360 Journal of the Royal Microscopical Society. London. 11373 Journal of Morphology (and Physiology). Boston. 11392 Journal of Neurology and Psychopathology. Bristol. 11428 Journal of Parasitology. Urbana, Illinois. 11431 Journal of Pathology and Bacteriology. London. 11454 Journal of Physiology. London and Cambridge. 11478 Journal fur Psychologie unci Neurologic. Leipzig. 11479 Journal of the Quekett Microscopical Club. London. 11560 Journal of State Medicine. London. 11571b See 4349. 11587 Journal of Tropical Medicine (and Hygiene). London. 11597 Journal of Urology. Baltimore. 11689 Kansas University Science Bulletin. Lawrence, Kansas. 11796 Kitasato Archives of Experimental Medicine. Tokyo. 11848 Kolloidchemische Beihefte (Erganzungschefte zur Kolloidzeitschrift). Dresden. 11976 Laboratory. Pittsburgh. 11977 Laboratory Journal. London. 11988 Lait. Lyons. 11995 Lancet. London. 13034 Medical Journal of Australia. Sydney. 13172 Medizinisc.he Klinik. Vienna. 13367 Memoirs of the College of Science, Kyoto Imperial LTniversity. Kyoto. 13461 Memorias do Instituto de Butantan. Sao Paulo. 13465 Memorias do Instituto Oswaldo Cruz. Rio de Janeiro. 13495 Memorie della Reale Accademia delle scienze dell'Istituto di Bologna. 13497 Memorie della Reale Accademia delle scienze di Torino. Turin. 13685 Military Surgeon. Washington, D. C. 14246 Mitteilungen aus der Zoologischen Station zu Neapel. Berlin. 14352 Monatsschrifte fiir praktische Dermatologie. Leipzig and Hamburg. 14370 Monatsschrift fiir Psychiatric und Neurologic. Berlin. BOOKS AND PERIODICALS CITED G79 Cited as Title 14425 Monitore zoologico italiano. Florence. 14555 Morphologisches Jahrbuch. Leipzig. 14674 Miinchener inedizinische Wochenschrift. Munich. 14706 Museums Journal. The Organ of the Museums Association. London. 14900 Nature. London. 14901 Nature. Paris. 14975 Naval Medical Bulletin. Washington, D. C. 15058 Neurologisches Zentralblatt. Leipzig. 15063 Nevraxe. Recueil de neurologic normale et pathologique. Louvain. 16035 Parasitology. Cambridge. 16059 Pathologica. Genoa. 16155 PflUgers Archiv filr die gesanitc Physiologic dcr Menschen und der Tiere. Bonn. 16157b Pharmaceutica Acta Helvetiae. Ziirich. 16185a Philadelphia Medical Journal. Philadelphia. 16273 Phytopathologj'. American Phytopathological Society. Ithaca. 16341 Polnisches Archiv fiir biologische und medizinische Wissenschaften. Lemberg. 16550 Presse medicale. Paris. 16592 Proceedings of the Royal Acadcmj- of Sciences. Amsterdam. 16599 Proceedings of the American Academy of Arts and Sciences. Boston. 16730a Proceedings of the Ro.yal Entomological Society of London. London. 16852 Proceedings of the New York Pathological Society. New York. 16913 Proceedings of the Society for Experimental Biology and Medicine. New York. 16916 Proceedings of the Royal Society of Medicine. London. 16953 Proceedings of the Washington Academy of Sciences. Washington, D. C. 16977 Processi verbali della Societa toscana di scienze naturali in Pisa. 17035 Progres medical. Paris. 17191a Protoplasma. Leipzig. 17302 Public Health Reports. Washington, D. C. 17510 Quarterly Journal of Microscopical Science. London. 17770a Recueil des travaux botaniques neerlandais. Socicte botanique neerlandaise, Nimegue. 18640 Report on (of Director of) Veterinary Research. Department of Agriculture, Union of South Africa, Pretoria. 18794 Revista de la Asociacion Medica Argentina. Buenos Aires. 19076 Revue generale de botanique. Paris. 19219 Revue neurologique. Paris. 19227a Revue de pathologic vegetale et d'entomologie agricole. Paris. 19288 Revue Suisse de zoologie et Annales de la Societe zoologique suisse et du Museum d'histoire naturelle de Geneve. Geneva. 19353 Ricerche del Laboratorio di anatomia normale della Reale Universita di Roma. Rome. 19443 Rivista di patologia nervosa e mentale. Florence. 19460 Rivista sperimentale di freniatria e medicina legale delle alienazioni mentali, Reggio-Emilia. 19704d Sang: Biologic et pathologic. Paris. 19938 Science. New York. 20080 Scmaine medicale. Paris. 20170 Sitzungsberichte der Kaiserliclien Akademie der \\'is.senschaftcn in Wien, Mathe- matisch-Naturwissenschaftliche Klasse, Abteilung I: Mineralogie, Krystallog- raphie, Botanik, etc. Abteilung. Ila: ^Lathematik, Astronomic, Physik, etc. Abteilung lib. Chemic. Abteilung III. Anatomic und Physiologic. N'icnna. 20181 Sitzungsberichte des Deutschen Naturwissenschaftlich-medizinischen Vereins fur Bohmen "Lotos" in Prag. Prague. 20188 Sitzungsberichte der Gesellschaft fiir Morphologic und i'hysiologic in Miinchen. Munich. 20189 Sitzungs])crichtc der Gesellschaft Xaturforschendcr Freundc zu Berlin. 20214 Skandinavischc Archiv fiir Physiologic. Leipzig. 20540b Stain Technology. Geneva, New York. 680 METHODS AND FORMULAS USED IN MAKING MICROSCOPE SLIDES Cited as Title 20796 Svenska Akademiens Handlingar. Stockholm. 20936 Technical Bulletin. New York State Agricultural Experiment Station, Geneva, New York. 21344 Trabajos del Laboratorio de investigaciones biol6gicas de la Universidad de Madrid. 21400a Transactions of the American Microscopical Society. Lancaster, Pennsylvania. 21458 Transactions of the Chicago Pathological Society. Chicago. 21559 Transactions and Annual Report. Manchester Microscopical Society, Manchester, England. 21652 Transactions of the Royal Society of Edinburgh. Edinburgh. 21654 Transactions of the Royal Society of South Australia. Adelaide. 21671 Transactions of the Royal Society of Tropical Medicine and Hygiene. London. 22073 University of California Publications in Botany. Berkeley. 22084 University of California Publications in Zoology. Berkeley. 22238 Verhandelingen der Koninklijke Akademie van wetenschappen: (a) Natuur- histonische geologische en medische wetenschappen. (b) Wis- en natuurkundige wetenschappen. Amsterdam. 22246 Verhandlungen der Anatomischen Gesellschaft. Jena. 22264 Verhandlungen der Deutschen Pathologischen Gesselschaft. Jena. 22302 Verhandlungen der Physikalisch-medizinischen Gesellschaft zu Wiirzburg. 22575 Virchows Archiv fiir pathologische Anatomic und Physiologie und fiir klinische Medizin. Berlin. 23053a Wissenschafthliche Ergebnisse der Deutschen Tiefsee-Expedition auf dem Damfer "Valdivia." 23253 Zapeske Kievskaho Obshchestva Estestvoesptateleye. Kiev. 23328 Zeitschrift fiir angewandte Mikroskopie und klinische Chemie. Leipzig. 23354 Zeitschrift fiir Biologie. Munich and Berlin. 23418 Zeitschrift fiir die gesamte Anatomie, Abteilung 1: Zeitschrift fiir Anatomic und Entwicklungsgeschichte. Abteilung 2: Zeitschrift fiir Konstitutionslehre. Abteilung 3: Ergebnisse der Anatomie und Entwicklungsgeschichte. Berlin. 23422 Zeitschrift fiir die gesamte experimentelle Medizin. Berlin. 23430 Zeitschrift fiir die gesamte Neurologic und Psychiatric. Berlin. 23454 Zeitschrift fiir Hygiene und Infcktionskrankheitcn. Leipzig. 23507a Zeitschrift fiir mikroskopisch-anatomische Forschung. Leipzig. 23543 Zeitschrift fur physikalischc Chemie, Stochiometrie und Verwandtschaftslehre. Leipzig. 23632 Zeitschrift fiir wissenschaftliche Mikroskopie und fiir mikroskopische Technik. Leipzig. 23635 Zeitschrift fiir wissenschaftliche Zoologie. Leipzig. 23639b Zeitschrift fur Zellforschung und mikroskopische Anatomie. Berlin. 23681 Zentralblatt flu- allgemeine Pathologic und pathologische Anatomie. Jena. 23684 Zentralblatt fiir Bakteriologie, Parasitenkunde und Infcktionskrankheitcn. Jena, 23720 Zentralblatt fiir Innerc Medizin. Leipzig. 23730 Zentralblatt fiir die mcdizinischen Wissenschaften. Berlin. 23732 Zentralblatt fiir NervenheUkundc. Berlin. 23820 Zoologica. Orginalabhandlungen. Hcrausgcgeben von C. Chun, Stuttgart. 23831 Zoologische Jahrbuchcr, Abteilung 1: Systematik (Okologie), Geographic und Biologic. Abteilung 2: Anatomic und Ontogcnie. Abteilung 3. Allgemeine Zoologie und Physiologic. Jena. 23833 Zoologischer Anzeiger. Leipzig. Index Abbott's method for bacterial spores, 484 Abbreviations, examples of use, 1-2 list of, 669 Abies balsamea, 640 Abopon mountant, 632 Accelerators for metal stains, 613-615 Accessory dye staining solutions, 514-521 decimal divisions of, 514 Accessory fixative solutions, 254-266 decimal divisions of, 254 Accessory metal staining solutions, 613-621 decimal divisions of, 612 general remarks on, 612 Acetaldehyde, fixative combination with for- maldehyde, 193 Acetic acid, as ingredient of, Alcorn and Yeager's preservatives, 177; Assier's preservatives, 76; Berlese's mountant, 631; Fol's adhesive for free sections, 659; Gage's adhesive, 661; Hogg's varnish, 653; macerating fluids, see under name of author, (262-263); Morrison's mountant, 632; Oudemann's preservative, 177; Pampel's preserva- tive, 177; Railliet's preservatives, 177; Robin's preservatives, 177; Seller's gela- tin cement, 655; Semmen's gum arable varnish, 652; Swan's mountant, 633; Wilson's Venice turpentine mountant, 638; Zirkle's balsam, 640; Zirkle's dex- trin mountant, 637; Zirkle's gelatin mountants, 635-636; Zirkle's pectin mountant, 637; Zirkle's Venice turpen- tine mountant, 638 as solvent, 626 effect on fixation, 189 for, clearing slides, 131; clearing whole- mounts, 56; fixing pollen grains, 309; swelling arthropods, 667 in fixative combinations with, alcohol, 189, 190; formaldehyde, 191; other ingredients, see under name of other ingredient; trichloroacetic acid, 190 physical properties of, 626 Acetic-iodine, as differentiator, 381 Aceto-carmines, 303 Acetone, as dehydrant in Lillie's stain, 381 as ingredient of, Bolsi's accelerator, 616; Cajal's de- velopers, 616, 617; Cajal's formalde- hyde accelerator, 615; Claverdon's fixa- tive, 224; Foley's developer, 617; Her- rara's formaldehyde accelerator, 613; Jahnel's developer, 618; Levaditi and Manouelian's developer, 618; Lobo's developer, 618; Michaelis's double stain, 345; Rodriguez's formaldehyde ac- celerator, 614; Riiyter's adhesive for nitrocellulose-paraffin ribbons, 658 fixative combinations with, formaldehyde, 193; other ingredients see under name of other ingredient for, dehydrating, 333, 345, 348; dispersing polyvinyl alcohol, 636 physical properties, 623 Achucarro's method for macroglia, 585 Acid alcohol, for, cleaning slides, 131, 666; differentiating stains, 519 Acid alizarine blue, in, Buzaglo's triple stain, 368; Korn- hauser's quadruple stain, 369, 370; Petersen's triple stain, 372 staining combinations with, alizarine viridine and quinalizarine, 368; anilin blue and orange G, 372; fast green FCF, orcein and orange G, 367, 370 Acid-alum-hematoxylins, 289, 290 Acid-fast bacteria, in, sections, 495-498; smears, 476-478 preparation of smear of, 469 urea for differentiating, 520 Acid fuchsin, as, nuclear stain, 319, 357-362; plasma stain, 320, 321 for staining, actinomyccs, (Lignifere), 504; adrenal cortex (Fujiware), 429; algae (Baum- gartel), 511; astrocytes (Beyer), 413; bacteria in sections (Fraenkel), 492; bacterial smears (Maneval), 473; bac- terial spores (Botelho), 485; blood 681 682 INDEX Acid fuchsin — {continued) for staining — (continued) (Buzard), 416; blood (Kahldeu and Laurent), 418; calcified tissues (Eros), 383; colloid in thyroid (Kraus), 456; elastic fibers (Gomori), 388; embryonic bone (von Korff), 384; Entamoeba histolytica (Doboll), 507; erythrocytes (Thompson), 420; fibroglia fibrils (Mal- lory), 456; flame cells (Coutelin), 430 fungus in plant tissue (Vaughn), 502 granules in mast cells (Bayard), 455 granules in Schwann cells (Reich), 457 intracellular "organisms" (Laidlaw), 461; microflora in scale insects (Mahdis- san), 504; Negri bodies (Petragnani), 467; nervous tissue (Alzheimer), 409; nervous tissue (MacConnail), 406; neu- roglia (Alzheimer), 411; neuroglia (Had- jiloff), 413; nuclei (Cooper), 435; nuclei in yeasts (Mancval), 512; Paneth cells (Klein), 452; paranuclear bodies (Sannomiya), 457; Phaeophyta (Johan- sen), 421; pituitary (Colin), 425 pituitary (Crook and Russel), 425 pituitary (Maurer and Lewis), 426 pituitary (Maxwell), 427; pollen tubes (Buchholz), 421; reticulum fibers (Kult- schitzky), 424; spirochetes (Weiss), 481; vaginal smears (Fuller), 430; vaginal smears (Papanicolaou), 431 in, Barbrow's triple stain, 359; Bensley's triple stain, 359; Biondi's triple stain, 356; Bohm and Oppcl's triple stain, 361; Brillmeyer's triple contrast, 336; Cason's double stain, 360; Crossmon's triple contrast, 336; Delamare's quad- ruple stain, 365; Delephine's double contrast, 328; Drew-Murray's triple stain, 369; Ehrlich's triple stain, 356; Fite's double contrast, 328; Foley's double stain, 356; Foley's triple stain 356; Fraenkel's simple contrast, 321 van Gieson's double contrast, 327 Goldner's quadruple contrast, 337 Guinard's double stain, 356; Hansen's double contrast, 328; Haythorne's triple contrast, 337; Houcke's quadruple stain, 352; Kingsbury and Johansen'a double contrast, 328; Krause's triple stain, 356, 357; Krichiski's triple stain, 360; KuU's triple stain, 353; Ladewig's quadruple stain, 365; Lendrum and McFarlane's quadruple contrast, 337; Lillie's double contrast, 328; Lillie's double stain, 370; McFarlane's quin- tuple contrast, 338; McFarlane's triple contrasts, 338; Male's double contrast, 328; Mallory's triple stain, 360; Mane- Acid fuchsin — (continued) in — {continued) val's simple contrast, 321; Maresch's triple stain, 361; Masson's double con- trast, 337-338; Masson's triple con- trast, 340; Masson's triple stain, 360; Mayer's triple stain, 357; Milligan's triple stain, 360; Millot's double con- trast, 341; Ohlmacher's double con- trast, 327; Oppel's triple stain, 357; Pasini's quadruple contrast, 338; Pia- nese's triple contrast, 341; PoUak's quadruple contrast, 338; Roskin's quad- ruple stain, 372; Roskin's triple stain, 361; Schaffer's double contrast, 327; Schneidan's double stain, 360; Scriban's triple contrast, 341; Squire's double stain, 357; Thome's double stain, 357; Unna's double contrast, 327; Wallart and Honette's double contrast, 339; Weigert's double contrast, 328; Wil- helmini's double contrast, 328 staining combinations with, acid violet, 426; anilin blue, 337, 360, 424; anilin blue, azocarmine, eosin B and orcein, 361; anilin blue, eosin B and orcein, 338, 361; anilin blue, eosin Y and orange G, 431; anilin blue, hema- toxylin and picric acid, 427; anilin blue and orange G, 336, 337, 359, 360, 425, 427; anilin blue, orange G, picric acid and ponceau 2R, 338; anilin blue and picric acid, 338; anilin blue and ponceau 2R, 338, 413; aurantia and methyl green, 450; aurantia and toluidine blue, 353, 394, 443 ; azophloxine, ponceau 2R, orange G and light green, 337 ; brilliant green, 481; brilliant green and picric acid, 341; carmine, methyl violet and picric acid, 389; crystal violet, 368, 481, 504; erythrosin and methyl green, 435; fast green FCG, 370, 421; fast green FCF and hematoxylin, 456; fast green FCF, orange G, picric acid and ponceau 2R, 337; fast yellow, 339; hematoxylin, 406; hematoxylin, magenta III and picric acid, 498; hematoxylin and methyl blue, 426; hematoxylin, methyl blue and orange G, 365; hematoxylin, new magenta and picric acid, 496; hematoxylin and orange G, 461; hema- toxylin, orange G and ponceau 2R, 430; hematoxylin, orcein and picric acid, 365, 372; indigocarmine and picric acid, 507; iodine green, 391; light green, 413, 421, 425, 485; light green, orange G and ponceau 211, 337, 338; light green and picric acid, 409, 411; malachite green and martius yellow, 341, 502; malachite green and orange G, 359; martius yel- INDEX G83 Acid fuchsin — (continued) staining combinations with — {continved) low, 329, 341; metanil yellow, 338; metanil yellow and picric acid, 340; methyl blue, 425, 442, 443; methyl blue and orange G, 3G0, 413, 423; methyl green, 35G, 357, 442; methyl green and orange G, 359, 418; methyl green and picric acid, 357, 361; methylene blue, 456, 492, 511; methylene blue and orange G, 359; methylene blue, thionine and toluidine blue, 352; methylene blue, trypan blue, 394; orange G, 329; orange G and brilliant cresyl blue, 361; orange G and light green, 336; orange G and methyl green, 356, 357; orange G and toluidine blue, 351, 359, 452; picric acid, 327, 328, 420; picric acid and hematoxylin, 365; picric acid and nile blue sulfate, 369; ponceau 2R, 429; thionine, 360; toluidine blue, 457; wool green FCF, 370 Acid fuchsin, see also Altmann's acid fuchsin Acid green, for staining spirochetes (Weiss), 481 staining combinations with, carmine, 392, 393; crystal violet, 481; magenta, 481; safranin, 481 Acid macerating agents, 262-263 Acid violet, for staining, mitochondria (Bailey), 411; pancreas (Baley), 428; pituitary (Maurer and Lewis), 426; pituitary (Severinghaus), 427 in Weiss's methods for spirochetes, 481 staining combinations with, acid fuchsin. 426; brilliant green, 481; magenta, 428, 481; magenta and methyl green, 427; safranin, 481 "Acidic" dyes, 270, 271 Acidophile cells, of pituitary, 425, 426 Acids, sec under Acetic, Phosphotungstic, etc. Acineta, 52 Acinous cells, 428, 499 Acridine red, in Stropeni's double stain, 357 Acrolein, as ingredient of Cajal's accelerator, 615 Actinomycetes, 494, 501, 504, 505 Adamkiewicz's method for nervous tissue, 409 Adam's method for acid-fast bacteria in sec- tions, 495 Addison's method for Nissl granules, 445 Adhesives, decimal divisions of, 650 for, free sections, 659; minute objects, 661; nitrocellulose sections, 659; paraffin sec- tions, 657-660 general remarks on, 656 Adrenal, methods for, 430 Adrenal — {continued ) nerves in, 558, 568 osmic stain for, 529 Adrenal cortex, Fujiware's method for, 429 Agar, as ingredient of, Chatton's embedding medium, 643; Gravis's adhesive for paraffin ribbons, 657; Samuel's embedding medium, 645 Agduhr's silver diammine stain, 575, 576 in Agduhr's method for neurofibrils, 581 Agduhr, see also Bielschowsky, 257 Agulhon and Chavennes' method for Pappen- heim's stain, 350 Aignier's fixative, 220, 238 Aladar-Anjeszky's method for bacterial spores, 485 Albert's method for diphtheria bacilli, 489 Albrecht's magenta, 315 method for acid-fast bacteria in sections, 495 Albumen, see Egg albumen Alcohol, as dehydrating agent, 622 as ingredient of, Baker's narcotic, 265; Golthard's de- hydrating mixture, 628; Lo Bianco's narcotic, 265 as macerating agent, 262 for, cleaning balsam moimts, 58; fixing nematodes, 36; narcotizing Crustacea, 45 recommended series for dehydration, 55, 96, 124 Alcohol-chloroform, for dehydrating nitro- cellulose sections, 152 Alcohol-glycerol preservatives, 176, 177 Alcohol-miscible mountants, 637-639 Alcoholic, accelerators, 614 borax-carmines, 301 "Bouin's fixative," 224 carmines, 301 differentiation of, 293 iron-hematoxylins, 281, 282 iron-mordant hematoxjdins, 282 plasma stains, 320 Alcohols, physical properties of, 623, 625, 626 Alcorn and Worley's method for perithecia of Erysiphacea, 511 Alcorn and Yeager's preservative, 177 Alexander's method for acid-fast bacteria, 476 Alexander and Jackson's method for acid- fast bacteria, 476 Alexander, see also Campbell, Doherty, 416, 417 Alfieri's method for bleaching, 261 Algae, aqueous wholemounts of, 23 collecting Crustacea from, 49 concentrating, 27, 28 dehydration of delicate, 65 Eckert's preservative for, 178 684 INDEX Algae — (continued) Emig's fixative for, 219 fixation of, 65 fluid wholemounts of, 27-29 Kirchner's preservatives for, 180 mounting individual, 67 Pfeiffer's preservatives for, 179 staining, 65 staining methods for, 511-573 wholemount in Venice turpentine, 64-66 Alimentary canal, nerve endings in, 535 Alizarine red S, as example of laking stain, 270 for staining, bone, 377, 383-385; bone and cartilage, (Williams), 386; bony scales, (Wil- liams), 386; erythrocytes, (Okajama), 418; fish embryo, (NoUister)," 384; macroglea, (Benda), 142; mitochondria, (Benda), 412; nervous tissues, (Schrot- ter), 411 in Szlitz's polychrome contrast, 329 staining combinations with, crystal violet, 442; hematoxylin, 382; methyl green, 383; toluidine blue, 384, 386, 412 Alizarine viridine, in Buzaglo's triple stain, 368 Alkaline macerating fluids, 264 Alkanet, as stain, 308 staining combination with, iodine green and chrome yellow, 393 Allen's fixatives, picric-chromic-formalde- hyde-acetic, 226, 238 recommended use, 95 picric-formaldehyde-acetic, 223, 238 Allen's mountant, 631 Allen and McClung's fixatives, 221 Alli's mordant, 515 in Alli's method for intestinal protozoans, 506 Allium, 433 Allyl alcohol, in Cajal's method for human brain, 552 physical properties of, 623 a-cells, of, pancreas, 420, 428, 429; pitui- tary, 426, 427 a-naphthol, as mordant in Graham's double stain, 417 Alphabetization, rules used, 4 Altmann's, acid fuchsin, 441 m. Bailey's method for mitochondria, 441; Bensley's triple stain, 359 Cann's method for mitochondria, 442 Hollande's method for mitochondria 443; Kiyono's differentiator for, 521 Kidl's method for mitochondria, 4415 Miller's method for muscle, 394 Milovidov's method for proplastids. Altmann's, acid fuchsin — (continued) in — (continued) mitochondria and starch, 450; Milovi- dov's method for bacteria and mito- chondria, 444; Schmorl's method for Schidde's granules, 457; Volkonsky's methods for mitochondria, 444 preparation of, 440 adhesive for free sections, 659 embedding wax, 646 fixatives, mercuric-acetic, 208, 238; mercuric- formic, 210, 238; osmic-dichromate. 202, 238, (in Altmann's method for mitochondria, 441) injection fluid, 662 method for mitochondria, 441 on olive oil injections, 163 Alum, see Chrome alum. Potassium alum, etc. Alum-carmines, 300-301 diluant for, 293 Alum-hematoxylins, 286-290 Aluminum acetate, as ingredient of, Haug's hematoxylin, 288; Pittfield's crystal violet, 483; Topping's preserva- tive, 181 as mordant for, Szlitz's alizarine, 329 Aluminum chloride as ingredient of, Cesares- Gil's mordant, 517 Cretin's mordant, 517 Mayer's carmines, 301, 307 hematoxylin, 292 Aluminum nitrate as ingredient of, Rawitz's carmine, 307 Rawitz's hematoxylin, 292 Aluminum stearate, as ingredient of Per- ruche's varnish, 654 Aluminum sulfate, as ingredient of, Langeron's adhesive for free sections 660; Peterssens gallocyanin, 313 in Kornhauser's quadruple stain, 369 Aluminum-carmines, 307, 454 Aluminum-hematoxylin, 292, 431, 454 Alveolar epithelium, 534 Alzheimer's methods for, granules in nerve cells, 454 nervous tissue, 401, 409 neuroglia, 411, 412 Alzheimer's stains, lithium-hematoxylin, 290 in Alzheimer's method for granules in nerve cells, 454 methyl blue-eosin, 371 in his method for nervous tissue, 401 Amber oil, as ingredient of Muir and Judah's cement, 656 Ambrosione's method for diphtheria bacilli, 489 Amebas, method of fixation, 52 parasitic, 508, 509 Amidol, see Diaminophenol hydrochloride INDEX G85 Ammann's dehydrating mixtures, G28 preservatives, 177, 178 in, Guegin's method for fungus in tissue scrapings, 504; Rivalier and Seydel's method for filamentous fvmgi, 512; Svvartz and Conant's method for fungus in tissue scrapings, 505 Ammerman's fixative, 228, 238 Amnion, horn of, 460 Ammonia, as ingredient of, metal stain accelerators, 013-015; Schridde's fixative removers, 250; silver diammine stains, 575-581 for bleaching muscle, 378 for cleaning, diatoms, 39; radiolaria, 19 Ammonia alum, as diluant for, alum car- mines, 293 in Westpahl's double stain, 373 Ammonia-carmines, 305, 307 Ammonium acetate, as ingredient of, Lenoir's fixative remover, 255; Thiersch's carmine, 307 in MacConaill's method for nervous tissue, 406 Ammonium alum as ingredient of, Boitard's preservative, 176 carmine stains, see under author's name (300-301) Gage's decalcifying fluid, 258 Goadby's preservatives, 176 hematoxylin stains, see under author's name (286-290) Ammonium bromide, as ingredient of for- maldehyde accelerators, 613-614 in Gans's method for microglia, 586 Ammonium carbonate, as ingredient of Bohm and Oppel's safranin, 314 Ammonium chlorate, as ingredient of Haug's carmine, 307 Ammonium chlorostannate, in Bensley's method for thyroid, 428 Ammonium chromate, as ingredient of Landois's macerating solution, 264 as fixative, 232 fixative combination with formaldehyde and acetic acid, 235 Ammonium dichromate, fixative combina- tions with, chromic acid, formaldehyde and acetic acid, 231; copper sulfate, 220; picric acid, formaldehyde and acetic acid, 227 in Vastarini-Crcsi's mordant, 517 staining combination with silver, 604 Ammonium hydroxide, see Ammonia Ammonium molybdate, as ingredient of, Besta's fixatives, 193; Clara's hematoxy- lin, 290; Clara's mordant, 515; Tur- chini's fixative, 227 Anunonium molybdate — (continued) as mordant, 371 in, Bensley and Bensley's method for al- veolar epithelium, 534; Lugaro's method for Nissl granules, 446; MacConaill's method for nervous tissue, 406; thiazin nerve stains, 401-402 Ammonium oxalate, as ingredient of, Herrara's formaldehyde accelerator, 613 in. Mucker's method for bacterial smears, 473; Rodriguez's method for oligo- dendria, 589 Ammonium picrate, danger of, 397 Ammonium sulfate, as ingredient of, Paquin and Goddard's hematoxylin, 286 Ammonium sulfide, in, Mallory's method for hemosiderin gran- ules, 456; Urechia and Nagu's method for reticulum, 595 Ammonium thiocyanate, as ingredient of, gold toning solutions, 620; Henneguy's stain, 364 Ammonium uranate, fixative combination with chromic and acetic acids, 232 preparation of, 232 Ammonium vanadate, as ingredient of, Heidenhain's hematoxylin, 291 in Azoulay's method for cerebellum, 609 Amphibian blood, 418 fixatives for, 193 Amphibian eggs, removal of albumen from, 33 Amphibian embryos, technique for section- ing, 133-136 Slater and Dornfeld's stain for, 367 see also Embryos, heavily yolked Amphibian tendons, nerve endings in, 540 Amphioxus, sections of, 357-359 softening Bouin-fixed, 358 Amprino's silver-chromic stain, 603 in Amprino's method for reticulum fibers, 607 Amyl acetate, physical properties of, 625 Amyl alcohol, as ingredient of, Pritchard's developer, 619 for dehydrating Masson's stain, 333 in Langeron's wax embedding method, 646 Peeter's wax embedding method, 647 Amyl carbinol, 625 Amyl nitrite, as vasodilator, 168 for killing animals before injection, 163 Amyloid, special methods for, 450-453 Andeer's decalcifying fluid, 256 Anderson's, chrome mordant, 515 embedding medium, 642 for frozen sections, 158ff, 159 fat insolubilizer, 514 686 INDEX Anderson's — {continued) iron-iodine mordant, 517 iron mordant, 515 methods for, degenerative changes, 528; myelin sheaths, 404, (applied to section of spinal cord, 397-399); neuroglia, 412; Nissl granules, 445 stains, acid-alum hematoxylin, 289; alum- carmine, 300, (in Anderson's technique for nervous tissues, 404) ; iron-hema- toxylin, 285 Andersson, see Hultgren, 233 Andre's mountant, 631 swelling fluid for arthropods, G67 Andriezen's, fixative, 203, 238 silver-osmic-dichromate method for nerv- ous tissues, 605 Androgenic cells, 429 Anesthetized nerves, differentiation from unanesthetized, 404 Angelucci's fixative, 217 Anglade and Morel's method for neuroglia, 412 Anguillula, 35 Anilin blue, as plasma stain, 320, 321 for staining, algae, 65; algae (Semmen), 512; as- trocytes (Beyer), 413; bacterial smears (Maneval), 473; centrosomes (Heiden- hain), 455; filamentous algae (Cham- berlain), 511; filamentous fungi (Lang- eron), 512; filamentous fungi (Rivalier and Seydel), 512; fungus in plant tissue (Ravn), 502; fungus in tissue scrapings (Guegin), 604; fungus in tissue scrapings (Schubert), 505; fungus in tissue scrap- ings (Swartz and Conant), 505; fungus in wood (Cartwright), 501; gastric cells (Hoecke and Sebruyn), 431; kidney (de Galantha), 423; myxosporids (Lang- eron), 508; nervous tissues (Foley), 567; nervous tissues (Prince), 411; nu- clei (Darrow), 435; pancreas (Baley), 428; pancreas (Gomori), 429; Perono- sporales (Lepik), 502; Peronosporales (Mangin), 502; pituitary (Berblinger and Bergdorf), 425; pituitary (Cleve- land and Wolfe), 425; pituitary (Daw- son and Fnedgood), 425; pituitary (Koneff), 426; pituitary (Perry and (Lochead), 427; pituitary (Romeis), 427; pituitary (Spark), 427; plas- modesma (Gardiner), 421; radulae (Langeron), 513; reticulum fibers (Kult- schitzky), 424; vaginal smears (Papa- nicolaou), 431 in, Bensley's simple contrast, 321; Ben- Anilin blue — {continued) in — {continued) sley's triple stain, 359; Brillmeyer's triple contrast, 336; Cason's triple stain, 360; Grossmen's triple contrast, 336; Haythorne's triple contrast, 337; Heidenhain's triple stain, 361; Ko- hashi's triple stain, 361; Koneff's double stain, 365; Kostowiecki's double con- trast, 327; Krugenberg and Thielman's triple stain, 370; Lendrum and Mc- Farlane's quintuple contrast, 337; Lil- lie's double contrast, 326, 337-338; Lillie's quadruple stain, 366; Lillie's triple contrast, 346; Lopez's triple stain, 371; McFarlane'a quintuple contrast, 338; McFarlane's triple contrast, 338; Maneval's simple contrast, 321; Mas- son's double contrast, 339; Masson's triple stain, 360; Milligan's triple stain, 360; Mollier's quintuple stain, 366; Paquin and Goddard's sextuple stain, 366; Pasini's quadruple contrast, 338; Petersen's triple stain, 372; Schleicher's triple stain, 372; Unna's double con- trast, 327; Unna's double stain, 365; Unna's quadruple stain, 364 staining combinations with, acid alizarine blue and orange G, 372; acid fuchsin, 337, 360, 424; acid fuchsin, azocarmine, eosin B and orcein, 361; acid fuchsin, eosin B and orcein, 338, 361 ; acid fuchsin, eosin Y and orange G, 431; acid fuchsin, hematoxylin and picric acid, 427; acid fuchsin and orange G, 359, 360, 427; acid fuchsin, orange G, picric acid and ponceau 2R, 338; acid fuchsin, orange G and ponceau 2R, 413; acid fuchsin, picric acid, 338; acid fuchsin and ponceau 2R, 338; azocar- mine and magenta, 427; azocarmine and orange G, 361, 372, 425, 426, 427, 429 Biebrich scarlet and hematoxylin, 366 Biebrich scarlet and picric acid, 340 Bismarck brown, hematoxylin, methyl violet and saffron, 431; carmine, creso- fuchsin and orange G, 425; carmine and picric acid, 508, 513; eosin, hematoxylin, orange G and phloxine, 366; eosin and orange G, 428; eosin, orcein and safra- nin, 431; eosin B and phloxine, 370; eosin Y, 387; erythrosin, hematoxylin and orange G, 425; erythrosin, magenta and methyl orange, 411; ethyl eosin, orcein and safranin, 364; fast green FCF, gallocyanin and protein silver, 567; hematoxylin, 365; magenta and methyl orange, 371; orange G, 327, 388; orange G and acid fuchsin, 336, 337; orceillin BB, 502; orcein, 327; phloxine, 340, INDEX G87 Anilin blue — {continued) staining combinations with — (conliniicd) 511; picric acid, 326, 421; picric acid and safranin, 393, 501; ponceau 2R, 339; safranin, 365, 395, 435, 502; Sudan III, 504 Anilin green, as ingredient of Zimmerman's shellac varnish, 654 Anilin hydrochloride, as stain for chitin (Bethe), 390 in, Kuhne's method for acid-fast bacteria in sections, 497; Langrand's method for actinomyces, 504 Anilin sulfate, as ingredient of Atkin's crj's- tal violet, 319 Aniline, as ingredient of. Cole's clearing mixture, 628; Babes safranin, 314; Dupres's magenta, 315; Goodpasture and Burnett's magenta, 315; Lendrum's softening fluid for em- bedded objects, 667; Maneval's ma- genta, 316; Sterling's gentian violet, 319; Weigert's clearing mixture, 628; Zwaademaker's safranin, 315 as solvent for dyes, 410 for differentiating, fungus hyphae, 500; Rubaschkin's methj'l violet, 414 Aniline's fixative, 223 Aniline-chloroform, for differentiating stains, 413 Aniline-xylene for differentiating, Anderson's Victoria blue, 412; Galescu's crystal violet, 413; Haythorn's crystal violet, 494; Kromayer's methyl violet, 424; Lehrmitte and Guccione's Vic- toria blue, 414; iNIerzbacher's Victoria blue, 414; Unna's crystal violet, 424; various stains, 458, 462 Anjeszky's method for bacterial spores, 485 Annelida, wholemounts of, 53 Annelida, see also Polj^chaetae, Earthworm, etc. Anonymous, celestin blue B, 313 for rat tongue, 324 in Proescher, Zapata and McNaught's technique, 372 decalcifying fluid, 256 method for plasmodium, 506 toluidine blue, 316 Anterior lobe, of pituitary, 426« Anthers, McClintock's method for chromo- somes, 437 preparation of smears from, 74 Anthony's method for bacterial capsules, 488 Anthozoa, narcotization and fixation, 53 Antiformin, 470 Antimony potassium tartrate, as ingredient of Zetnow's accelerator, 615 Antimony potassium tartrate — (continued) as mordant in Nemec's method for plas- tids, 450 Aoyama's fixative, 236, 238 in Aoyama's method for Golgi bodies, 608 Apdthy's, Canada balsam mountant, 639 cement, 655 for mounting nitrocellulose blocks, 145 clearing mixture for celloidin blocks, 628 fixatives, alcoholic mercuric chloride, 207, 238; mercuric-acetic, 208, 238; osmic-mer- curic, 196, 238 gelatin embedding medium, 642 gold-mercury method for nervous tissue, 538 macerating fluid, 262 method for nerve endings, 534 mountant, 631 nitrocellulose cement, 661 stains, acid-alum-hematoxylin, 289 ; dichromate mordant hematoxjdin, 283 Apathy and Boeke's fixative, 210, 211, 238 Apical meristem, cell walls of, 610 Aprobarbital, as ingredient of, de Castro's decalcifying fluids, 257 Volkonsky's narcotic, 266 Aquarium cement, Cigalas's, 655 Aqueous varnishes, 652 Aqueous wholemounts, 21-31 cell cements for, 21 cells for, 21 coverslip cements for, 23 Hanley's method of sealing, 31 of protozoa, 23 preservatives for, 23 sealing coverslip on, 24, 25ff, 26fT, 27 specific examples, 27-31 Spence's method of sealing, 26 technique of sealing, 29 Arcadi's, method for oligodendria, 585 silver diammine stain, 575, 576 Arcangeli's stains, alum-carmines, 300 boric-carmine, 306 picro-carmine, 302 Archibald and Marshall's, gum mountant, 636 preservative, 178 Argand's method for elastic fibers, 387 Argentophil cells, silver diammine methods for, 595, 596 Argentophil granules, 567 Armitage's, fixatives, formaldehyde-acetic, 191; picric-acetic, 221, 238 sandarac mountant, 638 Armuzzi and Stempel's developer, 616 in Armuzzi and Stempel's method for spirochetes, 560 688 INDEX Arndt's method for glycogen and fat, 451 Arnold's, fixative, 228, 238 macerating fluid, 264 triple stain, 351 Aronson's method for nervous tissue, 409 Arrangement of book, explanation of, 1 Arsenic-gold method for nervous tissue, 541 Arsenic trioxide, as ingredient of fixatives, arsenic-formaldeh_yde, 23G in, Krajian's method for acid-fast bacteria in sections, 497; Alartmotti's method for elastic fibers, 607 staining combinations with, potassium dichromate and silver nitrate, 607; silver nitrate, 605 Arteries, injection in chicken embryo, 166- 168 Arthropod blood, 417 Arthropod eyes, bleaching, 261 Arthropod nerves, gold-osmic method for, 541 Arthropods, Cole's clearing mixture for, 628 collecting small, 43-45 impregnating with glycerol, 32 mj'xosporids in sections of, 508 preservatives for, 177 reswelling dried, 667 Arthropods, see also Insects, Crustacea, etc. Artigas's mastic mountant, 637 Artschwager's adhesive for paraffin ribbons, 659 Ascoli's developer, 616 in Ascoli's method for leech nerves, 551 Ascospores, in yeasts, 512 Ashby's method for bacterial spores, 585 Aspergillus fumigatus, 503 Asphalt, as ingredient of, Beale's Brunswick black, 652; Benoit- Bazille's varnish, 652; Chevalier's var- . nish, 653; Davies's varnish, 653; Hood and Neill's cement, 656; Johnston's embedding wax, 646; IMuir and Judah's cement, 656; Robin's cement, 656 Asphalt varnish formulas for, 653 for sealing, dr,y wholemounts, 16; wholemounts, 20, 25ff, 26 Assier's preservative, 176 Assmann's double stain, 344 Astrangia, 85 Astrocytes, Merlaud's method for, 558 Be3^er's method for, 413 gold-chrome methods for, 541 del Rio-Hortega's methods for, 587, 588 Atkins's, crystal violet, 319 in Atkins's method for Gram-positive bacteria, 474 iodine mordant, 517 Atkins's — (continued) iodine mordant — (continued) in Kopeloff and Beerman's method for Gram-positive bacteria, 475 Attaching, celloiding sections to slide, 659 free sections to slide, 659-660 freshwater microfauna to slide, 67 labels to slide, 661 objects to coverslip, 661 objects to slide, 660-661 paraffin ribbons to slide, 657-659 protozoa to slide, 67, 660 Aubertin, see Laigret, 463 Auerbach, buds of, 552 Auerbach's double stain, 356 Auguste's magenta, 316 in Auguste's method for acid-fast bac- teria, 476 Aurantia, as plasma stain, 320 danger of, 439 for staining, bacteria and mitochondria (Milovidov), 444; gastric gland cells (Zimmerman), 432; mitochondria (Kull), 443; mito- chondria (Volkovsky), 444; Plasmodium (Hignami), 506; proplastids, mitochon- dria and starch, 450 in, Ehrlich's triple stain, 369; Hayem's double contrast, 328; KuU's triple stain, 353; simple solution, 320 staining combinations with, acid fuchsin and methyl green, 450; acid fuchsin and toluidine blue, 353, 394, 443; eosin W, 328; eosin Y and indulin, 369; hematoxylin, 432; magenta, 506 Aurin, for staining acid-fast bacteria (Pap- penheim), 477 Auxochromes, 270 Avian, blood parasites, 508 spirochetes, 561 Axis cylinders, dye staining methods for, 410-411 gold methods for, 535 gold-dichromate methods for, 541 gold-mercur\' methods for, 539 in sciatic nerve, 564-566 mercuric-dichromate methods for, 610 protein silver methods for, 567 silver diammine methods for, 581-583 Axons, in picric fixed material, 555 of meduUated fibers, 553 Azoacid B, in Menner's method for ganglia in wholemounts, 410 Azocarmine, for staining, bone marrow (Ralston and Wells), 431; chromaffin granules, (Gomori), 430; kidney (de Galantha), 423; pancreas (Gomori), 429; pituitary (Dawson and INDEX 689 Azocarmino, for staining — {continued) Fricdgoo(l), 425; pituitary (Konoff), 42G; pitviitary (Perry and Locliead), 427; pituitary (Romeis), 428; pituitary (Wallraff), 428; reticulum, collagen and myofibrils (Long), 597 in, Ileidenluiin's triple stain, 3G1; Ko- hashi's triple stain, 301; MoUier's quintuple stain, 366; Schleicher's triple stain, 372; Volkman and Strauss's triple stain, 373 staining combinations with, anilin blue and magenta, 427; anilin blue and orange G, 361, 372, 423, 425, 426, 427, 429; anilin blue, eosin B, acid fuchsin and orcein, 361; crystal violet and naphthol green, 373; hematoxylin, naphthol green B and orcein, 366; light green and silver diammine, 597; methyl blue and tartrazine, 430; toluidine blue, 428, 431 Azofuchsin in, Lillie's quadruple stain, 370 staining combinations with, brilliant pur- purin R, naphthol blue black and picric acid, 370 Azophloxine, for staining, bacteria in milk (Zaribnecky), 492; skin (Doublin), 567 in, Romeis's quadruple stain, 367 staining combinations with, coelestin blue B, 372; hematoxylin, light green, magenta and orange G, 367; light green and orange G, 337; napha- zarine, 492; ponceau 2R, orange G, acid fuchsin and light green, 337 Azoulay's method for, cerebellum, 609 myelin sheaths, 529 Azur, in, Blank's triple stain, 352; various double stains with eosin, 347-350 staining combinations with, eosin, 347; mercurochrome, 352; methy- lene blue, 506; methyl blue, methylene blue and orange G, 418; orange G, 464; orcein, 424; Sudan black B, 420 Azur A, for staining diphtheria bacilli (Kinyoun), 489 in, Cowdry's triple stain, 351; Gcschickter's double stain, 352; Kingsley's quadruple stain, 348, 349 staining combinations witli, eosin and thionin, 348; eosin B, 349; eosin Y, 349; eosin Y, methylene blue and methylene violet, 348, 349; erie garnet, 352; methylene blue and tolui- dine blue, 489; orange G and phloxine, 351 Azur B, for staining, Plasmodium (IIol)I) and Tlionipson), 508; Negri bodies (Jordan and Heather), 465 staining combinations with, eosin and methylene blue, 508; eosin Y and phloxine, 465 Azur C, in Holmes and French's triple stain, 352 staining combinations with, eosin B,; eosin Y, 349; eosin Y and Orange 11,352; ethyl eosin, 347 Azur I, for staining, intracellular "organisms" (Ilosokowa), 461; fungus in skin scrapings (Schlciff), 505; Paschen bodies (Craigie), 464 in, MacNeal's triple stain, 349; Turwitsch's method for Paschen bodies, 467 staining combinations with, crystal violet, eosin, and methylene blue, 461 ; eosin Y and methylene violet, 349; mercurochrome and methylene blue, 464; phloxine, 348 Azur II, for staining, acid-fast bacteria in sections (Douglas), 496; bacteria in sections, (Mallory), 493; blood (Ugruimow), 420; Plasmo- dium (Medalia, Kahaner and Singer), 509; Plasmodium (Villain and Comte), 510; Rickettsiae, (Lepine), 463 in, Houcke's triple stain, 351; Langeron's stain, 317 staining combinations with, eosin, 349, 350; eosin B, 510; eosin BA, 420; eosin Y, 349, 418; eosin Y and methylene blue, 349, 350; eosin Y, orange G, thionine and toluidine blue, 351; ethyl eosin, 347; methylene blue, 509; methylene blue and phloxine, 493; methylene violet, 319; safranin, 463 B Babes's safranin, 314 in, Laguesse's triple stain, 364; Land's method for plant sections, 392 Bachman's method for fungus in skin scrap- ings, 502 Bachuber's fixative, 227, 238 Background materials for dry wholemounts, 13 Backman's method for nuclei, 434 Bacsich's, chrome-iron mordant, 515 lithium-hematoxylin, 290 methods for, blood, 416; nervous tissue, 401 Bacteria, acid-fast, in sections, 495 690 INDEX Bacteria — (continued) differential staining of dead, 491 differentiation from mitochondria, 444 fat in, 491 Gram-positive, in sections, 493 in, leukocytes, 490, 491; milk, 490, 491, 492; plant tissues, 444, 498 sections, mentioned in, Holmes and French's triple stain, 352; Langeron's double stain, 353; Masson's double stain, 353; Masson's triple stain, 371; silver nitrate meth- ods for, 563 Neisser's granules in, 489-490 polar bodies in, 491 Bacterial capsules, in sections, 498 in smears, 487-489 Bacterial flagella, dye staining methods for, 481-484 silver diammine methods for, 598 silver-iron method for, 608 silver nitrate methods for, 563 silver-sulfate method for, 609 Bacterial smears, 473-492 acid-fast, 476-478 Gram-positive, 474-476 mountant for, 641 silver diammine methods for, 598 Bacterial spores, general remarks, 467-468 permanganate stain for, 611 stains for, 484-487 Badertscher's method for sebaceous glands, 422 Bailey's method for, mitochondria, 441 neuroglea, 412 Bailey's mordant, 517 in Bailey's method for bacterial flagella, 481 Baillif and Kimbrough's method for blood, 416 Baird's method for connective tissue spreads, 394 Baker, on selection of grasshoppers, 310 on spermatogenesis in Triturus, 272 Baker's fixatives, calcium-formaldehyde, 236, 238 osmic-chromic-acetic, 200, 238 Baker's, glycerol jelly, 633 method for, Golgi bodies, 442 mitochondria, 441, 443 narcotic, 265 softening fluid for embedded objects, 667 Baker and Crawford on osmotic pressure of fixatives, 187 Baker and Thomas's fixative, 202, 238 in Baker and Thomas's method for mito- chondria, 442 Balbiani's method for nuclei in ciliates, 434 Balbuena's alcoholic accelerator, 614 developer, 616 method for retina, 544-546, 551 silver staining solution, 550 toner, 620 Baley's fixative, 217, 238 in Baley's methods for pancreas, 428 Banard, machine for sealing glycerol mounts, 33 Bank and Davenport's fixative, 237 Barbadoes earth, separation of radiolarians from, 18 Barbrow's triple stain, 365 Bardelli and Cille's method for Zymonema, 503 Barium chlorate, as ingredient of bleaching solutions, 262 Barium chloride, as ingredient of, Frey's injection fluid, 663; Frey's injec- tion mass, 664; Hagmann's injection fluid, 664 in Forsgren's triple stain, 423 Barium eosinate, 348 Barium peroxide, for ripening hematoxylin, 284 Bark, collecting animals from, 44 Barker's method for microglea, 585 Barlow's ethyl cellulose embedding medium, 649 Barnabo on pH of fixatives, 188 Barnett, see Dawson, 567 Barret's fixatives, 199, 238 Barreto's method for Negri bodies, 464 Barrett's method for pollen mother cells, 435 Bartelmez's fixatives, 189, 238, 239 in Bartelmez's method for lorains in fish larvae, 551 Bartha, see Mitter, 437 Basal bodies in ciliates, 456, 559 Basement membrane, mentioned in Lillie's quadruple stain, 370 "Basic" dyes, 270, 271 Basic fuchsin, see Magenta Basophil cells, of pituitary, 425-428 Bassal, see Morel, 286, 516 Bastian's preservative, 178 Basu, see Knowles, 567 Batillon's fixative, 228, 239 Bauer's, developer, 616 in Bauer's method for spirochetes in tooth buds, 560 fixative, 223 method for, neuroglea, 412 nitrocellulose embedding, 647 silver staining solution, 550 Bauer and Leriche's triple stain, 352 Baumgartel's method for, algae, 511 leprosy bacilli in sections, 495 Bay oil, physical properties of, 624 INDEX G91 Bayberry wax, as ingredient of, Maxwell's embedding wax, ()4(); Pohl- man's embedding wax, 647; Waterman's embedding wax, 647 use in embedding media, 97 Bayerl's decalcifying lluid, 256 Bayne-Jones, see Zinsser, 464 Beach, see Davenport, 192 Beale's, ammonia-carmine, 305 cements, 652 French cement, 655 glycerol jelly, 633 gold size, 652 injection Huid, 662 as ingredient of Ranvier's injection- fluid, 663 marine glue, 655 preservative, 179 sealing wax varnish, 652 in Landois' macerating fluid, 264 varnishes, 652 Beams's method for Golgi bodies, 541 Bean's method for Nissl granules, 445 Beauchamp on narcotizing Vorticella, 265 Beauverie's method for diphtheria bacilli, 489 Beauverie and Hollande's differentiator, 520 as substitute for glyceric ethic, 466 in Langeron's technique, 317 Becher's stains, gallocyanin, 313 naphthopurpurin, 313 polychrome coelestin blue, 368 polychrome gallamin blue, 368 polychrome quinalizarine, 368 in Buzaglo's triple stain, 368 Becher and DemoU's, Canada balsam moun- tant, 639 fixatives, chrome-acetic, 228, 239; formaldehyde- acetic, 191, 239; mercuric-cupric-for- maldehyde-acetic, 213; mercuric-picric- acetic, 213, 239; osmic-mercuric-picric- acetic, 197, 239; picric-formaldehyde- acetic, 223; picric-hydrochloric, 223; platinic-mercuric, 206, 239 macerating fluids, 262 Bechtol's method for, bone, 382 cartilage, 382 Becker's method for spirochetes, 478 Becker, see also Crough, 506 Beckworth's method for nerves in teeth, 534 Becue, see Lemiere, 504 Beech, see Davenport, 554, 577 Beechwood creosote, 625 Beerman, see Kopeloff, 475 Beeswax, as ingredient of, Cigalas's cement, 655; Griffith's cement, 655; Kroenig's cement, 656; Martin's adhesive, 661; Martin's cement, 656; Mendeleef's cement, 656; Robin's in- Beeswax, as ingredient of — (continued) jection mass, 665; Vesseler's wax for dishes, 667; wax embedding media, 646- 647 Beetle, catalogue rules, 2-3 Beguin's fixative, 208, 239 Behrens's, cement for aqueous wholemounts, 23 copal varnish, 652 fixative, 219 for rat tongue, 324 method for bile capillaries, 422 Behrens, Kossel and Schiefferdecker's mac- erating fluid, 264 Belar's fixatives, osmic-chromic-acetic, 200, 239 zinc-acetic, 235, 239 Belezky's gelatin embedding medium, 643 silver diammine stain, 575, 576 in Belezky's method for neuroglia, 586 Bellido on, de-waxing shellac, 653 shellac, 651 Bellido's gelatin cement, use of, 39, 40 gelatin varnish, 652 method for mounting diatoms, 39, 40 Belling's, carmine mordant, 517 as ingredient of Belling's aceto-carmine, 302 cement, 655 fixative, 230, 239 in Hancock's method for nuclei, 435 used for lily bud, 149 stains, aceto-carmine, 302 for staining Chironomus salivary chro- mosomes, 299 in, Bradley's method for plant ovaries, 435; McClintock's method for nu- clei, 437; Semmen's mountant, 633; Zirkle's pectin mountant, 636 Belloni's alum-hematoxylin, 286 decalcifying fluid, 256 Bell's cement, 652 method for fat, 447 Benario's fixative, 190, 239 Benda's, fixatives, cupric - chromic -formaldehyde - acetic, 220; dichromate-nitric, 232, (in Benda's method for mitochondria, 442) ; osmic- chromic-acetic, 202, 239, (in Geither's method for nuclei, 435); osmic-dichro- mate-acetic, 202, 239 iron mordant, 515 as ingredient of Lee's iron carmine, 304 in Benda's iron-mordant hematoxylin, 281 method for, fat, 447; macroglia, 412; mitochondria, 442 "Benda's" safranin, 314 092 INDEX " Benda-uranic " fixative, 232 van Beneden's fixative, 208 recommended use, 95 van Beneden and Heyt's fixative, 189, 239 Bennliold's method for amyloid, 451 Benoit's fixatives, mercuric-dichromate-ura- nium, 219, 239 osmic-mercuric-dichromate-uranium, 198, 239 in Hollande's method for mitochondria, 443 Benoit-Bazille's asphalt varnish, 652 Bensley's, fixatives, mercuric-dichromate, 216; mercuric- dichromate-formaldehyde, 217, (in Ben- sley's method for canaliculi in plant cells, 454) ; osmic-dichromate-acetic, 204, 239, (recommended use, 95) injection fluid, 662 injection mass, 664 method for, canaliculi in plant cells, 454; mitochon- dria, 443; thyroid, 428 stains, acid fuchsin-orange G-anilin blue, 359; anilin blue-phosphomolybdic acid, 321, (in Bensley's method for thyroid, 428) ; brazilin, 308, (in Bensley's method for thyroid, 428) Bensley and Bensley's fixatives, mercuric-dichromate, 216, 239; mer- curic-dichromate-acetic, 216, 239; mer- curic-dichromate-formaldehyde, 217, 239; osmic-dichromate-acetic, 204, 239 method for, alveolar epithelium, 534; reticulum fibers, 591 on purifying Canada balsam, 639 silver diammine stains, 575, 577 stains, acid fuchsin-crystal violet, 368; chromic- mordant hematoxylin, 283 Benzamine, see Eucaine Benzene, as ingredient of Hollande's fixative, 225 as solvent for, gum damar, 640; rosin, 640 for clearing, 76 for clearing medusae, 298 physical properties of, 625 technique of clearing with, 129 Benzidine, for staining, blood vessels (Williams), 432; bone marrow (McJunkin), 456; capillaries (Campbell and Alexander), 416; capil- laries (Doherty, Suk and Alexander), 417; capillaries (Pickworth), 419; capil- laries (Slominski and Cunge), 420; capillaries (Ziegler), 421; invertebrate nervous system (Romeis), 411; oxidase Benzidine, for staining — (continued) granules (Brice), 455; oxidase granules (Graham), 417; peroxidase (Sato), 419 Benzine, 651 Benzoazurine for staining fungus in plant tissues (Langeron), 502 Benzol, see Benzene Benzopurpurin, as plasma stain, 320 Benzoyl peroxide, in Szecsi's tissue reviver, 515 Benzyl benzoate, as mountant, 380 Berberian's method for fungus in skin scrapings, 503 Berblinger and Bergdorf's method for pituitary, 425 Bergamot oil, as ingredient of, Eycleshymer's clearing mixture, 628; Gatenby and Painter's clearing mix- ture, 628 in Kedrovsky's method for mitosis, 434 physical properties of, 624 Bergdorf, see Berblinger, 425 ten Berge, see Heringa, 644 Berkely's fixative, 203, 240 silver-phosphomolybdic stain, 603 in Berkeley's method for nervous tissue, 603 Berlese funnel, 43, 44ff, 45 for nematodes in feces, 35 Berlese's mountant, 631 index of refraction of, 42 Bernhardt's mountant, 636 in Bernhardt's method for fungus in tissue scrapings, 503 Bertrand and Guillain's, formaldehyde ac- celerator, 613 method for oligoglia in ganglia, 586 silver diammine stain, 577 Bertrand and Mcdakovitch's method for acid-fast bacilli in sections, 496 Besson's fixative, 195 in Borrel's method for coccidia, 506 Besson's method for, Aspergillus fumigatus, 503 bacterial capsules, 487 bacterial spores, 485 Besta's fixatives, tin-formaldehyde, 236, 239 formaldehj'de-acetaldehyde, 193, 239 Besta's method for nervous tissues, 401 Best's carmine, 451 in, Arndt's method for glycogen and fat, 451; Best's method for glycogen, 451; Neukirch's method for glycogen, 453 ;8-cell, of pancreas, 428 i3-cells, of pituitary, 427, 428 Beta-pinenes, as ingredient of mountants, 641 Bethe's method for, frog brain, 395-397 nervous tissues, 401 INDEX 693 Bethe's stain for chitin, 390 Bethe and Monkeberg's method for neuro- fibriUae, 402 van Beust's decalcifying fluid, 256 Beyard's method for granules in mast cells, 455 Beyer's, embedding wax, 646 method for astrocytes, 413 " B " fixatives, 209 Bhaduri and Semmens' fixative, 231 Bhattacharji, see Singh, 318 Bianco, see Lo Bianco Bibliography, G70, 680 Biceps, motor end plates in, 554 Bidegaray's method for intestinal proto- zoans, 506 Biebrich scarlet, as plasma stain, 320 for staining, bone and cartilage (Bechtol), 382; pan- creas (Bowie), 428; pepsinogen granules (Bowie), 428; vaginal smears (Shoor), 431 Kefalas's double stain, 365; Lillic's double contrast, 337; Lillie's double stain, 370; Lillie's quadruple stain, 366; Lillie's triple contrast, 340; simple solu- tion, 320 staining combinations with, anilin blue (or methyl blue or wool green S), 366; anilin blue and picric acid, 340; Biebrich scarlet, 365; ethyl violet, 428; 455, fast green FCF, 337; fast green FCF and orange G, 431; methyl blue, 370; methylene blue, 382 Bielschowsky's methods for, nerve endings, 569-571 nervous tissues, 581 Bielschowsky's silver diammine stain, 575, 577 in, Achucarros's method for macroglia, 585; Bielschowsky's methods for nerv- ous tissues, 581; Cajal's method for peritubular sheath, 590; Doinikow's method for regenerating nerves, 582; Ferguson's method for spirochetes, 597; Kernohan's method for nervous tissues, 583; Orban's method for dentine, 596; Baton's method for embryonic fish nerves, 584; del Rio-Hortega's method for astrocytes, 587; Schutz's method for nervous tissues, 585; Snessarew'smethod for reticulum, 594; Szepsenwol's method for nervous tissue, 585; Wilder's metliod for reticulum fibers, 595 preparation of, 569, 570 Bielschowsky-Agduhr's method for decalci- fication, 257 Bigolow's fixative, 221, 240 Biggart's method for pituitarj', 425 Bignami's fixative, 208 in Bignami's method for Plasmodium, 506 Bigot's method for Zymonema, 503 Bile capillaries, dj^e staining methods for, 422, 423 silver diammine method for, 597 Bile duct, Forsgren's method for, 423 Bing and Ellerman's fixative, 193, 240 in their method for nervous tissues, 402 Biondi's triple stains, 356 for smears of Monocj'stis, 73 Biot's magenta, 315 Blot's method for acid-fast bacteria, 477 Birch-Hirschfeld's method for amyloid, 451 Birge and Imhoff's, decalcifying fluid, 260 method for decalcification, 256 Bismark brown, for staining, amyloid (Birch-Hirschfeld), 451; bac- terial spores (Tribondeau), 487; carti- lage (Semichon), 386; diphtheria bacilli (Ljubinsky), 490; diphtheria bacilli (Neisser), 490; diphtheria bacilli (Tri- bondeau and Dubreuil), 490; fat (Lillie), 448; gastric cells (Iloecke and Sebruyn), 431; pancreas (]\Iiiller), 429; plankton (Francotte), 513; Rickettsiae (Lepine), 463; Thallophyta (Johansen), 512; vaginal smears (Papinicolaou), 432 in, Bohm and Oppel's triple stain, 368; Lillie's triple stain, 366 staining combination with, anilin blue, hematoxylin, carmine and crystal violet, 389; crystal violet, 451, 487, 490; dahlia violet and methyl green, 368; eosin Y, light green and orange G, 432; fast green FCF, 393, 512; light green, 429; magenta, 389, 463; malachite green, 513; methyl violet and saffron, 431; methylene blue, 490; oil blue N, 448: thionine, 490 Bismark brown Y in staining combination with fast green FCF and hema- toxylin, 366 Bismuth nitrate, as ingredient of Groves's cement, 653 Bitter's method for bacterial spores, 485 Bizzozero's fixative, 193, 240 picro-carmine, 303 Bizzozero-Vassale's method for nuclei, 435 Black paper, as background for dry whole- mounts, 13 Black, see Stovall, 467 Blair and Davies's method for nerves in heart, 551 Blanc's fixative, 222, 240 Bland and Canti's method for intracellular "organisms," 461 Blank's triple stain, 352 694 INDEX Blank and McCarthy's Carbowax embed- ding medium, 643 Blayde's preservatives, 179 Bleaching methods, 261 for blood, 54 Bles's fixative, 191, 240 Blood, amphibian, 418 arthropod, 417 bleaching, 54, 262 fat in, 416, 420 fixatives for, 193 invertebrate, 418 preparing smears of, 71 mentioned in, Bauer and Leriche's triple stain, 352; Groat's quadruple stain, 349; Holmes and French's triple stain, 352; Lan- geron's double stain, 355; special stains for, 416-421; stained smear, 343-344; Twort's double stain, 372, 373 Blood, see also Erythrocytes, Leukocytes, etc. Blood parasites, silver diammine method for, 698 Blood parasites, see also Plasmodium, etc. Blood smears, liquid petrolatum for mount- ing, 34 preparation, 69, 70ff Blood vessels, endothelium of, 664 mentioned in Hubin's triple stain, 340 nerves in wholemounts of, 567 Bloom's method for bile capillaries, 423 Blueing hematoxylin, 284 Boccardi's developer, 616 method for nerve endings, 535 Bock's fixative, 233, 240 in his method for bone, 382 Bodecker's decalcifying fluid, 257 "Bodian" techniques, 564-568 Bodian's developer, 616 in, Bacsich's method for nervous tissue, 566; Bodian's method for nervous tissue, 566; MacFarland and Daven- port's method for nerves in adrenal, 567; McManus's method for spinal cord, 555; Rogoff's method for mos- quito brain, 568 toner, 620 Boeke's fixative, 190, 240 method for fungus in skin scrapings, 503 silver diammine method for nervous tis- sues, 581 Boeke, see also Apdthy, 210 Bogert's fixative, 208, 240 Bohm's method for nerve endings, 534 Bohm and Davidoff's, adhesive for paraffin ribbons, 657 fixatives, 234, 240 Bohm and Oppel's fixatives, dichromate-formaldehyde-acetic, 234, 240, (for fixing entire mouse, 137) ; mer- curic-formaldehyde-acetic, 212, 240; mercuric-picric-acetic, 214, 240 osmic method for myelin sheaths, 529 stains, acid fuchsin-orange G-brilliant cresyl blue, 361; alum-cochineal, 301; Bismark brown-dahlia violet-methyl green, 368; methylene blue-thionin-eosin B, 347; phenosafranin, 314; safranin, 314 Bohm and Oppel's triple stain, 361 staining combinations with, acid fuchsin and orange G, 361; eosin Y and methylene blue, 352; neutral red, 417 Bohmer's alum-hematoxylin, 284 as ingredient of Liengme's hematoxylin, 291 m. Galesesco and Bratiano's method for fat, 448; Haythorne's triple stain, 337; Kuhne's method for acid-fast bacteria in sections, 497; Letulle's method for acid-fast bacteria in sections, 497; Schmorl's method for nuclei, 437 Bohner's method for Rickettsiae, 462 Boiling point of, dehydrating agents, 623 synthetic clearing agents, 625, 626 universal solvents, 626 Boissezon's method for myelin sheaths, 410 Boitard's preservatives, 176 Bolcek's fixative remover, 255 Bolsi's accelerators, 615 in Bolsi's method for, macroglia, 586; neuroglia, 558 Bolton's, method for myelin sheaths, 404 silver dichromate method for nervous tis- sues, 604 Bond's method for Rickettsiae, 462 Bone, canaliculi in, 81 ground sections of, 87 Haversian canals, 81 in wholemounts, preserving embryos to show, 180 lamellae in, 81 mentioned in, Bohm and Oppel's triple stain, 361; Dupres's double contrast, 339; Gausin's triple stain, 351; Hubin's triple con- trast, 340; Masson's double contrast, 337; Patay's double contrast, 339; Schumway's triple stain, 372; Unna's orcein-anilin blue double contrast, 327 preparation of ground section of, 81-85 reticulum fibers in, 594 sawing slabs for sections, 82ff special stains for, 382-386 INDEX 605 Bone, see also Calcified structures Bone marrow, megakaryocytes in, 131, 432 methods for, 430, 43 1", 432 peroxidase granules in, 456 sections of in situ, 87 Boni's method for bacterial capsules, 487 Bonnet's, fixative, 200, 240 method for algae, 513 Bonney's triple stain, 3G8 Bonn's fixative, 200, 240 Books cited, 670-675 Borax-carmines, 307 Borax, see Sodium borate Bordeaux red, in. Hall and Powell's method for euglenoid fiagellates, 455; simple solution, 320; Sinton and Mulligan's method for Plasmodium, 510 staining combinations with, eosin and hematoxylin, 510; hematoxy- lin, 455 Boric acid, as differentiator for, Agulhon and Chavannes's stain, 350 as ingredient of, Arcangeli's alum-carmine, 300; Ar- cangeli's carmine, 306; Boitard's pre- servatives, 176; Foley's developer, 617; Francotte's carmine, 306; Goodrich's macerating fluid, 263; Grawitz's pre- servatives, 179 Boric-carmines, 306 Borine, see Moleshott, 264 "Borrers"blue, 317, 350 Borrel's double contrast, 325 fixative, 195, 240 method for coccidia, 506 Bostr0m's method for actinomycetes, 501 Botelho's method for bacterial spores, 485 Bottle, aspirator, in injection techniques, 164ff, 168, 169 Bottles, for storing nitrocellulose solutions, 143 Bouin and Bouin's fixative, 206 Bouin's fixatives, mercuric-formaldehyde, 211 mercuric-formaldehyde-acetic, 212 picric-formaldehyde-acetic, 224, 240 as mordant in Hubin's stain, 340; for fixing testis, 272, (plankton, 53) in, Anderson's technique for neuroglia, 412; Finel's method for nervous tis- sues, 583; Foley's method for axons, 555; Gluckman's method for argen- tophil cells, 595; Gormori's method for pancreas, 429; Kramer's method for insect muscles, 424; Laidlaw's method for reticulum fibers, 593 Margolena's method for pollen, 393 Masson's method for mucin, 454 Bouin's fixatives — (continued) picric-formaldehyde-acetic — (continued) in — (continued) Muller's method for pancreas, 430; Mulon's method for fat, 447 picric-formaldehyde-trichloroacetic, 225 platinic-acetic-formaldehyde, 205 platinic-mercuric-acetic, 206 platinic-mercuric-formaldehyde- formic, 206 Boule's, alcoholic accelerator, 614 developer, 616 fixative, 191, 240 method for nervous tissues, 551 Bourdon's polyvinyl acetate embedding medium, 649 Boveri's fixative, 221 Bowell's method for radulae of mollusca, 394 Bowhill's method for bacterial flagella, 481 Bowie's, fixative, 217, 240 in Bowie's method for pancreas, 428 method for pepsinogen granules, 455 Boxes, paper, for nitrocellulose embedding, 144ff, 150 Boye's double stain, 347 Bradley's fixative, 189, 240 in Bradley's method for plant ovaries, 435 Bragg's embedding method for heavily yolked embryos, 646 Brain, capillaries in, 416, 419 frog, 395-397 human, 552 Brain's, decalcifier, 257 in Brain's method for teeth, 649 gelatin-nitrocellulose-wax embedding method, 649 Brains, in fish larvae, 551 Branca's fixative, 214 Brand's method for neuroglia, 413 Brandt's glycerol jelly, 633 Brasil's fixative, 224 in Bigot's method for Zygonema, 503 Brass's fixative, 195, 207 Bratiano, see Galesesco, 448 Braun's fixative, 196 Braus's fixatives, chromic-formaldehyde, 230 dichromate-formaldehyde, 233 Brazilin, as stain, 308 in O'Leary's method for nervous tissues, 411 Brazille's preservative, 178 Brenn, see Brown, 319, 493, 494 Bretschneider's method for insect brains, 410 Brice's method for oxidase granules, 455 Brilliant black 3B, staining, combination with safranin, 394 Brilliant cresyl blue, for staining, Plasmodium (Field), 507; reticulocytes, G9G INDEX Brilliant cresyl blue, for staining — {con- tinued) 417, 419; salivary gland chromosomes (Mitter and Bartha), 437 Brilliant green, for staining, bacteria in sections (Krajian), 492; spirochetes (Weiss), 418 in Scriban's triple contrast, 341 staining combinations with, acid fuchsin, 481; acid fuchsin and pic- ric acid, 341; acid violet, 481; hematox- ylin and magenta, 492 Brilliant purpurin R, in Lillie's quadruple stain, 370 Brilliant yellow, for staining, acid-fast bac- teria (Doglio), 476 Brillmeyer's triple stain, 336 Brinkmann's fixative, 211 Bristol board, preparation of cells from, 11, 12 Broadhurst and Paley's method for bacteria in milk, 490 Brock's fixative, 199 Bromonaphthalene, for wholemounts, 32 mounting diatoms in, 37 Bromthymol blue, in Ferrari's method for fungi in roots, 501 Brooke's Brunswick black, 653 Brookover's osmic-silver-dichromate method for nervous tissues, 605 Brouardel, see Gouillart, 520 Brouha's fixative, 197 in Beam's method for Golgi bodies, 541 Brown and Brenn's crystal violet, 319 in Brown and Brenn's method for Gram- positive bacteria in sections, 493, 494 Brown's nitrocellulose-wax embedding method, 647 Brucke's injection fluid, 662 Bruere and Kaufmann's glycerol jellj^, 633 Bruesch, see Davenport, 567 Brumpt's preservative, 178 Bruner and Edwards's metliod for, bacteria in leukocytes, 490 bacterial spores, 485 Brun's preservative, 178 Brunotti's gelatin embedding medium, 643 Brunswick black as ingredient of, Eulen- stein's varnish, 653 Beale's, 652 flexible, 653 for sealing dry wholemounts, 16 Bryozoa, collecting, 59 Bubenaite's, method for Golgi bodies, 608 silver-dichromate stain for ganglia, 604 Buchholz's, method for pollen tubes, 421 sandarac mountant, 638 Buds of, Auerbach, 552 Held, 552 Buerger's method for bacterial capsules, 487 Bugnon's method for plant sections, 391 Biihlcr's fixative, 196, 240 Bujor's narcotic, 265 Bullard's alum-hematoxylin, 287 Bunge's, albumen embedding medium, 643 method for bacterial flagella, 481 Burckhardt on pH of fixatives, 188 Burckhardt's fixatives, chromic-dichromate- acetic, 231, 240 osmic-chromic-acetic, 200, 240 platinic-cupric-dichromate, 206 Burdon, Stokes and Kimbrough's method for fat in bacteria, 491 Burke on neutralizing formaldehyde, 190 Burke's, fixative, 190, 240 mordant, 517 in Burke's method for Gram-positive bacteria, 474 Burke, Dickson and Phillip's method for acid-fast bacteria, 476 Burnett, see also Goodpasture Busch's, decalcifying fluids, 257 fixative, 205, 240 in Anderson's method for degenerative changes, 528 Busch, see also Rossolino, 520, 528 Butanol, in Zirkle's schedule, 629 physical properties of, 626 Biitschli's, iron-mordant hematoxylin, 281 for staining spermatozoa, 72 Butterfly scales, 14 adhesives for, 661 Butyl alcohol, in Larbaud's wax embedding method, 647 in Seki's nitrocellulose-wax embedding method, 648 Butyl carbitol, physical properties of, 626 Butyl cellosolve, 627 Butyl chloride, physical properties of, 625 Buxton's method for insect brains, 551 Buzaglo's stains, acid alizarine blue-alizarine viridine-quinalizarine, 368 gallocyanin, 313 Buzard's stain for blood, 416 Buzzi's method for keratin and eleidin, 422 Caberla's preservative, 176 Cadmium chloride, as ingredient of fixative, 236 as mordant for Nakamura et al. magenta, 467 in Baker's method for Golgi bodies, 442 staining combination with silver nitrate, 608 Cadmium sulfate, as ingredient of, Robin's injection fluid, 663 Cain's method for mitochondria, 442 INDEX 697 Cajal's accelerators, 613, 614, 615 in, Balbuena's method for retina, 551 Barker's method for microglia, 585 Bolsi's method for neuroglia, 558 Cajal's method for Colgi bodies, 558 Cajal's method for nerve cells and processes, 551, 552, 553; Cajal's method for neuroglia, 539, 558, 586; Cajal's method for peritubular sheaths, 590; Cajal's method for reticulum fibers, 559; Cajal's method for Schwann cells, 590; Golgi's method for Golgi bodies, 559; Cluck's method for reticulum fibers, 592; Gurdjian's method for nerve cells and processes, 555; Ingleby's method for neuroglia, 586; Martinez's method for neuroglia, 607; Penfield's method for neuroglia, 571, 587; del Rio-Hortega's method for glioblasts, 589; del Rio-Hortega's method for gliosomes, 589; del RIo-Hortega's method for mitochondria, 591; del RIo- Hortega's method for neuroglia, 573- 575, 588, 589, 610; Urechia and Nagu's method for reticulum, 595 Cajal's decalcifying fluids, 259 Cajal's developers, 616, 617 in, Bartelmez's method for fish larvae, 551 ; Cajal's method for axons, 553; Cajal's method for brains of small mammals, 553; Cajal's method for cerebellum, 553; Cajal's method for nerve endings, 553; Cajal's method for nerve fibers, 553; Cajal's method for peritubular sheaths, 590; Cajal's method for reticu- lum fibers, 559; Cajal's method for Schwann cells, 590; Cajal's method for spirochetes, 560; de Castro's method for nerves in teeth, 554; Dogiel's method for corpuscles of Grandry and Herbst, 554; da Fano's method for nerve cells and processes, 554; Favorsky's method for nerve cells and processes, 554; Fello's method for nerve endings, 557; Golgi's method for Golgi bodies, 558, 559; Golgi's method for nerve endings, 605; de No's method for nerve endings, 556; Perez's method for Neissner bodies, 556; Walgren's method for nerve cells and processes, 557; Weatherford's method for Golgi bodies, 559 Cajal's fixatives, dichromate, 232, 240 dichromate-formaldehyde, 233, 241 in Cajal's method for neuroglia, 556 osniic-dichroniate, 203, 240 in, Cajal's method for nervous tissues, Cajal's fixatives — {continued) osmic-dichromate — (continued) in — (continued) 604; Justschenko's method for gan- glia, 605; KupfTer's method for liver, 607 platinic-formaldehyde, 205, 240 in Cajal's methylene blue technique, 402 TU'anium formaldehyde, 236 Cajal's fixer, 621 in Cajal's method for neuroglia, 536-538, 539 Cajal's methods for, brains of small mam- mals, 553 buds of Auerbach, 552 buds of Held, 552 cerebellum, 553 decalcification, 257 embryos of lower mammals, 552 golgi bodies, 558 human embryos, 552 invertebrate sensory nerve endings, 552 macroglia, 586 nerve cells and processes, 551, 552, 553, 604 nerve endings in tongue, 553 nerve fibers, 553 neuroglia, 536-538, 539, 556, 558, 586 pertibular sheath, 590 reticulum fibers, 559 Schwann cells, 590 spirochetes, 560 Cajal's stains, gold-mercury, 539 in Cajal's method for neuroglia, 536- 538, 539 osmic-silver-dichromate, 604 picro-indigocarmine, 325 for, Squalus embryo, 322-323 Borrel's method for coccidia, 506; Castro viejo's technique, 369; Do- bell's method for Entamoeba his- tolytica, 507; Fraenkel's method for elastic fibers, 387; Gallego's method for Negri bodies, 465; Langeron's method for acid fast bacteria in sec- tions, 497; Nageotte's method for Schwann cells, 415 silver diammine, 575, 577 in, Bolsi's method for macroglia, 586 Cajal's method for cerebellum, 581 Cajal's method for macroglia, 586 Cajal's method for peritubular sheaths, 590; Cajal's method for Schwann cells, 590; Miskolczy's method for axis cylinders, 584 silver-dichromate, 606 silver nitrate, 550 for 72-hr. chicken embryo, 546-553 698 INDEX Cajal's stains — (continued) silver nitrate — (condnued) in, Cajal's method for axons, 553; Cajal's method for cerebellum, 553; Cajal's method for human embryos, 552; Cajal's method for neurofibrils, 552; Cajal's method for neuroglia, 558; Cajal's method for spirochetes, 560 Cajal's toner, 620 in, Ascoli's method for leech nerves, 551; Cajal's methods for nerve cells and processes, 552; Masson's methods for argentophil cells, 595, 596 Cajal and de Castro's, accelerator, 615 in Cajal and de Castro's method for macroglia, 586 alum-hematoxylin, 287 in Nageotte's method for Schwann cells, 415 fixing solution, 621 method for, macroglia, 586; spirochetes, 562 Cajeput oil, as ingredient of, Gothard's de- hydrating mixture, 628 physical properties of, 624 Calcareous deposit, Roehl's method for, 385 Calcareous objects, cutting sections of, 80-87 Calcified tissues, Golgi bodies in, 559 nerve endings in, 557, 581 silver diammine methods for, 596 special stains for, 382-386 Calcium chloride, as ingredient of. Baker's fixature, 190; Chevalier's pre- servative, 179; Cretin's mordant, 517; Gemmelli's neutral red, 482; Mayer's cochineal, 301; Mayer's hematoxylin, 292; Squire's hematoxylin, 293 fixative combination with cobalt and for- maldehyde, 236 Calcium dichromate, as ingredient of Son- nenbrodt's fixative, 217 Calcium hypochlorite, as ingredient of, Anderson's acid-alum hematoxylin, 289; Anderson's alum-carmine, 300; Ander- son's iron-hematoxylin, 285; Ander- son's mordants, 515 for cleaning foraminiferan tests, 17 Calcium phosphate, as ingredient of decalci- fying fluids, 257 Calcium sulfate, as dehydrant, 623 Calcium-hematoxylins, 293 Calleja's, double contrast, 325 triple stain, 369 "Calsol," 260 Calvet's fixative, 241, 299 Campbell's method for leprosy bacilli in sec- tions, 496 Campenhout, see Simard, 621 Camphor as ingredient of, Claoue's adhesive, 657 Cox's sandarac mountant, 638 Denham's sandarac mountant, 638 Gravis's adhesive for paraffin ribbons, 657 Guyer's sealing fluid for vials, 667 Mohr and Wehrle's sandarac mountant, 638 Shepherd's sandarac mountant, 638 Camphor water, for wholemounts, 23 "Camphoral," 638 Camsal, 638 Canada balsam, applying coverslip, to sections in, 125ff ; to wholemount in, 57, 58fi^ as, cement in ground section technique, 83; embedding agent for ground sections, 80 ingredient of. Apathy's cement, 655; Carney's ce- ment, 653; Eulenstein's varnish, 653; Fant's cement, 655; Graw-Riitzon's varnish, 653; Griffith's cement, 655; Hood and Neill's cement, 656; Martin's adhesive, 661; Mohr and Wehrle's varnish, 654 cleaning from mounted sections, 125- 128 evaporation technique with, 298 for ringing Venice turpentine mounts, 66 general remarks on, 639 in alcoholic solution, 638 miscibility of, clearing agents with, 625, 626; essential oils with, 626; synthetic solvents with, 625, 626; "universal solvents" with, 626, 627 liiountants, 639, 640 solubility in dehydrating agents, 623 wholemounts in, 56 xylene solution, 639 Canada balsam-gum damar mountants, 640 Canada balsam-gum mastic mountants, 640 Canada resin, 639 Canaliculi, in bone, 81 in plant cells, 454 Canon's chlorazol black E, 369 Cansey's method for mitochondria in protozoa, 442 Canti, see Bland, 461 Capillaries, differential staining of, 416, 417, 419, 420, 421 injection with, carmine-gelatin, 170; India ink, 166-168; lead chromate, 169 staining in wholemounts, 432 Capillaries, sec also Bile capillaries INDEX C99 Caplan, see Huber, 63G Cappell's fixative remover, 255 Capri blue, staining combination with erythrosin, 392 Capsules of bacteria, in sections, 498 in smears, 487-489 Carany's cement, for aqueous vvholemounts, 23 Caraway oil, physical properties of, 624 Carazzi's, alum hematoxylin, 287 for, vvholemounts of embryos, 54; chicken embryo, 275; in Hubiu's tech- nique, 340 fixatives, mercuric-acetic, 208, 240; mercuric- acetic-nitric, 210, 240 Carbitol, 627 "Carbol-xylol," 629 Carbolic acid, see Phenol Carbon dioxide, as narcotic, 265 as refrigerant for frozen sections, 158 Carbon disulfide, as ingredient of Robin's cement, 654 physical properties of, 625 Carbon tetrachloride, for separating fora- menifera from sand, 17 physical properties of, 625 Carborundvim, use in grinding sections, 81, 84ff, 86 Carbowax, 643 Carboxymethyl cellulose, 42 Carchesium, 53 Cardboard, boxes for embedding, 100-101 Cardiac muscle, Dietrich's method for, 394 Cards, for storing slides, 128 Carious lesions, 563 Carleton and Leach's, decalcifying fluid, 257 fixative, 211, 240 recommended use, 95 glycerol jelly, 633 Carmalums, 300, 301 Carmine, as ingredient of, Beale's injection fluid, 662; Robin's in- jection fluid, 663; various injection masses, 664; Zirkle's balsam mountant, 240; Zirkle's gelatin mountants, 635; Zirkle's gum mountant, 633; Zirkle's pectin mountant, 637; Zirkle's Venice turpentine mountant, 638 gelatin injection mass, use of, 170 general remarks on, 272, 293, 294 in, Calleja's triple stain, 369; Lonnberg's triple stain, 370; Lowenthal's triple stain, 366; Lynch's double stain, 371; Merbel's double stain, 372; Norris and Shakespeare's double stain, 372; West- phal's double stain, 373 method of classification, 267 Carmine — {continued) staining combinations with, acid fuchsin, methyl violet and picric acid, 389; acid green, 392, 393; anijin blue, cresofuchsin and orange G, 425 anilin blue and picric acid, 508, 513 Bismark brown and crystal violet, 389 chlorophyll and hematoxylin, 451 crystal violet, 373, 420; gentian violet 503; gentian violet and picric acid, 501 hematoxylin, 509; hematoxylin and picric acid, 366; iodine green, 392 indigocarmine, 372; indulin, 371; ma genta, 421; methyl violet, 6B, 503 orange G, 448; osmic acid and protein silver, 607; picro-indigocarmine, 369 spirit blue, 370; Sudan black, 442 stains, aceto-carmines, 302; alcoholic-carmines, 301; alum carmines, 300-301; am- monia carmines, 305, 307; borax car- mine, 307; boric carmine, 306; chrome- carmine, 307; hydrochloric-carmines, 305, 306; iron-carmines, 304; lithium- carmine, 307; magnesia-carmines, 307; picro-carmines, 303-304; soda-carmines, 306; uranium-carmines, 307 Carminic acid, as ingredient of stains, see under Carmine Carnoy and Lebrun's fixative, 208, 240 in Jurray's method for embedding insects, recommended use, 95 Carnoy's, cement, 653 fixatives, acetic-alcohol, 189, 241 acetic-alcohol-chloroform, 189, 241; rec- ommended use, 95 in, Schleiff's method for fungi in skin scrapings, 505; Windle, Rhines and Rankin's method for Nissl granules, 447 Carother's fixatives, 224 Carpano's, method for Negri bodies, 464 stain, 321 in Grieves' method for dentine, 383 Carpenter and Nebel's fixative, 236 del Carpio's silver diammine stain, 575, 577 in del Carpio's method for reticulum fibers, 591 Carriere's method for nerve endings, 534 Carrot, use in freehand sections, 90, 9 Iff Carrots, for staining fat, 448 Carsten, see Lubkin, 644 Carter's, fixative, 208, 241 injection mass, 664 Cartilage, iron method for, 610 mentioned in, Becher's polychrome gallamin blue, 368; Bohm and Oppel's triple stains, 361, 368; Buzaglo's triple stain, 368; 700 INDEX Cartilage — {continued) mentioned in — {continued) Gausen's triple stain, 351; Haythorne's triple contrast, 337; HoUande's triple contrast; Hubin's triple contrast, 340; Lillie's triple stain, 366; Mann's double stain, 371; Patay's double contrast, 339; Semiclion's triple contrast, 329; Shumway's triple stain, 372 reticulum fibers in, 594 special methods for, 386-387 Cart Wright's method for fungi in wood, 501 Caruthers, see Wicks, 641 Casares-Gil's mordant, 517 in Casares-Gil's method for bacterial flagclla, 482 Cason's triple stain, 360 Cassia oil, 624 Castaneda's method for Rickettsiae, 462 Castile soap, in Nissl's method for his granules, 446 Castor oil as ingredient of, Claoue's ad- hesive, 657 Cox's sandarac mountant, 638 Gage's, cement, 655; clearing mixture, 628; varnish, 653 Steedman's embedding wax, 647 Thiersch's blue varnish, 654 Castor oil soap, as ingredient of Godfrin's embedding medium, 644 de Castro's, decalcifyer, 257 in de Castro's method for nerves in teeth, 554 fixative, 190, 241 de Castro, see also Cajal, 586 Castro viejo's double stain, 369 Cat, sciatic nerve, 564-566 Catcheside's fixative, 200, 241 CauUery and Mesnil's fixative, 195, 241 Cavity slide, sealing aqueous wholemount on, 25ff Cedarwood oil, as ingredient of, Apdthy's Canada balsam, 639; Apdthy's clearing mixture, 628; Bolcek's fixative remover, 255; Eycleshymer's clearing mixture, 628; Maxwell's clearing mix- ture, 628; Schmorl's storage fluid for celloidin blocks, 667 for clearing, earthworm, 331; wholemounts, 56 in cutting nitrocellulose sections, 146 physical properties of, 624 Celestin blue B, for rat tongue, 323-325 for staining intestine, 131 in staining combination with, azophloxine, 372; hematoxylin, ma- genta leucobase and orange G, 427 staining solutions, 313 Cell inclusions, Chura's solvent for, 514 Cell outlines, iron stain for, 611 of, endothelium of blood vessels, (KJ 1 ; vyn- thelia, 607, 608; invertebrate embryos, 564 Cell types, in adrenal, 429 pituitary, 425-428 pancreas, 428-430 Cell walls of, plant tissue, 391-393 pollen, 393 stem apex, 393 "Cellobalm," 654 "Celloidin," 142 techniques, see under Nitrocellulose "Cellosolve," 627 Cellosolve, methyl, uses of, see under Ethy- lene glycol monoethyl ether "Cellosolve acetate," 625 uses of, see under Ethylene glycol mono- ethyl ether, 265 Cells, attaching with marine glue, 22 for, aqueous wholemounts, 21; balsam mounts, 57; dry wholemounts, 11-12; glycerol jelly, 48 of dewaxed shellac, 40 preparation of, from Bristol board, 12; cement, 12, 29; mill- board, 12; plastic, 13; tin, 13 sealing aqueous wholemounts in, 26ff temporary, Perruche's varnish for, 654 tin, preparation, 21 vulcanite, preparation, 21 Harting's cement for, 656 warm table for attaching, 15 wholemount of insect in, 64 Celluloid, in Linstaedt's method for sec- tions, 659 mounting nitrocellulose sections on, 149 Cellulose, mentioned in Johansen's quadru- ple stain, 364 stains for, 391-393 Cellulose acetate lacquers, for ringing balsam mounts, 58 Cellulose ester varnishes, disadvantages of, 16 Cellulose nitrate, grades of, 142 Cement cell, for dry wholemounts, 11-12 preparation of, 11-12, 29 Cementing, cells to slides, 12-13 coverslips to dry wholemounts, 16 objects in dry wholemounts, 14-16 Cements, for, aqueous wholemounts, 21, 23; glass, 652; ringing, see Varnishes, 653 formulas for, 652-656 general remarks on, 651 Centrifuge for plankton, 28 Centrosomcs, 435 in nerve cells, 590 INDEX 701 Centrosomes — {continued) mentioned in Arnold's triple stain, 351 Cephalic ganglia, mosquito, 568 Cerebellum, Cajal's silver diammine method for, 581 human, 552 osmic-silver-dichromate methods for, 606 Schmultze's method for, 557 vanadium method for, 609 Cerebral cortex, neuroglia in, 399, 400 Cerebrum, human, 552 Schultze's method for, 557 Ceric oxide, use in polishing sections, 81 Cerrito's method for bacterial flagella, 482 "Certo," 636 Cervical ganglion, section of, 601-602 Cestodes, Bujor's method for, 265 reproductive system, 506 stains for, 302, 509 Chalk, separating foraminifera from, 17, 18 Chalmer's and ^Marshall's method for fungi in skin scrapings, 500, 503 Chamberlain's, fixative, 200, 240 method for, filamentous algae, 511; filamentous fungi, 511: plant sections, 391; plant skeletons, 380-382 stains, phloxine-anilin blue, for algae, 65 safranin, 314 for nitrocellulose section of lily bud, 151; in Smith's method for plant sections, 393 technique for dehydrating algae, 65 Venice turpentine technique, 65 Champy's fixatives, formaldehyde-trichloro- acetic, 192 in, Benda's method for mitochondria, 442; Maximow's fixative, 231 osmic-chromic-dichromate, 201, 240 in, Gatenby's method for Golgi bodies, 530; KuU's method for mitochondria, 443 ; KuU's method for pancreas, 439 osmic-chromic-dichromate-pyroligneous, 202, 240 recommended use, 95 Champy, Coujard and Coujard-Champy's method for nerve endings in gland cells, 529 Chan, see Johnson, 393 Chandler's method for pollen tubes, 421 Chang's method for nervous tissues, 402 Charipper's fixative, 197 Chat ton's, agar embedding medium, 643 double stain, 327 Chatton and Lwoff's adhesive, 660 in Chatton and Lwoff's method for ciliates, 559 Chavannes, see Agulhon, 350 Chelating agents, for decalcification, 256, 260 Chenopodium oil, jjhysical properties of, 624 Chenzinsky's double stain, 344 Chermock, see Miiller, 316, 477 Cherry gum, as ingredient of Salkind's em- bedding medium, 645 Chevalier's, asphalt varnish, 653 gelatin mountant, 634 gum arable mountant, 631 gum mastic mountant, 640 preservative, 179 Chicken embryo, injection of blood vessels, 166-168 neuroblasts and axons in, 546-548 removal from, egg, 275, 276; yolk, 546-547 sections of, 278-280 segmentation stages, 133 staining, 278 techniqiie of fixation, 277, 278 variations in apparent age, 275 wholemount of, 275-278 Chief cells, of stomach, 432 Chilesotti's method for axis cylinders, 410 Chiovenda's adhesive for free sections, 659 Chironomus, smear of salivary chromosomes, 299-300 Chitin, mentioned in, Chalton's double contrast, 327; Semi- chon's triple stain, 329 methods for softening, 215, 256, 261 special stains for, 390-391 Chlodkowsky's fixative, 208, 241 Chloral hydrate, as ingredient of, Amann's dehydrating mixtures, 628; Amann's preservatives, 177; Andre's swelling fluid for arthropods, 667; Bank and Davenport's fixative, 237; Becher and Demon's macerating fluids, 262; Belloni's hematoxylin, 286; Cajal's al- coholic accelerator, 614; Cajal's for- maldehyde accelerator, 613; de Castro's decalcifying fluids, 257; Cuccati's car- mine, 306; Denham's sandarac moun- tant, 638; Francotte's picro-carmine, 303; Frost's preservatives, 179; Gater's preservatives, 178; Geoffroy's gelatin mountant, 634; Gilson's glycerol jolly, 634; Gray's narcotics, 265; gum arable mountants, 631-633; Hoyer's injection mass, 665; Jores's preservatives, 180 Klatzo's formaldehyde accelerator, 613 Klotz and Coburn's preservatives, 180 Klotz and MacLachan's preservatives 180; Langeron's, hematoxylin, 289 Langeron's preservatives, 178; I-ee's hematoxylin, 288; Lhotka and Ferreira's fixative remover, 256; MacFarland's and Davenport's fbcatives, 193; Mal- lory's hematoxylin, 292; Maj^er's injec- 702 INDEX Chloral hydrate, as ingredient of — {con- tinued) tion fluid, 663; Meyer's carmine, 306; Mukerji's preservatives, 178; Nelson's hematoxylin, 406; Nollister's stain, 384; Police'shematoxylin, 292; Priestly 's preservatives, 178; del Rio-Hortega's fixative, 234; Salkind's fixative, 216; Tello's decalcifying fluid, 260; Weber's alcoholic accelerator, 615; Willard's accelerator, 615 for increasing index of refraction, 42 for narcotizing, Hydra, 78; medusae, 296; platyhel- minthes, 53; for softening chitin, 261 in, Cajal's method for Purkinje cells, 553; Cajal's silver-dichromate method for neuroglia, 606; Lewis's differentiator, 521; McManns's silver nitrate staining solution, 550; Perez's method for Neissner bodies, 556: Ungewitter's method for nervous tissues, 568 Chlorazol black E, as general stain, 369 for staining, nuclei (Conn), 435; plant sections (Dar- row), 392 "Chlorcarmin," 305 Chlorine, as ingredient of Harlow's macerat- ing fluids, 263 for bleaching, 261 Chlorobutanol, as ingredient of, Volkowsky's narcotic, 266 Chlorodioxyacetic acid, for softening chitin, 256 Chloroform, as clearing agent in double em- bedding, 155 as ingredient of, Apathy's clearing mixture, 628; Bar- telmez's fixative, 189; Bradley's fixa- tive, 189; Canada balsam mountants, 639; Carnoy's cement, 653; Cori's nar- cotics, 265; Dobell's fixative, 224; Eltringham's fixative, 222; Hethering- ton's fixative, 189; Gilson's fixative, 189; Jenkin's decalcifying fluids, 259; Kingsbury and Johannsen's fixative, 222; van Leeuwen's fixative, 224; Lendrum's fixatives, 189; Mahdissan's fixatives, 192; Monnig's preservative, 177; Sansom's fixative, 190; Schmorl's storage fluid for mitrocellulose blocks, 667; Schweitzer's fixative, 225; Sikora's fixative, 192; Waddington's narcotic, 266; Winge's fixatives, 214; Zacharias's fixative, 194 as narcotic, 265 as solvent for, gum mastic, 640; Holzer's crystal violet, 413; Landau's toluidine blue, 403 Chloroform — {continued) for, hardening nitrocellulose, 155; hardening nitrocellulose blocks, 145, 146, 151; narcotizing Crustacea, 49; removal of chlorophyll, 149 in nitrocellulose embedding methods, 647, 648 physical properties of, 625 Chloroform water, for wholemounts, 23 Chlorophyceae, 513 Chlorophyll, in, Arndt's method for glycogen and fat, 451; Blayde's preservatives for, 179 removal of, 149 staining combinations with carmine and hematoxylin, 451 Choquet's, decalcifying fluid, 259 method for dentine, 386 Chor's developer, 617 in Chor's method for motor end plates, 554 Chorine's method for Plasmodium, 506 Chromaffin granules, 430 Chromatin, see Nuclei Chrome alum as ingredient of, Ammerman's fixative, 228 Becher's, gallocyanin, 313 polychrome oxazines, 368 Benda's fixative, 220 Cole's gallocyanin, 313 Chura's, fixative, 227 mordant, 515 Kiyono's differentiator, 521 Fol's adhesive for free sections, 659 Fyg's carmines, 307 Gomori's hematoxylin, 291 Kirchner's preservative, 180 Marquez's mordant, 518 Masson's fixative, 226 Merkel's mordant, 516 Schweitzer's hematoxylin, 292 Yetwin's glycerol jelly, 635 Zikes's mordant, 519 Zwemer's glycerol jelly, 636 Chrome-carmines, 307 Chrome-hematoxylins, 291 Chrome yellow, staining combination with alkanet and iodine green, 393 Chromic acid, as fixative, 227 as ingredient of, Bacsich's mordant, 515; Bayerl's de- calcifying fluid, 256; Busch's decalcify- ing fluids, 257; Clara's mordant, 515; Drew's mordant, 515; Drost's macerat- ing fluid, 263; fixatives, see name of author or fixative combination (below) ; Haug's decalcifying fluids, 258; Katz's decalcifying fluids, 259; Marsh's de- calcifying fluids, 259; Ordonez's pre- INDEX 703 Chromic acid — (continued) as ingredient of — (continued) servatives, 180; Seiler's decalcifying fluids, 260; Semmen's mordant, 518; Trenkmann's mordant, 519; Waldeyer's decalcifying fluids, 260; WoUing's de- calcifying fluids, 260 as mordant, 395 as mordant for, Buzzozero-Vassale's gentian violet, 435; Gudden's hematoxylin, 405; Kockel's hematoxjdin, 423; Neuman's hema- toxylin, 407; Schneider's magenta, 315; Stockwell's triple stain, 364 basal fixative solution of, 237 fixative combinations with, acetic acid, 227-228; cobalt salts, 232; copper salts, 220; dichromates, 231; for- maldehyde, 230, 231; formic acid, 229; hydrochloric acid, 229; iodine, 232; mercuric salts, 215, 216; mercuric salts and dichromates, 216; mercuric salts and picric acid, 215; nitric acid, 229; osmic acid, 197, 199, 200, 201, 202; osmic acid and dichromates, 201, 202; osmic acid and mercuric salts, 197; osmic and picric acids, 198, 199; osmic acid and platinic chloride, 195, 196; osmic acid, platinic chloride and mer- curic salts, 195; osmic acid, platinic chloride and thorium salts, 196; osmic and trichloroacetic acids, 201; oxalic acid, 230; picric acid, 226; picric acid and dichromates, 227; platinic chloride, 207; platinic chloride, mercuric salts and picric acid, 206; uranium salts, 231, 232 for, differentiating crystal violet, 319; hard- ening, 52 in, Besson's method for bacterial spores, 485; Kolossow's method for nerve end- ings, 535; May's method for bacterial spores, 486; Moller's method for bac- terial spores, 486; Togby's method for chromosomes of Crepis, 438 prevention of discoloration of tissues by, 205 Chromic chloride, as ingredient of Mullen and McCarter's mordant, 516, 518 Chromic-formaldehyde fixatives, precau- tions necessary, 96 Chromic-mordant hematoxylin stains, 283 Chromic-silver stains, 603 Chromium fluoride, as ingredient of, Anderson's mordant, 515; Chura's mordant, 515; Schroder's mordant, 516; Weigert's chrome-copper mordant, 516, 517 Chromium fluoride — (continued) fixative combination with, chromic and dichromatc, 231; chromic, picric, dichromate and formaldehyde, 227; mercuric, chromic and dichromate, 215 Chromium sulfate, as ingredient of, Mas- son's fLxativc, 226 fixative combination with, chromic, cupric and formaldehyde, 220 Chromium trioxide, see Chromic acid Chromogenes, 270 Chromophobe cells, of pituitary, 425, 426 Chromophore, 270 Chromosomes, Belling's aceto-carmine for, 302 controlling differentiation of, 274, 275 in smear of Chironomus salivary gland, 299 maceration technique for, 264 mentioned in Hollande's triple stain, 339 of grasshopper, 310-312 plant, smear technique for, 74; squash tech- nique for, 77 special fixatives for, 196, 227, 230 Chromosomes, see also Nuclei Chromotrope 2R, as plasma stain, 320 for staining erythrocytes (Crossmon), 416 in, Gomori's double contrast, 339; simple solution, 320 staining combination with, fast green FCF, 339; methyl blue, 416 Chrysoidin, for staining, diphtheria bacilli (Ambrosioni), 489; diphtheria bacilli (Cowdry), 489; fat (Martinolti), 449 staining combinations with, crystal violet, 489; crystal violet and methylene blue, 489 Chura's, chrome mordant, 515 fixative, 227, 241 solvent for cytoplasmic inclusions, 514 Churchman and Emelianoff's method for bacterial capsules, 487 Ciacco's, fixative, 235, 241 method for nerve endings in tendons, 540 Cigalas's cement, 655 Ciliates, basal bodies, 559 cilia, cirri and basal bodies, 456 fixing and narcotizing, 53 Gray's narcotic for, 265 nuclei of, 434 Ciliates, see also under generic names Cill<5, see Bardelli, 503 Cinnamon oil, physical properties of, 624 Citric acid as ingredient of, Bensley's in- jection fluid, 662 704 INDEX Citric acid as ingredient of — (continued) Kramer and Shipley's decalcifying fluids, 260 Langeron's hematoxylin, 289 Citronella oil, physical properties of, 624 Cladocera, collecting from moss, 45 "Clairite," 641 as ingredient of Gray's wax embedding medium, 646 Clancy and Wolfe's method for Rickettsiae, 462 Claoue's, adhesive for paraffin ribbons, 657 lacquer, for saving loose sections, 125, 657 Clara's, molybdic-hematoxylin, 290 mordant, 515 in Clara's methods for bile capillaries, 422 Clarifying gelatin, 633 Clark's, gelatin embedding medium, 643 gelatin method for frozen sections, 160- 161 method for fat, 447 Clark and Sperry's method for Nissl gran- ules, 445 Clark and Ward's method for nervous tis- sues, 404 Clarke, see Dotti, 256 Claudius's method for bacterial smears, 473 Claussen's fixative, 228, 241 Claverdon's fixative, 224 Clay, separation of Foraminifera from, 18 Cleaning, balsam mounts, 57 diatoms, 38-39 foraminiferan tests, 17 gum mounts, 43 jelly mounts, 48 mounts of sections, 125-128 Radiolaria, 18-20 slides, 19, 69, 666, 667 Venice turpentine mounts, 66 Clearance angle, in cutting, 109 Clearing, capillary technique for, 298 flotation method for, 62 medusae, 298 mixtures, 627-629 objects for paraffin embedding, 98 Clearing agents, classification of, 622 general remarks on, 623, 624 physical properties of, 624, 625, 626 Cleveland and Wolfe's method for pituitary, 425 Clove oil, as ingredient of, Apdthj^'s nitrocellulose cement, 661; Dunham's clearing mixture, 628; Gies- brecht's adhesive, 660; Minot's clearing mixture, 628; Schallibaum's adhesive for free sections, 660; Stepanow's nitrocellulose medium, 648 as solvent for, erythrosin, 436; ethyl eosin, 347; fast Clove oil — (continued) as solvent for — (ronliniird) green FCF, 321, 393; orange II, 363, 364; orange G, 320, 340 for, clearing wholemounts, 56 differentiating, Bailey's double stain, 412; magenta, 312 flattening nitrocellulose sections, 152 in, Langeron's method for attaching nitro- cellulose sections, 659; Richardson's nitrocellulose embedding method, 648 physical properties of, 624 Coal dust, separation of diatoms from, 39 Cobalt chloride, fixative combination with, osmic acid, 205 staining combination with, silver nitrate, 559 Cobalt nitrate, as ingredient of, da Fano's formaldehyde accelerator, 613; Merland's formaldehyde accelera- tor, 613; Weber's alcoholic accelerator, 615 fixative combinations with, bromide and formaldehyde, 236; cal- cium salts and formaldehyde, 236 ; chro- mic and formic acids, 232; formaldehyde, 236 Cobe and Schoenfle's adhesive for paraffin ribbons, 657 Coburn's cement, 655 Coburn, see also Klotz, 180 Cocaine, for narcotizing Bryozoa, 60 Cocaine hydrochloride as ingredient of, Baker's narcotic, 265 Gater's mountant, 632 Langeron's narcotic, 265 Morrison's narcotic, 265 Rousselet's narcotic, 266 Waddington's narcotic, 266 Cocci in cell smears, 491 Coccidia, 506 Cochineal as ingredient of, Czokor's alum- cochineal, 300 Guyer's alum-cochineal, 300 Hansen's iron-carmine, 304 Kirkpatrick's alum-cochineal, 300 Mayer's alcoholic cochineal, 301 Partsch's alum-cochineal, 301 Peter's iron-cochineal, 305 Rabl's alum-cochineal, 301 Rawitz's aluminum-cochineal, 307 Spuler's, alcoholic-cochineal, 302 Spuler's iron-cochineal, 305 Coelenterates, narcotics for, 265 wholemounts of, 53 Coelenterates, see also Anthozoa, Medusae, Hydra, etc. Coelestin blue, see Celestin blue INDEX 705 Cohen's fixatives, formaldehyde-metaphos- phoric acid, 193, 241 in Cohen's method for fungi in plant tissues, 501 method for fungi in plant tissues, 501 picric-chromic-formaldehyde, 220 Cohen, see also Kopeloff, 474 Coker's fixative, 208, 241 Cole's, clearing mixture for arthropods, 628 fixative, 232, 241 gum arabic embedding medium, 643 methods for, nerve endings, 534; nervous tissues, 402; plant sections; 392; virus inclusion bodies, 464 mordant, 515 in, Cole's technique, 282; Knower's technique, 283 l)reservatives, 178, 179 stains, acid-alum hematoxylin, 289; ammonia- carmine, 305, (for plant sections, 392); gallocyanin, 313; iron-mordant hema- toxylin, 282 Coleman's magenta leucobase, 316 Colin's methods for pituitary, 425 Collagen, Long's method for, 597 mentioned in, Delamare's triple stain, 365; Drew- Murray's triple stain, 369; Dupres double contrast, 339; Freeborn's double stain, 369; Goldner's quadruple con- trast, 337; Heidenhain's triple stain 361; Hollands's triple contrast, 339 Holmes and French's triple stain, 352 Houcke's double stain, 353; Kalter's quadruple stain, 304; Kornhauser's quadruple stains, 309, 370; Lillie's triple stain, 366, 370; Mallory's triple stain, 360; Masson's double contrasts, 337, 340, 341; Matsura's polychrome- neutral red, 371; Mollier's quadruple stain, 366; Paquin and Goddard's quintuple stain, 366; Pasini's double contrast, 338; Patay's double contrast, 339; Romeis's quadruple stain, 367; Unna's quadruple stain, 304; Volkman and Strauss's triple stain, 373; Wallart and Honette's double contrast, 339; Walter's triple stain, 361; Waterman's triple stain, 361 Unna's method for, 395 Verocay's method for, 395 Collagen fibers, Hueter's method for, 394 in skin, 593 Jasswoin's method for, 394 Collecting, Bryozoa, 59 Cladocera, 45 copepods, 49 Crustacea, 48, 49 Collecting — {conlifiucd) diatoms, 37 foraminifcra, 17 liver flukes, 294 mites, 45 nematodes, 35, 45 oligochaetes, 45 ostracoda, 45 rotifers, 30 small arthropods, 43-44 CoUembola, 45 Collodion, definition, 142 in, Altmann's method for attaching free sections, 659; Cobe and Schoenflc's method for paraffin ribbons, 657; R^gand's adhesive for paraffin ribbons, 658; Zimmermann's method for attach- ing free sections, 660 Colombo's fixative, 197, 241 Colophonium, see Rosin Complex contrasts, general remarks on, 329 Complex stains, acid fuchsin-stained nuclei, 357-361 general remarks on, 341 hematoxylin stained nuclei, 365-367 methyl green stained nuclei, 353-357 miscellaneous, 367-373 safranin stained nuclei, 362-365 thiazin stained nuclei, 344-353 Comt6, see Villain, 510 Conant's quadruple stain, 363 for Ranunculus stem, 362-363 Conant, see also Swartz, 505 Concave slides, 21 Cone and Penfield's silver diammine stain, 575, 577 in Cone and Penfield's method for mi- croglia, 586 Congo red, as plasma stain, 320 for staining, amyloid, 451, 452 in, Gnanamuthu's double contrast, 327; simple solution, 320 staining combinations with, hematoxylin, 451, 452; picric acid, 327 Conklin's acid-alum hematoxylin, 289 Conn, on magdala red, 388 on pet names, 1 synonomy of dyes, 3 Conn's methods for, nuclei, 435; soil bacteria, 473 safranin, in Land's method for plant sec- tions, 392 Conn, see also Hucker, 474; Fisher, 482 Conn and Darrow's method for plant sec- tions, 392 Connective tissue mentioned in, Hecher's polychrome celestin blue, 368 Langeron's double stain, 353 706 INDEX Connective tissue mentioned in — (continued) Lillie's triple stains, 370 Twort's double stain, 372 Connective tissue, see also Collagen, etc. Connective tissue spreads, Baird's method for, 394 Cook's copper-hematoxylin, 290 Cook, see also Meeker, 218 Cooke's damar mountant, 640 Cooper's method for nuclei, 435 Copal varnish, Behren's, 652 Copepods, collecting, 49 Coplin jar, 119, 124ff for washing specimens in running water, 129 Copper, in protein silver techniques, 566- 568 Copper acetate, as ingredient of, Cole's preservative, 179; Dubuscq's thionin, 417; Eckert's preservative, 178; Faure's mordant, 516; Merkel's mor- dant, 516; Mitrophanow's mordant, 516; Morel and Bassal's hematoxylin, 286; Weigert's chrome-copper mordant, 517; Wood's preservative, 176, 177 as mordant for stains, Aronson's gallein, 409; Bensley and Bensley's hematoxylin, 283; Eppinger's hematoxylin, 453; MacCallum, et al. hematoxylin, 426; Neumen's hematoxy- lin, 407; Weigert's hematoxylin, 408 as mordant in, Alzheimer's method for nerve cell granules, 454; Bensley's method for canaliculi in plant cells, 454; Fischler's method for fat, 448; Harvey's method for parietal cell granules, 455 fixative combinations with, acetic acid, 210; chromic acid, 220; dichromates, 220, 221; formaldehyde and acetic acid, 219; osmic acid, 198; trichloroacetic acid, 219; picric acid, 220 Copper chloride, as ingredient of, Dubuscq's thionin, 417; Eckert's pre- servative, 178; Keefc's preservatives, 180 fixative combinations with, acetic acid, 219 Copper dichromate, fixative combinations with, formaldehyde, 221; mercuric salts and dichromates, 213; platinic chloride and dichromates, 206 Copper-hematoxylin, 290, 291 Copper hydroxide, fixative combinations with, chromic acid, 220; formaldehyde and proprionic acid, 219 Copper-mordant hematoxylin stains, 283 Copper nitrate, fixative combinations with, acetic acid, 219; formaldehyde, 219; mercuric salts, 213; nitric acid, 219; paranitrophenol and nitric acid, 221 Copper oleate, as ingredient of, Mohr and Wehrle's sandarac mountant, 638 Copper oxide, fixative combinations with, picric acid, 220 Copper sulfate, as differentiator in. Hiss's method for bacterial capsules, 488; Langeron's method for fungus in plant tissues, 502 as ingredient of, Blayde's preservatives, 179; Cook's hematoxylin, 290; Gordon's mordant, 518; Robin's injection fluids, 663; Woolman's crystal violet, 481 as mordant, for, Sato's safranin, 419 in, Goldsworthy and Ward's method for spirochetes, 479; Krajian's method for bacteria in sections, 492 basal fixative solution of, 237 fixative combinations with, dichromates, 220, 221; mercuric salts, 213; mercuric salts and dichromates, 213; osmic acid and mercuric salts, 197 staining combination with, potassium di- chromate and silver nitrate, 608 use in dehydration, 56, 129 Copper tetramine, in Roehl's method for calcareous deposits, 385 Coral, ground section of, 85-87 narcotizing, 85 Corbin's polychrome methylene blue, 317 in Corbin's method for plasmodium, 506 Cori's, fixatives, osmic-chromic-acetic, 200, 241; osmic, mercuric-chromic-acetic, 97, 241 narcotic, 265 for protozoa, 52 Cork, as ingredient of Balbuena's alcoholic accelerator, 614 Cornea, capillaries in, 421 Paschen bodies in, 467 silver diammine method for, 584 Cornheim's method for nerve endings, 535 Cornwall's, fixative, 209 method for fungi in wood, 501 Corpuscles of, Grandry, 554 Herbst, 554 Cotton blue, in Bernhardt's method for fungus in tissue scrapings, 503 Coujard, see Champy, 529 Coumarones, 640, 641 Counterstains, see Plasma stains Coupler, phenol as, 628 Coutelin's method for flame cells, 430 INDEX 707 Coverslip, applying salicylic acid from, 359 applying to, sections, 125ff balsam mounts, 57, 58ff as knife, 537 attaching diatoms to, 39, 40 attaching rhizopods to, 52 cementing to dry wholcmounts, 16 clip for, 57, 59ff for checking thickness of ground sections, 84 Langeron's pellets for support of, 656 sealing on aqueous wholemounts, 24, 25ff, 26ff, 27 Coverslips, origin of, 7 Coverslip cements, for aqueous whole- mounts, 23 Cowdry's, developer, 617 in Cowdry's method for nerve fibrils, 554 method for, diphtheria bacilli, 489; mitochondria, 442 stains, acid fuchsin-orange G-toluidine blue, 351; phloxine-orange G-azur A, 351 Cowdry, see also Gatenby, 189, 260 Cox's, fixatives, mercuric-dichromate, 216, 242, (in Cox's method for nervous tissues, 609) ; mercuric-formaldehyde-acetic, 212; osmic-mercuric-acetic, 197, 242; osmic- platinic-mercuric-acetic, 195, 242 method for, nervous tissues, 609; softening chitin, 261 mountant, 638 in Cox's method for nervous tissues, 609 Crabb's adhesive for paraffin ribbons, 657 "Craf" fixatives, 230-231 Craig's preservative, 179 Craigie's, method for Paschen bodies, 464 silver diammine stain, 580 in Craigie's method for bacterial smears, 598 Cramer's method for adrenal, 529 Crawford, see Baker, 187 Creosote, as ingredient of, Artigas's mastic mountant, 637; Cole's clearing mixture, 628; Gatenbj'^ and Painter's clearing mixture, 628; Goth- ard's, dehydrating mixture, 628; Kra- jian's magenta, 316; del Rio-Hortega's clearing mixture, 628; Weil's differen- tiator, 520 as solvent for Landau's toluidine blue, 403 physical properties of, 625 Crepis, chromosomes of, 438 Cresofuchsin, for staining, glycogen (Vastarini-Cresi), 453; pitui- tary (Berblinger and Bergdorf), 425 Cresofuchsin — {continued) staining combination with, anilin blue, carmine and orange G, 425; magenta and orange G, 453 Cresol, physical properties of, 626 Cresyl blue, see Brilliant cresyl blue Cresyl violet, as stain, 373 for staining, acid-fast bacteria in sections, (Spoerri), 498; mucin, (Merkel), 454; nervous tissues, (Tress and Tress), 411; Nissl granules, (Addison), 445; Nissl granules, (Keller), 446 in phenol solution, 321 staining combination with, thionin and toluidine blue, 498 Cretan origanum oil, physical properties of, 624 Cretin's, decalcifying fluid, 257 ferricyanide-hematoxylin, 291 fixatives, mercuric-picric-formaldehyde, 214, (in Cretin's method for starch, 451); picric- formaldehyde-nitric, 214, (in his method for bone, 382) ; picric-formaldehyde-tri- chloroacetic, 225; picric-trichloroacetic, 222 method for, bone, 382; starch, 451 mordant, 517 on pH of fixatives, 187, 188 Crocus, squash of microsporocyte of, 77 Crook and Russel's method for pituitary, 425 Crossmon's, method for erythrocytes, 416 triple stain, 336 Crough and Becker's method for oocysts of coccidia, 506 Crowell's method for sections of wood, 93 Crustacea, collecting, 48, 49 Daniel's method for muscles of, 394 mounting in glycerol jelly, 46, 48 narcotizing, 49 Cryptobranchus, 133 Crystal violet, for staining, acid-fast bacteria (Auguste), 476; acid- fast bacteria (Fontes), 476; acid-fast bacteria (Herman), 477; Actinomyces (Hutchins and Lutman), 501; Actino- myces (Langrand), 504; Actinomyces (Ligni^re), 504; amyloid, 451-453; bac- teria (Hucker), 468; bacterial capsules, 487-489; bacterial flagella, 482-484; bacterial smears (Hucker), 473; bac- terial spores (Tribondcau), 487; blood (Westphal), 420; Chlorophyceae (Yama- nouchi), 513; Crepis chromosomes (Togby), 438; diphtheria bacilli (Am- brosioni), 489; diphtheria bacilli (Cow- dry), 489; diphtheria bacilli (Ljubinsky), 708 INDEX Crystal violet, for staining — (continued) 490; elastic fibers (Goldman), 388; elastic fibers (Sheridan), 389; fungus in plant tissues (Cohen), 501; fungus in skin scrapings (Bachman), 502; Gram-positive bacteria, 474-475 ; Gram- positive bacteria in sections (Glynn), 494; intracellular "organisms" (Hoso- kowa), 461; mitochondria (Benda), 442; Negri bodies (Carpano) 464; neuroglia, 413; nuclei (Plancock), 435; nuclei (Smith), 437; pancreas, 429; pollen mother cells (Sax), 437; skin fibers (Unna), 424; spirochetes, 479-481; spirochetes in sections (Lustgarten), 498; Treponema (Du), 479; Zygonema (Bigot), 503 general remarks on, 309 in, Bensley and Bensley's double stain, 368; Conant's quadruple stain, 363; Foley's triple stain, 363; Kalter's quadruple stain 364; Laguesse's triple stain, 364; phenol solution, 321; Volk- man and Strass's triple stain, 373; Westphal's double stain, 373 staining combinations with, acid fuchsin, 368, 481, 504; acid green, 481; alizarin, 442; azocarmine, and naphthol green, 373; azur I, eosin and methjdene blue, 461; Bismarck brown, 451, 487, 490; carmine, 373, 420; car- mine and Bismark brown, 389; chrys- oidin, 489; chrysoidin, and methylene blue, 489; eosin, 477; eosin Y, 464; eosin Y and hematoxylin, 366; eosin Y and magenta, 504; erythrosin, 392, 503; fast green, orange II and safranin, 363, 364; hematoxylin, 452, 493; indigocar- mine, and methyl violet, 483 ; magenta, 387, 482, 494; magenta and methylene blue, 476; methylene blue, 479; orange G, 341, 429, 502; orange G and safranin, 363, 364, 513; orseillin BB, 501; phlox- ine, 495; picric acid, 480; safranin, 393, 474, 475; safranin and orange G, 364; safranin O, 481 CS 12, 13 moimtants, 633 CS 15 mountants, 639 CS 19 fixative, 232 CS 32 fixative, 235 CS 33 fixative, 231 Ctenoid scales, 386 Cuccati's carmine, 306 Culbertson, see Markey, 508; White, 475 Cumacea, 49 Cumming's fixative, 234, 242 Cunge, see Slominski, 236, 420 Cunningham's method for reticulocytes, 417 Cuprammonium complex, as mordant in Lagerberg's method for bacterial spores, 486 for polychroming methylene blue, 349 Curreri on Kallius's developer, 618 Curtis's, metliod for Saccharomyces in sec- tions, 503 salicylic balsam, 639 stains, black, 325 ; picro-naphthol picro-ponceau S, 327 Cushman, method for separating foraminif- eran tests, 17 Cutler's glycol stearate embedding medium, 643, 649 Cutting, theory of, 109 Cutting facet, effect of faulty, 120 on microtome knives, 109 Cyanin, staining combination with eryth- rosin, 391 Cyanophyceae, 511 Cycloid scales, 386 Cyrillic alphabet, transliteration of, 3 Cytoplasmic inclusions, mentioned in, Arnold's triple stain, 351; Drew-Mur- ray's triple stain, 369; Ehrlich's triple stain, 369; Holmes and French's triple stain, 352; Langeron's double stain, 355; Lillie's triple stains, 366; Sziitz's polychrome alizarin, 329; Unna's quad- ruple stain, 364 Cytoplasmic inclusions, see also Golgi, Mitrochondria, Virus, etc. Czaplewski's method for acid-fast bacteria in sections, 496 Czermak's fixative, 228, 242 Czokor's alum cochneal, 300 D Daddi's, method for fat, 447 Sudan III in Bell's method for fat, 447 Dagnelle, see Lison, 410 Dahlgren's, fixative, 217 method for muscle, 423 Dahlia violet, as ingredient of, Henking's fixative, 194 in, Bohm and Oppel's triple stain, 368; Roux's double contrast, 327 staining combinations with, Bismark brown and methyl green, 368; dahlia violet and methyl green, 327 Dalton's method for nuclei, 435 Damar, see Gum damar Dammen's method for cestodes, 506 Danchakoff's fixative, 218 Daniel's method for muscles of ciustacea, 394 Darlington, see Newton, 201 INDEX ■09 Darrow's, chlorazol black E, 369 methods for, nuclei, 1:55; plant sfctions, 32'.) Darzin's method for Rickettsiae, 4t)2 Davalos's magenta, 315 Davenport's developer, 617 in, Davenport's method for neurofibrils, 554; Foley's method for axons, 555 silver nitrate stain, 550 in, Foley's method for axons, 555 Davenport, see also Bank, 237; Heller, 403; MacFarland, 5G7; Swank, 205; Weil, 589 Davenport and Ivline's fixatives, acetic-trichloroacetic, 190; trichloro- acetic, 190 Davenport, McArthur and Bruesch's de- veloper, 617 in Davenport, McArthur and Bruesch's method for nervous tissue, 567 fixative, 190 Davenport, Windle and Beech's fixative, 192 in Davenport, Windle and Beech's methods for embryonic nervous tissues, 554, 582 silver diammine stain, 577 Davenport, Windle and Rhines's, developer, 617 in Davenport, Windle and Rhines's method for axis cylinders, 564-566, 567 fixative, 237 David's adhesive for paraffin ribbons, 657 decalcifying fluid, 257 method for bacterial flagclla, 609 silver sulfate stain, 608 in David's method for bacterial flagella, 609 tannic-mercuric mordant, 517 Davidoff's fixatives, mercuric-acetic, 209, 242 picric-acetic, 221, 242 Davies's asphalt varnish, 653 mountant, 631 Davies, see also Blair, 551 Dawson's method for, bone, 383; Negri bodies, 465 Dawson and Barnett's method for argento- phil granules, 567 Dawson and Friedgood's fixative, 211, 242 in Dawson and Friedgood's method for pituitary, 425 Dead black varnish, 656 Deane's glycerol jelly, 634 Debaisieux's fixative, 224 Debauche's embedding wax, 646 silver diammine stain, 580 in Debauche's method for invertebrate neurology, 582 De-alcoholizing agents, 623 Decalcification, teclmique for mouse, 138 Decalcifying fluids, 256-260 Decimal divisions of, accessory dye staining solutions, 514 accessory fixative formulas, 254 accessory metal staining solutions, 612 adhesives, 650 cements, lutes, and varnishes, 650 dye-stains of general application, 267-269 dye-stains of special application, 374 -37(5 embedding media, 642 fixatives, 182-185 injection media, 650 metal stains, 522-524 miscellaneous formulas, 650 mounting media, 630 preservatives, 175 solvents and oils, 022 various formulas, 650 Decimal references, explanation of, 1 Deegener's fixative, 222, 242 Deflandre's, Canada balsam-coumarone mountant, 641 coumarone mountant, 640 Dehydrating agents, classification of, 622 general remarks on, 622 selection for paraffin embedding, 96 Dehydration, capillary technique, 297 of, materials for paraffin embedding, 96, 97; nitrocellulose sections, 152 special jar for, 129 Deipolli and Pomerri's method for Nissl granules, 445 Dekhuyzen's, fixatives, osmic-dichromate, 203; osmic-dichro- mate-nitric, 204 method for blood, 417 Delafield's alum-hematoxylin, 287 as ingredient of, Conklin's hematoxylin, 289 for, amphibian embryos, 136; nitrocellulose sections of lily bud, 151 in, Bretschncider's method for insect brains, 410; Brillmeyer's technique, 336; Endi- cott's method for bone marrow, 430; Lubarsch's method for glycogen, 452; Maximow's triple stain, 349; Reeves's triple stain, 367; Reynold's method for nematodes, 509 Delamare's triple stain, 365 DeLameter, see Kligman, 504 Delepine's glycerol jelly, 634 double contrast, 328 reticulum fibers in, 594 5-cells of pituitary, 427 Demarbaix's fixative, 228, 242 710 INDEX DemoU, see Becher, 197 Dendrites, gold-merciiry method for, 539 mercuric-dichromate method for, 610 Dendrocometes, 52 Denham's sandarac mountant, 638 Dentine, Choquet's method for, 386 gold method for, 535 Grieves' method for, 383 Hanazawa's methods for, 383, 564 Must and Rose's method for, 386 Orban's method for, 596 Weil's method for, 385 Dependorf 's method for nerves in teeth, 535 Desmids, 513 Destin's fixative, 212, 230, 242 chromic-formaldehyde-acetic, 212, 242 Developers for metal stains, comparison with photographic, 615 formulas, 616-620 Dextrin, as ingredient of, Anderson's embedding medium, 642; Davenport's developer, 617; Meakin's adhesive, 661; Obregia's adhesive for free sections, 660; Webb's embedding medium, 645 Dextrose, as ingredient of, Berlese's mountant, 631; Doetschman's mountant, 631; Emig's mountant, 631; Landau's mountant, 632; Obregia's ad- hesive for free sections, 660 Diacetin, as solvent for, Gros's scarlet R, 448; Leach's Sudan black B, 448 Diacetone, 626 Diacetone alcohol, physical properties of, 626 Diamminophenol hydrochloride, as ingredi- ent of Davenport, McArthur and Bruesch's developer, 617 Diamond on iron-hematoxylin stains, 281 Diamond's iron-mordant hematoxylin, 281 Diaphenol, 256 Diatoms, arranging on coverslip, 39, 40 cleaning, 38-39 collecting, 37 concentrating, 37 drying and sorting, 40 fossil, 38 mounting dry, 37 mounting in bromonaphthalene, 40 Dibutyl phthallate, in Fleming's mountant, 641 Dichlorethyl ether, physical properties of, 625 Dichromate-silver stains, formulas, 603 general remarks on, 543 Dichromates, as albumen precipitants, 187 effect of pH on fixation by, 188 see also Potassium dichromate, ammonium dichromate, etc. Dick, see McCuUough, 491 Dickson's method for fungus in plant tissues, 501 Dickson, see also Burke, 476 Diercks and Tibbs' method for MacNeal's stain, 345 Dieterle's developer, 017 in Dieterle's method for spirochetes, 548- 550-560 Diethylamine barbiturate, as ingredient of de Castro's decalcifier, 257 Diethylene dioxide, physical properties of, 626 Diethylene glycol, as ingredient of Lebo- wich's soap embedding medium, 644 physical properties of, 623 Diethylene glycol monobutyl ether, as de- hydrant, 348 physical properties of, 626 Diethylene glycol monoethyl ether, physical properties of, 627 Diethylene glycol monoethyl ether acetate, physical properties of, 627 Diethylene glycol monomethyl ether, physi- cal properties of, 627 Diethylene glycol monostearate, as ingredi- ent of Steedman's embedding wax, 647 Dietrich's fixative, 191, 242 method for, fat, 447; heart muscle, 394 Differentiating hematoxylin, 274 Differentiators for, dye stains, 519-521 metal stains, 621 DifHugia, 52 Dileptus, 52 Dimethoxytetraethylene glycol, physical properties of, 627 Dinitroesorcinol, in Obersteiner's method for axis cylinders, 411 Dioxane, as ingredient of, Allen and McClung's fixatives, 221; Armitage's fixatives, 191, 221; Armi- tage's sandarac mountant, 638; Bauer's fixative, 223; Cretin's fixative, 222; Graupner and Weissberger's fixatives, 193; Mohr and Wehrle's sandarac mountant, 638; Potenza's fixatives, 190, 222; Puckett's fixative, 225; Roskin's fixatives, 221; Thomas' hematoxylin, 286, 293; Waterman's fixatives, 221, 224; Weber's alcoholic accelerator, 615 for dehydration, 96, 359 in, Snider's method for Nissl granules, 446; Weber's method for fatty tissues, 585 physical properties of, 626 Diphtheria bacilli, stains for, 489-490 Dipropylene glycol, physical properties of, 623 INDEX 711 "Direct" hematoxylin stains, 284-293 "Direct" staining, explanation, 271, 284 Disintegration of fossil deposits, 18 Dissecting dishes, wax for, 667 Dissociating agents, 262-264 Dissection of earthworm ovary, 526 Dobell's, fixatives, picric-acetic, 222, 242; picric-formalde- hyde-acetic, 224 iron-mordant hematin stain, 281 method for Entamoeba histolytica, 507 molybdenum-hematoxylin, 507 phosphotungstic hematoxylin, 507 triple stain, 369 Doetschman's "Berlese" mountant, 631 Dogiel's method for corpuscles of Grandry and Herbst, 554 Doglio's method for acid-fast bacteria, 476 Doherty, Suk and Alexander's method for blood capillaries, 417 Doinikow's method for regenerating nerves, 582 Doleris, see Morel, 357 Domagk's picro-thiazin red, 325 Dominici's fixative, 219, 242 in Nageotte's method for Schwann cells, 415 triple stain, 351 Donaggio's, method for neurofibrillae, 402 in hematoxylin, 291 in Donaggio's method for nerves, 404 Donaggio, see also Vassale, 605 Donaldson's iodine-eosin, 321 Dorner's method for bacterial spores, 485 Dornfield, see Slater, 367 Dotti, Papara and Clarke's method for de- calcification, 256 Double-embedded sections, 153-156 Double embedding, technique of making block, 155 Double stains, see under author or ingredient Doub row's method for acid-fast bacteria in sections, 496 Douglas's method for acid-fast bacteria in sections, 496 Doutrelepont's method for spirochetes, 479 Downey's, fixative, 211 method for megakaryocytes, 432 Downey, see also Slider, 350 Downs's polyvinyl alcohol mountant, 42, 636 Doyere's injection fluid, 662 injection method, 163 "DPX" mountant, 641 Dracunculus, larvae of, 509 Drasch's method for nerve endings in ali- mentary canal, 535 Drew's fixative, 236 in Drew's method for mitochondria, 443 Drew-Murray's triple stain, 369 Dried plants, reswelling, 667 "Drierite," 623 Driver's method for disintegration of fossil deposits, 18 Drosophila, salivary gland chromosomes, 437 Drost's macerating fluid, 263 Drueger, see Proescher, 318 Drliner's fixative, 197, 242 Dry wholemounts, 10-20 attaching cells for, 13 backgrounds for, 13-14 cementing coverslips to, 16 objects in, 14-16 definition, 10 of, bone, 85; Foraminifera, 17-20; Radio- laria, 17-20 sealing, 16 selection of, cell, 11-12; coverslip, 11; slide, 10 wooden slides for, 10 Dublin's method for, melanin, 568 skin, 567 "Dubosq," 314 Dubreuil's double contrast, 325 Dubreuil, see also Lancelin, 597; Tre- bondeau, 289, 484, 490 Dubuscq's method for arthropod blood, 417 Duesberg, see Meves, 201, 248 Duff, see MacCallum, 426 Dufrenoy's method for mitochondria and bacteria, 444 wax embedding method, 646 Duggan's fixative, 200, 242 Duggar's fixative, 210, 242 Dulaus, see Morel, 505 Dunham's clearing mixture for nitrocellulose sections, 628 Duperie's method for spirochetes, 479 Dupres's, differentiator, 520 in Dupres's technique, 315 stains, acid fuchsin-tolindine blue-orange G, 359; magenta, 315, (in Dupres's tech- nique, 339); toluidine blue-orange G, 339 Durig's, fixative, 233, 242 silver-dichromate method for nervous tissues, 604 Du's method for Treponema, 479 Duthie's fixative, 216 Dutton's method for bacterial spores, 485 Duval, see Rubens, 389 Dye, definition of, 269 Dye staining, definition, 269 Dye staining methods, see under name of author, object or ingredient Dye staining techniques, method of classifi- cation, 271 remarks on, 271 712 INDEX Dyes in mountants, Bernhardt's cotton blue, 503 Doetschman's magenta, 631 Semmen's aceto carmine, 633 Semmen's eosin balsams, 639 Semmen's Nile blue sulfate balsam, 639 Swartz and Conant's, 505 Zirkle's carmine-gelatin, 635 Zirkle's carmine-pectin, 636 Zirkle's carmine-pectin, 637 Zirkle's iron carmine, 633 Zirkle's orcem-gelatin, 636 Dyes, synonomy used, 3 Dyspontius, 49 E "EA" stains, 432 Ear, Fieandt and Sazen's decalcifying fluids for, 258 Golgi bodies in, 590 Earle, see Lillie, 285 Earthworm, embedding, 331 fixing, 331 narcotizing, 330 ovary for Golgi bodies, 525-527 removing grit from intestine, 330, 331 smears of monocj'stis from, 72, 73 spermatozoa, 73 staining sections of, 331, 332, 333 Eau de Javelle, for cleaning foraminiferan tests, 17 Eberspiicher's differentiator, 520 von Ebner's decalcifying fluids, 257 for mouse, 138 Echinoderm larvae, fixation, 154 sections of, 153-156 Eckert's preservative, 178 Eden's method for amyloid, 453 Edwards, see Bruner, 490 Egg albumen, as ingredient of, Heidenhain's adhesive for paraffin rib- bons, 657; Lillie's adhesive for paraffin ribbons, 658; Mayer's adhesive for paraffin ribbons, 658; Reinke's adhesive for paraffin ribbons, 658; Schncidan's adhesive for paraffin ribbons, 659 as injection medium, 163 for, attaching protozoa to coverslip, 52; clarifying gelatin, 633 Eggs, amphibian, 133 orienting in paraffin blocks, 135 insects, softening, 261 nematodes, 35 reptiles, recommended fixatives for, 214 Ehler's fixative, 228, 242 " Ehrlich-Biondi " stain, 356 Ehrlich's, methods for, bacterial smears, 473 granules in mast cells, 455 Ehrlich's stains, acid-alum-hematoxylin, 289 differentiating, 280 for staining, embryo sections of chicken, 278, 280; fat, (Clark), 447; Negri bodies, (Barreto), 464; nerves in muscle, (Sihler), 407; nerves in wholemounts, (Nelson), 406; nerves in whole- mounts, (Wharton), 408; pancreas, (Baley), 428; pituitary, (Cleveland and Wolfe), 425; vaginal smears, (Fuller), 430; vaginal smears, (Papa- nicolaou), 431, 432 in, Delamare's quadruple stain, 365; Pasini's triple stain, 338 ripening, 280 aurantia-eosin Y-indulin, 369 gentian violet, 473 for staining, acid-fast bacteria, (Fraenkel), 470; acid-fast bacteria, (Koch), 477; ac- tinomycetes, (Bostrom), 501; actino- mycetes, (^lallory), 504, 505; Asper- gillus fumigatus, (Besson), 503; bac- teria in sections, (Ollett), 493; bacteria in sections, (W^eigert), 495; bacterial capsules, (Buerger), 487; bacterial capsules, (Wadsworth), 489; bacterial capsules in sections, (Smith), 498; bacterial flagella, (Bowhill), 481; bacterial flagella, (Sclavo), 484; bac- terial flagella, (Trenkmann), 484; fungi in skin scrapings, (Chalmers and Marshall), 503; Gram-positive bacteria in sections, (Haythorne), 494; nuclei, (Buzzozero-Vassale), 435 methyl green-orange G-acid fuchsin, 356 in Morel and Doleris technique, 357 Eichorn's chromic moi'dant, 515 Einarson's method for Nissl granules, 445 Eisath's, differentiator, 519 in his method for neuroglea, 413 fixative, 233, 242 Eisig's fixative, mercuric-acetic, 209 platinic-chromic, 207, 242 as ingredient of, \\T3itman's fixative, 195 Elastic fibers, mentioned in, Buzaglo's quadruple stain, 368; Dela- mare quadruple stain, 365; Korn- hauser's qiuidruple stains, 369, 370; Matsura's polychrome neutral red, 371; Mollier's quintuple' stain, 366; Pasini's quadruple contrast, 338; Romeis's quin- tuple stain, 367; Unna's quadruple stain, 364; Volkman and Strauss's triple stain, 373; Walter's triple stain, 361 silver-arsenic-dichromate method for, 607 special dye stains for, 387-390 INDEX 713 Elastic tissue, differentiation from fat, Frencli's method, 448 mentioned in, Paquin and Cloddard's quintuple stain, 366; Wallart and Honetti's double con- trast, 339 Elder pith, for freehand sections, 89 Electric decalcification, 259 Eleidin, 422, 457 Ellerman's fixative, 218 in Ellerman's technique, 344 double stain, 344 Ellerman, see also Bing, 193 Ellermaner's method for blood, 417 Ellsworth, see MacCalluni, 426 Eltringham's fixatives, mercuric-acetic, 209, 242 mercuric-formaldehyde, 211 on softening chitin, 261 picric-acetic, 222 picric-nitric, 222, 242 for softening chitin, 261 Embedding, frog embryos for freehand sec- tions, 90 heavily yolked embryos, 646 in balsam, for ground sections, 86 Embedding media, agar, 643, 645 albumen, 643 carbowax, 643 classification of, 642 dextrin, 645 gelatin, 642-644 general remarks on, 642 glycol stearate, 643, 649 gum arabic, 643, 644, 645 isinglass, 643 nitrocellulose, 647-649 polyvinyl alcohol, 644 resinous, 649 soap, 643 water miscible, 642-644 wax, 645-647 Embedding methods for insects, Jurray's, 261 Embedding ovens, 98ff, 99ff, 106ff Embedding techniques, see under Paraffin, Nitrocellulose, etc. Embedding waxes, properties of, 97 Embryonic teeth, Van Beast's, method for, 256 von Korff's method for, 384 Embryos, brains of, 606 calcification centers in, 596 fish, nerves in, 584 heavily yolked, Ammerman's fixatives for, 228; Bragg's embedding method, 646; cutting sec- tions of, 133-136; Gregg and Puckett's method for, 134; Smith's method for, 134, 135; suggested fixatives for, 133 Embryos — (continued) nervous tissue in, 552, 554, 582 Nollister's luelliod for lione in, 384 preserving for bone staining, 180 recommended fixatives for, 95 staining before embedding, 300 vertebrate, wholemounts of, 54 wholemounts to show skeleton, 382, 383 Embryos, see «/.s-o Chicken, Frog, etc. Emelianoff, see Churchman, 487 Emig's fixative, 219 mountant, 631 stain for plant sections, 392 Enamel of teeth, Bodecker's decalcifying fluids for, 257 End plates, see Nerve endings Endicott's double stain, 348 in Endicott's method for bone marrow, 430 Endothelium of blood vessels, 664 Entamoeba, 507, 508 Enzyme macerating fluids, 264 Eosin, as stain, 321 ethyl, see Ethyl eosin, 320 for, coloring nitrocellulose blocks, 153 Eosin, for staining, acid-fast bacteria (Herman), 477; ac- tinomyces (Lemiere and Becue), 504; bacterial capsules (Muir), 488; bacterial spores (Abbott), 484; bacterial spores (Muzzarelli), 486; blood (Hayem), 418; bone marrow (McJunkin), 456; cocci in cell smears (Kahlden and Laurent), 491; fecal smears (Kofoid), 508; Herbst's corpuscles (Novak), 431; insect brains (Brctschneider), 410; insect muscles (Kramer), 424; Negri bodies (Barreto), 464; Negri bodies (Nakamura et al.), 467; Nissl granvdes (Bean), 445; oocysts of coccidia (Crough and Becker), 506; pancreas (Baley), 428; Plasmodumi (Hobbs and Thompson), 508; Rickett- siae (Bohner), 462; Rickettsiae (Bond), 462 m, Blank's triple stain, 352; Bohm and Oppel's triple stain, 347; Donaldson's simple contrast, 321; Friedlander's double stain, 365; Galiano's stain, 365; Gregg and Puckett's double contrast, 328; Houcke's quintuple stain, 351; McClean's simple contrast, 321; Mann's double stain, 371; MoUendorf's triple stain, 366; Paquin and Goddard's sex- tuple stain, 366; Semichon's triple con- trast, 328; Semmen's Canada balsam mountant, 639; Unna's quadruple stain, 364; various double stains with azurs, 347-350; various double stains with methylene blue, 344-345; various dou- 714 INDEX Eosin — (contin ued) in — (continued) ble stains with polychrome methylene blue, 345-347 staining combinations with, anilin blue, hematoxylin, orange G and phloxine, 366; anilin blue and orange G, 428; anilin blue, orcein and safra- nin, 431; azur, 347; azur I, crystal violet and methylene blue, 461; azur and mercurochrome, 352; azur and methylene blue, 506; azur, methyl blue, methylene blue and orange G, 418; azur and orange G, 464; azur and orcein, 424; azur and Sudan black B, 420; azur B and methylene blue, 508; azur II, 349, 350; Bordeaux red and hematoxy- lin, 510; crystal violet, 477; hematoxy- lin, 365; hematoxylin and methyl blue, 366, 464; iodine, 321; Janus green, 506; magenta, 467; methyl blue, 371, 372, 462, 466, 491; methylene blue, 394, 486; orange G, 328; thionin and azur A, 348 Eosin B, as plasma stain, 320, 321 for staining, blood (EUermaner), 417; bone (Bock), 382; Negri bodies (Petragnini), 466; Plasmodium (Sinton and Mulligan), 510; Plasmodium (Villain and Comte), 510 in, Jensen's simple contrast, 321; Krajian's simple contrast, 321; Krugenberg and Thielman's triple stain, 370; Pasini's quadruple contrast, 338, simple solu- tion, 320 staining combinations with, acid fuchsin, anilin blue and orcein, 338, 361; acid fuchsin, anilin blue, azocar- mine and orcein, 361; anilin blue and phloxine, 370; azur A, 349; azur C, 349; azur II, 510; hematoxylin and methy- lene blue, 466; methylene blue and thionin, 347; polychrome methylene blue, 346, 347; saffron, 329; toluidine blue, 348 Eosin BA, for staining, blood (Ugruimow), 420 Eosin W, for staining, amphibian blood (Moore), 418 in, Hayem's double contrast, 328 staining combinations with, aurantia, 328; methyl green, 418 Eosin Y as, plasma stain, 320, 322 for staining, actinomyces (Langrand), 504, am- phibian, blood (Liebmann), 418; bac- terial capsvdes in sections (Smith), 498; bacterial spores (Ruiz), 486; bacterial spores (Zeeti), 487; basal bodies (Wal- Eosin Y — (continued) for staining — (continued) lace), 458; blood (Saye), 419; bone (Bock), 382; fecal smears (Hollande), 508; fecal smears (Shortt), 510; fila- mentous fungi (Chamberlain), 511; fungus in skin scrapings (Chalmers and Marshall), 503; megakaryocytes (Kings- ley), 431; megakaryocytes (Wright), 432; Negri bodies (Carpano), 464; Negri bodies (Jordan and Heather), 465; Negri bodies (Lenz), 466; Negri bodies (Neri), 466; pituitary (Big- gart), 425; Plasmodium (Corbin), 506; skin (Pinkus), 424; vaginal smears (Papanicolaou), 431, 432; virus inclu- sion bodies (Hamilton), 465 m. Bauer and Leriche's triple stain, 352; Chatton's double contrast, 327; Do- bell's triple stain, 369; Dominici's triple stain, 351; Ehrlich's triple stain, 369; Groat's quadruple stain, 349; Guy- ler's double contrast, 328; Holmes and French's triple stain, 352; Hubin's triple contrast, 340; Kingsley's quadru- ple stain, 348, 349; Lendrum's quadru- ple contrast, 340; Lillie's double stain, 370; MacNeal's triple stain, 349; Pearson's simple contrast, 322; Re- naut's double stain, 367; Rhamy's triple stain, 353; simple solution, 320 staining combinations with, acid fuchsin, anilin blue, and orange G, 431; anilin blue, 387; aurantia and indulin, 369; azur A, 349; azur A, methylene blue, and methylene violet, 348, 349; azur B and phloxine, 465; azur C, 349; azur C and orange II, 352; azur I and methylene violet, 349; azur II, 341, 349, 418; azur II and methylene blue, 349, 350; azur II, orange G, thionine and toluidine blue, 351; Bis- marck brown, light green and orange G, 432; brilliant cresyl blue and methylene blue, 352; crystal violet, 464; crystal violet and magenta, 504; erythrosin, phloxine and tartrazine NS, 340; ethyl eosin and methyl blue, 465; gentian violet, 503; hematoxylin, 367; hema- toxylin and crystal violet, 366; hema- toxylin and light green, 508; hematoxy- lin and malachite green, 366; hematoxy- lin and methyl green, 498; hematoxylin and methyl violet, 458; indigo and car- mine, 328; isamine blue, 425; light green, 327; magenta and methylene blue, 353; methyl blue and victoria yellow, 329; methyl violet, 486; methylene blue, 344, 345, 347, 466, 487; methylene blue, INDEX 715 Eosin Y — (continued) staining combinations with — (continued) methyl violet 2B and thionine, 349; naphthol green B, 370; orange G and methyl blue, 369; orange G and safra- nin, 340; orange G and toluidine blue, 351; polychrome methylene blue, 34G, 347, 350; polychrome methylene blue and phloxine, 345; thionine, 419 "Eosinol," 321 Epidermis, mentioned in Dupres's double contrast, 339 Epistylis, 53 Epithelia, cell outlines of, 607, 608 mentioned in Patay's double contrast, 339 osmic stain for, 530 Epithelium, alveolar, gold method for, 534 ciliated, in unstained sections, 270 Eppinger's method for bile capillaries, 423 Epstein's method for blood, 417 Erb's method for bacteria in milk, 491 "Erenol," 640 Erie garnet B, in Geschickter's double stain, 352 Eriocyanine, in Lillie's method for pituitary, 426 Erlanger's fixative, 198, 242 Erlicki, see Erlitzky Erlitzky's fixative, 220 in, Aronson's method for nervous tissue, 409; Beckworth's method for nerves in teeth, 534; Kultschitsky's method for myelin sheaths, 405; Wolter's method for myelin sheaths, 408 recommended use, 95 van Ermengen's, developer, 617 in, Manouclian's method for spirochetes, 562; Seguin's method for spirochetes, 598 fixative, 194, 242 in, van Ermengen's method for bacterial flagella, 563 Eros' method for calcified tissues, 383 Erysiphaceae, 51 1 Erythrocytes, Grossman's method for, 416 "flagella" on, 457 mentioned in, Dupres's double contrast, 339; Gold- ner's quadruple contrast, 337; Hay- thorne's triple contrast, 337; Hol- lande's triple contrast, 339; Houcke's double stain, 353; Kornhauser's quadru- ple stains, 369, 370; Lillie's triple stains, 370; Mallory's triple stain, 360; Mas- son's double contrast, 337; Mollier's quintuple stain, 366; Pasini quadruple contrast, 338; Patay's double contrast, 339; Walter's triple contrast, 361 Erythrocytes — (continued) oxidase granules in, 455 specific stains for, 418 Erythrosin, as ingredient of, Semmen's Canada balsam mountant, 639 as plasma stain, 320, 321 for staining, fungus in plant tissue, (Margolena), 502; Gram-positive bacteria in sec- tions, (Haythorne), 494; mitochondria, (Held), 443; nervous tissues, (Prince), 411; nuclei, (Cooper), 435; nuclei, (Jo- hansen), 436; pituitary, (Cleveland and Wolfe), 425; Zygonema, (Bigot), 503 in, Lendrum's quadruple contrast, 340; McClean's simple contrast, 321; Mann's double stain, 371; Mann's triple stain, 352; Masson's double contrast, 328; Masson's triple stain, 371; White's double contrast, 326 staining combinations with, acid fuchsin and methyl green, 435 anilin blue-hematoxylin-orange G, 425 anilin blue-magenta-methyl orange, 411 capri blue, 392; crystal violet, 392, 503 cyanin, 391; eosin Y-phloxine-tartra- zine N.S., 340; gentian violet-hema- toxylin, 494; light green-orange G- thionin, 502; methyl violet, 213; orange G-toluidine blue, 352, 371; picric acid, 326; saffron, 329, 341; toluidine blue, 371 Espinasse's nitrocellulose-wax embedding methods, 648 Essential oils, for clearing, 56 general remarks on, 623 in, clearing mixtures, 628; paraffin em- bedding, 96 physical properties of, 624 Ethanol, physical properties of, 623 Ethanol, see also Alcohol Ethanolamine, as ingredient of, Lebowich's soap embedding medium, 644 Ether, as ingredient of, Behren's copal varnish, 652; Bujor's narcotic, 265; Gram-Rutzon's varnish, 653; Gray's fixatives, 214; Petrunke- witsch's fixatives, 219, 221; Wallart's alcoholic accelerator, 615; Waterman's fixatives, 221; Wilson's decalcifying fluids, 260 as, narcotic, 265, refrigerant for frozen sec- tions, 158; solvent for nitrocellulose, 647, 648; solvent for stains, Shultz's methylene blue, 491 for, flattening double embedded sections. 716 INDEX Ether — (continued) for — (contin ued) 156; narcotizing Platyhelminthes, 53 physical properties of, 625 specifications for nitrocellulose embed- ding, 143 Ethyl acetate, physical properties of, 625 Ethyl benzoate, physical properties of, 625 Ethyl cellulose, as ingredient of. Barlow's embedding medium. 649; Mohr and Wehrle's varnish, 654; Steed- man's embedding wax, 647 Ethyl eosin, as plasma stain, 320 , for staining, blood, (Sheehan), 420; chitin, (Smith), 391; Negri bodies, (Harris), 465; Negri bodies, (Lillie), 466; Negri bodies and Nissl granules, (Parson), 467; Negri bodies, (Stovall and Black), 467; Plas- modium, (Schmorl), 509 ; vu-us inclusion bodies, (Hamilton), 465 staining combinations with, anilin blue, eosin, orcein and safranin, 364; azur C, 347; azur II, 347; eosin Y-methyl blue, 465; hematoxylin, 466; methylene blue, 345, 465, 467, 509; Sudan black B, 420 Ethyl violet, for staining, neuroglia, (Bailey), 412; pancreas, (Bowie), 428; pepsinogen granules, (Bowie), 455 staining combination with, Biebrich scarlet, 428, 455; orange G, 412 Ethylamine, as ingredient of, Cajal's alcoholic accelerator, 614; Her- rera's silver diammine stain, 578 Ethylene diammine tetra acetic acid, tetrasodium, 260 Ethylene dichloride, physical properties of, 625 Ethylene glycol, as ingredient of, Thomas's hematoxylin, 293 as solvent for stain, 348 Ethylene glycol distearate, as ingredient of, Steedman's embedding wax, 647 Ethylene glycol monoethyl ether, as ingredi- ent of. Gray and Wess's mountants, 641; Hanley's narcotics, 265 as solvent for stains, Lendrum's quadruple contrast, 340 physical properties of, 627 Ethylene glycol monoethyl ether acetate, physical properties of, 625 Ethylene glycol monomethyl ether, as in- gredient of, Johansen's fast green, 321; Johansen's safranin, 314 in, Johansen's quadruple stain, 364 physical properties of, 623 Ethylene glycol monomethyl ether acetate, physical properties of, 627 Ethylene glycol monostearate, as ingredient of, Steedman's embedding wax, 647 Eucaine hydrochloride, as ingredient of nar- cotics, 265 Eucalyptol, as ingredient of, Mohr, and Wehrle's sandarac mountant, 638; Shepherd's sandarac mountant, 638 Eucalyptus oil, as ingredient of Bucholz's sandarac mountant, 638 physical properties of, 624 Eulenstein's Brunswick black, 653 "Euparal," 637, 638 Euphorbia, Phytomonas in, 511 Eusweller's method for plant squashes, 76 Evans and Krajian's decalcifying fluid, 258 Evaporation rate, of dehydrating agents, 623 synthetic clearing agents, 625, 626 universal solvents, 626 Ewald's fixative, 193, 242 Ewig's mountant, 631 Eycleshymer's clearing mixture, 628 Eyene and Sternberg's developer, 617 in Eyene and Sternberg's method for spirochetes, 561 Eyes, arthropod, method for bleaching, 261 Faberge's, macerating fluid, 264 method for plant squashes, 77. 78 Fabre-Domergue's, fixative, 194, 243 preservative, 178 Fajersztajn's, method for, axis cylinders, 583; myelin sheaths, 404 silver diammine stain, 575, 577 da Fano's, fixative, 236, 243 in, Chatton and Lwoff's method for ciliates, 559; Weatherford's method for Golgi bodies, 559 formaldehyde accelerators, 613 in da Fano's method for nerve cells and processes, 554 methods for, nervous tissues, 582; pericellular bas- kets, 583 1914 silver diammine stain, 575, 577 in, da Fano's methods for nervous tis- sues, 582 1919 silver diammine stain, 575, 577 in, Gridley's method for reticulum fibers, 592; Ingleby's method for neuroglia, 586; Perdrau's method for reticulum, 594 Fant's cement, 655 for use with dry wholemounts, 15 INDEX 717 Fant's cement — (continued) use of, 33, 34ff, 3G, 37 Farkas's fixative, 198, 243 Farmer and Shrove's fixatives, 189, 243 Farrants's mountant, 632 index of refraction, 42 Farrior and Warthin's developer, 617 in Farrior and Warthin's method for spirochetes, 561 Fasciola, see Liver fluke Fast green, FCF as stain, 320, 321 for staining, bacterial smears, (Alaneval), 473; gran- ules, breast and kidney, (Lendrum), 456; nervous tissues, (Foley), 567; nu- clei, (Dalton), 435; nuclei, (Kurnich and Ris), 436; plant tissues, (Johansen), 421; reticular fibers, collagen and myofibrils, (Long), 597; vaginal smears, (Shoor), 431 in, clove oil solution, 320; Conant's quad- ruple stain, 363; Gomori's double con- trast, 339; Johansen's quadruple stain, 364; Johansen's simple contrast, 321; Kalter's quadruple stain, 364; Korn- hauser's quadruple stains, 369, 370; Lillie's double contrast, 337; Lillie's double stain, 370; Lillie's triple stain, 366; Alilligan's triple stain, 360; Reeve's triple stain, 367 staining combinations with, acid alizarin blue, orcein and orange G, 369, 370; acid fuchsin, 370, 421; acid fuchsin and hematoxylin, 456; acid fuchsin, orange G, picric acid and pon- ceau 2R, 337; anilin blue, gallocyanin, orange G and silver protein, 567; anilin blue and orange G, 360; azocarmine and silver diammine, 597; Biebrich scarlet, 337; Biebrich scarlet and orange G, 431 ; Bismark brown, 393, 512; Bismark brown and hematoxylin, 366; chromo- trope 2R, 339; crystal violet, orange II and safranin, 363, 364; hematoxylin and Sflfraniu, 367; magenta, 497; methyl violet, orange G and safranin, 364; methyl violet and safranin, 421 ; orcein, 435, 436: violamiue R, 370 Fast yellow, for staining acid-fast bacteria, (Alexander and Jackson), 476 in, Wallart and Houette's double contrast, 339 staining combinations with, acid fuchsin, 339; light green, magenta and methylene blue, 476 Fat, dye staining methods for, 447-452 in bacteria, 491 mentioned in Williams's polychrome cresyl violet, 373 Fat — (conlin ued) osmic acid mctliods for, 530 Schridder's fixatives for, 205 Fat glands, in rodent skin, 450 Fat granules, in blood, 416, 420 P'atty tissues, mitochondria in, 518 nerves in, 585 technique of sectioning, 160-1(>1 Faulkner and Lillie's developer, 617 Faure's, mordant, 516 as ingredient of Faure's iron-heinatoxy- lin, 285 mountant, 632 Faussek's fixative, 228, 243 Favorsky's fixative, 189, 243 in Favorsky's method for nerve cells and processes, 554 silver diammine method for nervous tis- sues, 581 Fecal smears, various staining methods for, 506 Wheatley's method for, 339 Feces protozoa in, 321 separating nematodes from, 35 Felix's, fixative, 228 macerating fluid, 263 Ferguson's, fixative, 201 method for spirochetes, 597 Ferrari's method for fungi in plant tissue, 501 Ferreira, see Lhotka, 256 Ferreri's decalcifying fluid, 258 unsuitability for large objects, 138 Ferric acetate, as mordant for hematoxylin, 281, 283 Ferric alum, as ingredient of, Anderson's hematoxylin, 285; Anony- mous coelestin blue B, 313; Becher's naphthopurpurin, 313; Cajal's iron toning solution, 620; Cerrito's magenta, 482; Gordon's differentiator, 579; de- Groot's carmine, 304; Hansen's carmine 304; hematoxylin stains, 285-286, 406 Kupperman and Noback's fixative, 191 Lang's mordant, 516; Lendrum 's celes- tin blue, 313; Loffler's indigocarmine- methyl violet, 483; Peter's carmine, 305; Proescher and Arkush's celestin blue, 313; del Rio-Hortcga's formalde- hyde accelerator, 614; Strong's for- maldehyde accelerator, 614 as mordant for, acid fuchsin, 361, 420; azocarmine, 428; azur I, 467; brazilin, 308; carmine, 305; hematoxylin, 281-283, 412 in, Benda's method for mitochondria, 442; Cansey's method for mitochondria in protozoa, 442; Drew's method for mitochondria, 443; Fuller's method for 718 INDEX Ferric alum — {continued) in — {continued) acid-fast bacteria in sections, 496; Gordon's method for blood parasites, 598; Gordon and Sweets's method for reticulum fibers, 592, 593; Moore's method for fungus in plant tissue, 502; Shessarew's method for reticulum, 594 selection of crystals of, 273, 274 Ferric ammonium citrate, as mordant for carmine, 302 Ferric chloride, as ingredient of, Anderson's mordant, 517; Bacsich's mordant, 515; Bailey's mordant, 517; Beale's injection fluid, 062; Bensley and Bensley's fixative, 205; Brucke's injec- tion fluid, 662; Bunge's magenta, 481; Cajal's fixative, 205; Cole's mordant, 515; Cretin's mordant, 517; Faure's mordant, 516; Fisher and Conn's ma- genta, 482; French's crystal violet- magenta, 387; Kefalas's hematoxylin, 285; Kornhauser's acid alizarin blue, 369; Kozowsky's differentiator, 520; Krajian's hematoxylin, 285; LaManna's hematoxylin, 285; Landau's mordant, 516; Lillie's hematoxylin, 285; Mane- val's acid fuchsin, 321, 473; Maneval's anilin blue, 321; Maneval's magenta, 483; Mayer's injection fluid, 663; Morel and Bassal's hematoxylin, 286; Petragnini's mordant, 518; Robin's in- jection fluids, 663; Seidelin's hematoxy- lin, 286; Sheridan's crystal violet, 389; Shunk's mordant, 519; Thomas's hema- toxylin, 286; Weigert's magenta, 390; Weigert's hematoxylin, 286 as mordant for, carmine, 305; dinitroresorcinol, 411; hematoxylin stains, 282, 283, 423 as stain, 610, 611 for staining, cartilage, (Hogan), 010; cell outlines, (Leber), 611; cell walls of apical meristem, 610; histological prepara- tions, (Hogan), 610; nerves in teeth, (Pollaillon), 610; Tintinnopsids, (Fol), 611 in, Gallego's method for Negri bodies, 465; Giacomi's method for spirochetes, 479 Ferric nitrate, as ingredient of, Zirkle's carmine-balsam, 640; Zirkle's carmine dextrin mountant, 637; Zirkle's carmine gelatin mountants, 635; Zirkle's carmine pectin mountant, 637; Zirkle's carmine-venice turpentine mountant, 638; Zirkle's mountant, 633 Ferric oxide, as ingredient of, Belling's mordant, 517 Ferric sulfate, as mordant for pyrogallic acid, 390 Ferricyanide-hematoxylin, 291 Ferrous chloride, as ingredient of, Thomas's hematoxylin, 286 Ferrous sulfate, as ingredient of, Benda's mordant, 515; Kulp's magenta, 482; Lillie and Earle's hematoxylin, 285; Loffler's magenta, 482; Oliver's mordant, 518; Richardson's injection fluids, 663; Thiersch's injection mass. 666 in, Novel's method for bacterial flagella, 598 Fetus and fetal, see Embryo, Embryonic Feulgen and Rosenbeck's magenta leu- cobase, 316 in, Cretin's method for starch, 451; Klig- man, Mescon, and Delameter's method for fungus in skin sections, 504 Feyter's method for myelin sheaths, 403 Fiandt's method for gliosomes, 415 Fiandt and Sazen's decalcifier, 258 in Fiandt and Sazen's method for Golgi in ear cells, 590 Fibers, textile, 644 Fibrin, special methods for, 423, 424, 425 Fichet, see Tribondeau, 289, 484 Fick's fixative, 228, 243 Field's method for Plasmodium, 507 Field and Martin's nitrocellulose-wax em- bedding method, 648 Fielding, see Murray, 192, 562 Fiessinger and Laur's method for reticulo- cytes, 417 Filter paper, in fbcation of chicken embryo, 547 Finel's method for nervous tissues, 583 Fischer's, decalcifying fluid, 258 glycerol jelly, 634 method for elastic fibers, 390 milk injections, 163 Fischler's, fixatives, 200, 243 method for fat, 448 Fish, embryo, NoUister's method for bone in, 384 larvae, brains in, 551 nerves in, 584 scales, 43 Fish's, fixatives, dichromate-formaldehyde, 233, 243; mercuric-picric-acetic, 214, 243; osmic- dichromate-formaldehyde, 204, 243; zinc-formaldehyde, 236, 243 methods for nervous tissue, 605 Fisher and Conn's method for bacterial flagella, 482 Fite's picro-fuchsin, 327 in Fite's method for acid-fast bacteria in sections, 496 INDEX 719 Fitzpatrick, see Zinsser, 463 Fixation, flattening specimens during, 294 of, annelida, 63; chicken embryo, 277, 278; echinoderm larvae, 156; liver flukes, 294; smears from fluid material, 71; specimens for wholemounts, 51-54 suspension method for, 334 Fixative, acids, 18G agents, primary, 186 device for washing out, 129 modifiers, 186 removers, 254-256 Fixatives, as, coagulating agents, 187; macerating agents, 76 classification of, 185-186 decimal division of, 182-185 desirable qualities in, 187 distinction from preservatives, 175 effect of heating, 187 for, blood, 193; Bryozoa, 54; coelenterates, 53; leeches, 54; lily bud, 149; plankton, 53; protozoans, 52; root tips, 196 formulas for, see under name of author or ingredient method of presenting formulas, 188 osmotic pressure of, 187 oxidation-reduction, properties of, 188 selection of, 95 used as macerating agents, 262 Fixing solutions for metal stains, 621 Flagella of bacteria, dye staining methods for 481, 484 silver diammine methods for, 598 silver-iron method for, 608 silver nitrate methods for, 563 silver-sulfate method for, 609 "Flagella," on erythrocytes, 457 Flagellates, in fecal smears, 508 internal structure of, 455 Flame cells, 430 Flaming, slides, 666 smears, 468 Flater's method for lower vertebrate brains, 609 Flattening, double-embedded sections, 156 nitrocellulose sections, 148, 152 paraffin sections, 117, 118, 119ff rolled sections, 335-336 specimens during fixation, 294 Fleischer, see Safford, 608 Flemming's double stain, 340 Flemming's fixatives, chrome-acetic, 243, 288 osmic-chromic-acetic, 200, 243 as mordant for hematoxylin, 284 for, echinoderm larvae, 154; grasshopper chromosomes, 311 Flemming's fixatives — (continued) osmic-chromic-acetic — (continued) in, Alzheimer's method for nervous tis- sues, 409; Alzheimer's method for neuroglia, 411; Flemming's tech- nique, 340; Hadjioloff's method for neuroglia, 413; Henneguy's tech- nique, 315; Lee's osmic stain, 530; Unna's method for nuclei, 438 recommended use, 95 osmic-picric-acetic, 198, 243 Flemming's naphrax mountant, 641 Flemming's soap embedding medium, 643 "Flemming-uranic" fixative, 232 Flesch's fixative, 199, 243 Fletcher, see MacCallum, 426 Flexible asphalt cement, 654 Flexner's method for acid-fast bacteria in sections, 496 Flinn's method for bacteria in leukocytes, 491 Flotation method of, clearing, 62 separating foraminifera, 17 separating nematodes, 35 Floyd, see Parker, 191, 249 Fluid wholemounts, nonaqueous, 32-41 media for, 32 Fluorescein, for staining, acid-fast bacteria in sections, (Czaplew- ski), 496; bacteria in sections, (Kuhne), 492 staining combinations with, magenta and methyl blue, 496; methy- lene blue, 492 Foa's fixatives, 216, 243 Fol's, adhesive for free sections, 659 fixatives, chrome-acetic, 228; osmic-acetic, 194, 243; osmic-chromic-acetic, 200, 243; osmic-picric chromic. 199, 243, (recom- mended use, 35); picric-chromic-sul- furic, 226, 243 injection mass, 664 method for Tintinnopsids, 611 Foley's, alcohol accelerator, 614 in Foley's method for axons, 555 developer, 617 in Foley's method for nervous tissue, 567 fixer, 225 stains, methyl green-acid fuchsin, 356; methyl green-acid fuchsin-orange G, 354-355, 356; safranin-orange G-crystal violet, 363 Fontana's, accelerator, 615 fixative, 191, 243 in, Lancelin, Seguy and Debreuil's method for spirochetes, 597 720 INDEX Fon tana's — (continued) silver diammine stain, 575, 577 in, Fontana's arsenic-silvsr method for spirochetes, 608; Fontana's method for spirochetes, 597; Masson's method for argentaffin cells, 596; Masson's method for argentophil cells, 595; Novel's method for bacterial flagella, 598; Safford and Fleischer's method for bacterial flagella, 608; Seguin's method for spirochetes, 598 Fontes's method for acid-fast bacteria, 476 Foot's, formaldehyde accelerator, 613 silver diammine stains, 576, 577, 578 in, Foot's method for reticulum fibers, 592; Gordon and Sweet's method for reticulum fibers, 593; Oliveira's method for reticulum, 607; Pritch- ard's method for mitochondria and golgi bodies, 590; Wilder 's method for reticulum fibers, 595 toning solution, 620 Foraminifera, attaching to slide with traga- canth, 19 cementing in wholemounts, l-i-15 collection from sand, 17 disintegrating rocks containing, 18 mounting individual specimens, 20 preparation of, cell for, 19 separating from, chalk, 17, 18; clay, 18; marine deposits, 17; shale, 18 special slides for, 11 strewn slide of, 17-20 Forbes's glj'cerol jell}', 634 Formaldehyde, adjusting pH of, 190 as, developer in various silver methods, 550-609 fixative, 190 ingredient of. Blank's triple stain, 352; Bohm and Oppel's phenosafranin, 314; Bohm and Oppel's safranin, 314; Bujor's narcotic, 265; Buzaglo's gallocyanin, 313; Cajal's silver stain, 550; decalci- fying fluids, 256-260; developers, 616-620; EUerman's double stain, 344; Gage's alkaline macerating fluid, 264; Gallego's magenta, 315; pre- servatives, 176-181; Semichon's saf- ranin, 314 mordant, 359 effect of long storage in, 128 explanation of terms used for, 188 fixative combinations with, acetaldehyde, 193; acetic acid, 191; acetone, 193; formic acid, 192; meta- phosphoric acid, 193; nitric acid, 192; Formaldehyde — (continued) fixative combinations with — (continued) trichloroacetic acid, 192; other combi- nations, see under name of other ingredient for, aqueous wholemounts, 23 fixing Suctoria, 53 hardening, 52 hardening gelatin blocks, 161 killing, brj'ozoa, 60; rotifers, 29, 31 preserving, diatoms, 39; plankton, 49 in, Langeron's method for attaching ni- trocellulose sections, 659; Lebowich's method for attaching gelatin sections, 660; Szombathy's method for paraffin ribbons, 659 removal from tissues, 256 Formaldehyde accelerators for metal stains, 613-614 "Formahn," 188 Formamide, as ingredient of. Bank and Davenport's fixative, 237; Davenport, Windle and Rhines's fixa- tive, 237; MacFarland and Davenport's fixatives, 193 Formic acid, as ingredient of, Boccardi's developer, 616; decalcifying fluids, 256-260; Foley's alcoholic ac- celerator, 614; Pritchard's developer, 619; \Yeber's alcoholic accelerator, 615; Zirkle's mountant, 633 fixative combination with, formaldehj^de, 192; trichoroacetic acid, 190; other ingredients, see under other ingredient in, Dogiel's method for corpuscles of Grandry and Herbst, 554; gold impreg- nation methods, 534-536 "Formol," 188 Formulas, explanation of method of pre- senting, 2-3 general classification of, 173 specific classification of, see under Fixative, Dye Stain, etc. units used in, 173 Forsgren's method for bile duct, 423 Foshay's method for bacteria in sections, 492 Fossil diatoms, separating from rock, 38 Fossil foraminifera, collecting and cleaning, 17-20 Foster's method for, cell walls of apical meristem, 610 plant sections, 392 Foster and Gifford's softening fluid for plant materials, 667 INDEX 721 Fraenkel's, acid fuchsin, 321 in, Kraus's method for colloid in tliy- roid, 456 metliods for, acid-fast bacteria, 476; bacteria in sec- tions, 492; bacterial spores, 485; elastic fibers, 387 Francotte's, boric-carmine, 306 fixative, 199, 243 method for plankton, 513 picro-carmine, 303 preservative, 176 Freeborn's double contrast, 369 Freehand sections, 88, 93 adhcsivcs for, 659-660 hardening and fixing specimens for, 90 materials for supporting, 89 microtomes for, 89 of, leaf, 91-92; wood, 92-93 staining and mounting, 90 technique of cutting, 91if technique of supporting, 91fT Freezing method for, disintegration of fossil deposits, 18 sections, see Frozen sections Freitas's iron-mordant hematoxylin, 281 Freminean Canada balsam-gum mastic mountant, 640 French cement, 655 French's, method for, elastic fibers, 387; fat, 448 stains, crystal violet-magenta-resorcinol, 387; iron-mordant hematoxylin, 281 French, see also Scott, 955 Frenkel's fixative, 205, 243 Frenzel's fixative, 211 Freud's method for axis cylinders, 535 Freude's macerating fluid, 263 Frey's, injection fluids, 663 injection mass, 664 isinglass embedding medium, 643 preservative, 179 stains, ammonia-carmine, 305; borax-carmine, 307 varnish, 653 Friedenthal's fixative, 219 Friedgood, see Dawson, 211. 425 Friedlander's, fixative, 228, 243 stains, alum-hematoxylin, 287; hematoxylin- eosin, 365; picro-carmine, 303 Friedmann's fixative, 200, 243 Friefeld's method for toxic neutrophiles, 417 Frog, killing for, brain stain, 396 histological specimens, 129 section of brain, 395-397 staining skeleton of, 379 Frog — (conlin itcd) eggs and hirvac, fixative for, 133 Frog embryos, embedding for freehand sec- tions, 90 Frost's preservative, 179 Frotheringham's method for Negri bodies, 465 Frozen sections, 157-161 adhesivcs for, 659-660 embedding before, 157, 158 embedding media for, 642-644 from gelatin-embedded material, 160 161 microtome for, 157ff refrigerants for, 158 staining and mounting, 160 technique of cutting, 159ff using Anderson's medium for, 158fT, 159 "Fuchselin," 390 Fuchsin, basic, see Magenta Fuchsin, see Acid fuchsin or Magenta Fuelgen reaction, 309 Fujiware's method for adrenal cortex, 429 Fuller's method, for, acid-fast bacteria in sections, 496 vaginal smears, 430 Fulton, see Gough, 443, 518 and Schaeffer, 487 Fungi, parasitic, 499-505 preservatives for, 177 stains for, 501-505, 511-513 Fungus in, animal tissues, 502-505 plant tissues, 501-502 Fungus hyphae, mentioned in Johansen's quadruple stain, 364 "FWA" fixative, 199 Fyg's stains, chrome-carmine, 307 soda-carmine, 307 Gabbet's method for acid-fast bacteria, 477 Gadden's wax embedding medium, 646 Gage's, adhesive for minute objects, 661 cement, 655 use with dry wholemounts, 15 clearing mixtures, 628 decalcifying fluid, 258 fixative, 221, 244 label adhesive, 661 macerating fluids, 263, 264 method for chitin, 390 stains, alum-hematoxylin, 287; picro-carmine, 303 varnish, 653 Gairns's decalcifying agent, 258 de Galantha's, decalcifying fluid, 258 method for kidney, 423 Galescu's fixative, 197, 244 722 INDEX Galescu's fixative — (continued) in, Anglade and Morel's method for neu- roglia, 412; Galescu's method for neuroglia, 399-400, 413 Galesesco and Bratiano's method for fat, 448 Galiano's hematoxylin-eosin, 365 Gallamin blue, 368 Gallego's magenta, 315 in, Dupres's technique, 359 method for Negri bodies, 465 Gallego-Garcia's method for elastic fibers, 387 Gallein, for staining nervous tissues, 409 Gallic acid, as ingredient of, Bauer's developer, 616; van Ermengen's developers, 617; Karlson's double stain, 394; Lendrum's quadruple contrast, 340 in, Fol's method for Tintinnopsids, 611 Gallocyanin, as stain, 313 for staining Nissl granules, (Einarson), 445 staining combination with anilin blue, fast green FCF, orange G, and silver protein, 567 Gamassid mites, 45 T-cells of pituitary, 427 Ganglia, mentioned in Hubin's triple con- trast, 340 of invertebrates, 538 sympathetic, 605 Ganglion, cervical, 601-602 Ganglion cells, 604 Gans's method for microglia, 586 Gardiner's, method for plasmodesma, 421, 512 preservative, 179 Gardner's fixative, 198 Garlic, as ingredient of HoUande's adhesive for paraffin ribbons, 657 Garrett's method for fungi in roots, 501 Gaskell's gelatin embedding method, 645 Gastrotricha, 54, 67 Gatenby's, fixative, 231, 244 recommended use, 95 osmic methods for Golgi bodies, 530 Gatenby and Cowdry on acetic acid fixa- tives, 189 Gatenby and Cowdry's, decalcifying fluid, 260 stains, azur Il-methylene blue-eosin, 350 in Bland and Canti's method for intra- cellular "organism," 461 thionine, 318 Gatenby and Painter's, clearing mixture for nitrocellulose sections, 628 preservative, 176 Gatenby and Stern's silver diammine stain, 576, 578 Gater's, mountant, 632 preservative, 178 Gates's fixatives, chromic-acetic, 244 osmic-chromic-acetic, 200, 244 Gates and Latter's fixative, 228, 244 Gausen's double stain, 351 Gay and Clark's method for dead bacteria, 491 Gedoelst's fixative, 203 "GEEP" stain, 340 van Gehuchten's, fixative, 203, 244 in van Gehuchten's method for de- generative changes, 528 osmic method for degenerative changes, 528 Geiman, see Wenrich, 210 Geither's method for nuclei, 435 Gelarie's method for spirochetes, 479 Gelatin, as ingredient of, Bellido's varnish, 652; cements, 654- 655; Chatton and Lwoff's adhesive, 660; Cobe and Schoenfle's adhesive for paraffin ribbons, 657; Eyene and Stein- berg's developer, 617; Farrier and Warthin's developer, 617; Faulkner and Lillie's developer, 617; Fol's ad- hesive for free sections, 659; Gage's adhesives, 661; Heitzman's developer, 618; Heringa and ten Berge's adhesive for nitrocellulose sections, 659; Hogg's varnish, 653; label adhesives, 661; Landa and Rezek's [developer, 618; Langeron's adhesive for nitrocellulose sections, 659; Masson's adhesive for paraffin ribbons, 658; Moreau's adhe- sive for paraffin ribbons, 658; moun- tants, 46; Romanes's silver diammine stain, 580; Seiler's injection fluid, 664; Tandler's injection fluid, 664; various injection masses, 664-666; Warthin- Starry's developer, 620 for, attaching nitrocellulose sections, 148; embedding frozen sections, 160-161 in, Heringa's method for attaching free sections, 660; Lebowich's method for attaching free sections, 660; silver-di- chromate staining techniques, 605; Szombathy's method for paraffin rib- bons, 659 method of clarifying, 633 Gelatin embedding, 160-161 media, 642-644 Gelatin mcuntants, 633-636 Gelatin-saline, for transporting liver flukes, 294 Gelatin sections, 161-162 adhesives for, 659-660 Gelderd's fixative, 219 INDEX 723 Gelfand, see Richman, 259 Gemelli's motluxl for bacterial (l.-ijidla, 182 Gendro's fixativo, 22 t Gentian violet, as stain, 319 for staining, bacterial capsules, (Muir), 488, bac- terial smears, (Claudius), 473; bacterial smears, (Ehrlich), 473; bacterial smears, (Spehl), 474; bacterial spores, (Besson), 485; blood, (Price-Jones), 419; fungus in tissue scrapings, (Morris), 505; (jram-positive bacteria, (Konschegg), 475; Gram-positive bacteria, (Weiss), 475; Gram-positive bacteria in sections, (Brown and Brenn), 494; nuclei, (Her- mann), 436; nuclei, (Wing), 438; plas- modesima, (Gardiner), 512 general remarks on, 309 in, Flcmmings' double contrast, 339; Gram's phenol solution, 321; Reinke's double contrast, 341; Stockwell's triple stain, 364 staining combinations with, carmine, 503; carmine-picric acid, 501; eosin Y, 503; erythrosin-hematoxylin, 494; hematoxylin-magenta, 505; light green-neutral red, 493; magenta, 474, 489, 494; methylene blue, 476; orange G, 340, 341; orange G-safranin, 364; orcein, 481; phloxine, 504; safranin, 436 Geoffroy's gelatin mountant, 634 Georgi, see Skiles, 641 Gerhardt's fixative, 215, 244 for chicken embryos, 277 recommended use, 95 Gerlach's, ammonia-carmine, 305 fixative, 232, 244 methods for, Negri bodies, 465; nervous tissue, 539- 540; spinal cord, 540 Gerota's silver-dichromate method for nerv- ous tissues, 604 Geschickter's double stain, 352 Geschickter, Walker, Hjort and Moulton's triple stain, 348 Gherardini, see Kaiser, 465 Giacomi's method for spirochetes, 479 Gibbe's method for acid-fast bacteria, 477 Gibson's preservative, 179 Giemsa's, fixative, 207, 244 methods for, Plasmodium, 507; spirochetes, 479 stain, 347 for staining, blood, (Baillif and Kimbrough), 416; blood, (Kardos), 418; blood, (Sheehan and Storey), 420; blood, (Strumia), 420; larvae of Dracuncu- lus, (Moorthy), 509; Nissl granules. Giemsa's — (continued) stain — {continued) for staining — (continued) (Hansburg), 445; Phytomonas, (Strong), 511; Plasmodium, (Cho- rine), 506; Plasmodium, (Corbin), 506; Plasmodium, (Giemsa), 507; Plasmodium, (Ginrich), 507; Plas- modhim, (Hewitt), 507, 508; Plas- modium, (Knowles), 508; Plasmo- dium, (Sternberg), 510; Rickettsiae, (Gracian), 462; Rickettsiae, (Wol- bach), 464; skin, (Pinkus), 424; spi- rochetes, (Duperie), 479; spirochetes, (Giemsa), 479; spirochetes, (Hoff- man), 479; spirochetes, (Krauss), 479; spirochetes in sections, (Schmorl), 498; virus in elusion bodies, (Cole), 464 in, Langeron's technique, 348; Pappen- heim's technique, 349, 350 Southgate's mountant for, 640 Giesbrecht's, adhesive, 660 fixative, 198, 244 van Gieson's stain, 327 for staining, fat, (Schaffer), 449; macroglia, (Held), 413; rat tongue, 325 in. Drew- Murray's technique, 369; Foot's silver method for reticulum fibers, 592; Masson's technique, 340 Clifford's softening fluids for embedded ob- jects, 667 Gifford, see also Foster, 667 Gihei, on pH of fixatives, 188 Gilbert's fixative, 224 Gilson's fixatives, acetic-alcohol-chloroform, 189 mercuric-acetic-nitric, 210, 214 for fixing liver flukes, 294 recommended use, 52, 95 zinc-acetic-nitric, 236 Gilson's glycerol jelly, 634 Ginrich's method for Plasmodium, 507 Giordans, see Markey, 508 Gland cells, nerve endings in, 529 Glands, mentioned in, Lillie's triple contrast, 370; Mallory's triple stain, 360; Masson's double con- trast, 337 Glass-to-glass seals, Cigales's cement for, 655 de Grost's cement for, 654 Glees, Meyer and Meyer's method for nervous tissues, 583 silver diammine stain, 576, 578 "Gliabeize," 517 Glioblasts, 589 CJliosomes, 589 Fiandt's method for, 415 724 INDEX Globus, silver bromide technique, 543 Glomeruli, injection of, 168 Gluck's silver diammine stain, 576, 578 in Gluck's method for, argentophil cells, 595; reticulum fibers, 592 Gluconic acid, as ingredient of Zirkle's car- mine-gelatin mountant, 635 Glucose, as ingredient of, Gater's mountant, 632; Landau's moun- tant, 632; Womersley's mountant, 633 in, Arndt's method for glycogen and fat, 451; Neukirch's method for glycogen, 453 Glyceric ether, as differentiator for methy- lene blue, 317 in, Manouelian's method for Negri bodies, 466; Unna's differentiator, 521 Langeron's substitute for, 466 preparation of, 519 Glycerine, see Glycerol Glycerol, as clearing agent, 378 as dehydrant, 623 for algae, 65; in Wolf's nitrocellulose embedding method, 649 as ingredient of, Beale's injection fluids, 662; gelatin mountants, 633-636; gum arable moun- tants, 631-633; Hoyer's injection mass, 665; injection media, objections to, 163; label adhesives, 661; Lendrum's soften- ing fluid for embedded objects, 667; Liliie's adhesive for paraffin ribbons, 658; Lo Blanco's narcotic, 265, macerat- ing fluids, 262-264; Meakin's adhesive, 661; preservatives, 176-181; Reinke's adhesive for paraffin ribbons, 658; Robin's injection fluids, 663 ; Schneidau's adhesive for paraffin ribbons, 659; Szombathy's adhesive for paraffin rib- bons, 659; Thoma's injection fluid, 664 concentrating in specimens, 50 evaporation technique, 32 for, lowering surface tension in injection media, 162; wholemounts, 32 mounting nematodes in, 35-37 physical properties of, 623 sealing mounts in, 36 Glycerol-alcohol for storing, specimens, 96 wax blocks, 103 Glycerol jelly, as embedding medium, 642- 644 cells for, 48 example of mount in, 48-50 for mounting muscle to show nerve end- ings, 533 mountants, 633-636 Glycerol jelly — (continued) mounting Crustacea in, 48 repairing mounts in, 50 technique of mounting in, 46, 47ff tool for mounting in, 47ff wholemounts in, 46-48 Glycerol wholemounts, cements for sealing, 32, 33 sealing with petrolatum, 34 "Glychaemalum," 288 "Glychrogel," 636 Glycogen, Bauer's fixative for, 223 Cretin's fixative for, 222 Schabadasch's fixative for, 225 silver diammine method for, 595 special methods for, 450-453 Glycol stearate, as embedding medium, 649 in Cutler's embedding medium, 643 Glynn's method for Gram-positive bacteria in sections, 494 Gnanamuthu's picro-congo red, 327 Goadby's preservatives, 176 for aqueous wholemounts, 23 Goddard, see Paquin, 286, 366 Godfrin's gelatin-soap embedding medium, 644 Gold, as stain, general remarks on, 531 methods for, alveolar epithelum, 534; nerve endings, 534-536 staining solutions, 534 Gold chloride, as ingredient of toning solu- tions, 620-621 in combination with dye stains, 410 in various silver methods, 550-609 Gold-arsenic method for nervous tissue, 540 Gold-chromic methods for liver, 541 Gold-dichromate methods for nervous tis- sues, 540-541 Gold-mecury methods for, nerve cells and processes, 538 general observations on', 536 neuroglia, 539 Gold-osmic methods for, arthropod nerves, 541 Golgi bodies, 541 nervous tissues, 540-541 Gold size, as ingredient of, Benoit-Bazille's varnish, 652; Cheva- lier's varnish, 653; Eulenstein's varnish, 653; Kitton's cement, 654; Martin's black varnish, 656; Rousselet's varnish, 654 Beale's formula for, 652 cementing coverslip to dry wholemount with, 16 for, aqueous wholemounts, 22; attaching cell to slide, 19; cementing cells to glass, 13; sealing gum mounts, 43 INDEX 725 Gold size — {continued) general remarks on, 651 preparation of cell from, 11-12 Goldman's method for, elastic fibers, 388 intestinal protozoans, 507 Goldner's stain, 337 Goldsmith's fixative, 231, 244 Goldsworthy and Ward's method for spi- rochetes, 479 Golgi's, developer, 016 differentiator, 621 fixatives, dichromate, 232, 244 in, Golgi's method for Golgi bodies, 558 osmic-dichromate, 203, 244 in, Cajal's method for nervous tissues, 604; Golgi's method for nervous tissues, 604, 605; Salva's method for nervous tissues, 600 fixing solution, 621 gold-arsenic method for nervous tissue, 540 mercuric-dichromate method for axis cylinders and dendrites, 610 methods for, Golgi bodies, 558. 559; Purkmje cells, 599-601 "mixed process," 605 osmic-silver-dichromate methods for nerv- ous tissues, 604, 605 "quick process," 604 silver-arsenic method for nerve endings, 605 silver-dichromate methods, for nervous tissues, 604-605; mounting sections from, 600, 601 "slow process," 604 toner, 620 Golgi bodies, cadmium-silver method for, 608 dye staining methods for, 441-444 gold-osmic method for, 541 in, calcified structures, 559; cells of ear, 590; wholemounts, 559 osmic methods for, 530-531 silver diammine methods for, 590-591 silver-dichromate methods for, 008 silver nitrate methods for, 558 Golgi- Verratti'a method for Golgi bodies, 008 Golodetz, see Unna Gomori's, dye stains, chrome-hematoxylin, 291 in, Gomori's method for pancreas, 429, Wilson's method for fat, 450 chromotrope 2R-fast green FCF, 339 decalcifying fluid, see Gomori's metal stain Gomori's — {continued) methods for, calcium in tissues, 596; carious lesions in teeth, 563; chromaffin granules, 430; elastic fibers, 388; glycogen, 595; pan- creas, 429; reticulum fibers, 592 1933 silver diammine stains, 576, 578 in, Gomori's method for glycogen, 595; Gomori's method for reticulum fibers, 592 Gonococcus, Walton's method for, 491 Gooding and Stewart's, decalcifying fluid, 258 developer, 617 in, Gooding and Stewart's method for nerves in tooth pulp, 555 Goodpasture's, method for, bacterial smears, 473; Negri bodies, 465 polj^chrome methylene blue, 317 in Stafford's technique, 347 Goodpasture and Burnett's magenta, 315 in. Adam's method for acid-fast bacteria in sections, 495; Hertwig and Wolbach's method for Rickettsiae, 403 Goodrich's, fixative, 227 macerating fluid, 203 Gordon's, developer, 016 differentiator, 519 mordant, 518 in Gordon's triple stain, 345 silver diammine stain, 576, 578 in Gordon's method for blood parasites, 598 Gordon and Sweets's silver diammine stain, 576, 578 in Gordon and Sweets's reticulum fibers in spleen, 592, 593 Gore, see McNamara, 259 Gothard's, dehydrating mixture, 628 differentiator, 520 in, Anderson's method for NissI granules, 445; Gothard's method for Nissl granules, 445; Roussy and Lehrmitte's method for Nissl granules, 446 Goto's fixative, 209, 244 Gough and Fulton's mordant, 518 in Gough and Fulton's method for mito- chondria, 443 Gouillart and Brouardel's differentiator, 520 Goulard's "extract of Saturn," 645 Gower's alum-carmine, 300 Gracian's method for Rickettsiae, 462 Graff's, fixatives, chromic-oxalic, 230, 244 picric-formaldehyde, 223, 244 gelatin embedding method, 045 Graham's methods for oxidase granules in blood, 417 726 INDEX Gram's fixative remover, 255 Gramineae, pollen tubes, 422 Gram's iodine, 255 as ingredient of Tonkoff's spirit blue- iodine, 322 in, Albert's method for diphtheria bacilli, 489; Bailey's method for neuroglea, 412; Bensley's method for canaliculi in plant cells, 454; Besson's method for As- pergillus fumigatus, 503; Bigot's method for Zygonema, 503 ; Buerger's method for bacterial capsules, 487; Buzzozero-Vas- sale's method for nuclei, 435; Chalmers and Marshall's method for skin scrap- ings, 503; Hutchins and Lutman's method for actinomyces in potatoes, 501; Kemp's method for diphtheria bacilli, 490; Kuhne's method for bacteria in sections, 492; Langhan's method for amyloid, 452; Langrand's method for actinomyces, 504; Lubarsch's method for glycogen, 452: Mallory's method for actinomycetes, 504; Meyer's method for plasmodesma, 421; Morel and Dulaus's method for actinomycetes, 505; Mor- ris's method for fungi in tissue scrap- ings, 505; Muir's method for bacterial capsules, 488; Ollett's method for bac- teria in sections, 493; Kemy and Sugg's method for bacterial flagella, 484; Rubaschkin's method for neuroglia, 414; Schmorl's method for elastic fibers, 389; Smith's method for bacterial capsules in sections, 498; Strasburger's method for plasmodesma, 422; various methods for Gram-positive bacteria in smears, 474-475; various methods for Gram-positive bacteria in sections, 493- 495; Wadsworth's method for bacterial capsules, 489 Gram's method for Gram-positive bacteria, 474 Gram-positive bacteria, 474-475 in sections, 493-495 smear of, 468 Gram-Rlitzow's varnish, G53 "Gram-Weigert" methods, 493, 494 Grandis and Magnini's method for bone, 383 Grandry, corpuscles of, 554 Granules, hemafuchsin, 450 hemosiderin, 450 in. bone marrow, 450; breast, 456; chief cells, 455; diphtheria bacilli, 489, 490; erythrocytes, 455; gastric mucosa, 455; kidney, 450; mast cells, 455; Negri bodies, 405; nerve cell cytoplasm, 454; Paneth cells, 452; parietal cells, 455; Schwann cells, 457 Granules — (conlinued) peroxidase, 456 Schridde's, 457 Grasshopper, smear of chromosomes, 310- 312 Graupner and Weissberger's fixative, 193, 244 Graven 's method for nerve endings, 535 Gravis's adhesive, 657 in Zimmermann's method for free sections, 060 Grawitz's preservative, 179 Gray's acid alcohol for cleaning slides. 000 cement, 055 double contrast, 328 embedding was, formula, 040 properties of, 97 fixatives, 214 technique for use, 07 methods for, bacterial flagella, 482; bacterial spores, 480; bone, 383; bony skeleton, 377-379, minute fresh-water organisms, 07-08, stalked ciliates, 205 mordant, 518 in Gray's method for bacterial flagella, 482 narcotic, 205 for coelenterates, 53 wax embedding medium, 040 Gray on, basal fixative solutions, 237 fixative selection, 95 fixatives and narcotics for wholemounts, 52 immobilizing agents, 52 Miiller's fixative, 187 ringing balsam mounts, 9 Gray and Gra}% bibliography, 070 Gray and Wess's, polyvinyl acetate moun- tants, 041 polyvinyl alcohol mountant, 636 for gelatin sections, 161 recommended use, 42 Gregg and Puckett's, double contrast, 328 for amphibian embryos, 136 fixative, 212, 244 for fixing amphibian eggs, 133 technique for embedding amphibian eggs, 134 Grenadier's, method for bleaching, 261 stains, alcoholic borax carmine, 301 for, amphibian eggs, 134; dentine, 385; invertebrate larvae, 54; medusa, 297 alum-carmine, 300 as ingredient of. Legal 's picro-carmine, 303; Neu- man's picro-carmine, 303 INDEX '27 Grenacher's— (coH ///I wed) stsiins-— (continued) alum-carniine — {continued) in, Bcrblinger and Borsdorf's method for pituitary, 425; Kionka's method for yolk granules, 448; Langeron's method for myxosporids, 508; Langeron's method for plant sec- tions, 392; Lynch's technique, 371; Westphal's method for blood, 420 Gridlej^'s method for reticulum fil)ers, 592 Grieves's method for dentine, 383 Griflfitli's cement, 055 Grinding, materials for, 81 Groat's, quadruple stain, 349 resin mountant, 641 Groat, see also Koenig, 190 Grocott, see Tomlinson, 510 de Groot's, gelatin cement, 654 hematoxylin, 287 iron-carmine, 304 Gros-Schultze's silver diammine stain, 576, 578 in, Gros-Schultze's method for nervous tissues, 583; Landau's method for nervous tissues, 583; Miller's method for nerve trunk of oligochaetes, 556 Gross and Lohan's fixative, 235 Gross's method, for fat, 448; for smears of nervous tissues, 410 silver diammine stain, 578 in Finel's method for nervous tissues, 583 Grosso's, method for blood, 418 stains, methyl green-pyronin-orange G, 355 picro-methyl green, 326 Ground sections, 80-87 cementing to slide for grinding, 83ff cutting slabs for, 80, 82fT, 86 embedding specimens in balsam for, 86 grinding and polishing agents for, 81 Henrichs's method for, 80, 87 of, bone, 81-85; coral, 85-87 Grove's cement, 653 Grynfelt and Mestrezat's bleaching solution, 262 Guano, separating diatoms from, 37 Guarnieri bodies, 465 Guccione, see Lehrmitte, 200, 414 Gudden's method for myelin sheaths, 405 Guegin's methods for fungus in tissue scrap- ings, 504 Guild, see Hubcr, 555 Guillain, see Bertrand, 577, 586 Guinard's stains, alkanet, 308 methyl green-acid fuchsin, 356 Guinea dissection Horn of Ammon, 460 smear of Rickettsiae from scrutum, 458 Gulick's fixative, 222, 244 (luUand's fixative, 190, 244 Gum acacia, see Gum arabic Gum ammoniac, as ingredient of, Hogg's varnish, 653; Seller's gelatin cement, 655 Gum arabic, as ingredient of, Archibald and Marshall's mountant, ()3(); Bolsi's fixing solution, 621; ('ole's embedding medium, 643; I'^yene and Steinberg's developer, 617; Krantz's developer, 618; label adhesives, 661; Land's adhesive for paraffin ribbons, 658; Langeron's adhesive for free sec- tions, 660; Langeron's embedding me- dium, 644; Liesegang's developer, 618; mountants, 631-633 in, Bohm and Davidoff's method for par- affin ribbons, 657; formaldehyde fixa- tives, 190; Lebowich's method for at- taching free sections, 660; mounting media, 42 varnishes, 652 Gum damar, as ingredient of, Beale's cement, 652; Hogg's varnish, 653; James's cement, 653; Rousselet's varnish, 654; Semmens's varnish, 654; Woodhead's cement, 654 for, embedding ground sections, 80 mountants, 640 purification of, 640 Gum elemi, as ingredient of Southgate's mountant, 640 Gum mastic, as ingredient of, Belling's cement, 655; Frey's varnish, 653; Groves's cement, 653; Hogg's varnish, 653; Krajian's developer, 618; Steiner's alcoholic accelerators, 614, 615; Steiner's developer, 619; Wilson's varnish for knife edges, 667 in, Dieterle's method for spirochetes, 560; Krajian's method for spirochetes, 561; Steiner's method for spirochetes, 562 mountants, 637, 640 Gum mountants, formidas for, 631-633 method of mounting in, 45 objects suitable for, 43 wholemounts in, 42-45 Gum tragacanth, as ingredient of, Archibald and Marshall's mountant, 636; Marpinann's la})el adhesive, 661; Marshall's mountant, 632 for, dry wholemounts, 14; strewn slide of Foraminifera, 19 728 INDEX Gum turpentine, 624 Giinther's method for afid-fast l)acteria, 477 Gupta, see Knowles, 561 Gurdjian's, developer, 619 method for nerve cells and processes, 555 Guthrie's fixatives, mercuric-dichromate- formic, 217, 244 osmic-chromic-formic, 201, 244 Gutstein's method for, ascospores of yeasts, 512 bacterial capsules, 488 intracellular "organisms," 461 nuclei in yeasts, 512 Gutta percha, as ingredient of, Harting's cement, 656; Martin's ad- hesive, 661 in Altmann's method for attaching free sections, 659 Guyer's, fixative, 230, 244 glycerol jelly, 634 method for bacteria in sections, 492 sealing fluid for corks, 667 stains, alum cochineal, 300 in Reynold's method for hematodes, 509 picro-carmine, 303 Guyler's double contrast, 328 H Hadjioloff's method for, myelin sheaths, 407 neuroglia, 413 "Haemacalcium," 292 "Haemastrontium," 292 Haensel's fixative, 226 Haggquist's iron-mordant hematoxylin, 281 Hagmann's injection fluids, 664 Hagmann on injection media, 162 Hair, for handling diatoms, 40 wholemounts of, 43 Hall and Powell's method for euglenoid flagellates, 455 Haller's macerating fluid, 263 Hamazaki's fixative, 218 in Hamazaki's method for wandering cells, 431 Hamann's fixative, 194, 244 Hamilton's fixative, 232, 244 method for virus inclusion bodies, 465 osmic acid stain, 527 osmic method for degenerative changes, 528 Hamilton, see also M'llroy, 228 Hamperl's, fixative, 191 in Hamperl's method for chief cell granules, 455 method for granules in chief cells, 455 Hanazava's methods for dentine, 383, 535, 563 Hance's. iron-mordant hematoxylin, 281 wax embedding medium, 646 recommended for, 129 Hancock's method for nuclei, 435 Hanley's, method for wholemount of rotifer, 29 narcotic, 265 for, protozoa, 52; rotifers, 30 Hanna's phenol-sulfur mountant, 638 Hansberg's method for Nissl granules, 445 Hansen's stains, chrome-hematoxylin, 291 iron-carmine, 304 iron-hematoxylin, 285 in, Beyer's method for astrocytes, 413; Bock's method for bone, 382; Mollen- dorf's technique, 366 picro-fuchsin, 328 Hantzch's preservative, 177 Harlow's macerating fluids, 263 Harris's, method for, Negri bodies, 465; nervous tissue, 403; spirochetes, 479 mordant, 518 stains, acid-alum hematoxylin, 287 in, Albrecht's method for acid-fast bacteria in sections, 495; Fite's method for acid-fast bacteria in sections, 496; Foot's method for reticulum fibers, 592; Krajian's method for amyloid, 452; Krajian's method for bacteria in sections, 492; Martin's method for pituitary, 426; Slater and Dornf eld's tech- nique, 367; Tilden and Tanaka's method for acid-fast bacteria in sections, 498 alum-hematoxylin, 287 in. Brown and Brenn's method for Gram-positive bacteria in sections, 493 ; Campbell's method for leprosy bacilli in sections, 496; Koneff's technique, 365 Harris and Power's alum hematoxylin, 287 Hart's method for elastic fibers. 388 Harting's, cement, 22, 656 injection masses, 665 Hartley's gum arable embedding medium, 644 Hartvvig's fixative, 215 Hartz's fixative, 212, 244 Harvey's fixative, 218, 244 in Harvey's method for parietal cell granules, 455 Haug's, decalcifiers, 258 in Bock's method for bone, 382 fixative remover, 255 INDEX 729 riaug's — (continued) iodine, 255 stains, alum-hematoxylin, 288; amnionia-car- iniiic, 307 Haupt's adhesive for paraffin ribbons, 659 Haver's fixative. 234, 244 ffaversian canals, 81 llay, collecting from, 44 Hay's chloroform balsam, 639 Hayem's douI)le stain, 328 method for blood, 418 Haj^maker and Sanchez-Perez's silver diam- mine stain, 598 Haynes's stains, azur II (or C)-ethyl eosin, 347 azur I-phloxine, 348 Haythorne's, method for, acid-fast bacteria in sections, 497; Gram-positive bacteria in sections, 494 preservative, 179 triple contrast, 337 Heat, as, fixative, 187; immobilizing agent, 52; narcotic, 265 for fixation of, bacterial smears, 468; nematodes, 36; smears, 71 Heather, see Jordan, 448, 465, 648 Heidenhain's, adhesive for paraffin ribbons, 657 decalcifying fluid, see Heidenhain's picric- acetic-trichloroacetic fixative fixatives, trichloroacetic, 192, 245 mercuric-acetic-trichloroacetic, 210, 245 in, Fiandt's method for gliosomes, 415 mercuric-dichromate-formaldehyde-ace- tic, 218, 245 in, Fujiware's method for adrenal, 429; Maxwell's method for pitui- tary, 427; Organ's method for calci- fied tissues, 596; Perry and Loch- ead's method for pituitary, 427 mercuric-formaldehyde, 211, 245 mercuric-formaldehyde-acetic-trichloro- acetic, 212, 245 mercuric-saline, 207, 245 osmic-mercuric, 196, 245 "subtriessig," 210 "susa," 212 glycerol .jelly, 634 methods for, centrosomes, 455; striped muscle. 423 stains, azocarmine-orange G-soluble blue, 361 chromic-mordant hematoxylin, 283 iron-mordant lunnatoxyliti, 282 for rat testis, 273-275 Heidenhain 's — (continued) stains — (contin ucd) iron-mordant hematoxylin — (contin ued) in Heidenhain's method for centro- somes, 455 vanadium-hematoxylin, 291 Hein's fixative, 209 Heinz's fixative, 212, 245 Heintz's nitrocellulose embedding medium, for double embedding, 155 nitrocellulose-wax embedding method, 648 Held, butls of, 552 Held's, fixatives, dichromate-formaldehj'de-acetic, 234, 245; mercuric-acetone, 212, 245; mer- curic-dichromate-formaldehyde-acetic, 218, 245, (in Held's method for macrog- lia, 413) method for mitochondria, 443 stains, iron-phosphomolybdic-hematoxylin, 285; phosphomolybdic hematoxylin, 291, (in Bauer's method for neuroglia, 412) Helix pomatia, enzymes from, 77, 264 Heller-Robertson's method for myelin sheaths, 529 Heller, Thomas and Davenport's method for nerve fibers, 403 Helly's fixative, 218, 245 in, Cain's method for mitochondria, 442; Hamilton's method for virus inclusion bodies, 465; Hewitt's method for Plas- modium, 507; MacCallum et al. method for pituitary, 426; Martin's method for pituitary, 426, Masson's technique, 371; Miller's method for muscle, 394; Pap- penheim's techniques, 350; Pearse's method for pituitary, 427; Pritchard's method for Golgi bodies, 590; Schlei- cher's triple stain, 372 recommended use, 95 Hemafuchsin granules, 456 Hematein, as ingredient of stains, treated as Hematoxylin Hematoxylin, blueing, 284 differentiators for, 519-520 for staining, algae (Baumgartel), 511 ; bacteria in seC' tions (Fuller), 496; bone (Cretin), 382 bone and cartilage (Klaatsch), 384 diphtheria bacilli (Stottenberg), 490 elastic fibers (Verhoeff), 390; fat, 447- 450; fecal smears (Markey, Culbertson, and Giordano), 508; fecal smears (Nobel), 509; fecal smears (Tompkin and Miller), 510; gastric celLs (Hoecke and Sel)ruyn), 4)^1; intestinal pro- tozoans (Goldman), 507; macrogli 730 INDEX Hematoxylin — (ron/mvvpd) for staining — {contiriued) (Held), 413; mitrochondria (Drew), 443; nervous tissues, 403-409; nuclei, 482-493; Plasmodium (Schuffner), 509 general remarks on, 272, 309 in, Bprbrow's triple stain, 365; Delamare's quadruple stain, 365; Friedlander's double stain, 365; Galiano's double stain, 365; Kefalas's double stain, 365, Koneff's double stain, 365; Ladewig's quadruple stain, 365; Lillie's quadruple stain, 366; Lillie's triple stain, 366; Lowenthal's triple stain, 366; Masson's triple stain, 360; MoUendorf's triple stain, 366; Molher's quintuple stain, 366; Paquin and Goddard's sextuple stain, 366; Reeve's triple stain, 367; Renaut's double stain, 367; Romeis's quadruple stain, 367; Roskin's quad- ruple stain, 372 loss of color through oxidation, 275 mordants for, 515-517 ripening, 274, 278 staining combinations with, acid fuchsin, 406; acid fuchsin and fast green FCF, 456; acid fuchsin, magenta, and picric acid, 498; acid fuchsin and methyl blue, 426; acid fuchsin, methyl blue and orange G, 365; acid fuchsin, new magenta and picric acid, 496; acid fuchsin and orange G, 461; acid fuchsin, orange G and ponceau 2R, 430; acid fuchsin, orcein and picric acid, 372; alizarin red S, 382; anilin blue, 365; anilin blue, acid fuchsin and picric acid, 427; anilin blue, Bismark brown, methyl violet and saffron, 431; anilin blue, eosin, orange G and phloxine, 366; anilin blue, erythrosin and orange G, 425, aurantia, 432; azocarmine, naphthol green B and orcein, 366; azophloxine light green, magenta and orange G, 367; Biebrich scarlet, 365; Biebrich scarlet, anilin blue (or methyl bhie or wool green S), 366; Bordeaux red. 455; Bordeaux red and eosin, 510; brilliant green and magenta, 492; carmine and chlorophyll, 451; carmine and picric acid, 366; celestin blue B, magenta leucobase, and orange G, 427; Congo red, 451, 452; crystal violet, 452, 493; eosin, 128-132, 365, 509; eosin and methyl blue, 366, 464; eosin B and methjdene blue 466; eosin Y, 367; eosin Y and light green, 508; eosin Y and methyl green, 498; eosin Y and meth.vl violet, 458; erythrosin and gentian violet, 494; ethyl eosin, 466; fast green Hematoxylin— (continued) staining combinations with— (continued) FCF (or eosin Y), Bismark brown (or magenta or crystal violet or malachite green), 366; fast green FCF and saf- ranin, 367; fast green and safranin, 367; gentian violet and magenta, 505; iodine green and magenta, 497; light green and magenta, 429, 495, 496; light green and Sudan III, 391; light green and Sudan IV, 450; magenta, 494, 495, 497; ma- genta and orange G, 496, 497; magenta leucobase, 452; magenta leucobase and orange G, 424, malachite green and toluidine blue, 490; metanil yellow, 454; methyl violet and Victoria blue, 505; orange G and safranin, 429; phloxine, 456; picro-fuchsin, 365; picro, fuchsin, and orcein, 365; safranin, 392, 406, 437; Sudan I, 406 stains, 284-293 acid-alum-hematoxylins, 289-290 alum-hematoxylins, 286-289 aluminum-hematoxylins, 292 calcium-hematoxj'lins, 293 chrome-hematoxylins, 291 copper-hematoxylins, 290, 291 ferricyanide-hematoxylin, 291 iron-copper-hematoxylin, 286, 291 iron-hematoxylins, 284-286 lithium-hematoxylins, 290-291 molybdic-hematoxylin, 290 mordant after, copper, 283; dichromate, 283, 284; ferric acetate, 283; ferric alum, 281- 282; ferric chloride, 282, 283 phosphomolybdic-hematoxylins, 291, 292, 293 phosphotungstic-hematoxylins, 292 293, 294 tin-hematoxylin, 291 vanadium-hematoxylin, 291 Hemosiderin, 456 Hendey, hood for cleaning diatoms, 39 Henking's, fixative, 194 method for softening chitin, 261 Henneguy's stains, aceto-carmine, 302 magenta, 315 for chromosomes, 310, 312 safranin-methyl violet-orange G, 364 Henning's fixative, 215 Henocqne's method for nerve endings, 535 Henrichi's method for ground sections, 80, 87 Heptanol, physical properties of, 625 Herbst, corpuscles of, 431, 554 Heringa's method for attaching free sections, 660 Ilcriiiga and ten Berge's, adhesive, for serializing nitrocellulose sections. 148 INDEX 731 Heriiiga and ten Berge's — (continued) golatin (Miibcdding inotliod, (ill method tor nitrocellulose sections, OoU Hermann's, fixative, 195, 245 in, Hermann's method for nuclei, 436; Lee's osmic stain, 530 method for, acid-fast bacteria, 477; nuclei, 436 osmic method for histology, 529 TTorrera's, formaldehyde accelerator, 413 silver diammine stain, 576, 578 Herrera's method for microglia, 586, Rodriguez's method for oligodendria, 589 Hertvvig and Wolbach's method for Rickett- siae, 463 Hertwig's fixatives, chromic-acetic, 228, 245 mercuric-chromic-formaldehyde-acetic, 215, 245 osmic-acetic, 194, 245 picric-acetic, 222 Hertzman's developer, 618 in Hertzman's method for spirochetes, 561 Herxheimer's method for, elastic fibers, 388; fat, 448 Sudan IV in, Badertscher's method for sebaceous glands, 422; Mulon's method for fat, 447 Hetherington's, fixative, 189 in, Hetherington's embedding method for nematodes, 646 fluid for reswelling dried plants, 667 Hewer's developer, 619 Hewitt's method for, avian blood parasites, 508; Plasmodium, 507 Hexanol, physical properties of, 625 Heyt, see van Beneden, 189, 239 Hickson's brazilin, 308 Highman's, methods for, amyloid, 452; mucin, 453 mountant, 632 Hill's, fixative, 198, 245 osmic-silver-dichromate, method for nerv- ous tissues, 605 silver-formic staining solution, 603 Hill, see also Richman, 259 Hirsch and Bretschneider's method for mito- chondria, 443 Hirsch and Jacobs on role of sodium chloride in fixatives, 187 Hirschler's, fixative, 196, 245 in Hirschler's method for Golgi bodies, 530 osmic method for Golgi bodies, 531 Hiss's method for bacterial capsules, 488 Hitchcock's dewaxed shellac, 653 Hitchcock's dewaxed shellac — (continued) as ingredient of, Gage's cement, 655; Gage's varnish, 653 use of, 40 Hj0rt, see also Geschickter Hobbs and Thompson's method for Plas- modium, 508 Hoecke and Sel)ruyn's method foi| gastric gland cells, 431 Hoehl's, fixative, 204, 245 macerating fluid, 264 Hoft'mann's, fixative, 206, 245 method for spirochetes, 479 Hoffmann's violet, for staining, mast cells, (Ehrlich), 455 radulae, 394 Hofker's fixative, 190, 245 Hogan's method for, cartilage, 610 general histology, 610 Hogg's, mountant, 632 varnishes, 653 Hollande's, adhesive for paraffin ribbons, 657 fixatives, cupric-picric-formaldehyde-ace- tic, 220 in Hollande's method for fecal smears, 508 picric-formaldehyde-nitric, 225 in, Hollande's method for fecal smears, 508; Hubin's technique, 340 methods for, flagellates, in fecal smears, 508; mito- chondria, 443 stains, hydrochloric-carmine, 305; magenta- orange G-light green, 339 Holmer's method for bile capillaries, 423 Holmes's, developer, 617 method for invertebrate embryos, 564 silver diammine stain, 576, 578 in Holmes's method for nervous tissues, 583 Holmes and French's triple stains, 352 Holzer's method for neuroglia, 413 Honette, see also Wallart, 339 Honey, as ingredient of Dcane's gelatin mountant, 634 Hood and Neill's cement, 656 Hopewell-Smith's decalcifying fluid, 259 Hopkins' macerating fluid, 263 for Hydra, 78, 79 Horn, 329 Horn of Amnion, demonstration of Negri bodies in smears of, 75 Hornell's fixative, 191, 245 Hornyold's iodine-hematoxyliu, 291 Horvath's method for cilia, cirri and basal bodies, 456 Hoskin's fixative, 192 732 INDEX Hosokawa's fixative, 191 in Hosokawa's method for intracellular "organisms," 461 Hosokawa, see also Taniguchi, 467 Hotchkiss's, method for fungi in skin, 504 mordant, 515 in Pearse's method for pituitary, 427 Houcke's stains, methylene blue-rhodamine B, 353 methylene blue-toluidine blue-thionin- acid fuchsin, 352 toluidine blue-orange G-thionine-eosin Y-azur II, 351 Housefly, larva, 424 Howell's fixative, 277 Hoyer's, ammonia-carmine, 305 fixative, 216, 245 recommended use, 95 gum arable mountant, 632 injection mass, 665 mastic mountant, 637 method for mucin, 453 silver nitrate injections, 163 Hruby's method for nuclei, 436 Hsi, see Zinsser, 463 Hsii and Tang's embedding waxes, 646 Huber's fixative, 210, 245 in Huber's method for Nissl granules, 445 Huber and Caplan's mountant, 636 Huber and Guild's, developer, 619 method for nerve cells and processes, 555 Hubin's triple stain, 340 Hucker's, method for, bacteria, 468 bacterial smears, 473 oxalate-violet, 473 Hucker and Conn's method for Gram-posi- tive bacteria, 474 Hueppe, see Neisser, 486 Hueter's phosphotungstic hematoxylin, 292 in Hueter's method for collagen fibers, 394 Hultgren and Andersson's fixative, 233, 245 Humphrey's, alcoholic accelerator, 614 in Humphrey's method for nerves in blood vessels, 567 Huntingford, see Partington, 525 Huntoon's, method for bacterial capsules, 488 stains, ' crystal violet, 319; magenta, 315 Huseby's fixative, 237 Hutchins and Lutman's method for actino- myces in potatoes, 501 Hyalin, 456, 457 Hydra, dissociating cells of, 78, 79 fixing, 78 mounting dissociated cells of, 79 narcotizing, 78 squash of, 78, 79 staining dissociated cells of, 79 Hydrobromic acid in, Arcadi's method for oligondendria, 585 Cone and Penfield's method for microglia, 586 McCarter's method for neuroglia, 587 Penfield's method for microglia, 587 Winkler's method for microglia, 589 Hydrochloric acid, as ingredient of, decalcifying fluids, see under name of author; Freude's macerating fluid, 263; Konigstein's macerating fluids, 263; Ludwig's macerating fluids, 263; McDowell and Vassos's adhesive for paraffin ribbons, 658; Spoerri's adhesive for paraffin ribbons, 659 as macerating agent, 76 Hydrochloric-carmines, 305, 306 Hydrofluoric acid, for softening wood, 92 in Bertrand and Guillain's method for oligoglia, 586 Hydrogen disulfide, in Krohntal's method for nervous tissues, 610 Hydrogen peroxide, as ingredient of, Cajal and de Castro's accelerator, 615; Gray's narcotic, 265; Mallory's hema- toxylin, 292; Maneval's magenta, 483; Thomas' hematoxylin, 286, 293 for, bleaching, 262; narcotizing protozoa, 53 Hydrolysis, in preparation of squashes, 76 Hydroquinine, as ingredient of, developers, 616-620; Unna's quadruple stain, 364 in, Dublin's method for melanin, 568; Dublin's method for skin, 567; Knowles, Gupta and Basu's method for spiro- chetes, 561; von Recklinghausen's method for bone, 385; Schultze's method for cell outlines in epithelia, 608; Steiner's methods for spirochetes, 562 Hydroxylamine, as narcotic, 52 Hydroxylamine hydrochloride, 265 Hypodermic needles, conversion to injection needles, 165 Hypohysis, see Pituitary I de la Iglesia's fixative, 220 Imhof's fixative, 213 Imhoff, see also Birge, 260 Immobilizing agents, 52 Index, explanation of, 3-4 Index of refraction, 42 India ink, as injection medium, 166, 167 Indigocarmine, as ingredient of, Thoma's in- jection fluid, 664 for staining, bacterial flagella (Loffler), 483; bone INDEX 733 Indigocarmine — {continued) for staining — {continued) and cartilage (Kollikcr), 384; Negri bodies (Zottner), 467; nuclei (Hruby), 436 in, Cajal's double contrast, 325; Calleja's triple stain, 369; Castroviejo's triple stain, 369; Guyler's double contrast, 328; Krause's double contrast, 326; Masson's double contrast, 326; Merbel's double stain, 372; Norris and Shakes- peare's double stain, 372; Pol's double contrast, 326; Roskin's triple stain, 361; Waterman's triple stain, 361 staining combinations with, acid fuchsin and picric acid, 507; car- mine, 372; carmine and picric acid, 369; crystal violet and methyl violet, 483; eosin Y, 328; magenta, 369, 497; ma- genta and picric acid, 372, 436, 506; picric acid, 325, 326 "Indirect" staining, 271 explanation, 284 Indulin in, Ehrlich's triple stain, 369 Lynch's double stain, 371 Ingleby's fixative, 244, 245 in Ingleby's method for neuroglia, 586, 587 Injection, definition, 162 insertion of needles into vessels, 165, 166 killing animal for, 163 method of precipitation in situ, 163 methods for, 163, 164ff, 165 mounting, 166 needles for, 165 reasons for failure of, 166 selection of medium, 162 staining as substitute for, 162 Injection masses, 664-666 Injection media, 661-667 classification of, 650 egg white, 163 general remarks on, 661 lead chromate, 169 low surface tension in, 162 milk, 163 Injections of, chicken embryo, 166-168 intestine, 170 insect trachea, 664 rabbit kidney, 168-169 Inouye's method for bacterial flagella, 482 Insect brain, Buxton's silver nitrate method for, 551 Bretschneider's hematoxylin method for, 410 Rogoff's silver protein method for, 568 Insect, histology, mentioned in Millet triple stain, 341 larvae, fixatives for, 228 muscles in wholemounts of, 424 Insect — {continued) skeleton, stains for, 321, 390, 391 trachea, injection of, 664 Insects, fixation of, 261 Jurray's embedding technique for, 261 mounting in balsam, 56 skeletonizing, 63-64 special fixatives for, Henning's, 215; Eltringham's, 209, 211, 222; Kingsbury and Johannsen's, 215 stains for, 341 wholemounts, 62-64 Insects, see also Chitin Intestinal protozoa, special methods for, 506-510 Dobell's stain for, 369 Intestine, argentophil cells in, 595 of frog, section of, 128-136 Intracellular "organisms," methods for, 461 Intracerebral blood vessels, nerves in, 567 Invertebrate, blood, 418 preparing smears of, 71 embryos, cell outlines in, 564 ganglia, 538 larvae, fixation of marine, 53 mountant for, 636 mounting individual, 67 Pantin's method for fixation of, 187 sectioning for reconstruction, 154 stains for, 54 Donaldson's iodine-eosine, 321; Mayer's cochineal, 301 nerve fibers, 606 nerves, 582 nervous system, 411 sensory nerve ending, 552 striped muscle, 559 Invertebrates, fLxatives recommended for, 196 Iodine, as ingredient of, Arnold's macerating fluid, 264; Cretin's decalcifier, 257; Dominici's fixative, 219; fixative removers, 255; Gardiner's preservative, 179; Goodrich's fixative, 227; Hornyold's hematoxylin, 291; Kofoid's eosin, 508; Zeeti's eosin, 487 for hardening Salamander, 377 in, Anderson's mordant, 517; Atkin's mor- dant, 517; Gardiner's method for plas- modesma, 512; Kilduffe's mordant, 518 Iodine, see also Gram's iodine, Lugol's iodine Iodine green, for staining, acid-fast bacteria in sections (Letulle), 497; cell inclusions (Russel), 457; plant cell walls, 391-393 staining combinations with, acid fuchsin, 391; alkanet and chrome 734 INDEX Iodine green — (continued) staining combinations with — (continued) yellow, 393; carmine, 392; hematoxylin and magenta, 497; magenta, 457 Iron accelerator, in Fontana's method for spirochetes, 608 Iron alum, see Ferric alum Iron-carmines, 304 Iron-copper-hematoxylin stains, 291 Iron-hematoxylin stains, 284-286, 405-407, 435 for smears of plant tissue, 74 Iron-mordant hematoxylin stain, 281-283 Iron salts, see Ferric or Ferrous Iron toning solution, 620 Isamine blue, in, Biggart's method for pituitary, 425 Nicolau's phenol solution, 321 Isinglass, as ingredient of, Frey's embedding medium, 643; Klebs's mountant, 634; Roudanowski's moun- tant, 636 Isoamyl phthalate, as ingredient of, Graj' and Wess's mountant, 641 Isobutanol, as ingredient of, Denhams' sandarac mountant, 638 Isoelectric point of proteins, role in staining, 270 deir Isola's, fixative, 204, 245 method for nervous tissues, 604 Isopropanol, physical properties, 623 Isopropyl acetate, physical properties of, 625 Israel's method for actinomyces in plant tissues, 501 Ivory nut, 92 "J. A.'s" fixative, 211, 246 Jabonero's gold stain, 534 in Jabonero's method for myelin sheaths, 535 Jackson's, fixative, 191 method for plant sections, 392 preservative, 179 Jackson, see also Alexander, 476 Jacobs, see Hirsch, 187 Jacobson's method for argentaffin cells, 596 Jacobson, see also Pick, 474 Jacquiert on pH of fixatives, 188 Jadassohn's methylene blue, 317 in, Douglas's method for acid-fast bac- teria in sections, 496; Parson's method for Negri bodies and Nissl granules, 467 Jager's, fixative, 209, 246 in Jager's method for parasitic amebas, 508 preservative, 177 Jahnel's, developers, 618, 619 method for, nerve cells and processes, 555; spiro- chetes, 561 Jakob's method for neuroglia, 413 Jalowy's silver diammine stain, 576, 578 in Jalowy's method for reticulum, 593 James's cement, 653 Janssen's iron-hematoxylin, 285 Janus green, for staining, blood (Lightwood, Hawksley and Bailey), 418; blood (Sabin), 419; blood (Simpson), 420; oocysts of coccidia (Crough and Becker), 506 staining combinations with, eosin, 506; neutral red, 418, 419, 420 Japan wax, as ingredient of, Hsii and Tang's embedding waxes, 646 "Japanese method" for paraffin ribbons, 657 Jarisch, see Pfeiffer, 233 Jasswoin's method for collagen fibers, 394 Jasswoin, see also Yasvoyn Jaswart, see Singh, 318 "JBS" stain, 318 Jeffry's fixative, 213 Jelly mountants, use of, 46-48 Jenkin's, decalcifying fluid, 259 dehydrating mixture, 259 Jenner's double stain, 344 for staining intracellular "organisms" (Hosokawa), 461 in, Bauer and Leriche's technique, 352; Raadt's technique, 346; Slider and Downej^'s technique, 350 Jensen's stains, phenol-eosjn, 321 picro-carmine, 303 Jironch's method for leaves, 180 Johansen's, stains, crystal violet, 319 fast green, 321 in, Johansen's method for Phaeophyta, 421; Johansen's method for plant histology, 421; Johansen's method for Thailophyta, 512 gentian violet-orange G, 340 safranin, 314 for pollen grains, 309-310 in, Johansen's method for plant his- tology, 421; Johansen's method for plant tissues, 421; Johansen's technique, 364 unsuitabilitj- for nitrocellulose sec- tions, 151 safranin-methyl violet-fast green-orange G, 364 method for nuclei, 436 Johansen, see Kingsbury, 215 Johne's method for bacterial capsules, 488 INDEX 735 Johnson's, fixative, 196, 246 method for Nissl granules, 446 Johnson, see also Chan, 'MKi Johnston's embedding wax, 646 Jolly's fixative, 200, 246 Jones's, fixative, 191, 246 polyvinyl alcohol mountant, 636 Jordan and Heather's, method for Negri bodies, 465 nitrocellulose wax embedding method, 648 Jores' preservatives, formaldehyde-salts, 179 formaldehyde-chloral hydrate-salts, 180 Joseph's fixative, 234, 246 Journal reference, explanation of method, 2 Jousset's macerating fiuid, 264 Judah, see Muir, 656 Judes, see Roques, 246, 318 Juel's fixative, 235 Juge's method for bone and cartilage, 383 Jung, see Vogt, 640 Juniper oil, physical properties of, 624 Jurray's method for softening chitin, 261 Juschtschenko's silver-osmic stain, 603 in Juschtschenko's method for ganglia, 605 K "Kaformacet" fixatives, 234 Kahaner, see Medalia, 509 Kahlden and Laurent's, fixative, 226, 246 lithium-carmine, 307 methods for, bacterial capsules, 488; bacterial spores, 486; blood, 418; cocci in cell smears, 491 Kahle's fixative, 191, 246 Kaiser's, fixative, 209, 246 glycerol jelly, 634 method for myelin sheaths, 405 Kaiser and Gherardine's method for Guar- nieri bodies, 465 Kaiserling's preservatives, formaldehyde- salts, 178, 180 in von Recklinghausen's method for bone, 385 Kallius's developer, 618 Kalter's quadruple stain, 364 Kalwaryjski's silver diammine stain, 576, 579 in Kalwaryjski's method for Trichinella, 598 Kardos's method for blood, 418 Karlson's method for muscle, 394 "Karo," 636 Karpenchenko's fixative, 230, 246 Karusin, see Tschernyschen, 407 Katz's decalcifying fluids, 259 Kaufman's fixative, 201, 246 Kaufmann, see also Bruere, 633 Kaufmann and Lehmann, on Nair and Smith's method for fat, 450 Kedrovsky's method for nuclei, 433-434, 436 Keefe's preservative, 180 Kefaias's stains, hematoxylin-Biebrich scar- let, 365 iron-hematoxylin, 285, 365 Keil's method for spirochetes, 479 Keller's method for Nissl granules, 446 Kemp's method for diphtheria bacilli, 490 K(>ndall, see Kostoff, 220 Kenney's method for reticulum fibers, 610 Kenyon's fixative, 221 Keratin, mentioned in, Drew-Murray's triple stain, 369; Dupres's double contrast, 339; llay- thorne's triple contrast, 337; Hollande's triple contrast, 339; Mallory's triple stain, 360; Semichon's triple contrast, 329 special methods for, 422 Kernohan, method for nervous tissue, 583 mordanting before acid fuchsin-phopho- molybdic techniques, 359 Kerschner's fixative, 194, 246 in Kerschner's method for nervous tissues, 540 Kidney, de Galantha's method for, 423 injection of glomeruli, 168-169 isolation of tubules from, 263 Kihara, see Oguma, 201 Kilduffe's iodine mordant, 518 Killing, animals before injection, 163 cestodes, Bujor's method, 265 frog for histology, 129 mouse for, histological material, 334 sectioning, 136 rat, for spermatogenesis studies, 273 Kimbrough, see Bailiff, 416; Burdon, 491 King's accelerator, 615 fixative, 228, 246 method for Nissl bodies, 446 silver diammine stain, 576, 579 in King's method for neuroglia, 587 King, see also Slifer, 261 Kingsbury's fixative, 213 Kingsbury and Johannsen's, decalcifying fluid, 259 fixatives, 215, 222 formaldehyde accelerator, 613 in Kingsbury and Johannsen's method for striped muscle, 559 stains, hydrochloric-carmine, 306; methylene blue, 317; orange G-acid fuchsin, 328 Kingsley's quadruple stain, 348, 349 for blood smear, 343, 364 in Kingsley's method for megakaryocytes, 431 Kinyoun's, magenta, 315 in Campbell's method for leprosy bacilli in sections, 496 736 INDEX Kiny oun 's — {contin ued) method for diphtheria bacilli, 489 Kionka's method for yolk granules, 448 Kirchner's preservative, 180 Kirkman's method for Nissl granules, 446 Kirkpatrick's alum-cochineal, 300 Kirkpatrick and Lendrum's polystyrene mountant, 641 recommended use, 132 Kisser's, glycerol jelly, 634 method for sectioning wood, 93 Kitton's cement, 654 Kiyono's, differentiator, 521 method for mitochondria, 441 Klaatsch's method for bone and cartilage, 384 Klatzo's formaldehyde accelerator, 613 Klebs's, isinglass mountant, 634 macerating fluid, 263 Klein's fixative, 218, 227, 246 in Klein's method for Paneth cells, 452 KJeinenberg's, calcium-hematoxylin, 293 fixative, 222, 246 as ingredient of, Conklin's hematoxylin, 289; Patter- son's fixative, 222 for, earthworm, 331; softening chitin, 261 Klett's method for bacterial capsules, 488 Ivligman, Mescon and DeLameter's method for fungi in skin, 504 Kline, see Davenport Klotz and Coburn's preservatives, 180 Klotz and MacLachan's preservative, 180 Knife angle, effect of faulty, 122 in cutting paraffin sections, 114 Knife, see also Microtome knife Ivnower's iron-mordant hematoxylin, 283 Knowles's method for Plasmodium, 508 Knowles, Gupta, and Basu's method for avian spirochetes, 561 Koch's method for acid-fast bacteria, 477 Kockel's method for fibrin, 423 "Kodalk," 565 Koenig, Groat and Windle, gum-formalde- hyde fixation, 190 Kofoid's method for fecal smears, 508 Kofoid and Swegy's iron-mordant hema- toxylin, 282 Kohashi's quintuple stain, 361 Kohn's fixative, 217 recommended use, 95 Koinikow's method for Schwann cells, 415 KoUiker's method for bone, 384 Kollmann's fixative, 231 Kolmer's fixatives, dichromate-formalde- hyde-acetic-trichloroacetic, 235, 246 mercuric-dichromate-formaldehyde-acetic, 218, 246 Kolossow on Lee's method, 530 Kolossow's, developer, 618 in Kolossow's method for general his- tology, 530 fixatives, osmic-chromic, 203, 246;osmic-uranium, 205, 246, (in Kolossow's method for nerve endings, 535) osmic acid stain, 527 osmic-silver stain, 603 in Kolossow's method for ganglia, 605 osmic-silver-dichromate method for sym- pathetic ganglia, 601, 605, 612 Kolster's fixatives, mercuric-acetic, 209, 246 osmic-mercuric, 196 Komora, see Taniguchi, 467 Koneff's, double stain, 365 method for pituitary, 426 Konigsteins's macerating fluid, 263 Konschegg's method for Gram-positive bac- teria, 475 Kopciowska, see Nicolau, 461 Kopel, alcohol solvent for Giemsa's stain, 347 Kopeloff and Beerman's method for Gram- positive bacteria, 469, 475 Kopeloff and Cohen's method for Gram- positive bacteria, 475 Kopsch's, fixatives, chromic-acetic, 229 dichromate-formaldehyde, 233, 246 in Kopsch's method for nervous tis- sues, 605 method for Golgi bodies, 530 " Kopsch-Gatenby " technique, 530 von Korff's methods for, embryonic bone, 384 embryonic teeth, 384 Kornhauser's quadruple stains, 369, 370 Kossa's methods for general histology, 560 Kossel, see Behrens, 264 Kostanecki and Siedlecki's fixative, 211, 246 Kostoff and Kendall's fixative, 220, 246 Kostowiecki's double contrast, 327 Kozowsky's differentiator, 520 in Kozowsky's method for myelin sheaths, 407 Krajian's, alcoholic accelerators, 597, 614 developer, 618 methods for, acid-fast bacteria in sections, 497; amy- loid, 452; bacteria in sections, 492, 494; elastic fibers, 388; spirochetes, 561, 597 osmic method for fat, 530 silver diammine stain, 576, 579 in, Krajian's method for reticulum, 593 stains, eosin, 321; iron-hematoxylin, 285; mag- enta, 316, (in Krajian's method for Grampositive bacteria in sections, 494) Krajian, see also Evans, 258 INDEX 737 Kramer's method for insect muscles, 424 Kramer and Shipley's decalcifying fluid 260 Krantz's developer, 618 in Krantz's method for spirochetes, 561 Kraus's methods for, colloid of thyroid, 456 ■pituitary, 426 spirochetes, 479 Krause's injection mass, 665 stains, methyl green-orange G-acid fuch- sin, 356, 357 picro-indigo-carminc, 326 Kricheski's triple stain, 360 Kristensen's decalcifying fluid, 259 Kroenig's cement, 656 Krohntal's, fixative, 236 lead formate method for nervous tissues, 610 Kromayer's method for epithelial fibers, 424 Kronecker's preservative, 176 Krueger's fixative, 218, 246 Krugenberg and Thielman's triple stain, 370 Kufferath's method for ascospores in yeasts, 512 Kuga, see Taniguchi, 467 Kiihne's, methods for, acid-fast bacilli in sections, 497; bacteria in sections, 492; maceration, 263 stains, methylene blue, 317 in, Kuhne's method for bacteria in sections, 492; Nicolle's method for bacteria in sections, 493 polychrome methylene blue-eosin Y, 349 KuU's, method for mitochondria, 439-441, 443 triple stain, 353 Kulp's method for bacterial flagella, 482 Kultschitsky on fixing spinal cord, 398 Kultschitsky's, differentiator, 520 fixatives, cupric-dichromate-acetic, 220 recommended use, 95 mercuric-dichromate-acetic, 217, 246 hematoxylin, 283 in, Anderson's method for nervous tissue, 404; Bolton's method for myelin sheaths, 404; Clara's methods for bile capillaries, 422; Dietrich's method for fat, 447 ; Kraus's method for pituitary, 426 ; Kultschitsky's method for myelin sheaths, 405; Liber's method for mye- lin sheaths, 406; Mitroplanov's method for myelin sheaths, 406; Parat's method for mitochondria, 444 ; Sokolansky's method for neuroglia, 414; Tschernyschew and Karusin's method for myelin sheaths, 407 metliods for, elastic fibers, 388; reticulum fibers, 424 Kupffer's method for, astrocytes, 541 liver, 607 liver connective tissue, 541 Kupperman and Noback on fixative mor- dants, 191 Kupperman and Noback's fixative, 224 Kurnick and Ris's method for nuclei, 436 Kuskow's macerating fluid, 264 de Lachand, see Lacoste, 640 LaCoste and de Lachand's rosin mountant, 640 LaCour's, fixative, 202, 247 fixative remover, 255 gentian violet, 319 method for nuclei, 436 LaManna's fixative, 235 iron-hematoxylin, 285 in LaManna's method for myelin sheaths, 405 Label adhesives, 661 Labeling slides, 128 Lachi's fixative, 233, 246 silver-dichromate method for nervous tis- sues, 604 Lacquers, cellulose acetate, for ringing bal- sam mounts, 58 removing from slides, 666 Lactic acid, as ingredient of, Alcorn and Yeager's preservatives, 177; Amann's preservative, 177; Archibald and Marshall's mountant, 636; Dows's polyvinyl mountant, 636; Gansen's triple stain, 351; Gray and Wess's poly- vinyl mountant, 636; Hetherington re- swelling fluid for plants, 667; Huntoon's magenta, 488; Jones's polyvinyl moun- tant, 636; Lepik's anilin blue, 502; Moore's phenosafranin, 502; Rukhadze and Blajin's carmine, 509; Schubert's anilin blue, 505; Watkin's methyl blue, 422; Womersley's mountant, 633; Zir- kle's orcein gelatin mountant, 636 Lactochloral preservatives, 178 Lactophenol mounts, Semmens's varnish for, 654 preservatives, 177-178 Ladewig's quadruple stain, 365 Lageberg's method for bacterial spores, 486 Laguesse's, fixative, 200, 247 triple stain, 364 Laidlaw's, fixative, 209, 247 in Laidlaw's method for introcellular "organisms," 461 silver diammine stain, 576, 579 Laidlaw's method for reticulum, 593 Laigret and Auburtin's method for Rickett- siae, 463 738 INDEX Lake, definition of, 269 Lamellae in bone, 81 Schmorl's method for, 385 Lampblack, as ingredient of Martin's var- nish, 656 for baseline in reconstructions, 154, 155 Lancelin, Seguy and Debreuil's toner, 620 in Lancelin, Seguy and Debreuil's method for spirochetes, 597 Land's, adhesive for paraffin ribbons, 658 method for plant sections, 392 Landau's, differentiator, 520 in Landan's method for nervous tissues, 405 method for nervous tissues, 403, 405, 583 mordant, 516 mountant, 632 Landois's macerating fluid, 264 Landsteiner's fixative, 233, 247 Lane's fixatives, 216, 247 in Lane's method for pancreas, 429 Lang's, fixatives, mercuric-acetic, 209, 247 mercuric-picric-acetic-sulfuric, 214 mordant, 516 Langenbeck's fixative, 222 Langendorf's fixative, 200, 247 Langerhaus, islets of, 428, 429, 430 mountant, 632 Langeron on, acetic acid fixatives, 189 classification of dyes, 271 oxidation-reduction in fixatives, 188 platinic fixatives, 205 removal of thiosulfate, 538 staining, 269 Langeron's, adhesive for, free sections, 660 nitrocellulose sections, 659 bleaching solution, 262 decalcifying fluid, 259 fixatives, mercuric-acetic, 209, 247 picric-formaldehyde-acetic, 224 gum arable embedding medium, 644 lacto-phenol for swelling dried plants, 380 macerating fluid, 264 methods for, acid-fast bacteria in sections, 497; at- taching nitrocellulose sections, 659; filamentous fungi, 512; fungus in plant tissues, 502; myxosporids, 508; jilant sections, 392-393; radulac, 513; spiro- chetes, 597 narcotic, 265 preservative, 178 stains, acid-alum-hematoxylin, 289; azur II, 317; azur-eosin, 348; hydrochloric-car- mine, 306; methyl green-pyronin, 355; polychrome methylene blue, 317, (in Shortt's technic^ue, 350); polychrome methylene blue-orcein, 353; polychrome methjdene blue-tannin-orange G, 353; Langeron's — {continued) stains — (continued) saffron-erythrosin, 329; safranin, 314; thionine, 318 supporting wax, 656 Venice turpentine mountant, 637 wax embedding method, 646 wax for dishes, 667 Langhan's method for amyloid, 452 Langrand's method for actinomyces, 504 Lanolin, as ingredient of Plant's cement, 655 Noyer's cement, 656 Laporte, method of cleaning foraminifera tests, 17 Lapp's fixative, 209, 247 Larband's wax embedding method, 647 Lard, as ingredient of. Cigalas' cement, 655; Gadden's wax embedding medium, 646; Robin's injec- tion mass, 665 ; Ruffini's embedding wax, 647 Larvae of, Dracunculus, 509 Echinus, 153-156 housefly, 424 marine worms, 53 scale insects, microflora in, 504 Lataste's cement, 656 Latter, see Gates, 228 Lattice fibrils in liver, 607 Lauda and Rezek's, developer, 618 silver nitrate stain, 550 in Lauda and Rezek's method for kid- ney, 560 Launoy's alum-hematoxylin, 288 in Launoy's method for pancreas, 429 Laur, see Fiessinger, 417 Lavdowsky's fixatives, chromic-acetic, 229, 247 recommended use, 65, 95 formaldehyde-acetic, 191, 247 mercuric-dichromate-acetic, 217, 247 platinic-chrome-acetic, 207, 247 Lavender oil, as ingredient of Cox's sandarac mountant, 638 physical properties of, 624 Laveran's methylene blue, 317 Lawrentjew's, fixative, 236 in, Golgi's method for nerve endings, 605; Lawrentjew's method for nerve endings, 605 silver diammine stain, 576, 579 in Zhootsin's method for reticulum, 595 Lawson's method for bacterial capsules, 488 Laybourne's method for diphtheria bacilli, 489 Leach's method for fat, 448 Leach, see also Carleton, 211 Lead acetate, as ingredient of, Cajal and de Castro's fixing solution. INDEX 739 Lead acetate, as ingredient of — {continued) 621; Doyere's injection method, GG2; Frey's injection fluid, GG3; Ilarting's injection masses, GG5; Lison's fixative, 236; Petragnani's mordant, 518 Lead chloride, as monhmt, 406, 414 Lead chromate, injection of glomeruli with, 168-1G9 Lead formate, as ingredient of Krohntal's fixative, 23G in Krohntal's method for nervous tissues, 610 Lead nitrate, as ingredient of, MacConaill's hematoxylin, 406 Thiersch's injection mass, GGG Lead subacetate, as ingredient of Salkind's fixative, 236 in Salkind's cherry gum embedding me- dium, 645 Leaf, section of, 91 Leavitt, see Rosbach, 259 Leber's method for cell outlines, 611 Lebowich's, method for attaching gelatin sections, 660 soap embedding medium, 644 Lebrun, see Carnoy, 208, 240 Lee-Brown's triple stain, 360 Lee on, coagulant fixatives, 187 Kolossow's method, 530 Lee's, gold mercury method for nervous tis- sue, 538 osmic methods for histology, 530 stains, alum-hematoxylin, 288 iron-carmine, 304 Leech, narcotization and fixation of, 53 nerves in, 551 Leek, 433 van Leeu wen's fixative, 224 Legal's picro-carmine, 303 Legros's glycerol jellies, 634 Lehmann, see Kaufmann, 450 Lehrmitte and Guccione's fixative, 200, 247 in Lehrmitte and Guccione's method for neuroglia, 414 T>ehrmitte, see also Roussy, 446 Leigh-Sharpe's fixative, 217 Leishman's double stain, 346 in Pryce's technique for erythrocytes, 419 Lemiere and Bccue's method for actinomy- cetes, 504 Lemon juice, in Rauvier's method for nerve endings, 536 Lemon oil, physical properties of, 624 Lendrum's, fixative, 189 method for granules in breast and kidney, 456 softening fluid for embedded objects, 667 stains, acid fuchsin, 319, (in Lendrum's method for granules in breast and kidney, 456) ; Lendrum's — {continued) stains — {continued) eosin Y-erythrosin-i)hl(>xine-tartrazine NS, 340; phloxine-tartrazine, 340 Lendrum, see also Kirkpatrick, 641 Lendrum and McFarlane's, celestin blue B, 313 in Pearse's method for pituitary, 427 quintuple stain, 338 Lenhossek's fixatives, merciuic, 207, 247 mercuric-acetic, 209, 247 mercuric-picric, 213, 247 niercuric-jjicric-acetic, 214 platinic-mercuric-acetic, 206, 247 Lenoir's, differentiator, 521 fixative remover, 255 fixatives, chromic-iodide, 232, 247; picric-formal- dehyde-acetic, 224 ; picric-formaldehyde- dichromate-acetic, 227 Lenz's method for Negri bodies, 466 Lepik's, method for Peronosporales, 502 preservative, 178 Lepine's methods for, Negri bodies, 466 Rickettsiae, 463 Lepine and Sautter's fixative, 213 Leprosy bacilli, in sections, 495, 496, 497 Leptospira icterohaemorrhagica, 562 "Leptotriches" in sections, 495 Leriche, see Bauer, 352 Letulle's method for acid-fast bacteria in sections, 497 Leucobase, magenta, see Magenta leucobase Leukocytes, bacteria in, 490, 491 fat in, 420 mentioned in, Matsura's polychrome neu- tral red, 371 Pappenheim's double stain, 355 Levaditi's, developer, 618 in, Levaditi's methods for spirochetes, 561, 562; Nakano's method for spiro- chetes, 562; Para's method for spiro- chetes, 562; Warthin's method for spirochetes, 563 silver nitrate staining solution, 550 Levaditi and Manouelian's developer, 618 Levine's method for mast cells in parathy- roid, 431 Levi's, fixative, 198, 247 silver diammine stain, 576, 579 in Levi's method for reticidum, 593 Levitsky's fixative, 230, 247 Levulose, as ingredient of. Apathy's mountant, 631; Monk's pectin mountant, 636; Zirkle's carmine pectin mountant, 636, 637 Lewis's differentiator, 521 Lewis, see also Maurer, 426 Lewis and Miller's method for pituitarj-, 426 740 INDEX Lhotka and Ferreira's fixative remover, 256 Liber's method for myelin sheaths, 406 Lieb's, method for amyloid, 452 mountant, 632 Liebmann's method for, amphibian blood, 418 invertebrate blood, 418 Liefson's, method for bacterial flagella, 482 mordant, 518 Liengme's iron-copper-hematoxylin, 291 Liesegang's developer, 618 in Liesegang's method for nerve cells and processes, 555 Light green, as plasma stain, 320 for staining, acid-fast bacteria, (Alexander and Jack- son), 476; acid-fast bacteria in sections (Adam), 495; acid-fast bacteria in sec- tions (Doubrow), 496; acid-fast bacteria in sections (Fuller), 496; algae (Sem- mens), 512; bacterial smears (Maneval), 473; bacterial spores (Botelho), 485; collagen and myofibrils (Long), 597; fat (Wilson), 450; fecal smears (Hollande), 508; fungus in plant tissues (Dickson), 501 ; fungus in plant tissues (Margolena), 502; fungus in skin sections (Kligman, Mescon and DeLameter), 504; Gram- positive bacteria in sections (Male), 494; nervous tissues (Alzheimer), 409; nerv- ous tissues (Hadjiloff), 407; neuroglia (Alzheimer), 411; neuroglia (Hadjiloff), 413;nuclei (Geither), 435;nuclei (Mane- val), 436 ; nuclei (Semmens and Bhaduri) , 437; pancreas (Launoy), 429; pancreas (Moller), 429; pituitary (Colin), 425; pollen tubes (Buchholz), 421 ; skin (Dub- lin), 567; vaginal smears (Papanicolaou), 432 m, Chatton's double contrast, 327; clove oil solution, 320; Crossmon's triple con- trast, 336; Goldner's quadruple con- trast, 337; HoUande's triple contrast, 339; Minchin's double contrast, 326; Patay's double contrast, 339; Pollak's quadruple contrast, 338; Romeis's quad- ruple stain, 367; simple solution, 320; Twort's double stain, 372 staining combinations with, acid fuchsin, 413, 421, 425, 485; acid fuchsin, azophloxine, orange G and pon- ceau 2R, 337; acid fuchsin, orange G and ponceau 2R, 337, 338; acid fuchsin and picric acid, 409, 411; azocarmine and silver diammine, 597; azophloxine, hematoxylin, magenta and orange G, 367; azophloxine and orange G, 337; azophloxine, orange G, ponceau 2R and protein silver, 567 ; Bismarck brown, 429 ; Light green — {continued) staining combinations with — {continued) Bismarck brown, eosin Y and orange G, 432; eosin Y, 327; eosin Y and hema- toxj'lin, 508; erythrosin, orange G and thionin, 502; fast yellow, magenta and methylene blue, 476; gentian violet and neutral red, 493; hematoxylin and magenta, 429, 495, 496; hematoxylin and Sudan III, 391; hematoxylin and Sudan IV, 450 ; magenta leucobase, 437, 504; magenta and orange G, 339, 436; methyl violet and neutral red, 494; neutral red, 372; orange G and acid fuchsin, 336; phloxine, 501; picric acid, 326; ponceau 2R, 339; safranin, 392, 435 Lightwood, Hawksley and Bailey's method for blood, 418 Ligniere's method for actinomyces, 504 Lignified tissues, mentioned in Johansen's quadruple stain, 364 special stains for, 391, 392, 393 Lignum vitae, 92 Ligroin, as ingredient of Davies' asphalt varnish, 653 general remarks on, 651 Ligustrum, 91 Lilac oil, physical properties of, 624 Lilium, see Lily Lillie's, adhesive for paraffin ribbons, 658 differentiator, 520 fixatives, dichromate-formaldehyde-acetic, 235, 247; osmic-chromic-acetic, 201, 247; picric-formaldehyde-acetic, 224; picric- formaldehyde-formic, 225, 247 methods for, bone, 560; fat, 161, 448, 449; glycogen, 452; mucin, 254; myelin sheaths, 406; Negri bodies, 466; pituitary, 426; reticu- lum fibers, 424, 593 stains, acid-alum-hematoxylin, 290 ; azofuchsin- brilliant purpurin R-naphthol blue black-picric acid, 370; azur A (or C)- eosin B (or Y), 349; Biebrich scarlet- fast green FCF, 337; Biebrich scarlet- methyl bhie, 370; Biebrich scarlet-picro- anilin blue, 340; eosin Y-naphthol green B, 370; fast green {or wool green)-acid fuchsin {or violamine R), 370; hema- toxylin-Biebrich scarlet-aniline blue {or methyl blue or wool green S), 366; hema- toxylin-fast green FCF {or eosin Y)- Bismark brown Y {or magenta or new magenta or crystal violet or malachite green), 366; iron-hematoxylin, 285; magenta leucobase, 316, (in Lillie's method for glycogen, 452); methyl blue- phloxine-orange G, 370; picro-blue, 326; INDEX 741 Lillie 's — (contin ued) stains — {continued) picro-fuchsin, 328 ;picrif'-naphthol black, 32G, (in Lillie's method for myelin sheaths, 400, Lillie's method for reticu- lum, 593) Lillie and Ashburn's mountant, 632 in Lillie and Ashburn's method for fat, 449 in Wilson's method for fat, 450 Lillie and Earle's iron-hematoxylin, 285 in Weigert's stain, 285 Lillie and Pasternack's double stain, 34G Lily bud, fixation of, 149 impregnating with nitrocellulose, 150 orienting in celloidin block, 150 section of, 149-152 staining nitrocellulose sections of, 151 Lime, as ingredient of Beale's French cement, 655 Lim's double stain, 305 Linder's preservative, 177 Linin, 435 Linseed oil, as ingredient of, Beale's Brunswick black, 652; Coburn's cement, 655; gold size, 652; Mendeleef's cement, 656 Linstaedt's, celluloid sheet method for sec- tions, 659 method for serializing nitrocellulose sec- tions, 149 Lipoid, see Fat Lipp's methods for spirochetes, 480 Liquid petrolatum, for mounting, blood smears, 34 wholemounts, 32 sealing mounts in, 35 Lison's fixative, 236 method for argentaffin cells, 596 Lison and Dagnelle's method for myelin sheaths, 410 Literature cited, 670-680 Literature references, explanation of method used, 2 Litharge, as ingredient of Kitton's cement, 654 Lithium carbonate, as ingredient of, Alzheimer's hematoxylin, 290; anony- mous toluidine blue, 316; Bacsich's hematoxylin, 29; Foot's silver diammine stain, 578; Haug's carmine, 307; Kahl- den and Laurent's carmine, 307; Laid- law's silver diammine stain, 579; Long's silver diammine stain, 579; Loyez's hematoxylin, 291; Martinotti's methyl- ene blue, 318; del Rio-Hortega's silver diammine stain, 580; Schroder's hema- toxyhn, 292 for, polychroming methylene blue, 346 removing picric acid, 273 Lithium-carmine, 307 Lithium-hematoxylin, 290, 291, 408 Lithium sulfate, as ingredient of Fieandt and Sazen's decalcifying fluid, 258 Liver fluke, collection of, 294 wholemount of, 294-296 Liver, gold-chromic methods for, 541 lattice fibrils, 607 reticulum fibers in, 593 Ljubinsky's method for dijihtheria bacilli, 490 LoBianco's, fixatives, chrome-acetic, 229, 247; chromic-alco- hol, 227, 247; chromic-formaldehyde, 230, 247; mercuric-acetic, 209, 247; mercuric-chromic, 215, 247; mercuric- cupric, 213, (for fixing, coral, 85, medusae, 296); osmic-chromic, 199, 247, 248; osraic-dichromate, 203; picric- chromic-sulfuric, 226, 247 narcotic, 265 Lobo's, developer, 618 formaldehyde accelerator, 613 silver diammine stain, 576, 579 silver nitrate stain, 579 in Lobo's methods for, nervous tissues, 557 neuroglia, 558 Lochead, see Perry, 427 Loffler's, methods for bacterial flagella, 482, 483 stains, crystal violet-magenta, 483 in Mouye's method for bacterial fla- gella, 482 indigo carmine-methyl violet, 483 in, NicoUe and Morax's method for bacterial flagella, 483; Remy and Sugg's method for bacterial flagella, 483 polychrome methylene blue, 317 in, Abbott's method for bacterial spores, 484; Alexander's method for acid-fast bacteria, 476; Alexander and Jackson's method for acid-fast bacteria, 476; Beauverie's method for diphtheria bacilli, 489; Bitter's method for bacterial spores, 485; Castafieda's method for Rickettsiae, 462; Dawson's method for Negri bodies, 75, 465; Fraenkel's method for bacteria in sections, 492; Gay and Clark's method for dead bac- teria, 491; Gerlach's method for Negri bodies, 465; Goodpasture's method for Negri bodies, 465; Gor- don's technique, 345; Graham's method for oxidase granules, 417; Guyer's method for bacteria in sec- tions, 492; Kahlden and Laurent's method for bacterial spores, 486; 742 INDEX Loffler's — (continued) stains — {continued) polychrome methylene blue — (con- tinued) in — (continued) Kemp's method for diphtheria bacilli, 490; Krajian's method for acid-fast bacteria in sections, 497; Krajian's method for bacteria in sections, 492; Krajian's method for Gram-positive bacteria in sections, 494; May's method for bacterial spores, 486; Neelsen's method for acid-fast bacteria, 477; Noniewicz's method for bacteria in sections, 493; Proca's method for bacterial spores, 486; Roskin's method for Rickettsiae and Negri bodies, 463; Shunk's method for bacterial fla- gella, 484; Stvitzer's method for Rickettsiae, 463; Weiss's method for acid-fast bacteria, 478 Logwood, 290 Lohaus, see Gross, 235 Long's silver diammine stain, 576, 579 Long and Mark's fixative, 217, 247 Lonnberg's double stain, 370 Lopez's triple stains, 371 Lowenthal's, fixative, 203, 247 stains, picro-carmine, 303 picro-carmine-hematoxylin, 366 Lowit's, differentiator, 521 fixative, 207, 248 method for nerve endings in skin, 535 safranin, 314 Loyez's lithium-hematoxylin, 291 method for nervous tissue, 406 Lubarsch's method for glycogen, 452 Lubkin and Carsten's polyvinyl alcohol em- bedding medium, 644 Ludford's method for Golgi bodies, 525-527, 531 Ludwig's macerating fluid, 263 Lugaro's, method for, neuroglia, 609 Nissl granules, 446 silver-bromide-iodide-thiosulfate stains, 609 Lugol's, fixative remover, 255 iodine, 255 as ingredient of stains, Donaldson's iodine-eosin, 321 as mordant for stain, Mallory's, 406 for removing fixative from section, 127 in, Ambrosioni's method for diphtheria bacilli, 489; Anderson's method for neuroglea, 412; Anglade and Morel's method for neuroglia, 412; Bardelli and Cillc's method for Zymonema, 503; Beauverie's method for diph- Lugol's — (continued) iodine — (continued) in — (continued) theria bacilli, 489; Bidegaray's method for intestinal protozoans, 506; Car- pano's method for Negri bodies, 464 Darzin's method for Rickettsiae, 462 P'oley's stain, 363; Fonte's method for acid-fast bacteria, 476; Galescu's method for neuroglia, 413; Good- rich's macerating fluid, 263; Gray's method for bone, 383; Hermann's method for nuclei, 436; Krajian's method for elastic fibers, 388; Kro- inayer's method for epithelial fibers, 424; LaCour's technique, 319; Lan- geron's Giemsa technique, 348; Lehr- mitte and Guccione's method for neuroglia, 414; Ligniere's method for actinomyces, 504; McNamara's tech- nique, 349; Mann's staining tech- nique, 352; Manouelian's method for Negri bodies, 466; Merzbacher's method for neuroglia, 414; Neukirch's method for glycogen, 453; Newton's technique, 319; Peer's method for neurogUa, 414; Tribondeau's method for bacterial spores, 487; Tunnicliff's method for spirochetes, 481; Unna's method for skin fibers, 424; various methods for Gram-positive bacteria in, sections, 493-495, smears, 474-475; Verhoeff's method for elastic fibers, 390; Wallace's method for basal bodies, 458 Liiko's fixative, 192, 247 Lundvall's bleaching solution, 262 in Lundvall's method for, bone, 384; carti- lage, 386 Lung, alveolar epithelium, 534 fungus in, 503 Lustgarten's method for spirochetes in sec- tions, 498 Lutes, general remarks on, 651 Lutman, see Hutchins, 501 Lwoff, see Chatton, 559, 660 Lymph vessels in testes, 608 Lympliatic glands, reticulum fibers in, 593 Lymphocytes, Schridde's granules in, 457 Lynch's double stain, 371 for vertebrate embryos, 55 Lysol, as ingredient of Reinke's macerating fluid, 264 for cleaning slides, 666 M McArthur, see Davenport, 567 MacCallum's, injection mass, 665 macerating fluid, 263 INDEX 743 MacCallum, Fletcher, Duff and Ellsworth's method for pituitary, 42(') McCarter's method for oligodendria and microglia, 587 McCarter, see also Mullen, 518 McCarthy, see Blank, G43 Macchiavello's method for Rickettsiae, 458- 4G0, 4G;^ McClean's stains, :521 McClintock's method for nuclei, 437 McClung, on hardening brain, 572 McClung and Allen's fixative, 192, 248 MacConaill's method for nervous tissue, 406 McCuUough anil Dick's method for bacteria in leukocj'tes, 491 McDowell and \'assos's adhesive for paraffin ribbons, 658 recommended use, 131 Maceration, definition, 76 methods for squashes, 76-79 of, kidney, 263; plant tissues for squashes, 76; striped muscle, 263; wood, 263 Macerating agents, 260, 264 acid methods, 262-263 alkaline methods, 264 enzyme methods, 264 McFarlane's stains, picro-acid fuclisin-anilin blue, 338 picro-orange G-acid fuchsin-ponceau 2R- anilin blue, 338 MacFarland and Davenport's, developer, 618 in MacFarland and Davenport's method for nerves in adrenal, 567 fixative, 193 Mcllroy and Hamilton's fixative, 228 Mclndoo's silver diammine method for bile capillaries, 597 ]\IcJunkin's method for peroxidase granules in bone marrow, 456 INIcLachan, see Klotz, 180 Mc Mann's, fixative, 236 in Mc Mann's method for Golgi bodies, 443 method for, fungi in skin, 504 Golgi bodies, 443 silver stain, 550 in i\Ic Mann's method for spinal cord, 555 McXamara's double stain, 349 McXamara, Murphy, and Gore's decalci- fying fluid, 259 McXaught, see Proescher, 372 MacNeal's triple stain, 349 Macroglia, dye staining methods for, 411-415 gold-mercury methods for, 539 mixed metal methods for, 606, 607 silver diammine methods for, 585-590 silver nitrate methods for, 558 McWhorter's method for viroplasts, 466 von Maehrenthal's osmic staining method, 530 "Magdala red," 320 staining cond)ination with methylene blue, 388 Magenta, as nuclear stain, 315-316 differentiators for, 520-521 for staining, acid-fast bacilli in sections, 477-478, 495-498; actinomycetes (Mallory), 505; bacteria in milk (Broailhurstand Paley), 490; bacteria in sections (Krajian), 492; 1)acterial capsules (Ilvuitoon), 488; bac- terial capsules (Klett), 488; bacterial capsules (Wadsworth), 489; bacterial flagella, 481-483, 598; bacterial smears (Goodpasture), 473; bacterial smears (Spehl), 474; bacterial spores (Gray), 486; bacterial spores (Xeisser and Hueppe), 486; cell inclusions (Russel), 457; coccidia (Borrel), 506; elastic fibers (Manchot), 388; elastic fibers { Weigert), 390; Gram-positive bacteria in sections (Brown and Brenn), 494; Gram-positive bacteria in sections (Glynn), 494; Gram- positive bacteria (Weiss), 475; hema- fuchsin granules (Mallory), 456; mucin (Mallory), 454; Xegri bodies, 465-467; nervous tissues (Gross), 410; nervous tissues (Xissl), 410; nervous tissues (Prince), 411; nuclei (Hruby), 436; pan- creas (Baley), 428; Plasmodium (Big- nani), 506; pollen tubes (Chandler), 421 ; Rickettsiae (Clancy and Wolfe), 462; Rickettsiae (Macchiavello), 463; Rick- ettsiae (Xyka), 463; Rickettsiae and Negri bodies (Roskin), 463; Rickettsiae (Zinsser, Fitzpatrick and Hsi), 463; spirochetes (Lipp), 480; spirochetes (Noguchi), 480; spirochetes (Weiss), 481; wandering cells (Hamazaki), 431 general remarks on, 308 in, Castroviejo's triple stain, 369; Doetsch- mann's mountant, 631; Dupres' triple stain, 359; Gausen's triple stain, 351; Gray's mordant, 518; Hollande's triple contrast, 339; Lopez' triple stain, 371; Rhamy's triple stain, 353; Shumway's triple stain, 372; Waterman's triple stain, 361; Ziehl's jihenol solution, 321 staining ('ombinatit)ns with, acid green, 481; acid violet, 428, 481; acid violet and methyl green, 427; anilin blue and azocarmine, 427; anilin blue, erythrosin and methyl orange, 411 ; anilin blue and methyl orange, 371; aurantia, 506;aurin and methylene blue, 477; azophloxine, hematoxylin, light green and Orange G, 367; Bismarck 744 INDEX Magenta — {continued) staining combinations with — (continued) brown, 389, 463; brilliant green and hematoxylin, 492; brilliant yellow, 476; carmine, 421; cresofuchsin and orange G, 453; crystal violet, 387, 482, 494; crystal violet and eosin Y, 504; crj^stal violet and methylene blue, 476; eosin, 467; eosin Y and methylene blue, 353; fast green FCF, 497; fast green FCF and hematoxylin, 366; fast yellow, light green and methylene blue, 476; fluores- cein and methyl blue, 496; gentian vio- let, 474, 489, 494; hematoxylin, 494, 495, 497; hematoxylin and gentian violet, 505; hematoxylin and iodine green, 497; hematoxylin and light green, 429, 495, 496; hematoxjdin and orange G, 496 and 497; indigocarmine, 369, 497; indigo- carmine and picric acid, 361, 372, 436, 467, 506; iodine green, 457; light green and orange G, 436; malachite green, 485, 486, 496; methyl blue, 477; methyl violet, 463, 483; methyl violet and methylene blue, 389; methyl violet 6B, 475; methylene blue, 410, 417, 428, 462, 463, 465, 466, 467, 477, 478, 480, 485, 486, 488, 490, 492, 496, 497; methyl- ene blue and orange G, 351; methjdene blue and safranin, 466; methylene blue and thionin, 463; nigrosin, 485; Nile blue sulfate, 512; orange G, 428; orange G and light green, 339; picric acid, 478; picric acid and thionin, 478; safranin, 512; Sudan III, 448; thionin, 463; toluidine blue, 495 Magenta see also Ziehl's stain Magenta leucobase, for staining, nucleis, 316 pituitarj', 427 staining combination with, celestin blue B, hematoxylin, and orange G, 427; hematoxylin, 452; hematoxylin and orange G, 424; light green, 437, 504 jNIagenta III, as stain, 316 for staining leprosy bacilli in sections (Putt), 497 staining combinations with, acid fuchsin, hematoxylin and picric acid, 498 methylene blue, 497 Magnesia-carmine, 307 Magnesium acetate, as ingredient of Schiller's fixative, 218 Magnesium oxide, as ingredient of, Jensen's picro-carmine, 303 Kramer and Shipley's decalcifying fluids, 260 Magnesium picrate, as ingredient of Mayer's picro-carmine, 303 Magnesium sulfate, as narcotic, 265 as ingredient of Jores's preservatives, 179 Magnesium sulfate — (continued) for narcotizing, coelenterates, 53; coral 85; leeches, 53 Magnini, see Grandis, 383 Mahdissan's fixative, 192 in Mahdissan's method for microflora in scale insects, 504 Mahogany, 93 manufacture of slides from, 10 Mahrenthal's alcoholic carmine, 300 Malachite green, for staining, acid-fast bacteria, (Burke, Dickson, and Phillips), 476; acid-fast bacteria in sec- tions, (Bertrand and Medakovitch), 496; bacteria in leukocytes, (Bruner and Edwards), 490; bacterial capsules, (Gut- stein), 488; bacterial spores, 485-487 diphtheria bacilli, (Laybourne), 489 diphtheria bacilli, (Stottenberg), 490 fungus in plant tissue, (Vaughn), 502 plankton, (Fiancotte), 513 in, Dupres's triple stain, 359 Pianese's triple contrast, 341 staining combination with, acid fuchsin and Martins yellow, 341, 502; acid fuchsin and orange G, 359; Bismark brown, 513; eosin Y and hema- toxylin, 366; hematoxylin and toluidine blue, 490; magenta, 485, 486, 496; safranin, 485, 487, 490; toluidine blue, 489 Malarial parasite, see Plasmodium Malassez's picro-carmine, 303 Male's, double stain, 329 method for Gram-positive bacteria in sec- tions, 494 Mall's, method for reticulum fibers, 424 preservative, 180 in Dawson's method for bone, 383 Mallory's, methods for, actinomycetes, 504, 505; bacteria in sec- tions, 493; diphtheria bacilH, 489; En- taineba, 508; fibrin, 425; fibroglia fibrils, 456; Gram-positive bacteria in sections, 495; hemafuchsin granules, 456; hemo- siderin granules, 456; hyalin, 456; mucin, 454; nervous tissues, 406; neuro- glea, 414; tubercle bacilli in sections, 497 stains, acid fuchsin-anilin blue-orange G, 360 for, Amphioxus, 357-359 mouse, 141 in, Crook and Russel's method for pituitary, 425; Bretschneider's method for insect brains, 410; Jakob's method for neuroglia, 413; Torsgren's method for bile duct, 423 INDEX 745 Mallory's — {continued) stains- -{conlinued) alura-hematoxylin, 288 in, Haythorne's method for Gram- positive bacteria in sections, 49-4 Mallory's method for hyalin, 456 Mallory's method for mucin, 454 Thompson's method for erythro- cytes, 420 azur Il-methylene blue-phloxine, 471- 472 iron-mordant hematoxylin, 282 phosphomolybdic-hematoxylin, 292 in, Alzheimer's method for neuroglia, 412; Eisath's method for neuroglia, 413; Fiandt's method for gliosomes, 415; Mallory's method for neuroglia, 414; Peers's method for neuroglia, 414; Williamson and Pearse's method for thyroid, 430 polychrome methylene blue-eosin Y, 34G Malpighi's layer, mentioned in Dupres's double contrast, 339 Maltose, as ingredient of, Catcheside's fixa- tive, 200 Taylor's fixative, 201 Mammals, brains of, 553 Manchot's method for elastic fibers, 388 Manesse's preservative, 180 Maneval's, methods for, bacterial flagella, 483; bacterial smears, 473; nuclei, 436; nuclei in yeasts, 512 stains, acid fuchsin, 321; anilin blue, 321; magenta, 316, (in Maneval's method for nuclei, 436) Manfredi's method for nerve endings, 535 Manganese nitrate, as ingredient of Rojas's formaldehyde accelerator, 614 Mangin's method for Peronosporales, 502 " Mann-Kopsch " techniques, 530-531 Mann's, fixatives, mercuric-chromic, 215 mercuric-picric, 213 mercuric-picric-formaldehyde, 214 osmic acid, 193, 248 osmic-mercuric, 196, 248 in, Gatenby's method for Golgi bodies, 530; Ludford's method for Golgi bodies, 531; Weigl's method for Golgi bodies, 531 recommended use, 95 stains, acid-alum hematoxylin, 290 erythrosin-orange G-toluidine blue, 352 methyl blue-eosin, 371 in, Alzheimer's method for neuroglia, 412; Dobell's technique, 369; Mann's — (continued) stains — (conliniu-d) methyl blue-eosin — {conlinucd) in — (continued) Koinikow's method for Schwann cells, 415; Manouelian's method for Negri bodies, 466 toluidine blue-erythrosin, 371 Mann on silver-dichromate staining, 605 Manna, see LaManna Manou61ian's method for, Negri bodies, 466 spirochetes, 562 Marble, for neutralizing formaldehyde, 190 Marchi's fixative, 203, 248 in, Kaiser's method for myelin sheaths, 405; Koinikow's method for Schwann cells, 415; Marchi's method for degen- erative changes, 528 "Marchi methods," 528-529 Marchoux's fixative, 230, 248 Marchoux and Simond's fixatives, osmic- platinic-chromic-acetic, 196, 248 osmic-platinic-mercuric-chromic-acetic, 195, 248 Maresch's, silver diammine stain, 576, 579 in, Maresch's method for reticulum, 593 Ogata-Ogata's method for chromaffin cells, 596 triple stain, 361 Margolena's, double stains, 340 methods for, fungus in plant tissues, 499, 502 pollen, 393 Marie and Raleigh's double stain, 346 Marina's fixative, 230, 248 Marine deposits, separating Foraminifera from, 17 Marine glue, attaching cells with, 22 Beale's, 655 general remarks on, 651 Marjoram oil, physical properties of, 624 Mark, see Long, 217 Markey, Culbertson and Giordano's method for fecal smears, 508 Marpmann's label adhesive, 661 Marquez's mordant for mitochondria, 518 Marrassini's fixative, 216, 248 Marrow, see Bone marrow Marsh's, decalcifying fluid, 259 gelatin cement, 655 Marshall's mountant, 632 Marshall, see also Archibald, 636; Chalmers, 503 Martin's, adhesives, 661 cement, 656 dead black varnish, 656 label adhesive, 661 method for pituitary, 426 mountant, 632 746 INDEX Martin's — (continued) use of paper slide covers, 7 Martin, see also Field, 048 Martindale's glycerol jelly, 635 Martinez's silver-tungstic stain, 603 in Martinez's method for neuroglia, 607 Martinotti's, methods for, cell inclusions, 44-4; elastic fibers, 607; fat, 449 stains, toluidine blue, 318, (in Berberian's method for fungus in skin scrapings, 503); alum-hematoxylin, 288 Martius yellow, for staining, fungus in plant tissue, (Vaughn), 502 pollen tubes, (Nebel), 422 in, Male's double contrast, 328; Millot's double contrast, 341; Pianese's triple contrast, 341 staining combinations with, acid fuchsin, 329, 341; acid fuchsin and malachite green, 341, 502; resorcin blue, 422 Masson's, acid alcohol for cleaning slides, 666 adhesive for paraffin ribbons, 658 differentiator, 521 after Regaud's hematoxylin, 332 for pollen grains, 310 in Johansen's technique, 314 fixatives, 226, 248 in, Paquin and Goddard's technique, 366 Regaud's technique, 282 macerating fluid, 263 methods for, argentaffin cells, 595, 596; attaching free sections, 660; mucin, 454 stains, acid-alum hematoxylin, 290, (for mouse head, 336), (in Patay's technique, 340); acid fuchsin-anilin blue, 337, (for sec- tion of earthworm, 332); acid fuchsin- metanil yellow, 338; acid fuchsin-pon- ceau 2R-anilin blue, 338; erythrosin- orange G-toluidineblue, 371; erythrosin- saffron, 341; hematoxylin-acid fuchsin- soluble blue, 360; iron-mordant hema- toxylin, 282; metanil yellow-picro-fuch- sin, 340; picro-indigo carmine, 326; ponceau 2R-anilin blue, 339; saffron- erythrosin, 329; silver diammine, 576, 579, (in Masson's method for argentaffin cells, 596) ; thionin-picric acid, 353 Mast cells, granules in, 455 Matsura's polychrome neutral red, 371 Maurer and Lewis's method for pituitary, 426 Maximow's, double stain, 349 iu EUermancr's method for blood, 417 Maximow's — (continued) fixatives, chromic-dichromate-acetic, 231 mercuric-dichromate formaldehyde, 218 in EUermaner's method for blood, 417 osmic-dichromate-formaldehj'de, 204, 248 in Liebmann's method for inverte- brate, 418 recommended use, 95 Maxwell's, clearing mixture, 628 embedding wax, 646 in Maxwell's method for pituitary, 427 solvent, 628 May's method for bacterial spores, 486 May-Griinwald's double stain, 344, 345 in, Assmann's technique, 344; Baillif and Kimbrough's method for blood, 416; Ginrich's method for Plasmodium, 507; Kardos's method for blood, 418; Pap- penheim's technique, 349, 350; Pappen- heim's method for blood, 419; Slider and Downey's technique, 350; Strumia's method for blood, 420; Winkler's method for microglia and plasma cells, 589 Mayer's, adhesive, 658 as ingredient of, Krajian's developer, 618 Riiyter's adhesive for nitrocellulose paraffin ribbons, 658 for, attaching sections to slide, 116; at- taching squashes, 77; mounting dou- ble embedded sections, 156; paraffin ribbons, 658 in, Bacsich's method for nervous tissue, 566; Bohm and Davidoff's method, 657; Boni's method for bacterial cap- sules, 487; Gray's technique, 67; Langeron's method for nitrocellulose sections, 659 use with smears, 72 bleaching solution, 262 fixatives, picric-formaldehyde, 223; picric-hydro- chloric, 223; picric-nitric, 222; picric- sulfuric, 222, (recommended use, 95) injection fluids, 663 methods for, amyloid, 452 mucin, 454 stains, alcoholic-carmines, 301 alcoholic-cochineal, 301 alum-carmine, 300, 301 for staining liver fluke, 295 in. Baker's method for Golgi bodies, 442; Bethe's method for brain, 397; Langham's method for amyloid, 452 INDEX 747 Mayer's — (continued) stains — (continued) alum-hematoxylin, 288 as ingredient of Langcron's hema- toxylin, 289 in, Lendrum's method for granules in breast and kidney, 456; Pearse's method for pitnitary, 427; Petrag- nini's method for Negri bodies, 46G; Spark's method for pituitary, 427; Weiss's techni(nie, '.VM't ahuuinum-carmine, 251, ^07 ahuninum-hematoxylin, 454 in, IMasson's method for mucin, 254 Zimmerman's method for gastric gland cells, 432 aluminum-strontium-carmine, 301 for, echinoderm larva, 156; invertebrate larvae, 54; wholemounts, 61 calcium-hematoxylin, 292 hydrochloric-carmine, 306 magnesia-carmine, 307 methyl green-acid fuchsin-orange G, 357 magnesia-carmine, 307 as ingredient of Meyer's picro-car- mine, 303 picro-carmine, 303 Meakin's, adhesive for minute objects, 661 mechanical hair, 40 Medakovitch, see Bertrand, 496 Medalia, Kahaner, and Singer's method for Plasmodium, 509 Medusae, clearing, 298 collecting, 296 dehydration, 297 fixing, 296 getting into balsam, 298 wholemount of, 296-299 Meeker and Cook's fixative, 218, 248 Megakaryocytes, 431, 432 Meiosis, fixatives for, 223 Meissner bodies, 556 Melanin, protein silver method for, 568 Melnikow-Raswedenkow's preservatives, 180 Mendeleef's cement, 656 Menner's method for ganglia in whole- mounts, 410 Menthol, as ingredient of Gray's narcotic, 265 for narcotizing, Bryozoa, 54, 60; coelenterates, 53; liver flukes, 294; protozoa, 52 Merbel's double stain, 372 Merck Index, 3 Mercuric acetate, in Gough and Fulton's mordant, 518 Merctiric chloride, as fixative, 207 as ingredient of, Boitard's preservatives, 176; Cole's pre- Mercuric chloride — (continued) as ingredient of — (continued) servatives, 179; crystal violet, 413; fixa- tives, see fixative combinations (be- low); Foot's toning solution, 620; Frey's preservatives, 179; Gibson's preserva- tives, 179; Goadby's preservatives, 176; Hayem's eosin, 418; hematoxylin, 413, 414; McNamara, Murphy and Gore's decalcifying fluids, 259; Nastikow's gen- tian violet, 319; Ordonez's preserva- tives, 180; Pacini's preservatives, 180; Ralston and .Well's decalcifying fluids, 259 as mordant for, Victoria blue, 412, 414 basal fixative solution of, 237 effect of pH on fixation by, 188 fixative combinations with, acetic acid, 208-210; acetic and nitric acids, 210; acetic and trichloroacetic acids, 210; acetone, 212, 213; chromic acid, 215; chromic acid and dichromates, 215, 216; copper salts, 213; copper salts and dichromates, 213; dichromates, 216, 217, 218; dichromates and uranium salts, 219; dichromates, uranium, and magnesium salts, 218; formaldehyde, 211; formaldehyde and acetic acid, 211- 212; formaldehyde, acetone and acetic acid, 212; formaldehyde and trichloro- acetic acid, 212; formic acid, 210; hydro- chloric acid, 211; iodine, 219; nitric acid, 210; osmic acid, 196, 197; osmic and chromic acids, 197; osmic acid and cop- per salts, 197; osmic acid and dichro- mates, 197, 198; osmic acid, dichromates, and uranium salts, 198; osmic acid and iodine, 198; osmic and picric acids, 197; osmic acid and platinic chloride, 195; os- mic acid, platinic chloride and chromic acid, 195; osmic acid and uranium salts, 205; picric acid, 213, 214, 215; picric and chromic acids, 215; platinic chloride, 206; platinic chloride, picric and chromic acids, 206; trichloroacetic acid, 210, 212 in, David's mordant, 517; Gordon's mor- dant, 518; Gray's mordant, 518 removal from tissues, 255 Mercuric chloride-glucose, as fixative for glycogen, 453 Mercuric iodide in, Gelarie's method for spirochetes, 479 White and Culbertson's method for Gram- positive bacteria, 475 Mercuric oxide, as ingredient of, Bullard's alum-hematoxylin, 287 Harris's alum-hematoxylin, 287 Mercurochrome, in, Blank's triple stain, 352 Craigie's method for Paschen bodies, 464 748 INDEX Mercurochrome — {continued) staining combinations with, azur and eosin, 352 azur I and methylene blue, 464 Mercury-gold methods, see Gold-mercury methods, 536 Merieux's method for Gram-positive bac- teria, 475 Meriwether's, ammonia-carmine, 305 method for parasitic amebas, 509 Merkel's, fixative, 207, 248 method for mucin, 454 mordant, 516 in Behren's method for bile capillaries, 422 Merland's, fixative, 236 formaldehyde accelerator, 613 in, Meriwether's method for i^arasitic amebas, 509 Merland's method for astrocytes, 558 Merton's fixative, 197 Merzbacher's method for neuroglia, 414 Mescon, sec Kligman, 504 Mestrezat, see Grynfelt, 262 Metal staining, general observations, 524 Metal staining techniques, see under name of author, or ingredient or application Metal stains, method of classification, 524 Metanil yellow, for staining, mucin, (Masson), 454 Rickettsiae, (Nyka), 463 in, Lillie's double contrast, 338 Masson's triple contrast, 340 staining combinations with, acid fuchsin, 338; hematoxylin, 454; picric acid and acid fuchsin, 340 Metaphosporic acid, as ingredient of Cohen's fixative, 193 Methanol, as ingredient of, Bodecker's decalcifying fluids, 257 ; nar- cotics, 265, 266; Schiefferdecker's macer- ating fluids, 264; Schwarz's carmine, 302 as, narcotic, 52 solvent for nitrocellulose, 648 physical properties of, 623 Methenamine, as ingredient of fixatives, 235 in Gomori's silver diammine stain, 578 Methoxytriglycol acetate, 627 Methyl alcohol, see Methanol Methyl amj^l carbinol, 625 Methyl benzoate, as ingredient of, Riiyter's adhesive for nitrocellulose-paraffin ribbons, 658 as solvent for, nitrocellulose, 153, 648 physical properties of, 625 Methyl blue, as plasma stain, 320 for staining, acid-fast bacteria, (Gibbe), 477; acid- fast bacteria in sections, (Czaplewski), 496; blood, (Kardos), 418; chromaffin Methyl blue — {continued) for staining — {continued) granules, (Gomori), 430; cocci in cell smears, (Kahlden and Laurent), 491; cocci in cell smears, (Schwitz), 491; erythrocytes, (Crossmon), 416; fungi in wood, (Cornwall), 501; mitochondria, (Cain), 442; Negri bodies, (Barreto), 464; pituitary, (Colin), 425; pituitary, (Martins), 426; pollen tubes, (Watkin), 422; Rickettsiae, (Bohner), 462, Rick- ettsiae, (Bond), 462; virus inclusion bodies, (Hamilton), 465 in, Dobell's triple stain, 309; Dubreuil's double contrast, 325; Kricheski's triple stain, 360; Ladewig's quadruple stain, 365; Lillie's double contrast, 326; Lillie's double stain, 370; Lillie's quadruple stain, 366; Lillie's triple stain, 370; Mallory's triple stain, 360; Mann's dou- ble stain, 371; Mollendorf's triple stain, 366; Plehn's double stain, 372; Semi- chon's triple contrast, 328 staining combination with, acid fuchsin, 425, 442; acid fuchsin and hematoxylin, 426; acid fuchsin, hema- toxylin, and orange G, 365 ; acid fuchsin and orange G, 360, 413, 423; azocarmine and tartrazine, 430; azur, eosin, methyl- ene blue and orange G, 418; Biebrich scarlet, 370; Biebrich scarlet and hema- toxylin, 366; chroraotrope 2R, 416; eosin, 371, 372, 462, 466, 491; eosin and hematoxylin, 366, 464; eosin Y and orange G, 369; eosin Y and Victoria yellow, 329; ethyl eosin and eosin Y, 465; fluorescein and magenta, 496; magenta, 477; phloxine and orange G, 370; picric acid, 325, 326; picric acid and safranin, 501; safranin, 491 Methyl carbitol, 627 Methyl cellosolve acetate, 627 Methyl cellosolve, see Ethylene glycol mono- ethyl ether Methyl green, as, nuclear stain, 353-357; plasma stain, 320 for staining, amphibian blood, (Liebmann), 418; bac- teria in leukocytes, (Flinn), 491; bac- teria in sections, (Saathof), 493; bac- terial capsules in sections, (Smith), 498; blood, (Kahlden and Laurent), 418; blood, (Rossi), 419; bone and cartilage, (Juge), 383; diphtheria bacilli, (Albert), 489; fungus in skin sections, (Unna), 505; gonococcus, (Walton), 491; nuclei in ciliates, (Balbiani), 434; nuclei, (Cooper), 435, mitochondria, (Cowdry), 442; pituitary, (Severinghaus), 427; INDEX 749 Methyl green — {continued) for staining — {continued) proplastids, mitochondria and starch, (Milovidov), 450; spirochetes, (Keil), 479; spirochetes, (Lipp), 480 in, Auerbach's double stain, 356; Biondi's triple stain, 356; Bohm and Oppel's triple stain, 368; Dupres's triple stain, 359; Ehrlich's triple stain, 356; Foley's double stain, 356; Foley's triple stain, 356; Grosso's double contrast, 326; Grosso's triple stain, 355; Guinard's double stain, 356; Krause's triple stains, 356; 357; Maresch's triple stain, 361; Mayer's triple stain, 357; Opi)eirs triple stain, 357; Roux's double contrast, 327; Squire's double stain, 357; Stropeni's double stain, 357; Thome's double stain, 357; various double stains with pyronin, 355 staining combinations with, acid fuchsin, 356, 357, 442; acid fuchsin and aurantia, 450; acid fuchsin and erythrosin, 435; acid fuchsin and orange G, 356, 357, 359, 418; acid fuchsin and picric acid, 357, 361; acid violet and magenta, 427; acridine red, 357; alizarin red S, 383; Bismark brown and dahlia violet, 368; dahlia violet, 227; eosin W, 418; eosin Y and hematoxylin, 498; orange G, 339; picric acid, 326; pyronin, 355, 356, 491, 493, 505; pyronin and orange G, 355; pyronin and Victoria blue, 480, 479; toluidine blue, 489 Methyl methacrylate, mountant, comments on, 639 tool for spreading smears, 71 Methyl orange, for staining, nervous tis- sues, (Prince), 411; Nissl granules, (Bean), 445 in, Lopez triple stain, 371 staining combinations with anilin blue, erythrosin, and magenta, 411 anilin blue and magenta, 371 Methyl salicylate, as, mountant, 380; solvent for nitrocellulose, 648 in Hetherington's embedding method for nematodes, 646 physical properties of, 624, 626 Methyl violet, for staining, actinomycetes, (Morel and Dulaus), 505; amyloid, (Schmorl), 453; bacterial flagella, (Loffler), 483; bacterial flagella, (Muir), 483; bacterial spores, (Ruiz), 486; basal bodies, (Wallace), 458; bile capillaries, (Behren), 422; chief cell granules, (Hamperl), 455; diphtheria bacilli, (Rhyn), 490; fibrin, (Weigert), 425; gastric cells, (Hoeckeand Sebruyn), Mctliyl violet, for staining — {continued) 431; Gram-positive bacteria in sections (Male), 494; intracellular "organisms," (Gutstein), 461; neuroglia, (Rubasckin), 414; plasmodesma, (Meyer), 421; plas- modesma, (Strasburger), 422; Rickett- siae, (Nyka), 463; spirochetes, (Harris), 479 m. Bonney's triple stain, 368; Henneguy's triple stain, 364; Johansen's 'quadruple stain, 364 staining combinations with, acid fuchsin, carmine and picric acid, 389; anilin blue, Bismark ])rovvn, hema- toxylin and saffron, 431; crystal violet and indigo carmine, 483; eosin Y, 486; eosin Y and hematoxylin, 548; fast green FCF, orange G and safranin, 364; hematoxylin and Victoria blue, 505; light green and neutral red, 494; ma- genta, 483; magenta and methylene blue, 389; orange G and pyronin, 368; orange G and safranin, 364 Methyl violet 2B, for staining, nuclei, (Johansen), 436 plant tissues, (Johansen), 421 in Groot's quadruple stain, 349 staining combinations with, eosin Y, methylene blue and thionin, 349; erythrosin, 436; fast green FCF and safranin, 421 Methyl violet 6B, for staining, epithelial fibers, (Kromeyer), 424; Gram- positive bacteria, (Kopeloff and Cohen), 475; Saccharomyces in sections, (Curtis), 503 staining combination w^ith, magenta, 475 methyl violet and carmine, 503 Methylal, in Dufrenoy's wax embedding method, 646 Methylene blue, differentiators for, 520-521 for staining, acid-fast bacteria, 476-478; algae, (Baumgartel), 511; bacteria in leuko- cytes, (McCulIough and Dick), 491; bacteria in milk, (Broadhurst and Paley), 490; bacteria in milk, (Erb), 491; bacteria in milk, (Shult), 491; bacteria in sections, (Mallory), 493; bacterial capsules, (Klett), 488; bacterial cap- sules, (Muir), 488; bacterial spores, 485- . 487; blood, (Dekhuyzen), 417; bone and cartilage, (Bechtol), 382; desmid sheaths, (Taylor), 513; diphtheria ba- cilli, (Kinyoun), 489; diphtheria bacilli, (Xeisser), 490; leprosy bacilli in sections, (Putt), 497; mitochondria, (Ilollande), 443; Negri bodies, 465-467; nervous tissues, 401, 402, 403, 410; neutrophiles, 750 INDEX Methylene blue — (continued) for staining — (continued) (Freifeld), 417; Nissl granules, 445-447; nuclei in yeasts, (Gutstein), 512; pan- creas, (Baley), 428; Paschen bodies, (Craigie), 464; Plasmodium, 506-510; polar bodies in bacteria, (Weiss), 491; Rickettsiae, (Clancy and Wolfe), 462; Rickettsiae, (Zinsser and Bay ne- Jones), 464; Rickettsiae, (Zusser, Fitzpatrick and Hsi), 463; spirochetes, (Gelarie), 479; spirochetes, (Saboraud), 480; spiro- chetes in sections, (Nickiforoff), 498 in, Arnold's triple stain, 351; Bauer and Leriche's triple stain, 352; Bohm and Oppel's triple stain, 347; Dupres's triple stain, 359; Gausen's triple stain, 351; Groot's quadruple stain, 349; Houcke's double stain, 353; Houcke's quadruple stain, 352; Kingsley's quadi'uple stain, 348, 349; phenol solution, 321; Rhamy's triple stain, 353 ; ^Unna'sj^double stain, 353; various double stains with eosin, 344-345 polychroming with, silver hydroxide, 346; sodium carbonate, 346; ultraviolet rays, 346 staining combination with, acid blue and trypan blue, 394, acid fuchsin, 492; acid fuchsin, thionin, and toluidine blue, 352; aurin and magenta, 477; azur and eosin, 506; azur, eosin, methyl blue, and orange G, 418; azur A, eosin Y, and methylene violet, 348, 349; azur A and toluidine blue, 489; azur B and eosin, 508; azur I, crystal violet and eosin, 461; azur I and mercurochrome, 464; azur II, 509; azur II and eosin Y, 349, 350; azur II and phloxine, 493; Biebrich scarlet, 382; Bismark brown, 490; brilliant cresyl blue and eosin Y, 352; chrysoidin and crystal violet, 489; crystal violet, 479; crystal violet and magenta, 476; eosin, 394, 445, 486; eosin B and hematoxylin, 466; eosin B and thionin, 347; eosin Y, 344, 345, 347, 466, 487; eosin Y and magenta, 353; ethyl eosin, 345, 465, 467, 509; fast yellow, light green, and magenta, 476; fluorescein, 492; gentian violet, 476; magdala red, 388; magenta, 410, 417, 428, 462, 463, 465, 466, 467, 477, 478, 480, 485, 486, 488, 490, 492, 497; ma- genta III, 497; magenta and methyl violet, 389; magenta and safranin, 466; magenta thionin, 463; methyl orange, 445; methyl violet 2B, eosin Y and thionin, 349; orange G and acid fuchsin, 359; orange G and magenta, 351 ; orange Methylene blue — (continued) staining combination with — (continued) G and safranin, 351; orcein, 353; phlox- ine, 465, 506 ; picric acid, 418 ; propaeolin, 498; pyronin, 417; rhodamine B, 353; ruthenium red, 393; safranin, 462, 464, 485, 490, 491, 512; tropeolin, 493 staining solutions, Cobin's, 317; Goodpasture's, 317; Jadas- sohn's, 317; Kuhne's, 317; Langeron's, 317; Loffler's, 317; Manson's, 317: Man- well's, 318; Michaelis's, 318; Moschkow- sky's, 318; Muller and Chermock's, 318; Proescher and Drueger's, 318; Roques and Jude's, 318; Sahli's, 318; Stevenel's, 318; Terry's, 318; Unna's, 319 Methylene blue, see also Polychrome methyl- ene blue, Loffler, etc. Methylene blue-eosin stains, mounting in petrolatum, 34 Methylene violet, in, Kingsley's quadruple stains, 348, 349 MacNeal's triple stain, 349 stain combinations with, azur A, eosin Y and methylene blue, 348, 349; azur I and eosin Y, 349; azur II, 319 Mettler's fixatives, dichromate-formalde- hyde, 233, 248 osmic-dichromate, 203, 248 osmic method for degenerative changes. 528 Metzner's fixative, 203 Meunier and Vaney's method for plankton, 513 Meves's fixatives, osmic-chromic-acetic, 201, 248 osmic-platinic-acetic, 195 Meves's and Duesberg's fixative, 201, 248 Meyer's, fixative, 211 method for, neuroglia, 414 plasmodesma, 421 Meyer, see also Glees, 578 Michaelis's fixative, 214, 248 methylene blue, 318 methylene blue-eosin, 345 Michailow's molybdenum mordant, 518 Michelson's double stain, 346 Microcystis, fluid wholemount of, 27-29 Microglia, Bolsi's method for, 558 in. association with plasma cells, 589 rabbit brain, 571-573 silver diammine methods for, 585-590 Microglia, see also Neuroglia Micron, definition, 89 Microscope mounts, types of, 7-9 Microscope slides, origin of, 7 Microsporocytes, preparation of squash of, 77 " Microstomum, 53 INDEX 751 Microtome, definition, 89 faults in paraffin sections due to, 121 for, cutting wood, 93; freehand, 89ff, 9 Iff; frozen sections, 157ff, 158ft', 159ff; nitrocellulose sections, 147ff; paraffin sections, 103, 108ff Richards on, 89 sliding, 147ff cutting introccUulose sections on, 146- 148 cutting paraffin sections on, 140 Microtome knives, 108-1 i 1 cutting action of, 109, llOff effect of nicks in, 122 effects of fault}' sharpening, 120, 121 removal of nicks from. 111 sharpening, 109, 110, 11 Iff stropping, 11 Iff effect of faulty, 120 types of. 109, 11 Off varnish to conceal nicks in, 667 Mikele, see Randolf, 478 Milk, as injection medium, 163 stains for bacteria in, 490, 491, 492 Millboard, preparation of cells from, 12 Miller, method for, cartilage in wholemounts, 387 nerve endings, 536 nerve trunk of oligochaetes, 556 on "'Weigert-Pal" techniques, 404 Miller, see also Tompkins, 510 Milligan's triple stain, 360 Millot's double contrast stains, 341 Milovidov's, fixative, 444 method for, differentiating bacteria from mitochondria, 444 proplastids, mitochondria and starch, 450 Minchin's double contrast, 326 Mingazzini's fixative, 209, 248 Minot microtome, 103 Minot's solvent, 628 in Hoj'er's method for mucin, 453 Minouchi's solvent for cytoplasmic inclu- sions, 516 Minute objects, adhesives for, 661 Miracidia, 67 Miskolczy's method for axis cylinders, 584 Mislawsky's fixative, 204, 248 Mite, wholemount of, 43-45 Mitochondria, differentiation from, bacteria, 444; starch, 450 dye-staining methods for, 438-444 Chura's fixative for, 227 Gough and Fulton's mordant for, 518 Marquez's mordant for, 518 osmic method for, 531 Schridder's fixative for, 205 silver diammine methods for, 590-591 Mitosis in onion root, 433-434 Mitosis, see Nuclei, special methods for Mitotic figures, mentioned in, llollande's triph> stain, :}39 Johansen's quadruple stain, 364 Langeron's double stain, 353 Shumway's triple stain, 372 Mitrophanov's mordant, 517 in mitrophanov's for myelin sheaths, 406 Mitter and Bartha's method for nuclei, 437 Mixtures for clearing, 627-629 Mobius's fixative, 201 Mohr and Wehrle's, sandarac mountant, 638 varnish, 654 Moleschott's preservative, 177 Moleshott and Borine's macerating fluid, 264 Mollendorf's triple stain, 366 Moller's, fixative, 233, 249 method for bacterial spores, 486 MoUier's quadruple stain, 366 MoUusca, radula of, 394, 513 Molybdenum-hematoxylin, 290, 507 Monckeberg and Bethe's bleaching solution, 262 Monk's pectin mountant, 636 Monnig's preservative, 177 Monochloroacetic acid, as ingredient of Foley's fixative, 225 Monocystis, smear preparation of, 72-73 Monosodium phosphate in Langeron's Giemsa technique, 348 Moore's, injection mass, 665 use of, 170 method for, amphibian blood, 418 fungus in plant tissues, 502 preservative, 177 Moorthy 's method for larvae of Dracunculus, 509 Morax, see Nicolle, 483 Mordant-hematoxylin stains. 280-284 Mordants for, hematoxylin, 515-517 Moreau's, adhesive for paraffin ribbons, 658 fixative, 225 glycerol jelly, 635 IMoreland Bassal's, iron-copper-hematoxylin, 286 iron-copper mordant, 516 as ingredient of Liengme's hematoxvlin, 291 Morel and Doleris's triple stain, 357 Morel and Dulaus's method for actinomy- cetes, 505 Morel, see also Anglade, 412 Moritz's method for attaching gelatin sec- tions, 660 Morpugo's method for bone, 384 Morris's method for fungi in tissue scrapings, 505 Morris, see also Thomas, 187 752 INDEX Morrison's, mountant, 632 narcotic, 265 tannic mordant, 516 Moschkovsky's methylene blue, 318 in Moschkovsky's method for blood para- sites, 509 Moskowitz, method for protozoa, 568 on preparing protein silver, 564 Mosquito, cephalic ganglia, 568 Moss, collecting animals from, 45 dry wholemounts of spore cases, 16 Motor end plates, 553, 554 Mottier's fixative, 201, 249 Moulton, see Geschickter, 348 Mountants, alcohol miscible, 637-639 beta-pinene, 64 Canada balsam, 639 commarone, 640, 641 decimal divisions of, 630 definition, 42 distinction from preservatives, 175 function of glycerol in, 42 general remarks on, 630 gum elemi, 640 gum mastic, 640 gum mastic media, 637 giun sandarac media, 638 phenol-sulfur, 638 polystyrene, 641 polyvinyl acetate, 641 polyvinyl alcohol, 636 synthetic resin, 640-641 Venice turpentine media, 637, 638 with dye, Bernhardt's cotton blue, 503; Doetsch- mann's magenta, 631 ; Semmens's aceto- carmine, 633; Semmens's Canada bal- sam, 639 ; Semmens's eosin balsams, 639 ; Swartz and Conant's anilin blue, 505; Zirkle's carmine-gelatin, 635; Zirkle's carmine pectin, 636, 637; Zirkle's iron- carmine, 633; Zirkle's orcein-gclatin, 636 Mounting nitrocellulose sections, 148, 149 Mouse, fixative for, 334 pituitary, 427 section of entire, 136-141 section of head, 334-336 Mozejko's injection fluid, 663 "M 2.2.2.." 395 Miiche's fixative, 229, 249 Mucicarmine, 454 Mucihaematin, 454 Mucin, special methods for, 453-454 Mucus, mentioned in Lillie's triple stain, 366 Muhlpfordt's method for spirochetes, 480 Muir's, glycerol jelly, 635 methods for, bacterial capsules, 488 bacterial flagella, 483 mordant, 488 Muir and Judah's cement, 656 Mukerji's preservative, 178 Mullen and McCarter's chrome mordants, 516, 518 recommended use, 141 MuUer and Chermock's magenta, 316 in, MuUer and Chermock's method for, acid-fast bacteria, 477 acid-fast bacteria in sections, 497 methylene blue, 318 Mliller's double stain, 345 Miiller's fixatives, as ingredient of, An- gelucci's fixative, 217; Robin's pre- servative, 181 basal fixative solution for, 237 dichromate, 232, 249 in, Anderson's method for myelin sheaths, 404; Aronson's method for nervous tissues, 409; Beckworth's method for nerves in teeth, 534; Berkeley's method for nervous tissue, 603; Beyer's method for astrocytes, 413; Chilesolti's method for axis cylinders, 410; Golgi's silver-dichromate method for nervous tissues, 604; Hamilton's method for degenerative changes, 528; Hamilton's osmic stain, 527; Kaiser's method for myelin sheaths, 405; Koinikow's method for Schwann cells, 415; Kultschitzky's method for reticulum fibers, 424; Marchi's method for degenerative changes, 528 ; Marti- notti's method for elastic fibers, 607; Morpugo's method for bone, 384; Nyka's method for Rickettsiae, 463; O'Leary's method for nervous tissue 411; Orr's method for degenerative changes, 528; Reich's method for granules in Schwann cells, 457 ; Unna's method for nuclei, 438; Wolter's method for myelin sheaths, 408 role of sulfate in, 187 dichromate-formaldehyde, 233, 249 in, Azoulay 's method for myelin sheaths, 529 recommended use, 95 Mliller's method for pancreas, 429 Mulligan, see Sinton, 510 Mulon's method for fat, 447 Murdock's bleaching solution, 262 for leeches, 54 Murphy, see McNamara, 259 Murray's fixative, 204, 249 iron-mordant hematoxylin, 282 Murray and Fielding's fixative, 192, 249 method for Leptospira icterohaemor- rhagica, 562 Musclienkoff's method for nerve endings, 541 Muscle, Daniell's method for, 394 Dietrich's method for, 394 INDEX 753 Muscle — (continued) fixatives for, 218 Heideuham's method for contraetioii bauds in, 423 Karlsou's method for, 394 maceration of, 263 mentioned in, Becher's polychrome quinalyarine, 3()8; Bohni and Oppel's ciiiadruple stain, 361 ; Buzaglo's triple stain, 368; Dclamare's triple stain, 365; Dupres's double con- trast, 339; Foley's double stain, 356; Gausen's triple stain, 351; Haythorne's triple contrast, 337; Heideuham's triple stain, 361; Holmes and French's triple stain, 352; Houcke's double stain, 353; Kalter's quadruple stain, 364; Korn- hauser's quadruple stains, 369, 370; Lillie's triple stains, 366, 370; Mallory's triple stain, 360; Alasson's double con- trasts, 337, 341; Patay's double con- trast, 339; Romeis's quintuple stain, 367; Shumway's triple stain, 372; Volk- man and Strauss's triple stain, 373; Williams's polychrome cresyl violet, 373 Miller's method for, 394 of, insects, 424 Sihler's method for nerves in, 407 Sahlgren's stain for, 423 teasing, 533 Trichinella in, 599 Muscles, in wholemounts of Crustacea, 394 Must and Rose's method for dentine, 386 Muzzarelli's method for bacterial spores, 486 Myelin sheaths, Anderson's method for, 404 Bolton's method for, 404 Fajerstajn's method for, 404 Feyrter's methods for, 403 Gardden's method for, 405 Hadjioloff's method for, 407 Kaiser's method for, 405 Kozowsky's method for, 407 Liber's method for, 406 Lillie's methods for, 406 Lison and Dagnell's method for, 410 Mitrophanov's method for, 406 Olivecrona's method for, 407 osmic methods for, 529 protein silver method for, 568 Schroder's method for, 407 Schultze's method for, 407 Spielmeyer's method for, 408 Tschernyschew's method for, 407 Vassale's method for, 408 Weigert's method for, 408 Weil's method for, 408 Wolter's method for, 408 Wright's method for, 409 Myofibrils, 597 Myrcia oil," 624 Myxosi)orids. 508 N Nal)ias's method for ganglia of inverteljratcs, 538 Nageotte's method for Schwann cells, 415 Nagle and Pfau's method for Negri bodies, 466 Nagu, see Urechia, 595 Nair, see Smith, 449 Nakamura's fixative, 202, 249 Nakamura, sec also Tanigudii, 467 Nakano's metliod for si)irochetes, 5()2 Nan, see Yao-Nan, 231 Naphazarine, 492 Naphrax, 641 Naphtha, for defatting bone, 82 Naphthopurpurin staining solutions, 313 Naphthol blue black in, Curtis's double con- trast, 325 Lillie's double contrast, 326 Lillie's quadruple stain, 370 Naphthol green in Volkman and Strauss's triple stain, 373 Naphthol green B in, Lillie's double stain, 370 Mollier's quintuple stain, 366 Narcotics, 265-266 Narcotization, effect of pH on, 31 Narcotizing, bryozoa, 54, 60 cestodes, 265 coelenterates, 53 coral, 85 Crustacea, 45, 49 earthworm, 330 Hydra, 78 leeches, 53 liver flukes, 294 medusae, 296 oligochaetes, 53 platyhelminthes, 53 protozoa, 52 rotifers, 30 specimens for wholemounts, 51-54 Nassanow's fixative, 202, 249 in Nassanow's method for Golgi bodies, 531 Nastikow's gentian violet, 319 Navashin's fixative, 230, 249 for, chromosome squashes, 77 lily bud, 149 in, Sax's method for pollen mother cells, 437 n-butyl alcohol, for dehydration, 96 physical properties of, 626 n-butyl phthalate, as clearing agent, 348 Nebel's, fixatives, osmic-mercuric-uranium- formic, 198 754 INDEX Nebel's, fixatives — (continued) osmic-platinic-chroniium-thorinin, 196 method for pollen tubes, 422 Needles, glass, for injection, 166 hypodermic, conversion to injection nee- dles, 165 Neelsen's method for acid-fast bacteria, 469, 477 Negri bodies, mentioned in Krajian's method for bacteria in sections, 494 in section of guinea-pig brain, 460-461 preparation of smears of, 75 special methods for, 463, 464-467 Negrin's method for reticulum fibers, 594 Neill, see Hood, 656 Neisser's polychrome methylene blue, 490 in, Cowdry's method for diphtheria, 489 Neisser's method for diphtheria bacilli, 490 Neisser and Hueppe's method for bacterial spores, 486 Nelis's fixative, 213 Nelson's method for nerves in wholcmounts, 406 Nematodes, collecting from, feces, 35 moss, 45 combined fixative and dehydrant for, 189 fixation of, 36 Hetherington's embedding method for, 646 impregnating with glycerol, 32 mastic mountant for, 637 wholemount in glycerol, 35-37 Nemec's fixatives, chromic-formaldehyde, 230, 249 picric-acetic-sulfuric, 222 in Nemec's method for plastids, 450 Neoarsphenamine, as ingredient of Tron's accelerator, 615 in Fontana's method for spirochetes, 608 Nereids, 53 Neri's method for Negri bodies, 466 Nerve cells, centrosomes in, 590 mitochondria in, 591 Nerve endings, arsenic-silver method for, 605 gold methods for, 534-536 gold-dichromate method for, 541 in, calcified structures, 556, 557; gland cells, 529; male sex organs, 606; tendons, 540; tongue, 553 methylene blue method for, 403 osmic method for, 529 peripheral, 552, 606, 615 protein silver method for, 568 silver diammine method for, 581, 584 Nerve fibrils, 554 Nerve net, in ciliates, 559 Nerves, differentiation of anesthetized from normal, 404 in, oligochaetes, 556 Nerves — (continued) in — (continued) teeth, 534-535, 610 mentioned in, Hubin's triple contrast, 340; Mallory's triple stain, 360; Masson's double con- trast, 337; Patay's triple stain, 339; Shumway's triple stain, 372 regenerating, 582 Nervous tissues, dye-stains for, 395-416 gold-arsenic method for, 540 gold-dichromate methods, 540, 541 gold-mercury methods for, 538-539 gold-osmic methods for, 540, 541 hematoxylin methods for, 403-409 miscellaneous dye stains for, 409-411 osmic methods for degenerative changes in, 528-529 osmic-silver-dichromate methods for, 603- 607 protein silver methods, 566-568 reticulum fibers in, 595 silver nitrate methods for, 551-558 silver-dichromate methods for, 603-607 thiazin stains for, 401-403 Neubert's, double contrast, 326 method for smooth muscle, 424 Neukirch's method for glycogen, 453 Neuman's, double contrast, 303 method for nervous tissues, 407 Neurofibrils, Agduhr's method for, 581 Bethe's method for, 402 Cajal's method for, 552 Cowdry's method for, 554 Davenport's method for, 554 Donaggio's method for, 402 da Fano's method for, 583 in spinal cord, 555 Schultze and Stohr's method for, 557 Neurogenesis, 552, 553 Neuroglia, dye stains for, 411-415 gold-mercury methods for, 536-538, 539 in smears, 588 silver bromide-chloride-thiosulfate method for, 609 silver diammine methods for, 585-590 silver-dichromate methods, 606, 607 silver nitrate methods for, 558 silver-tungstic method, 607 Neuroglia, see also Macroglia, Astrocytes, etc. "Neutral" balsam, 639 "Neutral" dyes, 271 Neutral macerating fluids, 264 "Neutral mountants," 637, 638 Neutral red, as polychrome stain, 371 for staining, bacterial flagella, (Gemelli), 482; blood, (Lightwood, Hawksley and Bailey), 418; blood, (Sabin), 419; blood, (Simpson), INDEX 755 Neutral red — (continued) for staining — (continued) 420; (iram-positive bacteria in sections, (Male), 494; Xissl granules, (Bean), 445; Nissl granules, (Kirkman), 446; reticulocytes, (Fiessinger and T.aur\ 417 in Twort's double stain, 372 staining combinations with, brilliant cresyl blue, 417; eosin, 445; gentian violet and light green, 493; janus green, 418, 419, 420; light green, 372; light green and methyl violet, 494; methyl orange, 445 "Nevillites," as ingredient of Groot's moun- tant, 641 "Nevillite 5," as ingredient of Gray's wax embedding medium, 646 Newcomer's method for plant mitochondria, 531 New magenta, for staining acid-fast bacteria in sections (Fite), 496 staining combinations with, acid fuchsin, hematoxylin and picric acid, 496 fast green FCF and hematoxylin, 366 New methylene blue N, for staining mucin, (Highman), 453 Newmarch's preservative, 177 Newton's gentian violet, 319 Newton and Darlington's fixative, 201, 249 Nickiforow-Foa's fixative, 216 Nickiforow's aceto-carmine, 302 Nicolas's, fixative, 194, 249 gelatin embedding method, 644 Nicolau and Kopciowska's method for in- tracellular "organisms," 461 Nicolle and Morax's method for bacterial flagella, 483 NicoUe's, crystal violet, 475 in, Bidegaray's method for intestinal protozoans, 506; Merieux's method for Gram-positive bacteria, 475; method for Gram-positive bacteria, 475; Tribondeau and Dubreuil's method for diphtheria bacilli, 490 thionine, 318 in, Masson's technique, 353; Schmorl's method for cartilage and bone, 385; Spehl's method for acid-fast bacteria, 478 Nicotine, as ingredient of Cajal's alcoholic accelerator, 614 Niessing's fixatives, 195, 249 Nieuwenhuyse's gelatin mountant, 635 Nigrosin, for staining, bacterial spores, (Dorner), 485 keratin and eleidin, (Buzzi), 422 muscle, (Dahlgren), 423 Nigrosin, for staining — (continued) plant chromosomes, (van Rosen), 437 in Plitzer's double contrast, 326 staining combination with, magenta, 485 picric acid, 326, 369, 422, 423 Nikiforoff's, fixative, 216 in Nikiforoff's method for spirochetes in sections, 498 method for, nervous tissues, 410 Nile blue sulfate, as ingredient of Semmens's Canada balsam mountant, 639 as plasma stain, 321 for staining, ascospores in yeasts, (Kufferath), 512; bacteria in sections, (Foshay), 492; fat, (Smith and Mairj, 449: fat, (Smith), 449 in, Drew-Murray's triple stain, 369 AlcClean's simple contrast, 321 staining combinations with, acid fuchsin and picric acid, 369; ma- genta, 512; safranin, 492 Niobe oil, 625 Nissl's, method for, nerve cells and proc- esses, 410 nervous tissue, 403 Nissl granules, 446 stains, iron-mordant hematoxylin, 283 methylene blue, 446 in, Held's method for mitochondria, 443 Nissl's method for Nissl granules, 446 Nissl granules, 444-447, 467 Nitric acid, as ingredient of, Bolcek's fixative remover, 255; decalci- fying fluids see under author's name; fixatives, see under other ingredients; macerating fluids, see under author's name; Mayer's cochineal, 301; Robin's preservative, 187 as substitute for osmic acid, 193 for cleaning radiolaria, 19 Nitrobenzene, as deodorant for feces, 35 Nitrocellulose, as ingredient of, Apd,thy's cement, 661 Bodecker's decalcifying fluids, 257 Wilson's varnish for knife edges, 667 embedding retina in, 545 for moimting pollen grains, 309 general remarks on, 647 impregnating lily bud with, 150 in, decalcifying teclini()ues, 259 Obregia's method for attaching free sec- tions, 660 preparation of solutions of, 143 solubility in, dehydrating agents, 623; essential oils, 624; synthetic clearing agents, 625-626; "universal" solvents, 626 756 INDEX Nitrocellulose — {continued) storing solutions, 143 use on faulty paraffin ribbons, 122 Nitrocellulose blocks. Apathy's clearing mix- ture for, 628 attaching to wood, 661 casting, 145ff, 146, 150, 151 cutting under 70% alcohol, 151 Gage's clearing mixture for, 628 hardening, 145, 146, 151 mounting, 145, 150 storage fluid for, 667 technique of casting, 150 Nitrocellulose embedding, 143, 144ff, 145ff, 150 media, 647-649 removal of picric acid before, 255 Nitrocellulose sections, adhesives for, 659 dehydrating and clearing, 152 Dunham's clearing mixture for, 628 flattening, 148, 152 limitations of method, 142 method of cutting, 146, 147ff, 151 mounting on slide, 148 serializing on slide, 148 staining, 148, 151-152 Nitrocellulose sections, see also Double em- bedded sections de No's method for nerve endings in calcified structures, 556 Noback, see Kupperman, 191 Noble's method for fecal smears, 509 Nocht's stain, 345 Noguchi's developer, 618 in, Blair and Davies's method for nerves in heart, 551; Jahnel's method for spiro- chetes, 561; Noguchi's methods for spirochetes, 480, 562 Noguchi's formaldehyde accelerator, 562, 613 in Bolsi's method for neuroglia, 558 Nollister's method for bone in fish embryos, 384 Noniewicz's method for bacteria in sections, 493 Nordstedt's glycerol jelly, 635 Norris and Shakespeare's double stain, 372 Northen's method for plant sections, 393 Novak's, fixative, 215, 249 method for Herbst's corpuscles, 431 Novel's method for bacterial flagella, 598 Noyer's, cement, 656 use of, 33, 34ff, 36, 37 gum mastic mountant, 640 Nuclear stains, acid fuchsin, 319 alkanet, 308 brazalin, 308 carmine, 300-308 crystal violet, 319 Nuclear stains — {continued) general remarks on, 272 gentian violet, 319 hematoxylin, 481-493 magenta, 315-316 magenta leucobase, 316 oxazines, 313 safranin, 314-315 synthetic, 313-319 general remarks on, 308 thiazins, 316-319 Nuclei, in yeasts, 512 in yolky material, 319 protein silver method for, 568 remarks on staining, 433 special fixatives for, 223, 230 special methods for, 434-438 Nuclei, see also Mitosis Nucleoli mentioned in, Arnold's triple stain, 351; Houcke's double stain, 353 ; stains for, 434-438 Numerical designations of journals cited, 675-680 Nutrose, in Huntoon's method for bacterial capsules, 488 Nuttall's stain, 321 Nyka's methods for Rickettsiae, 463 O Oak, softening for sectioning, 92 Obersteiner's method for axis cylinders, 411 Objectives, water immersion, for controlling differentiation, 275 Obregia's method for attaching free sections, 660 Ochre, as ingredient of Mendeleef's cement, 656 Odontoblasts, 384 Ogata-Ogata's method for chromaffin cells, 596 Ogawa's method for nervous tissue, 538 Oguma and Kihara's fixative, 201 Ohlmacher's, double stain, 328 fixative, 209 Oil blue, as fat stain, 161 in Lillie's methods for fat, 448 Oil of bay, cloves, etc., see under Bay oil. Clove oil, etc. Oil of lilac, see Terpineol Oil red O, for staining fat, 449 Oils, classification of, 622 Oilstones, 111 Okada's developer, 619 in Okada's method for neurofibrils and pericellular nets, 556 Okajuna's method for erythrocytes, 418 O'Leary's method for nervous tissues, 411 Oleic acid, as ingredient of Zirkle's carmine balsam, 640 INDEX 757 Oligochaetes, collecting from moss, 45 narcotization and fixation, 53 nerve trunk of, 555 silver nitrate stain for, 564 Oligochaetes, see also Earthworm, etc. Oligodendria, in rabbit brain, 571-573 Oligodendria, special methods for, see under Neuroglia Olive oil, as ingredient of, Altman's injection fluid, 662 de Galantha's decalcifying fluid, 258 as injection medium, 163 Olivecrona's method for myelin sheaths, 407 Oliveira's, developer, 607 silver-dichromate stain, 603 in Oliveira's method for reticulum, 607 Oliver's mordant, 518 in Oliver's method for "flagella" on erythrocytes, 457 Ollett's method for bacteria in sections, 493 Olney on nitric acid as substitute for osmic acid, 193 "131" fixative, 218 Onion root tip, 433-434 Ono's method for spirochetes, 480 Oocysts of coccidia, 506 Opercularia, 53 Oppell's, method for lattice fiber in liver, 607 triple stain, 357 Oppler's picrocarmine, 303 in Oppler's method for eleidin, 457 Optical dead black, as background for dry wholemounts, 13 cementing objects to, 14-15 Orange G, as plasma stain, 320 for staining, acid-fast bacteria in sections (Hay- thorne), 497; astrocytes (Beyer), 413; bacteria in plant tissues (Stoughton), 498; blood (Kahlden and Laurent), 418; blood (Kardos), 418; Chlorophyceae (Yamanouchi), 513; embryonic bone (von Korff), 384; fungus in plant tissues (Margolena), 502; fungus in skin scrap- ings (Bachman), 502; glycogen (Vastari- Cresi), 453; intracellular "organisms," (Laidlaw), 461; kidney (de Galantha), 423; leprosy bacilli in sections (Camp- bell), 496; nervous tissue (Foley), 567; neuroglia (Bailey), 412; nuclei (Kedrovsky), 436; nuclei (Maneval), 436; pancreas (Baley), 428; pancreas (Gomori), 429; pancreas (Lane), 429; pancreas (Launoy), 429; Paneth cells (Klein), 452; pituitary, 425-427; Plas- modium (Tomlinson and Grocott), 510; reticulum fibers (Lillie), 424; skin (Dub- lin), 567; vaginal smears (Fuller), 430; vaginal smears (Papanicolaou), 431, 432; vaginal smears (Shoor), 431; virus Orange G — {continued) for staining — {continued) inclusion bodies (Cole), 464; yolk gran- ules (Kionka), 448 in, Arnold's triple stain, 351; Bensley's triple stain, 359; Biondi's triple stain, 356; Bohm and Oppel's triple stain, 361 ; Bonney's triple stain, 368; Brillmeyer's triple contrast, 336; Cason's triple stain, 360; clove oil solution, 320; Cowdry's triple stain, 351; Crossmon's triple con- trast, 336; Delephine's double contrast, 328;Dobeirstriplestain,369;Dominici's triple stain, 351; Dupres's double con- trast, 339; Dupres's triple stain, 359; Ehrlich's triple stain, 356; Flemming's double contrast, 339; Foley's triple stain, 363; Gausen's triple stain, 351; Goldner's quadruple contrast, 337; Gregg and Puckett's double contrast, 328; Grosso's triple stain, 355; Hay- thorne's triple contrast, 337; Heiden- hain's triple stain, 361 ; Henneguy's triple stain, 364; Hollande's triple contrast, 339; Houcke's quintuple stain, 351; Hubin's triple contrast, 340; Johansen's quadruple stain, 364; Kingsbury and Johansen's double contrast, 328; Korn- hauser's quadruple stains, 369, 370; Kostowiecki's double contrast, 327; Krause's triple stains, 356, 357; Krich- eski's triple stain, 360; Ladewig's quad- ruple stain, 365; Laguesse's triple stain, 364; Langeron's double stain, 353; Lendrum and McFarlane's quintuple contrast, 337; Lillie's triple stain, 370; McFarlane's quintuple contrast, 338; Mallory's triple stain, 360; Mann's tri- ple stain, 352; Margolena's double con- trast, 340; Masson's triple stain, 371; Mayer's triple stain, 357; Milligan's triple stain, 360; Paquin and Goddard's sextuple stain, 366; Petersen's triple stain, 372; Pollak's quadruple contrast, 338; Reinke's double contrast, 341; Romeis's quadruple stain, 367; Schleicher's triple stain, 372; simple solution, 320; Squire's double contrast, 328; Stockwell's triple stain, 364; Thome's double stain, 357; Unna's sim- ple contrast, 322 staining combinations with, acid alizarine blue and anilin blue, 372; acid alizarine blue, fast green FCF and orcein, 369, 370; acid fuchsin, 328, 329, 359; acid fuchsin and anilin blue, 427; acid fuchsin, anilin blue and eosin Y, 431; acid fuchsin, anilin blue, picric acid and ponceau 2R, 338; acid fuchsin, 758 INDEX Orange G — (continued) staining combinations with — {continued) anilin blue, ponceau 2R, 413; acid fuchsin, azophloxine, light green and ponceau 2R, 337; acid fuchsin, brilliant cresyl blue, 361; acid fuchsin, fast green FCF, picric acid and ponceau 2R, 337; acid fuchsin and hematoxylin, 461; acid fuchsin, hematoxylin and methyl blue, 365; acid fuchsin, hematoxylin and ponceau 2R, 430; acid fuchsin and light green, 336; acid fuchsin, light green and ponceau 2R, 337, 338; acid fuchsin, malachite green, 359; acid fuchsin and methyl blue, 360, 413, 423; acid fuchsin and methyl green, 356, 357, 359, 418; acid fuchsin and methylene blue, 359; acid fuchsin and toluidine blue, 351, 452; anilin blue, 327, 388; anilin blue and acid fuchsin, 336, 337, 359, 360, 425; anilin blue and azocarmine, 361, 372, 423, 425, 426, 427; aniUn blue, carmine, creso- fuchsin, 425; anilin blue and eosin, 428; anilin blue, eosin, hematoxylin and phloxine, 366; anilin blue, erythrosin and hematoxylin, 425; anilin blue and fast green FCF, 360; anilin blue, fast green FCF, and protein silver, 567; azo- phloxine, hematoxylin, light green and magenta, 367; azophloxine and light green, 337; azophloxine, light green, ponceau 2R and protein silver 567 ; azur and eosin, 464; azur, eosin, methyl blue and methylene blue, 418; azur A and phloxine, 351; azur II, eosin Y, thionine and toluidine blue, 351 ; Biebrich scarlet, fast green FCF, 431; Bismarck brown, eosin Y and light green, 432; carmine, 448; celestine blue B, hematoxylin and magenta leucobase, 427; cresofuchsin, 453; crystal violet, 341, 429, 502; crystal violet and safranin, 363, 364, 513; eosin, 328; eosin Y and methyl blue, 369; eosin Y and safranin, 340; eosin Y and toluidine blue, 351; erythrosin, light green and thionin, 502; erythrosin and toluidine blue, 352, 371; ethjd violet, 412; fast green FCF, methyl violet and safranin, 364; gentian violet, 340, 341; gentian violet, safranin, 364; hema- toxylin, magenta, 496, 497; hematoxylin and magenta leucobase, 424; hema- toxylin and safranin, 429; light green and magenta, 339 and 436; magenta, 428; methyl blue and phloxine, 370; method green, 339; methyl green and pyronin, 355 ; methyl violet and pyronin, 368; methyl violet and safranin, 364; methylene blue and magenta, 351; methylene blue and safranin, 351; Orange G — (continued) staining combinations with — (continued) phloxine and toluidine blue, 510; rose bengal and toluidine blue, 436; safranin, 393; tannin, 322; tannin and methylene blue, 353; thionine, 431, 498; toluidine blue, 339 Orange II, as plasma stain, 320 m. Conant's quadruple stain, 363; Gray's double contrast, 328; Holmes and French's triple stain, 352; simple solu- tion, 320 staining combinations with, azur C and eosin Y, 352; crystal violet, fast green and safranin, 363, 364; ponceau 2R, 328 Orban's mehod for dentine, 596 Orcein, as ingredient of, Zirkle's gelatin mountant, 636 for staining, actinomyces in plant tissue (Israel), 501 ; bacterial flagella (Bowhill), 481; elastic fibers, 387, 389; Herbst's corpuscles (Novak), 431; nuclei (Dalton), 435; nuclei (Kurnick and Ris's), 436; nuclei (LaCour), 436; skin (Pinkus), 424 Delamare's quadruple stain, 365; Ko- hashi's triple stain, 361; Kornhauser's quadruple stains, 369, 370; Langeron's double stain, 353; Mollier's quintuple stain, 366; Pasini's quadruple contrast, 338; Roskin's quadruple stain, 372; Unna's double contrast, 327; Unna's double stain, 353; Unna's quadruple stain, 364 staining combinations with, acid alizarin blue, fast green FCF, and orange G, 369, 370; acid fuchsin, anilin blue, azocarmine and eosin B, 361; acid fuchsin, anilin blue, and eosin B, 338, 361; acid fuchsin, hematoxylin, and picric acid, 372; anilin blue, 327; anilin blue, eosin, and safranin, 431; anilin blue, ethyl eosin, and safranin, 364; azocarmine, hematoxylin and naphthol green B, 366; azur and eosin, 424; fast green, 436; fast green FCF, 435; gentian violet, 481; hematoxylin, acid fuchsin and picric acid, 365; methylene blue, 353; polychrome methylene blue, 353 Ordonez's preservatives, 179-180 Oribatid mites, 45 Origanum oil, as ingredient of, Apdthy's clearing mixture, 628; Boloek's fixative remover, 255; Gatenby and Painter's clearing mixture, 628 physical properties of, 624 Orr's, fixative, 194, 249 INDEX 759 prr's — {continued) osmic method for degenerative changes, 528 Onseillin BB, for staining, fungi in plant tissues, (Cohen), 501; fungi in plant tissues, (Ravn), 502; Peronosporales, (Alangin), 502 in, combined preservative-stain, 177 staining combination with, anilin lilue, 502 crystal violet, 501 von Orth's, fixative, 233, 249 in, Endicott's method for bone marrow, 430; Koinikow's method for Schwann cells, 415; Masson's technique, 371; Pappenheim's technique, 350 recommended use, 95 stains, lithium-carmine, 307 as ingredient of, von Orth's picro- carmine, 304 Squire's picro-carmine, 304 in, Calleja's double contrast, 325 for staining, Aspergillus fumigatus, (Besson), 503; elastic fibers, (Hart), 388; elastic fibers, (Schmohl), 389 picro-carmine, 304 for staining Sacchromyces in sections, (Curtis), 503 Oschatz's cement, 656 Osgood and Wilhelm's method for reticulo- cytes, 419 Osmic acid, as, fixative, 193 histological stain, 529-530 immobilizing agent, 52 ingredient of, Becher and DemoU's macerating fluids, 262; Bethe's methylene blue, 401; Bethe and Monckeberg's tolui- dine blue, 402; Drew's mordant, 515; Drost's macerating fluid, 263; Du- buscq's thionine, 417; Harris's mor- dant, 518; Haug's decalcifying fluids, 258; Rossi's methyl green, 419; Zikes's mordant, 519 mordant for stains, Schultze's hema- toxylin, 407 stain, general observations, 525 basal fixative solution, 237 fixative combinations with, acetic acid, 193; acetic and tannic acids, 194; chromic acid, 199, 200, 201 ; chromic acid and dichromates, 201, 202; cobalt salts, 205; copper salts, 198; cupric and mercuric salts, 197; dichromates, 202- 205; iron salts, 205; formaldehyde and acetic acid, 194; formic acid, 194; iodine, 205; mercuric salts, 196, 197; mercuric salts and chromic acid, 197; mercuric salts and dichromates, 197, 198; roer- Osmic acid — {continued) fixative comliination with — {continued) curie salts, dichromates, and uranium salts, 198; mercuric salts and iodine, 198; mercuric salts and picric acid, 197; mercuric and uranium salts, 198; nitric acid, 194; palladium salts, 205; picric acid, 198; picric and chromic acids, 199; picric acid and silver salts, 199; platinic chloride, 195; platinic chloride and chromic acid, 195, 196; platinic chloride, chromic acid, and thorium salts, 196; platinic chloride and dichromates, 196; platinic chloride and mercuric salts, 195; platinic chloride, mercuric salts and chromic acid, 195; platinic chloride and picric acid, 195; uranium salts, 205 for fixing, protozoa, 52 smears, 71 for killing rotifers, 31 in Dekhuyzen's method for blood, 417 methods for, cell inclusions, 530-531; degenerative changes, 528-529; myelin sheaths, 529 removal from tissues, 256, 354 staining combinations with, carmine and protein silver, 607; picric acid and silver nitrate, 608; potassium dichromate, phosphomolybdic acid, and silver nitrate, 603; potassium dichromate and silver nitrate box, 608; silver nitrate, 608 storing solutions of, 193 Osmic-silver stains, 603 Osmium tetroxide, see Osmic acid Osmotic pressure, as affecting fixative qual- ity, 187 Osteol)lasts, 384 Ostracoda, collecting from moss, 45 Oudemann's preservative, 176 Ovaries, plant, 435 Ovary, of earthworm for CJolgi Ijodies, 525- 527 Overton's fixative remover, 256 Oxalic acid, as gold reducer in various silver methods, 550-609 as ingredient of, bleaching solution, 262; Boccardi's developer, 616; Cajal and de Castro's accelerator, 615; Cajal's iron toning solution, 620; macerating fluids, 264; MacFarland and Davenport's developer, 618; Pal's difi"erentiator, 520; stains, see under name of author or dye; Thiersch's carmine, 307; Thiersch's injection mass, 666 for, bleaching, 261 differentiating carmine stains, 295 Oxazine dyes, general remarks on, 272, 308 Oxidase granules, 417, 455 760 INDEX Oxidation-reduction, as fixative process, 188 Oxner's fixatives, mercuric-acetic, 209, 249 osmic-dichromate-acetic, 204, 249 osmic-dichromate-formic, 204, 249 von Pacaut's fixative, 206, 249 Pacini's preservative, 180 Painter's fixative, 227 Painter, see also Gatenby, 176, 628 Pal's differentiator, 520 in, Anderson's method for neuroglia, 412; Heller-Robertson's method for myelin sheaths, 529; nerve staining techniques, 404-409; Perdrau's method for reticu- lum, 594; Reich's method for granules in Schwann cells, 457; Schaffer's method for fat, 449 Pal on fixing spinal cord, 398 Paley, see Broadhurst, 490 Palladium chloride, as ingredient of, Choquet's decalcifying fluids, 259; Frenkel's fixative, 205; Katz's decalci- fying fluid, 259; Pampel's preservative, 177 Pancreas, dissection of, 439 mitochondria in, 439-441 paranuclear bodies in, 457 special methods for, 428, 429, 430 Pancreatin, as ingredient of macerating fluids, 264 in Wall's method for reticulum fibers, 424 "Panoptic" stain, 318, 343, 349 Pantin on heating fixatives for invertebrate larvae, 187 Papanicolaou's methods for vaginal smears, 431, 432 Papara, see Dotti, 252 Paper boxes for, nitrocellulose embedding, 144ff, 150 paraffin embedding, 102ff, 104ff, 105ff Paper cells for dry wholemounts, 12 Paper supports for fixing chicken embryos, 277 Pappenheim's, fixative, 197, 249 method for, acid-fast bacteria, 477 blood, 419 stains, methyl green-pyronin, 355 methylene blue-azur Il-eosin Y, 349, 350 Paquin and Goddard's stains, hematoxylin- anilin blue-eosin-orange G-phloxine, 366 iron-hematoxylin, 286 in Paquin and Goddard's technique, 366 Parabenzoquinone, in Baker's method for mitochondria, 441 "Paracarmine," 301 Parachlorophenol, as ingredient of, Amann's dehydrating mixtures, 628 Paradiaminobenzene hydrochloride, as in- gredient of Holmes's developer, 617 Paraffin, as ingredient of, Apdthj^'s cement, 655; Belling's cement, 655; embedding waxes, 646-647; Hood and Neill's cement, 656; Langeron's wax for dishes, 667; Lataste's cement, 656; Lebowich's soap embedding medium, 644 miscibility with, essential oils, 624; synthetic clearing agents, 626; "universal" solvents, 626 properties of, 97 Paraffin blocks, action of knife on, 1 lOff casting, 102, 103, 106ff, 107ff, 130 casting large, 139 choice of microtome for, 103 cooling, 103, 107, 135 effect on cutting, 120, 121 cutting hard materials in, 122 cutting ribbons, 114ff, 115ff effect of, faulty impregnation, 121, 122 using wrong wax, 121 lift ribbon when cutting, 123 making card box for, lOOff, 10 Iff, 102 making paper boxes for, 102ff, 104ff, 105ff method of orienting objects in, 135 mounting and trimming, 112, 113ff softening specimens in, 667 storing, 103 trimming with saw, 335 Paraffin embedding, impregnation of object, 100-101 infiltration methods, 99, 129, 130 of, embryos for freehand sections, 90 nitrocellulose blocks, 155 orienting frog's eggs during, 135 ovens for, 98ff, 99ff, 106ff selection of, clearing agent, 96; dehydrant, 96; wax, 97 technique of clearing before, 98 technique of dehydrating before, 97 technique with large objects, 139 transfer from solvent to wax, 99, 100 Paraffin-nitrocellulose ribbons, Riiyter's ad- hesive for, 658 Paraffin ribbons, see under Paraffin embed- ding and Paraffin sections Paraffin sections, adhesives for, 657-659 attaching to, each other, 279 slide, 131 cause of, failure to ribbon, 123 failure to stain, 127 checking adhesion to slide, 131 cleaning, 125 cutting on, rotary microtome, 114ff, 115ft' sliding microtome, 140 INDEX 761 Paraffin sect ions — {continued) defects in, granules or needles, 127 defects in ribbons, block lifts ribbon, I2;itf ; object shatt(>rs, 121ff; ribbon curved, 120iT; ribbon splits, 120ff ; sections alternate thick and thin, 121ff; sections bulge in middle, 121ff; sections fall out, 121; sections compress, 120ff ; sections has air bubbles, 126; sections bulges, 135; sections dis- torted, 127; sections wrinkled, 126 electrification of, 123 failure to adhere to slide, 126 fixation of materials for, 95 flattening, 117, 118, 119ff, 135 flattening large, 140, 1-41 judging dryness, 118 knives for, 108-111 labelling, 128 mounting in balsam, 125ff, 126 mounting on slide, 116, 117ff, 118ff, 135, 141 prevention of bubbles under, 118, 119 rehydrating, 124, 125 removing paraffin from, 119-124 squeezing to slide, 119 steps in preparation, 94 technique of, cutting, 114-119, 130; dehydrating, 132; staining, 131, 132 Paraldehyde, as ingredient of, Grapnuer and Weissberger's fixatives, 193; gum sandarac mountants, 638; Potenza's fixative, 190 in, Gomori's method for elastic fibers, 388 Smith and Rettie's mordant, 516 Paramecium, mounting in gum, 43 recommended fixative for, 197 Paramethylaminophenol sulfate, as ingredi- ent of, Ungewitte's developer, 619 Paraminophenol sulfate, as ingredient of, Rachmanov's developer, 619 Paranitrophenol, as ingredient of, Daven- port, Windle and Rhines' fixative, 237 Petrunkewitsch's fixative, 221 Paranuclear bodies, 457 Para's method for spirochetes, 562 Parasitic, amebas, 509 copepods, 49 fungi, 499-505 protozoa, 505-510 Parathyroid, mast cells in, 431 Parat's method for mitochondria, 444 Parietal cells, granules in, 455 of stomach, 432 Parinaud's conjunctives, 495 Parker and Floyd's fixative, 191, 249 Parlodion, 142 Pars intermedia, of pituitary, 426 Parson's method for Negri bodies and Nissl granules, 467 Partington and Iluntingford, on osmic acid stains, 525 Partsch's alum-cochineal, 301 Paschen bodies, special methods for, 467 Pasini's triple stain, 338 in, Kohashi's technique, 361 Walter's technique, 361 Pasteruack, see also Lillie, 346 Patay's double contrast, 339 for, mouse, 141 mouse head, 336 Paton's developer, 619 in Paton's method for embryonic fish nerves, 584 Patterson's fixative, 222 Patton's silver diammine stain, 576, 579 Pearl's, fixative, 209 injection fluid, 664 Pearse's method for pituitary, 427 Pearse, see also Williamson, 215, 430 Pearson's eosin, 322 Peck's preservative, 180 Pectin, as ingredient of mountants, 036-637 Pectinatella, wholemount of, 59-62 Peers's method for neuroglia, 414 Peeter's method for wax embedding, 647 Pelz, on de-waxing shellac, 653 Penfield's, fixative, 236, 249 method for, microglia, 587; neuroglia, 571-573; oligondendria, 587; perivascular nerves of pia mater, 584 silver diammine stain, 576, 579 Penfield, see also Conn, 577 Penicillium, in orange rind, 499 Pepsin, as ingredient of macerating fluids, 264 for softening chitin, 261 Pepsinogen granules, 455 Perdrau's, method for reticulum fibers, 594 mordant before methyl blue-eosin, 371 Perenyi's fixative, 229, 249 for, earthworm, 331 hydra, 78 in, Kingsbury and Johannsen's fixative, 215 Perez's, fixative, 230 method for Meissner's bodies in skin, 556 Pericellular nets, 556 Periodic acid, in, Gridley's method for reticu- lum fibers, 592 Kligman, Mescon and Deljameters method for fungi in skin sections, 504 Periodicals cited, 670-680 Peripheral nerve endings, 552, 58 1 , 605, 606 Peripheral nerves, 410 Schutze's method for, 557 762 INDEX Perithecia, 511 Peritubular sheath, 590 Perivascular nerves, 567 of pia mater, 584 Peronosporales, 502 Peroxidase granules, in bone marrow, 45G Sato's method for, 419 Perrin's method for spirochetes, 480 Perriraz's fixative, 199, 247 Perruche's varnish, 654 Perry and Lochead's method for pituitary of mouse, 427 "Pet names," 1 Peterfi's, method for double embedding, 153, 648 Peter's iron-cochineal, 305 Petersen's, gallocyanin, 313 in Neubert's method for smooth muscle, 424 triple stain, 372 Petit's method for plant sections, 393 Petragnini's, method for Negri bodies, 466, 467 mordant, 518 Petrolatum, for seaUng glycerol mounts, 34 in, Dufrenoy's wax embedding method, 646 Langeron's wax embedding method, 646 liquid, see Liquid petrolatum Petrunkewitsch's fixatives, cupric-nitric- paranitrophenol, 221 for frog intestine, 128 cupric-phenol-nitric, 219 in Rogoff's method for mosquito brain, 568 recommended use, 95 mercuric-acetic-nitric, 210 for onion root tip, 433 "PFA" fixatives, 223, 226 Pfau, see Nagle, 466 Pfaff and WilHam's method for blood vessels in wholemounts, 432 Pfeiffer's preservative, 179 Pfeiffer and Jarisch's fixative, 233, 249 Pfeiffer and Wellheim's Venice turpentine technique, 65 Pfitzner's, method for elastic fibers, 388 stains, picro-nigrosin, 326 safranin, 314 Pfuh's fixative, 215 pH, effect on narcotization, 31 role in, fixation, 187, 188 staining, 270 Phaeophyta, reproductive structures in, 421 Phenobarbital, as ingredient of, de Castro's decalcifying fluids, 257 Volkonsky's narcotic, 266 Pheno-glycerol, for improving cutting of paraffin blocks, 122 Phenol, as coupler in dehydration, 628 Phenol, as dehydrant, 627 as ingredient of, Albrecht's magenta, 315; Archibald and Marshall's mountant, 636; Auguste's magenta, 316; Bacsich's Sudan III, 416; Behren's fb^ative, 219; Brown and Brenn's crystal violet, 319; Champy's fixatives, 192; Conn's rose bengal, 473; Czokor's alum cochineal, 300; Davalos's magenta, 315; dehydrating mixtures, 628, 629; Downs's polyvinyl mountant, 636; Gatenby and Cowdry's thionine, 318; Goodpasture and Burnett's ma- genta, 315; Hamazaki's magenta, 431; Hanna's sulfur mountant, 638; Hether- ington's fbcatives, 189; Huntoon's ma- genta, 315, 488; Jensen's carmine-eosin, 321; Jones's polyvinyl mountant, 636; Kinyoun's magenta, 315; Krajian's eosin, 321; Kuhne's methylene blue, 317; Langeron's azur 11,317; Langeron's thionine, 318; Lendrum's acid fuchsin, 319; Lendrum's fixatives, 189; Lepik's anilin blue, 502; Moore's phenosafranin, 502; MuUer and Chermock's magenta, 316; Nicolle's thionine, 318; Petrunke- witsch's fixative, 219; Pottenger's ma- genta, 316; preservatives, 177-179; Ryo's crystal violet, 484; Schmorl's storage fluid for nitrocellulose blocks, 667; Schubert's anflin blue, 505; Schue- ninoff's hematoxylin, 292; Shortt's eosin, 510; Shortt's hemntoxylin, 282; Smith's ethyl eosin, 391; Stoughton's thionin, 318; Tilden and Tanaka's ma- genta, 316; Watkin's methyl blue, 422; Wilson's Venice turpentine mountant, 638; Womersley's mountant, 633; Zeeti's eosin, 487; Ziehl's magenta, 316; Zirkle's carmine-balsam, 640 as solvent for stains, 321 for softening chitin, 261 m, Gross's double stain, 410; Sheehan and Storey's method for fat, 420; Westphal's double stain, 420 physical properties of, 627 Phenosafranin, as ingredient of combined preservative stain, 177 in Moore's method for fungus in plant tissue, 502 Phenyl hydrazine, in Cretin's method for starch, 451 Phenyl salicylate, as ingredient of, Mohe and Wehrle's sandarac mountant, 638 Shepherd's sandarac mountant, 638 physical properties of, 626 Phillips, see Burke, 476 Phlobaphene, 519 INDEX 763 Phloroglucinol, as ingredient of, . Andeer's decalcifying fluids, 256; Fer- reri's decalcifying fluid, 258; Haug's decalcifying fluids, 258 limitations of, 138 Phloxine, as plasma stain, 320 for, algae, 65 demonstration of Negri bodies, 75 intestine, 131 staining, actinomycetes, (Mallory), 504; algae, (Semmens), 512; bacteria in sections, (Mallory), 493; filamentous algae, (Chamberlain), 511; fungus in plant tissue, (Dickson), 501; Gram-positive liacteria in sections, (Mallory), 495; Guarnieri bodies, (Kaiser and Gher- ardine), 465; hyalin, (Mallory), 456; Negri bodies, (Dawson), 465; Negri bodies, (Jordan and Heather), 465; pancreas, (Gomori), 429 ; Plasmodium, (Anonymous), 506; Plasmodium, (Tomlinson and Grocott), 510; viro- plasts, (Mc\^Tiorter), 466 in, Cowdry's triple stain, 307; Krajian's osmic method for fat, 530; Krugenberg and Thielman's triple stain, 370; Len- drum's quadruple contrast, 340; Lillie's triple stain, 370; Margolena's double contrast, 339; Paquin and Goddard's sextuple stain, 366; simple solution, 320 staining combination with, anilin blue, 340, 511; anilin blue and eosin B, 370; anilin blue, eosin, hema- toxylin and orange G, 366; azur A and orange G, 351; azur B and eosin Y, 465; azur I, 348; azur II and methylene blue, 493; crystal violet, 495; eosin Y, eryth- rosin and tartrazine NS, 340; gentian violet, 504; hematoxylin, 456; light green, 501; methyl blue and orange G, 370; methylene blue, 465, 506; orange G and toluidine blue, 510; polychrome methylene blue and eosin Y, 345; tartra- zine, 340; trypan blue, 466 Phosphate buffer, in Heller, Thomas and Davenport's method for nervous tissues, 403 Phosphomolybdic acid, action on acid fuch- sin, 357 as ingredient of, Bensley's anilin blue, 321; Held's hema- toxylin, 291, 413; Kostowiecki's double stain, 327; Lopez's double contrast, 371 ; Mallory 's hematoxylin, 292; Police's hematoxylin, 292; Schueninoff's hema- toxylin, 292; Thomas's heraatoxj'lin, 293 Phosphomolybdic acid — (continued) m. Alzheimer's method for neuroglia, 412; Anderson's mordant, 515; Buzaglo's triple stain, 368; Cretin's method for bone, 382; Ilolzcr's method for neurog- lia, 413; Jakob's method for neuroglia, 413; Koinikow's method for Schwann cells, 415; Lawson's method for bacterial capsules, 488; Matsura's polychrome neutral red, 371; Okajima's method for erythrocytes, 418; Oliveira's method for reticulum, 607; von Recklinghausen's method for bone, 385; Romeis's orange G, 367; Wilder's method for reticulum in spleen, 595 staining combinations with, acid fuchsin, 359-370; anilin blue and orange G, 359; magenta, orange G and light green, 339; methyl blue and orange G, 360; methyl green and orange G, 339; orange G and fast green FCF, 360; ponceau 2R and anilin blue, 339; pon- ceau 2R and light green, 339; toluidine blue and orange G, 339, 359 Phosphomolybdic-hematoxylins, 291, 292, 293, 413 Phosphotungstic acid, action on acid fuchsin, 357 as ingredient of, Bensley's brazilin, 308; Hueter's hema- toxylin, 292; Mai lory's hematoxylin, 292; Rawitz's fixative, 235 in, Crossmon's method for erythrocytes, 416; de Galantha's method for kidney, 423; Kornhauser's quadruple stains, 369, 370; Mollier's quadruple stain, 366; Morpugo's method for bone, 384;- Schleicher's triple stain, 372; Schmorl's method for bone, 385; Sclavo's method for bacterial flagella, 484; Volkman and Strauss's triple stain, 373 staining combinations with, acid fuchsin, anilin blue and orange G, 360; acid fuchsin and anUin blue, 338; acid fuchsin and eosin B, 361; acid fuch- sin, orange G, light green and ponceau 2R, 338; chromotrope and fast green, 339 Phosphotungstic-hematoxylins, 292, 293, 294, 507 Photographic process, compared to silver staining, 542 Physical properties of, essential oils, 624 universal solvents, 626 Phytomonas, 511 Pia mater, perivascular nerves in, 584 Pianese's, fixatives, chromic-cobalt-formic, 232; osmic- 764 INDEX Pianese's, fixatives — (continued) cobalt-formic, 205, 249; osmic-platinic- chromic-formic, 196, 249 triple stain, 341 Piccolyte, 641 Pick and Jacobson's method for bacterial smears, 474 Pickworth's embedding mass, 644 in Pickworth's method for brain capillaries, 419 Picric acid, as fixative, 221 Picric acid, as ingredient of, Chura's solvent for cell inclusions, 514; Gage's macerating fluids, 263; de Galan- tha's decalcifying fluid, 258; Jones's polyvinyl mountant, 636; Lowits differ- entiator, 521; Masson's differentiator, 521; Rogers's macerating fluids, 263; Safford and Fleischer's accelerator, 615 as stain, 321 basal fixative solution of, 237 fixative combinations with, acetic acid, 221; acetic and sulfuric acids, 222; chromic acid, 226; chromic acid and dichromates, 227 ; copper salts, 220; dichromates, 227; formaldehyde, 223 ; formaldehyde and acetic acid, 223- 225, 226; formaldehyde and formic acid, 225: formaldehyde and monochloro- acetic acid, 225; formaldehyde and nitric acid, 225; formaldehyde and trichloro- acetic acid, 225; hydrochloric acid, 223; iodine, formaldehyde and acetic acid, 227; mercuric salts, 213, 214, 215; mer- curic salts and chromic acid, 215. nitric acid, 222; osmic acid, 198; osmic and chromic acids, 199 ; osmic acid and mer- curic salts, 197; osmic acid and platinic chloride, 195; osmic acid and silver salts, 199; osmic and sulfuric acids, 198; platinic chloride, 206; platinic chloride, mercuric salts and chromic acid, 206; sulfuric acid, 222; trichloroacetic acid, 222 for differentiating, crystal violet, 319; hematoxylin, 281; safranin, 310 for staining, acid-fast bacteria, (Pottenger), 478; acid-fast bacteria, (Schulte-Tigge), 478, acid-fast bacteria, (Spengler), 478; algae, (Baumgartel), 511; blood, (Thompson), 420; bone and cartilage, (Klaatsch), 384; bone and cartilage, (Schmorl), 385, cestodes, (Dammin), 506; desmid sheaths, (Taylor), 513: fungi in wood, (Cartwright), 501; fungi in wood, (Cornwall), 501; Myxosporids, (Langeron), 508; spirochetes, (Renaux), 480 Picric acid — (continued) in, Barbrow's triple stain, 365; Caleja's triple stain, 369; Castroviejo's triple stain, 369; Delamare's quadruple stain, 365; double contrasts, 325-329; Drew- Murray's triple stain, 369 ; Lendrum and McFarlane's quintuple contrast, 337; Lillie's quadruple stains, 366, 370; Lil- lie's triple contrast, 340; Lowenthal's triple stain, 366 ; McFarlane's quintuple contrast, 338; Maresch's triple stain. 361; Masson's double stain, 353; Mas- son's triple contrast, 340; Nuttall's simple contrast, 321; Oppell's triple stain, 357; Paquin and Goddard's sextu- ple stain, 366; Roskin's quadruple stain, 372; Roskin's triple stain, 361; Scriban's triple stain, 341; Shumway's triple stain, 372; Waterman's triple stain, 361 removal from tissues, 254, 255 staining combinations with, acid fuchsin, 327, 328, 420: acid fuchsin and anilin blue, 338; acid fuchsin, anilin blue and hematoxylin, 427; acid fuchsin, anilin blue, orange G,and ponceau 2R, 338; acid fuchsin and brilliant green, 341; acid fuchsin, carmine and methyl violet, 389; acid fuchsin, fast green FCF, orange G, and ponceau 2R, 337; acid fuchsin, hematoxylin and magenta III, 498; acid fuchsin, hematoxylin and orcein, 372; acid fuchsin, indigo and carmine, 507; acid fuchsin and light green. 409. 411 ; acid fuchsin and metanil yellow, 340; acid fuchsin and methyl green, 357, 361; acid fuchsin and new magenta, 496; acid fuchsin and nile blue sulfate, 369; anilin blue, 336, 421; anilin blue and carmine, 508, 513; ani- lin blue and safranin, 393, 501 ; azofuch- sin, brilliant purpurin R, and naph- thol blue black, 370; Biebrich scarlet and anilin blue, 340; carmine and hema- toxylin, 366; carmine, indigo and car- mine, 368; Congo red, 327; crystal violet, 480; erythrosin, 326; hematein, 511; hematoxylin and acid fuchsin, 365; hematoxylin, acid fuchsin and orcein, 365; indigocarmine, 325, 326; indigo- carmine and magenta, 301, 372, 436, 506; light green, 326; magenta, 478; magenta and thionin, 478; methyl blue, 325, 326; methyl blue and safranin, 501 ; methyl green, 326; methylene blue, 402, 418; naphthol blue black, 325, 326; nigrosin, 326, 369, 422, 423; osmic acid and silver nitrate, 608; ponceau S, 327; spirit blue, 326; thiazin red, 325, 326; thionine, 353 INDEX 7G5 Picric Sicid— (continued) technique for decalcification with, 335 Picro-carmines, 303-304 recommendation for use, 293, 294 Pietschmann's fixative, 208, 250 Pigment, definition of, 269 Pineal, neuroglia in, 588 parenchyma of, 596 Pinene, physical properties of, 626 Pinkus's method for skin, 424 Pitch, as ingredient of Hood and Ncill's cement, 656 Pittfield's method for bacterial flagella, 483 Pituitary, differentiation of all types in, 425- 428 nerve fibers in, 556 Placoid scales. 323, 386 Planaria, narcotizing, 265 Plankton, bulk fixation of, 53 centrifuge for, 28, 37 concentrating, by, centrifuge, 28 phototropism, 30 fresh water, Gray's technique for, 67-68 net, 48 preserving, 49 stains for, 513 Planorbis, eggs of, 564 Plant cell walls, iron stain for, 610 Plant histology, methods for, 367, 391-393 Plant materials, softening, 667 Plant mitochondria, 531 Plant nuclei and chromosomes, see Nuclei, special methods for Plant ovaries, 435 Plant sections, skeletonizing, 381 Plant tissue, actinomycetes in, 501 bacteria in, 444, 498 fungal hyphae in, 501, 502 macerating techniques for, 263, 264 special methods for, 421-422 Plasma cells, in association with microglia, 589 Plasma stains, complex, general remarks on, 329 miscellaneous, 340-341 phosphotungstic-(molybdic-)acid fuch- sin, 336-339 phosphotungstic-(molybdic-)other dyes, 339-340 double contrast, for blue nuclei, 327-329 for red nuclei, 325-327 general remarks on, 319-320 single contrast, 319-322 Plasmodesma, methods for, 421, 422, 512 l^lasmodium, preparation of blood smears containing, 71 special methods for, 506-510 Plastic cells, preparation of, 13 Plastic tool for making smears, 71 Plasticizers, in Canada balsam, 639 Plastids, special methods for, 450 Plastin, 436 Platinic chloride, as fixative, 205 basal fixative solution of, 237 fixative combinations with, chromic acid, 207; copper salts and dichromates, 206; dichromates, 207; formaldehyde, 205; formaldehyde and acetic acid, 205; mercuric salts, 206; mercuric salts, picric and chromic acids, 206; osmic acid, 195, osmic and chromic acids, 195, 196; osmic acid, chromic acid and thorium salts, 196; osmic acid and dichromates, 196; osmic acid and mer- curic salts, 195; osmic acid, mercuric salts, and chromic acid, 195; osmic and picric acids, 195; picric acid, 206 in S(5guin's method for spirochetes, 598 Platyhelminthes, narcotizing and fixing. 53 Platyhelminthes, see also Turbellaria, Liver fluke, etc. Plehn's double stain, 372 Pluteus larva, 153-156 Podhradszky's, developer, 619 fixative, 192, 250 silver nitrate stain, 550 in Podhradszky's method for nerve cells and processes, 556 Podwj^ssozki's fixative, 197, 250 Pohlman's embedding wax, 647 Pol's triple stain, 326 Polar bodies, in bacteria, 491 Police's phosphomolybdic-hematojcylin, 292 Polishing, materials for, 81 Polishing sections, of balsam-embedded coral, 86 of bone, 84 Pollaillon's method for nerve fibers in teeth, 610 PoUak's, method for neuroglia, 587 quadruple stain, 338 Pollen grains, Margolena's method for, 293 strewn slide of, 309-310 Pollen mother cells, 435, 437 Pollen tubes, methods for, 421-422 Polychaetae, 53 Polychrome coelestin blue, 368 Polychrome methylene blue eosinates, 345- -s 347 in, Langeron's double stains, 353 Neisser's stain, 490 staining combinations with, orcein, 353; tannin and orange G, 353; various eosins, 345-347, 350 Polyethylene glycols, 643 Polysaccharides, Hotchkiss's mordant for, 515 Polystyrene mountants, 641 766 INDEX Polyvinyl acetate, as embedding medium, 649 as ingredient of Gray and Wess's moun- tants, 641 Polyvinyl alcohol, as ingredient of, moun- tants, 636 Nubkin and Carsten's embedding me- dium, 644 mounting in, 42 Polzam's soap embedding medium, 644 Pomerri, see DeipoUi, 445 Ponceau S, in Curtis's double contrast, 327 Ponceau 2R. as plasma stain, 320 for staining, adrenal cortex, (Fujiware), 429; astro- cytes, (Beyer), 413; skin, (Dublin), 568; vaginal smears, (Fuller), 430 in, Goldner's quadruple contrast, 337; Gray's double contrast, 328; Lendrum and McFarlane's quintuple contrast, 337; Lillie's double contrast, 338; INIcFarlane's quintuple contrast, 338; Masson's double contrast, 339; Patay's double contrast, 339; PoUak's quadruple contrast, 338; simple solution, 320 staining combinations with, acid fuchsin, 429; acid fuchsin and anilin blue, 338; acid fuchsin, anilin blue and orange G, 413; acid fuchsin, anilin blue, orange G and picric acid, 337, 338; acid fuchsin, fast green FCF, orange G and picric acid, 337; acid fuchsin, hema- toxylin and orange G, 430; acid fuchsin, light green and orange G, 337, 338; anilin blue, 339 ; azophloxine, light green, orange G and protein silver, 567; azo- phloxine, orange G, acid fuchsin and light green, 337; light green, 339; orange II, 328 Ponder's method for diphtheria bacilli, 490 Ponselle's iodine, 518 Popham, Johnson and Chan's method for cell walls of stem apex, 393 Post-chroming mitochondria, 590 Potassium acetate as ingredient of, Ham- pert's fixatives, 191 Highman's mountant, 632 IVIuir's glycerol jelly, 635 preservatives, 179-180 Potassium alum, as ingredient of, Cajal and de Castro's fixer, 621 Eros's acid fuchsin, 383 Gairns's decalcifying fluids, 258 hematoxylin stains, see under author's name, 286-290 Hopkins's macerating fluids, 263 Kingsbury and Johannsen's decalcifying fluid, 259 Potassium alum — (continued) preservatives, 176 Potassium aluminate, as ingredient of Rhyn's methyl violet, 490 Potassium antimony tartrate, as ingredient of, Menner's azoacid blue, 410 Yokata's mordant, 519 as mordant for, Rawitz's safranin, 314 Potassium bromide, as ingredient of Ingelby 's fixative, 224 in Holzer's method for neuroglia, 413 Potassium carbonate, as ingredient of, Good- pasture's methylene blue, 317 Kallius' developer, 618 Langeron's azur II, Loffler's methylene blue, 317 IMeriwether's carmine, 305 Sahle's methylene blue, 318 Terry's methylene blue, 318 Unna's methylene blue, 319 Volkonsky's methylene violet-azur II, 319 Williams's cresyl violet, 373 Potassium chlorate, as ingredient of, bleaching solutions, 262; Melinkow- Raswedenow's preservative, 180; Swank and Davenport's fixative, 194, 195 for cleaning diatoms, 38 Potassium chloride, as ingredient of, Meri- wether's carmine, 305 Peck's preservative, 180 Potassium chromate, as ingredient of, Cox's fixative, 216; Frey's injection fluid, 663; Robin's fixative, 229; Thiersch's injection mass, 666 in, Doyere's injection method, 662 Potassium dichromate, as fixative, 232 as ingredient of, Anderson's fat insolubilizer, 514; Ander- son's mordant, 515; Busch's decalcifying fluid, 257, Chura's mordant, 515; Clara's mordant, 515; Foley's alcoholic accel- erator, 614; Gage's neutral macera- ting fluids, 264; Gomori's hematoxylin, 291; Gordon's mordant, 518; Hansen's hematoxylin, 291; Harting's injection masses, 665; injection media, 163; Kiyono's differentiator, 521; KHatzo's formaldehyde accelerator, 613; Land's adhesive for paraffin ribbons, 658; Lan- dan's mordant, 516; Marquez's mordant, 518; Parat's method for mitochondria, 444; Ralston and Wells's decalcifying fluids, 259; Reuter's cement, 654; Robin's preservatives, 181; Schmorl's decalcifying fluids, 260; Schroder's mor- dant, 516; Schweitzer's hematoxylin, 292; Stafford's eosin, 347; Stockwell's mordant, 519; Thiersch's injection mass, 666; Weigert's chrome mordant, 516; Welling's decalcifying fluids, 260 INDEX 7G7 Potassium dichromate — (continued) as injection medium, 169 as mordant for, hematoxylin stains, 283, 284 as mordant in, Bailey's method for neurofrjia, 412; Bethe's method for chitin, 390; Dawson and Friedgood's method for pituitary, 425; Dietrich's method for fat, 447; Gracian's method for Rickettsiae, 462; Harris's method for nervous tissues, 403 ; Harvey's method for j^arietal cell }i;ran- ules, 455; Hewit's method for Plasmo- dium, 507, 508; Kraus's method for pituitary, 426; Lillie's method for nerv- ous tissues, 406; Milligan's triple stain, 360; Newman's method for nervous tis- sue, 407; osmic methods for degenera- tive changes, 528-529; Perry and Loc- head's method for pituitary, 427; Schaffer's method for fat, 449 ; Schultze's method for nervous tissue, 407; Sever- inghaus's method for pituitarj^ 427; Sokolansky's method for neuroglia, 414; Weigert's method for neuroglia, 415; Weigert's method for nervous tissue, 408; Weil's method for nervous tissues, 408 basal fixative solution of, 237 fixative combinations with, acetaldehyde, 235, acetic acid, 232; cal- cium salts, 235; chromic acid, 231; chromic acid and mercuric salts, 216; chromic and picric acids, 227; copper salts, 220, 221; formaldehyde, 232-235; mercuric salts, 216, 217, 218; mercuric salts and chromic acid, 215; mercuric and uranium salts, 219; mercuric, ura- nium and magnesium salts, 218; nitric acid, 232; osmic acid, 202, 203, 204, 205; osmic and chromic acids, 201, 202; osmic acid and iron salts, 205; osmic acid and mercuric salts, 177, 198; osmic acid, mercuric and uranium salts, 198; osmic acid and platinic chloride, 196; picric acid, 227; picric and chromic acids, 227; platinic chloride, 207; ura- nium salts, 235; zinc salts, 235 for, cleaning slides, 666 oxidation of methylene blue, 318 in, Brunotti's gelatin embedding method, 643 staining combinations with, arsenic trioxide and silver nitrate, 607; copper sulfate and silver nitrate, 608; dichromate and silver, 604-608; mer- curic chloride. 609, 610; osmic, phospho- molybdic and silver, 603; silver diaiu- mine, 607; silver nitrate, 608; silver nitrate and osmic acid, 604-608 Potassium dihydrogen phosphate as ingredi- ent of Cobin's methylene blue, 317 Potassium ferricyanide, as ingr(>dient of, Cajal's fixatives, 205; Harris's mordant, 518; Kultschitzky's differentiator, 520; Slonimski's fixative, 236; Smith and Rettie's diti'erentiator, 520; Weigert's differentiator, 520; Weil's differentiator, 520 in, Mallory's method for hemosiderin granules, 456 Pritchard's nu'thod for mitochondria and Golgi bodies, 590 Potassium ferrocyanide, as ingredient of, Beale's injection fluid, 662; Bnicke's in- jection fluid, 662; Landau's differ- entiator, 520; Mayer's injection fluid, 663; Richardson's injection fluids, 663; Robin's injection fluids, 663; Thiersch's injection mass, 666 as stain, 609, 610 in, Bensley and Bensley's method for alveolar epithelium, 534; Leber's method for cell outlines, 611; Rufini's method for nerve endings, 536 Potassium hydrosulfide, as ingredient of Liesegang's developer, 618 Potassium hydroxide, as ingredient of, Behrens et al. macerating fluids. 264; Belloni's hematoxylin, 286; Kingsbury and Johannsen's methylene blue, 317; Mall's preservatives, 180; Muller and Chermock's methylene blue, 318; Verocay's fixative remover, 256 for, cleaning, cells, 23; clearing skin scrap- ings, 500; hydrolyzing wholemounts, 406; radiolaria, 18-19; skeletonizing in- sects, 63-64; softening chitin, 261 in, Dawson's method for bone, 383 Gray's method for bone, 378 Potassium hypochlorite, for, hydrolyzing sputum, 470 skeletonizing plant tissues. 381 Potassium iodate, as ingredient of Cajal and de Castro's hematoxylin, 287 Potassium iodide, as ingredient of, Goodrich's fixative, 227; Krajian's hematoxylin, 285; Moore's injection mass, 665; Tandler's injection fluid, 664 in gold methods, 534-536 staining combinations with, iodine and chromic and, 232 iodide and formaldehyde, 236 "Potassium mercuric iodide," 475 Potassium metabi.sulfite, as ingredient of, Coleman's magenta leucobase, 316 de Tomasi's magenta leucobase, 316 7G8 INDEX Potassium metaborate, as ingredient of Davenport, Windle, and Rhlnes's developer, 617 Potassium nitrate, as ingredient of preserva- tives, 179-180 for cleaning diatoms, 39 Potassium oxalate, as ingredient of Rodri- guez's formaldehyde accelerator, 614 in, Herrera's silver diammine stain, 578 Pugsley's method for reticulocytes, 419 Potassium permanganate, as ingredient of, Gouillart and Bronardel's differentiator, 520; Kozowsky's differentiator, 520; Mallory's hematoxylin, 292; Pol's differ- entiator, 520 ; Stevenel's methylene blue, 318; Watson's acid-alum-hematoxylin, 290; Weil's differentiator, 520 as mordant, for Henneguy's magenta, 315 in Gemelli's method for bacterial flagella, 482 as stain, 611 for, bleaching, 261; cleaning diatoms, 39; differentiating, (carmine stains, 295, O'Leary's brazilin, 411); preserving osmic acid solutions, 193, 237 in Ono's method for spirochetes, 480 Potassium phosphate; tribasic, as ingredient of Landois's macerating fluid, 264 Potassium sulfate, as ingredient of, Braus's fixative, 233; EUermann's fixa- tive, 218; Fish's fixative, 204; Foa's fixative, 216; Guthrie's fixative, 217; Hamilton's fixative, 232; Heidenhain's fixative, 218; Hultgren and Andersson's fixatives, 233; Klotz and Coburn's pre- servatives, 180; Klotz and MacLachan's preservatives, 180; Krueger's fixative, 218; Landsteiner's fixative, 233; Maxi- mow's fixative, 204; Milller's fixative, 232; Murray's fixative, 204; von Orth's fixative, 233; Peck's preservatives, 180; Roques and Jude's methylene blue, 318; Schridder's fixative, 205; Spuler's fixa- tive, 217; Zenker's fixative, 217 Potassium sulfite, as ingredient of Pol's differentiator, 520 as mordant for Laguesse's triple stain, 364 Potato, actinomyces in, 501 Potenza's fixatives, paraldehyde-acetic, 190 picric-acetic, 222 Pottenger's magenta, 316 in Pottenger's method for acid-fast bac- teria, 478 Potter's method for macroglea, 414 Powell, see Hall, 455 Power, see Harris, 287 Pranter's method for elastic fibers, 388 Pregnancy cells, 425 Preservatives, 175-181 classification of, 175 definition, 175 explanation of classification, 175, 176 for, algae, (Eckerts), 178; algae, (Kirchner), 180; algae, (Pfieffer), 179; arthropoda, (Pampel), 177; fungus-infected plant tissues, 177; green plants, (Wood), 177; mammalian embryos, 180; parasitic protozoa, 178; ticks, (Monnig), 177 formaldehyde as, 175 formulas for, 176-181 Preservative-stain combinations, Alcorn and Yeagor's, 177 Moore's, 177 Price-Jones method for blood, 419 Priestly's preservatives, 178 in Priestly's method for fungus in skin scrapings, 505 Prince's method for nervous tissues, 411 Pritchard's, developer, 619 in Bohm's method for nerve endings, 534 fixative, 227, 250 method for mitochondria and Golgi, 590 Pritchard on strychnine as narcotic, 265 Proca's method for bacterial spores, 486 Procartilage, mentioned in Shumway's triple stain, 372 Kostowiecki's method for, 327 Schmorl's method for, 385 Proescher's method for fat, 449 Proescher and Arkush's celestin blue, 313 Proescher and Drueger's methylene blue, 318 Proescher, Zapata and McNaught's double stain, 372 Prokofieva's fixative, 230 Propaeolin for staining spirochetes in sec- tions, (Nickiforoff), 498 Propanol, see Isopropanol, 623 Proprietary compounds, 3 Proprionic acid as ingredient of, Wilson's Venice turpentine medium, 638 Zirkle's carmine balsam, 640 Zirkle's carmine-venice turpentine moun- tant, 638 Zirkle's fixative, 219 Propyl carbinol, 626 Propylene glycol, as solvent for Randolf and Mikele's magenta, 478 physical properties of, 623 Protargol, 564 Protein silver, general remarks on, 564 methods, 564-568 staining combinations with, azophloxine, light green, orange G and ponceau 2R, 567 osmic acid, and carmine, 607 Proteus vulgaris, smear to show flagella, 470 INDEX 7G9 Protozoa, aqueous wholemounts, 23 extra-nuoloar struntuics in, 5G8 fixation, 52 mitoc'hoiidiia in, 442 mounting individual, 67 narcotizing and fixing, 52, 53 parasitic preservative for, 178 special methods for, 506-510 special stains for, Butschli's hematoxylin, 281; Dobell's hematoxylin, 281; Donaldson's iodinc- eosin, 321; Gomori's, 339; Kofoid and Swegy's hematoxylin, 282 wholemounts of, 67 in gum moimtants, 43 Prowazeko's fixative, 208 Pryce's method for reticulocytes, 419 Pseudonavicellae, 73 Pseudoscorpionids, 45 Puckett's fixative, 225 in Slater and Dornfeld's technique, 367 Puckett, see also Gregg, 212 Pugsley's method for reticulocytes, 419 Pullinger's method for cornea, 584 Pumice, use in grinding sections, 81, 83, 86 Purification of, Canada balsam, 639 gum damar, 640 gum elemi, 640 Purkinje cells, 401, 553, 599-601 Purpurin, in Grandis and ]\Iangini's method for bone, 383 Putt's method for leprosy bacilli in sections, 497 Pyridine, as ingredient of, alcoholic accelerators, 614, 615; Belcz- sky's silver diammine stain, 576 ; Burke's fixative, 190; Cajal's decalcifying fluids, 257; Cajal's silver diammine stain, 577; formaldehyde accelerators, 613, 614; HoUande's carmine, 305; Jahnel's de- veloper, 618; Lobo's silver diammine stain, 579; del Rio-Hortega's silver diammine stain, 580; Romanes's silver diammine stain, 580; Shanklin's de- veloper, 619; silver nitrate staining solu- tions, 550-551 as solvent for Proescher's oil red, 449 for neutralizing formaldehyde, 190 in, Armuzzi and Stempel's method for spirochetes, 560; Bauer's nitrocellulose embedding method, 647; Bertrand and Quillam's method for oligoglia, 586; Cajal's method for brains of small mam- mals, 553; Cajal's method for neuro- genesis, 552; Chor's method for motor end plates, 554; Davenport, Windle and Beech's method for embryonic nervous tissue, 554; Davenport, Windle and Beech's method for nervous tissue, 582; Pyridine — (continued) in — {continued) Doinikow's method for regenerating nerves, 582; Donaggio's mcsthod for neurofibrillae, 402; Favorsky's method for nerve cells and processes, 554 ; Foley's methods for axons, 555; Foley's method for protein silver, 567; llertzman's method for spirochetes, 561 ; Iluber and Guild's method for nerve cells and proc- esses, 555; Jahnel's method for spiro- chetes, 561; Mclndoo's method for ])ile capillaries, 597; de No's method for nerve endings in calcified structures, 556; Ranson's method for nerve cells and processes, 556; Rasmussen's method for nerve fibers in pituithry, 556; del Rio-Hortega's method for neuroglia, 589; del Rio-Hortega's method for neu- rogUa in pineal, 588; Shanklin's method for pineal parenchyma, 596; Szatmari's method for nervous tissues, 583; Tello's method for nerve endings, 557 ; Weber's method for nervous tissues, 585 Pyrocatchol, in Steiner's method for bacteria in sections, 563 Pyrogallol, as ingredient of, developers, 616-620; Eisalh's differ- entiator, 519; Hoyer's injection mass, 665 for staining chitin, 390 in, Curtis's method for Saccharomyces m sections, 503; Heller-Robertson's method for myelin sheaths, 529 ; Hogan's method for general histology, 610; Karl- son's method for muscle, 394; Kossa's method for general histology, 560; Lee's osmic methods, 530; Murray and Field- ing's method for Leptospira ictero- haemorrhagica, 562; Podhradszky's method for nerve cells and processes, 556 Pyroligneous acid, as ingredient of, Benda's fixative, 202; Champy's fixa- tive, 202; Chevalier's gelatin mountant, 634 in, Herman's osmic method, 529 Severinghaus's method for pituitary, 427 Pyronin, for staining, bacteria in leukocytes, (Flinn), 491; bac- teria in sections, (Saathof), 493; fungus in skin sections, (Unna), 505; gonococ- cus, (Walton), 491; oxidase granules, (Graham), 417; spirochetes, (Keil), 479; spirochetes, (Lipp), 480 in, Bonney's triple stain, 368 Grosso's triple stain, 355 770 INDEX Pyronin — {continued) staining combinations with, methyl green, 355, 356, 491, 493, 505; methyl green and orange G, 355; methyl green and Victoria blue, 479, 480 ; methyl violet and orange G, 368; methylene blue, 417 Pyroxylin, as ingredient of, Claoue's adhesive, 657; Guyer's sealing fluid for vials, 667; Schallibaum's ad- hesive for free sections, 660 in Masson's method for attaching free sec- tions, 660 remarks on, 142 Pyxicola, 53 "Quad stains," 369, 370 Quadruple stains, see name of author or ingredient Quinalizarin, in Buzaglo's triple stain, 368 Quinone, for staining muscles in whole- mounts, 394 in Meunier and Vaney's method for plank- ton, 513 Quintuple stains, see author or ingredient R Raadt's double stain, 346 Rabbit, diplococci in liver of, 471-472 dissection of, brain, 536; cervical ganglion, 601-602; eye socket, 532 injection of, intestine, 170 kidney, 168-169 neuroglia, 399-400, 571-573 removal of embryos from, 602 suprarenal, 354-355 taste buds, 569 Rabl's, cochineal, 301 fixatives, chromic-formic, 229, 250; mercuric- picric, 213, 250; platinic-mercuric, 206, 250; recommended use, 95 Rachmanov's developer, 619 in Rachmanov's method for nerve cells and processes, 556 Racovitza's stain for chitin, 390 Radiolaria, cleaning, 18-20 preparation of strewn slide, 17-20 Radula, 394, 513 Raebiger's method for bacterial capsules, 488 Raileanu's, fixer, 621 formaldehyde accelerator, 613 gold-mercury stain, 538 in Raileanu's method for neuroglia, 539 Railliet's preservative, 177 Rait's method for bone in wholemounts, 384 Raleigh, see Marie, 346 Ralf's preservative, 176 Ralston and Wells' decalcifying fluid, 259 in Ralston and Wells' method for bone marrow, 431 Ramon y Cajal, see Cajal Rana, see Frog Randolf's fixative, 231 for lily bud, 149 Randolf and Mikele's method for acid-fast bacteria, 478 Rankin, see Windle, 447 Ranson's developer, 619 in, Foley's method for axons, 555; Gurd- jian's method for nerve cells and proc- esses, 555; Huber and Guild's method for nerve cells and processes, 555; Jahnel's method for nerve cells and processes, 555; Ranson's method for nerve cells and processes, 556 Ranimculus, section of stem, 362-363 Ranvier's gold stain, 534 injection fluid, 663 method for nerve endings, 532-533, 536 picro-carmine, 304 for Squalus embryo, 322-323 in Bostrom's method for actinomycetes, 501 Rasmussen's method for nerve fibers in pituitary, 556 Raswedenkow, see Melnikow, 180 Rat, connective tissue, 394 injection of blood vessels, 164ff section of tongue, 323-325 testis, 272-275 vom Rath's fixatives, osmic-mercuric-picric- acetic, 19ff, 250 osmic-picric-acetic, 198, 250 osmic-platinic-picric-acetic, 195, 250 mercuric-acetic, 209 platinic-picric-acetic, 206 Ravn's method for fungus in plant tissues, 502 Rawitz's, fixatives, osmic-acetic, 194, 250; osmic-picric- nitric, 198, 250; phosphotungstic-acetic, 235; picric-chromic-nitric, 226, 250, (recommended use, 95) stains, alum-carmines, 301; alum-hematoxy- lins, 288 ; aluminum-carmine, 307 ; alumi- num-hematoxylin, 292; safranin, 314 Razor, for cutting freehand sections, 91 Reagents, method of naming, 3 von Recklinghausen's method for bone, 385 Reconstruction from sections, 153, 154 Red lead, as ingredient of, Beale's gold size, 652 ' Kilton's cement, 654 INDEX 771 Red ochre, as ingredient of Griffith's cement, 655 Reeve's triple stain, hematoxyl in-fast green FCF-safranin, 367 References, explanation of method, 2 Refractive index of, essential oils, 624 synthetic clearing agents, 625-626 universal solvents, 626 R4gaud's, adhesive for paraffin ribbons, 658 fixative, 233, 250 in. Bailey's method for mitochondria, 442; Benda's method for mitochon- dria, 442; Bowie's method for pep- sinogen granules, 455; Cowdry's method for mitochondria, 442; Hirsch and Bretschneider's method for mito- chondria, 443; Lepine's method for Rickettsiae, 463; Milovidov's method for proplastids, mitochondria and starch, 450; Pritchard's method for mitochondria, 590; recommended use, 95 stains, iron-mordant hematoxylin, 282 for staining earthworm, 331 in, Cansey's method for mitochondria in protozoa, 442; Doubrow's method for acid-fast bacteria in sections, 496; McFarlane's technique, 338; Masson's techniques, 337-338, 341 ; Wolfe's and Cleveland's method for pituitary, 425 Regaud and Dubreuil's method for cell out- lines in epithelia, 607 Regenerating nerves, Doinikow's method, 582 Rehm's rosin mountant, 640 Reich's methods for granules in Schwann cells, 457 Reichardt and Wetzel's nitrocellulose-wax embedding method, 648 Reilhes's method for starch, 453 Reinke's, adhesive for paraffin ribbons, 658 in Chiovenda's method for free sections, 659 double stain, 341 macerating fluid, 264 Remy and Sugg's method for bacterial fla- gella, 483 Renault's osmic-picric-silver stain, 603 in Renault's method for lymph vessels in testis, 608 Renault's, double stain, 367 method for lymph vessels in testes, 608 Renaux's method for sjiirochetes, 480 Renaux and Wilmaers's method for spiro- chetes, 480 Reptile eggs, fixative for, 214 Resin, base exchange, 256 embedding media, 649 Resinous mountants, 639-641 mounting nitrocellulose sections in, 152 removing from slides, 666 wholemounts in, 51-68 Resorcin blue, in Nebel's method for pollen tubes, 422 Resorcin, in, Baley's method for pancreas, 428; Boecke's method for fungus in skin scrapings, 503; French's method for elastic fibers, 387; Krajian's method for elastic fibers, 388; Sheridan's method for elastic fibers, 389 fixative combination with formaldehyde, 237 Reswelling dried plants, 667 Reticular fibers, 597 Reticulosytes, 417-419 Reticulum fibers, antimony stain for, 610 dye staining methods for, 424 mentioned in Lillie's triple stain, 370 silver-chromic methods for, 607 silver diammine stains for, 591-595 silver nitrate methods for, 559 Retina, fixatives for, 217 Balbuena's method for, 544-546, 551 Uyama's method for, 557 Retterer's fixatives, chromic-acetic-formalde- hyde, 231 platinic-formaldehyde-acetic, 205 Rettie, see Smith, 450, 516, 520 Reuter's gelatin-dichromate cement, 654 Rexed and Wohlfart's triple contrast, 360 Reynold's method for nematodes, 509 Rezek, see Lauda, 550 Rhamy's triple stain, 353 Rhines, see Davenport, 237, 567; Windle, 447 Rhizopods, attaching to coverslip, 52 Rhizopods, see also under generic names Rhodamine B, in Honcke's double stain, 353 Rhyn's method for diphtheria bacilli, 490 Rich's method for viroplasts, 466 Richards on the microtome, 89, 119 Richards and Smith, on methacryate moun- tants, 639 Richardson's, injection fluids, 663 nitrocellulose embedding method, 648 Richman, Gelfand and Hill's decalcifying fluid, 259 Rickettsiae, in smear of guinea pig scrotum, 458-460 special methods for, 462-464 Rindfleisch's ahim-hematoxylin, 288 Ringing balsam moiuits, 9 cements, 652-654 Venice turpentine mounts, 66 del RIo-Hortega's, accelerators, 614, 615 fixatives, dichromate-formaldehyde, 234, 263 772 INDEX del Rio-Hortcga's^(conim?ie(^) fixatives, dichromate-formaldehyde — (continued) in del RIo-Hortega's silver dichromate method for neuroglia, 607 uranium-formaldehyde, 236, 250 methods for, astrocytes, 588; controsomes in nerve cells, 590; epithelial fibrils, 597; glio- blasts, 589; gliosomes, 589, 591; microg- lia, 588; mitochondria, 590, 591; neu- roglia, in pineal, 588; neuroglia in smears, 588; oligodendria, 588, 589; pathological microglia, 610; protoplas- mic astrocytes, 587; protoplasmic neu- roglia, 588; reticulum, 594 stains, ferrocyanide, 609 silver diammine, 576, 579, 580 in, Barker's method for microglia, 585; Fieandt and Sazen's method for Golgi bodies, 590; Gans's method for microglia, 586; Haymaker and Sanchez-Perez's method for tissue cultures, 598; McCarter's method for neuroglia, 587: Mclndoo's method for bile capillaries, 597; Negrin's method for reticulum fi- bers, 594; Penfield's method for mi- croglia, 587; Penfield's method for neuroglia, 571; Penfield's method for oligondendria, 587; Polak's method for neuroglia, 587; Pul- linger's method for cornea, 584; del Rio-IIortega's method for, (astrocytes, 588, epithelial fibrils, 597, glioblasts, 589, gliosomes, 589, 591, microglia, 573, 574, 575, 588, mitochondria, 590, 591, neuroglia, 588, 589, neuroglia, in pineal, 588, oligodendria, 588, 589; protoplas- mic neuroglia, 588; reticulum, 594) ; Shanklin's method for pineal par- enchyma, 596; Urechia and Nagu's method for reticulum fibers in nervous tissue, 595; Winkler's method for microglia, 589 silver nitrate, 551 solvent, 628 Ripart and Petit's, fixative, 219 for, aqueous wlioiemounts, 23 mounting algae, 27-29 in, Soulier's macerating fluid, 264 Ris, see Kurnick, 436 Ritchey, see Wicks, 641 Rivalier and Seydel's method for filamentous fungi, 512 Robb-Sniith's silver diammine stain, 576, 580 in Robb-Smith's method for reticulum, 594 Robin's, asphalt cement, 654 fixative, 229 injection fluids, 663 injection masses, 665 mountants, 632 preservative, 177, 181 silver nitrate injections, 163 Rock aggregates, separating diatoms from, 38 Rodent skin, fat glands in, 450 Rodriguez's formaldehyde accelerator, 614 in Rodriguez's method for oligondendria, 589 Roehl's method, for calcareous deposits, 385 Roger's, macerating fluid, 263 silver diammine stain, 576, 580 in Roger's method for axis cylinders, 584 Rogoff 's method for mosquito brain, 568 Rohl's fixative, 229, 250 Rojas's, developer, 619 in Rojas's method for Golgi bodies, 559 formaldehyde accelerator, 614 Romanes's, developer, 619 silver diammine stain, 576, 580 in Romanes's method for nervous tis- sues, 584 Romeis's, fixatives, dichromate-formaldehj^de-acetic, 235, 250; formaldehyde-acetic, 192, 250; mercuric-formaldehyde-trichloroacetic, 212, 250 gelatine embedding method, 645 method for, invertebrate nervous systems, 411 pituitary, 427 quintuple stain, 367 Roncoroni's fixative, 207 Roudanowski's, glycerol jelly, 635 isinglass mountant, 636 Roonwall, method for softening chitin, 261 Root nodules, 444 Root tips, method for, 435 recommended fixative for, 196 Roots, dried, swelling, 380 fungi in, 501 Roques and Judc's methylene blue, 318 Rosanilin hydrochloride, in Brown and Brenn's method for Gram-positive bacteria in sections, 493 in Casares-Gil's mordant, 517 Rosbach and Leavitt's decalcifying fluid, 259 Rose bengal, as plasma stain, 320 for staining, nuclei (Kedrovsky), 436 soil bacteria (Conn), 473 in, simple solution, 320 phenol solution, 321 staining combination with, orange G and toluidine blue, 436 van Rosen's method for nuclei, 437 Rosenbush's hematoxylin, 281 INDEX 773 Rosenthal's, fixative, 211 method for fat, 447 Rosin, as ingredient of, Cigalas's cement, 655; Coburn's ce- ment, 655; Faut's cement, 655; Grif- fith's cement, 655; Kroenig's cement, 656; Lacoste and de Lacliand's moun- tant, 640; Martin's adhesive, 661; Mar- tin's cement, 656; Mendeleef's cement, 656; Noyer's cement, 656; Rohm's mountant, 640; sealing wax, 652; Tomlinson and Grocott's orange G, 510; Wolbach's difTerentiators, 521 in, Steiner's method for spirochetes, 562 Roskin's, fixatives, cupric-paranitrophenol-ether, 221 ; form- aldehyde-acetic, 192, (in Roskin's method for oligochaetes, 564) ; mercuric- acetic, 209, 250; osmic-dichromate, 208, 250; picric-formaldehyde-acetic, 225, 250 ; picric-formaldehyde-trichloracotic, 225, 250 glycerol jelly, 635 methods for, oligochaetes, 564 Rickettsiae, and Negri bodies, 463 stains, acid fuchsin-hematoxylin-orcein-picric acid, 372; magenta-picro-indigo car- mine, 361; safranin, 314 Rossenbeck, see Feulgen, 316 Rossi's, method for blood, 419 mordant, 518 in Rossi's, method for bacterial flagella, 483 Rossi-Regaiid's cement, 661 Rossman's fixative, 223 Rossolino and Biisch's, differentiator, 520 osmic acid stain, 528 Rotary microtome, 103 Rothig's fixatives, mercuric, 208, 250 mercuric-chromic-formaldehyde-acetic, 215, 251 Rotifers, collection, 30 killing, 31 narcotics for, 265 narcotization, 30 wholemount of, 29-31 Roudabush, method for cleaning radiolaria, 18 Rouge, use in polishing sections, 81, 83, 86 Rousseau's decalcifying fluid, 259 Rousselet's, narcotic, 266 for, protozoa, 52, 53 rotifers, 29, 30 in Gray's method for ciliates, 265 varnish, 654 Roussy and Lchrmitte's method for Nissl granules, 44() "Roux'sblue," 327 Roux's double stain, 327 in Bohm and Oppel's technique, 368 Rowmanowski's double stain, 346 Rozas' iron-hematoxylin, 286 Rubaschkin's fixatives, 320 in Rubaschkin's method for neuroglia, 414 Ru])ber, as ingredient of, Beale's French cement, 655; Beale's glass cement; 652; Brook's Brunswick black, 653; ChevaHer's mountant, 640; Davies's asphalt varnish, 653; Frey's varnish, 653; Langeron's wax for dishes, 067; Lataste's cement, 656; marine glue, 655; wax embedding media, 646 use in embedding waxes, 97 Rubber-paraffin, recommended use, 129 Ruben-Duval's method for elastic fibers, 389 Ruffini's, embedding wax, 647 hxatives, mercuric-chromic-dichromate-acetic, 216, 251, (in Dupres's technique, 359); mercuric-formic, 210, 251 method for nerve endings, 536 Ruge's fixative, 192, 251 in, Becker's method for spirochetes, 478; Fontana's method for spirochetes, 608; Langeron's method for spirochetes, 597 Ruiz's method for bacterial spores, 486 Rukhadze and Blajin's method for cestodes, 509 Russel bodies, 457 Russel's, fixative, 235, 251 methods for cell inclusions, 457 Russel, see also Crook, 425 Ruthenium red, in Ferrari's method for fungi in plant tissue, 501 Rutherford's picro-carmine, 304 Riij'ter's, adhesive for paraffin ribbons, 658 cement, 655 for finishing glycerol jelly mounts, 48 sealing glycerol mounts with, 32 Ryo's method for bacterial flagella, 483 Saathof's method for bacteria in sections, 493 Sabin's method for blood, 419 Saboraud's method for spirochetes, 480 Ral)razes's, double stain, 345 methods for spirochetes, 480 Saccharomyces, 503, 504 Sadorsk3''s method for Nissl granules, 446 Safety-razor-blade holders, as microtome knives, 109 SalTord and Fleischer's accelerator in Safford and Fleischer's method for bacterial flagella, 608 774 INDEX Saffron, for staining gastric cells, (Hoecke and Sebruyn), 431 in Masson's double contrast, 328 staining combinations with, anilin blue, Bismark brown, hema- toxylin and methyl violet; 431; eosin B, 329; erythrosin, 329, 341 "Safranelin," 390 Safranin, differentiators for, 521 for staining, acid-fast bacteria (Weiss), 478; adrenal (Wiesel), 430; bacteria in leukocytes (Bruner and Edwards), 490; bacteria in sections (Foshay), 492; bacterial spores (Ashby), 485; bacterial spores (Bitter), 485; bacterial spores (Bruner and Edwards), 485; blood (Sato), 419; Chlo- rophyceae (Yamanouchi), 513; cocci in cell smears (Schwitz), 491; diphtheria bacilli (Kemp), 490; elastic fibers (Pfitzner), 388; fat in bacteria (Burdon, Stokes and Kimbrough), 491; fungi in wood (Cartwright), 501; fungi in wood (Cornwall), 501; Gram-positive bac- teria (Burke), 474; Gram -positive bac- teria (White and Culbertson), 475; Herbst's corpuscles (Novak), 431; Negri bodies (Lepine), 466; nervous tissues, 410; nervous tissues (Alzheimer), 409; nitrocellulose section of lily bud (Cham- berlain), 151; nuclei (Geither), 435; nu- clei (Hermann), 436; nuclei (Schmorl), 437; nuclei in yeasts (Gutstein), 512; Peronosporales (Lepik), 502; pituitary (Lillie), 426; plant sections (Foster), 392; polar bodies in bacteria (Weiss), 491; pollen grains, 309-310; Rickettsiae (Castaneda), 462; Rickettsiae (Lepine), 463; spirochetes (Weiss), 481; whole- mounts, 55 general remarks on, 308 in, Arnold's triple stain, 351; Conant's quadruple stain, 363; Foley's triple stain, 363; Henneguy's triple stain, 364; Hubin's triple contrast, 340; Johansen's quadruple stain, 364; Kalter's quad- ruple stain, 364; Laguesse's triple stain, 364; Reeve's triple stain, 367; Stock- well's triple stain, 364; Unna's double stain, 365; Unna's quadurple stain, 364 staining combinations with, acid green, 481; acid violet, 481; anilin blue, 365, 395, 502; anilin blue, eosin and orcein, 431; anilin blue, ethyl eosin and orcein, 364; anilin blue and picric acid, 393, 501; azur II, 463; brilliant black 3B, 394; crystal violet, 393, 474, 475; crystal violet, fast green and orange II, 363, 364; crystal violet and orange G, Safranin — (continued) staining combinations with — (continued) 363, 364, 513; eosin Y and orange G, 340; eriocyanine A, 426; fast green, and hematoxylin, 367; fast green FCF and hematoxylin, 367; fast green and methyl violet, 421; gentian violet, 436; gentian violet and orange G, 364; hematoxylin, 392, 406, 437; hematoxylin and orange G, 429; light green, 392, 435; magenta, 512; magenta and methylene blue, 466; malachite green, 485, 490; methyl blue, 491; methyl blue and picric acid, 501; methyl violet, fast green FCF and orange G, 364; methyl violet and orange G, 364; methylene blue, 462, 485, 490, 491, 512; nile blue sulfate, 492; orange G, 393; orange G and methylene blue, 351; Sudan black B, 491; toluidine blue, 430 Safranin O, as plasma stain, 320 for staining, bacterial spores (Schaeffer and Fulton), 487; bacterial spores (Shapiro), 487; Gram-positive bacteria (Hucker and Conn), 474; nuclei (Darrow), 435; Rickettsiae (Zinsser and Bayne-Jones), 464; spirochetes (Tunnicliff), 481 in, clove oil solution, 320 staining combinations with, anilin blue, 435; crystal violet, 481; malachite green, 487; methylene blue, 464 Safranin, see also Babes's safranin, etc. Sahli's, Canada balsam, 639 methylene blue, 318 Boeck's method for fungus in skin scrapings, 503; Mallory's technique, 346; Priestly 's method for fungi in skin scrapings, 505 Sainmont, see de Winiwarter, 341 Salamander, staining bony skeleton, 377-379 staining cartilagenous skeleton, 379-380 Sala's method for nervous tissues, 606 Salazar's mordant, 518 in Wallraff's method for pituitary, 428 Salicylic acid, as ingredient of, Anderson's hematoxylin, 289; Arcan- geli's alum-carmine, 300; Cohen's fixa- tive, 226; Kirkpatrick's cochineal, 300; macerating fluids, 263; Partsch's alum cochineal, 301 in balsam, 259, 639 Salicylic-balsam for mounting Masson's stain, 333, 338 Saling's fixative, 211, 251 Salivary chromosomes, smear of, 299, 437 Salkind's, cherry gum embedding method, 645 INDEX 775 Salkind's — (continued) fixatives, lead-acetic, 23G mercuric-dichromate, 21(), 251 Salol, physical properties of, 626 Samuel's agar embedding medium, 645 Sanchez's fixative, 234, 251 in Sanchez's method for invertebrate neurology, 606 Sanchez-Perez, see Haymaker, 598 Sand, separation of Foraminifera from, 17 Sand's toner, 620 in Sand's method for nerve cells and processes, 557 Sander's method for bacterial spores, 611 Sandiford's double stain, 355 Sannomiya's, fixative, 236 method for paranuclear bodies, 457 Sanson's fixative, 190, 251 recommended use, 95 Santtner, see Lepine, 213 Saponin, 202, 510 Sass's stains, acid-alum-hematoxylin, 290 alum-hematoxylin, 289 Sato's method for peroxidase granules in blood, 419 Satory's preservative, 177 Saturated solution, definition, 173, 174 Saturn, extract of, 645 Sawing paraffin blocks, 335 Saws, for cutting sections, 80, 82ff Sax's method for nuclei in pollen mother cells, 437 Saye's method for blood, 419 Sazen, see Fieandt, 258, 590 Scale insects, microflora in, 504 Scales, bony, Williams's method for, 386 of, butterfly, 14 fish, 43 Scarlet R, in Gros's method for fat, 448 Scathidium, 53 Schabadasch's, fixative, 225 method for nerve ending, 403 Schact's glycerol jelly, 635 Schaeffer and Fulton's method for bacterial spores, 487 Schaffer's, decalcifying fluid, 259 fixative, 191, 251 method for fat, 449 SchafTner's fixative, 229, 251 Schallibaum's adhesive for free sections, 660 Schaudinn's fixatives, 208, 251 for smears of monocytes, 73 in, Hall and Powell's method for euglenoid flagellates, 455; Langeron's Giemsa technique, 348; Markey, Culbertson and Giordano's method for fecal smears, 508; Moskowitz's method for protozoa, 568; Tompkins and Miller's method for Schaudinn's fixatives — (continued) in — (continued) fecal smears, 510; Tsuchiuya's method * for fecal smears, 510 recommended use, 95 Scheuring's fixative, 192, 251 Schiefferdecker's macerating fluid, 264 Schiefferdecker, see also Behrens, 264 Schiff's magenta leucobase, 309, 316 in, Lillie's method for reticulum fibers, 424 Verne's method for myelin, 411 Schiller's fixative, 218, 251 Schleicher's, method for bone marrow, 432 triple stain, 372 Schleiff's method for fungus in skin scrap- ings, 505 Schleifsteins' method for Negri bodies, 467 Schmaus's uranium carmine, 307 Schmidt's fixative, 194, 251 Schmorl's, decalcifying fluids, 260 fixatives, mercuric, 208, 251; mercuric-acetic, 210, 251; mercuric-formaldehyde, 211, 251; osmic-chromic-acetic, 201 methods for, amyloid, 453; bacterial capsules, 489; bone, 385; cartilage and bone, 385; elastic fibers, 389; fat and glycogen, 451 ; fibrin, 425; lamellae in bone, 385; nuclei, 437; Plasmodium, 509; Schridde's gran- ules, 457; spirochetes in sections, 498; storage fluid for nitrocellulose blocks, 667 Schneidau's, adhesive for paraffin ribbons, 659 double stain, 360 Schneider's stains, aceto-carmine, 302 in. Chandler's method for pollen tubes, 421 residue from preparation of, 300 magenta, 315 Schoenfle, see Cobe, 657 Schreiber's, fixative, 234, 251 method for nervous tissue, 605 Schridde's, fixative remover, 256 fixatives, dichromate-formaldehyde, 233, 251 osmic-dichromate-formaldehyde, 205, 251 granules, 457 Schroder's, chrome mordant, 516 lithium-hematoxylin, 292 in his method for mj^elin sheaths, 407 Schrotter's method for nervous tissues, 411 Schuberg's fixatives, osmic-picric-acetic, 198, 251 osraic-picric-sulfuric, 199, 251 Schubert's method for fungi in skin scrap- ings, 505 776 INDEX Schueninoff's phosphomolybdic-hematoxy- lin, 292 Schuffner's method for Plasmodium, 50U Schulte-Tigges's method for acid-fast bac* teria, 478 Schultze's, developer, 619 in Schultze's silver nitrate methods, 557 fixative, osmic-dichromate, 203, 251 picric-dichromate, 227, 251 method for, cell outlines of epithelia, 608; myelin sheaths, 407; nervous tissues, 584 osmic-chromic mordant-hematoxylin, 284 Schultze and Stohr's method, 619 for nerve fibers, 557 Schwann cells, granules, 457 special methods for, 415, 590 Schwarz's, alcoholic carmine, 302 fixative, 194 hydrochloric-carmine, 306 Schweitzer's, chrome-hematoxylin, 292 fixative, 225 mountants, 632 Schwitz's method for cocci in cell smears, 491 Sclavo's method for bacterial flagella, 484 Scott and French's double stain, 355 Scouring powder, for cleaning slides, 19, 69 Scriban's double contrast, 341 Sealing fluid for vials, 667 Sealing wax, as ingredient of Oschatz's cement, 656 general remarks on, 651 Sealing wax varnish, 652 disadvantages of, 16 Sebaceous glands, Badertscher's method for, 422 Sebruyn, see Hoecke, 431 Section of, amphibian embryo, 133-136 Amphioxus, 357-359 bone, 81-85 brain, for Negri bodies, 460-461; to show microglia, 573-575; to show oligoden- dria and microglia, 571-573 cerebellar cortex to show Purkinje cells, 599-601 cerebral cortex, 399-400 to show neuroglia, 536-538 chicken embryo, 278-280 to show neuroblasts, 546-548 coral, 81-85 earthworm, 330-333 ovary to show Golgi bodies, 525-527 echinoderm larva, 153-156 entire mouse, 136 fatty tissue, 160-161 frog brain, 395-397 leaf, 91 lily bud, 149-152 mitosis in onion root tip, 433, 434 Section of — {continued) mouse head, 334-336 orange rind to show mycelia of Penicillium, 499 pancreas for mitochondria, 439-441 post-mortem material to show spirochetes, 548-550 rabbit, embryo brain to sliow neurons and dendrites, 602-603 liver to show Diplocci, 471-472 Ranunculus stem, 362-363 rat, testes, 273-275 tongue, 323-325 retina to show nervous elements, 544-546 root, 380-382 sciatic nerve to show axis cylinders, 564- 566 spinal cord, 397-399 to show nerve cells, 539-540 Squalus embryo, 322-323 superior cervical ganglion, 601-602 suprarenal, 354-355 tongue to show nerve endings in taste buds, 569-571 wood, 92-93 Section lifter, 91 Section planes, definition, 88ff Sections, adhesives for, 657-660 definition of, 7, 88 determining which side of slide bears, 275 mounting on celluloid, 659 reconstruction from, 153, 154 techniques for preparing, see under Double- embedded Sections; Freehand Sec- tions; Frozen Sections; Ground Sections; Nitrocellulose Sections; Paraffin Sections Seguin's method for spirochetes, 598 Seguy, see Lancelin, 597 Sehrwald, on silver-dichromate staining, 605 Seidelin's iron-hematoxylin, 286 Seiler's, alcohol-balsam, 638 alcoholic carmine, 302 Canada balsam-gum damar mountant, 640 decalcifying fluid, 260 embedding wax, 647 gelatin cement, 655 injection fluid, 664 Seki's nitrocellulose-wax embedding method, 648 Semichon's, method for cartilage, 386 stains, aceto-carmine, 302; methyl blue-eosin Y-victoria yellow, 329; safranin, 314 Semmens's, chromic-uranic mordant, 518 dye-balsams, 639 fixatives, chromic-dichromate-formaldehyde- acetic, 231; chromic-uranium-acetic, INDEX 777 Semmens's — {continued) fixativrs — {continued) 232; dichromato-foniialdcliyde-acctic, 2;5o gum aral)i(; varnish, 652 method for algae, 512 moiintaiits, 033 preservatives, 178 in Semmens's method for algae, 512 Semmens and Bhaduri's, method for nurlei, 437 varnish, 654 Semmens, see also Bhaduri, 231 Senevet's double stain, 347 Sequestrene, 260 Serial section, definition of, 7 Setting knives, 109, 111 Severinghaus's fixative, 202, 251 in Severinghaus's method for pituitary, 427 Sex organs, nerve endings in, 600 Seydel, see Rivalier, 512 Shaffer's double contrast, 328 Shakespeare, see Norris, 372 Shale, separation of Foraminifera from, 18 Shanklin's, developer, 619 silver nitrate stain, 551 in Shanklin's method for pineal paren- chyma, 596 Shape, preservation of, 95 Shapiro's method for bacterial spores, 587 Sharman's method for cell walls of stem apex, 393 Sharpening stones for, grinding sections, 85, 87 microtome knives. 111 Sheath, peritubular, 590 Sheaths, of desmids, 513 Sheehan and Storey's method for fat in leukocytes, 420 Shellac, as ingredient of, Carnoy's cement, 653; Coburn's ce- ment, 655; Giesbrecht's adhesive, 660; Gram-Rlitzow's varnish, 653; Harting's cement, 656; marine glue, 655; sealing wax, 652; Thiersch's blue varnish, 654; Zimmerman's green varnish, 654 de-waxing, 653 for attaching, paper cells, 12 base to wooden slides, 10 general remarks on, 651 making cells with, 40 Shepherd's sandarac mountant, 638 Sheridan's method for elastic fibers, 389 Sherlock's fixative, 210 Sherman and Smith's embedding wax, 647 Shipley, see Kramer, 260 Shoor's method for vaginal smears, 431 Shorea wiesneri, 640 Shortt's, method for fecal smears, 510 Shortt's — {continued) stains, iron-mordant, hematoxylin, 282 in Siuton and Mulligan's nirthod fur Plasmodium, 510 Showalter's lixative, 201, 251 Shrove, see Farmer, 189, 243 Shumway's triple stain, 372 Shunk's mordant, 519 in Shunk's mclliod for vactcrial flagella, 484 Shutt's method for bacteria in milk, 491 Siedlecki, see Kostanecki, 21 1 Sihler's method for nerves in muscle, 407 Sikora's fixative, 192 Silica gel, 66 Silica, removal from wood before sectioning, 92 Silver-arsenic staining methods for spiro- chetes, 608 Silver-chromic staining solutions, 603 Silver diammine, as ingredient of Tribon- deau, Fichet and Dubreuil's alum- hematoxylin, 289 general remarks on, 575 staining combinations with azocarmine and light green, 597 potassium dichromate, 607 staining methods for, argentophil cells, 595-596; bacterial flagella, 598; bacterial smears, 598; blood parasites, 595; calcified tissues, 596; Golgi bodies, 590-591; mito- chondria, 590-591; nerve cells and proc- esses, 581-585; neuroglia, 585-590; peritubular sheath, 590; reticulum fibers, 591-595; Schwann cells, 590; spirochetes, 597-598; tissue cultures, 598: Trichinella, 599 staining solutions, 575-581 Silver hydroxide, as ingredient of Lillie and Pasternack's methylene blue, 346 for oxidation of methylene blue, 317 Silver nitrate, as ingredient of, Bensley's injection fluid, 662; Eyene and Steinberg's developer, 617; Farrier and Warthin's developer, 617; Faulkner and Lillie's developer, 617; Heitzman's developer, 618; Hoyer's injection mass, 665; Perreraz's fixative, 199; Robin's in- jection mass, 665; Seller's injection fluid, 664; silver diammine stains, 575-581; Steiner and Steiner's devel- oper, 619; Warthin-Starry's developer, 620 as injection medium, 163 staining combinations with, ammonium dichromate, 604; arsenic trioxide, 605; arsenic trioxide and potas- sium dichromate, 607; cadmium chlo- 778 INDEX Silver nitrate — (continued) staining combinations with — {continued) ride, 608; chromic acid, 607; cobalt nitrate, 559; copper sulfate and potas- sium dichromate, 608; dichromate, 604; dichromate, phosphomolybdic and osmic, 603; osmic acid, 608; osmic acid and picric acid, 608; osmic acid and potassium dichromate, 604-608; potas- sium dichromate, 608 staining methods for, bacteria in sections, 563; bacterial flagella, 563; bone, 560; cell outlines, 564; general histology, 560; Golgi bodies, 558-559; kidney, 560; lesions in teeth, 563; nerve cells and processes, 551-558; neuroglia, 558; oligochaetes, 564; reticulum, 559; spirochetes, 560- 563 staining solutions, 550-551 stains, 544-564 Silver oxide, for polychroming methylene blue, 350 Silver stains, general observations, 541-544 method of classification, 542 Silver-dichromate, staining methods for, Golgi apparatus, 608; neuroglia, 606- 607; reticulum fibers, 607 Silver-iron methods for spirochetes, 608 Silver-osmic staining solutions, 603 Silver-phosphomolybdic staining solutions, 603 Silver-protein, see Protein silver Silver-tungstic staining solutions, 603 Silver's method for nerve endings, 568 Simard and Campenhout's toning solution, 621 Simons's, fixative, 229, 251 method for Plasmodium, 510 Simpson's method for blood, 420 Singer, see Medalia, 509 Singh, Jaswart and Bhattacharji's methylene blue, 318 Sinton and Mulligan's method for Plasmo- dium, 510 Sjovall's osmic method for histology, 530 Skeletal tissues, special stains for, 377-395 arthropod, 390-391 plant, 391-393 vertebrate, 382-387 Skeletonizing, insects, 62, 63-64 plant sections, 381 Skiles and Georgi's vinylite mountant, 641 Skin, Dubhn's method, 567 Skin, eleidin in, 457 epithelial fibers in, 424 fat glands in wholemounts of, 450 keratin and eleidin in, 422 Meissner's bodies in, 556 mentioned in Unna's quadruple stain, 364 Skin^(conimued) nerve endings in, 535 Pinkus's method for, 424 reticulum fibers in, 593 special methods for, 423, 424 Skin scrapings, fungus in, 502, 505 Skovsted's fixative, 199 Slater and Dornfeld's triple stain, 367 Slavonic names, method of transliteration, 2 Slider and Downey's double stains, 350 in, McNamara's technique, 349 Pappenheim's method for blood, 419 Slides, attaching paper cells to, 12 attaching paraffin sections to, 116, 117ff, 118ff cleaning methods for, 19, 131, 666, 667 concave, 21 for, dry wholemounts, 10 foraminifera, 11 jars for staining, 119, 120, 124ff marking center of, 12 methods of cleaning, 69 mounting nitrocellulose sections on, 148 selection for aqueous wholemounts, 28 Slifer and King, method f or softening chitin, 261 Slonimski and Cunge's, fixative, 236 method for capillaries, 420 • Smear of, bacteria, 468 bacteria to show flagella, 470-471 blood, 69-70, 343-344 chironomus salivary chromosomes, 299- 300 Gram-positive bacteria, 468 grasshopper chromosomes, 310-312 monocystes, 72-73 Negri bodies, 75 Rickettsiae from guinea pig, 458-460 tubercle bacilli in sputum, 469 Smears, definition, 7, 69 fecal for protozoa, 506-510 fixatives for, 194 from, cut surfaces, 74-75 fluid material, 69-73 of, plant tissues, see also Squashes; Plas- modium in, 505-510; Spermatozoa, 72 silver diammine method for neuroglia in, 588 Smirnow's fixatives, dichromate-form alde- hyde, 234, 251 in Smirnow's method for cerebellum, 606 osmic-dichromate, 205, 251 Smith's, fixatives, dichromate-formaldehyde, 234, 252 dichromate-formaldehyde-acetic, 235, 252 for fixing amphibian eggs, 133 recommended use, 95 INDEX 779 Smith's, fixatives — (continued) osmic-chromic-dichromate-acctic, 202, 252 recommended use, 95 picric-formaldehyde-acetic, 225 methods for, bacterial capsules in sections, 498; chitin, 391; fat, 449; nervous tissues, 606; nuclei, 437; plant sections, 393 stains, ammonia-carmine, 305; picro-spirit blue, 326, (for sections of amphibian em- bryos, 135) Smith, see also Hopewell, 259; Sherman, 647 Smith and Mair's method for fat, 449 Smith and Quiegley's method for nervous tissue, 407 Smith and Rettie's, diflferentiator, 520 mordant, 516 in Smith and Rettie's method for fat, 450 Smooth muscle, 424 wandering cells in, 431 Snail, see also Helix, etc. Snesserew's method for reticulum, 594 Snider's method for Nissl granules, 446 Snyder's method for bacterial spores, 485 Soap, as ingredient of, Flemming's embed- ding medium, 643 Polzam's embedding medium, 644 Soda-carmine, 306 Sodium, for desiccating ether, 143 Sodium acetate, as ingredient of, Bauer's developer, 616; van Ermengen's developers, 617; Johansen's safranin, 314; Kornhauser's quadruple stain, 370; Melnikow-Raswedenkow'a preserva- tives, 180; Reeve's triple stain, 367 for disintegrating rock, 38 Sodium acid phosphite, in Gomori's method for carious lesions, 563 Sodium alum, as ingredient of Beecher's gal- lamin blue, 368 Sodium amytal, for killing mouse, 136 Sodium barbitol, for killing mouse, 334 Sodium bicarbonate, as ingredient of Gairns's decalcifying fluids, 258 Sodium bisulfite, as ingredient of Feulgen and Rossenbeck's magenta leuco- base, 316 Weil's differentiator, 520 explanation of, 283 in Cox's method for nervous tissues, 609 Sodium borate, as ingredient of, Balbuena's toning solution, 620; Boi- tard's preservatives, 176; Cole's car- mine, 305; Fischer's glycerol jelly, 634; Frey's carmine, 307; Harris's toluidine blue, 403; Jadassohn's methylene blue, 317; Krause's injection mass, 665; Sodium borate, as ingredient of — (con- tinued) Manson's methylene blue, 317; Mosch- kowsky's methylene blue, 318; Nicki- forow's carmine, 302; Rachinanov's de- veloper, 619; Schwarz's alcoholic car- mine, 302; Seller's alcoholic carmine, 302; Smith's carmine, 305 for polychroming methylene blue, 347 Sodium borate-HCl buffer, in Gross and Lohan's fixative, 235 Sodium bromide, as ingredient of Merland's fixative, 236 for polychroming methylene blue, 346 Sodium carbonate, as ingredient of, Bunge's embedding medium, 643; Hart- ing's injection mass, 665; Hoehl's mac- erating fluid, 264; Kuhne's methylene blue, 317; silver diammine stains, 575- 581 for cleaning Foraminifera, 17, 18 for polychroming methylene blue, 346 Sodium carminate, 410 Sodium chloride, role in fixation, 187 Sodium citrate, as ingredient of Evans and Krajian's decalcifying fluids, 258 Weber's developer, 620 Sodium cobaltinitrite, in Krajian's method of spirochetes, 561, 597 Sodium cyanide, in Negrin's method for reticulum fibers, 594 Sodium fluoride, as ingredient of, Frost's preservatives, 179 Jousset's macerating fluid, 264 Sodium formate, as ingredient of Ivristen- sen's decalcifying fluids, 259 Sodium hexametaphosphate, for decalcify- ing, 256 Sodium hydrosulfite, as ingredient of Cole's hematoxylin, 282 Sodium hydroxide, as ingredient of, Langeron's macerating fluids, 264; Lowenthal's picro-carmine, 303; Mi- chaelis's methylene blue, 318; silver diammine stains, 575-581; Squire's picro-carmine, 304 for cleaning, diatoms, 38 foraminiferan tests, 17 for clearing cestodes, 506 in, Held's method for macroglea, 413; Merzbacher's method for neuroglia, 414; Okada's method for neurofibrils and pericellular nets, 556 Sodium hypochlorite, for, removing albumen from amphibian eggs, 133; skeletonizing plant tissues, 381; softening chitin, 261 Sodium hypophosphite, in Gomori's method for calcified tissues, 596 780 INDEX Sodium iodate, as ingredient of, Busch's fixative, 205; Jjee's aluiu-lienia- toxyliu, 2S8; Lillic's lieinatoxylin, 2'.K); Mayer's alum-hematoXylin, 288; Sass's hematoxylin, 289, 290 Sodium iodide, as ingredient of Farkas's fixative, 198 Sodium lac.tnle, as ingredioTil of r.c^larie's crystal violet, 479 Sodium metabisulfite, as ingredient of, Cajal's fixing solution, 621; Knower's hematoxylin, 283; Lillie's magenta leucobase, 316 Sodium molybdate, as ingredient of Michai- low's mordant, 518 Sodium nitrate, as ingredient of Haug's de- calcifying fluid, 258 Sodium nitroprusside, for staining blood ves- sels, 416, 417, 419, 421, 432 Sodium perborate, as ingredient of Lange- ron's bleach, 262 Sodium peroxide, as ingredient of stains, Procscher and Drueger's methylene blue, 318 Sodium phosphate, as ingredient of Bensley's injection fluid, 662 dibasic, as ingredient of Cobin's methylene blue, 317 tribasic, for cleaning slides, 667 Sodium phosphomolybdate, as ingredient of Bethe's methylene blue, 401 Sodium potassium tartrate, as ingredient of, Steiner and Steiner's developer, 619 Steiner's silver diammine stain, 580 Sodium pyrosulfite, 283 Sodium pyruvate, as ingredient of Schaba- dasch's methylene blue, 403 Sodium salicylate, as ingredient of, Mayer's adhesive for paraffin ribbons, 658 Mozejko's injection fluid, 663 Sodium sulfantimonate, in Kenny's method for reticulum fibers, 610 Sodium sulfate, as dehydrant, 622 as ingredient of, Bock's fixative, 233; Bohm and Oppel's fixative, 234; Brucke's injection fluid, 662; Carleton and Leach's decalcifying fluids, 257; Charipper's fixatives, 197; Cole's fixative, 232; Danchakoff's fixa- tive, 218; David's decalcifying fluid, 257; Gage's neutral macerating fluid, 264; Haver's fixative, 234; Heringa and ten Berge's adhesive for nitrocellulose sections, 659; Jores's preservatives, 179; Klotz and Coburn's preservatives, 180; Klotz and MacLachan's preservatives, 180; LaCour's fixatives, 202; Landois's macerating solution, 264; Maximov's fixative, 218; Peck's preservatives, 180; Ralston and Wells's decalcifying fluids, Sodium sulfate — (continued) as ingredient of — {cnniinucd) 25!) ; Roljin's preservatives, 181; Puf- fiui's fixative, 216; Schaffer's decalcify- ing fluids, 259; Schmorl's decalcifying fluids, 260; Whitney's fixatives, 217 role in fixation, 187 Sodium sulfide, as ingredient of, Hanna's moutant, 638 Robin's injection fluid, 663 Sodium sulfite, as ingredient of, developers, 616-620; Harlow's macerat- ing fluid, 263; Rossolino and Busch's differentiator, 520 Sodium sulfocyanide, see Sodium thiocyanate Sodium thiocyanate, as ingredient of Sou- lier's macerating fluid, 264 Sodium thiosulfate, as ingredient of, Cajal's gold toning solution, 620; Cap- pell's fixative remover, 255; fixing solu- tions, 621; Golgi's toning solution, 620; Lancelin, Seguy and Dubreuil's toning solution, 620 in, various silver methods, 550-609 Sodium uranate, as ingredient of, Bhaduri and Semmens's fixative, 232; Semmens's fixative, 232; Semmens's mordant, 518 Soehr's gold stain, 534 Softening, chitin, 261 plant materials, 667 specimens in paraffin blocks, 667 wood for sectioning, 92, 93 Soil bacteria, 473 Soil collection of nematodes from, 35-36 Sokolansky's method for neuroglia, 414 Solubility of water in, clearing agents, 625, 626 "universal" solvents, 620 Solvent mixtures, 627-629 Solvents, classification of, 622 Somnifane, see Aprobarbital and Pheno- barbital "SI, S2" fixatives, 202 Sonnenbrodt's fixative, 217 Sorbitol, as ingredient of, Zirkle's carmine-dextrin movmtant, 637 Zirkle's carmine gelatin mountant, 635 Zirkle's carmine pectin mountant, 637 Zirkle's mountant, 633 role in mounting media, 42 Soulier's macerating fluid, 264 Southgate's gum elemi mountant, 640 Spark's method for pituitary, 427 Spec's embedding wax, 647 Spehl's method for, acid-fast bacteria, 478 bacterial smears, 474 Spcnce, method for sealing glycerol mounts, 34 on de-waxing shellac, 653 INDEX 781 Spencc — (continued) sealing aciucous wholcmouiits, 2(1 Sponco's vaiuisli, (153 Spencer, clinical microtome, 157ff rotary microtome, lOSlT, 112 Spengler's method for acid-fast bacteria, 478 Spermaceti, as ingredient of, Gray's wax embedding medium, 646; Robin's injection mass, 665; Water- man's eml)edding wax, 647 Spermatogenesis, in rat, method for, 272-275 Spermatozoa, staining smears of, 72 Sphagnum moss, collecting animals from, 45 Spielmeyer's, method for, myelin sheaths, 408 Nissl granules, 445 stains, iron mordant hematoxylin, 408 Spiess on proprionic acid in fixatives, 189 Spinal cord, 552 axis cylinders in, 583 gold-dichromate method for, 540 Hansberg's method for Nissl granules in, 445 nerve fibers in, 555 neuroblasts in embryonic, 546-548 section of, 397-399, 539-540 Spinal ganglia, oligoglia in, 586 Schutze's method for, 557 Spindle fibers, 435, 436 Spirit blue, as ingredient of Thiersch's var- nish, 654 as plasma stain, 320 in, Lonnberg's triple stain, 370; simple solution, 320; Smith's double contrast, 326; Tonkoff's sample contrast, 322 in staining combinations with, carmine, 370 picric acid, 326 Spirochetes, dye staining methods for smears, 478-481 in, section of post-mortem material, 548- 550 sections, 498 other silver methods for, 608 silver diammine methods for, 597 silver nitrate methods for, 560-563 Spirogyra, 65 Spirostomum, 52 Spleen, reticulum fibers in, 592, 593, 595 Spoerri's adhesive for, acid-fast bacteria in sections, 498 paraffin ribbons, 659 Spon's sealing wax, 652 Spores, bacterial, stains for, 484-487 Spuler's, fixatives, mercuric-acetic, 210; mercuric-dichro- mate-acetic, 217, 252; mercuric-form- aldehyde-acetic, 212, 252; mercuric- Spuler's, fixatives — {continued) picric-formaldehyde, 214, 252; osmic- picric-acetie, 198, 252 stains, alcoholic cochineal, 302 iron-carmine, 305 Sputum, hydrolyzing, 470 Squalus embryo, 322-323 Squash of Hydra, 78, 79 Squashes, 76-79 definition, 69 macerating before, 76 of microsporocytes of Crocus, 77-78 staining and mounting, 76-77 Squire on clearing agents, 96 Squire's, glycerol jelly, 635 stains, acid fuchsin-orange G, 329; ammonia- carmine, 305; calcium-hematoxylin, 293; methyl green-acid fuchsin, 357; picro- carmines, 304 Stafford's triple stain, 347 Stage's developer, 619 in Stage's method for nervous tissues, 568 Stain, definition of, 269 Staining, "direct," 271 "direct" and "indirect," 284 general observations on, 269 not always necessary, 270 purpose of, 270 Stains, see under name of author, dye, tissue or object Stannic chloride diammine, as ingredient of Donaggio's hematoxylin, 291 Stappers's fixatives, cupric-formaldehyde, 219 mercuric-cupric-formaldehyde-acetic-ni- tric, 213 Starch, as ingredient of, McDowell and Vassos's adhesive for paraffin ribbons, 658; Spoerri's adhe- sive for paraffin ribbons, 659; Warthin- Starry's developer, 620 fixative for, 222 grains, mentioned in Johansen's quad- ruple stain, 364 special methods for, 450-453 Steam, for, disintegration of fossil deposits, 18; fixing protozoans, 52; fixing smears, 71; softening wood for sections, 93 Stearic acid, as ingredient of, Barlow's embedding medium, 649; Gad- den's wax embedding medium, 646; Lebowich's soap embedding medium, 644; Steednum's embedding wax, 647; Waterman's embedding wax, 647 Steedman's embedding waxes, 647 Steil's method for blood, 420 Steiner's alcoholic accelerators, 614, 615 782 INDEX Steiner's alcoholic accelerators — (continued) in Steiner's method for bacteria in sec- tions, 563 developers, 619 methods for spirochetes, 562, 598 Steiner's silver diammine stain, 580 in Steiner's method for spirochetes, 598 Steiner and Steiner's developer, 619 in Steiner and Steiner's method for spiro- chetes, 563 Stempel, see also Armuzzi, 560 Stepanow's nitrocellulose embedding method, 648 Stern, see Gatenby, 578 Sternberg's method for Plasmodium, 510 Sternberg, see also Eyene, 561 Stevenel's methylene blue, 318 in, Boye's technique, 347 Lepine's method for Negri bodies, 466 Stewart's osmic method for degenerative changes, 528 Stewart, see also Gooding, 258, 555 Stieve's fixatives, mercuric-formaldehyde- acetic, 212, 252 picric-formaldehyde-trichloroacetic, 225, 252 Stilling's fixative, 234, 252 Stirling's, gentian violet, 319 in Mallory's method for Gram-positive bacteria in sections, 495 injection mass, 665 Stockwell's, chrome mordant, 519 in Stockwell's technique, 364 triple stain, 364 Stoehr's method for nerve endings, 536 Stokes, see Burdon, 491 Stomach, cell types, 432 chief cells, 455 differentiation of cells in, 431, 432 Storage fluid, for nitrocellulose blocks, 667 Storey, see Sheehan, 420 Storing, mounts, 128 specimens, 96 Stottenberg's method for diphtheria bacilli, 490 Stoughton's, method for bacteria in plant tissues, 498 thionin, 318 in Margolena's method for fungus in plant tissues, 502 Stovall and Black's method for Negri bodies, 460-461, 467 Strasburger's method for plasmodesma, 422 Strauss's fixative, 190, 252 Strauss, see also Volkman, 373 Straw, collecting animals from, 44 Strewn slides, 17 Striae of muscle. Miller's method for, 394 van der Stricht's fixative, 195, 252 Striped muscle, invertebrate, 559 Strong's fixatives, cupric-dichromate-form- aldehyde, 221 dichromate-formaldehyde, 234, 252 formaldehj^de accelerator, 614 in del RIo-Hortega's method for glio- somes, 591 method for Phytomonas, 511 silver-dichromate method for nervous tissues, 605 Strontium chloride, as ingredient of stains, Mayer's carmines, 301 Mayer's hematoxylin, 292 for differentiating carmine, 61 Strop, for polishing sections, 81, 83 Stropeni's double strains, 357 Stropping microtome knives, 11 Iff Strumia's method for blood, 420 Strychnine sulfate, as narcotic, 265 Studricka's method for reticulum fibers, 594 Stutzer's method for Rickettsiae, 463 Suberized tissue, mentioned in Johansen's quadruple stain, 364 stains for, 391, 393 "Subtriessig fixative," 210 Sucrose, as ingredient of, Highman's mountant, 632; Kleb's mac- erating fluid, 263; Lillie and Ashburn's mountant, 632 Suctoria, 52 Sudan black B, for staining, blood, (Baillif and Kimbrough), 416; blood, (Sheehan), 420; blood, (Sheehan and Storey), 420; fat, (Leach), 448; fat in bacteria, (Burdon, Stokes, and Kim- brough), 491; Golgi bodies, (Baker), 442; Golgi bodies, (McMann), 443; myelin sheaths, (Boissezon), 410; mye- lin sheaths, (Lison and Dagnelle), 410 staining combinations with, azur and eosin, 420; carmine, 442; ethyl eosin, 420; safranin, 491 Sudan brown, in Lillie's method for fat, 449 Sudan III, for staining, blood, (Bacsich), 416; fat, (Daddi), 447; fat glands, (Vrtis), 450; fat and elastic tissue, (French), 448; fungi in tissue scrapings, (Guegin), 504 staining combinations with, anilin blue, 504; hematoxylin, 406; hematoxylin and light green, 391; magenta, 448 Sudan IV, for staining, fat, (Clark), 447; fat, (Herxheimer), 448; fat, (Lillie and Ashburn), 440 staining combinations with hematoxylin and light green, 450 Sugg, see Remy, 483 Suitability of, clearing agents for wax em- bedding, 625, 626 INDEX 783 Suitability of — {continued) universal solvents in paraffin embedding, 626 Suk, see Doherty, 417 Sulfosalicylic acid, as ingredient of San- nomiya's fixative, 236 in Goniori's method for calcified tissues, 596 Sulfur, as ingredient of Hanna's mountant, 638 Sulfuric acid, as ingredient of, Anderson's iron-hematoxj'lin, 285; anon- ymous coelestin blue, 313; fixatives, see under name of other ingredient; Frey's injection fluid, 663; Klebs's macerating fluid, 263; Lendrum's celestin blue, 313; ]\Iichaelis's methylene blue, 318 for cleaning, diatoms, 38, 39 slides, 666 Suprarenal body, Foley's stain for, 354-355 Surface adsorption, in staining, 270 "Susa" fixative, 212 Svihla's double stain, 350 Swan on Berlese's mountant, 631 Swan's mountant, 633 Swank and Davenport on osmic methods for degenerative changes, 528 Swank and Davenport's fixatives, osmic- dichromate-formaldehyde-acetic, 205, 252 osmic-formaldehyde-acetic, 194, 195, 252 in Swank and Davenport's method for degenerative changes, 529 osmic method for degenerative changes, 529 Swartz and Conant's method for fungus in skin scrapings, 505 Swatman, cleaning diatoms, 39 method for disintegrating rock, 38 Sweets, see Gordon, 578, 592 Swegy, see Kofoid, 282 SW 16 accelerator, 615 Sj^mpathetic ganglia, 605 oligoglia in, 586 Schultze's method for, 557 Sympathetic nerve endings, 529 Synonomy of dyes, 3 Synthetic clearing agents, 625, 626 resin mountants, 640-641 Sypkens's fixative, 201, 252 Szatmari's method for nervous tissues, 583 Szecsi's tissue reviver, 515 Sz Gyorgy's fixative, 212 Szepsenwol's fixative, 192, 252 in Szepsenwol's method for nervous tis- sues, 585 Szombathy's adhesive for paraffin ribbons, 659 SzUtz's, alizarin red S, 329 fixatives, 206, 252 in Szutz's technique, 329 Tabias's gum arabic embedding medium, 645 Taenzer, see ITnna, 389 Takahashi's fixative, 201, 253 Tallow, as ingredient of, Bunge's embedding medium, 643; Mar- tin's adhesive, 661; Martin's cement, 656; Seiler's embedding wax, 647 Tanaka, see Tilden, 316 Tandler's injection fluid, 664 Tang, see Hsii, 646 Taniguchi et al. method for Negri bodies, 467 Tannic acid, as ingredient of, AlH's mordant, 515; Bailey's mordant, 517; Bunge's magenta, 481; Casares- Gil's mordant, 517; Cerrito's magenta, 482; David's mordant, 517; Eisath's differentiator, 518; van Ermengen's fixative, 194; Fisher and Conn's ma- genta, 482; Foot's formaldehyde ac- celerator, 613; Fontana's accelerator, 615; Foster's safranin, 392; Fraenkel's acid fuchsin, 321, 492; Gray's mordant, 518; Kulp's magenta, 482; Kolossow's developer, 618; Liefson's magenta; 482, Loffler's indigocarmine-methyl violet, 483; Loffler's magenta, 482; Maneval's magenta, 483; Masson's saffron, 329; Morrison's mordant, 516; Oliver's mor- dant, 518; Ordonez preservatives, 179; Petragnani's mordant, 518; Pittfield's crystal violet, 483; Rawitz's safranin, 314; del Rio-Hortega's accelerator, 615; Romanes's silver diammine stain, 580; Rossi's mordant, 518; Ryo's crystal violet, 484; Salazar's mordant, 518; Shunk's mordant, 519; Trenkmann's mordant, 519; Tribondeau, Tichet, and Dubreuil's crystal violet, 484; Unna's anilin blue, 365; Unna's orange G, 322; Weiss's mordant, 516, 519; Yamanoto's developer, 620; Yokata's mordant, 519; Zettnow's accelerator, 615; Zikes's mordant, 519 in, Achucarro's method for macroglia, 585; Amprino's method for reticulum fibers, 607; Azoulay's method for cerebellum, 609; Bauer's developer, 616; Becker's method for spirochetes, 478; Bensley and Bensley's method for reticulum fibers, 591; Bowhill's method for bac- terial flagella, 481; Foster's method for cell walls of apical meristem, 610; Gutstein's method for bacterial cap- sules, 488; Gutstein's method for nuclei in yeasts, 512; Hoffman's method for spirochetes, 479; Ingleby's method for neurolgia, 586; Maneval's method for 784 INDEX Tannic acid — (continued) in — {continued) nuclei in yeasts, 512; Milovidov's method for proplastids, mitochondria and starch, 450; Moschkovsky's method for blood parasites, 509; Nomec's method for plastids, 450; Nicolle's method for bacteria in sections, 493; Novel's method for l)acterial flagella, 598; Popham, Johnson and Chan's method for cell walls, 393; Renaux and Wilmaers's method for spirochetes, 480; del Rio-Hortega's method for astrocytes, 587; del Rio-Hortega's method for centrosomes, 590; del Rio-Hortega's method for mitochondria, 590; del Rio-Hortega's method for reticulum, 594; Sabrazes's method for spirochetes, 480; Schraorl's method for nuclei, 437; Sclavo's method for bacterial flagella, 484; Unna's method for nuclei, 438 Tartaric acid, as ingredient of Feyrter's thionin, 403 Tartrazine, in, Gomori's method for chro- maffin granules, 430 Lendrum's quadruple contrast, 340 staining combinations with, azocarmine and methyl blue, 430; eosin Y, erythrosin, and phloxine, 340; phloxine, 340 Taste buds, nerve endings in, 569-571 Taylor's, fixative, 201, 253 method for desmid sheaths, 513 smear technique for plant tissues, 74 Teak, softening for sectioning, 92 Teeth, Brain's embedding method for, 649 carious lesions in, 563 decalcifying fluids for, 256-259 embryonic, von Korff's method for, 384 nerve fibers in, 610 nerves in, 534-535, 554 nerves in pulp, 555 Teeth, see also Calcified structures, 581 Tello's decalcifyer, 260 in Tello's method for nerve endings, 557 Tellyesniczky's fixatives, dichromate-ace- tic, 232, 252 in Bell's method for fat, 447 mercuric-picric-acetic, 214, 252 recommended use, 95 Tellyesniczky's fixatives, see also Taiko, 192 Temporary cells, Perruche's varnish for, 654 Tergitol, as ingredient of, hematoxylin stains, 281 MuUer and Chermock's magenta, 316 Terpineol, as ingredient of Becher and J^emoll's Canada balsam, 639 for clearing, chicken eml)r.y()s, 278 wholemounts, 56 physical properties of, 626 Terry's methylene blue, 318 Testis, lymph vessels in, 608 of, grasshopper, 310 rat, 272-275 Tetrachlorethane, as solvent for, Broad- hurst and Paley's methylene blue- magenta, 490 "Tetrachrome" stain, 349 Textile fibers, embedding medium for, 644 Thallium nitrate, in MacFarlandand Daven port's method for nerves in adrenal, 567 Thallophyta, 512 Thermoplastic cements, 654-656 Thiazin dyes, fixatives for, 227 Thiazin-eosinates, acetone for dehydrating after, 345 formulas, see under name of dye general remarks on, 342 Thiazin red, for staining, muscle (Heiden- hain), 423 in, Domagk's double contrast, 325 Neubert's double contrast, 326 staining combination with, picric acid, 325, 326 thionin, 423 Tliiazins, general remarks on, 309 Thiersch's, blue shellac varnish, 654 injection masses, 665, 666 stains, ammonia-carmines, 307 Thionine, for staining, acid-fast bacteria in sections (Spoerri), 498; arthropod blood (Dubuscq), 417; bacteria in plant tissues (Stoughton), 498; blood (Saye), 419; bone (Morpugo), 384; bone (von Recklinghausen), 385; bone (Schmorl), 385; Entamoeba (Mal- lory), 508; mast cells (Levine), 431; mucin (Hoyer), 453; muscle (Heiden- hain), 423; nervous tissue, 401, 402, 403; Nissl granules, 445-447; Rickettsiae (Darzin), 462; Rickettsiae (Laigert and Auburtin), 463; Rickettsiae (Linseer, Fitzpatrick and Hsi), 463; Rickettsiae (Macchiavello), 463 in, Bohm and Oppel's triple stain, 347; Groat's quadruple stain, 349; Houcke's quadruple stain, 352; Houcke's quin- tuple stain, 351; Masson's double stain, 353; Nicolle's phenol solution, 321; Schneidau's double stain, 360; Stough- ton's phenol solution, 318 staining combination with, acid fuchsin, 360; acid fuchsin, methyl- ene blue and toluidine blue, 352; Bis- mark brown, 490; cresyl violet and toluidine bhie, 498; eosin and azur A, 348; eosin Y, 419; erythrosin, light green and orange G, 502; magenta, 463; INDEX 785 Thionine — {continued) staining combination with — {continued) magenta and methylene bkie, 463; ma- genta and picric acid, 478; methylene blue and eosin B, 347; orange G, 431, 498; picric acid, 353; thiazin red, 423 Thionine eosinate, 348 Thionyl chloride, in Kligman, Mescon and De Lameter's method for fungi in skin sections, 504 Thoma's, decalcifying fluid, 2G() ' injection fluid, 664 Thomas's iron-hematoxylin, 286 phosphomolybdic-hcmatoxylin, 293 Thomas, see also Baker, 203, 442; Heller, 403 Thomas and Morris on dichromates in fixa- tives, 187 Thome's triple stains, 357 Thompson's method for erythrocytes, 420 Thompson, see also Hobbs, 508 Thorium nitrate, as ingredient of, Nebel's fixative, 196 in, Krajian's method for Gram-positive bacteria in sections, 494 Thwaite's preservative, 181 Thyme oil, as ingredient of, Apdthy's clearing mixture, 628; Max- well's clearing mixture, 628; Minot's clearing mixture, 628; Schmorl's storage fluid for nitrocellulose blocks, 667 physical properties of, 624 Thyroid, special methods for, 428, 430 types of colloid in, 456 Thysanura, 45 Tibbs, see Diercks, 345 Tilden and Tanaka's magenta, 316 in Tilden and Tanaka's method for acid- fast bacteria in sections, 498 Timocheff's fixative, 203, 253 in Timocheff's method for nerve endings in sex organs, 606 Tin ammonium chloride, as ingredient of Besta's fixative, 236 Tin cells, preparation of, 13, 21 Tin-hematoxylin, 291 Tintinnopsidae, 611 Tissue cultures, silver diammine method for, 598 Togby's method for nuclei, 438 Tolu balsam, as ingredient of Carnoy's cement, 653 Toluene, as ingredient of. Cole's clearing mixture, 628 as solvent for, gum damar, 640; picric acid, 353; rosin, 640 for, clearing, 96 preserving enz_yme extracts, 77 physical properties of, 626 Toluidine blue, for staining, acid-fast bacteria in sections (Spoerri), 498; adrenal (Wiesel), 430; alveolar epitheUum (Bensley and Bensley), 534; blood (Epstein), 417; bone and cartilage (Lundvall), 384; bone and cartilage (William), 386; bone marrow (Ralston and Well), 431; cartilage (Lundvall), 386; cartilage (van Wijhe), 379; cell inclusions (Martinotti), 444; cilia, cirri and basal bodies (Ilorvath), 456; diph- theria bacilli (Albert), 489; diphtheria bacilli (Kinyoun), 489; diphtheria bacilli (Ponder), 490; diphtheria bacilli (Stot- tenberg), 490; granules in Schwann cells (Reich), 457; leprosy bacilli in sections (Baumgarten), 495; macroglia (Benda), 412; mitochondria (Kull), 463; mucin (Lillie), 454; nervous tissue, 401, 402, 403; Nissl granules, 445-447; nuclei (Kedrovsky), 436; Paneth cells (Klein), 452; pituitary (Walraff), 428; Plas- modium (Tomlinson and Grocott), 510; wholemounts (van Wijhe), 386 in, Dominici's triple stain, 351; Dupres's double contrast, 339; Dupres's triple stain, 359; Houcke's quadruple stain, 352; Houcke's quintuple stain, 351; Kull's triple stain, 353; Mann's double stain, 371; Mann's triple stain, 352; Masson's triple stain, 371 staining combinations with, acid fuchsin, 457; acid fuchsin, and aurantia, 353, 394, 443; acid fuchsin, methylene blue and thionine, 352; acid fuchsin and orange G, 351, 359, 452; alizarin red S, 384, 386; azocarmine, 428, 431; azvu* A and methylene blue, 489; azur II, eosin Y, orange G and thionine, 351; cresyl violet and thionine, 498: eosin B, 348; eosin Y and orange G, 351; erythrosin, 371; erythrosin and orange G, 352, 371; hematoxylin and malachite green, 490; magenta, 495; malachite green, 489; methyl green, 489; orange G, 339; orange G and phloxine, 510; orange G and rose bengal, 436; safranin, 430 Toluol, see Toluene de Tomasi's magenta leucobase, 316 in, Pcarse's method for pituitary, 427 Scmmen's and Bhaduri's method for nuclei, 437 Tomlinson and Grocott's method for Plas- modium, 510 Tompkins and Miller's method for fecal smears, 510 Tongue, nerve endings in, 553 sections of, 323-325 786 INDEX Tongue — (continued) taste buds, 569-571 Toning silver stains, general remarks, 543 Toning solutions for metal stains, 620-621 Tonkoff' s spirit blue-iodine, 322 Tooth buds, spirochetes in, 560 Tooth pulp, Orban's method for, 596 Topping's preservative, 181 Toxic neutrophiles, Freifeld's method for, 417 Trachea, insect, injection of, 664 Tragacanth, see Gum tragacanth, 14 Transliteration, rules used, 3 Trenkmann's mordant, 519 in Trenkmann's method for bacterial fiagella, 484 Treponema, 479 Tress and Tress's method for nervous tissues, 411 "Triacid" stain, 354, 356 Tribasic potassium phosphate, see Potassmm phosphate, tribasic Tribondeau's method for bacterial spores, 487 Tribondeau and Dubreuil's method for diphtheria bacilli, 490 Tribondeau, Fichet and Dubreuil's, alum- hematoxylin, 289 method for bacterial fiagella, 470-471, 484 Tricaine methanosulfonate, 395 Trichinella, silver diammine method for, 598 Trichloroacetic acid, as ingredient of decalci- fying fluids, 257-259 fixative combinations with, acetic acid, 190; formaldehyde, 192; formic acid, 190; other combinations, see under name of other ingredient Trichomonas, 507 Tricresyl phosphate, as ingredient of Kirk- patrick and Lendrum's mountant, 641 Triethanolamine, as ingredient of, Zeltnov's silver diammine stain, 580 Triethyl phosphate, physical properties of, 627 Triethylene glycol methyl ether acetate, physical properites of, 627 Triethylene glycol, physical properties of, 623 Trifluoroacetic acid, as ingredient of, Ros- bach and Leavitt's decalcifying fluids, 259 Trinidad asphalt, see Asphalt Trinitrophenol, see Picric acid "Tripiform" fixative, 225 Triple stains, see name of author or ingredient Tristearin, as ingredient of Altmann's em- bedding wax, 646 Triturus, spermatogenesis, 272 staining skeleton, 377-379 Tron's accelerator, 615 Tropeolin, in Noniewicz's method for bac- teria in sections, 493 True's method, for bone in wholemounts, 385 Trypan blue, as ingredient of Hagmann's injection fluid, 664 in McWhorter's method for viroplasts, 466 staining combination with, acid fuchsin and methylene blue, 394 phloxine, 466 Tschassownikow's fixative, 197, 253 Tschernyachinsky's nitroceUulose-wax em- bedding method, 648 Tschernyschen and Karusin's method for myelin sheaths, 407 Tsiminakis's method for Nissl granules, 446 Tsuchiuya's method for fecal smears, 510 Tuan's differentiator, 521 Tubercle bacilli, 469, 504 Tubes, for transferring objects between reagents, 55ff, 61 TunnicUff's method for spirochetes, 481 Turbellaria, 53 Turchini's fixative, 227 Turewitsch's method for Paschen bodies, 467 Turntable, for making cells, 29 use of, 11-12 Turpentine, as ingredient of, Cooke's damar mountant, 640; Cox's sandarac mountant, 638; Gage's clear- ing mixtures, 628; Robin's injection mass, 665; varnishes, 652-654 for clearing, insect skeletons, 64 wholemounts, 56 in, Gatenby's method for Golgi bodies, 530 Ludford's method for Golgi bodies, 531 physical properties of, 624 "2 B" fixative, 202 "2 BE" fixative, 202 Twort's double stain, 372 in OUett's method for bacteria in sections, 493 Tyler's method for bacterial capsules, 488 Tyroglyphid mites, 45 U Ugrimow's method for blood, 420 Ultraviolet rays, for bleaching fish embryos, 384 Umber, as ingredient of Beale's gold size, 652 Ungewitter's developer, 619 in Ungewitter's method for nervous tis- sues, 568 Universal solvents, general remarks on, 626 physical properties of, 626, 627 Unna's differentiator, 521 as ingredient of, Fraenkel's tannin-fuchsin, 321 INDEX 787 Unna's differentiator — {continued) in Fraenkel's method for bacteria in sec- tions, 492 Unna's, methods for, collagen, 395; elastic fibers, 389; fungus in skin sections, 505; mucin, 454; nuclei, 438; skin fibers, 424 stains, anilin blue-orcein, 327 in, Novak's method for Herbst's cor- puscles, 431 Pasini's technique, 338 methyl green-pyronin, 355 methylene blue, 319 in, Assmann's technique, 344; Goth- ard's method for Nissl granules, 445; Harris's method for Negri bodies; Kraus's method for colloid in thyroid, 456; MUovidov's method for bacteria and mitochondria, 444; Schmorl's method for amyloid, 453; Unna's method for mucin, 454; Unna's method for nuclei, 438; Unna's technique, 353; Volkon- sky's method for mitochondria, 444 methylene blue-orcein, 353 picro-fuchsin, 328 safranin-orcein -anilin blue-ethyl eosin, 364 safranin-tannin-anUin blue, 365 tannin orange, 322 in, Langeron's technique, 353 Volkonsky's method for mitochon- dria, 444 " Unna-Pappenheim " method, 493 Unna-TaDzer's, method for elastic fibers, 839 stain, in MoUier's technique, 366 Unna and Golodetz on oxidation-reduction in fixatives, 188 Upson's method for axis cylinders, 541 Uranium acetate, as ingredient of, Foley's accelerator, 614; Kolmer's fix- ative, 235; Schiller's fixative, 218 in Polak's method for neuroglia, 587 Uranium chloride, as ingredient of Nebel's fixative, 198 Uranium nitrate, as ingredient of, Benoit's fixative, 198, 219; Cajal's ac- celerators, 613; Cajal's fixative, 236; Keife's preservatives, 180; Kingsbury and Johannsen's formaldehyde accel- erator, 613; Kolossow's fixative, 205; Nebel's fijcative, 198; Oliveira's de- veloper, 619; Schmaus's carmine, 307; Steiner's alcoholic accelerators, 614, 615; Wilder's developer, 620 in. Chilesotti's method for axis cylinders, 410; Dieterle's method for spirochetes. Uranium nitrate — (continued) in — (continued) 560; Hertzman's method for spiro- chetes, 561; Jahnel's method for nerve cells and processes, 555; Jahnel's meth- od for spirochetes, 561; Lillie's method for reticulum fibers, 593; Oliveira's method for reticulum, 657; Para's method for spirochetes, 562; Steiner and Steiner's method for spirochetes, 563; Wilder's methods for reticulum, 595 Uranium sulfate, in Armuzzi and Stempel's method for spirochetes, 560 Uranium-carmine, 307 Urea, as ingredient of, Allen's fixatives, 223, 226; Carother's fixative, 224; Eberspacher's difi'erenti- ator, 520; Kostoff and Kendall's fixa- tives, 220; LaCour's fixatives, 202; Painter's fixative, 227; Penfield's fixa- tives, 236; Skovsted's fixatives, 199; Winge's fixative, 214 Urea nitrate, as ingredient of de Castro's fixatives, 190 Urechia and Nagu's method for reticulum fibers in nervous tissue, 595 Urethan, as ingredient of de Castro's de- calcifying fluids, 257 as narcotic, 52 Uyama's developer, 620 in Uyama's method for retina, 557 Vacuum ovens, 99 Vaginal smears, 430, 431, 432 Rickettsiae in, 463 Vanadium chloride, as mordant for Wolters's hematoxylin, 408 Vanadium-hematoxylin, 291 Vaney, see Meunier, 513 Various formulas, classification of, 650 general remarks on, 650 Varnish, asphalt for sealing dry whole- mounts, 16 optical dead black, 656 Varnishes, 652-654 aqueous, 652 general remarks on, 651 removing from slides, 666 Vassale's method for myelin sheaths, 408 Vassale, see also Buzzozero, 435 Vassale and Donaggi's fixative, 235, 253 silver-dichromate method for nervous tis- sues, 605 Vassos, see McDowell, 658 Vastarine-Cresi's, chrome mordant, 517 method for glycogen, 453 Vaughn's method for fungus in plant tissue, 502 788 INDEX Vaulx's stain for chitin, 390 Veins, injection in cliickon embryo, 168 Velvet, for polisliing sections, 80 Venetian soap, 446 Venice turpentine, as ingredient of, Gage's cement, 655; Gage's varnish, 653; Robin's cement, 654; sealing wax, 652; Vesseler's wax for dishes, 667 evaporation technique, 66 mountants, 637-638 mounting in, 66 testing for purity, 65 wholemount of alga in, 64-66 Veratti's fixative, 196, 252 in Golgi-Veratti's method for Golgi bodies, 608 Verhoeff's, fixative, 223 methods for, elastic fibers, 389 Leptotriches in sections, 495 Verne's method for myeUn, 411 Verocay's, fixative remover, 256 method for collagen, 395 "Versenate," 260 '"Versene," 260 "Vesuvelin," 390 Viallane's, fixative, 194, 253 method for nerves in arthropods, 541 Vialleton's fixative, 227, 253 Vials, seaHng fluid for, 667 Victoria blue, for staining, actinomycetes, (Morel and Dulans), 505; neuroglia, (Anderson), 412; neu- roglia, (Auglade and Morel), 412; neu- roglia, (Lehrmitte and Guccione), 414; neurogha, (Merzbacher), 414; spiroche- tes, (Goldsworthy and Ward), 479; spiro- chetes, (Keil), 479; spirochetes, (Lipp), 480; spirochetes, (Muhlpfordt), 480 Victoria blue 4R, staining combinations with, hematoxylin and methyl violet, 505 methyl green and pyronin, 479, 480 Victoria yellow, in Semichon's triple con- trast, 329 Viesseler's wax for dishes, 667 Vignal's picro-carmine, 304 Villain and Comte's method for Plasmodium, 510 Violamine R, in Lillie's double stain, 370 Virchow's fixative, 229, 253 Viroplasts, methods for, 466 Virus bodies, special methods for, 464-467 Viscosity, of nitrocellulose, 647 Vlakovic's fixative, 227, 253 Vogt and Jung's Canada balsam-damar mountant, 640 Volker's fixative, 214, 253 Volkman and Strauss's triple stain, 373 Volkonsky's, double stain, 319 method for mitochondria, 444 Volkonsky's — (continued) narcotic, 266 Vortex, 53 Vorticella, 53 narcotics for, 265 Voseler, on ringing Venice turpentine mounts, 66 Venice turpentine mountants, 637 Vrtis's method for fat glands in rodent skin, 450 Vulcanite cells, 21 attaching to slide, 19 Harting's cement for, 656 W Waddington's narcotic, 266 Wadsworth's methods for bacterial cap- sules, 489 de Waele's fixative, 198 Waldeyer's decalcifying fluid, 260 Walgren's method for nerve cefls and proc- esses, 557 Walker, see Geschickter, 348 Wallace's method for basal bodies, 458 Wallart's, alcoholic accelerator, 615 silver diammine stain, 576, 580 in Wallart's method for fatty tissues, 585 Wallart and Honette's double contrast, 339 Wallraff's method for pituitary, 428 van Walsem's embedding wax, 647 fixative, 226 Walter's quadruple stain, 361 Walton's method for gonococcus, 491 Warbritton, on neutralizing formaldehyde, 190 Ward, see Clark, 404; Goldsworthy, 479 Warm table, for attaching cells, 15ff, 22 Warthin's fixative, 236 in W^arthin's method for spirochetes, 563 Warthin, see also Farrier, 561 Warthin-Starry's developer, 620 in Warthin-Starry's method for spiro- chetes, 563 Washing, apparatus for, 129 Water, solubility in, clearing agents, 625, 626 "universal" solvents, 626 Water-immersion objectives, for controlling differentiation, 275 Water miscible, embedding media, 642-644 mountants, 631-637 Water stones. 111 Waterglass, 660 as ingredient of David's adhesive for paraf- fin ribbons, 657 Waterman on dioxane dehydration, 359 Waterman's, embedding wax, 647 fixatives, cupric-paranitro phenol, 221 picric-formaldehyde-acetic, 224 INDEX 789 Waterman's — (continued) triple stain, 361 \\'atho(l for pollen tubes, 122 \\'atson on ripening hematoxjdin, 284 Watson's acid-alum hematoxylin, 290 Wax embedding media, 615-G47 Wax sectioning technicjues, see Paraffin Weatherford's method for Golgi bodies in calcified structures, 559 Webb's dextrin embedding medium, 645 Weber's, alcoholic accelerator, 615 developer, 620 fixative, 231 silver diammiue stain, 576, 580 in Weber's method for nervous tissue, 585 Wehrle, see Mohr, 638 "Weigert-Pal" techniques, 403 Weigert's, clearing mixtures, 628, 629 differentiator, 520 in, Alzheimer's method for nervous tis- sue, 401; Aronson's method for ner- vous tissues, 409; Bauer's method for neuroglia, 412; Benda's method for fat, 447; Bensley and Bensley's tech- nique, 283; Bensley's method for canaliculi in plant cells, 454; Clara's method for bile capillaries, 422; Dietrich's method for fat, 447; Eppinger's method for bile capillaries, 423; Fischler's method for fat, 448- Harvey's method for parietal cell granules, 455; Kockel's method for fibrin, 423; Kraus's method for pitui- tary, 426; MacCallum et al. method for pituitary, 426; Meyer's method for neuroglia, 414; Mitrophanov's method for myelin sheaths, 406; Parat's method for mitochondria, 444; Potter's method for macroglea, 414; Roehl's method for calcareous deposits, 385; Weigert's method for myelin sheaths, 408; Weigert's method for neuroglia, 415 fixative, 231 in Weigerts' method for myelin sheaths, 408 " Mann-Kopsch " technique, 531 methods for, elastic fibers, 390; fibrin, 425; Gram- positive bacteria in sections, 495; myelin sheaths, 408; neuroglia, 415 primary mordant, 397, 516, 517 in, Alzheimer's method for nervous tis- sue, 401; Alzheimer's method for neuroglia, 412; Anderson's method for myelin sheaths, 404; Anderson's mordant, 515; Eppinger's method for Weigert's — (continued) primary mordant — (continued) in — (continued) bile capillaries, 123; de Galantha's method for kidney, 423; Heller- Robertson's method for myelin sheaths, 406; Meyer's method for neuroglia, 414; Potter's method for macroglea, 414; Weigert's method for neuroglia, 415 before, acid fuclisin-phosphomolybdic techniques, 359 stains, iron-hematoxylin, 286 in, Adam's method for acid-fast bac- teria in sections, 495; Bacsich's method for blood, 416; Barreto's method for Negri bodies, 464 I'^oot's silver method for reticulum 592; Goldner's technique, 337 Highman's method for amyloid 452; Ladewig's technique, 365 Laidlaw's method for intracellular "organisms," 461; Lillie's method for glycogen, 452; Lillie's method for reticulum fibers, 424; Lillie's techniques, 337, 366, 370; Meyer's method for neuroglia, 414; Mollier's technique, 366; Perdran's method for reticulum, 594; Potter's method for macroglea, 414; Roehl's method for calcareous deposits, 385; Romeis's technique, 367; Schaffer'g method for fat, 449; Schmorl's method for elastic fibers, 389; Wallart and Honette's technique, 339 lithium-hematoxylin, 408, 409, 415 in, Clark and Ward's method for ner- vous tissue, 404; Fischler's method for fat, 448; Kockel's method for fibrin, 423; Pol's method for myelin sheaths, 407 magenta, in, French's method for fat and elastic tissue, 448; Hart's method for elastic fibers, 388; Romeis's method for pituitary, 427; Romeis's technique, 367; Schmorl's method for elastic fibers, 389; Vastarini-Cresi's method for glycogen, 453 picro-carmine, 304 picro-fuchsin, 328 in Schmorl's method for elastic fibers, 389 "VesuveHn," 390 in Schmorl's method for elastic fibers, 389 Weigl's method for Golgi bodies, 531 790 INDEX Weil's, differentiators, 520 in, O'Leary's method for nervous tis- sues, 411 Weil's methods for myelin sheaths, 408 iron hematoxylin, 408 in, Lillie's method for myelin sheaths, 406 method for dentine, 385 Weil and Davenport's silver diammine stain, 576, 580 in Weil and Davenport's method for microglia, 589 Weiss's, method for, acid-fast bacteria, 478; Gram-positive bacteria, 475; polar bodies in bacteria, 491; spirochetes, 481 mordant, 516, 519 in, Weiss's method for acid-fast bac- teria, 478 Weiss's method for spirochetes, 481 triple stain, 336 Weissberger, see Grapnauer, 193 WeUheim's iron carmine, 305 Wellheim, see also Pfeiffer, 65 Welling's decalcifying fluid, 260 Wells, see Ralston, 259, 431 Wenrich and Geiman's fixative, 210, 253 Wess, see Gray, 636, 641 Westphal's, double stains, carmine-crystal violet, 373 method for blood, 420 Wetting agents in, injection media, 162 stains, 281, 316 Wetzel's fixative, 225 Wetzel, see also Reichardt, 648 Wharton's method for nerves in whole- mounts, 408 Wheatley's method for intestinal protozoa, 339 White's, decalcifying fluid, 260 double contrast, 326 White and Culbertson's method for Gram- positive bacteria, 475 White cedar oil, physical properties of, 624 White lead, as ingredient of, Beale's gold size, 652; Coburn's cement, 655; Kitton's cement, 654; Oschatz's cement, 656 White pine, 92 White rouge, use in polishing sections, 81, 83 White shellac, 651 White thyme oil, as ingredient of, Dunham's clearing mixture, 628 Whitehead's fixative, 234, 253 Whitman's fixative, 195 Whitman, see also Eisig, 207 Whitney's fixative, 217 Wholemount of, alga in Venice turpentine, 64-66 Wholemount of — (continued) Foraminifera, 17-20 48 hr. chicken embryo, 275-278 insect in deep cell, 64 insect skeleton, 62-64 liver fluke, 294-296 medusa, 296-299 Microcystis, 27-29 mite, 43-45 muscle to show nerve endings, 532-533 Pectinatella, 59-62 pollen grains, 309-310 Radiolaria, 17-20 Rotifer, 29-31 salamander, 377, 379, 380 Wholemounts, aqueous, 21-23 cell cements for, 21 cells for, 21 coverslip cements for, 23 Hanley's method of sealing, 31 preservatives for, 23 sealing coverslip on, 24, 25ff, 26ff, 27 specific examples, 27-31 capillaries in, 421 collagen fibers in, 394 crustacean muscles in, 394 definition of, 7 differentiating bone and cartilage in, 382, 386 double stains, 360, 370, 371 dry, 10-20 attaching cells for, 13 backgrounds for, 13-14 cementing, coverslips to, 16 objects in, 14-16 definition, 10 sealing, 16 selection of, cells for, 11-12; coverslip for, 11; slides for, 10 wooden slides for, 10 flattening specimens for, 60 fluid, media for, 32 Golgi bodies in, 559 in glycerol, cements for, 32 sealing with thermoplastic cements, 33, 34ff specific example, 35 in, glycerol jelly, 46-50 gum media, 42-45 nonaqueous fluids, 32-41 resinous media, 51-68 applying coverslip to, 57, 58ff cells for, 57 clearing, 56 dehydration, 55ff finishing, 57 narcotizing and fixing specimens for, 51-54 ringing, 58 INDEX 791 Wholemounts — (continued) in — (continued) resinous media — (continued) stains for, 54 Juge's method for bone and cartilage in, 383 Lundvall's method for bone and carti- lage, 384 Lundvall's methods for cartilage in, 386 Menner's method for gangha in, 410 Miller's method for cartilage in, 387 NoUister's method for bone for, 384 of, annelida, 53 Bryozoa, 54 coelenterata, 53 insects, 56 oligochaetes, 53 plant material, 55 platyhelminthes, 53 protozoa, in aqueous media, 23 protozoans, 52 vertebrate embryos, 54 pollen tubes in, 421, 422 Rait's method for bone in, 384 sebaceous glands in, 422 staining, capillaries in, 432 fat glands in, 450 insect muscles in, 424 nerves in, 406, 407, 408 perivascular nerves in, 567 technique of staining specimens, 61, 62 True's method for bones in, 385 undesirability of staining, 270 van Wijhe's method for cartilage in, 386 Williams's method for bone and cartilage in, 386 Whole organs, recommended fixatives for, 95 Wicks, Carruthers and Ritchey's mountant, 641 Wiesel's, fixative, 234 in Wiesel's method for adrenal, 430 method for adrenal, 430 van Wijhe's method for, cartilage in whole- mounts, 386 cartilagenous skeleton, 379-380 Wilder's developer, 620 in Wilder's method for reticulum, 595 Wilhelm, see Osgood, 419 fixative, 192, 253 Wilhelmine's double contrast, 328 Wilkes on decalcification, 256 Willard's, developer, 615 in Willard's method for adrenal, 557 Willebrand's double stain, 345 Williams's, cresyl violet, 373 method for, bone and cartilage, 386; bony scales, 386; Negri bodies, 467 Williams, see also Pfaff, 432 Williamson and Pearse's fixative, 215 Williamson and Pearse's fixative — (con- tinued) in Williamson and Pearse's method for thyroid, 430 Wilson's decalcifying fluid, 260 method for fat, 450 varnish for knife edges, 667 Venice turpentine mountant, 638 "Win-3000" for decalcification, 256 Windle, see also Davenport, 237, 554, 567, 577; Koenig, 190 Windle's, fixative, 204, 253 in Windle's method for embryonic brains, 606 silver-osmic-dichromate method for nerv- ous tissues, 602-603 Windle, Rhines and Rankin's method for Nissl granules, 447 Windleholz's adhesive for free sections, 660 Winge's fixatives, chromic-formaldehyde- acetic, 231, 253 mercuric-picric-acetic, 214 in Winge's method for nuclei, 438 de Winiwarter's fixative, 201, 253 de Winiwarter and Sainmont's double stain, 341 Winkler's method for microglia and plasma cells, 589 Wintergreen oil, physical properties of, 624 Wistinghausen's fixative, 223, 253 Witt on theory of staining, 270 Wittmaak's fixatives, 235, 253 in Fieandt and Sazen's method for Golgi bodies, 590 Wlassow's fixatives, mercuric-dichromate, 216, 253 osmic-dichromate, 204, 253 Wohlfart, see Rexed, 360 Wolbach's, differentiator, 521 in, Douglas's method for acid-fast bac- teria in sections, 496; Hayne's tech- nique, 348; McNamara's technique, 349; Mallory's technique, 346; Pap- penheim's technique, 350; Wolbach's method for Rickettsiae, 464 fixative, 216 in, Wolbach's method for Rickettsiae, 464 Wolbach, see also Hertwig, 463 Wolfe, see Clancy, 462; Cleveland, 425 Wolf's nitrocellulose embedding method, 649 Wolff's silver diammine stain, 576, 580 in Studricka's method for reticulum, 594 Woltereck's fixative, 210, 253 Welter's, fixative, 221 in, van Wijhe's method for cartilage, 379 Wolter's method for myelin sheaths, 409 hematoxylin, 283 792 INDEX Wolter's — {continued) hematoxylin — (continued) in Wolter's method for myelin sheaths, 408 vanadium aluminum mordant, 517 Womersley's mountant, 633 Wood alcohol, see Methanol, 623 Wood blocks, as microtome accessory, 113 for mounting nitrocellulose blocks, 145, 150 Wood, Crowell's method for sectioning, 93 cutting sections of, 92-93 embedding medium for, 644 fungus in sections of, 501 isolation of fibers from, 263 microtome for cutting, 93 slides, manufacture of, 10-11 softening before sectioning, 92-93 technique of cutting sections, 93 Wood's, glycerol jelly, 635 preservative, 176, 177 for aqueous wholemounts, 23 Woodhead's cement, 654 Wool green FCF, in Lillie's double stain, 370 Wool green S, in Lillie's quadruple stain, 366 Woolman's methods for spirochetes, 481 Worcester's fixative, 212, 253 World List of Scientific Periodicals, use of numbers from, 2 Worley, see Alcorn, 511 Wormseed oil, 624 Woronin's osmic stain for epithelia, 530 Wotton and Zwemer's chrome gelatin mountant, 636 Wright's, method for, megakaryocytes, 432 myelin sheaths, 409 stains, iron-mordant hematoxylin, 409 polychrome methylene blue-eosin, 347 in, Brice's method for oxidase granules in erythrocytes, 455; Churchman and Emelianoff' s method for bacte- rial capsules, 487; Cunningham's method for reticulocytes, 417; But- ton's method for bacterial spores, 485; Hausburg's method for Xissle granules, 445; Lawson's method for bacterial capsules, 488; Liel)mann's method for invertebrate blood, 418; Schleicher's method for bone mar- row, 432; Steil's method for blood, 420; Tsuchiuya's method for intes- tinal protozoa, 510; Wright's method for megakaryocytes, 432 Xylene, as ingredient of, Cole's clearing mixture, 628; Gage's clearing mixture, 628; Gatenby and Xylene — (continued) Painter's clearing mixture, 628; Goth- ard's dehydrating mixture, 628; Max- well's clearing mixture, 628; Nuttall's picric acid, 321; del Rio-Hortega's clear- ing mixture, 628; Weigert's clearing mixture, 628, 629 as solvent for, Canada balsam, 639 stains, 321 for, clearing before embedding, 96 de-waxing paraffin sections, 120, 123, 124 phj'sical properties of, 626 Xylene-aniline, see Aniline-xylene Xylene-balsam, 639 Xylol, see Xylene Yamanoto's developer, 620 in Yamanoto's method for spirochetes, 563 Yamanouchi's fixatives, chromic-acetic, 228 osmic-chromic-acetic, 201, 253 method for chlorophyceae, 513 Yao-Nan's fixative, 231 Yasvoyn's iron hematoxylin, 286 Yasvoyn, see also Jasswoin Yeager, see Alcorn Yeasts, 512 Yellow ochre, as ingredient of Beale'sg old size, 652 Yetwin's glycerol jelly, 635 Yokata's mordant, 519 in Yokata's method for bacterial flagella, 484 Yokum's fixative, 215 Yolk, Smith's stain for, 326 Yolk granules, 447-450 Young, on osmotic pressure of fLxatives, 187 Yucatan elemi, see Gum elemi Zacharias's, aceto carmine, 302 fixative, 194, 253 Zapata, see Proescher, 372 Zaribnecky's method for bacteria in milk, 492 Zebrowski on strychnine as narcotic for roti- fers, 265 Zeeti's method for bacterial spores, 487 Zeissig's method for Gram-positive bacteria in sections, 495 Zeitschmann's fixative, 204, 253 Zenker's fixative, 217, 253 as ingredient of, Eichorn's mordant, 515 Ralston and Wells's decalcifying fluid, 259 for, leeches, 54; mouse head, 334; rabbit liver, 472 INDEX 793 Zenker's fixative — (continued) in, Assmann's technique, 344; Bauer's method for neuroglia, 412; Doneggio's method for nerves, 404; Einarson's method for Nissl granules, 445; Foot's method for reticulum, 592; Galantha's method for kidney, 423; Gluck's method for reticulum fibers, 592; Ooodpasture's method for Negri bodies, 405; Hay- thorne's method for aciil-fast bacteria in sections, 497; llaythorne's technique, 337; Hewitt's method for avian blood parasites, 508; Langeron's Giemsa tech- nique, 348; Levi's method for reticulum, 593; McNamara's technique, 349; Mal- lory's method for fil)roglia fibrils, 456; Mallory's method for Gram-positive bacteria in sections, 495; Mallory's technique, 292; Masson's technique, 371 recommended use, 95 "Zenker-formol," 217, 218 Zettnow's, accelerator, 615 in, Craigie's method for bacterial smears, 598 silver diammine stain, 576, 580 in Zettnow's method for bacterial flagella, 598 Zhoskin's method for reticulum, 595 Ziegler's method for capillaries in cornea, 421 Zieglwallner's, fixative, 197, 253 method for fat and glycogen, 451 Ziehen's gold-mercur^y stain, 538 in Ziehen's method for axis cylinders and dendrites, 539 Ziehl's magenta, 316 in, Aladar-Anjeszk3''s method for bacterial spores, 485; Alcorn and Worley's method for Erysiphaceae, 511; Anjeszky's method for bacterial spores, 485; Bailej^'s method for bacterial flagella, 481; Bardelli and Cille's method for Zymonema, 503; Baumgarten's method for leprosy bacilli in sections, 495; Becker's method for spirochetes, 478; Besson's method for bacterial spores, 485; Boni's method for bacterial cap- sules, 487; Casares-Gil's method for bacterial flagella, 482; Castraviejo's technique, 369; l^eipolli and Pomerri's method for Xissl granules, 445; Dor- ner's method for bacterial spores, 485; Fisher and Conn's method for bacterial flagella, 482; Fraenkcl's method for bacterial spores, 485; Frief eld's method for toxic neutrophiles, 417; CJallego's method for Negri bodies, 465; Gallego's technique, 315; Gausen's technique, 351; Gay and Clark's method for dead Ziehl's — (continued) in — (continued) bacteria, 491; Gerlach's method for Negri bodies, 465; Giacomi's method for spirochetes, 479; Gray's method for bac- terial flagella, 482; Gutstein's method for ascospores in yeasts, 512; Hana- zava's method for dentine, 383; Hun- toon's method for bacterial capsules, 488; Kahlden and Laurent's method for bacterial spores, 486; Jvrajian's metliod for bacteria in sections, 492; Krajian's method for Gram-positive bacteria in sections, 494; Kuff'crath's method for ascospores in yeasts, 512; Lageberg's method for bacterial spores, 486; Langrand's method for actinomyces, 504; Launoy's method for pancreas, 429; Lepine's method for Rickettsiae, 463; Lipp's method for spirochaetes, 480; Lopez's technique, 371; Maneval's method for bacterial flagella, 483; MaA-'s method for bacterial spores, 486; MoUer's method for bacterial spores, 486; Muir's method for bacterial cap- sules, 488; Muir's method for bacterial flagella, 483; Nakamura el al. method for Negri bodies, 467; Nicolle and Morax's method for bacterial flagella, 483; Oliver's method for "flagella" on erythrocytes, 457; Perrin's method for spirochetes, 480; Proca's method for bacterial spores, 486; Renaux and Wil- maers's method for spirochetes, 480; Rossi's method for bacterial flagella, 483; Russel's method for cell inclusions, 457; Saboraud's method for spirochetes, 480; Sabrazes's method for spirochetes, 480; Schmorl's method for elastic fibers, 389; various methods for acid-fast bacteria, 476; various methods for acid-fast bacteria in sections, 495-498; Ziehl's method for bacterial smears, 474 ; Zottner's method for Negri bodies, 467 " Ziehl-Neelsen " methods, 476, 477 Zieler's method for May-Griinwald stain, 345 Zikes's method for bacterial flagella, 483 mordant, 519 Zilliacus's fixative, 213 Zimmer's fixative, 222 Zinuuerman on purification of Venice tur- pentine, 65 Zimmerman's fixatives, chromic-acetic, 229 picric-nitric, 222 green shellac varnish, t)5 1 methods for, attaching free sections, 600; gastric gland cells, 432; reticulum fibers, 494 794 INDEX Zinc acetate, as ingredient of Miiller's Bis- mark brown, 429 Zinc chloride, as ingredient of, Casares-Gil's mordant, 517; Gardiner's preservatives, 179; Sharman's safranin, 393 fixative combinations with, acetic acid, 235; acetic and nitric acids 236; dichromate and acetic acid, 235 dichromate and formaldehyde, 235 formaldehyde, 236 Zinc oxide, as ingredient of, de Groot's gelatin cement, 654; James's cement, 653; Woodhead's cement, 654 Zinc sulfate, as mordant in Krajian's method for bacteria in sections, 492 Zinsser and Bayne-Jones's method for Rickettsiae, 464 Zinsser, Fitzpatrick and Hsi's method for Rickettsiae, 463 Zirconium oxychloride, as mordant in Gelarie's method for spirochetes, 479 Zirkle, on pH of fixatives, 188 Zirkle's, butanol schedule, 629 carmine-balsam, 640 carmine-dextrin mountant, 637 Zirkle's — (continued) carmine-gelatin mountants, 635 carmine-pectin mountants, 636 carmine-pectin mountant, 637 carmine- Venice turpentine mountant, 638 fixatives, cupric-chromic-formaldehyde, 220, (in, Newcomer's method for plant mito- chondria, 531); cupric-formaldehyde- proprionic, 219 gum arable mountant, 633 orcein-gelatin mountant, 635 Zoothamnion, 53 Zottner's method for Negri bodies, 467 Zwaademaker's safranin, 315 in, Henneguy's technique, 364 Launoy's method for pancreas, 429 Zweibaum's fixative, 202, 253 in Leach's method for fat, 448 Zwemer's, chrome-glycerol jelly, 636 gelatin embedding method, 645 glycerol jelly in Bacsich's method for blood, 416 Zwemer, see also Wotton, 636 Zymogen granules, 428 Zymonema, 503