r LT) m un i; s D m THE MICROTOMIST'S VADE-MECUM BOOKS ON THE MICROSCOPE Critical Microscopy. How to get the Best out of the Microscope By A C. COLES, M.D., D.Sc., F.R.S. (Edin.) 8 Illustrations. 7.9. 6d. net. The Microscope and its Revelations By W. B. CARPENTER, F.R.S. Edited by W. H. DALLINGER, F.R.S. Eighth Edition. 23 Plates and 800 Text Figures. 1 Vol., 36-9. net. Separate Volumes, 20.s. net each. • J. & A. CHURCHILL THE MICROTOMIST'S VADE-MECUM A HANDBOOK OF THE METHODS OF MICROSCOPIC ANATOMY BY ARTHUR BOLLES LEE, Hon. F.R.M.S. EIGHTH EDITION EDITED BY J. BRONTE GATENBY, B.A., B.Sc., D.Phil. (Oxon.), D.Sc. (Lond.), F.R.M.S., SOMETIME LECTURER IN HISTOLOGY, OXFORD; LECTURER IN CYTOLOGY AND SENIOR ASSISTANT IN ZOOLOGY, UNIVERSITY COLLEGE, LONDON, AND SENIOR DEMY, MAGDALEN COLLEGE, OXFORD. With the collaboration of W. M. BAYLISS, M.A., D.Sc. (Oxon.), F.R.S., F.R.M.S., PROFESSOR OF GENERAL PHYSIOLOGY IN UNIVERSITY COLLEGE, LONDON ; G. DA FANO, M.D., F.R.M.S.,- L.D. ON MORBID ANATOMY, UNIVERSITY OF PAVIA (ITALY) ; LECTURER IN HISTOLOGY, KING'S COLLEGE, LONDON; A. DREW, D.Sc. (Lond.), F.R.M.S., IMPERIAL CANCER RESEARCH FUND, LONDON ; W. CRAMER, Ph.D., D.Sc., M.R.C.S., L.R.C.P., F.R.M.S., IMPERIAL CANCER RESEARCH FUND, LONDON ; AND J. THORNTON GARTER, F.R.M.S., F.Z.S., HON. RESEARCH ASSISTANT, UNIVERSITY COLLEGE, LONDON. LONDON J. & A. CHURCHILL 7, GREAT MARLBOROUGH STREET 1921 PREFACE TO EIGHTH EDITION. IN the preparation of this new edition of Dr. Bolles Lee's well- known book I have received the assistance of Professor W. M. Bayliss, Dr. C. Da Fano, Dr. A. Drew, Dr. W. Cramer, and Mr. J. Thornton Carter. It has been almost entirely due to these workers that my plans for the new edition have been able to be carried out in the way I wished. Thanks to them, this book may be considered a most complete and stimulating book of reference for the research worker. Professor W. M. Bayliss, to whom I am especially grateful, has rewritten Chapter XI. on " Staining." Dr. C. Da Fano has revised, and in some cases almost completely rewritten, the difficult chapters on " Neurological Techniques," which constitute indeed a special branch of microtomy. Dr. A. Drew has completely rewritten the valuable section on " Protozoa " ; while from Dr. W. Cramer's pen has come most of the article on " Fatty Substances," which will bring to the notice of embryologists and histologists the newest advances in the micro-chemistry of this subject. Mr. J. Thornton . Carter has revised the section on " Teeth and Bone." I have personally been responsible for the rest of the book, besides having written special chapters or sections on " Chromatin, Chromosomes, Nucleoli, Glycogen, Iron, Yolk, Fat, Mitochondria, Golgi Apparatus and Benzidine Dyes " ; I have completely rewritten the section on "Mammalian Embryological Methods." In the sections dealing with the cytoplasmic inclusions, most of the various structures, known as "attraction sphere rodlets," " idiozome, " " Golgi-Kopsch apparat," " nebenkern batonettes," etc., are grouped under the term " Golgi apparatus," as now seems justifiable in view of the results of modern researches on the subject. A small, yet very important, addition to the present volume has been the inclusion of two new methods for staining bacteria in tissue (§§ 089, 761). The histologist and cytologist are often puzzled to know whether certain enigmatic bodies they find are or are not bacterial in nature. vi PREFACE. An addition which is likely to be welcomed, and to make the book more useful, is a special chapter for students (Chapter XXXVII. ). I had especially desired to have a chapter dealing with Tissue Culture," and had asked my friend Mr. H.'M. Carleton, of Oxford, to undertake it ; but as illness prevented his carrying out the work, I was myself obliged at the eleventh hour to write the article. Dr. A. Drew was most helpful here, and Dr. Strangeways, of Cambridge, lent me some literature on the subject. For advice and encouragement in this arduous task I have to thank my many friends. Dr. Bolles Lee sent me his blessing and three hundred references ; Professor J. P. Hill placed his note- books and experience at my disposal ; Dr. J. A. Murray, of the Cancer Research Fund, proved a perfect mine of suggestions, and I dug freely at this source. I am very grateful to both Dr. Murray and his amiable staff, of wrhom Dr. Drew and Dr. Cramer have helped me most. Professor Starling, of University College, Pro- fessor E. S. Goodrich, of Oxford, Professor Sherrington, of Oxford, Professor Boycott, of University College Medical School, Professor E. B. Wilson, of Columbia University, and Professor D. M. S. Watson, of University College, London, helped me in one way or another. Professor W. M. Bayliss and Dr. Oscar Brady gave me valuable information on certain chemical aspects. My friends at the Rothamsted Station, especially Mr. Ward Cutler and Dr. Imms, were very helpful. Dr. Allen, of Plymouth, and Dr. Orton kindly answered inquiries. Great care has been taken in the preparation of the Index, and I have to thank my sister for much assistance. In writing a book of this kind, it is difficult at times to ascertain exactly who did originate some one method, and if we have occa- sionally overlooked the original source we will be glad to have such lapsus calami rectified in some future edition. Finally, it must be remarked that the arrangement and method of exposition of the new material closely follows that of Dr. Bolles Lee? while the bulk of the book is still largely due to him. J. BRONTE GATENBY. UNIVERSITY COLLEGE. LONDON. CONTENTS. PAET I. PAGE CHAPTER I. INTRODUCTORY ......... 1 * CHAPTER II. KILLING . . . . . . . . . .11 CHAPTER III. FIXING AND HARDENING . . . . . . .18 CHAPTER IV. FIXING AND HARDENING AGENTS — MINERAL ACIDS AND THEIR SALTS .......... 29 CHAPTER V. FIXING AND HARDENING AGENTS — CHLORIDES, ORGANIC ACIDS, AND OTHERS ......... 44 CHAPTER VI. DE-ALCOHOLISATION AND CLEARING AGENTS .... 65 CHAPTER VII. » IMBEDDING METHODS — INTRODUCTION . . . . .71 CHAPTER VIII. IMBEDDING METHODS : PARAFFIN AND OTHER FUSION MASSES . 76 Paraffin, 76 ; Gelatin, 92. CHAPTER IX. COLLODION (CELLOIDIN) AND OTHER IMBEDDING METHODS . 95 Collodion or Celloidin, 95 ; other Cold Masses, 106 ; Grind- ing Masses, 108 ; Freezing, 109. viii CONTENTS. PAGE CHAPTER X. SERIAL SECTION MOUNTING . . . . . . .111 Methods for Paraffin Sections, 111 ; Methods for Watery Sections, 116 ; Methods for Celloidin Sections, 116. CH AFTER XT. STAINING .......... 120 CHAPTER XII. CARMINE AND COCHINEAL STAINS . . •. . . .135 Theory of Carmine Staining, 135 ; Aqueous Carmines, Acid, 136 ; Neutral and Alkaline, 140 ; Alcoholic Carmines and Cochineals, 141. CHAPTER XIII. H,EMATEIN (H^MATOXYLIN) STAINS . . . . .145 Theory of Staining with Haematoxylin, 145 ; Iron-haematem Lakes, 147 ; Aluminium -hsematein Lakes, 151 ; other HaBina- tei'n Compounds, 155. CHAPTER XIV. NUCLEAR STAINS WITH COAL-TAR DYES . . . . .159 Progressive Stains, 159 ; Regressive Stains, 162. CHAPTER XV. PLASMA STAINS WITH COAL-TAR DYES . . . . .171 CHAPTER XVI. METHYLEN P>LUE 186 r CHAPTER XVII. METALLIC STAINS (IMPREGNATION METHODS) . . . .197 Silver, 198 ; Gold, 202 ; other Metallic Stains, 208. CHAPTER XVIII. OTHER STAINS AND COMBINATIONS . . . . . .211 Carmine Combinations, 212 ; Hsematein Combinations, 213. CHAPTER XIX. EXAMINATION AND PRESERVATION MEDIA . . . .216 Aqueous Liquids, 217 ; Mercurial Liquids, 220 ; other Fluids, 220 ; Glycerin Media, 222 ; Glycerin Jellies, 223 ; High Refractive Liquids, 224 ; Resinous Media, 225. CONTENTS. ix PAGE CHAPTER XX. CEMENTS AND VARNISHES .... 229 PAET II. SPECIAL METHODS AND EXAMPLES. CHAPTER XXI. INJECTION — GELATIN MASSES (WARM) ..... 232 Carmine, 234 ; Blue, 236 ; other Colours, 237. CHAPTER XXII. INJECTIONS — OTHER MASSES (COLD) ...... 238 CHAPTER XXIII. MACERATION, DIGESTION, AND CORROSION .... 243 Maceration, 243 ; Digestion, 248 ; Corrosion, 249. CHAPTER XXIV. DECALCIFICATION, DESILICIFICATION, AND BLEACHING . .251 Decalcification, 251 ; Desiiiciflcation, 255 ; Bleaching, 255. CHAPTER XXV. EMBRYOLOGICAL METHODS ....... 258 Mammalia, 263 ; Aves, 271 ; Reptilia, 274 ; Amphibia, 275 ; Pisces, 279 ; Tunicata, 281 ; Bryozoa, 282 ; Mollusca, 282 ; Arthropoda, 284; Venues, 288. CHAPTER XXVI. CYTOLOGICAL METHODS 292 Glycogen, 294 ; Iron, 297 ; Chromosomes, 303 ; Cytoplasmic Inclusions, 316. CHAPTER XXVII. TEGUMENTARY ORGANS ........ 339 CHAPTER XXVIII. MUSCLE AND TENDON (NERVE-ENDINGS) ..... 344 Striated Muscle, 344 ; Electric Organs, 345 ; Tendon, 347 ; Smooth Muscle, 348. M. h x CONTENTS. PAGE CHAPTER XXIX. CONNECTIVE TISSUES 359 Connective Tissue, 350 ; Elastic, 352 ; Plasma Cells 354 ; Fatty Substances, 356 ; Bone and Cartilage, 369 ; Skele- tons of Embryos, 377. CHAPTER XXX. BLOOD AND GLANDS 379 Blood, 379 ; Benzidine Dyes, 388 ; Glands, 391. CHAPTER XXXI. NERVOUS .SYSTEM — GENERAL METHODS 397 CHAPTER XXXII. NERVOUS SYSTEM — SPECIAL METHODS, CHIEFLY CYTOLOGICAL . 410 Cells, 410 ; Cells and Fibres, 416 ; Golgi's Internal Apparatus, 435 ; Medullary Sheath, 439. CHAPTER XXXIII. MYELIN STAINS (WEIGERT AND OTHERS) .... 442 CHAPTER XXXIV. AXIS-CYLINDER AND DENDRITE STAINS (GOLGI AND OTHERS) . 454 CHAPTER XXXV. NEUROGLIA AND SENSE ORGANS . . . 479 Neuroglia, 479 ; Retina, 493 ; Inner Ear, 496. CHAPTER XXXVI. METHODS FOR INVERTEBRATES .... 499 Tunica ta, 499 ; Molluscoida, 500 ; Mollusca, 500 ; Arthropoda, 504 ; Vermes/509 ; Echinodermata, 518 ; Coelenterata, 521 ; Porifera, 525 ; Protozoa, 526. CHAPTER XXXVII. CULTIVATION OF TISSUE " IN VITRO " AND ITS TECHNIQUE . . 550 CHAPTER XXXVIII. A GUIDE FOR STUDENTS OF MICROTOMY 556 APPENDIX. . . 563 564 THE MICROTOMIST'S VADE-MECUM. PART I. CHAPTER I. INTRODUCTORY. 1. The General Method. — The methods of modern microscopic anatomy may be roughly classed as General and Special. There is a General or Normal method which consists in carefully fixing the structures to be examined, staining them with a nuclear stain, dehydrating with alcohol, and mounting series of sections of the structures in balsam. It is by this method that the work is blocked out and very often finished. Special points are then studied, if necessary, by Special Methods, such as examination of the living tissue elements, in situ or in indifferent ' media ; fixation with special fixing agents ; staining with special stains ; dissociation by teasing or maceration ; injection ; impregnation ; and the like. There is a further distinction which may be made, and which may help to simplify matters. The processes of the preparation of tissues may be divided into two stages, Preliminary Preparation and Ulterior Preparation. Now the processes of preliminary preparation are essentially .identical in all the methods, essential divergences being only found in the details of ulterior preparation. By prelimi- nary preparation is meant that group of processes whose object it is to get the tissues into a fit state for passing unharmed through all the ulterior processes to which it may be desired to submit them. It comprehends the operations of (1) killing ; (2) fixing ; (3) the washing and other manipulations necessary for removing the fixing agent from the tissues, and substituting for it the preservative liquid or other reagents which it is desired to employ. Ulterior preparation comprehends the processes sketched out in §§ 3 et seq. 2. Preliminary Preparation. — The first thing to be done with any structure is to fix its histological elements. (This statement applies equally to all classes of objects, whether it be desired to cut them into sections or to treat them in any other special way.) Two M. 1 2 INTRODUCTORY. things are implied by the word " fixing " : first, the rapid killing of the element, so that it may not have time to change the form it had during life, but is fixed in death in "the attitude it normally had during life ; and second, the hardening of it to such a degree as may enable it to resist without further change of form the action of the reagents with which it may subsequently be treated. Without good fixation it is impossible to get good stains or good sections 9 or prepara- tions good in any way. The structure, having been duly fixed by one of the processes described in the chapter on Fixing Agents, is, except in special cases, washed in order to remove from the tissues as far as possible all traces of the fixing reagent. The kind of liquid with which washing out is done is not a matter of indifference. If corrosive sublimate (for instance), or osmic acid, or a solution into which chromic acid or a chromate enters, have been used for fixing, the washing may be done with water. But if certain other agents, such as picric acid, have been used, the washing should be done with alcohol. The reason for this difference is that the first-named reagents (and, indeed, all the compounds of the heavy metals used for fixing) cause certain of the elements of the tissues to become less reactive, and partly or wholly insoluble in water. The combinations of picric acid and the elements of the tissues, on the other hand, are generally much more soluble in watery fluids. These operations having been duly performed, two roads become open. The object may be further prepared by what may be termed the wet method, in which all subsequent operations are performed by means of aqueous media. Or it may be further prepared by the dehydration method, which consists in treatment with successive alcohols of gradually increasing strength, final dehydration with absolute alcohol, imbibition with an essential oil or other so-called clearing agent which serves to remove the alcohol, and lastly either mounting at once in balsam or other resinous medium or imbedding in paraffin for the purpose of making sections. The dehydration method is the course which is generally preferred, chiefly because of its great superiority as regards the preservation of tissues. For the presence of water is the most important factor in the conditions that bring about the decomposition of organic matter, and its complete removal is the chief condition of permanent preservation. 3. Dehydration. — The further course of preparation by the dehydration method is as follows : — At the same time that the superfluous fixing agent is being removed from the tissues, or as soon as that is done, the water of the tissues must be removed. This is CHAPTER I. 3 necessary for two reasons : first, in the interest of preservation, as above explained ; and secondly, because all water must be removed in order to allow the tissues to be impregnated with the imbedding material necessary for section-cutting, or with the balsam with which they are to be finally preserved. This dehydration is per- formed as follows : — The objects are brought into weak alcohol, and are then passed through successive alcohols of gradually increased strength, remaining in each the time necessary for complete satura- tion, and the last bath consisting of absolute or at least very strong alcohol. In dealing with delicate objects, it may be necessary to take special precautions in order to avoid injury to them through the violent diffusion -currents that are set up in the passage from water to alcohol, or from one bath of alcohol to another of considerably different density. Some kinds of diffusion-apparatus may conveniently be used in these cases. The objects may be placed with some of their liquid in a tube corked at one end and closed at the other by a diaphragm of muslin or chamois skin or other suitable membrane, the tube being then immersed in a vessel containing the grade of alcohol that it is desired to add to the liquid in the tube, and the whole allowed to remain until by diffusion through the diaphragm the two liquids have become of equal density. Or, COBB'S differentiator (Proc. Linn. Soc., N.S.W., v, 1890, p. 157 ; Journ. Boy. Mic. Soc., 1890, p. 821) may be employed. Or, the apparatus of HASWELL (Proc. Linn. Soc., N.S.W., vi, 1891, p. 433 ; Journ. Eoy. Mic. Soc., 1892, p. 696). Or that of CHEATLE, described in Journ. Patliol. and Bacteriol., i, 1892, p. 253, or Journ. Eoy. Mic. Soc., 1892, p. 892. See also SCHULTZE (Zeit. wiss. Mik., ii, 1885, p. 537) ; and SUSUKI, ibid., 1909, p. 211 ; KOLSTER (ibid., xvii, 1900, p. 294). The " Siebdosen," or sieve-dishes of STEINACH, ZIMMERMANN, and SUCHANNEK (vide Zeit. wiss. Mik., iv, 1887, p. 433, and vii, 1890, p. 158), are useful for many purposes. See also TISCHATKIN, ibid., xxiii, p. 45. FAIRCHILD'S perforated porcelain cylinders for washing (ibid., xii, 1896, p. 301) seem to be a very neat idea. See also the similar device of SCHAFFER (ibid., xvi, 1900, p. 422 ; Journ. Eoy. Mic. Soc., 1900, p. 394). For EWALD'S section -washing apparatus, see Zeit. Biol., xxxiv, 1897, p. 264. That of SCHOEBEL (ibid., xx, 1903, p. 168) is simple and efficient ; as also that of KREIGBAUM (ibid., xxvii, 1910, p. 504). A capillary siphon for the aspiration of liquids in the fixing, staining, and washing of suspended blood-corpuscles, sperm -cells, protozoa, and the like, is described by EWALD, ibid., p. 253. It is sometimes stated that it is necessary that the last alcohol- bath should consist of absolute alcohol. This, however, is incorrect, a strength of 95 per cent, being sufficient in most cases. For the small amount of water that remains in the tissues after treatment with these grades of alcohol is efficiently removed in the bath of 1—2 4 INTRODUCTORY. clearing agent if a good clearing agent be employed. Oil of cedar will remove the remaining water from tissues saturated with 95 per cent, alcohol ; oil of bergamot will ' clear ' from 90 per cent, alcohol, and anilin oil will clear from 70 per cent, alcohol. I am not aware of any substance that can entirely take the place of alcohol for dehydration and preservation. Acetone and methylal have been substituted for alcohol in the dehydration of methylen- blue preparations (PARKER, Zool. Anz., 403, 1892, p. 376), and anilin oil can be made to dehydrate watery sections if they be first mopped up with blotting-paper ; but a really efficient substitute for alcohol in general work remains yet to be discovered. 4. Preservation. — Considered as a mere dehydrating agent, alcohol fulfils its functions fairly well. But considered as a histo- logical preservative agent, it is far less satisfactory. If tissues be left in alcohol for only a few days before further preparation, injurious effects will perhaps not be very disagreeably evident. But it is otherwise if they are put away in it for many weeks or months before the final preparation is carried out. The dehydrating action of the alcohol being continuously prolonged, the minute structure of tissues is sometimes considerably altered by it ; they become over- hard and shrink, and become brittle, and their capacity for taking stains well becomes seriously diminished. KULTSCHITZKY (Zeit. wiss. Mik., iv, 1887, p. 349) has proposed to remedy this by putting up objects after fixation and washing out with alcohol in ether, xylol, or toluol. FLEMMING (Arch. mik. Anat., xxxvii, 1891, p. 685) advises putting up objects after fixation in a mixture of alcohol, glycerin, and water, in about equal parts, pointing out that objects thus preserved may be at any moment either prepared for sectioning by treatment with pure alcohol or softened for dissection or teasing by a little soaking in water, and that they do not become so hard and brittle as alcohol specimens, and retain their staining power much better. After extensive experience of this plan I can recom- mend it, and would only further suggest that the action of the liquid seems to me to be in many cases much improved by addition of a little acetic acid (say 0-5 to 0-75 per cent.). For material that is intended only for section-cutting, I find that by far the best plan is to clear (next §) and imbed at once in paraffin. This affords, as far as I can see, an absolutely perfect preservation. Cedar-wood oil is, I find, nearly, if not quite, as good as paraffin, so far as the preservation of the tissues is concerned, but of course it is not so handy for storage. CHAPTER I. 5 5. Removal of Alcohol; Clearing. — The water having been sufficiently removed, as described in § 3, the alcohol is in its turn removed from the tissues, and its place taken by some anhydrous substance, generally an essential oil, which is miscible with the material used for imbedding or mounting. This operation is generally known as Clearing. It is very important that the passage from the last alcohol to the clearing agent be a gradual one. This is effected by placing the clearing medium under the alcohol. A sufficient quantity of alcohol is placed in a tube (a watch-glass will do, but tubes are generally better), and then with a pipette a suffi- cient quantity of clearing medium is introduced at the bottom of the alcohol. Or you may first put the clearing medium into the tube, and then carefully pour the alcohol on to the top of it. The two fluids mingle but slowly. The objects to be cleared, being now quietly put into the supernatant alcohol, float at the surface of separation of the two fluids, the exchange of fluids takes place gradually, and the objects slowly sink down into the lower layer. When they have sunk to the bottom, the alcohol may be drawn off with a pipette, and after some further lapse of time the objects will be found to be completely penetrated by the clearing medium. This method of making the passage from one fluid to another applies to all cases in which objects have to be transferred from a lighter to a denser fluid — for instance, from alcohol, or from water, to glycerin. This is a convenient stage for carrying out minute dissections, if any such have to be done, a drop of clearing agent being a most helpful medium for carrying out such dissections (see § 9). At this point the course of treatment follows one of two different roads, according as the object is to be mounted direct in balsam (§ 8), or is first to be sectioned (§ 6). 6. Imbedding, and Treatment of Sections. — The objects are now imbedded. They are removed from the clearing medium, and soaked until thoroughly saturated in the imbedding medium. This is, for small objects, generally paraffin, liquefied by heat, and for large objects either paraffin or a solution of collodion or " celloidin " (in this last case the clearing may be omitted and the tissues be imbedded direct from the alcohol). The imbedding medium con- taining the object is then made to solidify, and sections are made with a microtome through the imbedding mass and the included objects. The sections are then mounted on a slide by one of the 6 INTRODUCTORY. methods described in the chapter on Serial Section Methods, the imbedding material is removed from them (in the case of paraffin), they are stained in situ on the slide, dehydrated with alcohol, cleared, and mounted in balsam or damar. Or they may be stained, washed, dehydrated, and cleared in watch-glasses, and afterwards mounted as desired — the imbedding medium being first removed if desirable. Or. the material may be stained in bulk, before cutting the sections. In this case the object, after having been fixed and washed out, is taken from the water, or while still on its way through the lower alcohols (it should not be allowed to proceed to the higher grades of alcohol before staining, if that can be avoided), and passed through a bath of stain, then dehydrated with successive alcohols, passed through a clearing medium into paraffin, cut, and treated as above described, the sections in this case being mounted direct from the chloroform, xylol, or other solvent with which the paraffin is removed, If aqueous staining media be applied (and this is sometimes desir- able), the structures should either be stained in toto immediately after fixing and washing out, or sections may be stained on the slide, the objects, if delicate, being passed through successive baths of alcohol of gradually decreasing strength before being put into the aqueous stain. In my opinion it is generally advisable not to stain in bulk material that is intended to be sectioned ; by staining it as sections the staining can be much better controlled, and many excellent stains can in this way be employed that are not available for staining in bulk ; and of course sections can be stained much more rapidly than material in bulk. Balsam mounts of which the stain has faded, or which it may be •/ desired to submit to some other staining process, or mount in some other medium, may often with great advantage be re-stained and re-mounted. All that is necessary is to put the slide into a tube of xylol or benzol till the cover falls off (about two days), wash well for some hours in clean xylol, and pass through alcohol into the new stain. Since this was pointed out to me by Dr. Henneguy I have unmounted and re-stained a large number of old preparations, and have succeeded in every case with series of sections mounted on Mayer's albumen, or by the water method. For shellac-mounted series, see E. MEYER, Biol. Centralb., x, 1890, p. 509, or last edition. The most convenient vessels, I find, in which to perform the various operations of staining, differentiating, dehydrating, clearing, etc., on the slide, are flat-bottomed corked glass tubes. I have mine made 10 CHAPTER I. 7 centimetres high and 27 millimetres internal diameter. Each of these will then take two slides, English size, placed back to back. 7. Resume of the General Method.— To sum up, you may either fix, wash out, stain, wash, dehydrate, clear, imbed, cut sections, clear and mount them in balsam ; or fix, wash, dehydrate, clear, imbed, cut, stain, wash, dehydrate, clear, and mount — according to choice. 8. Preparation of Entire Objects, or of Material that is not to be sectioned. — The treatment of objects which, can be studied without being cut into sections is identical with that above described, with the omission of those passages that relate to imbedding processes. Its normal course may be described as fixation, washing out, staining, treatment with successive alcohols of gradually increasing strength, final dehydration with absolute alcohol, clearing, and mounting in balsam. In the preparation of entire objects or structures that are intact and covered by an integument not easily permeable by liquids, special care must be taken to avoid swelling from endosmosis on the passage of the objects from any of the liquids employed to a liquid of less density, or shrinkage from exosmosis on the passage to a liquid of greater density. This applies most specially to the passage from the last alcohol into the clearing medium. A slit should be made in the integument, if possible, so that the two fluids may mingle without hindrance. And in all cases the passage is made gradual by placing the clearing medium under the alcohol, as described (§ 5). Fluids of high diffusibility should be employed as far as possible in all the processes. Fixing agents of great penetrating power (such as picric acid or alcoholic sublimate solution) should be employed where the objects present a not easily permeable integument. Washing out is done with successive alcohols, water being used only in the case of fixation by osmic acid, or the chromic mixtures or other fixing solutions that render washing by water imperative. Staining is done by preference with alcoholic staining media. The stains most to be recommended are Grenacher's borax-carmine, or one of Mayer's alcoholic carminic acid or hsematein stains. Aqueous stains are more rarely indicated, though there are many cases in which they are admissible, and some in which they are preferable. 9. Minute Dissections. — These are best done, if necessary, in a drop of clearing agent. I recommend cedar- wood oil for this purpose as it gives to the tissues a consistency very favourable for dissection, whilst its viscosity serves to lend support to delicate structures. Clove oil has a tendency to make tissues that have lain in it for r.ome 8 INTRODUCTORY. time very brittle. The brittleness is, however, sometimes very helpful in minute dissections. Another property of clove oil is that it does not easily spread itself over the surface of a slide, but has a tendency to form very convex drops, and this also makes it frequently a very convenient medium for making minute dissections in. If it be desired to dissect in a watery fluid, such as glycerin, it may be well to prepare the slide by spreading on it a thin layer of MAYER'S albumen, and on this place a small drop of glycerin, or other dissect- ing medium. As soon as the dissection has been accomplished, a cover may be let fall, horizontally, on to the preparation to keep the parts in place, and a weight placed on it. Then the mount may be filled up with glycerin, or other mounting medium, run in under the cover, and closed, if desired, or instead of the albumen a solution of gelatin may be taken, and hardened in formol with the objects on it. For a balsam mount, after clove or cedar oil, SCHALLIBAUM'S collodion may be taken, and the organs fixed in situ on this by adding xylol. 10. Instruments. — For all that concerns the mechanism and manipulation of the Microscope, see vol. i of CARPENTER'S The Microscope, eighth edition, 1891 ; paying particular attention to all that is said concerning the English and the Continental Models, pp. 254 to 261, the Substage, pp. 184 to 189, Condensers, pp. 289 to 316, and Tube Length, pp. 158 to 159. For information concerning the principles of construction and manipulation of the Microtome, see also CARPENTER'S The Micro- scope. Microtomes are instruments for the accurate production of thin slices of tissues. They are used both for cutting tissues that have acquired a certain favourable consistency through having been imbedded in paraffin, and also for cutting tissues that have been imbedded in softer masses, such as collodion, and tissues that have not been imbedded at all. Not all microtomes are equally well adapted for all these three classes of work. The microtome of the zoologist should at all events be one that is well adapted for cutting imbedded material. Now there are two methods of imbedding in general use — the paraffin method and the celloidin method. In the paraffin method the object is cut dry, frequently with the knife set square to the line of section. In the celloidin method, as in the cutting of unimbedded tissues, it is generally cut wet, and always with the knife set slanting. Some microtomes that are well adapted for the paraffin method are ill adapted for the celloidin method or the cutting of unimbedded CHAPTER I. 9 material, and vice versa. It may be well to possess the two sorts of instrument ; but if only one can be afforded it should be such as will give good work in either way. Microtomes fall further into two classes according as the knife and the surface of section of the object are (A) in a horizontal plane, or (B) in a vertical plane. The former offer greater facility for the orientation of the plane of section, which is an important point for the zoologist and embryologist. Amongst these may be mentioned (a) The " Sliding " Microtomes, in which the knife is carried on a sledge and moved against the object (those of THOMA, SCHANZE, REICHERT, and others). The THOMA, of medium size, as made by R. Jung, Hebelstrasse, Heidelberg (No. 56 of his catalogue for 1911, which may be obtained from Mr. C. Baker, 244, High Holborn, London), is very suitable for the zoologist. It works equally well with either paraffin or celloidin, and can be adapted as a freezing microtome. But this (as is the case with the others above mentioned) will not always furnish work of the highest accuracy ; for the knife being only clamped at one end is liable to spring, and to give sections of unequal thickness. This defect is remedied in (6), a type of sliding microtomes in which the knife is clamped at both ends and is a fixture, the object being carried on a sledge and moved against it (CAMBRIDGE SCIENTIFIC INSTRUMENT COMPANY'S large microtome, the MINOT precision microtome, LEITZ'S, DE GROOT'S, JUNG'S Tetrander." This last seems to be near perfection ; see the description by MAYER in Zeit. iviss. Mik., xxvii, 1910, p. 52 ; but is more cumbrous than is desirable for ordinary work. Class A also includes some instruments in which the knife is carried on a horizontal arm and swung against the object by a rotary movement (JuNG, ROY, FROMME, REICHERT, THATE, and others). I know nothing of these personally, but doubt their constant accuracy. Class B contains some very fine instruments, admirably adapted for the production of continuous ribbons of sections by the paraffin method, but not so well adapted for celloidin or other work in the wet way, or for soft objects. Amongst these are the New and Old Rocking Microtome, made by THE SCIENTIFIC INSTRUMENT Co., Cambridge, or by SWIFT & SON, or by JUNG, or by VAN DER STAD, Amsterdam ; the MINOT, made by BAUSCH &-LOMB and the SPENCER Model, or by BECKER (Gottingen), or by Zimmermann (21, Emilien- strasse, Leipzig) ; the REINHOLD-GILTAY, made by J. W. GILTAY, Delft. For descriptions of the multitudinous models on the market see 10 INTRODUCTORY. the reports in the Zeit. wiss. Mik. and Journ. Roy. Micr. Soc., and the price lists of the instrument makers. 11. Staining Reagents and Chemicals. — For some years it has been difficult to obtain good dyes, but recently several new firms have succeeded in manufacturing materials suitable for the most critical work. The pre-war continental dyes were generally impure products, and since the new dyes being made are mostly purer, it is often necessary to make some slight alteration in the quantity of dye added to "a staining mixture before the optimum staining condition is reached. Details of such alteration necessary for British dyes should be sent to the Journal of the Royal Microscopical Society. I advise the reader to get his reagents and dyes from some well-known British firm which has specialised in the matter. Great Britain. — Flatters and Garnett, Ltd., 309, Oxford Road, Manchester (all sorts of chemicals, dyes and apparatus ; makers of Gilson's " Euparal ") ; the British Drug Houses, Ltd., Graham Street, City Road, London, N. 1 (makers of many sorts of dyes) ; The British Dyestuffs Corporation, Ltd., 70, Spring Gardens, Manchester (important makers of dyes ; London agents are, Baird and Tatlock, 14, Cross Street, Hatton Garden, E.G. 1) ; Hawksley & Sons, 83, Wigmore Street, Cavendish Square, W. 1 ; and Charles Baker, 244, High Holborn, W.C. (both firms are agents for Griibler, and for most microscope and accessory apparatus makers). For incubators, etc., for embedding and tissue-culture work, Charles Hearson & Co., Ltd., 235, Regent Street, W. 1. United States of America. — Eimer and Amend, 205 — 211, Third Avenue, New York ; Palo Company, 90, Maiden Lane, New York ; Edward Pennock, 3609, Woodland Avenue, Philadelphia, Pa. ; General Biological Supply House, 5508, Kimback Avenue, Chicago, 111. ; The Will Corporation, Rochester, N.Y. ; Paul Weiss, 1620, Arapahoe Street, Denver, Colorado. Germany. — G. Griibler and Hollborn, Chemiker, Leipzig, Germany. CHAPTER II. KILLING. 12. IN the majority of cases, the first step in the preparation of. an organ or organism consists in exposing it as rapidly and as com- pletely as possible to the action of one of the Fixing Agents that are discussed in the next chapter. The organ or organism is thus taken in the normal living state ; the fixing agent serves to bring about at the same time, and with sufficient rapidity, both the death of the organism and that of its histological elements. It should be noted that narcotisation generally implies some change in the cells, and most narcotics have to be applied for a long time. Such treatment is absolutely barred in material destined for careful cytological study. This applies especially to ether and chloroform, which are extremely injurious to cells : in the case of larger mammals like the cat and dog a preliminary treatment in ether or chloroform may be necessary, but directly after anaesthesia the animals' throats should be cut or they should be killed by a blow, if possible. Coal gas chambers are good for killing all mammals, and I do not believe carbon monoxide is hurtful to cells. Amphi- bians killed by chloroform are often completely spoilt for cytological purposes ; if the brain is not wanted, pith the animal. For birds the time-honoured custom of wringing their necks is recommended. In the case of small lizards, newts and such live stock it is a good plan to cut off their heads quickly with strong scissors. If the material is wanted for chromosome or mitochondria work look up these sections for special directions. But these methods are by no means applicable to all cases. There are many animals, especially such as are of a soft consistence, and deprived of any rigid skeleton, but possessing a considerable faculty of contractility, which if thus treated contract violently, and die in a state of contraction that renders them unfit for study. In these cases special methods of killing must be resorted to. Speaking generally, there are two ways of dealing with these difficult cases. You may kill the animal so suddenly that it has not time to con- tract : or you may paralyse it by narcotics before killing it. See also under " Chromosomes," § 652, and " Mitochondria," § 673. 12 KILLING. Sudden Killing. 13. Heat.- -The application of Heat affords a means of killing suddenly. By it the tissues are more or less fixed at the same time that somatic death is brought about. The difficulty consists in hitting off the right temperature, which is of course different for different objects. I think that 80° to 90° C. will generally be amply sufficient, and that very frequently it will not be necessary to go beyond 60° C. An exposure to heat for a few seconds will generally suffice. Small objects (Protozoa, Hydroids, Bryozoa) may be brought into a drop of water in a watch-glass or on a slide, and heated over the flame of a spirit-lamp. For large objects, the water or other liquid employed as the vehicle of the heat may be heated beforehand and the animals thrown into it. As soon as it is supposed that the protoplasm of the tissues is coagu- lated throughout, the animals should be brought into alcohol (30 to 70 per cent, alcohol) (if water be employed as the heating agent), An excellent plan for preparing many marine animals is to kill them in hot fresh water. Some of the larger Nemertians are better preserved by this method than by any other with which I am acquainted. 14. Slowly Contracting Animals. — Animals that contract but slowly, such as Alcyonium and Veretillum, and some Tunicates, such as Pyrosoma, are very well killed by throwing them into some very quickly acting fixing liquid, used either hot or cold. Glacial or very strong acetic acid (VAN BENEDEN'S method) is an excellent reagent for this purpose ; it may be used, for example, with some Medusae. After an immersion of a few seconds or a few minutes, according to the size of the animals, they should be brought into alcohol of at least 50 per cent, strength. Lemon juice employed in this way has given me very good results with small Annelids and Hirudinea. Corrosive sublimate is another excellent reagent for this purpose. Narcotisation. 15. Narcotisation is performed by adding some anesthetic sub- stance very gradually, in very small doses, to the water containing the animals, and waiting patiently for it to take effect slowly. Menthol. — Now used with great success for anaesthetising large marine animals. Place latter in clean vessel, and sprinkle over surface of water, menthol crystals. As the latter dissolve the animals expand. In from twelve to twenty-four hours they may be transferred to a fixer. Very good for Anemones, Holothuria, Ascidia and many Mollusca. (Personal communication from Dr. E. J. ALLEN, Plymouth.) 16. Nicotin in solution (ANDRES, Atti R. Accad. dei Lincei, v, 1880, p. 9). Andres employs a solution of 1 gramme of nicotin in a litre CHAPTER II. 13 of sea water. The animal is placed in a jar containing half a litre of sea water, and the solution of nicotin is gradually conducted into it by means of a thread, acting as a syphon, of such a thickness as to be capable of carrying over the whole of the solution of nicotin in twenty- four hours. See also Mitth. Zool. Stat. Neapel, Bd. ii, 1880, p. 123. 17. Chloroform may be employed either in the liquid state or in the state of vapour. The animals being extended, a watch-glass containing chloroform may be floated on the surface of the water in which they are contained, and the whole covered with a bell-glass. As soon as they have become insensible they are killed by means of hot sublimate or chromic acid solution plentifully poured on to them. (KOROTNEFF, Mitth. Zool. Stat. Neapel, v, 1884, p. 233.) Liquid chloroform is employed by squirting it in small quantities on to the surface of the water containing the animals. A syringe or pipette having a very small orifice, so as thoroughly to pulverise the chloroform, should be employed. Small quantities only should be projected at a time, and the dose should be repeated every five minutes until the animals are anaesthetised. I have seen large Medusae very completely ansesthetised in ex- tension in an hour or two by this method. ANDRES finds that it does not succeed with Actiniae, as with them maceration of the tissues supervenes before anaesthesia is established. PREYER (Mitth. Zool. Stat. Neapel, Bd. vii, 1886, p. 27) recommends chloroform water for star-fishes. WADDINGTON employs a mixture of equal parts of 1 per cent, sol. of cocaine (or eucain) and saturated sol. of chloroform in water (sea or fresh), according to the habitat. 18. Ether and Alcohol may be administered in the same way. ANDRES has obtained good results with Actiniae by the use of a mixture (invented by SALVATORE LO BIANCO) containing 20 parts of glycerine, 40 parts of 70 per cent, alcohol, and 40 parts of sea water. This mixture should be carefully poured on to the surface of the water containing the animals, and allowed to diffuse quietly through it. Several hours are sometimes necessary for this. EISIG (Fauna u. Flora Golf. Neapel, xvi, 1887, p. 239) benumbs Capitellidae by putting them into a mixture of 1 part of 70 per cent, alcohol with 9 parts of sea water. OESTERGREN (Zeit. wiss. Mik., xix, 1903, p. 300) makes a saturated (7 to 8 per cent.) solution of ether in sea or soft water, and uses it either concentrated or diluted to about 1 per cent., and finds it to succeed with all classes of aquatic animals, 14 KILLING. CORI (Zeit. wiss. Mik., vi, 1890, p. 438) recommends a mixture composed of 10 c.c. methyl-alcohol (of 96 per cent, strength), 90 c.c. water (fresh or sea water), and 0-6 grm. of sodium chloride (to be added only when fresh water is taken, the addition of the salt having for its object to prevent maceration). It may be well to add to this mixture a very few drops of chloroform (for Cristatella ; Zeit. wiss. Zool, Iv, 1893, p. 626). 19. Chloreton (Aceton Chloroform) is recommended for inverte- brates and larvae of Rana by RANDOLPH (Zool. Anz., xxiii, 1900, p. 436). KRECKER (Zeit. wiss. Zool., xcv, 1910, p. 383) takes solutions of J to 1 per cent, for Oligochseta. SULIMA (Zeit. Biol. Techn., Strasburg, i, 1909, p. 379) takes a mixture of 99 parts of sea water and 1 of 10 per cent. sol. of chloreton in absolute alcohol, for Scyllium and Anguilla. For Bryozoa, see BESSIE GREEN, Journ. Roy. Mic. Soc., 1914. 20. Hydrate of Chloral. — FOETTINGER (Arch, de Biol., vi, 1885, p. 115) operates by dropping crystals of chloral into the water containing the animals. For Alcyonella he takes 25 to 80 centi- grammes of chloral for each hundred grammes of water. It takes about three-quarters of an hour to render a colony sufficiently insensible. He has obtained satisfactory results with marine and fresh-water Bryozoa, with Annelida, Mollusca, Nemertians, Actiniae, and with Asteracanthion. He did not succeed with Hydroids. Lo BIANCO (Mitth. Zool. Stat. Neapel, Bd, ix, 1890, p. 442) employs for various marine animals freshly prepared solutions of chloral in sea water, of from -^ to ^ per cent, strength. I have never had the slightest success with Nemertians. VERWORN (Zeit. wiss. Zool., xlvi, 1887, p. 99) puts Cristatella for a few minutes into 10 per cent, solution of chloral, in which the animals sooner or later become extended. KUKENTHAL (Jena Zeit. Naturw., Bd. xx, 1887, p. 511) has obtained good results with some Annelids by means of a solution of 1 part of chloral in 1,000 parts of sea water. The chloral method gives rise to maceration with some subjects, as I can testify, and has been said to distort nuclear figures. 21. Cocaine (RICHARDS, Zool. Anz., cxcvi, 1885, p. 332). — Richards puts a colony of Bryozoa into a watch-glass with 5 c.c. of water, and adds gradually 1 per cent, solution of hydrochlorate of cocaine in water. After five minutes the animals are somewhat numbed; half a cubic centimetre of the solution is added, and ten minutes later the animals should be found to be dead in a state of extension. This method is stated to succeed with Bryozoa, Hydra, and CHAPTER II. 15 certain worms. It is the best method for Rotifers (ROUSSELET). It has also been recommended for Aplysia. It has been pointed out (by GOBI, in the paper quoted § 18) that, unfortunately, when fixing agents, such as sublimate solution, are added to the animals, the cocaine is thrown down on them as a white precipitate. This precipitate, however, may be redissolved afterwards in alcohol (EisiG). Cocaine solutions cannot be depended on to keep for more than a few days. 22. Eucain. HARRIS (Journ. Roy. Mic. Soc., 1900, p. 404) recommends a 1 per cent, solution of eucain hydrochloride, as giving far better results, with Vorticellidse, Rotatoria, and Vermes. ROUSSELET (ibid.) reports favourably as to its action on Flosculariae. It is stated to be perfectly stable in aqueous media. It dissolves in sea water to about 0-5 per cent. 23. Hydroxylamin. — HOFER (Zeit. wiss. Mik., vii, 1890, p. 318). Either the sulphate or, preferably, the hydrochlorate may be used. This should be dissolved in water (spring or sea water, according to the habitat) and exactly neutralised by addition of carbonate of soda. The organisms are placed in a solution diluted to about 0-1 per cent., for thirty minutes or less (as for Infusoria), to 0-25 per cent., for from fifteen minutes to one hour (Hydra), 1 per cent., one half to two hours (Hirudo), or as much as ten to twenty hours (Helix and Anodonta). Hydroxylamin is a powerful reducing agent, and should therefore be well washed out before treating with easily reducible fixing agents. 24. Chloride or Sulphate of Magnesium. — TULLBERG (Arch. Zool. Exper. et Gen., x, 1892, p. 11). For Actiniae, a 33 per cent, solution of the chloride should be very slowly added to the water containing the expanded animal, until the vessel contains 1 per cent, of the salt (thus for 1 litre of sea water 33 c.c. of the solution must be added). The addition must be completed within half an hour, and thirty minutes later the animal may be fixed. For terrestrial and fresh-water Invertebrates rather stronger solutions should be used. REDENBAUGH (Amer. Natural., xxix, 1895, p. 399) takes the sulphate, either added in crystals to the sea water containing the animals until a saturated solution is obtained, or in the shape of a saturated solution into which they are thrown (Annelids). See also MAYER, Bid. Butt. Wood's Hole, xvii, 1909, p. 341 (puts direct into sol. of f strength). 25. Poisoning by small doses of some fixing agent is sometimes good. Lo BIANCO kills Ascidia and Rhopalwa in an extended state (Mitth. Zool. Stat. Neapel,ix, 1890, p. 471) by pouring a little 1 per cent. 16 KILLING. chromic acid on to the surface of the water containing them, and allowing it to diffuse slowly into it. About twelve to twenty-four hours is necessary. He kills dona in a similar way with a mixture of 1 part of 1 per cent, chromic acid and 9 parts of 49 per cent, acetic acid. Osmic acid, or Kleinenberg's solution, is sometimes employed in the same way. I have seen Medusae killed in a satisfactory manner by means of crystals of corrosive sublimate added to the water containing them. Morphia, Curare, Strychnin, Prussie Acid, and other paralysing drugs, have also been employed. 26. Asphyxiation may be sometimes successfully practised. Terrestrial Gastropods may be killed for dissection by putting them into a jar quite full of water that has been deprived of its air by boiling, and hermetically closing it. After from twelve to twenty- four hours they are generally found dead and extended. The effect is obtained somewhat quicker if a little tobacco be added to the water. Good results are sometimes obtained with aquatic animals by simply leaving them to exhaust the oxygen of the water in which they are contained. I have sometimes succeeded with Holothuriae and other Echinoderms in this way. WAKD (see Amer. Nat., xxv, 1891, p. 398) has succeeded with Hydroids, Actinise, and similar forms, and UEXKULL (Mitth. Zool. Stat. Neapel, xii, 1896, p. 463) with Echinids. Marine animals are sometimes successfully killed by simply putting them into spring water. Warm Water will sometimes serve to immobilise and even kill both marine and fresh-water organisms. Carbonic Acid Gas has been recommended (by FOL, Zool. Anz., cxxviii, 1885, p. 698). The water containing the animals should be saturated with the gas. The method is stated to succeed with most Ccelenterata and Echinodermata, but not with Molluscs or Fishes. I have had most excellent results with small Annelids and Hirudinea. It is not necessary to employ a generator for obtaining the gas. It suffices to take an ordinary ' soda-water ' ' syphon, and squirt its contents into the water containing the animals. Narcotisation is very rapidly obtained with very small animals, but much more slowly with larger ones. For instance, Stylaria proboscidea, I find, is paralysed in a few seconds ; a small Nephelis of 15 or 20 millimetres in length, will require about five minutes ; and a large Nephelis, of from 10 to 15 centimetres, will require as many hours. UEXKULL (Mitth. Zool. Stat, Neapel, xii, 1896, p. 463) has paralysed CHAPTER II. 17 Echinids very rapidly with carbonic acid, likewise a small Teleostean fish ; whilst Scyllium and Crustaceans were affected much more slowly, and mussels not at all. 27. Peroxide of Hydrogen. — VOLK (Zool. Anz., xix, 1896, p. 294) kills Rotatoria by means of one or two drops of a 3 per cent, solution added to 1 c.c. of the water containing them. M. CHAPTER III. FIXING AND HARDENING, 28. The Functions of Fixing Agents. — The meaning of the term " fixing ' has been explained above (§ 2). Here is an example showing the necessity of fixation. If a portion of living retina be placed in aqueous humour, serum, or other so-called ' ' indifferent ' medium, or in any of the media used for permanent preservation, it will be found that the rods and cones will not preserve the appearance they have during life for more than a very short time ; after a few minutes a series of changes begins to take place, by which the outer segments of both rods and cones become split into discs, and finally disintegrate so as to be altogether unrecognisable, even if not totally destroyed. Further, in an equally short time the nerve-fibres become varicose, and appear to be thickly studded with spindle- shaped knots ; and other post-mortem changes rapidly occur. If, however, a fresh piece of retina be treated with a strong solution of osmic acid, the whole of the rods and cones will be found perfectly preserved after twenty-four hours' time, and the nerve-fibres will be found not to be varicose. After this preliminary hardening, portions of the retina may be treated with water (which would be ruinous to the structures of a fresh retina), they may even remain in water for days without harm ; they may be stained, acidified, hardened, imbedded, cut into sections, and mounted in either aqueous or resinous media without suffering. This example shows that one of the objects aimed at in fixing is to impart to tissues the degree of hardening necessary to enable them to offer such mechanical resistance to post-mortem change and to the processes of after-treatment as not to suffer change of form. Another important function of fixing is to render insoluble elements of cells and tissues that would otherwise be more or less dissolved out by the liquids employed in the after-treatment. A third and highly important function of fixing agents consists in producing optical differentiation in structures. By coagulating the elements of tissues and cells, fixing agents alter their indices of refraction, raising them in varying degrees. They do not act in an equal degree on all the constituent elements of cells and tissues, but raise the index of CHAPTER III. 19 some more than that of others, thus producing optical differentiation where there was little or none before. Compare the aspect of the epithelium of the tail of a living tadpole, observed in water, with its aspect after the action of a little diluted solution of Flemming. In the living state the protoplasm of its cells has a refractive index little superior to that of water, and consequently so low an index of visibility that hardly any structure can be made out in the object. But as soon as the protoplasm has been sufficiently coagulated by the reagent the refractive indices of some of its elements will have been raised to above that of balsam, the chromatin of the nuclei will be brought out, and other structures be revealed where none was visible before. 29. The Action of Fixing Agents consists in coagulating and rendering insoluble certain of the constituents of tissues. This is effected sometimes without any chemical action being involved, as when alcohol is employed, which acts by simple withdrawal of the water of the tissues. But in the majority of cases the fixing agents enter into chemical combination with certain of the elements of the tissues. The compounds thus formed are sometimes unstable and soluble, so that they are removable by washing, as is the case with several of those formed by picric acid. It is found in practice, however, that those formed by chromic acid and its salts, and the salts of the heavy metals, as mercury, iron, platinum, gold, and silver, are mostly insoluble. The insolubility of these bodies is an advantage in that it ensures that the tissues shall not be robbed of their essential constituents, nor deprived of their desired consistency and optical differentiation, by the reagents subsequently employed. It is also sometimes an advantage in that certain of the compounds in question have the property of combining with certain colouring matters, and thus affording important stains which could not otherwise be obtained ; or in other words, of acting as mordants. But it is sometimes a disadvantage, inasmuch as these same compounds which render possible the production of some stains are hindrances to the production of others. Tissues that have been fixed with osmic or chromic acid or their salts are in general not easily to be stained with carmine or similar colouring matters, unless the metals have been previously removed by special chemical treatment ; though they may generally be stained with hsemalum, or, after sectioning, with iron hsematoxylin or tar colours. According to FISCHER (Fixirung, Fdrbung, und Ban des Proto- 2 — 2 20 FIXING AND HARDENING. plasmas, Jena, G. Fischer, 1899), the coagulation which constitutes fixation is, in the case of the liquid and semi-liquid constituents of tissues, always a phenomenon of precipitation. The more solid constituents (such as fibrils that are visible during life, nucleoli, and the like) he admits may be acted on by fixing reagents without the formation of any visible precipitates. But all the liquid ones, in so far as they are fixed at all, are visibly precipitated in special precipi- tation forms, which vary according to the precipitant. Each fixing agent gives its own characteristic fixation image, which may be more or less lifelike, but can never be absolutely so. Fischer gives copious descriptions of the precipitation forms of the chief organic compounds found in tissues, and of the precipitation powers of the chief fixing agents, which the reader will do well to study. It seems to be a consequence of Fischer's theory of fixation by precipitation that the most energetic fixing agents should always be found amongst the most energetic precipitants. But on the showing of his experiments this is not so. For instance, it is allowed on all hands that osmic acid is a most energetic fixative. But Fischer finds (op. cit., pp. 12 — 14, 27) that it is a very incomplete and weak precipitant. Or, to take a contrary instance, he finds that picric acid is an energetic precipitant of the majority of cell constituents ; but surely every cytologist must admit that it is not a highly energetic fixative ! It would seem to follow, from these instances and from other similar ones, that Fischer's tables of precipitating power cannot be taken as a measure of the fixing power of the reagents. And further, the study of the fixation images of tissues afforded by osmic acid, formaldehyde, and other reagents, seems to show that the coagula- tion brought about by them is in part accompanied by the formation of visible precipitates, but in part not so, and that they may do their work to a larger extent than he seems to admit through a homogeneous coagulation. Fischer, studying the effects of certain fixatives on albumose, states that mixing 10 per cent, slightly acid deutero- albumose with Altmann's bichromate-osmic fluid causes a precipitate of granules of from 1 to 3 ^ in diameter, while corrosive sublimate of 7 per cent, causes granules of 0-4 to 1 ju. in size ; one might be led away, as was Fischer, to consider that Altmann's fluid used on cells therefore causes artifacts to appear. As a matter of fact corrosive sublimate is much more dangerous than Altmann's fluid, in this respect, while Altmann's fluid merely preserves cell granules which are visible intra vitam. The ground protoplasm after corrosive is more granular and coarsely reticulate than after Altmann ; this CHAPTER III. 21 does not apply to mitochondria or Golgi elements which are often very badly preserved with corrosive. Fischer (op. cit.) says, " Many kinds of cell contents, indeed the majority, have an alkaline reaction, and are thereby quite inacces- sible to the precipitating action of certain agents, such as osmic acid, or bichromate ; and the action of certain other fixatives, such as platinum chloride or chromic acid, is more or less hindered by the presence of free alkalies. For neither the chromic acid (of the Flemming), nor the platinum chloride (of the Hermann) would be adequate to act as acidifiers to the osmic acid of the mixtures." My own experience is directly contrary to these conclusions of Fischer : it is common knowledge among modern workers that a cell fixed in acetic acid-containing solutions has a more " raked out ' appearance than when the acetic acid is omitted : this applies not only to cell granules, but to the appearance of the ground cytoplasm, nucleoli, and chromatin filaments. The statement that osmic acid must be acidified before it will fix all parts of the cell is also contrary to general experience. Formalin neutralised gives a gentler and more precise fixation than acid formalin. While Fischer's results may be excellent so far as concern his theoretical conclusions on the fixation of weak solutions of egg-white, etc., too much attention should not be paid to one who is not thoroughly acquainted with practical cytology and histology. But from his very suggestive observations it certainly appears that the formation of visible precipitates is a very widespread, if not universal concomitant of fixation ; and that the wider the precipitating power of a fixative (i.e. the greater the number of organic liquids that it can precipitate), the greater will be the number of artifacts to which it can give rise. It has lately been pointed out by some workers (e.g. UNNA, Arch, f. Mikr. Anat., Ixxviii), that many of the fixing reagents come under the category of oxidisers ; Unna places great importance on the fact that some of the most successful fixers are oxidisers, e.g. Os04, K2Cr207, Cr03 ; but formalin, admittedly a splendid reagent, is a reducer, while picric acid and corrosive sublimate are feeble oxidisers. and that only under special conditions. Of ^he ten common reagents used for fixing, only four are marked oxidisers, and Unna's generalities with reference to the significance of oxidisers may not be the correct explanation. Generalities such as made by Unna with reference to the role of oxidisers in fixation resemble like claims which have been made with reference to the supposed necessity for the constant use of an acid in fixatives (and preferably acetic acid which is really a dangerous reagent). 22 FIXING AND HARDENING. With regard to the relative values of oxidisers or reducers in fixing cytoplasm or nucleus, formalin (reducer)* and Os04 (oxidiser) are both famous cytoplasm fixers, while acetic acid (neither oxidiser nor reducer), or alcohol (reducer) and Cr03 (Oxidiser) are well-known nuclear fixatives. Kelly's fluid, formol-bichromate or formol-Flemming are all splendid fixatives, and mixtures of both oxidisers and reducers ; it is difficult to see how Unna's theories can apply here. In the case of Flemming fluid, without acetic acid, it is certain that the fixation process in so far as it concerns the Os04 in this mixture, is not solely an oxidising process, at least of the same nature as the fixation reaction by the chromic acid (Cr03). In a word, fixation of the cell by various kinds of chemical reagents is an extremely complicated matter concerning a large number of organic substances whose re- actions to the chemical used are probably different in most cases. 30. The Characters of the Usual Fixing Agents. — These agents are as follows :- 1. Osmium tetroxide, 2. Formaldehyde gas, 3. Chromium trioxide, . 4. Bichromate of potassium, 5. Platinum chloride, 6. Mercury bichloride, all in water. r 7. Picric acid in water, 8. Alcohol, "R - I 9. Nitric acid and , 10. Acetic acid in water. Chloroform and urea are also used. In the group marked A are arranged the more valuable reagents, in B the less valuable or destructive ones. Good fixatives can be made from the substances in group A without using any of the reagents in group B. The latter contain most of the reagents useful for chromosome work, the former, reagents useful for fixing the cytoplasm and ' resting ' nucleus. From group A have been made the following mixtures : Altinann, Champy, and Flemming and Hermann-without-acetic acid ; these are among the best mixtures known. Then there are forrnol (5 per cent, to 10 per cent.), Regaud, Helly, formol-Muller and formol- Flemming, which are so good for mammals. Good general micro- anatomical fixatives from both groups A and B, are Zenker, Benin, * See, however, Blum, Enzykl. d. mikr. Tech., 1910. CHAPTER III. 23 Gilson-Petrunkewitsch and corrosive acetic, but these all destroy much of the cell-contents, and give an incorrect picture of the cell, excepting of chromatinic structures, for which they are indicated. A good fixing agent should first of all preserve all the elements it is desired to fix. But that is not enough ; it should also give good optical differentiation, and should have sufficient power of penetra- tion to ensure that small pieces of tissue be equally fixed by it throughout. No single substance or chemical compound fulfils all that is required of a good fixing agent ; hence it is that all the best fixing agents are mixtures. Osmic acid, for instance, fulfils some of these conditions, but not all of them. It kills rapidly and preserves admirably the elements of cytoplasm, but nuclei not so well. But 'the optical differentiation that it gives, though sometimes good, is often very inferior. For osmic acid, by coagulating in nearly equal degrees alike the spongioplasm (the plastin reticulum) and the hyaloplasm (the enchylema) of the cell-body, and the chromatin of nuclei, raises alike the refractive indices of all of them ; so that if the 'fixing action have been in the least degree overdone, the cells acquire a homogeneous aspect in which the finer details are obscured by the general refractivity of the whole. If now, instead of Using it pure, it be used in combination with chromic acid, a better differ- entiation is obtained ; for chromic acid, whilst enhancing, and at all events not interfering with the fixation of chromatin, serves to facilitate penetration and to counteract the excessive action of the osmic acid on the protoplasm, so that the cells come out less homo- geneous and with more detail observable in them. Descriptive embryologists often use strange illogical mixtures containing both reducible substances and violent reducers, both fat-solvents and fat-preservers, mixed together without regard for the chemistry of fixation. It is only the logically planned fixative that is found generally useful, and which stands the test of time. Fixation falls under three broad headings :- 1. Micro-anatomical, in which correct preservation of cell aggre- gates, without shrinkage or expanding, is the desideratum. Such is the aim of most descriptive embryologists. 2. Cytological from the point of view of the chromosome or nucleus. 3. Cytological from the point of view of fixing the cell in a state which most resembles its condition when alive ; also so as to identify the cell elements, especially in the cytoplasm. In most cases the results attained by workers belonging to sections 1 and 2 can truly be said to give a caricature of the cell intra vitam. I give below a general classification of fixatives, those in (a) being 24 FIXING AND HARDENING. fixatives causing the maximum disturbance and destruction in the individual cell, those in (c) the least. A great deal, however, depends on the accessibility of the cells to the fixative, and as to whether vertebrate or invertebrate material is being used. (a) Carnoy, Petrunkewitsch, alcohol, Gilson, picro-nitric, etc. Fat, mitochondria, Golgi apparatus, and often delicate yolk discs do not show after these. (Using alcohols and xylol subsequently.) (6) Bouin, Zenker, corrosive acetic, Flemming-with-acetic acid, etc. Mitochondria and Golgi apparatus rarely show after these, except possibly in mammals, where these cell inclusions are more resistant than in invertebrata. Fats show with the last-mentioned fixative. (c) Osmic acid, Flemming- without-acetic, Champy, Altmann,' formalin, Mann's mercury-osmic liquid, SjovalFs method, etc. Preserve all formed granules (except glycogen). (Using fluids subsequently as above.) In section (c) the formol alone will not preserve fat ; but see Sjovall's method (§ 696). The fixatives have not been classed according to how they them- selves alone affect the contents of the cell, but according to how they preserve the cell preparatory to its treatment in the liquids necessary for embedding and sectioning. Injurious liquids which should never be used in cytological fixation (3, vide supra) are acetic acid, chloroform and alcohol. Acetic acid is nearly the most destructive to delicate lipins, and its use, except where chromosomes are being studied, is rarely indicated ; any worker who uses acetic acid in his fixing mixtures cannot hope to get a correct picture of any part of his cell, possibly excepting the chromo- somes (not the resting nucleus). The most valuable fixatives are osmium - tetroxide, bichromate of potassium, chromium - trioxide, and formaldehyde, possibly in the order named ; the most valuable mixtures are Muller-formol (or Helly), Flemming- without-acetic, Altmann, and Champy ; the three latter approach as near perfection as present-day technique allows. Altmann's fluid (K^Cr^O; + Os04) I find to be a splendid mixture. In no case, except in small invertebrates, do these fixatives (excluding formol) give a true fixation of cell aggregates ; this is due to their inferior penetrating powers, and to an unevenness of penetration. Small invertebrates, both marine and fresh- water, and small pieces of tissue, are usually exquisitely preserved in chrome- osmium mixtures, but are not then generally suitable for staining and mounting whole, especially for staining in carmine mixtures. CHAPTER 111. 25 For routine zoological work Bourn's picro-formalin-acetic is recommended. Gilson-Petrunkewitsch is a fixative which is easy to work and generally better than corrosive sublimate acetic. For routine vertebrate histological work Zenker and Helly's Zenker-formol are indicated. I think the beginner should avoid such things as liquid of FLEMMING and similar mixtures. Picric acid gives a fair though weak fixation, with very good penetration, is easy to manage, and does not make tissues brittle, which sublimate easily may do. Pure formol is not bad, and very easy to manage. Speaking generally, osmic acid, chromic acid, bichromates, chloride of platinum, and the majority of the compounds of the heavy metals, are hindrances to staining ; whilst heat, alcohol, trichloracetic acid, formol, corrosive sublimate, picric acid, and acetic acid, are neutral, or even favourable, in this respect. 31. The Practice of Fixation. — See that the structures are per- fectly living at the instant of fixation, otherwise you will only fix pathological states or post-mortem states. Some observers have made special observations on the effect of delay in fixation; J. THORNTON CARTER (Phil. Trans. Roy.Soc., Series B,vol. ccviii, 1917) has made some interesting experiments on the finely granular ameloblasts in the developing teeth of the pike. He noticed that the cytoplasm gave evidence of marked changes unless fixed within three minutes of " death " ; these changes were manifested by the behaviour of the cytoplasmic granules to stains ; the selectivity of the latter was progressively altered as the rapid post-mortem changes were set in action. Fixation is generally performed by immersion of the objects in the fixing liquid. In this case, everything should be done to facilitate the rapid penetration of the fixing agent. To this end let the struc- tures be divided into the smallest portions that can conveniently be employed, and if entire organs or organisms are to be fixed whole, let openings, as large as possible, be first made in them. The penetration of reagents is greatly facilitated by heat. You may warm the reagent and put it with the object to be fixed in the paraffin stove, or you may even employ a fixing agent heated to boiling-point (as boiling sublimate solution for certain corals and Hydroids, or boiling absolute alcohol for certain Arthropods with very resistent integuments). But this should only be done as a last resource. Let the quantity of fixing agent employed be many times 26 FIXING AND HARDENING. the volume of the objects to be fixed. If this precaution be not observed the composition of the fixing liquid may be seriously altered by admixture of the liquids or of the soluble substances of the tissues thrown into it. For a weak and slowly acting fixing agent, such as picric acid, the quantity of liquid employed should be in volume about one hundred times that of the object to be fixed. Reagents that act very energetically, such as Flemming's solution, may be employed in smaller proportions. But fixation may also be performed by injection of the fixing liquid into the objects, thus ensuring a more rapid and thorough penetration of voluminous objects. See for this practice the methods of fixation by injection of GOLGI, DE QUERVAIN, MANN, and others, given under Nervous System. BRAUS and DRUENER (Jena Zeit. Naturw., Bd. xxix, 1895, p. 435) fix fishes by injection through the bulbus aortce. The vessels are first washed out with normal salt solution, and the fixing liquid is then thrown in. KOLMER (Anat. Anz., xlii, 1912, p. 47) fixes thus even large mammals (Chimpanzee, Goat). He first washes out with KINGER'S solution. It is well not to leave specimens in fixing liquids longer than is sufficient to obtain the desired reaction. Sublimate, for instance, soon makes tissues brittle. But long immersion may be neces- sary to produce the desired optical differentiation with some reagents. Careful washing out (by which is meant the removal from the tissues of the excess of uncombined fixative) is necessary in order to get tissues to stain properly. But it is not always equally impera- tive. Alcohol and formaldehyde do not require washing out before staining ; acetic and picric acid only for some stains ; sublimate will allow of staining even if not washed out, but allows of a sharper stain if well washed out ; all osmic, chromic, and platinic liquids require very thorough washing out. It is important to use the appropriate liquid for washing out the fixing agent after fixation. It is frequently by no means a matter of indifference whether water or alcohol be employed for washing out. Sometimes water will undo the whole work of fixation (as with picric acid). Sometimes alcohol causes precipitates that may ruin the preparations. Objects fixed in alcohol, formol, acetic acid, picric acid, or nitric acid require to be washed out with alcohol, or at least with some hardening liquid, whilst those that have been fixed with osmic or chromic acid, or with one of the other compounds of the heavy metals, require in general to be washed out with water. Sublimate, however, is best washed out with alcohol. CHAPTER III. 27 Use liberal quantities of liquid for washing. Change the liquid as often as it becomes turbid, if that should happen. The process of washing out is greatly facilitated by heat. Picric acid, for instance, is nearly twice as soluble in alcohol warmed to 40° C. as in alcohol at the normal temperature (Fol). 32. Fixation of Marine Animals. --The tissues of marine organisms are as a general rule more refractory to the action of reagents than those of corresponding fresh-water or terrestrial forms, and fixing solutions should in consequence be stronger (about two to three times). Marine animals ought to be freed from the sea water adherent to their surface before treating them either with alcohol or any fixing reagent that precipitates the salts of sea water. If this be not done, the precipitated salts will form on the surfaces of the organisms a crust that prevents the penetration of reagents to the interior. Fixing solutions for marine organisms should therefore be such as serve to keep in a state of solution, and finally remove, the salts in question. If alcohol be employed, it should be acidified with hydro- chloric or some other appropriate acid. Picro-nitric acid is a fixing reagent that fulfils the conditions here spoken of. (On this subject see MAYER, in Mitth. Zool. Stat. Neapel, ii (1881), pp. 1 et seq., and ALLEN and BROWNE in " Science of the Sea," John Murray, 1912). 33. Hardening.- -The process of hardening is distinguished from that of fixing as being directed to the attainment of a degree of consistency sufficient to allow of soft tissues being cut into sections without imbedding. It is an after-process, and only ranks as a special method. Methods of imbedding have now been brought to such a degree of perfection that the thorough hardening of soft tissues that was formerly necessary in order to cut thin sections from them is, in the majority of cases, no longer necessary. But there are some excep- tions. Such are, for instance, the cases in which it is desired to cut very large sections, such as sections of the entire human brain. The reagents employed for hardening are for the most*part of the same nature as those employed for fixing. But it does not follow that all fixing agents can be employed for hardening. Corrosive sublimate, for instance, would be most inappropriate as a hardening agent. 34. The Practice of Hardening. — Employ in general a relatively large volume of hardening liquid, and change it very frequently. If 28 FIXING AND HARDENING. the volume of liquid be insufficient, its composition will soon become seriously altered by the diffusion into it of the soluble substances of the tissues ; and the result may be a macerating instead of a harden- ing liquid. Further, as soon as, in consequence of this diffusion, the liquid has acquired a composition similar in respect of the propor- tions of colloids and crystalloids contained in it to that of the liquids of the tissues, osmotic equilibrium will become established, and diffusion will cease ; that is to say, the hardening liquid will cease to penetrate. This means, of course, maceration of internal parts. On the other hand, it appears that a certain slight proportion of colloids in the hardening liquid is favourable to the desired reaction, as it gives a better consistency to the tissues by preventing them from becoming brittle. Hence the utility of employing a certain proportion of hardening agent. Hardening had better be done in tall cylindrical vessels, the objects being suspended by a thread, or muslin bag, or otherwise, at the top of the liquid. This has the advantage of allowing diffusion to take place as freely as possible, whilst any precipitates that may form fall harmlessly to the bottom ; or, they may be laid on a layer of cotton- wool, or filter-paper, or spun glass. In general, begin hardening with a weak reagent, increasing the strength gradually, as fast as the tissues acquire a consistency that enables them to support a more energetic action of the reagent. Let the objects be removed from the hardening fluid as soon as they have acquired the desired consistency. CHAPTER IV. FIXING AND HARDENING AGENTS — MINERAL ACIDS AND THEIR SALTS. 35. Osmic Acid. --The tetroxide of osmium (Os04) is the substance commonly known as osmic acid, though it does not possess acid properties. It is extremely volatile, and in the form of an aqueous solution becomes partially reduced with great readiness in presence of the slightest contaminating particle of organic matter. It is generally believed that the aqueous solutions are reduced by light alone, but this is not the case : they may be exposed to the light with impunity if dust be absolutely denied access to them. The solution of osmic acid in chromic acid solution is not, like the solution in pure water, easily reducible, but may be kept without any special precautions. I therefore keep the bulk of my osmium in the shape of a 2 per cent, solution of osmic acid in 1 per cent, aqueous chromic acid solution. This solution serves for fixation by osmium vapours, and for making up solution of Flemming, which is the form in which osmium is most generally employed. A small quantity of osmic acid may also be made up in 1 per cent, solution in distilled water, and kept in a drop-bottle with grooved stopper, from which quantities can be obtained when required without removing the stopper. CORI (Zeit. wiss. Mik., vi, 1890, p. 442) finds that solutions in distilled water keep perfectly if there be added to them enough permanganate of potassium to give a very slight rosy tint to the liquid. From time to time, as the solution becomes colourless, further small quantities of the salt should be added, so as to keep up the rosy tint. BUSCH finds that the addition of sodium iodate hinders reduction (N enrol. Centralb., xvii, 1898, p. 476). PINTNER finds that a slight addition of corrosive sublimate has the same effect, e.g. 10 drops of 5 per cent, solution of sublimate added to 100 c.c. of 1 per cent, solution of osmic acid. For the Kopsch, Mann-Kopsch and Sjovall methods the osmic acid solution must be free from all traces of chrome and platinum salts, etc. 30 FIXING AND HARDENING AGENTS. For the so-called c regeneration ' of reduced solutions, see previous editions. Osmic acid is met with in commerce in the solid form in sealed tubes. The assigned weights should be checked, as they may vary greatly. Fixation by the Vapours. — This is indicated in most of the cases in which it is possible to expose the tissues directly to the action of the vapour. The tissues are treated as described in § 695. Very small objects, such as isolated cells, are simply placed on a slide, which is inverted over the mouth of the bottle. They remain there until they begin to turn brown (isolated cells will generally be found to be sufficiently fixed in thirty seconds : whilst in order to fix the deeper layers of relatively thick objects, such as retina, an exposure of several hours may be desirable). It is well to wash the objects with water before staining, but a very slight washing will suffice. For staining, methyl-green may be recommended for objects destined for study in an aqueous medium, and, for permanent preparations, alum-carmine, picro-carmine, or hsematoxylin. In researches on nuclei, it may be useful to employ the vapours of a freshly prepared mixture of osmic and formic or acetic acid (Gilson, La Cellule, i, 1885, p. 96). The reasons for preferring fixation by the vapour are that osmic acid is more highly penetrating in vapour than in solution ; that the arduous washing out required by the solutions is done away with ; and that all possibility of deformation through osmosis is elminated. See also under. "Cramer's Method," § 695. Fixation by Solutions. — Osmic acid is now very seldom used pure in the shape of solutions. When, however, it is so employed it is used in strengths varying from ^ to 2 per cent. I should say myself that, as a rule, not more than 0-1 per cent., and never more than 2 per cent., should be used. On account of its feeble penetrating power the objects to be fixed should be as small as possible. The solutions should be kept protected from bright light during the immersion of tissues. (This precaution is not necessary if Flemming's or Hermann's solution be used.) If the immersion is to be a long one the tissues must be placed with the solution in well- closed glass-stoppered vessels. The objects may be deemed to be fixed as soon as they have become brown throughout. But see " Mann-Kopsch Methods," §693. After-Treatment. — The excess of osmic acid must be well washed out before proceeding to any further steps in preparation ; water should be used for washing. Notwithstanding the greatest care in CHAPTER IV. 31 soaking, it frequently happens that some of the acid remains in the tissues, and causes them to blacken in time, and in any case hinders staining. To obviate this blackening it has been advised to wash them out in ammonia-carmine or picro-carmine (not very effectual), or to soak them for twenty-four hours in a solution of bichromate of potash (Miiller's solution or Erlicki's will do), or in 0-5 per cent, solution of chromic acid, or in Merkel's solution. The treatment with bichromate solutions has the great advantage of highly facili- tating staining with carmine or haematoxylin. Max Schultze recommended washing, and mounting permanently in acetate of potash ; Fol, treatment with a weak solution of carbonate of ammonia. But the best plan of all is to properly bleach the prepara- tions. See " Bleaching." This may be done by means of peroxide of hydrogen. OVERTON (Zeit. wiss. MiL, vii, 1890, p. 10) finds that it is completed in a few minutes in a mixture of 1 part commercial peroxide with 10 to 25 parts 70 per cent, alcohol. (The commercial peroxide, slightly acidulated with HC1, will keep well in the dark ; but the mixture with alcohol must be made fresh for use.) Accord- ing to BRISTOL (Amer. Natural, xxvii, 1893, p. 176) the peroxide acts best in the sun. BINET (Journ. de I'Anat. et de la PhysioL, xxx, 1894, p. 449) has successfully used permanganate of potash. MANN (Methods, etc., p. 83) takes a solution of 0-25 per cent., and treats the browned tissues with 1 part of saturated solution of sulphurous acid to 9 of normal salt solution. . . . MONCKEBERG and BETHE (Arch. Mik. Anat., liv, 1899, p. 135) have succeeded in satisfactorily restoring the staining susceptibility of osmium material by means of sulphurous acid (obtained by adding hydrochloric acid to bisul- phite of sodium, 2 to 4 drops of the acid added to 10 c.c. of a 2 per cent, solution of the salt). FOL (Lehrb., p. 174) recommends a weak aqueous solution of ferri- cyanide of potassium. I find the sulphate of iron solution used in Benda's hsematoxylin stain has a marked bleaching effect, and so also, though in a less degree, the iron alum of Heidenhain's process. ALTMANN (Die Elementarorganismen, pp. 33 and 35) puts sections overnight into gold chloride of 2 per cent., and reduces in formic acid in the sun, and removes the gold by iodised alcohol. But perhaps the best plan is the chlorine method of MAYER, or his magnesium peroxide, for both of which see " Bleaching." The same stains recommended for objects fixed by the vapours will be found useful here. For sections, of course, in both cases safranin and other anilin stains may be employed with advantage, may hsematoxylin. 32 FIXING AND HARDENING AGENTS. In general, osmic acid, especially when used in the form of vapour, fixes protoplasm very faithfully, nuclei badly. It is pre-eminently a fixative of the hyaloplasm or enchylema of cells. The penetrating poiver of the solution is very low, so that if any but very small pieces of tissue be taken the outer layers become over-fixed before the reagent has penetrated to the deeper layers. Over-fixed cells have a certain homogeneous, glassy, or colloid look, and are unfit for study, and attention should be confined to cells four or five layers deeper down, which will generally be found to present the required intensity of fixation. In these the fixation is admirable, with no shrinkage and next to no swelling of anything. * 36. The Osmium Tetroxide Reaction. — MANN believed that during the osmic reaction on fatty substances the Os04 was reduced to osmium tetra-hydroxide Os(OH)4. Other observers have assumed the reaction to be the reduction of the Os04 to some lower oxide. The matter has recently been reviewed by Professor J. R. PARTINGTON and Mr. D. B. HUNTINGFORD, who find that the reduced substance is a hydrated form of Os02, possibly Os02, 5H20, or Os02, 6H20. In all probability, Professor PARTINGTON informs me, the amount of water is not definite. (See also § 768 on " Fat.") 37. Osmic Mixtures. — NICOLAS (Intern. Monatsschr., 1891, p. 3) adds ^ per cent, of osmic acid to nitric acid of 3 per cent. I have employed a similar mixture and not had good results, though I find the mixture keeps perfectly. BUSCH (Neural. Centmlb., xvii, 1898, No. 10, p. 476 ; Zeit. wiss. Mik., xv, p. 373) finds that the penetration of osmic acid is enhanced by combining it with iodate of sodium, which by hindering its too rapid decomposition in the tissues ensures a more energetic action in the deeper layers. He adds 3 per cent, of sodium iodate to a 1 per cent, solution of osmic acid. UNNA (Monatschr. praJct. Derm., xxvi, 1898, p. 602) adds 1 per cent, of alum to a 1 per cent, solution. For some mixtures of KOLOSSOW, see 5th ed., or Zeit. iviss. Mikr., v, 1888, p. 51, and ix, 1892, p. 39. See also under " Cytology," § 677. 38. Chromic Acid. — Chromic anhydride, Cr03, is found in com- merce in the form of red crystals that dissolve readily in water, forming chromic acid, H2Cr04. These crystals are very deliquescent, and it is therefore well to keep the acid in stock in the shape of a 1 per cent, solution. Care must be take*i not to allow the crystals to be contaminated by organic matter, in the presence of which the anhydride is readily reduced into sesquioxide. Chromic acid is generally employed in aqueous solution. Some CHAPTER IV. 33 observers (KLEIN ; URBAN PRITCHARD ; PERENYI) have recom- mended alcoholic solutions ; but this is evidently irrational. For in the presence of alcohol chromic acid has a great tendency to become reduced to chromous oxide or sesquioxide, neither of which appears to have any fixing power. The most useful strengths in which it is employed in aqueous solution are from 0-1 to 1-0 per cent, for a period of immersion of a few hours (structure of cells and ova). For nerve tissues weaker solutions are taken, -^ to J per cent, for a few hours. Stronger solutions, such as 5 per cent., .should only be allowed to act for a few seconds. Washing out- -The general practice is to wash out very thoroughly with- water (by preference running water, for many hours) before bringing into alcohol or any staining liquid. For if the objects are put direct into alcohol it is found that after a short time a fine precipitate is thrown down on the surface of the preparations, thus forming an obstacle to the further penetration of the alcohol. Previous washing by water does not prevent the formation of this precipitate, and changing the alcohol does not prevent it from forming again and again. It has, however, been found by HANS VIRCHOW (Arcli. MiL Anat., xxiv, 1885, p. 117) that it may be entirely prevented by simply keeping the preparations in the dark. The alcohol becomes yellow as usual (and should be changed as often as this takes place), but no precipitate is formed. If this precaution be taken, previous washing with water may be omitted, or at all events greatly abridged. MAYER (Grundzuge, 1st ed., p. 28) proceeds as follows :--The hxed material is merely rinsed in water and brought direct into 70 per cent, alcohol. It is washed therein, preferably in the dark, until after several changes the alcohol remains colourless. It is then either passed through higher alcohols and imbedded in paraffin, the chromous oxide (or whatever chrome compound it may be that is present in the tissues) being removed from the sections after these are made ; or this necessary removal is performed at once. If this be preferred, the material is brought into sulphuric acid diluted with twenty volumes of water, or into nitric acid diluted with ten volumes of water. After at most a few hours therein, it will have become of a light greyish green, and on removal of the acid may be readily stained. If it be preferred to treat the sections, it is sufficient to put them into the usual hydrochloric acid alcohol (4 to 6 drops of HC1 to 100 c.c. of 70 per cent, alcohol), in which after a short time they become almost white, and will stain excellentlv with any of the M. 34 FIXING AND HARDENING AGENTS. usual stains. So also EDINGER (Zeit. wiss. Mik., i, 1884, p. 126 ; nitric acid 1 : 20 for five minutes). UNNA (Arch. Mik. Anat., xxx, 1887, p. 47) holds that the chrome is present in the tissues in the form of chromic chromate, and removes it by treatment with per- oxide of hydrogen. OVERTON (Zeit. wiss. Mik., vii, 1890, p. 9) employs a weak solution of sulphurous acid, which converts it into a sulphate. See also the directions for bleaching osmic acid prepara- tions, § 35. Tissues that have been fixed in chromic acid may be stained in aqueous solutions, as water does not have an injurious effect on them. The best stain for chromic material that has not been treated by Mayer's special process, or by a similar one, is hsematoxylin, or,- for sections, the basic tar colours. Chromic acid is not a very penetrating reagent, and for this reason, as well as for others, is now seldom used pure ioT fixing. For prolonged hardening it is generally employed in strengths of i to J per cent., the immersion lasting a few days or a few weeks, according to the size and nature of the object. Mucous membrane, for instance, will harden satisfactorily in a few days ; brain will require some six weeks. Large quantities of the solution must be taken (at least 200 grammes for a piece of tissue of 1 centimetre cube — Eanvier). In order to obtain the best results you should not employ portions of tissue of more than an inch cube. For a human spinal cord you should take 2 litres of solution, and change it for fresh after a few days. Six weeks or two months are necessary to complete the hardening. I think it is frequently useful to add a little glycerin ; there is less brittleness. The solution should be taken weak at first, and the strength increased after a time. The objects should be removed from the solution as soon as they have acquired the desired consistency, as if left too long they will become brittle. .They may be preserved till wanted in alcohol (95 per cent.). It is well to wash them out in water for twenty-four or forty-eight hours before putting them into the alcohol. After a time they generally become green in the alcohol. They may be bleached if desired. Chromic acid is a most powerful and rapid hardening agent. (By it you may obtain in a few days a degree of hardening that you would hardly obtain in as many weeks with bichromate, for instance.) It has the defect of a great tendency to cause brittleness. CHAPTER IV. 35 • 39. Chromo-acetic Acid (FLEMMING, Zellsbz., Kern. u. ZelltJi., p. 382). Chromic acid . . . 0-2 to 0-25 per cent. Acetic acid . . .0-1 per cent, in water. Flemming found this the best reagent for the study of the achromatic elements of karyokinesis. You can stain with hsematoxylin, or the basic anilin dyes. The following has been recommended for Annelids by EHLERS : — To 100 c.c. of chromic acid of 0-5 to 1 per cent, add from 1 to 5 drops of glacial acetic acid. The acetic acid is said to be sufficient to counter- act any shrinkage due to the chromic acid. Fix overnight, wash out several hours in water. Similar to this is the " chromo -acetic acid, Xo. 1," of Lo BIANCO (Mitth. Zool. Shit. Neapel, ix, 1890, p. 443), viz. 1 part 50 per cent, acetic acid and 20 parts 1 per cent, chromic acid, which is found very useful for fixing marine animals. 40. Chromo-formic Acid (RABL, Morph. Jahrb., x, 1884, pp. 215, '2 16). — Four or 5 drops of concentrated formic acid are added to 200 c.c. of 0-33 per cent, chromic acid solution. The mixture must be freshly prepared at the instant of using. Fix for twelve to twenty-four hours, wash out with water. Used by Rabl for the study of karyokinesis. 41. Chromo-aceto-osmic Acid (FLEMMING, Zettsubstanz, Kern und ZeUtheilung, 1882, p. 381). FIRST or WEAK formula : Chromic acid . . . 0-25 per cent. ) Osmic acid . . . .0-1 ,, in water. Glacial acetic acid . .0-1 „ ) MKVES (Enc//d. mikr. Techit., 1, p. -175) .sometimes adds 1 per cent, of sodium chloride. FOL (Lelirb. d. vet'fjl. mik. Anal., 1884, p. 100) recommends the following variant : 1 per cent, chromic acid . . . .25 vols. 1 per cent, osmic acid . . . 2 ,, 2 per cent, acetic acid . . . 5 „ AVater 68 „ — that is to say, a mixture weaker in osmium than Flemming's. A mixture still weaker than this in osmium, viz. with 1 vol. osmic acid solution, instead of 2, has been recommended by Com (Zeit. wiss. Mik., vi, 1890, p. 441). SECOND or STRONG formula (Zeil. tciv*. Mik., J, 1884. p. 349) : 1 per cent, chromic acid . . . .15 parts. 2 per cent, osmic acid . . . 4 „ Glacial acetic acid ..... 1 part. If this mixture be kept in stock in large quantities, it may go bad, on account of the large proportion of organic acid contained in it. 3—2 36 FIXING AND HARDENING AGENTS. I therefore recommend that the osmic and chromic acid be kept ready mixed in the proportions given, and 5 per cent, of acetic acid added at the moment of using. WEAKER FORMULA. — More recently, FLEMMING has been making up the mixture with only 2 parts of the osmic acid instead of 4, and has spoken of this modification as ' weaker osmium mixture ' (MEVES, in Encyd. Mikr. Techn., p. 476). MEVES (loc. cit.) takes for delicate objects 15 parts of chromic acid of only 0-5 per cent., 2 or 4 of osmic acid of 2 per cent., and 1 of acetic acid, and thus gets less shrinkage. Under "Cytology' Sections, § 678, see BENDA and GATENEY modifications. PODWYSSOZKI recommends (for glands especially) the following modification : 1 per cent. CrOs dissolved in 0-5 per cent, solution of corrosive sublimate . . . . .15 c.c. 2 per cent, osmic acid solution .... 4 c.c. Glacial acetic acid . . . . . 6 to 8 drops. The sublimate is said to augment the penetration of the osmium, but is unfavourable to staining (ZIEGLER'S Beitrdge z. path. Anat., i, 1886 ; Zeit. wiss. Mil\, iii, 1886, p. 405). The first or weak liquid is the better for very small objects, the second or strong one for larger ones, as it has better penetration. These liquids may be allowed to act for many hours or clays, or according to some workers even weeks or months ; but this exagge- rated fixation is clearly only justifiable in very special cases, if at all. For chromosome studies some workers fix for only one hour. Others recommend cooling the FLEMMING on ice before using. Wash out ,very thoroughly in water (running, twenty-four hours), or treat as directed for chromic acid, § 38. Stain with alum hsematoxylin if you wish to stain in toto (staining in this way with other reagents is possible, but difficult). Stain sections with safranin or other basic coal-tar colour, or with iron haema- toxylin. For fixing with the strong mixture you need only take a bulk of liquid of some 4 times the volume of the objects (but with the weak mixture the proportion should be increased). Both of them are first-rate fixatives of cellular structures, both as regards their preser- vation and as regards their optical differentiation. But they must be properly used, and not applied to objects for which they are not fitted. For instance, their power of penetration is extremely bad ; they will not fix properly, even in a loose-celled tissue, through more than a layer of about five cells thick. They are therefore suitable CHAPTER IV. 37 only for very small objects or for very small pieces of tissue, such as suffice for cytological or histological work. The strong liquid especially has not the character^of a general reagent. As a matter of fact it was recommended by FLEMMING in the first instance merely for a very special purpose, the hunting for karyokinetic figures, and not for general purposes. It is still very much used, but in my opinion unadvisedly. In most cases, Bouin's picroformol v, ill do all that it is intended to do, without its disadvantages. It may be used for prolonged hardening, e.g. of small pieces of nervous tissue, and is very good for that purpose. Fat is blackened (or browned) by it. See § 35. Chromatin is mordanted by it for basic anilin dyes, enabling them to give peculiarly sharp and powerful stains. 42. Osmic Acid and Bichromate. — ALTMAXN (Die Elementar- organismen, Leipzig, 1890) takes for his " bioblasts " a mixture of «M j ual parts of 5 per cent, solution of bichromate of potash and 2 per cent, solution of osmic acid. The bichromate ought not to contain anv free chromic acid. Refer to §680. Lo BIANCO (Mitth. Zool Stat. Neapel, ix, 1890, p. 443) employs for marine animals a mixture of 100 c.c. of 5 per cent, solution of bichromate and 2 c.c. of 1 per cent, osmic acid. HOEHL (Arch. Anat. Phys., Anat. Abth., 1896, p. 31) recommends a mixture of 80 c.c. of 3 per cent, bichromate, 20 c.c. of 1 per cent. o.Mnic acid, and 2 c.c. of glacial acetic acid. 43. Bichromate-chromic-osmic Acid. — CHAMPY (Arch, de Zool. Exper.. 1913). — Mixture of 7 parts of 3 per cent, bichromate of potash, 7 parts of 1 per cent, chromic acid, 4 parts of 2 per cent, osmium tetroxide. This mixture keeps well. Fix for from six to twenty -four hours. Wash out in running water about the same time. You can stain in iron hsematoxylin. or less well in ALTMANN or V ' BEND A. See § 681 for a description of mordanting after Champy's fluid. This fluid is extremely useful, and I nearly always use it in addition to FLEMMING. 44. Osmic, Bichromate, and Platinic Mixture (LINDSAY JOHNSON'S Mixture). — Latest formula, 1895, communicated by Dr. Lindsay Johnson : Bichromate of potash (2-5 per cent.) . .70 parts. Osmic acid (2 per cent.) . . . 10 ,, Platinum chloride (1 per cent.) . . . 15 ,, Acetic or formic acid . 5 ,, 38 FIXING AND HARDENING AGENTS. HENNEGUY, who has worked a great deal with this reagent, and recommends it highly, says (Lecons sur la Cellule, p. 61) that it is well only to add the acetic or formic acid just before using, as it frequently reduces the osmium and platinum very rapidly and energetically. He finds that it contracts the more spongy sorts of protoplasm less than mixture of FLEMMING. I think highly of it- for certain objects. Twelve hours is probably the optimum time for fixation. Wash out in water. 45. Platino-aceto-osmic Acid (HERMANN'S) Solution (Arch. Mik. Anat., xxxiv, 1889, p. 58). — One per cent, platinum chloride 15 parts, glacial acetic acid 1 part, and 2 per cent, osmic acid either 4 parts or only 2 parts. Hermann found that protoplasm structures are thus better preserved than with the chromic mixture. As with Flemming, the optimum time is from twelve to sixteen hours. Wash out at least three hours in running water. The after-treatment and staining should be the same as for object* o j treated with Flemming's solution. RENGEL (Zeit. wiss. Zoo/., Ixiii, 1898, p. 454) washes out for half an hour to an hour with •saturated aqueous sol. of picric acid, which he thinks facilitates the staining, especially of nuclei. The action of this fixative is, roughly, similar to that of Flemming's. Like Flemming's, it mordants chromatin for staining with " basic ' colours, with which it affords equally fine nuclear stains. But, owing to the platinum in it, it diminishes more than Flemming's the colorability of tissues with ' acid ' colours, so that it is extremely difficult to obtain good plasma stains after its action. It causes a notable shrinkage in chromatin. It gives a full fixation of cyto- plasm, to which it gives a much more fine-grained aspect than liquid of Flemming does. Leaving out the acetic acid, the solution may be used for mito- chondria as in § 679. 46. Rawitz (Zeit. wiss. Mikr., xxv, 1909, p. 386) takes 4 parts of Kahlbaurn's Phospho-Tungstic acid, 5 of alcohol, and 1 of acetic acid, added just before use, fixes for twenty-four hours, and washes out the sections before staining with water containing a little calcium acetate. 47. Nitric Acid (ALTMANN, Arch. Anat. Phys., 1881, p. 219).- Altmann employs for fixing embryos dilute nitric acid, containing from 3 to 3| per cent, pure acid. Such a solution has a sp. gr. of about 1 -02. Stronger solutions have been used, but do not give such good final results. After extensive trial I find ALTMANN'S solution to be a second- rate reagent, giving a weak and thin fixation. His (ibid., 1877, p. 115) recommended a 10 per cent, solution. CHAPTER IV. 39 Flemming at one time employed solutions of 40 to 50 per cent, for the ova of Invertebrates. TELLYESNICZKY (Arch. mik. Anat., lii, 2, 1898, p. 222) thinks that " for general cell-fixing " the proper strength is 2 to 2| per cent., as stronger grades act too energetically on the superficial layers. MAYER has had good results with 5 per cent, solution. Nitric acid has the valuable property of hardening yolk without making it brittle. Pure water should in no case be used for washing out ; the prepara- tions should be brought direct into alcohol. Some persons take absolute, but I should say 70 per cent, is more generally indicated. Rabl has employed a 1 or 2 per cent, solution of alum. For prolonged hardening, strengths of from 3 to 10 per cent, are sometimes employed. A strength of 12 per cent., allowed to act for two or thr>e weeks, is said to afford very tough preparations of the encephalon. BENDA (Verh. Anal, Ges., 1888 ; Ergeb. d. Anat., i, 1891, p. 7) fixes for twenty-four to forty -eight hours in 10 per cent, nitric acid, and then hardens in bichromate of potash. Fol's Mixture (verbally communicated to me). — Three vols. of nitric acid, with 97 vols. of 70 per cent, alcohol. 48. Chromo-nitric Acid (PERENYI'S formula, Zool. Anzeig., v, 1882, p. 459) : 4 parts 10 per cent, nitric acid. 3 parts alcohol. 3 parts 0-5 per cent, chromic acid. Fix for four to five hours and pass into alcohol of 70 per cent. This mixture has been criticised (see previous editions) as irrational, the alcohol reducing the chromic acid and itself becoming etherised by the nitric acid. Some workers reject it, especially for ova, for which it is specially intended. But others speak highly of it. I myself have used it extensively for preparing objects for dissection and museum specimens, and found it admirable for these purposes. But preparations made to test its value from a cytological point of view have given me only second-rate results. It is now little used. 49. Chromic Acid and Platinum Chloride (MERKEL'S Macula lutea des Menschen, Leipzig. 1870, p. 19). — Equal volumes of 1400 solution of chromic acid and 1400 solution of platinum chloride. Objects should remain in it for several hours or even days. After washing out with alcohol of 50 to 70 per cent., objects stain excellently. If objects that have been fixed by osmic acid be put into it for some hours, blackening is said to be effectually prevented. This is an excellent hardening medium for delicate objects. Merkel allowed from three to four days for the action of the fluid for the retina ; for Annelids Eisig employs an immersion of three to five hours, and transfers to 70 per cent, alcohol ; for small leeches 40 FIXING AND HARDENING AGENTS. Whitman finds one hour sufficient, and transfers to 50 per cent, alcohol. A similar mixture, with the addition of 0-25 to 0-1 per cent, of acetic acid, is recommended by BRASS for Protozoa ; and LAV- DO WSKY has used for nuclei a mixture of 10 parts of 1 per cent, chromic acid, 5 of 1 per cent, platinum chloride, and 100 of 5 per cent, acetic acid. Whitman recommends for the hardening of pelagic fish ova a stronger mixture (due, I believe, to Eisig), viz.- 0-25 per cent, solution of platinum chloride . . 1 vol. 1 per cent, solution of chromic acid . . 1 „ The ova to remain in it one or two days (WHITMAN, Methods in Micro. Anat., p. 153). Salts. 50. Chromates. — The chromates are amongst the oldest and best tried of hardening agents. The bichromate of potash especially was at one time universally employed for hardening all sorts of tissues. FLEMMING (Arch. mik. Anat., xviii, 1880, p. 352) pointed out that though it preserves cytoplasm well it causes chromatin to swell, and therefore should not be employed for the study of nuclei. But, duly corrected with acetic acid, it affords a correct and fine fixation of nuclei ; whilst preserving hyaloplasm and its inclusions, secretions, etc., much better than chromic acid. For an elaborate study of the action of chrome salts on nucleus and cytoplasm, see BURCKHARDT, La Cellule, xii, 1897, p. 335. He finds that the bichromates of sodium, ammonium, magnesium, strontium, and zinc have the same destructive action on nuclei that the bichromate of potassium has ; but that the bichromates of barium, calcium, and copper have not. He concludes that acetic acid ought always to be added, not only to ensure the correct fixation of nuclei, but also to enhance penetration and the good preservation of cytoplasm. The following is recommended by him as a good combination for tin1 fixation both of cytoplasm and nucleus : Bichromate of barium, 4 per cent, solution . . 60 vols. Bichromate of potassium, 5 per cent, solution . . 30 ,, Glacial acetic acid . . . . . . 5 ,, (Instead of the barium you may take. 4 per cent, solution of bichromate of calcium, or 6 per cent, solution of bichromate of copper.) For the demonstration of the achromatic figure of cell division he recommends— Chromic acid, 1 per cent, solution . . . .60 vols. Bichromate of potassium, 5 per cent, solution . . 30 ,, Glacial acetic acid . 5 ,, CHAPTER IV. 41 51. Bichromate of Potash. — Perhaps the most important of all known hardening agents, sensu stricto. It hardens slowly, much more so than chromic acid, but it gives an incomparably better consistency to the tissues. They may remain almost indefinitely exposed to its action without much hurt. The strength of the solutions employed is from 2 to 5 per cent. As with chromic acid, it is extremely important to begin with weak solutions and proceed gradually to stronger ones. About three weeks will be necessary for hardening a sheep's eye in solutions gradually raised from 2 to 4 per cent. Spinal cord requires from three to six weeks, a brain at least as many months. After hardening, the objects should be well soaked out in water before being put into alcohol, or be treated as directed for chromic acid, § 38. They had better be kept in the dark when in alcohol. See § 38. (Bomi and OPPEL [Taschenbuch, 3 Auf., 1896, p. 22] fix in the dark.) // you wish to have a good stain with carmine you should not put the objects into alcohol at all, even for a second, until they have been stained, You may stain either with carmine or hsematoxylin, as well as with tar colours. Bichromate objects have an ugly yellow colour which cannot be removed by mere soaking in water. It is said that it can be removed by washing for a few minutes in a 1 per cent, solution of chloral hydrate. Prof. GILSON writes me that alcoholic solution of sulphurous anhydride (S0.2) is very convenient for the rapid decoloration of bichromate objects. A few drops suffice. See also § 38, and " Bleaching." To facilitate staining with hsematoxylin, WOLFF (Zeit. wiss. Mik., xv, 3, 1899, p. 311) first stains in Boehmer's hsematoxylin for twenty-four hours, and then for a few minutes in the same hsematoxylin to which lias been added 1 drop per watch-glassful of 5 per cent, solution of oxalic acid. The simple aqueous solution of bichromate is hardly to be recom- mended as a fixing agent, because not only does it not preserve nuclei properly, but also because it penetrates very slowly. The first of these defects may be overcome entirely, the second to some extent by addition of acetic acid ; whence the liquid of TELLYES- NICZKY, next §. 58. Acetic Bichromate (TELLYESNICZKY, Arch. mik. Anat., Hi, 1889, p. 242) : Bichromate ...... 3 grms. Glacial acetic acid ..... 5 c.c. Water ....... 100 ,, Smaller objects to remain in the fluid for one or two days, larger 42 FIXING AND HARDENING AGENTS. ones longer. Wash well in plenty of water, and pass through alcohols of increasing strength, beginning with 15 per cent. Mixtures of bichromate with osmic acid have been given above, §§ 42, 43 and 44. 53. MULLER'S Solution.- Bichromate of potash . . . 2 — 2J parts. Sulphate of soda .... 1 part. Water . . . . . .100 parts. The duration of the reaction is about the same as with the simple solution of chromic salt. Recent authors find the action of this liquid to be identical with that of plain bichromate, and doubt whether the sulphate in it has any effect whatever as regards its hardening properties. Fol says that for mammalian embryos, for which it has been recommended, it is worthless. 54. ERLICKI'S Solution (Warscliauer med. Zeit., xxii, Nos. 15 and 18 ; Progres Medical, 1897, No. 31) : Bichromate of potash . . . 2-5 parts. Sulphate of copper .... 1-0 part. Water ...... 100-0 parts. Here the addition of the cupric sulphate is intelligible, for this salt is itself a hardening agent of some energy. As a matter of fact, ; Erlicki ' hardens very much more rapidly than either simple bichromate or Miiller's solution. A spinal cord may be hardened in it in four days at the temperature of an incubator, and in ten days at the normal temperature (FoL, Lehrb. d. vergl. mik. Anat., p. 106). Human embryos of several months may be conveniently hardened in it. Nerve-centres that have been hardened in Eiiicki's fluid frequently contain dark spots with irregular prolongations, simulating ganglion - cells. These are now known to consist of precipitates formed by the fluid. They may be removed by washing with hot water, or with water slightly acidified with hydrochloric acid, or by treating the specimens with 0-5 per cent, chromic acid before putting them into alcohol (TscHiscH, Virchow's Arch., Bd. xcvii, p. 173 ; EDINGER, Zeit. wiss. Mik., ii, p. 245 ; LOEWENTHAL, Rev. med. de la Suisse romande, 6me annee, i, p. 20). 55. KULTSCHITZKY'S Solution (Zeit. wiss. Mik., iv, 1887, p. 348). — A saturated solution of bichromate of potash and sulphate of copper in 50 per cent, alcohol, to which is added at the instant of using a little acetic acid, 5 or 6 drops per 100 c.c. To make the solution, add the finely powdered salts to the alcohol in CHAPTER IV. 43 excess, and leave them together in total darkness, for twenty-four hours. Fix for twelve to twenty-four hours in the dark. Then treat with strong alcohol for twelve to twenty-four hours. 56. DEKHUYZEN'S Liquids (0. R. Acad. Sci., cxxxvii, 1903, pp. 415 and 445). — (A) 250 c.c. of 2-5 per cent. sol. of bichromate in sea -water, 25 c.c. of 6-3 per cent, nitric acid, and 54 c.c. of 2 per cent, osmic acid. For general use with marine animals. (B) 173-1 c.c. of the bichromate sol. and 26-9 of 2 per cent. sol. of osmic acid. For objects containing calcareous elements that it is desired to preserve. These liquids are stated to be isotonic with sea-water. 57. Bichromate and Sublimate (KULTSCHITZKY, Arch. f. mik. Anat., xlix, 1897, p. 8). — Two parts bichromate, £ part corrosive sublimate, 50 parts 2 per cent, acetic acid, and 50 parts 96 per cent, alcohol. The mixture should be filtered after twenty-four hours. Tissues of verte- brates may remain in it for four to six days. LAVDOWSKY (Zeit. wiss. Mik., xvii, 1900, p. 301) takes 500 c.c. of 1 per cent, acetic acid, 20 to 25 grins, bichromate, and 5 to 10 c.c. saturated solution of sublimate in water. 58. Bichromate of Ammonia. — This salt is in considerable favour for hardening. Its action is very similar to that of the potassium salt. Fol says that it penetrates somewhat more rapidly, and hardens some- what more slowly. It should be employed in somewhat stronger solutions, up to 5 per cent. 59. Neutral Chromate of Ammonia is preferred by some. It is used in the same strength as the bichromate. Klein has recommended it for intestine, which it hardens, in 5 per cent, solution, in twenty-four hours. 60. Bichromate of Calcium.— SONNENBRODT (Arch. mikr. Anat., Ixxii, 1908, p. 416) fixes ovaries of Gallus in 20 parts of 2 per cent, sol. of calcium bichromate with 10 of 2 per cent. sol. of sublimate and 1 of acetic acid. 61. Bichromates and Alcohol.— Mixtures of bichromate of potash or ammonia with alcohol may be employed, and have a more rapid action than the aqueous solution. Thus HAMILTON takes for hardening brain a mixture of 1 part methylated spirits with 3 parts of solution of Miiller ; see also KULTSCHITZKY'S Mixture, ante, § 55). Preparations should be kept in the dark during the process of hardening in these mixtures. 62. Sulphurous Acid. — WADDINGTON (Journ. Roy. Mic. Sac., 1883, p. 185) uses a saturated solution of sulphurous acid in alcohol for fixing infusoria. OVERTON (Zeit. wiss. Mik., vii, 1890, p. 9) uses the vapours of an aqueous solution for fixing algas. CHAPTER V. FIXING AND HARDENING AGENTS — CHLORIDES, ORGANIC ACIDS, AND OTHERS. % Chlorides. 63. Bichloride of Mercury (Corrosive Sublimate). — Corrosive sublimate is soluble in about 16 parts of cold and 3 of boiling distilled water. It is more soluble in alcohol (1:3) or in ether (1:4) than in water. Its solubility in all these menstrua is augmented by the addition of hydrochloric acid, ammonious chloride, or camphor. With sodium chloride it forms a more easily soluble double salt ; hence sea-water may dissolve over 15 per cent.' The simple aqueous solutions should always be made with distilled -not spring— water. The HgCL2 in them is partly split up by hydrolysis into Cl, H, and (HgCl)2, or HgClOH (see Chem. Central!)., 1904, i, p. 571 ; the statements of MANN [Methods, pp. 22, 77] are incorrect). These solutions should give an acid reaction with litmus paper, whilst those made with strong sodium chloride solution are neutral. For fixing, corrosive sublimate may be used pure ; but in most cases a finer fixation will be obtained if it be acidified with acetic acid, say about 1 per cent, of the glacial acid. I find that a saturated solution in 5 per cent, glacial acetic acid is a very good formula for marine animals ; for others I should take the acid weaker. KAISER'S solution consists of 10 grms. sublimate, 3 c.c. glacial acetic acid, and 300 c.c. distilled water (from Zeit. wiss. Mik., xi, p. 378). VAN BENEDEN has used a saturated solution in 25 per cent, acetic acid, and Lo BIANCO (Mitih. ZooL Stat. Neapel, ix, 1890, p. 443) a mixture of 2 parts saturated solution with 1 part of 49 per cent, acetic acid. It is sometimes advisable to take the most concentrated solution obtainable. For some very contractile forms (coral polypes, Planaria), a concentrated solution in warm or even boiling water should be employed. For Arthropoda alcoholic solutions are frequently indicated. Delicate objects, however, may require treatment with weak solutions. CHAPTER V. 45 Objects should in all cases be removed from the fixing bath as soon as fixed, that is, as soon as they are seen to have become opaque throughout, which may be in a few minutes or even seconds. Wash out with water or alcohol. Alcoliol is almost always prefer- able. Alcohol of about 70 per cent, may be taken, and (MAYER, Intern. Monatsschr. Anat. Phys., iv, 1887, p. 43) a little tincture of iodine may be added to the liquid, either alcohol or water, used for washing, enough to make it of a good port-wine colour, and the mixture be changed until it no longer becomes discoloured by the objects. APATHY (Mikrotechnik, p. 148) takes a 0-5 per cent, solution of iodine in strong alcohol, leaves the objects in it (suspended) until they have become of about the colour of the solution, and then washes for twenty-four hours in pure alcohol. In obstinate cases solution of iodine in iodide of potassium (e.g. LUGOL'S) may be taken. MAYER (Zeit. wiss. Mik., xiv, 1897, p. 28) makes it by dissolving 5 grms. of iodide of potassium in 5 c.c. of distilled water and mixing this with a solution of 0-5 grin, of iodine in 45 c.c. of 90 per cent, alcohol, but seldom uses the mixture concentrated, merely adding as much, of it as is required to the alcohol or water containing the objects. The important point is, that the iodine and iodide be employed together. The iodine may be washed out in obstinate cases with magnesia water. Similarly APATHY (Mitth. Zool. Stat. Neapel, xii, 1897, pp. 729, 730). It has been objected to this process that iodine in potassic iodide precipitates corrosive sublimate instead of dissolving it. That is true, but the precipitate is soluble in excess of the precipitant. The iodide of potassium process should be employed with care, for the iodide may partly redissolve the precipitated compounds formed by the sublimate with the albuminoids, etc., of the tissues, and it may be well not to begin adding the iodine till the objects have been brought into fairly strong alcohol, 70 or 80 per cent. It is important that the sublimate be thoroughly removed from the tissues, otherwise they become brittle, and will not stain so well. They will also become brittle if they are kept long in alcohol. It may happen that if the extraction of the excess of sublimate from the tissues in bulk has been insufficient, crystals may form in the sections after they have been mounted in balsam. This may easily be prevented by treating the sections themselves with tincture of iodine for a quarter of an hour before mounting. Some workers hold that this does away with the necessity of treating the tissues in bulk with iodine, which is frequently a very long process. Thus, MANN (Zeit. wiss. Mik., xi, 1894, p. 479) prefers treating the sections rather than the tissues in bulk, on the ground that the iodine makes thenTsoft, so that they shrink on corning into paraffin. SCHAPER 46 FIXING AND HARDENING AGENTS. (Anal. Anz., xiii, 1897, p. 463), hou'ever, has shown that neglect to extract the sublimate from the tissues in bulk may give birth to serious artifacts, ichich appear to arise during the imbedding process. So also LOYEZ (Arch. Anat. Micr., viii, 1905, p. 71). HEIDENHAIN (Zeit. in'ss. J//A'., xxv, 1909, p. 398) removes the iodine from sections by means of sodium thiosulphate. You may stain in any way you like. Carmine stains are peculiarly brilliant after sublimate. The solutions must not be touched with iron or steel, as these produce precipitates that may hurt the preparations. To manipu- late the objects, wood, glass or platinum may be used ; for dissecting them, hedgehog spines, or ijuill pens, or cactus spines. When properly employed, sublimate is for general work un- doubtedly a most useful fixing agent. It is applicable to most classes of objects. It is perhaps less applicable, in the pure form, to Arthropods, as it possesses no great power of penetrating chitin. For cytological work it is, according to my experience, not to be trusted, and only to be recommended where more precise fixing agents are counter-indicated by reason of their lack of penetration, or the like. Amongst other defects it has that of frequently causing very serious shrinkage of cells. 64. Sublimate with Salt. — A solution containing 5 grms. sublimate, O5 grm. sodium chloride, and 100 c.c. water has been quoted as " solution of GAULE." A | per cent, aqueous solution of sodium chloride saturated whilst, hot with sublimate was much recommended by HEIDENHAIN (Festschrift /. Koelliker, 1892, p. 109). The addition of sodium chloride allows a stronger solution to be obtained than can be made with pure water, and also, it is stated, enhances the penetration of the sublimate. But the fixation -precipitates (§ 29) formed by the double salt are (according to SPULER, Enci/l. mil,-. Technik., p. 1274) for the most part soluble in water, thus giving rise to imperfect preservation. Concentrated (i.e. over 20 per cent.) solution in sea-water is recom- mended for some marine animals. STOELZNER (Zeit. wiss. Mikr., xxiii, 1906, p. 25) recommends saturated solution of sublimate in sugar solution of 44 per cent., as isotonic (for warm-blooded animals). Liquid of Lang (ZooL Anzeigrr, 1878, i, p. 14). — For Phuniria.- Distilled water . . . . . 100 parts. Chloride of sodium . . . .6 to 10 ,, Acetic acid . . . . 6 to 8 ,, Bichloride of mercury . . . . 3 to 12 „ (Alum, in some cases . . . . | part.) 65. Alcoholic Solutions. — APATHY (Mikrotechnik, p. Ill) rccom- CHAPTER V. 47 mends a solution of 3 to 4 grms. of sublimate and 0-5 grm. sodium chloride in 100 c.c. of 50 per cent, alcohol for general pur- poses. OHLMACHER (Journ. Ejrper. Medicine, ii, 6, 1897, p. 671) takes- Absolute alcohol . . . . .80 parts. Chloroform . . . . . 15 „ Glacial acetic acid . . . . 5 ,, Sublimate to saturation (about 20 per cent.). " Ordinary pieces " of tissue are sufficiently fixed in fifteen to thirty minutes. Entire human cerebral hemispheres, subdivided by Meynert's section, take eighteen to twenty-four hours. For liquids containing a much higher proportion of acetic acid, see Acetic Alcohol. * 66. Acetone Solution. — HELD (Arch. Anat. Phys., Anat. Abth., 1897, p. 227) fixes nerve-tissue in a 1 per cent, solution of sublimate in 40 per cent, acetone, and washes out through increasingly con- centrated grades of acetone. 67. Phenol Solution. — PAPPENHEIM (Arch. Path. Anat., clvii, 1899, p. 23) shakes up carbolic acid with aqueous sublimate solution and filters. 68. Ciaccio (Arch. Ital. Anat. Embr., vi, 1907, p. 486) has an irrational mixture of sublimate, iodine, and formol. 69. Mercuro-nitric Mixtures. — FRENZEL (Arch. mik. Anat., xxvi, 1885, p. 232) recommends a half-saturated solution of sublimate in 80 per cent, alcohol, to which is added nitric acid in the proportion of 1 drop to 1 or 2 c.c. Objects of the size of a pea to be fixed in it for five or ten minutes, then hardened in the same sublimate alcohol without the acid, and finally in 90 per cent, alcohol. It is said that the nitric acid renders after-treatment with iodine un- necessary. GILSON'S Mixture (GILSON, in litt. 1895). Nitric acid of 46° strength (this would be sp. gr. 1-456, or 80 per cent., nearly) .... 15 c.c. Glacial acetic acid . . . . 4 „ Corrosive sublimate ... 20 grms. 60 per cent, alcohol . . . 100 c.c. Distilled water .... 880 ,, When required for marine animals add a few crystals of iodine, which will prevent the formation of precipitates of sea salts. If in any case the preparations should show a granular precipitate, this may be removed by washing with water containing a little tincture of iodine. 48 FIXING AND HARDENING AGENTS. I find that it affords in general a faithful and delicate fixation, and gives to tissues an excellent consistency. Objects may remain in it for a considerable time without hurt. It has a high degree of penetration. A treatment for a few days with it will serve to remove the albumen from the ova of Batrachians. This liquid may be recommended to beginners, as it is very easy to work with. For some objects, as I found, the proportion of sublimate may be in- creased with advantage. KOSTANECKI and. SIEDLECKI (Arch. mik. Anat., xlviii, 1896, p. 181) take a mixture of saturated sublimate solution and 3 per cent, nitric acid in equal parts, or a mixture of equal parts of sublimate solution, 3 per cent, nitric acid, and absolute alcohol, fix for twenty-four hours, and wash out in iodine-alcohol. PETRUNKEWITSCH (Zool. Jahrb. Abth. Morpk., xiv, 1901, p. 576) takes water 300, absolute alcohol 200, glacial acetic acid 90, nitric acid 10, and sublimate to saturation. Both this and Grilson's have been much used lately. 70. Picro-sublimate Mixtures. — EABL'S (Zeit. wiss. Mik., xi, 1894, p. 165). — Sublimate, saturated solution in water, 1 vol. ; a similar solution of picric acid, 1 vol. ; distilled water, 2 vols. Embryos may be left in it for twelve hours, washed for two hours in water, and brought into weak alcohol. 0. vom BATH (Anat. Anz., xi, 1895, p. 268) takes cold saturated solution of picric acid, 1 part ; hot saturated solution of sublimate, 1 part ; glacial acetic acid, -J to 1 per cent. Also the same with the addition of 10 per cent, of 2 per cent, osmic acid solution. 71. Osmio-sublimate Mixtures. — MANN'S (Zeit. wiss. Mik., xi, 1894, p. 481) consists of a freshly prepared mixture of equal parts of 1 per cent, osmic acid solution and saturated solution of sublimate in normal salt solution (for nerve-centres). See " Mann-Kopsch Method," § 693. 72. Chromo-sublimate. — Lo BIANCO (Mitth. Zool. Stat. Neapel, ix, 3, 1890, p. 443). — Concentrated sublimate solution, 100 parts ; 1 per cent, chromic acid, 50 parts. MANN (Verh. Anat. Ges., 12, 1898, p. 39) takes for nerve-cells equal parts of 5 per cent, sublimate and 5 per cent, chromic acid. 73. Sublimate and Bichromate. — ZENKER'S Mixture (Miinchener med. Wochenschr., xxiv., 1894, p. 534 ; quoted from MERCIER, Zeit. wiss. MiL, xi, 4, 1894, p. 471). — Five per cent, of sublimate and 5 per cent, of glacial acetic acid dissolved in solution of MULLER. CHAPTER V. 49 Fix for several hours or overnight, wash out with water, treat the tissues in bulk, or the sections with alcohol containing tincture of iodine. Refer to § 684. See also RETTERER, Journ. Anat. Phys., xxxiii, 1897, p. 463, and xxxvii, 1901, p. 480. // the objects be allowed to remain too long in the fluid there may be formed precipitates, which it is very difficult to remove. SPULER (Encycl. mik. Technik., 1st ed., p. 1280) says that they may be avoided by removing the objects as soon as penetrated, and com- pleting the hardening in liquid of MULLER. I recommend this method. HELLY (Zeit. wiss. Mik., xx, 1904, p. 413) omits the acetic acid, and adds, immediately before use, 5 per cent, of formol. This is a splendid fixative for vertebrate material. Fix overnight, wash out in running water for several hours. See footnote to § 684. MAXIMOW (ib., xxvi, 1909, p. 179) adds 10 per cent, of formol and sometimes 10 per cent, of osinic acid of 2 per cent, (fix in the dark). FOA (Quart. Journ. Mic. Sci., 1895, p. 287) takes equal parts of saturated solution of sublimate in normal salt solution, and of liquid of Miiller, or 5 per cent, solution of bichromate. HOTEB, (Arch. Mikr. Anat., liv, 1899, p. 97) takes 1 part 5 per cent sublimate and 2 of 3 per cent, bichromate. KOHN (ib., Ixx, 1907, p. 273) takes 5 parts 5 per cent, sublimate, 15 parts 3£ per cent, bichromate, and 1 part acetic acid. 74. Sublamin (Ethylendiamm Sulphate of Mercury) is recommended in 5 per cent, solution by KLINGMULLEB and VEIEL (Zeit. iviss. Mikr., xxi,-1904, p. 58). 75. Platinum Chloride. — The substance used and intended by the authors who have recommended this reagent is not the true platinic chloride, or tetrachloride, PtCl4, but the compound H2PtCl6, that is, platinochloric, or hydro-chloro-platinic acid, by custom called platinum chloride. It occurs as brown-red crystals, easily soluble in water and very deliquescent. For this reason it had better be stocked in the form of a 10 per cent, solution, kept in the dark (weak solutions — 0-5 per cent. — may be kept in the light). It appears that some authors have stated that they were using platinous chloride, PtCl2, but that is not possible, as this salt is not soluble in water. RABL (Morph. Jahrb., x, 1884, p. 216) employed an aqueous solution of 1 : 300. The objects remained in it for twenty-four hours, and were then washed out with water. Well-washed preparations give good chromatin stains with the " basic ' tar colours ; but I find, as do others, that plasma-staining with the "acid" colours is 50 FIXING AND HARDENING AGENTS. rendered extremely difficult. It causes a certain shrinkage of chromatin. It is now almost always employed in the form of mixtures. For these see §§ 44, 45, 49, 76, as well as the mixtures given under " Picric Acid" and "Formol." 76. RABL (Zeit. wiss. Mikr., xi, 1894, p. 165) takes for embryos of vertebrates, and also for other objects, 1 vol. of 1 per cent, platinum chloride, 1 of saturated sublimate, and 2 of water. LENHOSSEK (Arch. mikr. Anat., li, 1898, p. 220) takes 20 parts of 1 per cent, platinum chloride, 20 of 5 per cent, sublimate, and 1 of acetic acid. 77. Palladium Chloride (SCHULZE, Arch. mik. Anat., iii, 1867, p. 477). — Used by Schulze as a hardening agent in a 1 : 800 solution, acidified with hydrochloric acid. CATTANEO has used it in solutions of 1 : 300, 1 : 600, or 1 : 800 strength, for from one to two minutes, for Infusoria. FRENKEL (Anat. Anz., viii, 1893, p. 538) recommends for connective tissue a mixture of 15 parts 1 per cent, palladium chloride, 5 parts 2 per cent, osmic acid, and a few drops of acetic acid. 78. Iridium Chloride (EISEN, Zeit. wiss. Mik., xiv, 1897, p. 195).- Solution of \ or \ per cent., acidified with 1 per cent, of glacial acetic acid. With the ovotestis of the snail, I have obtained about the worst fixation I have ever seen, but with the testis of Triton much better results. 79. Osmium Chloride (EISEN, Journ. of Morph., xvii, 1900). — Solution of | to ^ per cent. From specimens I have seen I should say it is useless. 80. Perchloride of Iron (For,, Zeit. wiss. Zool., xxxviii, 1883, p. 491, and Lehrb. d. vergl. mile. Anat., p. 102). — Fol recommends 1 vol. of Tinct. Ferri Perchlor. B.P. diluted with 5 to 10 vols. of 70 per cent, alcohol. The tincture diluted with 3 to 4 vols. of either alcohol or water has been recommended for fixing medullated nerve by PLATNER ( Zeit. wiss. Mik., vi, 1889, p. 187). 81. Iron Alum. — STRONG (Journ. comp. Neur., xiii, 1903, p. 296) fixes (and decalcifies) heads of young AcantMas in 9 parts of 5 per cent, solution of iron alum with 1 of formol, for about two weeks. 82. Chloride of Zinc is sometimes used for hardening brain (see Part II). GILSON (La Cellule, vi, 1890, p. 122) has used it as a fixative for the silk glands of Lepidoptera, as follows : Glacial acetic acid . . . . . 5 c.c. Mtric acid of 46° (or 80 per cent, nearly) . 5 ,, Alcohol of 80 per cent. . . . 100 „ Distilled water 300 „ Dry chloride of zinc .... 20 grms. CHAPTER V. 51 83. Iodine. — KENT (Manual of the Infusoria, 1881, p. 114) uses it for fixing Infusoria. Prepare a saturated solution of potassic iodide in distilled water, saturate this solution with iodine, filter, and dilute to a brown-sherry colour. A very small portion only of the fluid is to be added to that containing the Infusoria. Or you may use LUGOL'S solution : Water . . . . . . .100 parts. Iodide of potassium . . . . 6 „ Iodine ....... 4 ,, Or for small marine animals, a solution of iodine in sea-water. Personally I have found it very useful for the examination of sperma- tozoa. See also under Goodrich's lodine-Bouin method. Very small objects may be instantaneously fixed by means of vapour of Iodine. Crystals of iodine may be heated in a test-tube till the vapours are given off ; then on inclining the tube the heavy vapours may be made to flow over the objects arranged on a slide. The slide should then be warmed to about 40° C. for one to three minutes in order to evaporate the iodine from the objects, which may then be mounted or otherwise treated as desired (OVERTON, Zeit. wiss. Mile., vii, 1890, p. 14). Organic Acids, and other Agents. 84. Acetic Acid. — A substance most injurious to the finer elements of the cytoplasm ; in some cases it is indicated for a study of the nuclear elements. Flemming, who has made a special investigation of its action on nuclei, finds (Zellsubstanz, etc., p. 380) that the best strength is from O2 to 1 per cent. Strengths of 5 per cent, and more bring out the nuclein structures clearly at first, but after a time cause them to swell and become pale, which is not the case with the weaker strengths (ibid., p. 103). The strong acid is, however, a valuable fixative of certain objects, which it kills with the utmost rapidity, and leaves fixed in a state of extension. The modus operandi of VAN BENEDEN is as follows : — Pour glacial acetic acid in liberal quantity over the organisms, leave them until they are penetrated by it — which should be in five or six minutes, as the strong acid is a highly penetrating reagent — and wash out in frequent changes of alcohol of gradually increasing strength. Some persons begin with 30 per cent, alcohol, but this appears to me rather weak, and I think 70 per cent, or at least 50 per cent, should be preferred. Other energetic reagents may be combined with the glacial acetic acid if desired. Dr. LINDSAY JOHNSON (in litt.) has found that one of the best fixatives for retina is a mixture of equal parts glacial acetic acid and 2 per cent, osmic acid. S. Lo BIANCO adds to his "concen- trated " (49 per cent.) acid one-tenth of a 1 per cent, solution of chromic acid. He finds that even this small proportion of chromic 4—2 52 FIXING AND HARDENING AGENTS. acid serves to counteract in a marked degree the softening action of the acetic acid. Acetic acid, used alone, is only a fixative for a limited time. If its action be prolonged, it becomes a swelling agent. Its function in mixtures is, besides that of killing, the valuable one of counteracting the shrinking action of the ingredients with which it is combined, and by its swelling action enhancing the penetration of the mixture ; whilst by clarifying tissues it adds to the optical differentiation of their elements. The proportions in which it should enter into mixtures in general seem to me to be from 0-5 per cent, to 5 per cent, of the glacial acid ; higher strengths, such as 25 per cent, to 100 per cent., being only indicated in cases in which the highest possible penetration is the chief consideration. Throughout this work, wherever acetic acid is mentioned, it is the glacial acid that is meant unless the contrary is stated. All liquids containing a large proportion of this acid (e.g., §§ 85, 86) should only be allowed to act for a very short time. 85. Acetic Alcohol (CARNOY, La Cellule, iii, 1886, p. 6 ; and ibid., 1887, p. 276; v. BENEDEN et NEYT, Bull. Ac. Sri. Belg., xiv, 1887, p. 218 ; ZACHARIAS, Anat. Anz., iii, 1888, pp. 24 — 27 ; v. GEHUCH- TEN, ibid., 8, p. 227). — CARNOY has given two formulae for this important reagent. The first is- Glacial acetic acid .... 1 part. Absolute alcohol . . . .3 parts. The second is- Glacial acetic acid . . .1 part. Absolute alcohol . . , . 6 parts. Chloroform . . . . 3 ,, The addition of chloroform is said to render the action of the mixture more rapid. V. BENEDEN and NEYT take equal volumes of glacial acid and absolute alcohol. ZACHARIAS takes- Glacial acetic acid . . .1 part. Absolute alcohol . . . .4 parts. Osmic acid . . . . .a few drops. Acetic alcohol is one of the most penetrating and quickly acting fixatives known. It preserves both nuclei and cytoplasm, and admits of staining in any way that may be preferred. It was employed by all of the authors quoted for the ova of Ascaris- proverbially one of the most difficult objects to fix, — but I have found that it is applicable to many other objects. Wash out with alcohol, and avoid aqueous liquids as far as possible in the after- treatment. CHAPTER V. 53 Acetic Alcohol with Sublimate. — CARNOY and LEBRUN (La Cellule, xiii, 1, 1887, p. 68, due to GILSON). Absolute alcohol ...... 1 vol. Glacial acetic acid . . . . . 1 ,, Chloroform . . . . . . 1 „ Sublimate to saturation. (The mixture does not keep long, forming ethyl acetate, which precipitates). Isolated ova of Ascaris, even though furnished with a shell, are fixed in twenty-five to thirty seconds. Entire oviducts take about ten minutes. The liquid is therefore one of the most penetrating and rapidly acting of any, if not the most. Wash out with alcohol until all traces of odour of the acetic acid have disappeared (I myself wash out with alcohol containing tincture of iodine). I consider this a very fine reagent. For Ohlmacher's mixture see § 65. G. S. SANSOM'S Carnoy Modification. - Absolute alcohol . . . . .65 c.c. GUac; acetic acid . . . . 5 „ Chloroform . . . . . 30 ,, Corr. subl. to saturation. Leave ten minutes to half an hour ; wash in iodine absolute, then absolute. (Personal communication.) Eminently suitable for study of vertebrate material. I have seen some really brilliant results obtained by the use of this fluid (§ 590). 87. Trichlor-acetic Acid (HOLMGREN, Anat. Hefte, xviii, 1901, H. 2). — Five per cent, solution in water. Fix (nerve-cells) for eight to twenty-four hours, wash out with alcohol. See also HEIDENHAIN, Zeit. iviss. Mikr., xxii, 1905, p. 321, and xxv, 1909, p. 405, who makes a mixture of 6 per cent, sublimate solution with 2 per cent, of trichlor- acetic and 1 per cent, of acetic acid, which he calls " Subtriessig." 88. Trichlor-acetic Fluid for Batrachia (CHAMP Y, Arch. d. Zool. Exper. et Gen., t. lii, 1913).- Carbolic acid cryst. in sat. aq. sol. .- .15 parts. Formol, 40 per cent. . . . 4 ,, Trichlor-acetic acid, 20 per cent. . . 1-5 „ Outside of tissue often bad, inner parts better. 89. Salicylic Acid (HEIDENHAIN, Arch. mik. Anat., liv, 1899, p. 186).- Saturated solution in one-third alcohol. A trial has given me simply atrocious results. 90. Chloride and Acetate of Copper (Ripart et Petit' s Liquid, CARNOY, La Biologie Cellulaire, p. 94).- Camphor water (not saturated) . . 75 grms. Distilled water . . . . . 75 „ Crystallised acetic acid .... 1 grin. Acetate of copper . . . . . O30 „ Chloride of copper ..... 0-30 „ 54 FIXING AND HARDENING AGENTS. This is a very moderate and delicate fixative, extremely useful for objects that are to be studied in as fresh a state as possible in aqueous media. Objects fixed in it stain instantaneously and perfectly- with methyl green. Osmic acid may be added to the liquid to increase the fixing action. For cytological researches a valuable medium. 91. Nitrate of Copper (G-iLSON, from GELDERD, La Cellule, xxv, 1909, p. 12). — Nitrate of copper 200, formol 500, sea-water 200. Seven parts of this solution to be diluted with 100 of sea-water. For Crustacea. 92. Acetate of Uranium (SCHENK, Mirth. Embri/ol. Inst. Wien, 1882, p. 95 ; cl. GILSON, La Cellule, i, 1885, p. 141) has a mild fixing action, and a hi^h decree of penetration, and may be combined with methyl green. FRIEDENTIIAL (Sitzb. Ges. Nat. Freunde Berlin, 1907, p. 209) recom- mends equal parts of saturated solution of the acetate and trichlor-acetic acid of 50 per cent. 93. Picric Acid. — Picric acid in aqueous solution should be em- ployed in the form of a strong solution whenever it is desired to make sections or other preparations of tissues with the elements in situ, as weak solutions macerate ; but for dissociation preparations or the fixation of isolated cells, weak solutions may be taken. Flemming finds that the fixation of nuclear figures is equally good with strong or weak solutions. The saturated solution is the one most employed. (One part of picric acid dissolves in about 86 parts of water at 15° C. ; in hot water it is very much more soluble.) Objects should remain in it for from a few seconds to twenty-four hours, according to their size. For Infusoria one to at most two minutes will suffice, whilst objects of a thickness of several millimetres require several hours. Picric acid should always be washed out with alcohol, that of 70 per cent, being mostly indicated. Staining should be performed by means of alcoholic solutions, or if with aqueous, then with such as are themselves weak hardening agents, such as haemalum, carmalum, methyl green. Washing out is facilitated by heat, the extraction being about twice as rapid at 40° C. as at the normal temperature (FoL). It has been found by JELINEK (Zeit. iviss. Mik., xi, 1894, p. 242) that the extraction is greatly quickened by the addition of a base to the wash-alcohol. He recommends carbonate of lithia, A feu- drops of a saturated solution of the salt in water are added to the alcohol : a precipitate is formed. The objects are put into the turbid alcohol, which becomes clear and yellow in proportion as the picrin is ext racted. Further quantities of carbonate are added from time to time until the colour has been entirely extracted. CHAPTER V. 55 Tissues fixed in picric acid can be perfectly stained in any stain. It is seldom necessary to remove the picric acid by washing out before staining. Paracarmine, Boraxcarmine, or Hsemacalcium may be recommended for entire objects. The most important property of picric acid is its great penetration. This renders it peculiarly suitable for the preparation of chitinous structures. 94. Picric Alcohol (GAGE, Proc. Amer. Soc. Nicr., 1890, p. 120).- Alcohol (95 per cent.), 250 parts ; water, 250 parts ; picric acid, 1 part. 95. Picro-acetie Acid. — BOVERI (Zellenstudien, 1, 1887, p. 11) dilutes a concentrated aqueous solution of picric acid with two volumes of water and adds 1 per cent, of acetic acid. According to my experience, the results are miserable. ZIMMER'S mixture (from DEEGENER, Zool. Jahrb., Alrtli. Morph., xxvii, 1909, p. 634). — Saturated aqueous solution of picric acid, 10 parts ; absolute alcohol, 9 ; acetic acid, 1. 96. Picro-sulphuric Acid (KLEINENBERG, Quart. Jo urn. Mic. Sci., April, 1879, p. 208 ; MAYER, Mitt. Zool. Stat. Neapel, ii, 1880, p. 2).- MATER takes distilled water, 100 vols. ; sulphuric acid, 2 vols. ; picric acid, as much as will dissolve. Liquid of KLEINENBERG is made by diluting the concentrated picro- sulphuric acid prepared as above with three times its volume of water. I hold that the concentrated solution is generally preferable. This particularly applies to marine organisms. Wash out with successive alcohols, beginning with 70 per cent., never with water. Warm alcohol extracts the acid much more quickly than cold, without which weeks may be* required to fully remove the acid from chitinous structures. This liquid may still be useful for Arthropoda, on account of its great power of penetrating chitin ; and for some embryological purposes. For a fuller account see early editions. 97. Picro-nitric Acid (MAYER, Mitth. Zool. Stat. Neapel, 1881, p. 5)- Water ....... 100 vols. Nitric acid (of 25 per cent. N205) . . 5 „ Picric acid, as much as will dissolve. Properties of this fluid similar to those of pier o -sulphuric acid, with the advantage of avoiding the formation of gypsum crystals, and the disadvantage that it is much more difficult to soak out of the tissues. Mayer states that with eggs containing a large amount of yolk material, like those of Palinurus, it gives better results than nitric, picric, or picro-sulphuric acid. I myself consider it distinctly superior to picro-sulphuric for most things. See Hill's fluid, § 586. 56 FIXING AND HARDENING AGENTS. 98. Picro-hydrochloric Acid (MAYER, ibid.).- Water 100 vols. Hydrochloric acid (of 25 per cent. HC1) 8 „ Picric acid, as much as will dissolve. 99. Picro-chromic Acid (FoL, Lehrb., p. 100).- Picric acid, sol. sat. in water 10 vols. 1 per cent, chromic acid solution . 25 ,, Water . . . • • 65 ,, I have seen Fol's formula, with the addition of a trace of acetic acid, quoted as " liquid of Haensel." Lo BIANCO takes equal parts of picro- sulphuric acid and chromic acid of 1 per cent. RAWITZ (Leitfaden, 1895, p. 24) takes 1 part of picro-nitric acid, and 4 parts 1 per cent, chromic acid. Wash out in 70 per cent, alcohol. 100. Piero-osmic Acid. — FLEMMING (Zells. Kern u. ZeUth., p. 381) has experimented with mixtures made by substituting picric for chromic acid in the chromo-osmic mixtures (§ 42), and finds the results identical, so far as regards the fixation of nuclei. The fixation of cytoplasm is in my preparations decidedly inferior. 0. VOM RATH ( Anat. Anz., xi, 1895, p. 289) adds to 200 c.c. of saturated aqueous solution of picric acid, 12 c.c. of 2 per cent, solution of osmic acid, and 2 c.c. of glacial acetic acid. KAWITZ (Leitfaden, p. 24) takes picro-nitric acid, 6 vols. ; 2 per cent, osmic acid, 1 vol. Fix for half to three hours. Transfer direct to 70 per cent, alcohol. 101. Picro-platinie and Picro-platin-osmic Mixtures. — 0. VOM RATH (loc. cit., last §, pp. 282, 285) makes a picro -platinic mixture with 200 c.c. saturated aqueous solution of picric acid, 1 grm. of platinic chloride (dissolved in 10 c.c. of water), and 2 c.c. of glacial acetic acid. The picro-platin-osrnic mixture, which is, in my opinion, much superior, is made by adding to the foregoing 25 c.c. of 2 per cent, osmic acid. Other PICRIC MIXTURES. See §§ 70 and 110 to 112. Other Fixing and Hardening Agents. 102. Alcohol. — For fixing only two grades of alcohol should be employed — very weak, or absolute. Absolute alcohol ranks as a fixing agent because it kills and hardens with such rapidity that structures have hardly time to get deformed in the process ; very weak, because it possesses a sufficiently energetic coagulating action and yet contains enough water to have but a feeble dehydrating action. The intermediate grades do not realise these conditions, and therefore should not be employed alone for fixing. But they may be very useful in combination with other fixing agents by CHAPTER V. 57 enhancing their penetrating power ; 70 per cent, is a good grade for this purpose. Table for diluting alcohol (after GAY-LUSSAC). — To use this table, find in the upper horizontal row of figures the percentage of the alcohol that it is desired to dilute, and in the vertical row to the left the percentage of the alcohol it is desired to arrive at. Then follow out the vertical and horizontal rows headed respectively by these figures, and the figure printed at the point of intersection of the two rows will show how many volumes of water must be taken to reduce one hundred volumes of the original alcohol to the required grade. Weaker grade required. ORIGINAL GRADE. 90 p. 100. 85 80 p. 100. p. 100. 75 p. 100.. 70 p. 100. 65 p. 100. 60 55 p. 100. p. 10i>. 50 p. 100. p. 100. 85 6-56 80 13-79 6-83 75 21-89 14-48 7-20 . 70 31-05 23-14 15-35 7-64 65 41-53 33-03 24-66 16-37 8-15 • 60 53-65 44-48 35-44 26-47 17-58 8-76 55 67-87 57-90 48-07 38-32 28-63 19-02 9-47 50 84-71 73-90 63-04 52-43 41-73 31-25 20-47 10-35 45 105-34 93-30 81-38 69-54 57-78 46-09 34-46 22-90 11-41 40 130-80 117-34 104-01 90-76 ! 77-58 64-48 51-43 38-46 25-55 35 163-28 148-01 132-88 117-82 102-84 87-93 73-08 58-31 \ 43-59 30 206-22 188-57 171-05 153-61 136-04 118-94 101-71 84-54 67-45 Alcohol is an easily oxidisable substance. Chromic acid, for instance, easily oxidises it, first into aldehyde, and then into acetic acid. It follows that alcohol should not be combined in mixtures with oxidising agents of notable energy. Further, alcohol is a reducing agent, and therefore should not be combined with easily reducible substances. These remarks particularly apply to chromic acid. See §§ 38, 39, 48. For fixing, alcohol is a very third-class reagent, only to be used alone where better ones cannot be conveniently employed, though it enters as a useful ingredient into many mixtures, in which it serves to enhance the power of penetration. For hardening it is an im- 58 FIXING AND HARDENING AGENTS. portant one. 90 to 95 per cent, is the most generally useful strength. Weaker alcohol, down to 70 per cent., is often indicated. Absolute alcohol is seldom advisable. You ought to begin with weak, and proceed gradually to stronger, alcohol. Large quantities of alcohol should be taken. The alcohol should be frequently changed, or the tissue should be suspended near the top of it (§ 34). Many weeks may be necessary for hardening large specimens. Small pieces of permeable tissue, such as mucous membrane, may be sufficiently hardened in twenty-four hours. */ 103. Absolute Alcohol.- -This is sometimes valuable on account of its great penetrating power. Mayer finds that boiling absolute alcohol is often the only means of killing certain Arthropoda rapidly enough to avoid maceration. It is important to employ for fixing a very large proportion of alcohol. Alum-carmine is a good stain for small specimens so fixed. For preservation, the object should be put into a weaker alcohol, 90 per cent, or less. As to the supposed superiority of absolute alcohol over ordinary M rong alcohol, see last § ; and amongst authors upholding its superiority, see besides RANVIER, MAYER (Mitth. Zool. Stat. Neapel, ii, 1880, p. 7) ; BRUEL (Zool. Jahrb., Abth. Morpli., x, 1897, p. 569) ; and VAN KEES (ibid., Hi, 1888, p. 10). Absolute alcohol is a product that it is almost impossible to preserve in use, on account of the rapidity with which it hydrates on exposure to ;iir. Fol recommends that a little quicklime be kept in it. This absorbs part at least of the moisture drawn by it from the air. Ranvier prepares a sufficiently " absolute ' alcohol as follows : — Strong (95 per cent.) alcohol is treated with calcined cupric sulphate, with which it is shaken up and allowed to remain for a day or two. It is then decanted and treated with fresh cupric sulphate, and the operation is repeated until the fresh cupric sulphate no longer becomes conspicuously blue on contact with the alcohol ; or until, on a drop of t lie alcohol being mixed with a drop of turpentine, no particles of water can be seen in it under the microscope. The cupric sulphate is prepared by calcining common blue vitriol in a porcelain capsule over a flame until it becomes white, and then reducing it to powder (see Proc. Acad. \ is used here to denote the true fats and the lipoids. TRUE FATS are esters of the alcohol, glycerol, with the -higher fatty acids, chiefly palmitic acid, stearic acid and oleic acid. The fatty acids may be " saturated " as, for instance, palmitic and stearic acid, or they may be " unsaturated" as, for instance, oleic acid. * By Dr. W. Cramer, Imper. Cancer Research Bureau, and partly by J. B. G. CHAPTER XXIX. 357 LIPOIDS. This term was used originally to denote substances having solubilities similar to those of the true fats, i.e., substances which may be present, together with the true fats in alcohol, ether and chloroform extracts of tissues. This definition is, however, too crude, and the term as now used is restricted to certain chemi- cally well-defined groups of substances, which in fact constitute the bulk of such extracts, namely, cholesterol and its esters, phospha- tides, cerebrosides and phosphorised cerebrosides. The phos- phorised cerebrosides are ' compound lipoids." Their molecule is very large and consists of a chemical combination of several groups of simple lipoids, e.g., cerebrosides and a phosphatide in protagon. The existence of such compound lipoids is denied by some authors, who look upon them merely as mixtures of cerebrosides and phos- phatides. These groups of substances, although differing in their chemical constitution from each other and from true fats, frequently occur together in the tissues, and may, therefore, be presumed to have a similar physiological significance. They also resemble each other and true fats in their general staining reactions, but some of them exhibit characteristic differences in this respect. Some authors use the term " lipoid " as including the true fats. Others, morphologists in particular, use the term ; fat ' so as to include lipoids. It seems advisable, however, to separate the true fats from lipoids, as is done here, since the two groups of substances fulfil different physiological functions. The term ; LIPIN ' has been proposed by some authors to denote certain groups of lipoids. The advantage of this nomenclature is not obvious, and since almost every author who has used this term has given it a new definition it will not be used here. For a detailed account of the chemistry of these substances the reader is referred to the larger text-books and monographs, such as HAMMARSTEN'S Text-book of Physiological Chemistry, ABDER- HALDEN'S Biochemisches Handlexicon, Vol. iii, MACLEAN'S monograph on Lecithin and the Allied Substances. The table on p. 358 gives only a few elementary data concerning the chemical constitution of these substances and their solubilities, which are of importance in connection with their staining reactions. In the following, the term " true fats " will always be applied to mixtures of unsaturated and saturated fats, since in the tissues these substances always occur together. The table shows that all fatty substances occurring in the tissues contain somewhere in their molecule a double linkage (marked thus ii in the table overpage), and are therefore unsaturated compounds. This fact is of importance, 358 CONNECTIVE TISSUES. Table of Chemical Constitution of Fatty Substances. Group. True fats . Simple lipoids. Sterines. Phospha- tides (phos- phorised fats). Cerebro- sides. Compound lipoids. Phospho- rised cerebro- sides. Examples. Palmitin. Stearin. Olein. (A.) Cholesterin (Cholesterol). (B.) Cholesteri- nesters. Lecithin. Kephalin. Cerebron (Cerebrin, Phrenosin). Homocerebrin (Kerasin). Protagon. Characteristic central groups. Glycerine and fatty acids. 'ii (A.) Cholesterin free. ii (B.) Cholesterin and fatty ii acids, ii Glycerine, phosphoric acid, and fatty acids, 'ii Galactose and a fattv acid. A chemical combination of the two cerebrosides, cerebrin and homocere- brin (vide supra), and a substance sphingomye- lin, allied to the phos- phatides (see next column). Additional groups. None. None. A nitrogenous base, e.g., cholin in the case of lecithin. The nitrogenous base sphingosin. ii Sphingomyelin, which contains phosphoric acid, a fattv acid, sphingosin and cholin ii ii Solubilities. Readily soluble in ace- tone, ether, chloro- form and benzene. (A.) Same as true fats. (B.) Not readily soluble in alcohol and ace- ton, soluble in ether, chloroform and ben- zene. Not soluble in acetone, readily soluble in alcohol, ether, chloro- form and benzene. Kephalin, when pure, is insoluble in alcohol. Insoluble in fatty sol- vents in the cold ex- cept pyridine. Soluble in hot alcohol, ben- zene, chloroform. Same as cerebrosides. as on it depends most of the characteristic staining reactions for fatty substances. The characteristic staining and other reactions for fatty sub- stances fall into five groups :- (1) Staining with Sudan III., or Scharlach R. (2) Blackening with osmic acid, either, with the acid itself or mixed with bichromate solution. (3) Staining with hsematoxylin after mordanting with bichromate. (4) Their behaviour in polarised light. (5) Staining with Nile blue. The rationale of these methods will now be considered. (1) SCHARLACH R, SUDAN III. — Mixtures of the true fats, as they occur in the tissues, are readily stained by these dyes. Most lipoids, when pure, do not take this stain at all, or only slightly, except cholesterinesters, which take the stains, although not as readily as the true fats. The staining is probably a purely physical process and depends on the solution of the stain in the fatty material. Such solution occurs only when the fatty material is fluid, and this condition is fulfilled in the tissues where mixtures of the true fats CHAPTER XXIX. 359 are always present together, as, for instance, in the cells of adipose tissue, or in cells showing fatty degeneration. Mixtures of the true fats and lipoids which constitute, for instance, many of the fat globules of the adrenal cortex also take these stains. It is to be noted, however, that Scharlach R and Sudan III. are applied in alcoholic solution, and in the process of staining with these dyes some of the fatty substances tend to be dissolved out. The true fats are apparently not so readily dissolved as some substances, which show a double refraction, presumably cholesterin — fatty acid mixtures. The method of Herxheimer, in which Scharlach R is used in strongly alkaline solution, has been recommended on the ground that it is a more ' ' energetic ' ' fat stain for fatty substances in the sense that it stains many cell inclusions which are not stained by Scharlach or Sudan alone. The chemistry of the reaction has not been worked out. It depends probably on the saponifying action of the alkali, which liberates some fatty acids and then produces mixtures of fatty acids and lipoids, which are more readily stained by Scharlach. The range of staining by this method is, therefore, probably as wide as that of osmic acid alone, and will be found to comprise most fatty substances, but for the purpose of differentiating between the different groups of fatty substances has lost the advantage of restricted staining, which the ordinary staining with Sudan III. and Scharlach R alone possesses. (2) OSMIC ACID METHODS.- -The true fats and the lipoids are all blackened by osmic acid. This blackening indicates a reduction of osmic acid to a lower oxide. It is due to the fact that all these fatty substances have a double linkage (marked ii in table on p. 358) in their molecule and are, therefore, more or less easily oxidised. But the various groups of substances differ in the readiness with which they are oxidised, and consequently in the rapidity with ivhich they are blackened by osmic acid and the depth of the blackening produced. The true fats are blackened most rapidly and most deeply, the phospha- tides, lecithin and kephalin, come next in order, while cholesterin, the cerebrosides and phospho-cerebrosides are least susceptible to the oxidising action of osmic acid. In fact, these substances when solid are not blackened at all, but are so when dissolved in an appro- priate solvent, such as chloroform. In the myelin sheath of peri- pheral nerves they are present in the form of a colloidal solution, since it can be shown that they contribute to the blackening of the sheath by osmic acid. This difference in the reducing power of the various fatty sub- stances can be accentuated by using osmic acid together with bichro- 360 CONNECTIVE TISSUES. mate solutions, as in the Marchi method. The bichromate, which is itself an oxidising agent, acts in the various double linkages, and prevents the osmic acid from being reduced except by the substances having the strongest reducing power ; these are the true fats and mix- tures of cholesterin and unsaturated fatty acids. Since the latter show double refraction in polarised light (see below), while the true fats do nob, it is possible to differentiate these two groups of substances. The behaviour of mixtures of cholesterin and fatty acids is para- doxical, since cholesterin alone and fatty acids alone do not blacken with osmic acid after bichromate. The same paradoxical behaviour is exhibited by these cholesterin fatty acid mixtures in their staining reaction with hsematoxylin after mordanting with bichromate (see next paragraph). One must assume that cholesterin is present in these mixtures in a special physical state, in which it exhibits a greater reducing power. The myelin sheath of normal nerves does not contain true fats and gives, accordingly, no blackening with bichromate-osmic acid treatment. In the early stages of degeneration globules of true fat are formed, which stain black with bichromate-osmic acid and then give the positive Marchi reaction. With other tissues rich in lipoids, such as the adrenal cortex, no such clear distinction can be obtained because the lipoids are present therein, not only together with true fats, but also mixed with them, so that a globule of fatty material frequently contains both groups of substances. (3) STAINING WITH H^MATOXYLIN AFTER MORDANTING WITH BICHROMATE. — This method, which was introduced originally by Weigert for the staining of the medullary sheath of nerves, is applicable to all fatty substances. Like the preceding method, it also depends on the presence of a double linkage in the molecule, as pointed out by Thorpe. Substances having a double linkage are oxidised by the bichromate solution and, in this process of oxidation, a chromium compound is formed which is insoluble in fat solvents and which at one stage of the oxidation has the property of forming a dark blue lake with hsematoxylin. If oxidation be continued, however, hsematoxylin will again cease to stain. The rate of oxidation with bichromate varies, as might be expected, with the concentration of the solution used and with the temperature at which it is carried out. Working under similar conditions, it is found again that the different fatty substances vary in the readiness with which they are oxidised by bichromate and, consequently, reach the stage of staining with hsematoxyiin after different periods CHAPTER XXIX. 361 of mordanting with bichromate. The different lipoids, when pure, differ greatly in the ease with which the stainable chromium com- pounds is formed ; thus the cerebrosides and protagon stain after a short mordanting ; the unsaturated true fats require a slightly longer time, while lecithin, and especially cholesterin, are very resistant and require prolonged mordanting. But mixtures of cholesterin and the various fatty substances show quite a different behaviour and reach the stainable stage very rapidly. This appears to be due to the fact that such mixtures form colloidal solutions in a peculiar physical condition (fluid crystals). It will be clear that a histochemical identification of the various lipoids by means of this method is not possible. Its value lies in the possibility of demonstrating, first, the presence of fatty substances by a method which gives good histological details, and secondly, by comparison with normal tissues, the occurrence of chemical changes in these substances under pathological conditions. As introduced by Weigert, the method stains the lipoids of the normal myelin sheath. By prolonging the bichromating the degenerating nerve fibres may be made to stain in the early stage of degeneration, or the fat droplets in fatty organs may be stained. The same principle underlies Altmann's method for the staining of mitochondria, which are by some believed to consist of a central core of protein covered by an envelope of fatty material. The original method consists in mordanting with a bichromate solution and staining with acid fuchsin, just as Weigert originally used acid fuchsin for the staining of the myelin sheath. In the staining of mitochondria, the acid fuchsin can again be replaced by hsematoxylin (Heidenhain's hsematoxylin). (4) BEHAVIOUR IN POLARISED LIGHT. — The true fats and the fatty acids are isotropic, i.e., show no double refraction in polarised light, so that with crossed nicols the field appears dark. The lipoids are anisotropic. In fresh teased preparations they can be seen with crossed nicols as luminous droplets with a varying degree of brilliancy. The double refraction disappears on gentle heating to about 60° and reappears on cooling. In formol fixed frozen sections the aniso- tropic lipoids appear chiefly in the form of needles and as droplets. Heating and cooling produces the effect mentioned above. The behaviour in polarised light is, therefore, an easy and im- portant means of differentiating the isotropic true fats from lipoids. (For a detailed description of the technique of the polarisation microscope, see AMBRONN, Anleitung zur Benutzung des Polarisa- tions Mikroskopes bei Histologischen Untersuchungen ; ADAMI, 362 CONNECTIVE TISSUES. The Myelin and Potential Fluid Crystalline Bodies of the Organism, 1906 ; KAISERLING and OGLER, " Uber das Auftreten von Myelin inZellen, etc.," Virchow's Archiv., clxvii.). (5) NILE BLUE. — When a solution of Nile blue is boiled with sulphuric acid the solution contains, in addition to the original basic oxazine dye, which unites with fatty acids to form a blue compound, a red oxazone dye, not basic in character, but soluble in liquid fat and giving it a red colour. The stain, therefore, enables us to distinguish neutral fat and fatty acid. If a given globule contains neutral fat and no fatty acid, it will be coloured red ; if it contains fatty acid only, it will be coloured blue ; but if it contains both neutral fat and fatty acid, it will be coloured a tint between blue and red, depending on the proportion of neutral fat and fatty acid. The principal histochemical group reactions are summarised in the following table :- Table of some important Histochemical Group Reactions. Sudan. Double refrac- tion. Osmic acid. Osmic after bi- chromate. Solubility in cold Acetone. Alcohol. Chloro- form. True fats + + + - + + + + + + + Cholesterin 1 + _ + + Cholesterinesters + 4- — — — -(- Phosphatides . 4- + — — + + Cerebrosides . — -)- -)- — — — -j- Phospho-cerebrosides + + — — — + Cholesterin - fatty ) acid mixtures. j n Although different groups of fatty substances differ in their behaviour towards different staining methods, it is, nevertheless, necessary to proceed with caution in interpreting the results obtained when these methods are applied to tissues. In the tissues the various groups of fatty substances are mixed, so that one globule may contain two or three different substances. The various substances may then form a special kind of colloidal solution, which modifies their physical state and alters their staining reaction completely. The behaviour of mixtures of cholesterin and fatty acids is an example and has been referred to above. Or one substance encloses another substance belonging to a different group. In such a case the staining reactions of the globule would be those of the material which constitutes the envelope. In the adrenal cortex, for instance, CHAPTER XXIX. 363 many globules contain a core of lipoids. surrounded by true fats. In such a case examination in polarised light is helpful. Differentia- tion by means of different solvents is also possible within limits. Again, the size of the globules is, for obvious reasons, an important factor in the methods which involve mordanting with bichromate. But even if due consideration is given to these reservations, the application of these staining reactions has yielded important results and has profoundly modified our conceptions of the part taken by the fatty substances as constituents of protoplasm. In the first place, it is important to realise that no one single method is a specific staining method, either for all fatty substances or for any one group of them. Thus, Sudan III. or Scharlach R. do not stain all fatty substances, as a glance at the table of the group reactions shows. They stain intensely the true fats, and less intensely cholesterinesters and cholesterin-fatty acid mixtures. But many lipoids are not stained by these dyes, and an examination in polarised light is necessary to detect their presence. Osmic acid alone has the widest range as a reagent for fatty substances, and stains all the different groups. With some substances, however, such as chole- sterin or cerebrosides, it may give negative results if they are not present in a state of colloidal solution in the cytoplasm. But since osmic acid stains also substances which are not fatty in nature, e.g., adrenalin in the cells of the adrenal medulla, it cannot be looked upon as a specific stain. In the case mentioned, a differentiation can still be effected by immersing the section in turpentine, which dissolves the fatty substances, even after osmication. The method giving the highest degree of specificity is the use of osmic acid after bichromate. This will stain only the true fats or cholesterin-fatty acid mixtures. Or, expressed in terms of everyday histological technique, the presence of black cell globules in material fixed either in bichromate-osmic mixtures, such as Altmann's, Champy's or Flemming's fixatives, or first in bichromate fixatives, such as Midler's or Zenker's fluids, and post-osmicated (§ 691) indicates the presence either of true fats or of cholesterin-fatty acid mixtures. These two can then be differentiated by examination in polarised light. It is perhaps equally important to be able to draw the opposite con- clusion, when other facts have indicated the presence of fatty substances. The absence of blackening of the globules under the conditions just mentioned definitely excludes the presence of true fats. Thus, true fats can be proved to be absent from the myelin sheath of normal nerves. The significance of the staining of fatty substances after mordanting with bichromate has already been 364 CONNECTIVE TISSUES. referred to as revealing the essential similarity of Weigert's method for the myelin sheath of nerves and Altmann's method for mito- chondria. The staining reactions of the Golgi apparatus — blacken- ing after prolonged action of osmic acid, staining with hsematoxylin after prolonged mordanting with bichromate — suggest that it is made up of fatty substances, and, further, that these substances are not readily oxidised. They thus correspond in their behaviour to lipoids such as cholesterin. From what has been said, it is clear that for the study of fatty substances several methods must be applied to the tissues. In so far as the selection of a fixative is concerned, it follows that fixatives containing alcohol or chloroform must be avoided. A fixative such as Carnoy, or such mixtures containing strong alcohol or chloroform, are themselves fat solvents, as well as lipoid solvents, and they dissolve away all fat-vacuoles and shrink up cell organs which may be formed partly of other ' fatty ' sub- stances, e.g., mitochondria. Other fixatives, such as picric acid or corrosive sublimate, while in themselves not fat or lipoid solvents, are unable to prevent the fat from being dissolved away in any subsequent dehydration and clearing of the tissue. Very few reagents are known which are able to form with fat substances insoluble or scarcely soluble in alcohol and a clearing medium, like xylol ; osmium tetroxide and, to a less extent, potassium bichromate, are thus valuable reagents to the histologist. Various oils act differently towards these fat Os02 compounds ; xylol and chloro- form do not easily disintegrate them, but an oxidising oil like tur- pentine-will quickly do so. The " compounds " of Os04 with fats and lipoids are ill understood, but Partington and Huntingford have recently shown (see § 772) that the reduced black substance is a hydrated form of Os02. Martinotti (see § 772) has recently introduced a new method which may prove of great importance. According to this observer the orange yellow dye chrysoidin (phenyl-azo-m-phenylene-diamide), when applied to fatty tissue and then treated with an oxidising agent, such as bichromate of potash, has the power of preventing the fat globules from being dissolved away in alcohol, benzol and xylol. In preparations the fat looks a brown orange colour. A complete histochemical investigation of fatty cell inclusions comprises, thus, the following methods :- (1) In fresh preparations : (a) Examination in polarised light. (6) Staining with Sudan III. or Scharlach R. CHAPTER XXIX. 365 (c) Staining with osmic acid in solution or as vapour. (2) In frozen sections, material fixed in formol : (a) Examination in polarised light. (b) Staining with Sudan III. or Scharlach R. (c) Staining with Nile blue. (d) Repeat (b) and (c) after immersing sections in cold acetone or cold alcohol for a few minutes. (3) In paraffin sections : (a) Fixation in osmic acid. (b) Fixation in bichromate osmic acid mixtures or fixation in bichromate solutions and post-osmication. (c) Prolonged fixation in bichromate and staining with Sudan III. (Ciaccio, Bell). (d) Staining with hsematoxylin (Weigert, Lorrain, Smith, Dietrich). (e) Treatment with chrysoidin and subsequent fixation in bichromate (Martmotti). Such a plan of investigation refers especially to vertebrate materials, but with certain small modifications is applicable to the study of fatty substances in developing eggs, embryos, and to the tissues of invertebrates. Paragraphs 1 and 3 are both applicable to invertebrate tissues and embryos, while in the case of para- graph 2 the tests can be used after the whole embryos or eggs have been fixed in neutralised and suitably diluted formalin. It should be remembered that in all animal cells (and possibly plant cells, too) there exist two categories of cytoplasmic inclusions, Golgi apparatus and mitochondria, which are partly formed of lipoid materials, and which we now know may produce fats, or may metamorphose into fats. In nearly all eggs where fat granules are present, exami- nation has revealed the fact that such " yolk " is derived either from Golgi elements or mitochondria (GATENBY and WOODGER, Journ. Roy. Micr. Soc.} 1920), and in certain cells of vertebrate tissues we know that mitochondria may change into fat (MURRAY, Scientific Report Cancer Research Fund, 1919). Reference should be made to the sections on ' Mitochondria," "Golgi Apparatus," "Fat," and "Yolk" (§§ 673—713), and especially to the tables in §§ 702, 708, 710 and 712, where some attempt has been made to illustrate the behaviour of the various inclusions after the application of certain well-known techniques. It is always necessary to ascertain exactly the condition and behaviour of the mitochondria and Golgi apparatus in tissues or cells being investigated for fatty and lipoid substances, particularly 366 CONNECTIVE TISSUES. in view of any inter-relationship which may exist between the former and the latter. See also BELL (Journ. Med. Research, xxiv, 1911, p. 539 ; Journ. of Pathol. and Bad. xix, 1914, p. 105. CIACCIO (Centralblatt f. ally. Pathol. and Path. Anatomic, xx, 1909, p. 771 ; Arch. f. Zellforschung, v, 1910, p. 235. CRAMER, FEISS and BULLOCK (Proceed. Phys. Soc., 1913 ; Journ. of Physiology, xlvi, p. 51. International Congress of Medicine, London, 1913. Section of Pathology). DIETRICH (Erg. d. Allg. Pathologic und Patholog. Anat., xiii, 1909, pt. 2, p. 283 ; Deutsche Patholog. Gesellsch., xiv, 1910, p. 263). KAWAMURA (Die Cholesterinester verfcttung. Jena. Gustav Fischer. 1911). SMITH and MAIR (Journ. Pathol. and Bact., xiii, 1909, p. 14 ; Skand. Arch. f. Physiol., xxv, 1911, p. 247). 769. Fixing and Staining.- -The choice o': the fixative depends on the question whether the material is to be examined in frozen sections or in paraffin sections. In any case all fixatives containing acetone, alcohol, chloroform or other fat solvents are excluded. For paraffin sections the material may be fixed in osmic acid alone (1 per cent, in solution, or 2 per cent, if fixed in vapour), or in osmic acid mixed with bichromate solution (see fixatives of Flemming, Altmann, Champy). Or it may be fixed in formol bichromate and treated subsequently with a bichromate-osmic mixture (see methods of Schridde and Marchi). As stated in the general part (see p. 363) the different methods give different results with the various groups of fatty substances. For all these methods very small pieces of tissue must be used. For the effects of alcohol on the blackening of certain fatty substances by osmic acid, see HAND- WERCK, Zeit. iviss. Mik., xv, 1898, p. 177 ; MULON, ibid., xxii, 1905, p .138 ; GOLODETZ, ibid., xxviii, 1911, p. 213 ; and Chem. Rev. Fett u. Harzindustrie, xvii, 1910, p. 70 ; LOISEL, C. R. Soc. Biol., 1903, p. 826. Another method consists in fixing and mordanting with strong bichromate solution and subsequent staining with Sudan (see below, Bell's method, also Ciaccio) or with hgematoxylin (methods of Weigert for nervous system ; also Lorrain Smith, Dietrich). For examination in frozen section the tissue may be fixed in formol saline or formol-bichromate, or the stain may be applied directly to the fresh tissue after teasing. For fine cytological work, the formol should be neutralised by shaking with solid calcium carbonate. HAYS BULLARD (Amer. Journ. Anat., xix, 1916) recom- mends neutralisation and distillation method of GUSTAV MANN (Physiological Histology, Oxford, 1902) : neutralise commercial CHAPTER XXIX. 367 solution with sodium or lithium carbonate, and freshly distil. A 20 per cent, solution is then prepared and rendered isotonic : •75 gins, of NaCl to 100 c.c. of fluid. With short fixation (thirty-five minutes to five hours), the quantity of fat usually does not differ from that seen in fresh tissue (Bullard). Cut on freezing microtome, stain by one of the methods given below, preferably Herxheimer's alkaline scarlet red. As control use also fresh tissue. For quinolein blue, see § 322. DADDI (Arch. Ital. BioL, xxvi, 1896, p. 413) stains fat in tissues by treating for five to ten minutes with concentrated alcoholic solution of Sudan III. washing for the same time with alcohol, mopping up with blotting paper, and mounting in glycerin. Similarly RIEDER, see Zeit. wiss. Mik., xv, 1898, p. 211. The alcohol for making the stain should be of 70 per cent., according to most authors, though SATA (Beitr. path. Anat., xxviii, 1900, p. 461 ; Zeit. wiss. Mik., xviii, 1901, p. 67) employs 96 per cent. ROSENTHAL (ibid., xix, p. 469 ; Verh. path. Ges., September, 1899, p. 440) insists that the washing-out be done with alcohol of exactly 50 per cent. MICHAELIS (Virchoiv's Arch., clxiv, 1901, p. 263) recommends Scharlach R (syn. " Fettponceau "). Stain for fifteen to thirty minutes in a saturated solution in 70 per cent, alcohol, and mount in glycerin or levulose. Other authors also commend this stain. HERXHEIMER (Deutsche med. Wochenschr., xxvii, 1901, p. 607 ; Zeit. wiss. Mik., xix, 1902, p. 66) makes a solution of 70 parts of absolute alcohol, 10 of water. 20 of 10 per cent, caustic soda, and Scharlach R to saturation. This makes a stronger solution, and stains in a couple of minutes. Wash out with alcohol of 70 per cent. With either solution the staining must be done in a covered vessel or the stain will precipitate. Similarly BELL, Amer. Jo-urn. Anat., ix, 1909, p. 401, and Anat. Rec., iv, 1910, p. 199. HERXHEIMER also (Centralb. (dig. Path., xiv, 1903, p. 841 ; Zeit. wiss. Mik., xxi, 1904, p. 57) recommends a saturated solution of the dye in a mixture of equal parts of acetone and 70 per cent, alcohol. He also (Deutsche med. Wochenschr., xxvii, 1909, p. 607 ; Zeit. wiss. Mik., xix, 1902, p. 67) has had very fine results by staining for 20 minutes in a saturated solution of Indophenol in 70 % alcohol. MOLLISON (Zeit. wiss. ZooL, Ixxvii, 1904, p. 529) has had good results by staining gelatin sections for a few minutes in strong 368 CONNECTIVE TISSUES. extract of Alkanna in 96 per cent, alcohol, and mounting in glycerin or syrup. LORRAIN SMITH (Journ. Path. Bact., xii, 1907, p. 1) finds that Nile blue stains fatty acids blue and neutral fats reddish. Similarly EISENBERG (Virchow's Arch., cxcix, 1910, p. 502), who recommends aqueous solution of Nilblau BB. BENDA (ibid., clxi, 1900, p. 194) finds that free fatty acids can be detected by Weigert's neuroglia mordant. See also BERNER, ibid., clxxxvii, 1907, p. 360, and FISCHLER, Zeit. wiss. Mik., xxii, 1905, p. 263. OK A JIM A (ibid., xxix, 1912, p. 67) extracts red capsicum berries for some days with alcohol, and evaporates down to one fifth. This stains only fatty bodies : amongst them, myelin. See also KINGSBURY, Anat. Rec., v, 1911, p. 313. 770. Removal of Fatty Substances. — If not treated with osmic acid or mordanted with strong bichromate, alcohol, ether, chloroform, pyridine, xylol, will readily dissolve fatty substances. Osmicated fats and lipoids are more resistant, especially if osmic acid and bichromate have been combined. It can then be removed in a few hours by alcoholic hydrogen peroxide (10 per cent. H202 in 80 per cent, alcohol) or in twenty-four hours by oil of turpentine. Ether, creosote, xylol, clove oil and chloroform will also remove osmicated fats and lipoids if allowed to act sufficiently lone;. See also FLEMMING in Zeit. wiss. Mikr., 1889, pp. 39, 178. 771. Differentiation between Fats and various Lipoids. — Fix in for- mol and prepare frozen sections. Stain some with Sudan or Scharlach (see above § 769), others with osmic acid. Leave some unstained. Globules which stain with Sudan or Scharlach and osmic acid, and which in unstained sections show no double refraction, can be identified with certainty as true fats. This may be confirmed in paraffin sections by fixing in bichromate and subsequent treatment with osmic acid as in the methods of Schridde and Marchi. These globules should then reduce osmic acid. But the presence of double refraction must not be taken as excluding the presence of true fats since the globules may be a mixture of true fats and double refracting lipoids. The deduction that true fats are absent can be made when tissue containing fatty material as indicated by blackening with osmic acid fails to give this blackening after previous treatment with bichromate, as for instance in normal peripheral nerve. The histochemical differentiation between true fats and lipoids is much more difficult when these substances are mixed in one and the same globule than when one cell contains several globules of which some are composed entirely of true fats while others contain lipoids. In the latter case methods may be applied which depend on differences in the solubility in various solvents. DEFLANDBE (Journ. Anat. Phys., 1904, p. 80) fixes in formol of 4 per CHAPTER XXIX. 369 cent, and brings into acetone, in which fat is dissolved, but not lecithin, which can then be stained by osmium. See also CIACCIO, Arch. Zellforsch, v, 1910, p. 235 ; and FISCHLER, Zeit. wiss. Mik., xxii, 1905, p. 262 ; LOISEL, C. E. Soc. Biol., Iv, 1903, p. 703. BELL with a modification of DIETRICH'S and CIACCIO'S methods (Journ. Path, and Bad., xix) claimed to be able to distinguish between fat drops mainly of triolein and those that principally contain " lipoids." The former appear in annular shape, the latter are quite solid. In the former case the centre of the droplet is not chromated, and therefore dissolves out in xylol used for imbedding. Fix at 45° C. to 50° C. in 10 per cent, aqueous K2Cr207, 100 c.c., acidified by 5 c.c. of acetic. Wash, dehydrate and imbed in paraffin. Fasten 3 to 5 ^ sections to slide with albumen water. Treat sections in xylol, absolute alcohol, and transfer to freshly prepared solution of Sudan III in 80 per cent. alcohol for ten minutes. Rinse off excess stain in 50 per cent, alcohol ; transfer to water to stop action of alcohol. Counterstain in Delafield's hsematoxylin, wash in water, differentiate in acid alcohol, wash and mount in glycerin gum-arabic. 772. Mounting Fat. — After treatment with osmic acid sections can generally be mounted in balsam without special precautions. Many lipoids, however, fade even after osmication, if kept for a long time in balsam. In some cases it may suffice to avoid absolute alcohol and essences as much as possible, and mount direct in alcohol balsam or euparal, or clear with cedar oil, which has little solvent action. For very delicate fats it may be necessary to avoid alcohol of more than 70 per cent., or avoid it altogether, and mount in glycerin or levulose. Chrysoidin (L. MARTINOTTI, Zeit. Physiol. Chem., xci, 1914) fixes tissue in 10 per cent, formol, sections on a freezing microtome, washes in aq. dest., and immerses for five to ten minutes up to several hours in a 1 per cent, aqueous solution of chrysoidin. Wash not longer than one minute in aq. dest., and treat in 10 per cent. K2Cr207 or Cr03 for one minute, wash, dehydrate in benzol and xylol, and mount in neutral balsam. Bone.* 773. Bone, Non-decalcified. — RANVIER (Traite, p. 297) has the following : Bones should be plunged into water, without being allowed to dry, as soon as the surrounding soft parts have been removed, and should be divided into lengths with a saw whilst wet. The * For a detailed review of the whole subject, see the paper of SCHAF- FER in Zeit. wiss. Mik., x, 1893, p. 167, or the article " Knochen und Zahne " in Encycl. mile. Technik. | This section has been revised by J. Thornton Carter, Esq., F.R.M.S., of the Zoological Department, University College, London. M. 24 370 CONNECTIVE TISSUES. medulla should then be driven out from the central canal by means of a jet of water ; spongy bones should be treated as follows : An epiphysis having been removed, together with a small portion of the diaphysis, a piece of caoutchouc tubing is fixed by a ligature on to the cut end of the diaphysis, and the free end of the piece of tubing adapted to a tap through which water flows under pressure ; they are then put to macerate for several months, the liquid being changed from time to time. As soon as all the soft parts are per- fectly destroyed, the bones may be left to dry. Thin sections may then be cut with a saw and prepared by rubbing down with pumice-stone. Compact pumice-stone should be taken and cut in the direction of its fibres. The surface should be mois- tened with water and the section of bone rubbed down on it with the fingers. When both sides of the sections have been rubbed smooth in this way, another pumice-stone may be taken, the section placed between the two, and the rubbing continued. As soon as the section is thin enough to be almost transparent it is polished by rubbing with water (with the fingers) on a Turkey hone or litho- graphic stone. Spongy bone should be soaked in gum and dried before rubbing down (but see VON KOCH'S copal process and EHRENBAUM'S colophonium process). SCHAFFER (Zeit. wiss. Mik., x, 1893, p. 171) grinds and polishes on stones of graduated fineness. For the process of WEIL for bones and teeth see § 180. ROSE (Anat. Anz., vii, 1892, pp. 512-519) follows Koch's process. He penetrates first with a mixture of cedar oil and xylol, then with pure xylol, and imbeds in solution of Damar in chloroform or xylol. The method can bs combined with Golgi's impregnation. FANZ (Anat. Record, xiv, 1918, p. 493) employs sand or carborundum paper of different grades of coarseness for grinding, using the back or smooth side of a piece of sandpaper for polishing the section. He recommends shellac in preference to balsam for attaching the section to the glass slip. WHITE (Journ. Roy. Mic. Soc., 1891, p. 307) recommends the following : Sections of osseous or dental tissue stlould be cut or ground down moderately thin, and soaked in ether for twenty- four hours or more. They should then be put for two or three days into a thin solution of fuchsin in collodion, then into spirit to harden the collodion. After this they are ground down to the requisite thinness between two plates of old ground glass, with water and pumice powder, and mounted, surface dry, in stiff balsam or styrax, care being taken to use as little heat as possible. Lacunae, canaliculi, and dentinal tubuli are found infiltrated by the coloured collodion. CHAPTER XXIX. 371 HANAZAWA (Dental Cosmos, lix, 1917, pp. 125 et seq.) gives a number of methods for staining giound and decalcified sections of dentine to demonstrate its minute structure. MATSCHINSKY (Arch. mik. Anat., xxxix,.1892, p. 151, and xlvi, 1895, p. 290), after grinding, impregnates with nitrate of silver. For similar method of EUPRECHT, see Zeit. wiss. Mik., xiii, 1896, p. 21, wherein see also quoted (p. 23) a method of ZIMMERMANN. CSOKOR (Verh. anat. Ges., 1892, p. 270) describes a saw which will cut fresh bone to 120 p. ; and ARNDT (Zeit. wiss. Mik., xviii, 1901, p. 146) a double saw which will also give very thin sections. 774. Mounting. — To show lacunae and canaliculi injected with air, take a section, or piece of very thin flat bone, quite dry. Place on a slide a small lump of solid balsam, and apply just enough heat to melt it. Do the same with a cover glass, place the bone in the balsam, cover, and cool rapidly. When thin ground sections of enamel are mounted in Canada balsam it is found often that they appear almost structureless. To demonstrate the enamel pattern of such sections they may be etched by immersion in -6 per cent, of hydrochloric acid in 70 per cent, alcohol, or in a weak aqueous solution of picric acid, and mounted in Camsal balsam' or Euparal, media which, on account of their low index of refraction, will be found to disclose the structure of the enamel more easily. 775. Sections of Bones or Teeth showing the Soft Parts. — A developing tooth with its epithelial enamel-organ, its mesodermal dentinal papilla, and its layers of partially calcified enamel and den- tine, is made up of very delicate structures of different consistency and so peculiarly liable to unequal shrinkage, with consequent distortion during ^the period of fixation and in the subsequent processes passed through in the preparation of sections. Further, post-mortem changes in the ameloblasts occur within a very few minutes after death leading to a less precise behaviour to stains than is found in the case of cells which are fixed immediately after death. For the examination of developing teeth in situ, jaws may be fixed in corrosive-formalin-acetic mixture, in Bouin's picro-formol, in Zenker's mixture or Helly's modification thereof, or in Sansom's modification of Carney's mixture (§ 86). For the study of the micro-anatomy of the enamel-organ and the dentinal papilla, a young pup or a kitten, two or three days old, is killed, preferably by a blow on the head. The jaws are removed and the bone of the under-surface of the mandible pared away by a sharp scalpel until the bases of the tooth-germs are almost exposed. 24—2 372 CONNECTIVE TISSUES. The muco-periosteum is grasped with a pair of forceps and stripped from the bone, when the tooth-germs will come away attached thereto. Sansom's modification of Carnoy's mixture, employed at blood- heat, is particularly effective when the tooth-germs have been exposed in the manner outlined above, fixation therein being com- plete in from five to ten minutes. They are then passed through successive baths of alcohol of 30 per cent, and 50 per cent., each for fifteen minutes ; 70 per cent., to which is added tincture of iodine, for four hours ; 90 per cent, for thirty minutes ; and into two changes of absolute alcohol, each for fifteen minutes or longer. The tooth-germs are then transferred to a mixture of equal parts of absolute alcohol and carbon disulphide for one hour, two changes of pure carbon disulphide, each of fifteen minutes, then for thirty minutes into carbon disulphide saturated with paraffin at 30° C., transferred to carbon disulphide saturated with paraffin at 42° C. for a like period, and finally into two baths of paraffin, in each half an hour. Imbed for cutting in pure paraffin. By the employment of this method the amount of shrinkage in the tissues is extremely slight and the dentine does not become hardened, so that the tooth-germs of the incisors may be cut without decalcification. In the case of the canine and molar tooth-germs a short period of decalcification may be necessary, and for this purpose a rapid and delicate method lies in the employment of ZEIGLER'S method (Festschr. f. Kupffer, 1899, p. 51), in which, by the use of a 5 per cent, solution of sulphurous acid, the insoluble tricalcium phosphate is changed into the readily soluble mono- calcium phosphate. To demonstrate cytological detail no stain equals iron-hsema- toxylin followed by a counter-stain of picric-lichtgrun or of Rubin S in picrate of ammonia. It cannot be too strongly emphasised that the precision of staining methods depends on the rapidity ivitli ivhich fixation of the tissues is effected after death. Refer to § 31. For large jaws imbedding in celloidin, or, when serial sections are required, double imbedding in celloidin, parlodion or photoxylin and paraffin is recommended (§ 171). MUMMERY (Phil. Trans. B., ccviii, 1917, p. 258) deprecates the employment of paraffin for imbedding the tooth-germs of fishes, considering the heat employed to be very injurious to the delicate enamel organs, and advocates the use of the freezing method in obtaining sections. See carbon disulphide method above. NEALEY (Amer. Mon. Mic. Journ., 1884, p. 142 ; Journ. Eoy. Mic. CHAPTER XXIX. 373 Soc., 1885, p. 348) says that perfectly fresh portions of bone or teeth may be ground with emery on a dentist's lathe, and good sections, with the soft parts in situ, obtained in half an hour. HOPEWELL- SMITH (Journ. Brit. Dent. Ass., xi, 1890, p. 310; Journ. Roy. Mic. Soc., 1890, p. 529) says that for preparing sections of teeth showing odontoblasts in situ the bast plan is to take embryonic tissues. A lower jaw of an embryonic kitten or pup may be taken, and hardened in solution of Miiller followed by alcohol, then cut with a freezing microtome. WEIL (loc. cit., § 180) fixes pieces of fresh teeth in sublimate, stains with borax -carmine, brings them through alcohol into chloroform and chloroform -balsam, and after hardening this by heat proceeds to grind as usual (§ 177). See also EOSE, § 773. 775A. — For the study of the vessels in teeth, LEPKOWSKY (Anat. Hefte, viii, 1897, p. 568) injects with Berlin blue, hardens the teeth with a piece of the jaw for one or two days in 50 per cent, formol, decalcifies in 10 per cent, nitric acid (eight to fourteen days, change frequently) and makes celloidin sections. For decalcification of teeth, see also § 546 (ROUSSEAU, BODECKER and FLEISCHMANN). Bodecker finds Rousseau's process not appli- cable to human teeth : the acid must be added to the fluid celloidin. For the study of the lymphatics in the dental pulp, DEWEY and No YES (Dental Cosmos, lix, 1917, pp. 436 — 44) first inject the blood vessels with carmin-gelatin. Then 2 grms. of Prussian blue (oil colour in tubes) is stirred with 3 grms. of turpentine oil in a glass mortar for five minutes ; 15 grms. of sulphuric ether is added, and this fluid filtered through flannel or chamois skin. After the injection of this fluid the head is placed for twenty-four hours or longer in 20 per cent, formalin, and then the injected teeth are carefully removed and the pulps examined. Later it was found that more constant results were obtained when the injection of the blood vessels followed that of the Prussian blue. Prussian blue injected directly into the pulps and trypan blue or lithium carmine injected intravenously or intraperitoneally were also employed. See §§ 780 and 790. WELLINGS (Proc. Sixth Internat. Dent. Cong., pp. 47 et seq.) demon- strated intra-vitam staining of dental and adjacent tissues by means of trypan blue (§ 780). MUMMERY (Phil. Trans. B., ccii, 1912), for the fixation of the nerve-tissue of the dental pulp, finds formalin to be preferable to all other fixing agents, employing 10 parts of the 40 per cent, com- mercial formalin to 90 parts of water. Decalcification is effected by means of 33-3 per cent, formic acid. After thorough washing he leaves for twenty-four hours in a strong 374 CONNECTIVE TISSUES. solution of dextrin (which he finds preferable to gum arabic), and sections are cut on the freezing microtome, by the employment of which he is able usually to obtain thinner sections than when paraffin is used for imbedding. The sections are stained either by means of iron and tannin, iron-hsematoxylin (Benda), Congo red, Ranvier's modification of L6 wet's gold chloride process, or by Cajal's method, where :- (1) Small pieces of the decalcified tooth, not more than 4 milli- metres thick, are placed in 50 c.c. of rectified spirit, to which 3 or 4 drops of ammonia may be added, and kept in this solution for from four to six hours. (2) Transfer to absolute alcohol for twenty-four hours. (3) Rinse with distilled water. (4) Place in a large quantity of 1-5 per cent, solution of silver nitrate, and keep in warm incubator at about 35° C. for five or six days. (5) Rinse in distilled water for a few seconds. (6) Place in the following solution for twenty-four hours :- Hydrokinone . . . . 1 to 1-5 grm. Distilled water ..... 100 c.c. Formol . . . . . . 5 to 10 c.c. Rectified spirit . . . . 10 to 15 c.c. (7) Wash in water for some minutes. (8) Cut sections, and mount. The presence of nerve-end cells in the dental pulp was demon- strated by MUMMERY (Phil. Trans. B., ccix, 1920), by means of a modification of the gold method of Beckwith. Teeth, immediately after extraction, are placed in a solution of formol and water or of formol and normal salt solution, preferably 4 per cent, of formol. This is, after a few days, changed to a 10 per cent, solution, and the teeth kept in this for at least a fortnight. Decalcification is effected by means of a 33-3 per cent, solution of formic acid in distilled water, to which 5 per cent, of formol may be added. (Mummery states that neither he nor Dependorf has ever procured good nerve preparations of teeth which have been decalcified in the mineral acids.) Wash in running water for twenty-four hours, then for a few minutes in distilled water. The pieces are taken from the distilled water and suspended by threads in a large quantity of a weak solution of gold chloride (1 in 5,000). Each piece should be suspended in at least 100 c.c. CHAPTER XXIX. 375 of the solution, in which it is left in the dark for from four days to one week, according to its size. On removal from the gold solution it is washed for a few minutes only in distilled water. Reduction is effected by placing the pieces in a 20 per cent, solution of caustic soda for four minutes, then rinsing in water and placing in a 10 per cent, solution of potassium carbonate for from half an hour to an hour. This is then drained off, and the pieces are placed in a 10 per cent, solution of potassium iodide for a short time — usually five to ten minutes. As soon as seen to darken, the pieces are removed from this solution to water, placed in gum for twelve hours, and sections cut on the freezing microtome. After dehydration the sections are mounted in camsal (propylic) balsam. 776. VIVANTE (Intern. Monasschr. Anit. u. Phys., ix, 1892, p. 398) impregnates portions of frontal bone ot four to six months calves, which are not more than 3 to 4 millimetres thick, by Golgi's rapid bichromate and silver process. After impregnation the specimens should be decalcified in von Ebner's mixture (§ 562), well washed with water, and brought into solution of carbonate of soda, and finally imbedded in paraffin. For his quinolein blue method see fourth edition. For UNDERWOOD'S gold process for teeth, and for that of LEPKOWSKI, see third edition, or Anat. Anz., 1892, p. 294. LAW (Proc. Eoy. Soc. Med., i, 1908, p. 45) studies nerve-endings in teeth of mammals by treating paraffin sections of decalcified tissue with BETHE'S molybdenum toluidin blue (details in Journ. Roy. Micr. Soc., 1908, p. 518). VAN DER STRICHT (Carnegie Instil. Embryol. Contrib., No. 21) fixes the isolated cochlea in a 5 per cent, aqueous solution of tri- chloracetic acid, or in Bouin's or Zenker's fluid, and stains, before imbedding, in borax-carmine. The sections are afterwards stained in iron-hsematoxylin, Congo red and light green. He obtained the best results with the membrana tectoria by making one or two openings in the bony wall of the fresh cochlea and exposing the piece for fifteen minutes to the vapours from an aqueous solution of osmic acid or by submerging it in a 1 per cent, solution of the same for one hour. Afterwards fixation was completed by immersion in trichloracetic acid, Bouin's fluid or Zenker's fluid, and the series of sections therefrom stained as above. By this method some of the turns of the cochlea give very good preparations of the structure of the membrana tectoria. The mitochondria are also visible within osteoblasts, osteoclasts, connective-tissue cells, all epithelial cells, and the sensor ial elements. Mitochondria in odontoblasts and osteoblasts may be demon- strated by fixation in Flemming-without-acetic followed by staining 376 CONNECTIVE TISSUES. in iron-haematoxylin (§ 679), and the Golgi apparatus in these cells is well shown by the employment of Golgi's method, Cajal's method, or of Da Fano's modification thereof (§§ 844, 849), though a negative image of this cell-element is clearly shown when the tissues are fixed in Sansom's modification of Carnoy's mixture. 777. Bone, Decalcified (FLEMMING, Zeit. wiss. Mik., 1886, p. 47).- Sections of decalcified bone are soaked in water, dehydrated with alcohol under pressure, dried under pressure and mounted in hard balsam melted on the slide. They show the lacunar system injected with air as in non-decalcified sections. 778. Stains for Cartilage and Decalcified Bone. — See hereon SCHAFFER in Zeit. wiss. Mik., v, 1888, p. 1 ; and Encycl. mik. Technik., art. " Knochen." KOLLIKER (Zeit. wiss. Zoo?., xliv, 1886, p. 662) treats sections of decalcified bone with concentrated acetic acid until they become transparent, and then puts for one quarter to one minute into a concentrated solution of indigo-carmine, washes and mounts in glycerin or balsam. The fibres of Sharpey appear red, the remaining bone substance blue. SCHAFFER (Zeit. wiss. Mik., v, 1888. p. 17) employed at one time a safranin method modified from BOUMA (Centralb. med. Wiss., 1883, p. 866), for which see previous editions. He now (Encycl. mik. Tech., 1910, i, p. 762) stains sections for twenty-four hours in a bath of 20 c.c. of water with 1 drop of 1 per cent, solution of safranin (or thionin) and (apparently) mounts in balsam. The safranin stain will keep if the material is cartilage which has been fixed in picro- sublimate ; otherwise it must be fixed with ammonium molybdate of 5 per cent, before dehydrating. SCHMORL (Centralb. allg. Path., x, 1899, p. 745) stains in a mixture of 2 c.c. concentrated solution of thionin in alcohol of 50 per cent. and 10 c.c. of water for ten minutes, rinses and puts into saturated aqueous picric acid for thirty to sixty seconds. Rinse and pass through graded alcohols into origanum oil or carbol-xylol and balsam. Matrix yellow, cells red, fat-cells violet. He also describes a more complicated method with thionin and phosphotungstic or phosphomolybdic acid. MOLL (Centralb. Physiol., xiii, 1899, p. 225) stains embryonic cartilage for six to twenty-four hours in orcein 0*5 gr., alcohol 40, water 20, hydrochloric acid 20 drops, and mounts in balsam. Matrix blue, nuclei red. KALLIUS (Anat. Hefte, xxx, 1905, p. 9) stains first with borax carmine or alum-carmine, then (sections) for ten minutes in satu- CHAPTER XXIX. 377 rated solution of thionin, and washes out with alcohol of 70 per cent. Said to be specific for embryonic cartilage. VASTARINI-CRESI (Aft. Accad. med.-chir. Napoli, 1907, p. 4) stains sections of embryonic cartilage with borax carmine, then with muchsematein (alcoholic solution without acid), and then with Orange G. in alcohol. BAYERL'S method for ossifying cartilage (Arch. mik. Anat., 1885, p. 35) : — Portions of ossified cartilage are decalcified as directed, § 555, cut in paraffin, stained in Merkel's carmine and indigo- carmine mixture, and mounted in balsam. MAYER (Grundziige, LEE and MAYER, 1910, p. 393) prefers to all these resorcin fuchsin, § 758, the precipitate being freed from iron chloride by washing before dissolving in the alcohol. Aqueous solution of benzoazurin has been commended as a stain for ossifying cartilage by ZSCHOKKE, see Zeit. wiss. Mile., x, 1893, p. 381. A process of BAUMGARTEN'S has been given, § 388. MOERNER (Skandinavisches Arch. Physiol., i, 1889, p. 216 ; Zeit. wiss. Mik., vi, 1889, p. 508) gives several stains for tracheal cartilage, chiefly as microchemieal tests, for which see third edition. See also a critique of these methods by WOLTERS in Arch. mik. Anat., xxxvii, 1891, p. 492 ; and on the whole subject of cartilage see SCHIEF- FERDECKER'S Gewebelchre, p. 331. FUSARI (Arch. Ital. Biol., xxv, 1896, p. 200) makes sections of fresh cartilage, puts them for twenty-four hours into 1 per cent, nitrate of silver, washes, dehydrates, and exposes to the light in balsam. See also DISSE, Anat. Anz., xxxv, 1909, p. 318, a stain for dentine (haemalum followed by a mixture of Saurerubin and Orange G) ; and RETTERER and LELIEVRE, C. E. Soc. Biol., Ixx, 1911, p. 630. Skeletons of Embryos. 779. Cartilaginous Skeletons of embryos (VAN WIJHE, Proc. K. Akad. Wetensch. Amsterdam, 1902, p. 47) may be studied by staining embryos for a week in a solution of 0-25 grm. methylen blue in 100 c.c. of 70 per cent, alcohol with 1 per cent, of hydrochloric acid. Wash out in alcohol with 1 per cent, of hydrochloric acid until no more colour comes away (about a week) and mount in balsam. The cartilage remains blue, all the other tissues being colourless. Similarly, LUNDVALL (Anat. Anz., xxv, 1904, p. 219, and xl, 1912, p. 639), using toluidin blue. Thionin blue also may be used. Similarly also BAKAY (Verh. Anat. Ges., 1902, p. 248), with Bismarck brown (the embryos having been previously treated with nitric acid of 3 per cent.). For fish embryos, Professor E. S'. Goodrich, of Oxford, informs me that thionin is excellent. For the Spalteholz method of clearing such preparations see § 592. 378 CONNECTIVE TISSUES. 780. Demonstration of Centres of Osteoblastic Activity by Trypan Blue (P. G-. SHIPLEY and C. C. MACKLIN, Anat. Record, x, 1915 — 16). -If an azo dye like trypan blue be administered to a very young animal, the bones are stained quickly and very intensely with vital colour. The dye is injected in a 1 per cent, solution into the peri- toneal cavity (less preferably subcutaneously). The animal is killed forty-eight hours after staining, and the tissues are fixed by 10 per cent, neutral formalin injected through blood vessels, followed by immersion in 10 per cent, formalin for twenty-four to forty-eight hours. Bones are washed thoroughly, hardened in ascending grades of alcohol, after which the soft parts are dissected away. Clear in benzol and then in oil of wintergreen. Study with dissecting microscope. Refer also to J. Thornton Carter, § 775A. 780A. Potash Method for Osteoblastic Centres (SCHULTZE, Grundriss d. Entwickl. d. Menschens, 1897, and F. P. MALL, Amer. Journ. Anat., v, No. 4, 1905-6). Embryos of mammals after fixation in alcohol may be cleared, for the study of the ossification centres, by means of weak potash. For alcohol specimens Mall considers that Schultze's solution is too strong, and uses instead a 1 per cent. KOH solution for a few hours. With weak solutions the tissues of the smaller embryos remain firm, and, in the end, the specimen is transparent, with the bones held in place. After treatment with the potash, the embryo is placed in the following solution for days, or even months : — Water ........ 79 Glycerin ....... 20 Potash ........ 1 From time to time the embryo may be returned to a 3 per cent, solution of potash for a number of hours to hasten the clearing process ; then returned to the glycerin solution, which helps to hold the parts together. When properly cleared, upgrade gradually to pure glycerin, in which they may remain. MALL (op. cit.) clears formalin embryos in 10 per cent, potash for about a month or longer. Formalin renders the connective tissues very tough, and this strong KOH solution is necessary. Refer also to § 592, CHAPTER XXX. BLOOD AND GLANDS. Blood. 781. Fixing and Preserving Methods. — The school of Ehrlich used to fix by heat. A film of blood was spread on 8 cover-glass and allowed to dry in the air, and then fixed by passing the cover a few times, three to ten or twenty, through a flame, or by laying it face downwards on a hot plate kept for several minutes or as much as two hours at a temperature at which water not only boils, but assumes the spheroidal state (110° to 150° C.). For details see GULLAND, Scottish Med. Journ., April, 1899, p. 312 ; RUBINSTEIN, Zeit. wiss. Mik., xiv, 1898, p. 456 ; ZIELINA, ibid., p. 463. But I believe they have now well-nigh abandoned this barbarous practice. In wet methods either the blood is mixed at once, on being drawn, with some fixing and preserving medium, and studied as a fluid mount, or films are prepared and put into a fixing liquid before they have had time to dry, or after drying in the air without heat for a few seconds (at most ten to thirty). To make a film, place a very small drop of blood on a perfectly clean slide. Bring down on to the slide the edge of another slide held over it at a slope ; move this along till it touches the edge of the drop and the blood runs along the angle between the two slides. Then move the second slide away from the drop, and the blood will follow it and be drawn out into a film without being crushed. Simi- larly with two cover-glasses, to make a cover-glass film, which can be floated face down on to fixing or staining liquids in a watch-glass. Some persons make films by flattening blood between two cover- glasses which are afterwards separated by sliding the one over the other ; but that produces an injurious kneading of the cellular elements. Most of the usual fixing agents are applicable to blood. But it is often necessary to employ only such as are favourable to certain stains. Those most recommended in this respect are alcohol, formol, sublimate (should not be too strong), osmic . acid in very light fixation, or absolute methyl alcohol, which is an energetic fixative of dried films. 380 BLOOD AND GLANDS. Air-dried films ought to be fixed before putting into aqueous or glycerin stains, else they will wash off ; but this is not necessary for alcoholic stains. 782. Fixing and Preserving in Bulk. — Most morphologists are agreed that by far the most faithful fixing agent for blood-corpuscles is osmic acid. A drop or two of blood (BiONDi recommends two drops exactly) is mixed with 5 c.c. of osmic acid solution, and allowed to remain in it for from one to twenty-four hours. As a rule the osmic acid should be strong — 1 to 2 per cent. Fixed speci- mens may be preserved for use in acetate of potash solution (MAX FLESCH, Zeit. wiss. Mik., v, 1888, p. 83). GRIESBACH also (ibid., 1890, p. 328) combines the osmic acid with certain stains. He mentions methyl green, methyl violet, crystal violet, safranin, eosin, Saurefuchsin, rhodamin, and iodine in potassic iodide. Eossi (ibid., vi, 1889, p. 475) advises a mixture of equal parts of 1 per cent, osmic acid, water, and strong solution of methyl green, permanent mounts being made by means of glycerin cau- tiously added. EWALD (Zeit. Biol., xxxiv, 1897, p. 257) mixes 3 to 4 drops of blood of amphibia or reptiles with 10 c.c. of a solution of 0-5 per cent, osmic acid in 0-5 per cent, salt solution (for mammals 0-6 to 0-7 per cent, salt), siphons off the supernatant liquid after twenty- four hours with his capillary siphon (§ 3, p. 4), and substitutes water, alum-carmine, etc., and lastly, 50 per cent, alcohol. WEIDENREICH (Arch. mik. Anat., Ixxii, 1908, p. 213) lays a cover with a drop of blood on it on a layer of agar-agar (1 per cent, in salt solution of 0-8 per cent.), and after five minutes runs in osmic acid of 1 per cent., and after five minutes more removes the cover. DEKHUYZEN (Anat. Anz., xix, 1901, p. 536) recommends a mixture of either 3 or 9 volumes of 2 per cent, osmic acid with 1 of 6 per cent, acetic acid, containing J per cent, of methylen blue, which he calls " Osmacet." The mercurial liquids of Pacini (§ 414) used to be considered good. HAYEM (" Du Sang" etc., Paris, 1889 ; see also Zeit. wiss. Mik., vi, 1889, p. 335) has the following formula : sublimate 0-5, salt 1, sulphate of soda 5, and water 200. This should be mixed with blood in the proportion of about 1 : 100. Eosin may be added to it. LOWIT'S formula (Sitzb. k. Akad. Wiss. Wien, xcv, 1887, p. 144) consists of 5 c.c. cold saturated sublimate solution, 5 grms. sulphate of soda, 2 grms. salt, and 300 c.c. water. Mosso finds that both of these are too weak in sublimate. CHAPTER XXX. 381 DUBOSCQ (Arch. Zool. Exper., vi, 1899, p. 481) uses (for blood of Chilopoda) a solution of acetic acid, copper acetate, copper chloride, osmic acid, thionin, 1 grm. each, water 400, which, mixed with the blood, fixes and stains in about two minutes. Formol has lately been used. MARCANO (Arch, de Med. Exper., xi, 1899, p. 434) mixes fresh blood with a mixture of 100 parts of sodium sulphate of sp. gr. 1-020 and 1 of formol ; or with water 85 to 100 parts, sodium chloride 1, and formol 1. KIZER (Journ. Roy. Mic. Soc., 1990, p. 128) simply mixes 1 drop of blood with 3 of 2 per cent, formalin, and allows to stand for an hour. SCHRIDDE (Hcemat. Techn. Jena, 1910, p. 17) lets blood drop into a mixture of 1 part of formol, 9 of liquid of Miiller, and 10 of water, fixes therein for two to four hours at 40° C., filters, washes and brings through alcohol and chloroform into paraffin for sectioning. 783. Fixing and Preserving in Films. — MUIR (Journ. of Anat. and Phys., xxvi, 1892) makes cover-glass films and drops them into saturated sublimate solution, and after half an hour washes, dehy- drates, and passes through xylol into balsam. GULLAND (Br'it. Med. Journ., March 13th, 1897 ; Scottish Med. Journ., April, 1899) makes cover-glass films, and after a few seconds drops them face downwards into a solution of- Absolute alcohol saturated with eosin . . 25 c.c. Pure ether . . . . . . 25 ,, Sublimate in absolute alcohol (2 grms. to 10 c.c.) ...... 5 drops. After three or four minutes they are washed, stained, and mounted in balsam. For JENNER'S fixing and staining and staining method, see next section. Many recent authors fix wet films with formol. BENARIO (Deut. med. Wochenschr., 1895, p. 572) mixes 1 part of 10 per cent, formol with 9 of7. alcohol (the mixture must be freshly prepared), and plunges films into it for a minute. Similarly GULLAND, with 1 part of formol to 9 of alcohol. Similarly WERMEL (see Zeit. wiss. Mik., xvi, 1899, p. 50), who combines various stains (methylen blue, eosin, gentian, etc.) with the formol. EDINGTON (Brit. Med. Journ., 1900, p. 19) exposes films for fifteen to thirty minutes to vapour of formol under a bell-jar. SCOTT (Journ. of Path, and Bacter., vii, 1900, p. 131) exposes 382 BLOOD AND GLANDS. films to the vapour for about five seconds and drops into absolute alcohol, and after fifteen minutes stains and mounts. A short exposure (thirty seconds) to vapour of osmium has also been recommended. SZECSI (Deutsch. med. Wochschr., 1913, p. 1584) has recommended Lucidol for blood smears, and smears of fseces containing protozoa and cysts. The formulae for an acetone and a pyridin solution will be found on p. 59, § 107, and also of an acetone-xylol solution for subsequent washing of the smears. It is best to keep a sufficient quantity of the fixing solutions in staining jars. Make a smear, allow it to dry, and place it in the acetone peroxide of benzol solution for fifteen minutes ; transfer to the acetone xylol solution for ten minutes in order to remove the lucidol ; wash off in pure methyl alcohol ; the slide is now ready for staining. It will be found that most of the current stains used for such smears will act successfully after the lucidol fixation. Pappen- heim's panoptic method (§ 784) is recommended. For smears of fseces a fixation of twenty minutes in the pyridin- benzol peroxide solution is used ; wash as above, in acetone-xylol, or pyridin-xylol, and then in methyl-alcohol. Possibly the substitution of pure acetone for the methyl alcohol bath might prove advantageous in some ways. 784. Stains for Blood. — Fresh (unfixed) blood can be stained on the slide. See also § 1008, et seq. TOISON (Journ. Sci. med. de Lille, fev., 1885 ; Zeit. wiss. Mik., 1885, p. 398) recommends that it be mixed with the following fluid : Distilled water . . . . 160 c.c. Glycerin (neutral, 30° Baume) . 30 „ Pure sulphate of sodium ... 8 grammes. Pure chloride of sodium ... 1 gramme. Methyl violet 5 B . . . . 0-5 „ (The methyl violet is to be dissolved in the glycerin with one half of the water added to it ; the two salts are to be dissolved in the other half of the water, and the two solutions are to be mixed and filtered.) This mixture stains leucocytes sharply, which facilitates enumeration. BIZZOZERO and TORRE (Arch. Sci. Mediche, 1880, p. 390) dilute a drop with normal salt solution containing a little methyl violet, which stains nuclei intensely, cytoplasm less intensely. Similarly GiGLio-Tos (Zeit. wiss. Mik., 1898, p. 166), diluting with saturated solution of neutral red in salt solution, which stains CHAPTER XXX. 383 hsemoglobigenous granules in five to ten minutes. This is also recommended by EHRLICH and LAZARUS. See § 309. Similarly also Ross (Trans. Path. Soc., 1907, p. 117), using polychrome methylen blue. LEVADITI (Journ. Phys. path. Gen., Paris, 1901, p. 425) allows solution of Brillantkresylblau in alcohol to dry on a slide, puts a drop of blood on the dried layer, and covers. Similarly CESARIS- DEMEL (Arch. path. Anat., 1909, p. 92), with a mixture of this dye and Sudan III ; and NAKANISHI (Centralb. Bakt., 1901, p. 98), with methylen blue BB. Fixed films may be treated with the usual tissue stains, eosin being an important one, as it stains rose-red all parts of blood-cells that contain haemoglobin. EHRLICH' s acid hsematoxylin, with 0-5 gr. of eosin dissolved in it, is a good general stain. Or, stain with hsemalum, and then with eosin (0-5 per cent, in alcohol or water). EHRLICH'S triacid (§ 296) gives good general views, and demon- strates neutrophilous granules. His mixture for eosinophilous cells has been given (§311). PAPPENHEIM'S panoptic triacid (on sale by Griibler) is Ehrlich's triacid with methylen blue in place of the methyl green. CHENZINSKI'S mixture, which is good, has been given (§ 313). Stain for six to twenty-four hours in a stove. This gives rise to precipitates. To avoid them (WILLEBRAND, Deutsch. med. Wochenschr., 1901, p. 57) you may make a mixture of equal parts of 0-5 per cent, solution of eosin in 70 per cent, alcohol and saturated solution of methylen blue in water, and add acetic acid of 1 per cent, drop by drop till the mixture begins to turn red, and filter before use. Or (MICHAELIS, ibid., 1899, No. 30) make (a) a mixture of 20 parts 1 per cent, aqueous methylen blue with 20 of absolute alcohol, and (6) a mixture of 12 parts 1 per cent, aqueous eosin with 28 of acetone, and for staining mix equal parts of these and .stain for half a minute to ten minutes. JENNER (Lancet, 1899, No. 6, p. 370) mixes equal parts of 1-2 to 1-25 per cent, water-soluble eosin (Griibler's) and 1 per cent, methylen blue, filters after twenty-four hours, washes the precipitate on the filter, dries it, and dissolves it in 200 parts of absolute methyl alcohol (the solution can be had ready made from Griibler and Hollborn). (Or, simply mix 125 c.c. of 0-5 per cent, solution of the eosin in methyl alcohol with 100 c.c. of 0-5 per cent, solution of methylen blue.) Cover-glass films are floated on to this, in which they are fixed and stained in three minutes. Wash off the stain with a little 384 BLOOD AND GLANDS. water (not under the tap), dry, and mount in balsam. Erythrocytes red, all nuclei blue, parasites blue, but with unstained nuclei. The methods of MAY and GRUNWALD are closely similar to this. ASSMANN (Munch, med. Wochenschr., 1906, No. 28 ; " Das eosinsaure Methylenblau," Leipzig, 1908, p. 35) treats fresh films for half a minute to three minutes in a Petri dish with a few drops of Jenner's solution (from Griibler and Hollborn), then pours on 20 c.c. of distilled water with 5 drops of ^ per cent, solution of lithium carbonate, leaves for five minutes, rinses in distilled water, dries with blotting paper, and mounts in neutral balsam. The foregoing mixtures give a stain — seemingly due to the forma- tion of an eosinate of methylen blue — in which the nuclei of blood- cells are blue and their plasma red to violet. It was made out by ROMANOWSKY (St. Peter sburger med. Wochenschr., 1891) that under certain conditions mixtures of these two dyes give a stain which is in some respects the inverse of this, blood-cells being stained in divers hues, according to their kinds, and any protozoan parasites that may be present showing red nuclei and blue plasma, which greatly facilitates their detection and diagnosis. This reaction appears to be due to the formation of an eosinate — not of methylen blue, but — of Methylenazur (§ 377). The method, only vaguely indicated by Romanowsky, has undergone, at the hands of ZIEMANN, ZETTNOW, NOCHT, REUTER, MICHAELIS, RUGE, MAURER, LEISHMAN, GIEMSA and others^ numerous modifications which have culminated in the establishment of a process worked out by GIEMSA as perhaps the most trustworthy and efficient of : Romanowsky " stains. This is as follows : GIEMSA'S Azur-eosin process. You start with a mixture of eosin with methylenazur (instead of methylen blue). This mixture is very troublesome to prepare, and is best obtained ready made from Griibler and Hollborn (their" Giemsa'sche Loesung f tir Romanowsky- faerbung "*). Air-dried films '(Deutsch. med. Wochenschr., 1907, No. 17) are fixed in alcohol or in methyl-alcohol (two to three minutes), and dried with blotting paper. They are treated for ten to fifteen minutes with a dilution of 1 drop of the stock mixture to 1 c.c. of water, washed under a tap, dried with blotting paper, and again dried in the air and mounted in balsam, or (preferably) pre- served unmounted. All reagents, especially the balsam, must be strictly free from acid. * To make this up from Griibler's powders, dissolve 3 grms. of Azur II-eosin and 8 decigrammes of Azur II in 125 grms. of glycerin and 375 of methyl-alcohol. CHAPTER XXX. 385 Wet- films (ibid., 1909, p. 1751) are treated as follows : Fix them for twelve to twenty-four hours in a mixture of 2 parts saturated aqueous solution of sublimate with 1 of absolute alcohol. Wash and treat for five to ten minutes with a mixture of 2 parts of iodide of potassium, 100 of water, and 3 of Lugol's solution. Wash, and treat for ten minutes with 0-5 per cent, solution of sodium thio- sulphate. Wash, and stain as above (changing the stain for fresh after half an hour), for one to twelve hours. Then pass through mixtures of acetone with first 5, then 30, then 50 parts per cent, of xylol into pure xylol, and mount in cedar oil. This process is applicable to sections. Or (ibid., 1910, p. 2476) a slide is placed in a Petri dish and covered with a mixture of equal parts of methyl-alcohol and stock mixture. After half a minute this is poured off and enough distilled water poured in to cover the slide, and the whole is rocked to mix the two. After three to five minutes, wash in running water, dry, and mount in cedar oil. By any of these processes nuclei (red) are demonstrated not only in hsematozoa, but in many bacteria, spirochsetse, coccidia, sar- cosporidia, etc. See also, for paraffin sections, SCHUBERG, in Deutsch. med. Wochenschr., xxv, 1909, No. 48, or Zeit. wiss. Mik., xxvii, 1910, p. 161, who passes through acetone and xylol into balsam. The older Romano wsky stains published by the authors men- tioned above, as also Laveran's ' ; Bleu Borrel ' ' seem to be super- seded by Giemsa's. LEISHMAN'S Romanowsky Stain (Brit. Med. Journ., March 16th and September 21st, 1901) is as follows : To a 1 per cent, solution of Griibler's medicinal methylen blue in water add 0-5 per cent, of sodium carbonate, heat to 65° C. for twelve hours and let stand for ten days. Then add an equal volume of 0-1 per cent, solution of Griibler's Eosin extra B, let stand for six to twelve hours, collect the resulting precipitate on a filter, wash it until the wash water comes off colourless, dry and powder. For staining, dissolve 0- 15 grm. in 100 c.c. of pure methyl alcohol. Stain cover-glass films (air- dried) for five to ten minutes ; flood the film with water for one minute, and examine, or dry (without heat) and mount in xylol balsam. Nuclei in shades of red, cytoplasm bluish, parasites blue with ruby red chromatin. RAADT (Munch, med. Wochenschr., 1911, No. 27 ; Zeit. wiss. Mik., 1912, p. 236) obtains a Romanowsky stain of blood and parasites with JENNER'S solution. Films fixed with alcohol and ether are M. 25 386 BLOOD AND GLANDS. first stained for five to ten minutes in solution of 1 part methy- lenblau med. puriss. Hoechst, 0-5 part of lithium carbonate and 100 of water, kept for at least three weeks and diluted with 10 volumes of water. Rinse with water, dry with blotting paper; flood with JENNER'S solution diluted with 2 or 3 volumes of water, and stain for five to ten minutes. Wash, dry with blotting paper, and mount. See also SCOTT, Folia Haem., xii, 1911. 785. PAPPENHEIM (Anat. Anz., xlii, 1912, p. 525) recommends the following for sections of licemopoietic tissues, and also of kidney, liver, hypophysis, suprarenals, lung, intestinal epithelium and central nervous system. Fix in Orth's Form ol-Mii Her, stain sections for twenty minutes in a stove in " aqueous diluted alcoholic " solution of MAY-GRUNWALD or JENNER diluted with 8 volumes of water ; after-stain for forty minutes in the stove in " aqueous GIEMSA solution (15 c.c. of water with 10 drops of glacial acetic acid) " ; differentiate in 100 c.c. of water with 5 to 6 drops of acetic acid ; wash, dry between blotting-paper ; dehydrate in mixture of equal parts of acetone and absolute alcohol, and mount in neutral balsam. The result is not a Komanowsky stain, but a pale methylen-blue-eosin stain. See also WRIGHT, Pub. Massachusetts Gen. Hosp., in, 1910, p. 1, or Journ. E. Micr. Soc., 1910, p. 783. For the special technique of eosinophilous cells see MARTINOTTI in Zeit. wiss. Mikr., xxvi, 1909, p. 4 (alphabetical bibliography of eight pages). 786. Demonstration of Blood-plates of Bizzozero (KEMP, Studies from the Biol. Lab. Johns Hopkins Univ., May, 1886, iii, No. 6 ; Nature, 1886, p. 132). — A somewhat large drop of blood is placed on a slide, and quickly washed with a small stream of normal salt solution. The blood-plates are not washed away, because they have the property of adhering to glass. They may be stained with solution of 0-02 per cent, of methyl violet or 1 : 3,000 of gentian violet, in salt solution. To make permanent preparations of them, they should first be fixed, by putting a drop of osmic acid solution on the finger before pricking it. They may also be stained in films,, especially by the Romanowsky method. According to PAPPENHEIM (Farbchemie, p. 107) Wasser- blau is almost specific for them. WRIGHT (Journ. Morph., xxi, 1910, p. 274) studies them in tissues, after fixation with formol or sublimate (not Zenker) by staining with a modified Giemsa stain, and bringing through acetone and oil of turpentine into turpentine colophonium. Details loc. cit. or Journ. Roy. Mic. Soc., 1910, p. 783. See also DEKHUYZEN. Anat. Anz., xix, 1901, p. 533 ; KOPSCH, CHAPTER XXX. 387 Intern. Monatschr. Anal. Phys., xxi, 1904, p. 344, and xxiii, 1906, p. 359 ; DEETJEN, Zeit. phys. Chem., Ixiii, 1909, p. 1. 787. Demonstration of a New Body in Red Blood Corpuscles (GOLGI, Boll. Soc. Med. Chir. Pavia, 1919, xxxi ; Hcematologica (Napoli), 1920). This original communication of Golgi gives two methods of interest to workers on blood : (1) Blood-films are fixed twenty-four to forty-eight hours in equal parts of saturated solutions of mercury chloride and potassium bichromate. They are then transferred into equal parts of 2 per cent, mercury chloride and potassium bichro- mate, to which 5 to 10 c.c. of 1 per cent, gold chloride and 5 to 10 drops of acetic acid are added. The films are observed in glycerol, starting from the second or third day after the last treatment until the fifteenth or twentieth day ; (2) drops of blood are fixed in a watch-glass by means of a fluid composed of 2 per cent, mercury chloride 60 c.c., saturated solution of picric acid 20 c.c., 1 per cent, osmic acid 10 c.c., acetic acid 5 drops, with the addition either immediately or after eight to twenty-four hours of 10 c.c. of 1 per cent, gold chloride for every 50 c.c. of fixative. Preparations are made from the sediment with some glycerol, from the second until about the tenth day after fixation. Both methods show within the red blood corpuscles a peculiar body with a diameter of about one-third that of adult corpuscles and of about half that of foetal ones. The body occupies the central part of the erytrocytes, and particularly by means of the second process it appears to have a fine, sometimes more fibrillar, sometimes more reticular structure. Its contours, though clearly defined, are irregular, and there is no indication whatever of a limiting membrane. This ' reticulo-fibrillar apparatus ' is not a nucleus, as the latter remains colourless by the new methods, not only in the white, but also in the nucleated red corpuscles, in which the apparatus appears concentrically arranged round the unstained nucleus. According to Golgi this apparatus does not correspond to any of the structures already described within the red corpuscles, but it reminds one a little of the endoglobular body recently demonstrated by Petrone, by means of a lead impregnation method, and not to be confused with the well-known Petrone's bodies. Golgi is convinced that the images obtainable by his two new methods are the expression of a real structure situated within the red blood corpuscles, but he does not feel able at present to give any opinion about their significance. The new methods stain also the centrosome in the white corpuscles of the blood. 25—2 388 BLOOD AND GLANDS. 788. WEIUERT'S Fibrin Stain (Fortschr. d. Med, v, 1887, No. 8, p. 228). — Sections (alcohol material) are stained in a saturated solution of gentian or methyl violet in anilin water (§ 286). They are brought on to a slide and mopped up with blotting-paper, and a little Lugol's solution is poured on to them. After this has been allowed to act for a sufficient time they are mopped up with blotting- paper, and a drop of anilin is poured on to them. The anilin soon becomes dark, and is then changed for fresh once or twice. The anilin is then thoroughly removed by means of xy]ol, and a drop of balsam and a cover are added. This stain may be applied to celloidin sections without previous removal of the celloidin. See also the modifications of this method by KROMAYER (§ 656) ; BENECKE (§ 690) ; UNNA (Monatssch. prakt. Dermat., xx, 1895, p. 140) ; WOLFF (Zeit. wiss. Mik., xv, 1899, p. 310) ; and one of -another sort by KOCKEL (Centmlb. allg. Path., x, 1899). 789. Elective Staining of Erythrocytes (K. OKAJIMA, Anat. Record, xi, 1917).- -This stain is based on the fact that the phosphomolybdic acid lake of alizarin stains, shows a special affinity for haemoglobin. Fix material in formol, sublimate, chrome, etc. Transfer sections on slide to aq. dest. ; mordant in 10 per cent, phosphomolybdic acid solution for thirty seconds to two minutes ; wash in water ; stain in this mixture for twenty minutes to twenty hours : — sodium sulfalizarinate, saturated aqueous solution, 100 c.c. ; and 10 per cent, phosphomolybdic acid, aq. solution, 30 c.c. (10 to 50 c.c.) ; wash in water ; alcohols, xylol, balsam. Erythrocytes go bright yellow orange. Counter-staining may be done in Ehrlich's hsema- toxylin. The completeness of the " specificity " of this method is open to doubt, but it gives interesting results. 790. Intravital Staining with Benzidine Dyes. — BOUFFARD (Ann. de VInst. Pasteur, xx), then GOLDMANN (Beit. z. Klin. Chir., Ixiv, have shown that animal tissues may be " stained ' intra vitam by the injection of several benzidine dyes. There are some categories of cells in the body which seem to show a special affinity for phagocy- tosing or, at least, ingesting in some wray granules of certain of these colloid dyes. These various cells are often called pyrrhol cells (Goldmann), macrophages (Evans), histiocytes (Aschoff-Kiyono), or resting wandering cells (Tschaschin). According to HAL DOWNEY (Anat. Eecord, xii, 1917) the process of " staining " is one of storage or ingestion, and not of true staining, and attempts to classify cells according to their reactions to these colloidal CHAPTER XXX. 389 dyes are a failure. This author believes that blood cells behave towards these dyes just as they do to any foreign matter. Other authors do not agree. " To dismiss these cells (pyrrhol cells) as scavengers is to do them an injustice, for, however important this function may be, their service to the body is a far greater one " (P. G. SHIPLEY, Amer. Journ. Physiol. , xlix, 1919, p. 300). EVANS and SCIIULEMANN (Science, N.S., 1914), believe that vital staining with azo dyes is the result of " phagocytosis " of ultra-micro- scopic dye particles, existing in a state of fine dispersion as an hydrosol. In using the term " phagocytosis ' Evans does not quite mean an engulfing by pseudopodia as with amoeba. P. G-. SHIPLEY (Amer. Journ. Physiol., 1919, p. 285) points out that some cells which are most active in phagocytosing bacteria and other coarse particles, take no part, under ordinary conditions, in the segregation of vital dyes in the body of the living animal. It has been stated that the benzidine dyes are not characterised by a propensity for staining the mitochondria, as are Janus green or dahlia. The granules in cells which store ultramicroscopic pa.rticles of the benzidine dyes seem to be something apart from the mitochondria. Only occasionally the mitochondria, as such, take up a benzidine dye like trypan blue. In tissue cultures from forty to sixty hours old many of the cells are seen to contain large greyish granules which were either not present in the early stages or were not very noticeable. Such granules (" segregation granules " of Shipley, and possibly degeneration or "neutral red " granules of the Lewises, Amer. Journ. Anat., 1915), stain red in neutral red and deep purple in cresyl blue, and by using a combination of trypan red and Janus green, it can be shown, according to Shipley, that the mitochondria (green in the Janus) and the segrega- tion granules which take up the azo red dye, are separate entities. This opinion is not shared by TSCHASKIN (Fol. Rcem., 1914), by LEVY (E. Accad. d. Lincei, 1916), and by MAXIMOW (Arch. Russ. d'Anat. d. Hist. etd'Embryol,, 1916). Trypan blue is also used for demonstrating areas of osteoblastic activity (Shipley and Macklin). See § 780. The Methods of using Benzidine Dyes are as follows : — Trypan blue and pyrrol-blue of 1 per cent, strength in Ringer's solution are injected subcutaneously, intraperitoneally or into the blood vessels. Whereas 1 c.c. of a 1 per cent, solution per 20 grms. of the animal's body weight injected subcutaneously has no ill-effect on the animal, no more than 0-5 c.c. of the same solution should be used for intra- vascular work. In the latter case coloration sets in speedily, increases up to the second day, but rapidly fades after the fourth day, in any case, quicker than when gradual absorption of the stain takes place through the lymphatic channels. It is undoubtedly safest and best for histological study to inject the staining fluids subcutaneously. Injections of 1 c.c. of a 1 per cent, solution per 20 grms. 'body weight may be repeated many times once a week. 390 BLOOD AND GLANDS. In some cases Goldmann has given fifteen consecutive injections. These remarks refer especially to small animals like the rat ; for bigger animals, such as the rabbit or ape, intraperitoneal injections are preferable to subcutaneous : use the standard of 1 c.c. of a 1 per cent, solution per 20 grms. of animal's weight. Trypan blue, isamin blue and pyrrol blue allow of fixation in 10 per cent, formalin solution (injected intravenously if possible), but it is only tissues stained in trypan blue that allow ordinary processes of histological technique, but even for trypan-blue, the fixative should contain a little formalin. Sections are cut with a freezing microtome from tissue fixed in 10 per cent, formalin not less than forty-eight hours. Stain as necessary in alum carmine or hsemalum, etc. Pappenheim's pyronin and methyl green are good for connective tissues, Ehrlich's triacid for hsemopoetic tissues (GOLDMANN, Proc. Roy. Soc., Ixxxv, 1912), G. B. WISLOCKI and H. DOWNEY (Anat. Record, xii, 1917), after staining, fix in Zenker or formol, upgrade in alcohols, imbed in wax and section. Counter- stain in hsemalum. GOLDMANN (loc. tit.) mentions the following ' vital stains," apart from those given above : Trypan violet, benzopurpurin, diamin blue B.B., diamin black B.H., vital ' neu rot," vital " neu orange," vital " neu gelb," dianil blue R (Griibler). These are used in 1 per cent, strength in salt solution. Trypan blue, trypan red, Congo red, azo blue, and benzopurpurin can be used on tissue cultures by introducing some of the dye into the culture medium (SHIPLEY, Amer. Journ. Physiol., 1919, p. 287). Apart from the references given above see also, HOFFMAN ( Fol. Hcem., 19U), RENAULT (Arch. d'Anat. Micy., 1907) ; LOELE (Fol. Hcem., 1913) ; BATCHELOR (Proc. Amer. Assoc. Anat., 1914, Anat. Record, 1914). 791. Microchemical Tests for the Oxygen Place in Tissues.- Recently certain workers have claimed to be able to locate centres or regions of oxidation in the cell by means of some substances sensitive to free oxy- gen. Unna's method is to use a solution of rongalit white, which is a solution of the leucobase of methylen blue kept in a state of reduction by excess of rongalit, an absorption product of formaldehyde with sodium sulphite. See UNNA (Die Reduktionsorte und Saiierstofforte des tierschen Gewebes, Arch. f. Mikr. Anat., Ixxviii, 1911). A. N. DRURY (Proc. Boy. 8oc., 1914) has shown that Unna's claim is inadmissible, and consequently his theory of staining by oxidation and reduction is not proven. GRAHAM (Journ. Med. Research, Boston, xxxv, 1916) claims to have demonstrated by means of H.202 and naphthol, that the granules of leucocytes and myelocytes contain a peroxidase of the peroxide type. Schultze in his Oxydase Reaction uses a -naphthol and dimethyl-p-phenylen-diamin (Merck). Blood and marrow smears fixed in formalin vapour are treated firstly in the a -naphthol solution ; pre- CHAPTER XXX. 391 pared by melting ] grin, of naphthol on the surface of 100 c.c. of aq. dest., and adding potassium hydrate till the naphthol dissolves. After a few minutes in this solution (cooled) the smears are transferred to a 1 per cent, solution of the dimethyl — for the same time, when a blue colour is seen to appear where the oxydases lie. Mount in glycerine jelly, but blue colour fades. Myelocytes and not lymphocytes are said to give a positive reaction. It is doubtful how far these various colour indicators for oxygen place in cells and tissues are reliable. It has been claimed that by means of the last-mentioned method, it is possible to show that the staining (oxygen place) appears especially around the nucleus of the cell. This has not been confirmed. Glands. 792. Mucin. — HOYER (Arch. mik. Anat., xxxvi, 1890, p. 310) finds that the mucin of mucus cells and goblet cells stains with basic tar colours and with alum hsematoxylin, but not with acid tar colours. He obtained his best results by means of thionin, and good ones with toluidin blue, both of these giving a metachromatic stain- tissues blue, mucin reddish — and also with methylen blue (which is particularly useful from its power of bringing out the merest traces of mucin), safranin, etc. Tissues should be fixed for two to eight hours in 5 per cent, sublimate solution, and paraffin sections stained for five to fifteen minutes in a very dilute aqueous solution of the dye (2 drops of saturated solution to 5 c.c. of water). Hyaline cartilage, the jelly of Wharton, and the Mastzellen of Ehrlich give the same reactions with basic dyes as mucin does. See also SUSSDORF, Deutsche. Zeit. Thiermed., xiv, pp. 345, 349 (Zeit. wiss. Mik., vi, 1889, p. 205) ; BIZZOZERO, Atti. R. Accad. di Sci. di Torino, 1889 to 1892 (reports in Zeit. wiss. Mik., vii, 1890, p. 61 : and ix, 1892, p. 219) ; also UNNA, ibid., xiii, 1896, p. 42. The safranin reaction is not obtained with all brands of the dye ; that of Bindschedler and Busch, in Bale, gives it, whilst safranin 0 of Griibler does not. UNNA employs chiefly polychrome metnvjen blue. As regards the thionin stain, see HARI, Arch. Mik. Anat., Iviii, 1901, p. 678. BRUNO (Bull. Soc. Nat. Napoli, 1905, p. 220) fixes and stains the skin of the frog in a mixture of 100 c.c. of formol of 1-25 per cent. with 8 c.c. of 1 per cent, solution of thionin. Mucus glands red. KULTSCHIZKY (Arch. mik. Anat., xlix, 1897, p. 8) fixes in his mixture (§ 57), and stains sections either in safranin with 2 per 392 BLOOD AND GLANDS. cent, acetic acid, or in a similar solution of neutral red (two to three days, washing out with alcohol). MATER (Mitt. Zool Stat. Neapel.,xii, 1896, p. 303, or last edition) gives the following two formulse for mixtures that stain exclusively mucus. 793. MAYER'S Mucicarmine (op. cit., last §).— One gramme of carmine, and 0-5 grm. of aluminium chloride with 2 c.c. of distilled water heated over a small flame for two minutes, and made up to 100 c.c. with 50 per cent, alcohol. This gives a stock solution, which is as a rule to be diluted for use tenfold with distilled or tap water. MATER'S Muchsematein (ibid.).— Hsematein 0-2 grm., aluminium chloride 0-1 grm., glycerin 40 c.c., water 60 c.c. An alcoholic solution may be made by dissolving in 100 c.c. of 70 per cent, alcohol, with or without the addition of 2 drops of nitric acid. 794. Mucicarminic Acid (KAWITZ, Anat. Anz., xv, 1899, p. 439).- One gramme of carminic acid, 2 of aluminium chloride, and 100 c.c. of 50 per cent, alcohol. 795. Goblet Cells. — So far as these contain mucin they give the reactions above described. See PANETH, Arch. mik. Anat., xxxi, 1888, pp. 113 et seq. ; LIST, ibid., xxvii, 1886, p. 481 ; and GUYEISSE, C. R. Soc. Bid., 1907, p. 1212. For intestinal epithelium, especially the cells of PANETH, see also MARTIN, Unters. ueb. Oberfldclien u. Driisenepithel, Leipzig, 1910 ; and KULL, Arch. mik. Anat., Ixxvii, 1911, p. 541 (sections stained with alum hsematoxylin, treated for twenty to thirty seconds with tincture of iodine, stained a few minutes with Victoria blue, then with eosin). 796. Salivary Glands. — SOLGER (Unters. z. Naturlehre d. Menschen xv, 5 and 6, pp. 2 — 15 ; Festschr. f. Gegenbaur, ii, 1896, p. 21T demonstrates the granules in serous cells and half-moons of the submaxillary gland by hardening in a 10 per cent, solution of formol, and then making sections and staining with hsematoxylin of Delafield or of Ehrlich, the granules taking the stain. KRAUSE (Arch. mik. Anat., xlv, 1895, p. 94) stains sections either with Heidenhain's iron heematoxylin or with Ehrlich-Biondi mixture or thionin. See also KRAUSE, ibid., xlix, 1897, p. 709 ; and MULLER, Zeit. wiss. Zool., 1898, p. 640. 797. Gastric Glands. — KOLSTER (Zeit. wiss. Mik., xii, 1895, p. 314) differentiates the two kinds of cells in stomach glands by over-staining with hsematoxylin, washing out with alcohol containing 1 per cent, of HC1, blueing with alcohol containing 1 per cent, of ammonia, and, after washing, staining for one to five minutes in a CHAPTER XXX. 393 weak solution of Saurefuchsin. Peptic cells blue, parietal cells red. Osmic material cannot be employed. CADE (Arch. Anat. Micr., iv, 1901, p. 4) stains material fixed with Bouin's picroformol in Victoria blue of 1 per cent. R. and L. MONTI (Rich. Lab. Anat. Roma, ix, 1902) demonstrate ducts and canaliculi of delomorphous cells by Golgi's bichromate and silver impregnation, especially with rejuvenated material (see SACERDOTTI), leaving it for five or six days in half -saturated sulphate of copper, then for twenty-four hours in the osmic-bichromate mixture. You can embed in paraffin (rapidly). 798. Intestine. — BENSLEY (Amer. Journ. Anat., v, 1906, p. 323) stains sections of glands of Lieberkiihn in a mixture of equal parts of saturated solutions of orange G- and Saurerubin, and then with toluidin blue, and mounts in balsam. 799. Liver. — BRAUS (Denkschr. Med. Nat. Ges. Jena, v, 1896, p. 307) demonstrates the bile capillaries by the rapid method of GOLGI, hardening in a mixture of 1 part formol with 3 parts liquid of Miiller or J per cent, chromic acid. EPPINGER (Beitr. path. Anat., xxxi, 1902, p. 230) studies them by means of a complicated modification of WEIGERT'S neuroglia stain, and CIECHANOWSKI (Anat. Anz., xxi., 1902, p. 426) by means of WEIGERT'S myelin stain (the 1885 method). OPPEL (Anat. Anz., v, 1890, p. 144 ; vi, 1891, p. 168) puts pieces of liver or spleen (alcohol material) for twenty-four hours into a solution of neutral chromate of potash (J to 10 per cent.), then for twenty-four hours into a f per cent, solution of silver nitrate, washes, dehydrates and cuts without embedding. The lattice fibres are only stained near the surface, so that tangential sections must be made. Similarly BERKLEY, ibid., 1893, p. 772, fixing in picric acid, then in an osmium bichromate mixture, and then silvering. See also RANVIER, Journ. de Microgr., ix, x, 1885-6 ; IGACUSCHI, in Arch, patli. Anat., xcvii, p. 142, or Zeit. wiss. Mik., 1885, p. 243 (gold process for study of fibrous networks) ; KUPFFER, Sitzb. Ges. f. Morph., etc., Miinchen, Juli, 1889, or Zeit. wiss. Mik., vi, 1889, p. 506 ; KRAUSE (Arch. mik. Anat., xlii, 1893, p. 57) ; and TIMOFEJEW, Anat. Anz., xxxv, 1909, p. 296 (sections of frozen tissue stained with methylen blue). 800. Spleen. — For lattice fibres, see OPPEL, last §. KULTSCHITZKY (Arch. mik. Anat., xlvi, 1895, p. 675) studies the musculature in sections (of material from liquid of Miiller) stained for a day or more in a solution of lakmoid in ether and mounted in balsam. 394 BLOOD AND GLANDS. For elastic fibres he puts .sections for half an hour or a day into a mixture of 800 parts 96 per cent, alcohol, 40 parts 1 per cent, solution of carbonate of potash, 2 parts Magdala red, and 1 part methylen blue. For the blood vessels he puts sections of Miiller material for a few minutes into a solution of 1 or 2 parts of Saurerubin in 400 parts of 3 per cent, acetic acid, washes out in 2 per cent, acetic acid, and after-stains in a similar solution of helianthin or Wasserblau until the red only remains in the erythrocytes. See also WHITING (Trans. Boy. Soc., Edinburgh, xxxviii, 1896, p. 311) ; SCHUMACHER (Arch. mil:. Anat., Iv, 1899, p. 151) ; WEIDENREICH (ibid., Iviii, 1901, p. 251). 801. Lymphatic Glands. — For lattice-fibres especially, see ROESSLE and YOSHIDA, Beitr. path. Anat., xlv, 1909, p. 110, or Zeit. wiss. Mik., xxvi, 1909, p. 295. Sections .stained with hsematoxylin and eosin, or Weigert's iron hsematoxylin, or Bielschowsky's neurofibril stain as applied by MARESCH, loc. tit., § 752. The sections should not remain for more than fifteen to thirty minutes in the oxide bath. See also for the thymus some very complicated methods of SAL- KIND, Anat. Anz., xli, 1912, Nos. 6 and 7. 802. Kidney. — SAUER (Arch. mik. Anat., xlvi, 1895, p. 110) finds that for the renal epithelium the best fixative is Carnoy's acetic alcohol with chloroform (three to five hours, washing out with absolute alcohol). A mixture of 9 parts alcohol with 1 of nitric acid -is also good, as is liquid of Perenyi. He stains with iron haematoxylin, and after-stains in a very weak solution of Saurerubin in 90 per cent, alcohol, which stains the ciliary plateau. He macerates with iodised serum or one-third alcohol, staining after- wards with dahlia. ARNOLD (Anat. Anz., xxi, 1902, p. 417) employs intra vitam staining methods for the study of the granules of the epithelium cells. Sections of fresh kidney are cut with a Valentin's knife, and brought into a very dilute solution of neutral red, or methylen blue, in which the granules stain in a few minutes or hours. Or saturated solutions of the dyes, or of indigo carmine, may be injected sub- cutaneously during life, at intervals of fifteen to twenty minutes, and after two to five injections the organ may be excised and sections made and examined (see §§ 208 and 342 to 344). 803. Thyroid. — BENSLEY (Amer. Journ. Anat., xxix, 1916) uses brazilin and water blue. Fix gland in Zenker-formol. Section in paraffin and fix sections to slide with water alone, or very little CHAPTER XXX. 395 albumen ; pass through toluol, absolute alcohol, water, iodise, and place in this brazilin solution for several hours : — • Phosphotungstic acid . . . . 1-0 grm. Distilled water ..... 100-0 c.c. Brazilin . . . . . . 0-05 grm. The brazilin is first dissolved in a small quantity of distilled water by the aid of heat and added to the phosphotungstic acid solution. This solution goes bad after three days. After staining in the brazilin, wash in water, and place for five minutes in this mixture : — Phosphomolybdic acid .... 1-0 grm. Wasserblau . . . . . . 0-2 ,, Water ....... 100-0 c.c. Wash rapidly in water, dehydrate in absolute alcohol, clear in toluol, and mount in balsam. Cytoplasm stains blue to lilac, nuclear chromatin deep red, contents of thyroid vacuoles sky blue, and colloid droplets of Hiirthle deep blue to deep red. 804. Pancreas. — Most of the techniques given under the heading of " Mitochondria " and " Golgi apparatus," etc. (§§ 673—712) give important results with the zymogen granules of the pancreas. The methods of Bensley-Cowdry (§ 686), Regaud (§ 685), Benda (§ 683), and Schridde (§ 687), all apply here. For the Golgi apparatus Cajal's formalin silver nitrate method may be used (§ 847). BENSLEY'S Neutral Red Method (Amer. Journ. Anat.,xii, 1911- -12). -Animal killed by bleeding ; a cannula introduced into aorta and a solution of neutral red in isotonic salt solution, containing 1 in 15,000 neutral red, is injected. Immediately after the pancreas has assumed a faint rosy tint a part of the organ is removed — the islets of Langerhans stain intense yellow red, the rest faint rosy-pink. In a short time after mounting the islets remain the only stained elements, owing to bleaching in the acini. Method applicable to the counting of the islets of Langerhans. Janus Green Method. See § 702. — Islets deep blue on a red background. Pyronin Method for Ducts. — Inject a 1 in 1,000 solution of pyronin, as above, for neutral red method. The ducts stain intensely red. Double stains may be made by injecting mixed Janus green and pyronin (BENSLEY, op. cit.). Methylen blue, 1 in 10,000 may also be used for this purpose. After injection fix in 5 per cent, ammonium molybdate, for which see also Chapter XVI. GRAND-MOURSEL and TRIBONDEAU (C. R. Soc. BioL, liii, 1901, 396 BLOOD AND GLANDS. p. 187) recommend for pancreas NTCOLLE'S " thionine pheniquee," which stains the insulse of Langerhans hardly at all, the rest strongly. LANE'S Methods for Demonstration of A Cells of the Islets of Langerhans. (1) Fix tissue for from two to four hours in equal parts of saturated alcoholic solution of mercury chloride, and 2J per cent, potassium bichromate. Wash in 50 per cent, alcohol, then upgrade and embed ; 3 i^ sections are stained in neutral gentian, obtained by precipitation of equivalent solutions of gentian violet (crystal violet) and orange G. If the correct quantity of the latter is added to the former, a practically complete precipitation is obtained. The precipitate is soluble in alcohol or acetone. For staining add the stock alcohol solution to 20 per cent, alcohol until a solution having the colour of good hsemalum is obtained. Allow to stand for twenty-four hours. Stain for twenty-four hours, blot, dehydrate in acetone, toluol, differentiate in absolute alcohol 1 part, oil of cloves 3 parts, wash in toluol, and mount in balsam. (2) Fix in 70 per cent, alcohol, then stain in neutral gentian as above. LANE'S Methods for Demonstration of B Cells of Islets of Langerhans. Fix for four to twenty-four hours in :- KsCr207 ...... 2*5 grms. HgCl2 5-0 „ Aq. dest. ...... 100 '0 c.c. Dehydrate, clear, embed, and section ; stain in neutral gentian as above. Formalin Bichromate Method for Fixation.- -This gives a very regular and reliable fixation, and is suitable where one is carrying out observations which necessitate a successful routine method. BENSLEY (op. cit.) uses 10 c.c. of neutral formalin to 90 c.c. of Zenker's fluid without acetic acid, for twenty-four hours. Stain in neutral gentian, acid fuchsin and toluidin blue, iron hsematoxylin or Mallory (§ 314). HOMANS (Journ. Med. Research, xxx, 1914) used Bensley's modified Altmann fixative (Os04 of 4 per cent., 2 c.c. ; potassium bichromate of 2*5 per cent., 8 c.c. ; glacial acetic acid, 1 drop), Lane's methods (vide supra), and ordinary hsematoxylin and eosin. Very pretty results are obtainable by using Mallory 's polychrome methylen blue and eosin (§ 314). See also BABKIN, RUBASCHKIN and SSAWITSCH, Arch. f. Mikr. Anat., Bd. 74 ; HELLY, ibid., Bd. 67 ; LANE, Amer. Journ. Anal. vii, 1907, SAGUCHI, ibid., Vols. 26 and 28, and § 713. CHAPTER XXXL* NERVOUS SYSTEM — GENERAL METHODS. 805. Introduction. — The microscopic investigation of the nervous system pursues two ends. Either it is desired to elucidate the intimate structure of nerve-cells, nerve-fibres and their supporting tissues, or to study the morphology of nerve-cells, their distribution in the grey matter, their connections with each other, and with the nerve fibres which chiefly constitute the white matter, and lastly to investigate the architectural arrangement of both nerve-cells and nerve-fibres in the various regions of the central nervous system. The processes employed in the first case form a group of cytological methods, whilst the processes used in the second instance are spoken of as the anatomical methods. The processes used in the study of nervous tissue in peripheral organs having been described in the chapter on " Methylene Blue," ' Impregnation Methods," " Tegumentary Organs," and " Muscle and Tendon," the following chapters are chiefly devoted to the description of methods for the investigation of the central nervous system. Fixation. 806. Fixation by Injection. — Fixation, in the proper sense of the word, is of course out of the question for the human subject. But in the case of the lower animals it is possible to inject fixing fluids into their nervous centres when still in an almost living state. The practice ensures a very rapid penetration into and even distribution within the tissues of the fixing agents, and has, moreover, the capital advantage of greatly helping to prevent distortion of the nerve- tissues during their subsequent treatment. And as in most instances the practice does not meet with special difficulties, it should be adopted as far as possible also in the case of human subjects, but particularly for a preliminary fixation and hardening of the very soft cerebral mass of young individuals, which is particularly liable to much injury and distortion in the process of removing it from the brain case. * Revised and in great part rewritten by Dr. C. Da Fano, King's College, University of London. 398 NERVOUS SYSTEM— GENERAL METHODS. The choice of the fluid to be injected depends upon the object in view and the subsequent treatment to which the tissues are to be submitted. In the case of animals it is a good practice to warm the fixing fluid to body- temperature before injecting it, and, whenever possible, to wash out the blood by first injecting physiological solution as suggested by Mann. The injection can be carried out through the carotids if the fixation is to be limited to the encephalon, and through the aorta if it is desired to fix the spinal cord too. The above applies to higher vertebrates and particularly to mammals ; in the case of lower vertebrates, fixation by injection has not, as a rule, the same importance, and one must have recourse to special methods. See on this subject GOLGI, op. cit., in § 880 ; GEROTA, § 811 ; DE QUERVAIN, Virchow's Arch., cxxxiii, 1893, p. 481 ; MANN, Ztschr. wiss. Milcr., xi, 1894, p. 482 ; STRONG, Anat. Anz., xi, 1886, p. 655 ; Journ. Comp. Neurol., xiii, 1903, p. 291 ; McFARLAND, Journ. App. Micr., ii, 1899, p. 541. Hardening. 807. Hardening by the Freezing Method. — This phrase has often given rise to confusion and should, therefore, be clearly understood. One can harden by freezing either fresh tissues, or material already fixed and consequently also a little hardened. In the first instance small pieces of fresh tissue, immediately after removal and without any previous treatment, are hardened on a freezing microtome. The sections are generally floated on to water, and immediately after- wards treated for a minute on the slide with a 0-25 per cent, solu- tion of osmic acid ; or otherwise treated according to the object of one's investigation. In this case the ether freezing method should be preferred, bearing in mind, however, that there is considerable difficulty in obtaining sufficiently good sections, and that the results attainable are very limited particularly since BRODMANN (Journ.Psychol. Neurol., ii, 1903 — 4, p. 211) has shown that formalin material can be used even for investigations by polarised light. (See also p. 361.) The hardening by freezing of already fixed material may be also attended with some difficulty, but this will be easily overcome if pieces are relatively small, the fixing agent properly washed away, and one has, eventually, recourse to one or other of the processes described in § 183. Material fixed in formalin, however, does not, as a rule, require any soaking in gum, or syrup, or the like, and is easily cut if the formalin has been washed away. In this case the C02 freezing microtome is in my opinion to be preferred. CHAPTER XXXI. 399 The hardening and section cutting by the freezing method of very large pieces require special apparatus and special methods, for which see NAGEOTTE, C. R. Soc. BioL, Ixvii, 1909, p. 542. 808. Hardening by Reagents. — If large pieces of nervous tissue are to be hardened, it is necessary to take special precautions in order to prevent them from being deformed by their weight during the process. The spinal cord or small portions of any region of the encephalon may be cut into thin slices, laid out on cotton wool in a vessel into which the hardening fluid is poured. The specimens may also be suspended in the liquid (§ 34). Another good plan consists in adding to the hardening fluid just enough glycerine or sodium chloride to make tissues float. If several pieces are placed in the same vessel, they should never be put on top of each other. Voluminous organs to be hardened in toto should be at least incised as deeply as possible in the less important regions. With the exception of the dura mater, the membranes are not generally removed at first, as they serve to protect the tissues. They can be removed partially or entirely later on when the hardening has made some progress. In the case of material intended for Golgi's methods it is best not to remove them at all. The spinal cord, medulla oblongata and pons Varolii may be hardened in toto, and the preparation hung up in a cylindrical vessel with a weight attached to its lower end to prevent it from becoming distorted. The cerebrum should have light plugs of cotton wool in the fissure of Sylvius, and as far as possible between the convolutions. If it is desired not to open the lateral ventricles, the hardening fluid may be injected into them. Unless there are special reasons to the contrary, the brain should be divided into two portions by a middle frontal section, or better .into two symmetrical halves by a sagittal cut passing through the median plane of the corpus callosum. The cerebellum should be treated in the same manner. The action of most hardening fluids is greatly enhanced by heat. But in the judgment of most histologists this rapid hardening is not, as a rule, attended by good results, and one should have recourse to it only for particular reasons and special purposes after a tentative experiment, whenever possible, at establishing the degree of tem- perature at which the desired results may be obtained without otherwise injuring the delicate structure of the nervous tissues. 400 NERVOUS SYSTEM— GENERAL METHODS. On the other hand the hardening action at room temperature of certain reagents, such as solutions of chromic salts, proceeds so slowly that decomposition may set in before the fluid has had time to act effectively. For this reason voluminous preparations which are to be hardened in toto in solutions of chromic salts, and were not injected as described in § 806, should be put away in a very cool place or in an ice-chest. A human cerebral hemisphere may require eight or nine months for hardening in this way. The volume of the fluid should always be very large in proportion to that of the pieces of tissue and to their number. It should be taken in solutions as weak as is consistent with the proper preserva- tion of the tissues. It should be frequently changed and its strength gradually increased. MARIE'S method of fixing and hardening in situ is highly recom- mended ; for its indications and contra-indications, see SAINTON and KATTWINKEL (Deutsche Arch. klin. Med., Ix, 1898, p. 548) and PFISTER (N enrol. Centrbl, xvii, 1898, p. 643). 809. The Reagents to be Employed. — As in the case of the fixation by injection one should bear in mind that the preservation of tissues for neiiro-histological investigations greatly depends upon the purpose in view. Fixing and hardening fluids which are excellent for cytological investigations are very often unsuitable for anatomical methods. (See § 805.) On the other hand, material collected and prepared for cyto-architectonic or fibro- architectonic studies can hardly be used to elucidate questions regarding the intimate structure of nerve-cells or nerve-fibres. Alcohol, formalin * and chromic salts are most frequently used because they are generally ready at hand, and because they are useful for carrying out afterwards either a great number of methods, or certain methods, under constant conditions of hardening and staining. 810. Alcohol. — It is generally employed in the strength of 94 to 96, per cent., penetrates well and hardens quickly ; but as it rapidly * AVrong as it is, I find it expedient to use the term " formalin " or " formol " in the generally accepted sense, viz., as if it were a chemical reagent, while it is only a commercial denomination which ought not to have been introduced in the histological terminology. See § 108. As is well known, the commercial formalin is only a 40 per cent, solution of formaldehyde ; but when in this and the following chapters on the Cuervo us system a 5, 10 or 20 per cent, solution of formalin is mentioned, it is intended to mean 5, 10 or 20 parts of commercial formol, and 95, 90 or 80 parts of water, respectively, while, e.g., a 20 per cent, solution of formaldehyde is the commercial formalin diluted with half its volume of water. — C. d. F. CHAPTER XXXI. 401 absorbs water from the tissues the latter shrink considerably, whilst the alcohol loses its fixing and hardening properties through hydra- tion. It has consequently to be changed soon for a fresh supply and used in quantities exceptionally large in proportion to the size of the pieces, which ought to be neither too small nof too large. For this reason one seldom hardens in alcohol voluminous organs, and its use has become on the whole very restricted. Alcohol, however, remains the principal fixing and hardening reagent for cytological investiga- tions by Nissl's method (see § 826), and for carrying out some of Ramon y Cajal's reduced silver processes (§ 827), its shrinking influence being counteracted by having recourse for the first fixation to weaker dilutions of alcohol (60 to 70 per cent.) to be raised gradually up to 95 or 96 per cent, within the first nine to twelve hours, and to be changed once or twice or more often in the next few days. 811. Formalin. — Since the time when it was introduced into histological technique by F. BLUM (Ztschr. wiss Mikr., x, 1893, p. 314) ; J. BLUM (Zool. Anz., xvi, 1893, p. 434) ; HERMANN (Anat. Anz., ix, 1893, p. 112) ; HOYER, jun. (Anat. Anz., ix, 1894, p. 236) ; LACHI (Monit. Zool. ItaL, v, 1895, p. 15) and many others, its use has been steadily increasing because of the many advantages it offers. As a matter of fact it penetrates more quickly than solutions of chromic salts, and even than alcohol ; it is not likely to over- harden ; it allows of the most various after-treatments and methods of staining, including neurofibril stains and Goigi's impregnation method. Several writers have insisted that for nervous tissue it should not be acid, but some prefer it acid. See " Retina." For neurofibrils it should be preferably neutral. To neutralise it, it is generally suffi- cient to prepare its solutions with spring water, but one may shake it with magnesium or sodium or calcium carbonate. Some authors prefer to neutralise with ammonia. (See also § 108.) The strength of the formalin solutions generally used for fixing and hardening nervous tissues varies considerably with the quality of the material in hand, but particularly with the age of the subjects. As a rule the more delicate the material and the younger the subject, the weaker should be the formalin solutions to be employed at first. Generally, however, one starts with a 3 or 5 per cent, solution in the case of very soft tissues, gradually increasing the strength up to 10 or 12 per cent. An adult human encephalon can be very well preserved in a 10 or 15 or 20 per cent, solution with two changes of M. 26 402 NERVOUS SYSTEM— GENERAL METHODS. the fluid during the first days of fixation and hardening. See further on this subject, § 108. Formalin can be associated with, or followed by, alcohol (§ 109) or other reagents. Thus FISH (Proc. Am. Micr. Soc., xvii, 1895, p. 319) recommends :- Water 2,000 c.c. Formalin . . . . . 50 ,, Sodium chloride .... 100 grms. Zinc chloride . . . . . 15 „ Brains are left in the mixture eight to ten days or longer, and then transferred into a mixture of water 2,000 c.c. and formalin 50 c.c., in which they may remain indefinitely if the jar is kept tightly stoppered. PARKER and FLOYD (Anat. Anz., ix, 1896, p. 156) advise for sheep's brains a mixture of 6 volumes of 95 per cent, alcohol and 4 volumes of 2 per cent, formol. Brains may be kept in the mixture for months. FLATAU (Anat. Anz., xiii, 1897, p. 323) finds that brain increases in weight slightly in 10 per cent, formol (spinal cord somewhat more) ; whilst in 1 per cent, solution it may increase as much as 24 per cent. GEROTA (Int. Monatschr. Anat., xiii, 1896, p. 108) puts human brains into a 5 or 10 per cent, solution of formol, and after twenty- four hours removes the pia mater, and changes the liquid ; this is also further done every five to seven days, and in one or two weeks the hardening is complete. In the case of foetal brains of Canis, Felis and Homo, he first injects the vascular system with a 10 to 15 per cent, solution of formol in 85 per cent, alcohol, and then brings the heads into the 5 to 10 per cent, watery solution ; after one or two days he removes the brains from the skull and puts them back for fifteen to twenty days into the formol. KADYI (Poln. Arch. Biol. Med. Wiss., i, 1901, p. 80) takes 5 parts of formol, 100 of water, and 2 of bicarbonate of soda, for four to ten days. HERDLICKA (Proc. U. S. Nat. Mus., xxx, 1906, p. 304) takes 3 parts of formol, 25 to 45 of water, and 72 to 52 of 95 per cent, alcohol. STRECKER fixes small pieces for twenty-four to forty-eight hours in equal parts of 10 or 20 per cent, formol and Ehrlich-Biondi triacid mixtures, and imbeds in paraffin, thus getting a stain at the same time as a fixation. Similarly with toluidine blue fixing it with ammonium molybdate. See Ztschr. wiss. Mikr., xxviii, 1911, p. 17, and the literature discussed therein. CHAPTER XXXI. 403 812. Chromic Salts. — The most commonly used is potassium bichromate. The liquid of Erlicki has a more rapid action than other solutions of chromic salts, but it has been generally abandoned because of the alterations it very often produces. SAHLI (Ztschr. wiss. Mikr., ii, 1885, p. 1), after investigating the action of the usual solutions, concludes that the best hardening agent for fresh tissues is pure potassium bichromate, in 3 or 4 per cent, solution, the hardening being done in a cold place. He rejects the liquid of Erlicki on account of the precipitates to which it so fre- quently gives rise. OBERSTEINER is of the same opinion, and recommends pure bichromate for general hardening purposes ; whilst for the study of delicate structural details he recommends fixing in Fol's modifica- tion of Flemming's fluid (§47) for twenty-four hours, followed by washing with water and hardening in 80 per cent, alcohol. In view of the slowness of penetration of chromic salts, it is often advisable to treat the preparations for twenty-four hours or more with 80 to 90 per cent, alcohol, or better, for a few days with a formol solution before putting them into the hardening fluid, or to add formol (say 3 per cent.) to it, in order to avoid maceration of the deeper layers of the tissues. In this case, however, the fluid must be changed after twenty-four to forty-eight hours. Potassium bichromate should be employed at first of not more than 2 per cent, strength ; this is then gradually raised to 3 or 4 per cent, for the cord and cerebrumt and as much as 5 per cent, for the cerebellum. Ammonium bichromate should be employed of half the strength recommended for potassium bichromate at first ; it may be raised to as much as 5 per cent, for cerebellum towards the end of the hardening. NISSL (Enzycl. Mik. Technik., ii, 1910, p. 245) uses, for rapid hardening, large quantities of Miiller's fluid 100 parts, formol 3 parts, and enough glycerine to make the tissues float. If the solution is often changed, even entire brains are in a few days sufficiently hardened to be safely transferred into pure Miiller's fluid, or potassium bichromate of about the same strength. BETZ'S method (Arch. mik. Anat., 1873, p. 101). Brain and spinal cord are first hardened, for some days or weeks, in 70 to 80 per- cent, alcohol containing enough iodine tincture to give it a light brown coloration (as soon as the alcohol becomes colourless, more iodine must be added.) They should then be definitely hardened in 3 per cent, potassium bichromate for spinal cord, medulla 26-2 404 NERVOUS SYSTEM- GENERAL METHODS. oblongata, and pons, 5 per cent, for cerebellum, and 4 per cent, for cerebrum. The methods of BEVAN LEWIS (op. cit.) and HAMILTON (Journ. Anat. and Physiol., 1878, p. 254) can be considered as superseded, chiefly because they are based on a fixation with methylated spirit (Bevan Lewis) or mixtures of Miiller's fluid and methylated spirit (Hamilton), which cannot be used any longer for histological pur- poses, owing to the excess of impurities it now contains. Also the methods for encephala of DUVAL (Robin's Journ. de I' Anat., 1876, p. 497) and DEECKE (Journ. R. Micr. Soc., 1883, p. 449) can be considered as obsolete. ORTH uses formol-Muller changed every few days. See § 113. BONVICINI (Ztschr. wiss. Mikr., xxvi, 1909, p. 412)' puts entire human brains into 10 per cent, formol (first injected through the carotids or into the ventricles) for six to eight days, cuts them into 1 cm. thick slices by means of a special macrotome, and transfers them into a mixture of 4 parts of potassium bichromate and 2-5 parts of chromium sulphate in 100 of water, and keeps them therein, in the dark, for two months for cerebral hemispheres, twelve to fourteen days for medulla and pons, five or six days for spinal cord. In the case of slices of brain, the fluid must be changed every week. KAWITZ (ibid., p. 338) puts formol material for exactly five days into alcohol with 10 per cent, tinctura iodi P. G-., then for eight to ten into saturated solutions of potassium bichromate changed after the first day, and lastly into 95 per cent, alcohol for three days in the dark. 813. Other Reagents. — Osmic acid is hardly useful for specimens of more than 2 or 3 mm. thickness. Chromic acid is rarely used alone. Its action is rapid, but uneven, and causes shrinkage and brittleness. A very little (e.g. 3 to 5 drops of a 1 per cent, solution to every 100 c.c. of fluid), added to bichromate solutions will do no harm and quicken the hardening. Nitric acid, has been and still is employed in strengths of 10 to 12 per cent. Neutral acetate of lead affords, according to KOTLAKEWSKI (Ztschr. uriss. Mikr.,iv, 1887, p. 287), an excellent preservation of ganglion cells. Corrosive sublimate solutions either alone or mixed with other re- agents (see Chapter V.), have been very often used for cytological studies. Similarly acetic alcohol. MANN (op. cit.) for cell studies, puts small pieces for twenty-four hours into a solution of 5 parts of potassium iodide and 25 of iodine in 100 parts of water, and then into 70 per cent, alcohol. OLMACHER (§ 65) recommends his mixture. KODIS (Arch. mikr. Anat., lix, 1901, p. 212) fixes tissues in a saturated solution of cyanide CHAPTER XXXI. 405 of mercury, brings them into 10 per cent, forinol, and makes sections by the freezing method. NELIS (.BwW. Ac. Sc. Belg., 1899-1900) fixes spinal ganglia for twenty- four hours in a solution of 20 grms. of copper sulphate and corrosive sublimate to saturation in a litre of 7 per cent, formol .with 5 c.c. of acetic acid. KING (Anat. Rec., iv, 1910, p. 213) after trying over twenty-five methods on brains of rats, concludes that the best is Ohlmacher's. The brain should be put into it for two to three hours, then for one into 85 per cent, alcohol, then into 70 per cent, with iodine for at least twenty- four hours, then passed through alcohols of ascending strength and alcohol-ether into 2 per cent, celloidin for two to three days, and through chloroform and benzol into paraffin. In her opinion, Bourn's is the best of the formol liquids ; Tellyesnicky's is the only one of the bichromate mixtures that equals it. All sublimate mixtures fix the nuclei well, but vacuolise the cytoplasm. See further particulars on this subject in the original papers of TRZEBINSKI, Virchow's Arch., cvii, 1887, p. 1 ; DIOMIDOFF, ibid., p. 499 ; FISH, The Wilder Quarter -Century Book, 1893, p. 335 ; DONALDSON, Journ. Morphol., ix, 1894, p. 123 ; MARINA, Neurol. Centrbl., xvi, 1897, p. 166 ; TIMOFEEW, Intern. Monatschr. Anat., xv, 1898, p. 259. 814. Nervous Centres of Reptiles, Fishes and Amphibia. — MASON (Central Nervous System of Certain Reptiles, etc. ; WHITMAN'S Methods, p. 196) recommends iodised alcohol, six to twelve hours ; then 3 per cent, bichromate, changed once a fortnight until the hardening is sufficient (six to ten weeks). BURCKHARDT (Das Gentralnervensystem von Protopterus, Berlin, 1892 ; Ztschr. wiss. Mikr., ix, 1893, p. 347) recommends a liquid composed of 300 parts of 1 per cent, chromic acid, 10 parts of 2 per cent, osmic acid, and 10 parts of concentrated nitric acid, in which brains of Protopterus are hardened in twenty-four to forty-eight hours. FISH (Journ. of Morphol., x, 1895, p. 234) employed for Desmog- nathus a mixture of 100 c.c. of 50 per cent, alcohol, 5 c.c. of glacial acetic acid, 5 grms. of corrosive sublimate, and 1 grm. of picric acid, fixing for twelve to twenty-four hours, and passing through the usual alcohols. STRONG (Journ. com}}. Neurol., xiii, 1903, p. 296) fixes (and decalcifies at the same time) the heads of young Acanthias in a mixture of 9 parts of 5 per cent, iron alum and 1 part of formol, for about two weeks, makes paraffin sections, stains with hsematoxylin, and differentiates in 1 or 2 per cent, iron alum. JOHNSTON (Morphol. Jahrb., xxxiv, 1905, p. 150) recommends for nerves of Petromyson to make paraffin section from Zenker material, and stain them with a mixture of saturated solution of nigrosin, saturated solution of picric acid, and 1 per cent, acid fuchsin, in water mixed in proportions arrived at by trial. Sections. 815. Imbedding is by no means always necessary, and is objected to in some cases. Indeed sections can be obtained from any part 406 NERVOUS SYSTEM— GENERAL METHODS. of the central nervous system without it, if the tissues are well hardened. Material hardened in alcohol, or in chromic solutions, or treated according to Golgi's methods may be glued on to a piece of wood or hard cork (or still better to a glass cube) by means of a rather thick solution of gum arabic. As soon as it begins to stick to the support the whole is put into 70 to 80 per cent, alcohol to harden the gum, and then fixed in the obj ect-holder of the microtome and cut. Or one can simply make a clean cut at the bottom of the specimen, dry it with blotting paper and stick it on the support with sealing wax or paraffin of high melting point. For section cutting the knife should be wetted with alcohol or water ; if the latter is used some soap may be added to it to prevent it from running into drops on the knife. Formalin material is preferably cut by the freezing method, this being very largely used since the introduction of C02 microtomes, by means of which many and relatively very thin sections can be rapidly obtained with great economy of time and imbedding reagents. Imbedding in paraffin is not advised for the nervous system in general, particularly after fixation in alcohol, and bichromate solutions. One should have recourse to it only for special cytological methods, taking care not to use paraffin of too high a melting point. Imbedding in celloidin is very largely used, and to great advantage, for many purposes. If, notwithstanding every precaution, the celloidin has not thoroughly penetrated the tissues, good sections may still be obtained by DUVAL'S method of collodionising the sections. The cut surface of the block is dried by blowing on it, and is covered with a thin layer of collodion laid on it with a brush. As soon as this layer has somewhat dried, which happens very rapidly, a section is cut, and the cut surface collodionised as before, and so on for each section. This process gives very good results, and may be advantageously employed even with material that has been successfully imbedded, as it gives greater consistency to brittle or otherwise delicate tissues. The above applies to section cutting of small, medium-sized and even relatively large pieces. Also unusually large pieces, entire human hemispheres, and brains of high vertebrates can be cut into thin, and, if necessary, serial sections both without, and after im- bedding either in celloidin or paraffin or by mixed methods. The processes used for the purpose do not differ essentially from those above-mentioned and fully described in Chapters VII., VIII. and CHAPTER XXXI. 407 IX., but (particularly for cy to-architectonic and fibre- architectonic studies) special apparatus and installations are needed, the description of which is outside the province of this book. See FEIST, Ztschr. wiss. Mikr., viii, 1891, p. 492 ; DEECKE, op. cit. ; DEJERINE, Anat. Centres Nerveux ; STRASSER, Ztschr. wiss. Mikr., ix, 1892, p. 8 .- BRODMANN, Journ. Psychol. u. Neurol., ii, 1903-4, p. 206 ; WARNKE, ibid., p. 221 ; LIESEGANG, Ztschr. wiss. Mikr., xxvii, 1910 p. 369 ; VENDEROVIC, Anat. Anz., xxxix. 1911, p. 414. General Stains. 816. Carmines. — Ammonia- carmine is good for general views. Stain very slowly in extremely dilute solutions. Bichromate material should be brought direct into the stain without passing through alcohol (see § 51). Picro-carmine has much the same action, but gives a better demonstration of non-nervous elements. Bolles Lee (see 1913 ed.) prefers carmalum with formol material as giving a more delicate stain. He finds it better then paracarmine. The best way of staining formol material with ammonia carmine, carmalum, picro- carmine and the like, consists in my opinion in cutting sections by one or the other of the freezing methods, and transferring them for a few hours either in Miiller's fluid, or 0-5 per cent, chromic acid as suggested by SCHWALBE (Centrbl. allg. PathoL, xii, 1901, p. 881). Sections are then washed for a longer or shorter time according to the amount of mordant one wishes to extract, proceeding afterwards to stain with one of the above-mentioned carmine solutions. On the other hand sections of non-imbedded material fixed and hardened in one or the other of the fluids mentioned in §§ 810 to 812 may be stained not only with carmines, but also with a great variety of dyes if one so desires (see Chapter XL). The same applies to sections of imbedded material, though the after-treatment to which it has been submitted may render more or less difficult the carrying out of certain general stains. But one should remember that in any case the results thus obtained are not very instructive, and by no means comparable with those attainable by the rational use of the special methods described in the following chapters. For other carmine processes of staining, see SCHMAUS (Munch, med. Wochenschr., 1891, p. 147) ; UPSON (Neurol. Centralb., vii, 1888, p. 319) ; FREEBORN (Journ. Roy. Mic. Soc., 1889, p. 305) ; KADYI (Neurol. Centralb., xx, 1901, p. 687) ; CHILESOTTI (ibid., xix, 1902, p. 161, and Centralb. allg. Pathol., xiii, 1892, p. 191). 408 NERVOUS SYSTEM—GENERAL METHODS. 817. Nigrosin and Anilin-Blue-Black. — Nigrosin lias given useful results in some hands. Anilin-blue-black has been much recommended by SANKEY (Lancet, 2, 1875, p. 82) ; BEVAN LEWIS (Human Brain, p. 125, and Quart. Journ. Micr. Sc., 1876, pp. 73-75) ; VEJAS (Arch. f. Psych: xvi, 1885, p. 200) ; MARTINOTTI (ii, 1885, p. 478) ; JELGERSMA (Ztschr. v:iss, Mikr., 1886, p. 39) ; SCHMAUS (Munch, med. Wochenschr., 1891, p. 147), and others. And see also previous editions. 818. Picronigrosin. — MARTINOTTI (loc. cit., 1885, p. 478) stains for two or three hours or days in a saturated solution of nigrosin in saturated solution of picric acid in alcohol, and washes out in a mixture of 1 part of formic acid with 2 parts of alcohol. 819. KAISER (Ztsehr. wiss. Mikr., vi, 1889, p. 471) stains sections of spinal cord for a few hours in a solution of 1 part of naphthylamin brown , 200 of water, and 100 of alcohol, washes with alcohol, clears with origanum oil and. mounts. 820. Alizarine.— SCHR OTTER (N enrol. CentrbL, xxi, 1902, p. 338) stains sections for twenty-four hours in a 1 to 2 per cent, solution of sulphalizarinate of soda, differentiates for half to one minute in tap-water, dehydrates, and mounts. This is a general stain, but demonstrates Nissl bodies and other internal details. 821 MALLORY'S Phosphomolybdic Acid Hsematoxylin and KODIS' modification, see § 271. For AUERBACH'S modification, see Neurol. CentrbL, xvi, 1897, p. 439. 822. Hsematoxylin and Acid Fuchsin. — FINOTTI (Virchow's Arch., fxliii, 1896, p. 133) stains in hsematoxylin, counterstains for three minutes with 0'5 to 1 per cent, solution of acid fuchsin, and differen- tiates in 75 per cent, alcohol containing a very little caustic potash. VAN GIBSON'S haematoxylin and picro-fuchsin (§ 398) may give useful general views of nerve-cells, axis-cylinders, and neuroglia. 823. Other General Stains, — ALT (Munch, med. Wochenschr., 1892, No. 4) stains for a couple of hours in a solution of Congo red in absolute alcohol, and washes out with pure alcohol. This is useful for peripheral axis-cylinders and other elements. SCARPATETTI (Neurol. CentrbL, xvi, 1897, p. 211) stains sections of formol material for five minutes in 1 per cent, hsematoxylin, treats for five minutes with concentrated solution of neutral copper acetate, differentiates with Weigert's borax-ferricyanide, treats with con- centrated solution of lithium carbonate, washes and mounts. Myelin is not stained. EOTIIIG (Folia NeurobioL, ii, 1909, p. 385) fixes and stains for about four weeks in saturated solution of methylenasur I., in 10 per cent, formol, puts for ten to fifteen minutes into acetone, then for twelve hours into chloroform, and imbeds in paraffin. He also has a process with trichloracetate of lead and methylenazur. CHAPTER XX XL 409 RAWITZ (Ztschr. iciss. Mikr., xxvi, 1909, p. 341) has some compli- cated methods with Indulin, 1 'ndaminblau , and Azosdureblau, which take twenty-eight days; and (ibid., xxviii, 1911, p. 1) others with fuchsin and azofuchsiu which take over thirty-six days. ARIENS KAPPERS (ibid,, xxviii, 1911, p. 417) describes a staining method with extract of elderberries for material fixed and hardened in Mutter's fluid or similar solutions. It is very simple and particularly recommended for photographic purposes ; it should be carried out as follows : Stain celloidin or paraffin sections overnight in neutralised elderberries extract (obtained by fermentation at 20° to 25° C. ), to which 1 per cent, carbolic acid has been added. Wash in water. Differentiate in 3 per cent. Liquor ferri sesquichlorati P.G-., wash, dehydrate, and mount. CHAPTER XXXII. * NERVOUS SYSTEM — SPECIAL METHODS, CHIEFLY CYTOLOGICAL. 824. Introduction. — The ordinary methods of cytology are, of course, available for nerve cells ; but there are certain constituents of these cells, as well as of nerve fibres, which require, for minute study, special methods, such as the following :- A. Methods for Cells, demonstrating Tigroid Substance and other Granular Materials. 825. Tigroid substance or bodies, chromophilic or chromatophilic substance or material or granules, Nissl's bodies or granules, etc., are all denominations for a markedly basophil substance which appears as blocks, granules or irregular patches within the cytoplasm of nerve cells under certain conditions of fixing and staining. It is now almost universally admitted that this substance exists in the living cells as a fluid or semi-fluid ' plasm rich in nutritive value," and that the blocks, granules or patches are appearances chiefly due to the coagulation of this plasm, as brought about by the fixing agents employed for their demonstration. As, however, these bodies or granules appear always the same under constant optical conditions in healthy cells fixed and stained in a constant manner, they are said to be the equivalent of such healthy cells during life. " It follows that if the cells, prepared by the same method and examined under the same conditions, show a difference from the equivalent or symbol of healthy cells, the difference is the measure of some change that has occurred during life." See HALLIBURTON, Handbook of Physiology, London, 1920, p. 194. This is pointed out here to make it quite clear why NISSL has always insisted that his method should be carried out according to his suggestions, and in a constant manner. At first (N enrol. Centrbl., iv, 1885, p. 500) he used to stain sections of material fixed in alcohol with a warmed watery solution of magenta red or dahlia violet or vesuvine, and to differentiate them with alcohol. Later (Allg. Ztschr. Psych., xlviii, 1892, p. 197) he suggested floating sections on a warmed solution of methylene blue (B patent), with * Re-written by Dr. C. Da Fano, King's College, University of London. CHAPTER XXXII. 411 subsequent differentiation with a 10 per cent, solution of anilin oil in 96 per cent, alcohol. The present form of the method was published in 1894 (N enrol. CentrbL, xiii, p. 507) ; but NISSL con- tinued to introduce into it slight modifications, as one understands from many of his papers, to which due attention was paid when preparing the following account. It must be added here that Nissl's method has been, and still is, extremely useful for the study of nervous tissue under various physiological and pathological conditions, and that it stains, when properly carried out, not only the tigroid substance and the basophil parts of nuclei of nerve cells, but also the nuclei and certain parts of the cytoplasm of neuroglia cells and connective tissue elements normally or abnormally present in the nervous tissue. 826. NISSL'S Methylene-blue Method. — Not too small pieces of fresh tissue are fixed in 96 per cent, alcohol and hardened therein for a few days. They should not be allowed to fall to the bottom of the bottle, but kept floating by means of some filter paper or cotton wool. The alcohol must be in large quantities in proportion to the number of pieces, and repeatedly changed. The pieces are cut without embedding and the sections collected in 96 per cent, alcohol, from which they are directly floated on some stain filtered into a watch glass at the moment of using it. The stain should be at least three to four months old, and shaken at the moment of filtering the quantity needed. It is prepared by carefully dissolving 1 -75 grms. of Venetian soap in 1 litre of distilled water and adding to it 3 -75 grms. of methylene blue (B patent). It is a good practice to vigorously shake the bottle from time to time, and to re-filter into the same bottle the amount of stain left in the watch-glass after staining one or more sections. The watch-glass containing the stain with the section floating on it is warmed carefully over a flame until small bubbles rise to the surface. The section, which should not have fallen to the bottom of the watch-glass, is immediately transferred into a mixture of 10 parts of anilin oil and 90 parts of 96 per cent, alcohol, and as soon as no more colour is given off (it often takes only some seconds), it is lifted on to a slide, pressed with smooth filter paper, and cleared with a few drops of pure anhydrous cajeput oil. Care should be taken not to dry the section excessively with the filter paper and to pour the cajeput oil on to the section very quickly. The cajeput oil not only clears the section, but stops the differentiation ; it is, therefore, advisable to renew it after a little 412 NERVOUS SYSTEM—SPECIAL METHODS. while on the section. As soon as this has become quite transparent, the cajeput oil is dried of? with filter paper, the section thoroughly washed with benzol and covered with a drop of thick xylol-colo- phonium, rendered more fluid by passing the slide carefully over a flame, and quickly covering the section with a thin cover-glass before the colophonium sets again by cooling. 827. Suggestions regarding the Carrying-out of Nissl's Method (as deduced from NISSL'S papers ; see chiefly Em. d. mikr. Techn., ii, 1910, pp. 252 — 280, and the references therein quoted at p. 287).- For the fixation of tissues alcohol should be almost exclusively used. Formalin, mixture of formalin and alcohol, sublimate and mixture of the same with alcohol or picric acid and the like, nitric acid may be occasionally employed, but no particularly good results can be expected from them. These are, however, somewhat better if tissues are placed for some time in alcohol after fixation with one or the other of the above reagents. This applies particularly to formalin material, which can be kept with advantage for many weeks, and even months, in repeatedly changed 96 per cent, alcohol. The bichromates of potassium and ammonium and mixtures con- taining chromic' salts, though useful for other purposes, should be entirely avoided for cytological investigations in Nissl's sense. See on this subject also BURCHARDT, La Cellule, xii, 1897, p. 337. If tissues are too brittle to be sectioned without embedding, or if embedding is for any other reason desirable, one should have recourse to celloidin, paraffin being used only when unavoidably necessary or for special purposes. Pieces to be embedded in celloidin are not to be passed through alcohol-ether, but directly from absolute alcohol into thin celloidin. Embedding should, in any case, be carried out as quickly as possible. Sections of material which was not fixed in alcohol and of em- bedded tissues, however fixed, stain, as a rule, very poorly by Nissl's soap-methyl ene- blue method ; but good and even excellent results can be obtained by staining such sections with watery solutions (generally 0-5 to 1 per cent.) of toluidine blue, thionine, Unna's polychrome methylene blue, dahlia violet, vesuvine, neutral red, magenta red, Azur I, Azur II3 and the like. If one of such stains is used, it need not be warmed until bubbles come to the surface, but only until vapour arises. For the differentiation pure 96 per cent, alcohol, viz., without any addition of anilin oil, should be used. In this connection I find that very good results can be obtained from material embedded in CHAPTER XXXII. 413 celloidin if the sections are, whenever possible, freed from the celloidin before staining them, and if they are re-stained a second and a third time after having been each time completely differentia ted. See DA FANO, Proc. Physiol. Soc., Journ. PhysioL, liv, 1920-1. All preparations stained by Nissl's method keep badly, but they keep a little better and may last almost unchanged even for years : (1) if the anilin-alcohol or alcohol used for the differentiation is properly washed away with pure benzol ; (2) if the xylol-colo- phonium for the mount is prepared with pure xylol, and of the thickness needed ; (3) if sections of material embedded in celloidin are submitted to the above-mentioned succession of staining and differentiation ; (4) if preparations are carefully protected from light. 828. Modifications of Nissl's Method. — REHM (Munch, med. Wochenschr., xxxix, 1892, p. 217) floats sections for half a minute to a minute on a hot 0-1 per cent, solution of methylene blue, differentiates them in 96 per cent, alcohol and clears them with origanum oil. LENHOSSEK (Fein. Bau. d. Nervens., 1895) stains sections of formol material in a concentrated aqueous solution of thionine, rinses them with water and mounts them, like Nissl. LUXENBURG (Neural. CentrbL, xviii, 1899, p. 629) stains paraffin serial sections either with Nissl's methylene blue or with thionine as Lenhossek. JULIUSBURGER (Neurol. CentrbL, xvi, 1897, p. 259) stains sections of material fixed in Orth's fluid and embedded in celloidin, either with Nissl's methylene blue or with warmed neutral red. ROSIN (Deutsche med. Wochenschr., xxiv, 1898, p. 615) treats sections of formol material similarly. LENHOSSEK (Neurol. CentrbL, xvii, 1898, p. 577) stains paraffin or celloidin sections of spinal ganglia fixed in Carnoy's fluid with a concentrated watery solution of toluidine blue overnight, rinses with water, differentiates quickly with alcohol and clears with xylol or carbol-xylol. POLUMORDWINOW (Ztschr. iviss. Mikr., xvi, 1899, p. 371), uses 1 part of 1 per cent, solution of toluidine blue to 119 of distilled water and 1 of sodium carbonate. VAN GEHUCHTEN and NELIS (La Cellule, xiv, 1898, p. 374) recom- mend fixing spinal ganglia in Gilson's mixture. VAN GEHUCHTEN (see 1913 ed.) uses paraffin sections mounted on slides by the water method and stains them for five to six 414 NERVOUS SYSTEM— SPECIAL METHODS. hours in Nissl's methylene blue solution in the incubator at 35° 1 o 40° C. GOTHARD (C. R. Soc. BioL, v, 1898, p. 330) stains celloidin sections for twenty-four hours in Unna's polychrome methylene blue, and differentiates them with a mixture of 5 parts of creosote, 4 of cajeput oil, 5 to 8 of xylol, and 16 of absolute alcohol. The mixture is removed with absolute alcohol and sections mounted in xylol dammar after clearing with cajeput oil. LUITHLEN and SORGO (N enrol. CentrbL, xvii, 1898, p. 640) diffe- rentiate in Unna's glycerin-ether mixture, remove this with absolute alcohol, and clear with origanum oil. Similarly LENNHOFF (ibid., 1910, p. 20) ; or, polychrome methylene blue two minutes, distilled water quickly, carbol-pyronin-methyl green twenty minutes ; distilled water quickly, absolute alcohol, oil, balsam. LORD (Journ. Ment. Sc., xliv, 1898, p. 693) makes sections from frozen fresh tissues, treats them for a few seconds with a mixture of equal parts of 6 per cent, formaldehyde and saturated solution of picric acid, then rinses them with distilled water and stains them in 5 per cent, solution of methylene blue B pat. 829. BIELSCHOWSKY and PLIEN'S Cresyl Violet Method (Neural. CentrbL, xix, 1900, p. 1141). — Celloidin or paraffin sections of material fixed either in alcohol or formalin, or sections made by the freezing method from formalin material, are stained for twenty-four hours in a very diluted solution of cresyl violet R.R., prepared by adding 6 to 8 drops of a concentrated aqueous solution to every 50 c.c. of distilled water. After a quick wash in distilled water sections are brought through the ascending series of alcohols, cajeput oil, and xylol, into balsam. The preparations are said to keep better than those stained with thionine or toluidine blue. 830. Picrocarmine has been successfully used by MESSNER (Journ. Psycliol. Neural., xviii, 1912, p. 204, and xx, 1913, p. 256). Sections of alcohol material, embedded in celloidin or not, are washed in water and then stained for five minutes in a warmed diluted solution of Ranvier's picrocarmine. After a quick wash, they are differen- tiated in 3 per cent, hydrochloric acid, dehydrated and mounted as usual. In the case of the spinal cord, medulla oblongata and pons the method succeeds also if material was fixed in formalin. 831. Other Methods for Tigroid Substance and Basophil Granules in General. — See GOLDSCHEIDER u. FLATAU, Norm. u. path. Anat. d. Nervenz, Berlin, 1898, or Ztschr. wiss. Mikr., xvi, 1899, p. 102, and CHAPTER XXXII. 415 NISSL'S remarks thereon, Deutsche Ztschr. Nervenheilk, xiii, 1899, p. 348 ; ILBERG, Neurol. Centrbl, xv, 1896, p. 831; Cox, Anat. Hefte, x, 1898, p. 75 ; Int. Monatschr. Anat., xv, 1898, p. 241 ; AUERBACH, Monatschr. Psych. Neurol., iv, 1898, p. 31; MYERS, Anat. Eec., ii, 1908, p. 434; SAVINI, E. u. TIL, Centrbl. Bakt., I Abth., xlviii, 1909, p. 697 ; MOSSE (argentamin stain), Arch. mikr. Anat., lix, 1902, p. 403 ; MENTZ v. KROGH, Centrbl. Bakt., I Abth., Iviii, 1911, p. .95 ; JOHNSTON, Anat. Eec., xi, 1916, p. 287. 832. HELD'S Methylene Blue and Erythrosin Method (Arch. Anat. Phys., Anat, Abth., 1895, p. 399 ; 1897, pp. 226—233, 273—385, Supplementband). — Material may be fixed in alcohol, but preferably either in picro-sulphuric acid, or in van Gehuchten's mixture of alcohol, chloroform and acetic acid, or 1 per cent, corrosive sublimate in 40 per cent, acetone. Tissues should be carefully embedded in paraffin and sections stuck to slides by the water method. They are stained with the aid of gentle heat for one to two minutes in a solution of 1 grm. erythrosin in 150 of distilled water acidulated with 2 drops of glacial acetic acid. After washing with water the slides are transferred into a mixture of equal parts of Nissl's methylene blue solution and 5 per cent, acetone, warming until all odour of the latter has disappeared. Differentiation is carried out after cooling by means of a 0 -1 per cent, solution of alum until sections are reddish. Rinse in distilled water, dehydrate as rapidly as possible in absolute alcohol, wash in xylol and mount in balsam. BOCCARDI (Monit. Zool. Ital, x, 1899, p. 141) uses a mixture of erythrosin 0-1 grm., toluidine blue 0-2 grm., and water 100 c.c., and differentiates in 0 -5 per cent, alum solution. By means of Held's method, besides the tigroid substance, other granules — viz., Held's neurosomes— become stained. It may, there- fore, be considered as a typical example of double staining of nerve- cells. Other double stains demonstrating basophil, acidophil and other granules have been repeatedly proposed and may be easily obtained by the combination of an acid and a basophil dye. One generally uses watery solutions, e.g., of acid fuchsin, and methylene or toluidine blue, and one stains first with the acid dye and then with the basophil one, differentiation being carried out with alcohol. One may also have recourse to EHRLICH'S triacid (§ 296) as originally proposed by ROSIN (Neurol. Centrbl, xii, 1893, p. 803), or to one or other of the methods used for staining blood films (§ 784), such as PAPPENHEIM'S panoptic triacid stain, Jenner's mixture, Leishman's Romanowsky stain, Pappenheim's method as described in § 785, and so on. See on this subject COWDRY, Int. Monatschr. Anat., xxix, 1913, 416 NERVOUS SYSTEM— SPECIAL METHODS. p. 673, and for pathological specimens ALZHEIMER'S methods 5, 6 and 9 in Histol. u. Histopath. Arb. uber d. Grosshirnr, iii, 1910, pp. 406 — 412, which may be useful for the study of nerve-cells though originally pro- posed for the investigation of neuroglia. B. Methods for Cells and Fibres, demonstrating Neitrofibrils. 833. Neurofibrils ; General Characters. — Nerve cells and the fibres into which they are prolonged contain, in addition to the chromatic constituents shown by the methods already dealt with, a character- istic so-called achromatic element, consisting chiefly of very fine and fairly refractive fibrils which can only be seen with great difficulty in the unstained state, but may be fixed with osmic acid and made out in thin sections of medullated nerve fibres observed in diluted glycerin or water, and may be to a certain extent isolated by macera- tion. For their demonstration, however, one or the other of the methods chronologically described in the following paragraphs must be employed. They are all regarded as giving true stains of neuro- fibrils. For the method of KUPFER (Sitzb. math. Kl. Akad. Wiss. Munchen, xiii, 1884) see former edition. 834. APATHY'S Methods. — The gold method (" Nachvergoldung ") has been given in § 371. The stain is very sharp, but good results are obtained only in certain invertebrates, and even in these with considerable difficulty. The hmmateine method (Mitth. ZooL Stat. Neapel, xii, 1897, p. 712) has the same advantages and disadvantages, and has been little used since the discovery of the Cajal and Bielschowsky processes. Material may be fixed with corrosive sublimate, Zenker's fluid, picro-sulphuric acid, or any other mixture which is not inimical to staining with alum hsematoxylin, and should be preserved in 90 per cent, alcohol. Portions, no more than i cm. thick, are stained for at least forty- £t ** eight hours in hsemateine I. A. (§ 259), and then washed for up to twenty-four hours in absolutely pure distilled water, or preferably suspended therein. Before the stain has become washed out of the neurofibrils entirely, it is fixed by putting the preparations for three to five hours into spring water, after which they are put back for not more than two hours into distilled water, dehydrated as rapidly as possible by hanging them up in absolute alcohol, and embedded in paraffin or celloidin, after clearing with chloroform, and carefully protecting them from light whilst in chloroform or celloidin. The sections are mounted either in a resin or in neutral glycerin. CHAPTER XXXII. 417 835. BETHE'S Molybdenum-Toluidine Blue Method (Ztschr. wiss. Mikr., xvii, 1900, p. 13). — Pieces of the central nervous system of vertebrates are fixed for twenty-four hours in 3 to 7*5 per cent, nitric acid, and then brought directly into 96 per cent, alcohol for a day or longer. They are afterwards put for twelve to twenty-four hours in a mixture of 1 part of ammonia (of sp. gr. 0 -95) with 3 of distilled water and 8 of 96 per cent, alcohol ; for six to twelve hours into pure alcohol ; for twenty-four hours into a mixture of 1 part of concen- trated hydrochloric acid, 3 of distilled water, and 8 to 12 of alcohol ; for ten to twelve hours into pure alcohol ; for two to six hours into water. They are now mordanted with 4 per cent, ammonium molybdate, washed again, dehydrated and embedded in paraffin. The sections, 8 to 10 ju, thick, are seriated on slides by means of egg albumen, but without ivater, then passed through xylol and alcohol and " differentiated," viz., covered with water poured on the sections so as to form over them a layer 1 -5 to 2 mm. deep, and put into an incubator at 55 to 60° C. for ten minutes. They are then rinsed with water, covered with a 1 : 3000 solution of toluidine blue, stoved for another ten minutes, rinsed with water, and lastly treated with 96 per cent, alcohol till no more colour comes away. After dehydration with absolute alcohol they are mounted in the usual way. The method is also applicable to invertebrates for which other fixing agents besides nitric acid are admissible, and the impregnation with ammonium molybdate may be done on the sections instead of previously on the uncut tissues. For LUGARO'S modification see Riv. pat. nerv. ment., x, 1905, p. 265. 836. DONAGGIO'S Methods (Riv. Sper. Frenialr., xxx, 1904, p. 397, and xxxii, 1906, p. 394). — There are five methods of Donaggio. By the first two, pieces are stained in bulk before embedding, but results are not so good as by the other three, the most important of which is — Method III. — Good for spinal cord, pons, medulla oblongata, spinal and sympathetic ganglia. Thin slices of tissues are fixed for five to six days in pure pyridine changed at least once, and then treated with repeatedly changed distilled water until the pyridine has been entirely eliminated. The surfaces of pieces are smoothed by means of a sharp razor, and the pieces brought for twenty-four hours into 4 per cent, ammonium molybdate to which 4 drops of hydrochloric acid have been added. After a quick wash, they are rapidly dehydrated in 95 per cent, and absolute alcohols, and em- M. 27 418 NERVOUS SYSTEM—SPECIAL METHODS. bedded in paraffin. The sections, which must be rather thin (3 to 6 n), are brought through xylol, absolute and 95 per cent, alcohols into distilled water and here washed. This is the crucial point of the method because, by washing, ammonium molybdate becomes extracted from the sections, and the success of the subsequent staining depends almost entirely on carrying out the extraction up to the right point. I find that the only way of ensuring this consists in proceeding by trials, which must be repeated for every series of sections. Once the right amount of washing has been decided upon, one can proceed to stain even many slides at the same time by means of a 1 : 10,000 solution of thionine, to be freshly prepared every time from a less diluted stock solution. The staining is a ' progressive ' one, and must be controlled under the microscope. It generally takes about twenty minutes to obtain it, at the end of which time the grey substance has a red- purple tone whilst the white substance appears bluish. If the staining is right the preparations can be quickly washed, dehydrated and mounted. But if the neurofibrils are not quite sharply stained, the preparations can be ' differentiated ' for another fifteen to twenty minutes in the ammonium molybdate solution used for mordanting the pieces, or for ten seconds in a diluted solution (1 : 10 to 1 : 20) of " pink salt " (C. Erba, Milano). Preparations last only a few months, but are sometimes of great interest. See DA FANO, Ziegler's Beitrdge, xliv, 1908, p. 495. Method IV, which is particularly useful for the demonstration of neurofibrils in the cells of the cortex cerebri and cerebelli, differs from Method III only in regard to a preliminary fixation of pieces for twenty-four hours in a mixture of pyridine nitrate 10 grms., and pyridine, 100 c.c. ; they are then transferred for another thirty-six hours into pure pyridine, proceeding as in Method III. Method V may be used for the demonstration of ' both Nissl's substance and neurofibrils. Pieces are fixed in a saturated solution of corrosive sublimate ; after a day they are treated for twenty-four hours with distilled water to which a few drops of iodine tincture have been added, then for two to three hours with pure distilled water ; and lastly passed for forty-eight hours into pure pyridine, this being changed at least once. The rest as in Method III. PARAVICINI (Boll. Mus. Z. Anat. Comp. Torino, xx, 1905, p. 1) fixes and mordants in the dark, and differentiates after staining with extremely weak hydrochloric acid. TOMASELLI (Ztschr. wiss. Mikr., xxiii, 1906, p. 421) fixes spinal ganglia for six to seven hours in absolute alcohol 100 c.c. with four to five drops of ammonia, and then transfers them for two days into pure CHAPTER XXXIL . 419 pyridine to be repeatedly changed, the vessel with the pieces being kept at 36° to 37° C. After washing for two to three hours in running tap water, he continues as in Donaggio's method III. For the criticism of JADERHOLM, see Arch. mikr. Anat., Ixvii, 1906, p. 108 ; and for that of MONTANARI, Ztschr. wiss. Mikr., xxviii, 1911, p. 22. 837. RAMON Y CAJAL'S Methods. Introductory. — It has been said by some authors that Cajal's methods were originally only modifica- tions of the photographic process of Simarro. The criticism is unjust because even the first formula of Cajal differs so profoundly from Simarro' s process as to form an entirely new method. One cannot, however, deny the existence of a certain similarity of con- ception between the two processes in so far as both are based on the silver-reducing power of certain photographic reagents. For this reason it has been thought expedient to briefly describe here Simarro's process, which though uncertain in its results, may still be of some value to elucidate certain histological questions. SIMARRO'S Process (Rev. Trim. Micr., v, 1900, p. 45) consisted in poisoning animals with subcutaneous injections of solutions of sodium or potassium bromide or iodide in order to impregnate their living nervous tissues with one or the other of these salts. As soon as the animals showed that the poisoning had reached its maximum they were killed and their central nervous system removed in the photographic dark room. Small pieces were then immersed in a solution of silver nitrate, which, by combining with the bromine or iodine with which the tissues were impregnated, gave rise to the formation of silver bromide or iodide, which is easily affected by light. Sections were then made (always in the dark room), best by means of a freezing microtome, and exposed for a little while to light. There remained only treating them with a photographic developer, such as hydroquinone, pyrogallol or the like, and fixing them with sodium hyposulphite and so on, as if they were photographic plates ; they were lastly washed, dehydrated and mounted in the usual way. One can easily understand the many drawbacks of such a method and the reason for which it was abandoned as soon as Cajal published in 1903 his first reduced silver methods. From that time onwards, Ramon y Cajal continued improving them and adding new formula?, which he himself summarised in a special article of his Trab. Lab. Invest. BioL, Madrid, viii, 1910, on which the following account is based. The numbering is that of Ramon y Cajal. Formula la. — Small pieces of fresh tissue are directly put into 1-5 per cent, silver nitrate and kept therein for three to four and 27—2 420 NERVOUS SYSTEM—SPECIAL METHODS. even to five days at a temperature of about 35° C. In summer, with a temperature constantly over 22° C., the stove may be dispensed with, provided the impregnation is prolonged for two to three days more. The tissues are known to be ripe for reduction when a freshly cut surface shows a brownish-yellow colour. They are then washed for one to two minutes in distilled water and put into- Pyrogallol or hydroquinone . . . 1 — 2 grms. Distilled water ..... 100 c.c. Formalin ...... 5 — 10 c.c. The formol is not necessary but useful. One may use pyridine instead (1 to 3 per cent.). The addition of a small quantity of sodium sulphite (0-2 to 0-5 per cent.) has been abandoned by Cajal. The stronger the pyrogallol, the greater the contrast, so that it may be useful to take, sometimes, as much as 3 per cent., but then the over-impregnation of the outer layers will be increased. Hydro- quinone reduces more energetically than pyrogallol. The pieces remain in the reducing fluid for about twenty-four hours and are then quickly washed, hardened in alcohol and embedded in paraffin or celloidin. The sections (15 to 20 ^ thick) are mounted in dammar after toning with a solution of gold chloride if the reaction is rather weak, without toning if the impregnation is a good one. Faintly impregnated sections can be advantageously toned with— Distilled water ..... 100 c.c. Ammonium sulphocyanide ... 3 grms. Sodium hyposulphite . . . 3 „ 1 per cent, gold chloride ... a few drops. If subsequently found to be too dark they can be bleached by Veratti's potassium permanganate and sulphuric acid mixture (see § 846). The sections from the outer layer are generally too dark for study, those from the innermost too pale, whilst those from the intermediate layer are good. The over-staining of the outer layer can be diminished by diluting the silver nitrate with 1 volume of water for the last twelve hours. The method has the defect of giving an imperfect fixation of the nervous tissue and of impregnating, almost exclusively, cell bodies and dendrites. It is not good for ganglia and large cells of adult subjects, but excellent for small and medium-sized cells of very young subjects and early embryos. Formula la, A. — As the last, but pieces are fixed in 3 to 6 per cent. CHAPTER XXXII. 421 silver nitrate. This formula gives better fixation, and was success- fully used by DOGIEL (Anat. Anz., xxv, 1904, p. 558, and Arch, mikr. Anat., Ixvii, 1906, p. 638) for the study of Grandry's corpuscles and other sensory nerve endings, by KOLMER^^. Anz., xxvi, 1905, p. 560) for the epidermis of Lumbricus, etc., and by other authors for the ganglionic chain of Hirudinea. Formula la, B. — As above, but taking 0-75 per cent, silver nitrate and very small pieces, preferably from embryos and new-born sub- j ects. Poor fixation, much shrinkage, but vigorous stain of the neuro- fibrils, nucleolar granules and the intranuclear rodlet of Roncoroni. Formula la, C. — As above, but tissues are fixed in 2 per cent, silver nitrate to which one-fourth of absolute alcohol or acetone has been added. Better fixation than with pure silver nitrate. Results very similar to those obtainable by Formula la with dog, cat and rabbit, and better results with human cerebrum and cerebellum. Formula 2a. — Fixation for twenty-four hours in 96 per cent, alcohol. Tissues not washed, but mopped with blotting paper and put into 1-5 per cent, silver nitrate for seven days at 35° C., or six days at 40° C. The rest as Formula la. Good impregnations of nerve centres of adults, of peripheral nerve endings, of regenerating nerves, of early embryos, and of young fishes. It impregnates medullated and many non-medullated fibres, large and medium nerve cells, the basket fibres of Purkinje's cells, etc. Results fairly constant, but sometimes showing a granular precipitate of unknown origin. To hinder this precipitate and, at the same time, to hasten the impregnation, it is well to add to the alcohol certain substances which Cajal calls " accelerators.'' Such are chloral hydrate, veronal, pyridine, nicotine, ethylamine, antipyrine, and others. Hypnotics, particularly veronal and chloral, and in a less degree pyridine and ammonia, also act as " rejuvenators," reviving the susceptibility of impregnation in tissues which have lain too long in alcohol. Formula 2a, A. — Fixation for twenty-four to forty-eight hours in 96 per cent, alcohol with 2 per cent, of chloral hydrate. Silver bath of 1 -5 per cent, for five days in the stove. The rest as usual. Veronal (same proportion) gives the same result, as do also sulphonal, trional, hedonal, etc. The results are very constant. Medullated fibres well shown. Formula 2a, B. — Fix for twenty-four hours in 96 per cent, alcohol with 10 to 20 per cent, of pyridine ; wash for some hours in pure alcohol and transfer pieces into 1 -5 silver nitrate for five days. 422 NERVOUS SYSTEM— SPECIAL METHODS. I understand from Cajal's pupil, Del Rio Hortega, that this formula may be successfully employed for the study of peripheral nerve endings. In this case material is better fixed for twenty-four hours in pyridine to which one-third its volume of distilled water or 96 per cent, alcohol has been added. Pieces should be washed in running tap-water overnight and then transferred for six hours into pure 96 per cent, alcohol. Impregnation, reduction, embedding, etc., as above. Results are good, but pieces become extremely hard even if dehydrated very quickly, and are consequently difficult to cut. See also Formula 5a. Formula 2a, C. — Fix for twenty-four hours in 50 c.c. of alcohol with 10 drops of nicotine. Mop up with blotting paper, without washing, and silver as usual for five days (or four at 40° C.). Good results with adult tissues, especially spinal cord. Good penetration and less shrinkage than with pure alcohol. Formula 2a, D. — Fix for twenty-four hours in allyl alcohol (the industrial product will do). Wash for some hours in several changes of water. Put for a day into 50 c.c. of alcohol with 4 drops of ammonia. Silver for four days at 35° to 38° C., and reduce as usual. Good for human tissues, especially for fibre plexuses of cerebrum and cerebellum. Instead of allyl alcohol one may take acetal or acetone. Put for six hours into acetone with 25 per cent, of water, then for twenty-four into pure acetone, wash in water, etc., as above. Formula 3a. — Fixation in ammoniacal alcohol for twenty to forty- eight hours. The most generally useful formula is 50 c.c. of 96 per cent, alcohol with 4 to 5 drops of ammonia (of 22° strength). But for cerebrum not more than 1 to 3 drops ; for cerebellum, ganglia, spinal cord and regenerating tracts, 4 drops ; for neurofibrils of the large nerve cells of the medulla oblongata and spinal cord, 9 to 10 drops. To avoid shrinkage, it is well to begin by putting the pieces for six hours into 70 per cent, alcohol, then in 85 per cent., without ammonia ; then for the rest of the time into the ammoniacal alcohol. Do not wash, but mop up with blotting paper before putting into the silver. Silver for four to four and a half days (small specimens) at 40° C., or medium to large (3 to 4 mm. thick) for five days at 32° to 35° C. So long as the tissues are only yellowish- white, they are not ripe for reduction ; light grey indicates ripeness ; dark grey over-ripeness. Reduce as by Formula la. Specimens may be decalcified, after reducing and washing, in 96 per cent, alcohol to which a few drops of nitric acid have been added. For the delicate impregnation of the neurofibrils of the large and CHAPTER XXXII. 423 medium nerve cells this formula is superior to all others. It gives good results with the majority of nerve centres, and is particularly good for non-medullated fibres, peri-cellular baskets of cerebellum, buds of Held and Auerbach in the oblongata, for human sympathetic, and for the study of regenerating nerve fibres. Formula 3a, A. — Fix in 50 c.c. of alcohol with 10 grms. of glycerin and 6 to 10 drops of ammonia. Good for retina and non-medullated fibres, but especially for the buds of Held and Auerbach. Formula 3a, B. — Fix in 50 c.c. of alcohol with 1 -5 c.c. of a 33 per cent, alcoholic solution of ethylamine. Results the same as with ammoniacal alcohol. Formula 4a. — Pieces of tissue of not more than 4 mm. in thickness are fixed for six to twelve hours in 15 per cent, formol. Wash for six or more hours in running tap-water. Put for twenty-four hours into 50 c.c. of alcohol with 5 drops of ammonia. Wipe with blotting paper, silver for five days (or four if the stove is at 38° to 40° C.). The rest as usual. Sharp impregnation of the finer fibres of nerve centres and of the terminal buds of pericellular nests. Adult tissues give better results than young ones. Energetic stain of the arborisa- tions of the moss fibres of the cerebellum. Formula 4a, A. — Fix in " a mixture of formol and alcohol." Wash out thoroughly with running tap-water, silver, and reduce as usual. Fixation more rapid and better ; results similar to those of 3a. Formula 5a. — This is characterised by a preliminary fixation in pyridine as originally suggested by HELD (Arch. Anat. PhysioL, Anat. Abth., 1905, p. 77; Anat. Anz., xxix., 1906, p. 186). He used to fix tissues in pure pyridine, but Cajal finds that this is likely to cause much shrinkage, and he recommends fixing small pieces first for six to eight hours in a mixture of equal parts of distilled water and pyridine, then for eighteen to twenty-four hours into pure pyridine. Wash for several hours in running water, and put for a day into 90 per cent, alcohol. Wipe, and put for four to five days into 1 -5 per cent, silver nitrate at 35° to 38° C., and reduce as usual. Not very good for adult organs, but superior to. all others for the earliest phases of neurogenesis, and good for regenerative processes, as well as for peripheral nerve endings. Formula 6a. — Put for twenty-four hours into 50 c.c. of water with 5 grms. of chloral hydrate, rinse, and put into 50 c.c. of 96 per cent, alcohol with 5 drops of ammonia (time not stated). Wipe with blotting paper ; put for four to five days at 35° to 38° C. into 424 NERVOUS SYSTEM— SPECIAL METHODS. 1-5 per cent, silver nitrate, and reduce as usual. Kesults very constant, without shrinkage. Good for the fine plexuses of cere- brum, bulb and cord, the baskets of Purkinje's cells, and moss fibres ; also for motor plates and for regenerating nerves. Formula 6a, A. — Fix for twenty-four hours in 10 per cent, chloral hydrate, wash for six, and put direct into the silver. Stove for four days. Kesults similar to those of Formula la. Medullated fibres well stained. Formula 7a. — Fix for twenty-four hours in Merck's fibrolysine, wash for six, put for twenty-four into 50 c.c. of alcohol with 5 drops of ammonia. The rest as by other formulae. Instead of fibrolysin, lysidme may be taken. 838. Application of CAJAL'S Methods to different Objects. — (1) For the study of the evolution of neuroblasts and nerve fibres in very early embryos it is necessary to avoid fixing with formol, or alcohol with an accelerator, or ammoniacal liquids. The best formulae are 2a and 5a which are applicable to all vertebrates, but preferably to embryos of birds and fishes. (2) For late embryos and fatus of mammals. Besides the above formulae, 3a, 6a and alcohol with an accelerator. Best subjects, embryos of chick from the fifth day, and of rabbit from the tenth to the twelfth day ; or new-born birds, with ammoniacal alcohol, or 5a. (3) For sympathetic ganglia. Formula 3a, or pure alcohol, or 4a and 5a. Best with man. Dog, cat, and rabbit give mostly weak reactions. The visceral ganglia are the most difficult. (4) Sensory ganglia. Formula 2a or 3a. Easy. (5) Cerebellum. For Purkinje cells, la or 3a. For the baskets, climbing fibres, and medium and small dendrites, 2a or its variants. For terminal rosettes and collaterals of moss fibres and for the plexuses of the granular layer, 4a or sometimes 5a or 6a. For the stellate cells of the molecular layer, 2a and 3a. The best subject for the latter is the dog. (6) Cerebrum. In general, the same formulae as for the cere- bellum, especially la for pyramids of young dogs and cats (of eight to twenty days). In Formula 3a the proportion of ammonia should be diminished. For fine plexuses, 4a, 5a, and 6a. (7) Spinal cord and bulb. All the formulae are applicable. For neurofibrils of motor cells the best subject is the dog of four to fifteen days, with Formula 3a, with a large dose of ammonia (10 drops) ; also the alcoholic fixatives with an accelerator. For medullated CHAPTER XXXIL 425 fibres, large and small, 2a or 6a. For buds of Held and Auerbach and for fine plexuses, 4a, 3a, A, or 5a. (8) Ganglia of invertebrates. For the medicinal leech (not for other leeches), la, A. For Hcemopis, Aulostomum, Pontobdella and Glossiphonia, 2a or, better, 3a, with not more than 2 to 5 drops of ammonia and 3 per cent, silver nitrate, stoving three or three and a half days. For further details see SANCHEZ, Trab. Lab. Invest. .BioL, Madrid, vii, 1909, pp. 42—47. Lumbricus is generally refractory to Cajal's methods. BOULE (Le Nevraxe, x, 1908, p. 15) obtained good impregnations by acidi- fying the fixatives. He takes : (a) 25 per cent, formol with 5 per cent, of acetic acid ; or (b) the same with 0 -5 per cent, of ammonia ; or (c) 100 c.c. of alcohol, 25 c.c. of formol, 5 c.c. of acetic acid, and 0-5 c.c. of ammonia. For the impregnation he uses. 3 per cent, silver nitrate with 15 per cent, of alcohol, and reduces in the usual hydroquinone-formol solution, with the addition of 15 c.c. of alcohol. These results are confirmed by KOWALSKI (La Cellule, xxv, 1909, p. 292, and by SCHUTZ (Anat. Anz., xlii, 1912, p. 262). Kowalski gets impregnations also by simply starving worms for several days, or exposing them to cold (- - 5° C.) for a quarter of an hour. (9) Regenerating nerve tissue. For nerves operated a month or more previously, Formula 2a or 3a, with not more than 3 drops of ammonia, will stain equally the old and the new fibres ; for nerves operated not more than two to ten days previously, Formulae 3a with 4 to 6 drops of ammonia, 5a with pyridine, and 4a, also some- times 6a ; for regeneration in cord, cerebrum, and cerebellum, 3a with 3 drops of ammonia, or 5a, or pure alcohol. 839. Modifications of EAMON Y CAJAL'S Methods. — DA FANO (Ziegler's Beitr., xliv, 1908, p. 495) recommends using solutions of silver nitrate and hydro quinone in 1 : 10,000 gelatin in order to obtain a deeper and sharper impregnation. KAT6 (Folia neurobiol, ii, 1908) fixes in 10 to 15 per cent, formol, and silvers for one to five days at 35° C. in 5 per cent, argentamin to which 3 per cent, of silver nitrate has been added in such a way as to have an impregnating fluid with a little argentamin in excess ; or argentamin eight to ten parts, with 3 per cent, potassium bichromate thirty parts and distilled water 100 parts. For the reduction he uses 10 per cent, formol with 1 per cent, hydroquinone. PUSATERI (see AMATO, Virchow's Arch., clxxxv, 1908, p. 547) fixes for three to six days at 35° to 38° C. in a mixture of 45 c.c. of tachiol (10 per cent, silver fluoride) and 155 of distilled water. BESTA (Eiv. pat. nerv. ment. Firenze, xv, 1910, p. 333) fixes for forty- eight hours in alcohol with 5 per cent, nitric acid, neutralised in alcohol with ammonia. 426 NERVOUS SYSTEM— SPECIAL METHODS. LIESEGANG (Kolloidchemie, Beihefte, iii, 1911, H. 7 ; Ztschr. wiss. Mikr., xxviii, 1912, p. 369) makes sections of formol material by the freezing process, and silvers them until yellow. He then adds to the silver bath an equal volume of 50 per cent, solution of gum arabic and the same amount of saturated solution of hydroquinone. After one or two minutes the sections are brought into 10 per cent, solution of sodium hyposulphite, washed and mounted. Results said to be the same as by the usual process. ASCOLI (Boll. Soc. med. chir., Pavia, 1911, p. 177) recommends for the sympathetic nervous system of Hirudinea the following : The animals cut open at the back are stretched on a piece of cork and fixed in a solution prepared by dissolving over a flame 5 gr. of pulverised crystals of silver nitrate in 100 c.c. of 95 per cent, alcohol. After a few minutes the animals may be detached from the cork and put back in the same fixative for twenty-four to forty-eight hours in an incubating stove. They are then transferred for another twenty-four to forty-eight hours into a '10 per cent, watery solution of silver nitrate, to be kept also in the incubator. After a quick wash they are reduced for five to eight hours in Amidol- Hauff 0-5 gr., sodium sulphite cryst. 10 grms., distilled water 100 c.c., and lastly passed into glycerin. Preparations are made by teasing, the thinner ones being toned and counterstained as usual. For mounting he prefers Apathy's syrup. HANSON (Anat. Anz., xlvi, 1914, p. 522) has the following for the demonstration of non-medullated nerve fibres in cranial and peripheral nerves : Fix in absolute alcohol containing 1 per cent, of strong ammonia for forty.-eight hours ; rinse in distilled water, put in pyridine for twenty -four hours, wash in many changes of distilled water for twenty -four hours, place in 2 per cent, silver nitrate at 35° C. in the dark for three days, rinse in water, and place for one day in a 4 per cent, solution of pyrogallic acid in 5 per cent, formalin. As suggested by HABER and GUILD (Anat. Rec., vii, 1913, p. 253) the results can be improved by a preliminary injection of 95 per cent, alcohol, containing 1 per cent, of ammonia, through the arteries till tissues are thoroughly saturated, after which they are dissected out and placed in a similar arnmoniated alcohol solution for from two to three days. Huber and Guild have found this method of use for the study of cranial nerves of small animals and embryos, since the entire heads can, after fixation, be decalcified by means of 7 per cent, nitric acid, brought through 80, 90, and 95 per cent, alcohols, each containing 1 percent, of ammonia, and finally treated as above. 840. BIELSCHOWSKY'S Methods. Introductory. — It is well known that, if ammonia be poured into a solution of silver nitrate, a pre- cipitate is formed which is redissolved by the addition of some more ammonia. If an alkaline solution of formaldehyde be slowly added to this easily reducible di-ammoniacal silver nitrate (N(NH4)AgH2N03), metallic silver is immediately precipitated and deposited on the walls of the test tube. Both FAJERSTAJN (N enrol. CentrbL, xx, 1901, p. 98) and BIELSCHOWSKY (ibid., xxi, 1902, p. 579) CHAPTER XXXII. 427 thought of taking advantage of this reaction for histological purposes with the object of finding out a silver impregnation of the nervous tissue similar to that which characterises Golgi's method. The results of their attempts were different : Fajerstajn was able to obtain only a difficult method for staining axis-cylinders which is now superseded ; Bielschowsky also published, at first, a complicated silver method for impregnating axis-cylinders very similar to that of Fajerstajn, but, through successive modifications of his first process, was led to the discovery of a new method, which is as important as Cajal's reduced silver methods from an histological point of view, but is of still greater advantage than the latter for histopathological investigations. Moreover, Bielschowsky's method is applicable to any formol material, even if very old. BAYON (Die Untersuchungsmeth, etc.) succeeded with four-year-old material, and I with brains which had been left in formalin for more than eleven years. There are at present three Bielschowsky methods : one for sections, one for peripheral nerve-fibres and axis-cylinders, and one for pieces. It seems better to describe them separately in the following account which is based on the original papers of Bielschowsky, as well as on some personal experience I gained through a visit paid to him when in Berlin. BIELSCHOWSKY'S Method for Sections (Journ. Psychol. NeuroL, iii, 1904, p. 169 ; and xii, 1909, p. 135). — Pieces from central nervous organs, fixed in 15 to 20 per cent, formalin, are washed for some hours in running tap-water and then cut by means of a C02 freezing microtome. The sections are collected in distilled water, thoroughly washed therein and passed in a 2 or 3 per cent, solution of silver nitrate where they are left for twenty-four hours in a dark place, and at room temperature. The sections can also be passed first into pure pyridine for twenty -four to forty -eight hours, washed in many changes of distilled water until the pyridine has been completely eliminated and then transferred into 2 to 3 per cent, silver nitrate as above. The pyridine bath is optional and has the advantage of ensuring a sharper stain of axis-cylinders whilst neuroglia, which is more or less coloured when the pyridine bath is dispensed with, remains unstained. Also connective tissue and nuclei are generally very faintly stained after the pyridine treatment. Intracellular neuro- fibrils, however, are not always so well shown as by the direct passage of sections into the silver nitrate solution. Before proceeding further, one should prepare the Bielschowsky 428 NERVOUS SYSTEM— SPECIAL METHODS. ammoniacal silver nitrate-and-oxide bath as follows : — Pour 5 c.c. of a 20 per cent, solution of silver nitrate into a measuring cylinder and add to it first 5 drops of a 40 per cent, solution of NaOH, and then ammonia, drop by drop, until the brown precipitate formed disappears ; dilute to 25 c.c. with distilled water, and filter through paper washed with the same water. For staining take sections one by one from the silver nitrate bath, quickly wash them in distilled water and transfer them into the ammoniacal silver bath. Here they remain for about ten minutes when they become yellowish-brown and should be, once more, quickly washed in distilled water and placed in 20 per cent, formalin prepared with spring water. The reduction takes place immediately, and if one works with a number of sections it is advisable to re- transfer them into a fresh bath of 20 per cent, formalin. At the end of half an hour and even less, the reduction can be considered as accomplished and sections can be washed in distilled water and toned with a diluted (0-2 per cent.) solution of gold chloride. This may be slightly acidified with acetic acid if one wishes to obtain a faintly purple background, or neutralised with a few drops of a diluted solution of sodium or lithium carbonate if one prefers greyish- white backgrounds. Instead of gold chloride one can use a slightly acid solution of chloroplatinic acid. After toning there remains only the washing once more of sections in distilled water, and the passing of them for a few minutes in a 5 per cent, solution of sodium hyposulphite, or any diluted fixing bath for photographic plates. Wash again, dehydrate in alcohols of increasing strength up to 95 per cent., clear in carbol.-xylol, and mount in balsam. For other details about the toning and fixing of sections see the original papers of BIELSCHOWSKY (op. cit. and Journ. Psychol. N enrol., iv, 1904 — 5, p. 227), as well as WOLFF (Eiol. CentrU., xxv, 1905, p. 683), and DA FANO (Proc. Physiol. Soc. Journ. Physiol., liii, 1920). Bielschowsky states that this method is also suitable for sections of celloidin or paraffin blocks of formol material, but he does not recommend the practice, and I have no experience of it. BIELSCHOWSKY'S Method for Peripheral Nerve-fibres (Journ. Psychol. Neurol, iv, 1904—5, p. 227). — This method can be applied to the study of spinal and sympathetic ganglia, peripheral nerve- endings, and end-organs in normal conditions, but its chief applica- tions belong to the domain of histopathology. According to my experience good results are rarely obtained, and the method requires important modifications to become as useful as the above and following ones. CHAPTER XXXII. 429 The staining is carried out on sections of formol material in the same way as described above. There is only this difference that the staining in the ammoniacal silver bath is carried on a few minutes longer, viz., until the sections have taken a decidedly brown colour, after which they are washed in 10 c.c. of distilled water acidified with 5 drops of acetic acid, when they acquire (sometimes in a few seconds) a yellowish tinge. They should then be immediately transferred into the usual 20 per cent, solution of formalin. For the toning a neutral gold bath is necessary : sections should be left therein until red-violet. In the finished preparations axis-cylinders are black, myelin red- violet, connective tissue violet or blue-violet. The washing in acidified water and the prolonged toning both answer for the purpose of creating a sharp contrast between nerve fibres and connective tissue fibres, which might otherwise become stained almost as black as the axis-cylinders. Bielschowsky has also a method for central nerve-fibres. Sections made by freezing from formol material are placed for twenty-four hours or longer in a 4 per cent, solution of copper sulphate or Weigert's mordant for neuroglia stain (§ 910). After washing they are placed for a few seconds in the usual ammoniacal silver bath and then washed, reduced, toned and fixed as above. The preparations are similar to those obtainable by the methods of Fajerstajn, Strahiiber and Kaplan. BIELSCHOWSKY'S Method for Pieces (op. tit.). — Good for peripheral nerve-endings and embryonic material, and also for small specimens of adult subjects. This method has been described by Bielschowsky in various ways, probably because of the difficulty of giving fixed rules in a case in which the greatest freedom had to be left to histo- logists to adapt the method to the quality of their material and the purpose of their investigations. In what follows two forms of the method are described : one without and one with pyridine treatment of pieces. A. Method for Pieces without Pyridine Treatment. --Thin slices or small pieces of formol material are washed for some hours, first in running tap-water and afterwards in distilled water. They are then placed in a 2 per cent, solution of silver nitrate for from one to eight days in the dark. The use of an incubator at 35° to 37° C. is optional. After a wash in several changes of distilled water (to be prolonged for some minutes up to some hours according to the length of time during which pieces have been kept in the silver bath, and if in an incubator or not) they are transferred into an ammoniacal solution of silver nitrate prepared as in the method for sections, but diluted 430 NERVOUS SYSTEM— SPECIAL METHODS. up to 100 c.c. They are kept therein for from an hour up to six, washed once more in distilled water, passed for twelve to twenty-four hours into the usual 20 per cent, solution of formalin. Wash, dehydrate quickly, embed, preferably in paraffin, tone sections as described above, counterstain, if necessary, mount in balsam. B. Method for Pieces with Pyridine Treatment. — Pieces of formol material, up to 1 cm. thick for adult tissue, and up to 5 cm. long for embryos, is put for two, three or four days into pure pyridine, washed for some hours in several changes of distilled water and put for three to five days into 3 per cent, silver nitrate at 36° C. Wash in distilled water and transfer into the diluted ammoniacal silver bath as above, but leaving pieces therein for twenty-four hours. Wash for about two hours in several changes of distilled water, reduce in 20 per cent, formalin. The rest as above. 841. Modifications of BIELSCHOWSKY'S Methods. — FAVORSKY (Journ. Psycliol. NeuroL, vi, 1906, p. 260) uses 10 per cent, silver nitrate for the first silver bath instead of 2 or 3 per cent. PATON (Mitth. Zool Stat. Nea-pel, xviii, 1907, p. 576) fixes fish embryos in 4 per cent, formaldehyde neutralised with carbonate of magnesia. For the first silver bath he uses 0-75 to 1 per cent, silver nitrate and keeps material therein four days in summer, five to seven in cooler weather. To make the ammoniacal silver nitrate-and- oxide bath he takes 20 c.c. of 0 -75 to 1 per cent, silver nitrate, adds to it 4 drops of 40 per cent, caustic soda and then ammonia drop by drop in the usual way. The embryos are first washed in distilled water, then kept for five to fifteen minutes in 10 c.c. of water acidified with 5 drops of acetic acid, washed once more in pure water, and trans- ferred for twelve hours into a reducing fluid consisting of 1 per cent, hydroquinone 20 c.c., neutralised formalin, 2 c.c. After embedding in paraffin, the sections are toned as usual and counterstained with 1 per cent, eosin in absolute alcohol. SCHUTZ (NeuroL CentrbL, xxvii, 1908, p. 909) finds that the times given by Bielschowsky are too short and washes sections for twenty- four hours after the 2 per cent, silver nitrate bath, leaves them thirty to forty minutes in the ammoniacal silver bath, and twenty-four hours in the 20 per cent, formalin. For toning he puts them for ten minutes into 10 c.c. of water with 2 drops of acetic acid, then for thirty to forty-five minutes into 10 c.c. of water with 3 drops of a 1 per cent, gold chloride solution (until blackish-grey). BOEKE (Anat. Anz., xxxv, 1910, p. 193) has .obtained excellent results by the use of Bielschowsky's method for pieces when applied CHAPTER XXXII. 431 to the study of peripheral nerve endings. He fixes in 10 per cent, formalin prepared with 60 per cent, alcohol, changes the fluid two or three times, and then either leaves material therein until wanted or keeps it in 70 to 80 per cent, alcohol. For staining, pieces are brought into 10 to 12 per cent, formalin, and left in it until they are quite free from alcohol. The rest as by Bielschowsky's method for pieces, with or without pyridin treatment. Boeke finds that the method succeeds also after other kinds of fixation. SCHLEMMER (Ztschr. wiss Mikr., xxvii, 1910, p. 22) makes the ammoniacal silver nitrate-and-oxide bath by adding to any silver nitrate solution, 40 per cent, caustic soda, drop by drop, until no more precipitate is formed. He then washes the precipitate by repeated decantation until the wash water no longer gives an alkaline reaction, takes it up with the smallest possible quantity of ammonia, and filters through glass wool. This concentrated solution keeps for many days unaltered, and should be diluted ten times its volume before using it. DEL Rio-HoRTEGA (Trab. Lab. Invest. Biol., Madrid, xiv, 1916, p. 181) has made known a similar method used in those laboratories for preparing the ammoniacal silver nitrate bath. Forty drops of 40 per cent, caustic soda are added to 30 c.c. of 10 per cent, silver nitrate, and the precipitate washed ten to twelve times by means of about a litre of distilled water. Fifty cubic centimetres of water are then added to it, and ammonia, drop by drop, until the precipitate is dissolved. The solution, brought finally to 150 c.c. and filtered into a dark brown bottle, keeps well for many months. I find that the ammoniacal silver bath thus prepared can be further diluted with one, two, up to five times its volume of water, and usefully employed for Bielschowsky's method for pieces, particularly for the study of peripheral nerve endings. AGDUHR (Ztschr. wiss. Mikr., xxxiv, 1917, pp. 1-99), who has exhaustively investigated almost all questions relating to the results obtainable by Bielschowsky's method for pieces, has come to the conclusion that material is best fixed in neutral or slightly acid 20 per cent, formaldehyde (50 per cent, formalin). Pieces should then be washed in distilled water for many days until the wash-water is free from substances reducible by an ammoniacal silver nitrate solution used as test. For the first silver bath he uses 3 per cent, silver nitrate, and for the second a solution obtained by adding to 10 c.c. of 10 per cent, silver nitrate, first 20 drops of 25 per cent. NaOH, then from 200 up to 600 c.c. of distilled water, and lastly ammonia enough to dissolve the precipitate. For the reduction he 432 NERVOUS SYSTEM—SPECIAL METHODS. uses again 20 per cent, formaldehyde. To avoid an excessive im- pregnation of the connective tissue he also finds it useful to wash pieces in acidified distilled water (see the Bielschowsky method for peripheral nerve-fibres), but he uses as much as five times the amount suggested by Bielschowsky. 842. DA FANG'S Modifications. — An important point of this series of modifications of Bielschowsky 's method for sections is the use of distilled water, re-distilled on potassium permanganate, with the object of ensuring elimination of any trace of organic matter from the ordinary distilled water and of avoiding the forma- tion of precipitates. Da Fano's first modification (Mod. 1) (Atti. Soc. Lomb. Sc. Med. BioL, Milano, iii, 1914) was meant for the study of recticular tissue of spleen, lymph glands, and other organs, and is to be carried out as follows : — (1) Fix small pieces of fresh tissue in 10 to 20 per cent, formalin or in Kayserling's first fluid (forty-eight hours at least), or in Orth's fluid (twenty-four to forty-eight hours). (2) Wash pieces in running tap-water for twenty-four to thirty hours, and then in distilled water for another twenty-four hours. (3) Wash sections made by the freezing method in re-distilled water (twenty-four hours), and then place them in filtered 2 per cent, silver nitrate (prepared with redistilled water) in a Petri dish, taking care that they do not touch each other. Here they are kept in the dark and at room temperature from six hours to three days. (4) Treat sections for twenty to thirty minutes with Bielschowsky's ammoniacal silver nitrate solution prepared with only 2 drops of 40 per cent, caustic soda and diluted with redistilled \vater to 40 to 70 c.c. (5) Reduce, tone, counterstain, and mount as by Biel- schowsky's method for sections. Mod. 2 (Proc. Physiol. Soc. Journ. Physiol., Hi, 1919) consists in an application to nervous tissues of Mod. 1. The use of redistilled water and the mode of preparing the ammoniacal silver bath are the same, but Da Fano lays stress on the following points : — (1) Nervous tissue must be fixed in 10 up to 20 per cent, formalin for at least three weeks, better still for two months. Attempts to obtain a rapid fixation with 10 to 20 per cent, formalin at 37° C. gave bad results. (2) Sections of nervous tissues may be placed, after washing in redistilled water, in anhydrous pyridine (six to twelve hours), then repeatedly washed and left overnight in redis- tilled water, to get rid of all pyridine. This treatment appears to render neurofibrils a little thinner and, consequently, a little CHAPTER XXXII. 433 sharper, but increases the length and cost of the method, and may cause precipitates to form, especially where much myelin is present. (3) It is possible to keep sections, which cannot be stained imme- diately, for some days or even a fortnight, in redistilled water to which a few drops of formalin have been added. Thorough washing with redistilled water is then imperative before they are transferred into the 2 per cent, silver nitrate solution. (4) Sections of nervous tissues must not remain in the 2 per cent, silver nitrate more than forty-eight hours, or precipitates may form. The longer their stay there, the longer must be the washing before staining ; this, however, must not, as a rule, exceed five minutes. (5) The volume to which the ammoniacal silver nitrate is diluted should be 35 to 45 c.c., and the sections remain in it fifteen to twenty minutes. The subsequent washing before transferring the sections into 20 per cent, formalin should not occupy more than ten to fifteen seconds, and their stay in the final formalin solution (especially for cerebral cortex) should not exceed two to three hours. The other eight Da Fano modifications (Proc. Physiol. Soc., Jo-urn. Physiol., liii, 1919-20) were all proposed for the study of cortex cerebelli, and are characterised by a special treatment of the sections (cut by the freezing method) with various reagents before transferring them into the 2 per cent, silver nitrate solution, nothing having been changed, however, in regard to the long fixation of material in formalin and the use of redistilled water. They may be summarised as follows :- Mod. 3. Place sections, after washing in redistilled water, in 2 to 3 per cent, silver nitrate at 36° to 37° C. for about twenty-four hours ; wash quickly ; stain in ammoniacal silver nitrate solution diluted to 40 c.c. for thirty minutes. Wash, reduce, tone, and mount as usual. Mod. 4. Place sections in 50 per cent, pyridine for six to eighteen hours ; wash in redistilled water for twenty-four to forty-eight hours ; 2 per cent, silver nitrate at 37° C. for twenty-four hours, etc., as in Mod. 3. Mod. 5. Place sections in pure pyridine for four to twelve hours. Wash in redistilled water overnight. Transfer sections into 20 per cent, formalin prepared with redistilled water for about twenty-four hours. Wash again in redistilled water overnight ; 2 per cent, silver nitrate at 37° C., etc., as before. Mod. 6. Sections are treated first with 20 per cent, formalin, and then with pure pyridine, in the reverse order of Mod. 5. Mods. 1 and 8. The same as Mods. 5 and 6, but replacing the M. 28 434 NERVOUS SYSTEM-SPECIAL METHODS. pyridine with a mixture of 3 parts of methyl-alcohol and 2 parts of water. Mod. 9. Place sections in a mixture of equal parts of 20 per cent, formalin and methyl alcohol for twenty-four hours ; wash in redistilled water for six to twenty-four hours ; 2 per cent, silver nitrate at 37° C. for twenty-four hours, etc., as before. Mod. 10. Place sections into 20 per cent, formalin for twenty- four hours, transfer them, without washing, into a mixture of equal spart of 20 per cent, formalin and methyl alcohol, etc., as in Mod. 9. Mod. 3 is particularly suitable for human material of young individuals : Mod. 4 for adult subjects. Mods. 5 and 6 are useful for the study of neurofibrils in the various elements of the cortex cerebelli and for the staining of the granules. Mods. 7, 8 and 9 are to be preferred for the demonstration of pericellular baskets and nervous processes. Mod. 10 gives very complete stainings, and is the most certain of all ; preparations are, however, fairly dark, and, therefore, more suitable for general view. PERDRAU (Journ. Pathol. Bact., xxiv, 1921) has worked out a modification which appears to be particularly suitable for the study of the connective tissue in nervous organs. He washes pieces and sections as in Da Fano's modifications, but without having recourse to redistilled water. He then places sections for about ten minutes in 0 -25 per cent, potassium permanganate, washes, and treats them as by Pal's modification of Weigert's myelin stain (see § 857). After another wash in distilled water, he transfers sections into 2 per cent, silver nitrate, and continues as in Da Fano's Mod. 1. Nerve cells, nerve fibres, neuroglia, etc., unstained ; connective tissue and elastic fibres stained in various shades of purple-grey to black. 84-3. Neurofibrils ; Other Methods.— Cox's Method for fibrils of spinal ganglion cells ; see Ztschr. wiss. Mikr. xiii, 1896, p. 498, and Anat. Hefte, x, 1898, p. 98. S. MEYER'S Berlin blue, see Anat. Anz., xx, 1902, p. 535. LUGARO'S collargol (colloidal silver) method, see Monit. Zool. Ital., xv, 1904, p. 353. JORIS' colloidal gold method has not been received with favour ; see Bull. E. Acad. Med. Belg., xviii (S. iv), 1904, p. 293. SAND (C.R. Ass. Anat. Bruxelles, 1910 ; Bibliogr. Anat. Supp., 1910, p. 128, or Ztschr. wiss. Mikr., xxviii, 1911, p. 500) gives the following as entirely certain for man, dog, cat, and rabbit. Speci- mens of not more than 5 mm. in thickness are fixed for forty-eight hours in a freshly prepared mixture of 90 parts of acetone and 10 of nitric acid, to be changed for fresh after half an hour, and once CHAPTER XXXII. 435 again within twenty-four hours. Wash out for at least six hours in pure acetone, changed two or three times. Make paraffin sections and bring them through xylol and acetone into distilled water ; silver for three days at about 37° C. in 20 per cent, solution of silver nitrate. Put for ten minutes into a mixture (at least three days old) of 1000 parts of water, 10 of sodium acetate, 5 of gallic acid, and 3 of tannin (to be changed if it becomes turbid). Mount at once or tone until grey (five minutes) in 80 parts of water with 17 of 2 per cent, ammonium sulphocyanide and 3 of 2 per cent, gold chloride ; fix for a few seconds in 5 per cent, sodium hyposulphite. Neurofibrils grey- violet, shown in cells, dendrites, and axons. Terminal buds of Held also clearly shown, and nothing else stained. One may counterstain in any way, even by Weigert's or Benda's methods for neuroglia stain. The methylene blue intra vitam method is important, and may be usefully employed for the study of neurofibrils. See the processes of Apathy, Dogiel, and Bethe in Chapter XVI. C. Methods for the Demonstration of Golgi's Internal Apparatus. 844. Introduction. — The discovery of the " apparato reticolare interno " was made by Golgi in 1898 by means of his rapid process (see Chapter XXXIV). Soon afterwards he had recourse to a mixture due to Veratti (see next paragraph), and Negri, Pensa, and others of Golgi's pupils found that the internal apparatus is not a peculiarity of nerve cells. In 1902 Kopsch showed that the apparatus can be stained by a simple immersion of nervous tissues (spinal ganglia) into 2 per cent, osmic acid for eight to ten days. Since then the apparatus was shown to exist in almost every kind of cells, and new processes proposed for its demonstration in nervous and other tissues by Sjovall, Golgi, Ramon y Cajal, Gatenby, Da Fano. As the methods of Kopsch and Sjovall, the Mann-Kopsch method, and Gatenby's Mann-Kopsch-Altmann combination have already been fully discussed in Chapter XXVI, and particularly described in §§ 692 — 694 and 696, there remain to be described here only those methods which are particularly suitable for the study of the internal apparatus in nervous tissues, with exception of Golgi's rapid process, for which see Chapter XXXIV, § 882. 845. GOLGI- VERATTI' s Method (see GOLGI, Anat. Anz. Verh. Anat, Ges., xiv, 1900, p. 174). — Small pieces are hardened for a time 28—2 436 NERVOUS SYSTEM— SPECIAL METHODS. varying from a few hours to ten days or longer in Veratti's mixture, consisting of- 5 per cent, potassium bichromate . . 30 parts. 0-1 per cent, chloroplatinic acid . 30 ,, 1 per cent, osmic acid . . . 15 to 30 „ From time to time pieces are put in one or other of Golgi's rejuvenating fluids (as described in § 888), and thence into 0 -8 to 1 per cent, silver nitrate. Sections are cut and mounted as by Clolgi's bichromate and nitrate of silver method (see § 882). 846. GOLGI'S Arsenious Acid and Silver Nitrate Method (Arch. Ital. BioL, xlix, 1908, p. 272). — Small pieces of quite fresh tissues are fixed for three, six, eight or twelve hours in equal parts of 20 per cent, formalin, saturated solution of arsenious acid, and 96 per cent, alcohol. After a quick wash with distilled water, they are passed for some hours (or days) into 1 per cent, silver nitrate, and then treated with a reducing fluid, usually Cajal's hydroquinone mixture (hydroquinone 20 grms., sodium sulphite 5 grms., formalin 50 c.c., water 1000 c.c.). Wash quickly, dehydrate, and embed either in celloidin or paraffin. The sections are toned with equal parts of 1 per cent, gold chloride and a mixture consisting of water 1000 c.c., with 30 grms. each of sodium hyposulphite and ammonium sulphocyanide, and then rapidly bleached by the following method, due to VERATTI : — Wash the toned sections in distilled water and transfer them for one, two or three minutes into potassium per- manganate 0-5 grm., distilled water 1000 c.c., sulphuric acid 1 c.c. ; wash again ; transfer into 1 per cent, oxalic acid until the yellowish colour imparted to the sections by the potassium permanganate has disappeared ; wash thoroughly in repeatedly changed distilled water ; counterstain, dehydrate, and mount as usual. 847. RAMON Y CAJAL'S Uranium Nitrate and Silver Nitrate Method (Trab. Lab. Invest. BioL, Madrid, xii, 1914, p. 127).— (1) Small pieces of quite fresh tissues are fixed for ten to fourteen hours in a mixture of neutralised formalin 15 c.c., distilled water 85 c.c., uranium nitrate 1 grm. Instead of uranium nitrate, uranium acetate, as suggested by Del Rio-Hortega, may be sometimes used. Should a very fine reaction be desirable, the following formula may be employed : — Uranium nitrate 1 grm., ethyl or methyl alcohol 30 c.c., distilled water 80 c.c., neutralised formalin 15 to 20 c.c. (2) After a quick wash in distilled water, pieces are transferred into 1 -5 per cent, silver nitrate and kept therein for thirty-six to forty- eight hours at room temperature. If the pieces are only a few and CHAPTER XXXII. 437 small, 1 per cent, silver nitrate will be sufficient. (3) Wash quickly and reduce for eight to twenty-four hours in hydroquinone 1 to 2 grms., formalin 15 c.c., distilled water 100 c.c., sodium sulphite 0-5 grm. (4) Wash quickly, embed in paraffin or celloidin, or make sections by the freezing method. (5) Tone and counterstain sections if desirable. Dehydrate and mount as usual. Best results are obtained from vertebrates, preferably kittens and young rabbits. The method may be applied to human material, if available in a sufficiently fresh condition. From invertebrates results are not so good, and rather uncertain, so that Cajal advises a simple fixation in formalin or formalin-acetone, followed by impregnation with silver nitrate, as by his reduced silver methods for neurofibrils. 848. DA FANG'S Cobalt Nitrate Modification (Proc. Physiol Soc., Journ. Physiol., liii, 1920 ; Journ. R. Micr. Soc., 1920, p. 157).- Small pieces of quite fresh tissues are fixed for six to eight hours at room temperature in cobalt nitrate 1 grm., distilled water 100 c.c., formalin 15 c.c. The solution can be prepared beforehand, and keeps unaltered for months. The formalin need not be neutralised unless strongly acid or containing free sulphuric acid, in which case it is necessary to neutralise it by one of the usual methods. For the fixation of embryonic organs and in all cases in which a shrinkage of delicate tissues is to be feared, the quantity of the formalin may be reduced to 10, 8, or 6 c.c. for every 100 c.c. of distilled water. The time of fixation should be shortened to three to four hours or even less in the case of very small pieces, such as spinal ganglia of mice and rats, the pituitary body of the same animals, etc. Pieces of spinal cord, cerebrum, cerebellum of adult animals give better results if fixed for about eight to ten hours. The fixation may be prolonged in special cases to twelve to twenty hours, but should not exceed twenty-four hours. The fixation in an incubator at a temperature varying between 25° and 37° C. has been attempted with success in the case of tissues of adult subjects, but it leads to a staining of both the internal apparatus and intracellular formations, which, according to their morphology, are to be considered as mitochondria. For the impregnation, Da Fano quickly washes the pieces in distilled water, makes their surfaces smooth if necessary, and then places them into 1 -5 per cent, silver nitrate in the dark for twenty- four to forty-eight hours at room temperature. For very small fragments, 1 per cent, silver nitrate may be used, whilst for pieces of 438 NERVOUS SYSTEM—SPECIAL METHODS. spinal cord of adult subjects, 2 per cent, should be preferred. For the reduction he uses Cajal's hydroquinone-formalin mixture, taking care in further recutting the pieces, before transferring them into the reducing fluid, so that their thickness should not exceed 2 mm. He dehydrates and embeds pieces, preferably in paraffin, or he makes sections by the freezing method. He usually tones these by means of 0-2 per cent, gold chloride, fixes with 5 per cent, sodium hypo- sulphite, counterstains and mounts as usual. The method gives good results also with material from lower vertebrates and invertebrates.* 849. Other Methods and Modifications. — BESTA (Anat. Anz., xxxvi, 1910, p. 477) fixes for two days in 20 parts of formol with 2 of acetic aldehyde and 80 of water, washes for twenty-four hours in distilled water changed seven or eight times, and puts for two days in 4 per cent, solution of ammonium molybdate, makes paraffin sections, stains in a 1 : 1000 solution of thionin, differentiates in 3 parts of creosote to 1 of absolute alcohol, and passes through pure creosote and xylol into neutral balsam. Recommended for Purkinje cells and spinal ganglia of young animals. SUCHANOW (Neurol. Gentrbl., xxi, 1902, p. 777) has obtained good results by the use of Golgi-Veratti mixture, keeping pieces of spinal cord and spinal ganglia for twenty to thirty days in the mixture and for two to three days in the rejuvenating fluid. LEGENDKE (Anat. Anz., xxxvi, 1910, p. 209) omits the toning and bleaching by Golgi's arsenious acid method, and embeds in paraffin. Similarly COLLIN ET LUCIEN, Bibliogr. Anat. Supp., 1909, p. 238. SAVAGNONE (Pathologica, i, 1909) silvers pieces fixed in Golgi's arse- nious acid mixture with 30 c.c. of tachiol (10 per cent, silver fluoride) in 100 of water. CAKLETON (Journ. E. Micr. Soc., 1919, p. 321) reduces pieces treated according to Cajal's uranium nitrate method for only two hours in the usual hydroquinone mixture. PENFIELD (Brain, xliii, 1920) has successfully employed Cajal's uranium nitrate method for his experimental investigations on the alterations of Golgi's apparatus in nerve cells of spinal cord and spinal ganglia of young cats. He adds 20 c.c. (instead of 15) to Cajal's fixing fluid and as much as 1-5 grms. of sodium sulphite to the hydroquinone-formalin solution. He finds it imperative to dehydrate pieces very quickly before embedding them in paraffin. In order to obtain perfect fixation of the spinal cord he sometimes performs a laminectomy in the lower lumbar region of the ansesthe- * Da Fano's method has been used by me with great success for the study of gametogenesis of many invertebrata (J. B. G.). CHAPTER XXXII. 439 tised animal, passes a needle in the subarachnoid space, and allows the fixative to flow in "under a gravity pressure of 75 cm." The heart stops about a minute after the beginning of the injection, which is continued for twenty hours. At the end of this time the cord is removed, pieces cut and dropped directly into the silver bath. For counterstaining Penfield finds it particularly useful to immerse untoned sections into a diluted solution of Unna's polychrome- methylene blue for one to four hours, this being followed by passage through alcohols of increasing strength and differentiation in absolute alcohol. By this method, also Holmgren's, trophospongium is sometimes stained. But for the study of the relationship between the latter and Golgi's apparatus, Penfield (in litteris) prefers to make drawings of the apparatus from certain selected cells, subsequently removing the coverslip and bringing the slides through graded alcohols into 5 per cent, iron alum for twelve to twenty-four hours. This removes all silver from the cells as well as the count erstain, and at the same time mordants the tissues for further staining by Heiden- hain's iron-hsematoxylin method. If the proper amount of diffe- rentiation has been secured of the particular cells already drawn, the trophospongium will be found stained with great detail. Addendum. HOLMGREN'S Method for Trophospongium. — Fix small pieces or ganglia in trichlorolactic acid for twenty-four hours. Dehydrate and embed in paraffin as usual. Stain thin sections for twenty-four hours in Weigert's resorcin fuchsin solution for elastic fibres (see § 758) recently prepared and a little diluted. Dehydrate and mount as usual. • D. Methods for the Demonstration of the Sustaining Apparatus of Medullary Sheaths, Neurokeratin, etc. 850. Methods demonstrating Funnels and Spiral Filaments.- GOLGI (see KEZZONICO, Arch. p. 1. Sc. Med., iv, 1880, p. 78 ; GOLGI, Opera Omnia I, p. 163) puts small pieces of spinal cord in 2 per cent, potassium bichromate for eight to fifteen days in summer, or a month in winter. After a quick wash he transfers them into 0 -50 to 0 -75 per cent, silver nitrate for two or three days in summer, or eight, ten or more in winter. -The pieces are then washed in 95 per cent, alcohol, dehydrated in absolute alcohol, cleared in oil of turpentine and teased therein. The preparations, mounted in dammar, must be exposed to sunlight for eight to ten days ; or to diffused daylight for twenty to forty days. 440 NERVOUS SYSTEM— SPECIAL METHODS. For peripheral nerve fibres, GOLGI (Op. Omnia I, p. 162) has proposed two methods. Of these the first is a modification of his rapid process (see § 882), and should be carried out as follows :- Tracts of peripheral nerves are cut with care not to stretch them, and put in a mixture of 10 parts of 2 per cent, potassium bichromate and 2 of 1 per cent, osmic acid. After about one hour the tract or tracts of nerves are sufficiently hardened to be further recut in pieces of about | cm. in length, which are put back in the same mixture. After another three hours, and successively at intervals of three hours during twenty-four hours, pieces are transferred into 0 «5 per cent, silver nitrate where they may remain for any time, but no less than eight hours. Preparations are made and mounted as above. The other method is a modification of that used for central nerve fibres, the only difference consisting in keeping the pieces in the bichromate for a much shorter period, i.e., for from four hours to at most two days, and in transferring specimens into the silver bath at intervals of about three hours. After twelve to twenty-four hours preparations can be made as described above. The preparations made by the first method show the spiral filaments very clearly, but do not keep well. The preparations made by the second method do not show the spiral filaments so completely, but are more useful for the demonstration of the funnels and last longer. CATTANI (Arch. Ital. Biol., vii, 1886, p. 345) either fixes in Flem- ming's fluid and teases and mounts in glycerine, or puts pieces into Golgi's bichromate and osmic acid mixture, dehydrates and passes into oil of turpentine to be changed until it remains colourless. The turpentine dissolves the myelin and leaves funnels and spiral fila- ments visible. Cattani also has a modified Golgi method, now superseded. SALA (Verh. Anat. Ges. Anat. Anz., 1900, p. 176) employs the Golgi- Veratti method for the intracellular network (see § 845). See also concerning these methods, MONDINO, Arch. p. I. Sc. Med., viii, p. 45. GALLI (Ztschr. wiss. Mikr., iii, 1886, p. 467) hardens peripheral nerves for eighteen to twenty days into Miiller's fluid, cuts out pieces 5 to 6 mm. long, and keeps these in Miiller's fluid diluted with 2 parts of water for another two days. He then stains for fifteen to twenty minutes in aqueous solution of China blue, washes out in alcohol, clears in essence of turpentine, and mounts in damar. RAMON Y CAJAL has successfully employed some modifications of CHAPTER XXXII. 441 his reduced silver and uranium nitrate methods, for which see Trab. Lab. Invest. Biol., Madrid, x, 1912, p. 221. 851. Methods demonstrating Neurokeratin Network. — PLATNER (Ztschr. wiss. Mikr., vi, 1889, p. 186) fixes for several days in a mixture of 1 part of Liq. Ferri Perchlor. (Ph. G., ed. 2) and 3 to 4 parts of water or alcohol, washes out well in water and stains for several days or weeks in a concentrated solution of " Echtgriin' (dinitroresorcin) in 75 per cent, alcohol. See also BEER, Jahrb. Psychiatric, ii, 1893. Cox (Aiiot. Hefte, i, 1898, p. 102, note) fixes nerves in 2 per cent, osmic acid (rabbit) or 1 per cent, (frog), washes, dehydrates, clears with bergamot oil, and mounts in balsam. The bergamot oil dissolves out the myelin, and leaves the neurokeratin visible. It may be necessary to leave the nerves for forty-eight hours in the oil. CORNING (Anat. Am., xvii, 1900, p. 309) studies the neurokeratin network in the sciatic of the frog by means of sections of sublimate material strongly stained with iron ha3matoxylin. KAPLAN (Arch. Psychiatr., xxxv, 1902, p. 825) stains sections with acid fuchsin and differentiates them by Pal's method. GEDOELST (La Cellule, v, 1889, p. 136) has the following : (a) A nerve is treated with liquid of Perenyi, either pure or with addition of a trace of osmic acid, and examined in glycerin. By this treat- ment the myelin loses its excessive refractivity and the neurokeratin network comes out clearly. (6) Silver nitrate. Good images, but uncertain, (c) Treatment with a mixture of 1 per cent, osmic acid and absolute alcohol. The network comes out black. CHAPTEK XXXIII.* MYELIN STAINS. 852. Iron Haematoxylin. — According to A. Bolles Lee (see 1913 Ed.) the simplest way of staining myelin is to make paraffin sections of formol material and stain them with iron hsematoxylin exactly as for central corpuscles (say, twelve to fourteen hours in the mordant, six in the hsematoxylin, and a few minutes for the differentiation). Sections best not over 15 /x. One may counterstain the cells with carmalum, but not for more than half an hour, or the haematoxylin will be attacked. The stain is not so aesthetic as Weigert's, but quite as sharp. Axis cylinders are not shown. Similarly REGAUD (C. R. Acad. Sc., cxlviii, 1909, p. 861), but adding a chrome mordantage either concurrently with the formol fixation, or subsequently. Also NAGEOTTE (C. R. Soc. Biol., Ixvii, 1909, p. 542), with sections of formol material by the freezing method ; HOUSER (Journ. Comp. Neurol., x, 1901, p. 65), and BROOKOVER (ibid., xx, 1910, No. 2) ; SPIELMEYER (Neurol. CentrbL, xxix, 1910, p. 348) ; and his Technik d. mikrosl. Untersuch. d. Nervensy stems, 1911, p. 87, with sections of 25 to 35 /x, by the freezing method ; LOYEZ (C. R. Soc. Biol., Ixix, 1910, p. 511), who differentiates first lightly, till the grey matter begins to appear, in the iron alum, then washes, and differentiates further in Weigert's borax ferricyanide ; GILBERT (Ztsch. wiss. Mikr., xxviii, 1911, p. 279), who mordants with iron alum, stains with molybdic acid hcematoxylin, and differentiates with the borax ferricyanide ; STOELTZNER (ibid., xxiii, 1906, p. 329), who mordants celloidin sections of formol material for five minutes in Liq. ferri sesquichlorati, stains in 0 -5 per cent, hsematoxylin, and differentiates in the mordant or in borax ferricyanide ; and KODIS (Arch. mik. Anat., lix, 1902, p. 211), who fixes for one or two days in saturated solution of mercury cyanide, hardens in 10 per cent, formol, and stains sections, made by the freezing method, with Heidenhain's iron hsematoxylin. 853. WEIGERT'S Methods*- -There have been in all three methods of WEIGERT: — the 1884 method, the 1885 method, and the 1891 method. * Kevised by Dr. C. Da Fano, King's College, University of London. CHAPTER XXXIII. 443 The 1884 method (Fortschr. d. Med., ii, 1884, pp. 120, 190 ; Ztschr. wiss. Mikr., i, 1884, pp. 290, 564), which depends on the formation of a chrome lake of hsematoxylin, may be considered as superseded. Not so the two others, which depend on the formation of a copper lake in addition to the chrome lake. 854. WEIGERT'S 1885 Method (Fortschr. d. Med., iii, 1885, p. 236 ; Ztschr. wiss. Mikr., 1885, pp. 399, 484 ; Ergebn. Anat., vi, 1896 (1897), p. 10).- -The tissues are hardened in potassium bichro- mate. WEIGERT takes (Ergebn., p. 10) a 5 per cent, solution, and if time is an object hardens in a stove. (Other bichromate mixtures will do, £.., p. 130). After 496 NEUROGLIA AND SENSE ORGANS. removing the vitreous, the retina is cut away around the papilla with a punch or fine scalpel, and separated from the choroid. It is then rolled up (after being cut into quadrants or not), so as to. form a solid block. This is painted with 2 per cent, celloidin, which is allowed to dry for a few seconds, and the whole is put into the bichromate mixture, and further treated as a solid mass of tissue. RAMON also employs his neurofibril silver method, see Intern. Monatsschr. Anat. Phys., xxi, 1905, p. 393. GOLGI'S sublimate impregnation (Cox's form) has also been successfully employed by KRAUSE and RAMON. The bichromate and silver method serves for the study of the fibres of Miiller and neuroglia cells, as well as neurones. Weigert's neuroglia stain does not give good results. LENNOX (Arch. f. Ophthalm., xxxii, 1886, 1 ; Zeit. iviss. Mik., Hi, 1886, p. 408) has used Weigert's hsematoxylin method. KuHNT(Jew. Zeit. Naturw., Bd. xxiv, 1890, p. 177) employs Pal's modification. Similarly SCHAFFER (Sitzb. Akad. wiss. Wien., xcix, 1890, Abth. 3, p. 110; Zeit. wiss. Mik., viii, 1891, p. 227). These methods give a differential stain of rods and cones. For the zonula and ciliary body see MA WAS, Arch. d'Anat. micr., xii, 1910, p. 103. 9£4. Dissociation. — For maceration preparations you may use weak solutions (0*2 to 0-5 per cent.) of osmic acid for fixation, and then macerate in 0-02 per cent, chromic acid (M. SCHULTZE), or in iodised serum (M. Schultze), or in dilute alcohol (LANDOLT), or in Miiller's solution, or (RANVIER, Traite, p. 957) in pure water, for two or three days. THIN (Journ. of Anat., xiii, 1879, p. 139) obtained very good results by fixing for thirty-six to forty-eight hours in one-third alcohol, or in 25 per cent, alcohol, and then staining and teasing. SCHIEFFERDECKER macerates fresh retina for several days in the methyl mixture, § 543. KRAUSE (Intern. Monatsschr. Anat., i, 1884, p. 225) recommends treatment for several days with 10 per cent, chloral hydrate solution ; the rods and cones are well preserved. Inner Ear. > 925. Inner Ear, Dissection. — For the dissection of the human ear see POLITZER, " Die anatomische u. histologische Zergliederung d. men- schlichen Gehoroganes," Stuttgart (Enke), 1889 (Zeit. wiss. Mik., vii, 1890, p. 364). Amongst the lower mammalia, the guinea-pig is a favourable subject, as here (as with some other rodents) the cochlea projects freely into the cavity of the bulla, and may be easily removed CHAPTER XXXV. 497 with a scalpel and brought into a fixing liquid, and opened therein. With fishes and amphibia also the membranous labyrinth may easily be got away. 926. Preparation. -- SCHWALBE (Beitr. z. Phys. (C. Ludwig's Festschr), 1887, p. 200). — Fix (cochlea of guinea-pig) for eight to ten hours in " Flemming," wash in water, decalcify (twenty-four hours is enough) in 1 per cent, hydrochloric acid, wash the acid out, dehydrate, and embed in paraffin. PRENANT (Intern. Monatsschr. Anat., ix, 1892, p. 28). — Open the cochlea in solution of Flemming or of Hermann, and fix therein for four to five hours. Avoid decalcification as far as possible, but if necessary take 1 per cent, palladium chloride. Make paraffin sections. Isolation preparations of the stria vascularis may be made by putting a cochlea for a day into 1 per cent, solution of osmic acid, then for four to five days into 0-1 per cent, solution ; the stria may then be got away whole. KATZ (Zeit. wiss. Mik., xxv, 1908, p. Ill) fixes the inner ear, opened, for one or two hours in 30 c.c. of 0'5 per cent, osmic acid with 5 drops of acetic acid, then adds 10 drops of acetic acid and 60 c.c. of chromic acid (or platinum chloride) of 0-5 per cent, and leaves it for four days therein. He then rinses, puts for twelve to twenty-four hours into pyroligneous acid or pyrogallol or tannin solution, decalcifies (not necessary for mice) in 200 parts of water with 1 of chromic acid and 4 to 10 of nitric or hydrochloric acid, and embeds in celloidin or sometimes paraffin. Similarly WITTMAACK, see § 799. BIELSCHOWSKY and BRUEHL (Arch. mik. Anat., Ixxi, 1908, p. 27) fix the petrous in formol of 20 per cent., decalcify it in nitric acid of 5 per cent., wash this out, and put back for a few days into the formol, cut by the freezing method, and silver by the neurofibril method (§ 840 — twenty-four hours in nitrate of 4 per cent., but only a few minutes in the oxide bath). Similarly MULLENIX (Bull. Mus. Comp. Zool. Harvard Coll., liii, 1909, p. 215). STEIN (Anat. Anz., xvii, 1900, p. 398) decalcifies in celloidin by the method of ROUSSEAU. So also KISHI (Arch. mik. Anat., lix, 1902, p. 173). For staining, RANVIER (Traite, p. 991) employs his gold and formic acid method. The bichromate and silver method of GOLGI may be employed with fatal or new-born subj ects. The methylen blue intra vitam method 31. 32 498 NEUROGLIA AND SENSE ORGANS. has given good results. For the higher vertebrates the injection method should be employed. The Encyd. mik. Technik., i, p. 511, recommends injection of 1 c.c. of 0-5 to 1 per cent, solution every five minutes through the vena femoralis until the death of the animal. The cochlea then to be got out, exposed to the air for fifteen or thirty minutes, and fixed for some hours (overnight) in 10 per cent, ammonium molybdate with a little osmic acid. Then decalcified in trichloracetic acid of 5 per cent, with a trace of platinum chloride, washing for twenty-four hours and got into paraffin. For fishes and amphibia the immersion method will suffice. 927. Other Methods. — WALDEYER, Strieker's Handb., p. 958 (decalci- fication either in 0-001 per cent, palladium chloride containing 10 per cent, of HC1, or in chromic acid of 0-25 to 1 per cent.). URBAN PRITCHARD (Journ. Eoy. Mic. Soc., xii, 1872, p. 380). — Decal- cification in 1 per cent, nitric acid. LAVDOWSKY ( Arch. mik. Anat., xiii. 1877, p. 497). — Fresh tissues (from the cochlea) are treated with 1 per cent, solution of silver nitrate, then washed for ten minutes in water containing a few drops of 0-5 or 1 per cent, osmic acid solution, and mounted in glycerin. MAX FLESCH (Arch. mik. Anat., xvi, 1879, p. 300) ; TAFANI (Arch. Hal. de Biol., vi, 1884, p. 207) ; EICHLER ( Abh. math-phys. Cl. Sachs. Ges. Wiss, xviii, 1892, p. 311 ; Zeit. wiss. Mik., ix, 1892, p. 380 (injection of blood-vessels of the labyrinth) ) ; SIEBENMANN (Die Blutgejdsse im Labyrinthe des menschlichen Ohres, Wiesbaden, Bergmann, 1894 ; Zeii. wiss. Mik., xi, 1894, p. 386) ; GRAY (Journ. Anat. Phys., xxxvii, 1903, p. 379) ; SCOTT (ibid., xliii, 1909, p. 329). 9.28. Olfactive Nerve-endings, Tactile Corpuscles, etc. — Besides the gold method, Chapter XVII, and the methylen-blue method, Chapter XVI, the rapid bichromate and silver method of GOLGI should be employed, and for the olfactive mucosa gives the best results. See VAN GEHUCHTEN, La Cellule, vi, 1890, p. 405. For intra-epidermic nerve-endings, besides the methods given in Chap- ter XXVII, the GOLGI method should be employed. According to VAN GEHUCHTEN (La Cellule, ix, 1893, p. 319) it gives much better results than gold methods. He uses the rapid process. For tactile corpuscles, etc., besides the methods given in §§ 721 and 722, see RAMON Y CAJAL'S neuro-fibril methods. CHAPTER XXXVI. METHODS FOR INVERTEBRATES. Tunicata. 929. Fixation of Tunicata. — A method of Lo BIANCO * for killing simple Ascidians in an extended state has been given, § 25. Some forms, such as Clavellina, Perophora, Phallusia, Molgula, Cynthia, etc., should first be narcotised by treatment for from three to twelve hours with chloral hydrate (1 : 1000 in sea water), then killed in a mixture containing chromic acid of 1 per cent. 10 parts, acetic acid of 50 per cent. 100 parts, and finally hardened in 1 per cent, chromic acid. The compound Ascidians with contractile zooids may be left in clean sea water till the zooids have become fully extended, then fixed by VAN BENEDEN'S acetic acid process, § 84 (steel instruments being avoided for manipulating them). I strongly recommend this process. S. Lo BIANCO recommends for this group the chloral hydrate process, followed by fixation with sublimate or chromo-acetic acid. CAULLERY (Bull. Sc. France Belg., xxvii, 1895, p. 5) first stupefies the animals with cocaine (LAHILLE, a few drops of 5 per cent, solution to 30 c.c. of sea water), then fixes in liquid of Flemming or acetic acid. Most small pelagic Tunicates are very easily fixed with osmic acid or acid sublimate solution. I have found the acetic acid process very good for Pyrosoma. Lo BIANCO puts them for a quarter of an hour into 50 per cent, alcohol containing 5 per cent, of hydrochloric acid, then into successive alcohols, beginning with 60 per cent. He kills the hard forms of Salpa with acetic acid of 10 per cent., the semi-hard ones with 1 per cent, chromic acid containing 5 per cent, acetic acid, the soft ones with 1 per cent, chromic acid containing -^ per cent, osmic acid, or 10 parts of 1 per cent, chromic acid, with 1 of formol and 9 of sea water, Doliolida3 with sublimate, or the above osmic mixture, or a * References to methods of Lo BIANCO in this Chapter are all to his paper in Mitth. Zool. Stat. Neapel, ix, 1890, p. 435. 32—2 500 METHODS FOR INVERTEBRATES. * mixture of 10 parts 10 per cent, solution of sulphate of copper with 1 part concentrated sublimate solution, or the formol mixture. Molluscoida. 930. Bryozoa. — For some methods of killing and fixing see §§13, 20, and 21. S. Lo BIANCO employs for Pedicellina and Loxosoma the chloral hydrate method, fixing with sublimate. For Flustra, Cellepora, Bugula, Zoobothrium, he employs the alcohol method of EISIG, § 18. For Cristatella see §§ 16, 20. See also BRAUN. ; CONSER (Trans. Amer. Mic. Soc., xvii, 1896, p. 310) kills the fresh water forms with cocaine, puts them for an hour into 1 per cent, chromic acid, and passes through water into alcohol, etc. Similarly CALVET (Hist. Nat. Bryozoaires, Montpellier, 1900, p. 15), for marine forms. ZSCHIESCHE (Zoo?. Jafirb., xxviii, 1909, p. 6) fixes larvee of Alcyonidium (settled down on a layer of celloidin or paraffin) with 8 parts of sublimate and 2 of acetic acid to 90 of sea water, for twenty-five to thirty minutes. 931. Brachiopoda. — Lo BIANCO kills small animals in 70 per cent, alcohol, larger ones being first narcotised with alcohol and sea water. BLOCHMANN (Untersuch.fein. Ban Brachiopoden, Jena, 1892, p. 5) fixes principally with sublimate, macerates by the HERT WIGS' method, § 526, decalcifies with 1 per cent, chromic acid (for thick shells add a little hydrochloric or nitric acid), or with nitric acid in alcohol of 50 to 70 per cent., and embeds in paraffin or celloidin. See also EKMAN, Zeit. iviss. Zoo/., Ixii, 1896, p. 172. Mollusca. 932. Fixation. — To kill Mollusca extended for dissection make up stock solution A : 90 parts absolute alcohol, 10 parts turpentine ; 10 per cent, of A to 90 per cent, water. Leave in twelve to twenty- four hours. Lo BIANCO narcotises Lamellibranchs for six to ten hours or more with alcohol, § 18, and then kills them. LIST (Fauna Flora Golf. Neapel, xxvii, 1902, p. 292) narcotises Mytilidse with 2 per cent, of cocaine in sea water, and (for preserva- tion of cilia) fixes in sea water, with 10 per cent, of formol. Lo BIANCO advises that Prosobranchiata, and, amongst the Heteropoda, Atlantidse, be narcotised with 70 per cent, alcohol, § 18. For Opisthobranchiata I recommend sudden killing with liquid of Perenyi, or the acetic method, § 929. Aplysia may first be narcotised CHAPTER XXXVI. 501 by subcutaneous injection of about 1 c.c. of a 5 to 10 per cent, solution of hydrochlorate of cocaine (ROBERT, Bull. Sclent, de la France, etc., 1890, p. 449 ; Zeit. iviss. mik., ix, 1892, p. 216), or (SCHONLEIN, Zeit. BioL, xxx, 1893, p. 187) 1 c.c. of 4 per cent, solution of Pelletierin. For Lo BIANCO 's various methods see the original, p. 467. For Pteropoda in general, liquid of Perenyi. Creseis is a difficult form. Lo BIANCO advises the alcohol method, § 18. For the Gymnosomata he narcotises with 0-1 per cent, chloral hydrate. For terrestrial Gastropods see §§ 23 and 26. MARCHI (Arch. mik. Anat., 1867, p. 204) gets rid of the mucus of the integument of Limax, which may be an obstacle to preparation, by putting the living animal into moderately concentrated salt solution, in which it throws off its mucus and dies in a few hours. LANG (Anat. Hefte, 1902, p. 84) puts Helix into water with enough chromic acid to make it of a Rhine-wine colour, with an air-tight cover to the vessel, and when the animals are extruded injects into them a quarter to a half of a Pravaz syringe of 1 per cent, cocaine, and after five to fifteen minutes dissects and fixes. HEYMANS (Bull. Acad. Belg., xxxii, 1896, p. 578) injects ethyl bromide under the skin of Cephalopoda. Lo BIANCO uses for fixing them his chromo-acetic acid, No. 1 (§ 39), with a double quantity of acetic acid, for twenty-four hours. 933. Liver of Mollusca. — ENRIQUES (Mitth. Zool. Stat. Neapel, xv, 1901, p. 289) fixes the liver of Octopus and Sepia with sublimate. For Aplysia (especially in summer) alcohol, formol, and chromic mixtures are counter-indicated, on account of the carbohydrates in the cell. Sublimate is best. 934. Nervous System of Pulmonata. — B. de NABIAS (Act. Soc. Linn. Bordeaux, 1894 ; Rech. Hist, centres nerveux des Gasteropodes, 1894, p. 23) opens the animals and fixes the ganglia for one hour in a mixture of 6 parts glacial acetic acid to 100 of 90 per cent, alcohol, or for fifteen to twenty minutes in 5 per cent, sublimate with 5 per cent, acetic acid. He stains in bulk, with Renaut's hsematoxylic eosin, or R. Heidenhain's hsematoxylin, or a copper hsematoxylin of Viallanes, and embeds in paraffin. He also stains by the rapid method of GOLGI, embedding, however, the ganglia in celloidin directly after the hardening in osmic acid and bichromate, and treating the sections with the silver (p. 458). He stains with methylen blue by treating the ganglia in situ for twelve to twenty- four hours with a 1 per cent, solution. 502 METHODS FOR INVERTEBRATES DREYER (Zeit. wiss. Zoo/., xcvi, 1910, p. 380) narcotises Nudi- branchs with, cocaine, and for studying the nerves fixes them with MAYER'S picro-formol, puts for a week into a mixture of 1 grm. of iron alum with 2 c.c. of formol and 40 of water, makes sections and stains with iron hsematoxylin. See also, for nerve-cells, McCLURE, ZooL Jahrb., 1898, p. 17 (MANN'S methyl blue and eosin, or BENDA'S safranin and Lichtgriin), and LEGENDRE, Arch. mic. Anat., x, 1909, p. 312. 935. Eyes of Gastropoda (FLEMMING, Arch. mik. Anat., 1870, p. 441). — To obtain the excision of an exserted eye, make a rapid cut at the base of the peduncle, and throw the organ into very dilute chromic acid, or 4 per cent, bichromate ; after a short time it will evaginate, and remain as completely erect as if alive. Harden in 1 per cent, osmic acid, in alcohol, or in bichromate. SMITH (Bull. Mus. Comp. Zool. Harvard, xlviii, 1906, p. 238) macerates eyes for at least two days in 9 parts of water with 1 of weak mixture of Flemming, followed by glycerin of 10 per cent. He bleaches them (in sections) with nitric acid and chlorate of potash. 936. Eyes of Cephalopoda and Heteropoda (GRENACHER, AbJi. naturf. Ges. Halle-a.-S., Bd. xvi, 1896, p. 213). — Depigment with hydrochloric acid (in preference to nitric acid). The mixture § 574 may also be used. If you stain with borax-carmine and wash out in this mixture, the pigment will be found to be removed quicker than the stain is washed out. LENHOSSEK (Zeit. wiss. Zool., Iviii, 1894, p. 636 ; Arch. mik. Anat., xlvii, 1896, p. 45) applies the method of GOLGI to the eyes of Cephalopods. Similarly KOPSCH (Anat. Anz., xi, 1895, p. 362), but using formol instead of the osmic acid. HESSE (Zeit. wiss. Zool., Ixviii, 1900, p. 418) fixes eyes of Hetero- poda with 1 of formol to 4 of water, and (p. 257) bleaches those of Cephalopoda by the methods of GRENACHER and that of JANDER, §575. See also MERTON, ibid., Ixxix, 1905, p. 326. 937. Eyes of Lamellibranchiata. — See PATTEN, Mitth. Zool. Stat. Neapel, vi, 1886, p. 733, and RAWITZ, Jena. Zeit. Naturw., xxii, 1888, p. 115, and xxiv, 1890, p. 579 (bleaches with caustic soda) ; see § 576. HESSE (op. cit., last §, p. 380) employs the method of JANDER for Area. He fixes the eye of Pecten in 10 per cent, formol for five minutes, followed by sublimate or picro-nitric acid. CHAPTER XXXV I. 503 938. Shell. — Sections of non-decalcified shell are easily obtained by the usual methods of grinding, or, which is often a better plan, by the methods of v. KOCH or EHRENBAUM. MOSELEY (Quart. Journ. Mic. Sci. (2), xxv, 1885, p. 40) decalcifies with nitric acid of 3 to 4 per cent, and then makes sections. This method serves for the study of the eyes of CHITONID^E. 939. Injection of Acephala (FLEMMING, Arch. mik. Anat., 1878, p. 252). — To kill the animals freeze them in a salt and ice mixture, and throw them for half an hour into lukewarm water. They will be found dead, and the injection-pipe may be tied in the heart, and the entire animal filled and covered up with plaster of Paris, which serves to occlude cut vessels that it is not possible to tie. As soon as the plaster has hardened the injection may be proceeded with. See also DEWITZ, Anleit. zur Anfert. zootom. Prdp., Berlin, 1886, p. 44 (Anodonta) and p. 52 (Helix). DAKIN (Liverpool Mar. Biol. Comm., xvii, 1909, p. 76) narcotises by adding alcohol and glycerin for eighteen to twenty-four hours, puts for half an hour into formol of 5 per cent., and injects from a branchial vessel. MOZEJKO (Zeit. wiss. Mik., xxvi, 1909, p. 353, and 1910, p. 542) puts for half an hour into water at 40° to 50° C., removes the shell, and injects carmine by auto-injection through the heart. For occluding vessels he takes cotton-wool soaked with gelatin and plaster of Paris. He takes for a vaso-dilator a saturated solution of peptonum siccum. 940. Maceration Methods for Epithelium. — ENGELMANN (Pfliiger's Arcti., xxiii, 1880, p. 505) macerates the intestine of Cyclas in osmic acid of 0-2 per cent, (after having warmed the animal for a short time to 45° to 50° C.), or in concentrated boracic acid solution. C'^m.--The entire intra-cellular" fibre apparatus may be isolated by teasing fresh epithelium from the intestine of a Lamellibranch (e.g., Anodonta) in either bichromate of potash of 4 per cent, or salt solution of 10 per cent. To get good views of the apparatus in situ in the body of the cell, macerate for not more than an hour in concentrated solution of boracic or salicylic acid. Very dilute osmic acid (e.g., 0-1 per cent.) gives also good results. The " lateral cells " of the gills are best treated with strong boracic acid solution (5 parts cold saturated aqueous solution to 1 part water). Dr. ORTON uses borax carmine and picro-nigrosin (in liter a). BELA HALLER'S Mixture, see § 532; BROCK'S Medium, § 523; MOBIUS'S Media, § 527 ; the second of these is much recom- 504 METHODS FOR INVERTEBRATES. mended by DROST (Morphol. Jahrb., xii, 1866, p. 163) for Cardium and My a. PATTEN (Mitth. Zool. Stat. Neapel, vi, 1886, p. 736) takes sulphuric acid, 40 drops to 50 grms. of water. Entire molluscs, without the shell, may be kept in it for months. BERNARD (Ann. Sci. Nat., ix, 1890, p. 191) macerates the mantle of Prosobranchs in a mixture of 1 part each of glycerin and acetic acid, 2 parts each of 90 per cent, alcohol and 0-1 per cent, chromic acid and 40 parts water, which acts in from a quarter of an hour to three hours. He also (pp. 102, 306) uses a weak solution of chloride of ruthenium, especially for nerve-tracts, mucus-cells and cilia. Alcohol material may be macerated in a mixture of 1 part. glycerin, 2 of acetic acid and 40 of water. 941. Mucus Glands. — RACOVITZA (Arch. Zool. exper. [3], ii, 1894, p. 8) studies these in Nudibranchs (and Annelids) by killing with acetic acid, staining in toto with methyl green dissolved in liquid of RIPART and PETIT, and after three to six days, when only the glands show the stain, examining in mixture of equal parts of glycerin and the liquid. Arthropoda. 948. General Methods for Arthropoda. — As general methods for the study of chitinous structures, the methods worked out by Paul Mayer (see §§ 8, 96 and 97) are excellent. It is, at all events, absolutely necessary, in the preparation of entire organisms or unopened organs, that all processes of fixation, washing and staining should be done with fluids possessing great penetrating power. Hence picric acid combinations should in general be used for fixing, and alcoholic fluids for washing and staining. Concentrated pier o -sulphuric acid (or picro-nitric) is the most generally useful fixative, and 70 per cent, alcohol is the most useful strength for washing out. Alcoholic picro- sulphuric acid may be indicated for fixing in some cases. But if the animals or organs can first be properly opened, the usual methods may be employed. 942A. Mounting Small Arthropods. — Dr. A. D. Imms informs me that for mounting and clearing aphids and other small insects, etc., the following formula as used by Professor Beiiese for Acarina gives good results : — H20 ....>.. 20grs. Chloral hydrate . . . . 160 ,, Gum arable . . . . . 15 ,, Glucose syrup . . . . . 10 ,, Acetic acid . . . . . . 5 „ CHA PTER XXX VI. 505 The living specimens may be placed direct on the medium on the slide, or may be killed by a short immersion in 10 per cent, acetic acid, or boiling water. If the specimens are in alcohol they should be washed in 10 per cent, acetic acid before mounting. After the cover glass is put on, gently warm the slide, then allow to cool, and leave for one or two weeks to dry and set. Ring the slide with a waterproof substance and finally ring with a layer of Canada balsam. Personally, I often kill in 90 per cent, or absolute alcohol, leave for a few days, and mount in Euparal (see § 449). 943. Crustacea. — Some forms are very satisfactorily fixed with sublimate. Such are the Copepoda and the larvae of Decapoda. It is sometimes indicated to use the sublimate in alcoholic solution. Some Copepoda, however (Copilia, Sapphirina), are better preserved by means of weak osmic acid, and so are the Ostracoda. In many cases the osmic acid will produce a sufficient differentiation of the tissues, so that further staining may be dispensed with ; so for Copilia and Phyllosoma. The pyrogallic process (§ 374) may be useful. GIESBRECHT takes for marine Copepods a concentrated solution of picric acid in sea water, to which a little osmic and acetic acid may be added. For fresh-water forms, ZACHARIAS (Zool. Anz., xxii, 1899, p. 72) takes chromo-acetic acid. GriESBRECHT fixes larvae of Stomatopoda for five to ten minutes in formol of 10 per cent, warmed to 40° or 50° C., opens them in sea water and puts for one and a half to two and a half hours into formol 1 part and sea water 5 parts, and brings into alcohol of 70 per cent. STAFFERS (La Cellule, xxv, 1909, p. 356) fixes Sympoda in GILSON'S copper formol, § 115, or in HORNELL'S mixture of 100 parts of 5 per cent, formol with 40 of alcohol ; and for softening the chitin puts for twelve to thirty-six hours into 3 per cent, solution of sublimate with 5 per cent, of nitric acid. NETTOVITCH (Arb. z. Inst. Wien, xiii, 1900, p. 3) fixes Argulus with liquid of Telly esniczky, § 52, warmed to 50° C. For FISCHEL'S intra-vitam stain of Cladocera with alizarin, etc., see § 207. 944. Tracheata.— KENYON (Tufttj^olL Stud., No. 4, 1896, p. 80) fixes Pauropoda in Carnoy's acetic alcohol and chloroform, § 85, cuts them in twTo for staining, etc., and embeds in celloidin followed by paraffin. HENNINGS (Zeit. wiss. Mik., xvii, 1900, p. 311) takes — Nitric acid 16 parts, chromic acid of 0-5 per cent. 16 parts, sublimate saturated in 60 per cent, alcohol 24 parts, picric acid saturated in water 12, and absolute alcohol 42, fixes for twelve to twenty-four 506 METHODS FOR INVERTEBRATES. hours, and washes out with iodine alcohol. He says that this mixture not only fixes, but softens chitin enough to allow of paraffin sections being made through hard parts. HAMANN (Site. Naturw. Freunde Berlin, 1897, p. 2) fixes small Tracheata in 10 per cent, formol and finds the chitin sufficiently soft for sections to be made. VAN LEEUWEN (Zool. Am., xxxii, 1907, p. 318) takes for larvae of Hexapoda 12 parts of 1 per cent, solution of picric acid in absolute alcohol, 2 of chloroform, 2 of formol, and 1 of acetic acid. HOLLANDE (Arch. d'Anat. mic., xiii, 1911, p. 171) takes 12 parts of saturated solution of picric acid in formol of 40 per cent., 54 of absolute alcohol, 3 of benzene, and 1 of nitric acid, and finds that this fixes quickly enough not to make chitin too hard. NUTTALL, COOPER and ROBINSON (Parasitology, 1908, i, p. 163) fix for a few minutes in hot picrosulphuric acid. 945. Methods for Clearing and Softening Chitin.— The methods of Looss have been described § 545, those of HENNINGS and HAMANN last §. LIST (Z&it. wiss. Mik., 1886, p. 212) treats Coccidse (after harden- ing) for eighteen to twenty-four hours with eau de Javette, diluted with 4 volumes of water. After washing out they may be embedded in paraffin, and good sections obtained. SALING (Dissert. Marburg., 1906, p. 11) boils larvse of Tenebrio for some minutes in eau de Labarmque, the heat serving to fix the soft parts, which in successful cases are well preserved. Wash out with warm water, then alcohol. SAZEPIN'S method for antennae of Chilognatha (Mem. Acad. Imp. St. Petersb., xxxii, 1884, pp. 11, 12) consists in steeping antennae (that have been dehydrated with alcohol) for twenty-four hours in chloroform containing a drop of fuming nitric acid (shake occasion- ally). BETHE (Zool. Jahrb., viii, 1895, p. 544) puts telsons of My sis for eight to fourteen days into 40 per cent, alcohol, to which nitric acid is gradually added, so that by the end of that time they have been brought into alcohol containing 20 per cent, of the acid. This softens the chitin, and somewhat breaks down the structure of the otolith, so that good sections through it are occasionally obtained. Similarly HERBST, Arch. Entwickelungsmech, ix, 1899, p. 291. See also the depigmentation processes, §§ 567 to 576. 945A. Double Embedding of Insects.— -Dissolve 1 -5 grs. desiccated celloidin chips in 50 c.c. clove oil, or, better, add the celloidin in CHAPTER XXXVL 507 an ordinary ether-alcohol solution and evaporate off the solvent in the oven overnight. It takes many days to dissolve thoroughly, but the time may be shortened by keeping it at 90° F. Fix objects in absolute alcohol ; bring them into clove oil, and allow this to clear the preparations, then transfer to the clove oil celloidin. The time in this must be gauged according to the size and nature of the insect ; fleas, if a rupture is made in the chitinous covering, are penetrated in twenty- four hours or less. When ready to embed, dip a cover-glass in melted paraffin wax, to get a smooth surface on which the celloidin solution will not spread, but forms a thick drop. Place the insect on the prepared glass slip in a drop sufficient to completely cover it, and arrange in any desired position. Invert the cover-glass and float on to some chloroform ; leave for half an hour or longer, according to the size of the drop. The drop of celloidin should fall away from the glass. Transfer to melted wax, and allow time for the wax thoroughly to permeate (twenty minutes is long enough for fleas and lice). The result will be a small tablet-shaped mass of spongy celloidin impregnated with wax ; this can be at once embedded, or may be put away for future use. (Professor Boycott informs me that he has never left his preparations for more than two or three weeks at a time, so that he has no data as to how long the same could be kept, but they could probably be stored indefinitely.) 945s. Carbon Bisulphide Embedding of Insects, etc. — HEIDENHAIN many years ago recommended carbon bisulphide as a medium for embed- ding in wax. This fluid is both smelly and dangerous and should only be used in electric thermostats. The wax is dissolved in carbon bisulphide, and dehydrated insects, etc., are placed in some of the fluid which is allowed to evaporate at a gentle warmth. Subsequently the material is rapidly treated in pure wax in the thermostat. This method cer- tainly curtails the length of time in the thermostat, and overheating is a serious matter when one is working at chitinous or brittle organisms. 946. Test for Chitin (ZANDER, Pfliigers Arch., Ixvi, 1897, p. 545).- Treat for a short time with a drop of freshly prepared solution of iodine in iodide of potassium and add a drop of concentrated chloride of zinc. This is then removed with water as far as possible, and the violet reaction is obtained. See also WESTER, Zool. Jahrb., Abth. SysL, xxviii, 1910, p. 531. 947. BETHE'S Stain for Chitin (loc. tit., § 945).— Sections are put for three or four minutes into a freshly prepared 10 per cent, solution of anilin hydrochloride, to which has been added 1 drop of hydrochloric acid for every 10 c.c. They are then rinsed in water, and the slide is put with the sections downwards into 10 per cent, solution of bichro- 508 METHODS FOR INVERTEBRATES. mate of potash. The stain is at first green, but becomes blue in tap water or alcohol containing ammonia. MAYER simply uses a solution of pyrogallol in alcohol or glycerin ; and HOFMANN (Zeit. uiss. Zool., Ixxxix, 1908, p. 684) puts for a day or more into raw pyroligneous acid. Dr. ORTON writes to me that he simply uses picro-nigrosin and borax carmine. 948. Tracheae may be studied by the Golgi bichromate and silver process. MARTIN (C. R. Soc. Philomath., 1893, p. 3) injects them with indigo white (through the body cavity), and puts into hot water from which the air has been expelled by boiling. Tracheae blue. 949. Brain of Bees. — KENYON (Journ. Comp. N enrol., vi, 1896, p. 137 ; Journ. Roy. Mic. Soc., 1897, p. 80) treats by the GOLGI process (seldom successful), or hardens in a mixture of 1 part formol and 2 of 5 per cent, sulphate of copper, followed by staining in Mallory's phospho-molybdic hsernatoxylin. JONESCU (Jena. Zeit., xlv, 1909, p. Ill) has employed the silver methods of Ramon y Cajal and Bielschowsky and Wolff. 950. Ventral Cord. — FLOYD (Mark. Anniv. vol., 1904, p. 355) fixes the ganglia of Periplaneta for eighty minutes with vapour of formol, and brings into alcohol. See also BINET, Journ. Anat, Phys., xxx, 1894, p. 469. 951. Eyes of Arthropods. — For the methods of LANKESTER and BOURNE (Quart. Journ. Mic. Sci., 1883, p. 180 : Limulus) ; HICKSON (ibid., 1885, p. 243 : Musoa) ; PARKER (Bull. Mm. Harvard Coll., xx, 1890, p. 1 ; Zeit. wiss. Mik., viii, 1891, p. 82 : Homarus) see early editions. PARKER (Mitth. Zool. Stat. Neapel, xii, 1895, p. 1) also applies the methylen blue method to the retina and optic ganglia in Deca- pods, especially in Astacus. He injects 0-1 c.c. of a 0-2 per cent, solution into the ventral sinus. After twelve to fifteen hours the animals are killed, the ganglia quickly dissected out, and the stain fixed as described, § 343. For his method for eyes of Scorpions see § 575. For the methods of PURCELL for the eyes of Phalangida see Zeit. wiss. Zool., Iviii, 1894, p. 1. He has the following stain. The cephalothorax is removed and brought for twenty minutes into 50 per cent, alcohol warmed to 45° or 50° C., and saturated with picric acid. The pigment dissolves in this solution and stains the nuclei and some other parts of the rhabdoms, so that no further stain is required. CHAPTER XXXVI. 509 HENNJNGS (Zeit. wiss. Mik., xvii, 1900, p. 326) depigments sections by putting them for ten minutes (Musca) to twelve hours (Myriopoda) into a mixture of 2 parts of 80 per cent, alcohol with 1 of glycerin and 2 per cent, of nitric acid, best kept at 35° C. The elements are well preserved. WIDMANN (Zeit. wiss. ZooL, xc, 1908, p. 260) makes the lens of Arachnida fit for sectioning by putting for a day or so into alcohol with 10 to 15 per cent, of nitric acid ; and bleaches sections with 1 part of chlorine water to 2 of alcohol. See also ROSENSTADT, Arch. mik. Anat., xlvii, 1896, p. 478 ; VIALLANES, Ann. Sci. Nat., xiii, 1892, p. 354 ; and DIETRICH, Zeit. wiss. ZooL, xcii, 1909, p. 465 (fixes in alcoholic formol, and bleaches with dilute aqua regia). 952. Injections (Arachnida and Crustacea especially). — AIME SCHNEIDER (Tablettes Zool., ii, 1892, p. 123) recommends lithographic Indian ink, the animals being narcotised with chloroform, then injected and thrown into strong alcohol. Similarly CAUSARD (Bull. 8c. France Belg., xxix, 1896, p. 16). 953. Aretiseoida (DOYERE, Arch. mik. Anat., 1865, p. 105). —Examina- tion of living animals after partial asphyxiation in boiled water. See early editions. Vermes. 954. Chsetopoda : Cleansing Intestine. — KUKENTHAL (Journ. Roy. Mic. Soc., 1888, p. 1044) puts Lumbricus into a glass vessel filled with bits of moistened blotting-paper. They gradually evacuate the earthy particles from the gut, and fill it instead with paper. VOGT and YUNG (Traite d'Anat. Comp. Prat., v) recommend coffee-grounds instead of paper, as they cut better after embedding. JOEST (Arch. Entwicklungsmech., v, 1897, p. 425) simply keeps the worms for a few days in moist linen, and finds the gut empty. PEARL (Journ. appl. Mic., iii, 1901, p. 680) injects alcohol of 6 per cent, through the gut of narcotised worms. 955. Chsetopoda : Fixation. — Lumbricus may be anaesthetised by putting the animals into water with a few drops of chloroform. PERRIER puts them into water in a shallow dish, sets up a watch-glass with chloroform in the corner of it, and covers the whole. CERFONTAINE (Arch, de Biol., x, 1890, p. 327) injects interstitially about 2 c.c. of a 1 : 500 solution of curare. 510 METHODS FOR INVERTEBRATES. JAQUET (Bib, Anat., iii, 1895, p. 32) kills Lumbricus in extension in 1 part of nitric acid to 125 of water. COLLIN (Zeit. wiss. Zool.j xlvi, 1888, p. 474) puts Criodrilus lacuum into a closed vessel with a little water, and hangs up in it a strip of blotting-paper soaked in chloroform. KUKENTHAL (Die mik. Technik, 1885 ; Zeit. wiss. Mik., 1886, p. 61) puts Annelids into a glass cylinder filled with water to the height of 10 cm., and then pours 70 per cent, alcohol to a depth of 1 to 2 cm. on to the water. For Opheliadae he also employs 0-1 per cent, of chloral hydrate in sea water. Many marine Chaetopoda may be successfully narcotised (Lo BIANCO) in sea water containing 5 per cent, of alcohol, or by means of the mixture, § 18. The Polychceta sedentaria may sometimes be satisfactorily fixed by bringing them rapidly into corrosive sublimate. Cold, not hot, solutions should be taken, as heat frequently shrivels up the branchiae. Eunice and Onuphis may be treated in the same way. Lo BIANCO advises killing Chaetopteridae, Sternaspidae, Spiro- graphis, Protula, by putting them for half an hour into 1 per cent, chromic acid. Some of the sedentaria may be got protruded from their tubes by leaving them for some hours in 0 -1 per cent, chloral hydrate in sea water. «/ For EISIG'S methods for Capitellidaa see Fauna u. Flora Golf. Neapel, xvi, 1887, p. 295. See also § 14 (lemon juice), and the methods §§ 20 to 26, 39 and 49. 956. Blood-vessels of Annelids (KUKENTHAL, Zeit. iviss. Mik., 1886, p. 61).- -The animals should be laid open and put for two or three hours into aqua regia (4 parts of nitric acid to 2 of hydrochloric acid). Vessels black, on a yellow ground. BERGH (Anat. Hefte, xlv, 1900, p. 392, and xlix, 1900, p. 599) puts small Annelids for a week or more into equal parts of 1 per cent, nitric acid and 1 per cent, nitrate of silver, or into 50 parts of nitrate, 25 of formic acid, and 25 of water, dissects out the organs and exposes to light. Marine forms may be treated by HARMER'S process. 957. Nerves of Annelids. — Note the methylen blue method and the bichromate of silver method of Golgi (the rapid method). For the latter see v. LENHOSSEK (Arch. mik. Anat., xxxix, p. 102). LANGDON (Journ. Comp. Neur., x, 1900, p. 4) injects strong solution of methylen blue into the body cavity of Nereis, and puts the animal for some hours into sea water in the dark, fixes the stain by Bethe's method, and makes paraffin sections. CHAPTER XXXVI. 511 See also M. LEWIS, Anat. Anz., xii, 1896, p. 292 ; ATHESON, ibid., xvi, 1899, p. 497 ; and the methods of APATHY §§ 342, 368, 371, and 834. 958. Hirudinea. — For the methods of killing see those given for Lumbricus in § 955, also §§ 20 to 26, and 49. WHITMAN (Meth. in mic. Anat., p. 27) recommends that they be killed with sublimate. I have obtained better results myself by narcotising with carbonic acid (§ 26), and fixing with liquid of Flemming. I have also found that lemon juice kills them in a state of very fair extension. APATHY succeeds with alcohol of 40 per cent. GRAF (Jen. Zeit., 1893, p. 165) has obtained good results by nar- cotising with a decoction of tobacco. 959. Injection. — WHITMAN (Amer. Natural., 1886, p. 318) states that very perfect natural injections may often be obtained from leeches that have been hardened in weak chromic acid or other chromic liquid. JACQUET (Mitth. Zool. Stat. Neapel, 1885, p. 298), for artificial injections, puts leeches into water with a very small quantity of chloroform, and allows them to remain a day or two in the water before injecting them. 960. Nervous System. — Impregnation with gold. BRISTOL (Journ. of Morph., xv, 1898, p. 17) kills in formic acid of 15 to 20 per cent., puts for twenty-five minutes into 1 per cent, gold chloride, reduces in formic acid of 1 per cent, (twelve to eighteen hours), and embeds in paraffin. See also §§ 342, 368, 371 and 834. 961. Nephridia. — SHEARER (Quart. Journ. Micr. ScL, Iv, 1910, p. 288) stains Histriobdella intra vitam with a very weak solution of Methyl blue, which allows the course of the nephridia to be made out. 962. Gephyrea. — VOGT and YUNG (Anat. Comp. Prat., p. 373) direct that Siphunculus nudus be kept for some days in perfectly clean basins of sea water, changed every day, in order that the intestine of the animals may be got free from sand, and then anaesthetised with chloroform. WARD (Bull. Mus. Comp. Zool., Cambridge, Harvard Coll., xxi, 3, p. 144) puts them into a shallow dish with sea water and pours 5 per cent, alcohol in a thin film on to the surface of the water, and as soon as they make no contractions on being stimulated removes to 50 per cent, alcohol. 512 METHODS FOE INVERTEBRATES. Lo BIANCO says killing with 0-5 per cent, chromic acid or with 04 per cent, chloral hydrate in sea water may be tried. Phascolo- soma and Phoronis should be treated by the alcohol method, larvae of Sipunculus with cocaine, § 21. APEL (Zeit. wiss. Zool., xlii, 1885, p. 461) puts Priapulus and Halicryptus into a vessel with sea water and heats on a water bath to 40° C. ; or they may be thrown into boiling water, which paralyses them so that they can be quickly cut open and thrown into J per cent, chromic acid or picro-sulphuric acid. 963. Rotatoria. — For quieting them for study in the living state, WEBER (Arch, de Biol., viii, 4, 1888, p. 713) finds that 2 per cent, solution of hydrochlorate of cocaine gives the best results. Warm water gave him good results for large species, such as those of Hydatina and Brachionus. HARDY (Journ. Roy. Mic. Soc., 1889, p. 475) recommends thick syrup added drop by drop to the water. HUDSON (ibid., p. 476) mentions weak solution of salicylic acid. VOLK (Jahrb. Hamburg, wiss. Anst., xviii, 1901, p. 164) quiets them in quince mucilage, 40 grm. of the seeds to 1 litre of water. Qf. § 1018. HIRSCHFELDER (Zeit. wiss. Zool., xcvi, 1910, p. 211) studies them living in neutral red of 1 : 50,000. See also §§ 23, 24 and 27. Methylene blue, § 339, may be found useful. Permanent preparations may be made by the method of ROUSSELET (Journ. Quekett Mic. Club, v, March, 1895, p. 1) : The animals are got together in a watch glass and are narcotised by adding to the water at intervals a few drops of the following mixture : Hydrochlorate of cocaine 2 per cent, solu- tion ....... 3 parts. Methylated spirit . . . . .1 Water ....... 6 As soon as the cilia have ceased to beat, or are seen to be on the point of ceasing to beat, they are fixed by adding a drop of liquid of Flemming or of \ per cent, osmic acid. After half a minute or less the animals are taken out with a pipette, and thoroughly washed by passing them through two or three watch glasses of distilled water. They are then definitely mounted in a mixture of formol 2J parts, distilled water 37J parts. ZOGRAF (Comptes Rend., cxxiv, 1897, p. 245) narcotises as ROUSSE- LET, but without the spirit, fixes with osmic acid for two to four: 55 55 CHAPTER XXXVI. 513 minutes, then replaces this by raw pyroligneous acid diluted with 8 to 10 volumes of water, and after five to ten minutes washes in several changes of water, and passes through successive alcohols into glycerin or balsam. LENSSEN (La Cellule, xiv, 1898, p. 428) for the embryology of Hydatina, kills with hot saturated sublimate, dehydrates, stains lightly, embeds in paraffin and stains with heemalum. HIRSCHFELDER (op. tit., supra) narcotises with cocaine, and fixes with Fol's picro-chromic acid. BEAUCHAMP (Arch. Zool. Exper., iv, 1906, p. 29) finds 1 per cent, stova'ine better than cocaine for some forms. He (ibid., x, 1909, p. 77) fixes for five to ten minutes in 4 parts of 1 per cent, osmic acid, with 1 of 6 per cent, sublimate, and 5 of 5 per cent, bichromate of potash, and 1 drop of acetic acid for each 2 c.c., and embeds in celloidin, and then through chloroform in paraffin (three to ten minutes). See also TOZER (Journ. Roy. Micr. Soc., 1909, p. 24). 964. Acanthocephali. - - SAEFFTIGEN (Morph. Jahrb., x, 1884, p. 120) obtained the best results by killing gradually with 0-1 per cent, osmic acid ; the animals placed in this contract during the first hours, but stretch out again and die fully extended. Similarly with 0-1 per cent, chromic acid ; Ecninorhynci live for days in it, but eventually die fully extended. HAM ANN (Jen. Zeit., xxv, 1890, p. 113) has succeeded with sub- limate, and also with alcohol containing a little platinum chloride. KAISER (Biblioth. Zool., H. vii, 1 Halfte, 1891, p. 3) found that a saturated aqueous solution of cyanide of mercury, warmed to 45° to 50° C., and allowed to act for from fifteen to sixty minutes, and then washed out with 70 per cent, alcohol, was the best of all fixing media. 965. Nematodes. — The impermeable cuticle is a great obstacle to preparation. According to Looss (Zool. Anz.} 1885, p. 318) this difficulty may be overcome in the manner described in § 545. Wash in 1 per cent, saline (if necessary) and fix in boiling 70 per cent, alcohol ; store in fresh 70 per cent, for examination. If this method is properly applied the worms will die extended and straight. For examination transfer to 70 per cent, alcohol made up with 5 per cent, glycerine. Place small bottle of this fluid, plus worms, on incubator at 60° C., and allow to evaporate slowly for about twenty- four hours or even two days, which finally leaves the worms in viscid, almost pure, glycerine. Examine in pure glycerine, or glycerine M. 33 514 METHODS FOR INVERTEBRATES. jelly. For rapid examination after killing in alcohol, transfer to absolute alcohol for thirty minutes, and clear in " white " creosote. (LEIPER, in Science of the Sea, London. John Murray. 1912.) For fixing, most recent authors recommend sublimate solutions ; chromic solutions seem to have a tendency to make the worms brittle. But, according to ZUE STRASSEN (Zeit. wiss. Zool., liv, p. 655), Bradynema rigidum ought to be fixed for at least twelve hours in mixture of Flemming. AUGSTEIN (Arch. Naturg., Ix, 1894, p. 255) takes for Strongylus filaria Mayer's picro-nitric acid. VEJDOVSKY (Zeit. wiss. Zool., Ivii, 1894, p. 645) advises for Gordius 0-5 per cent, chromic acid (twenty-four hours). Lo BIANCO employs for marine forms concentrated sublimate or picro-sulphuric acid. Looss (Zool. Anz., xxiv, 1901, p. 309) prefers hot (80° to 90° C.) alcohol of 70 per cent. GLAUE (Zeit. wiss. Zool., xcv, 1910, p. 554) kills Ascaris in a hot mixture of 100 parts of saturated sublimate, 100 of alcohol, and 1 of acetic acid. Staining is frequently difficult, and sometimes alcoholic carmine §234A, is the only thing that w^ll give fair results. BRAUN (see Journ. Eoy. Mic. Soc., 1885, p. 897) recommends that small unstained Nematodes be mounted in a mixture of 20 parts gelatin, 100 parts glycerin, 120 parts water, and 2 parts carbolic acid, which is melted at the moment of using. Canada balsam, curiously enough, is said to sometimes make Nematodes opaque. Demonstration of living Trichinae. — BARNES (Amer. Mon. mik. Journ., xiv, 1893, p. 104) digests trichinised muscle (of the size of a pea) in a mixture of 3 gr. of pepsin, 2 dr. of water, and 2 minims of hydrochloric acid, kept at body temperature for about three hours. The flesh and cysts being dissolved, the fluid is poured into a conical glass, and allowed to settle ; the trichinae are drawn off from the bottom with a pipette, got on to a slide with water and examined on a hot stage. GRAHAM (Arch* mik. Anat., 1, 1897, p. 216) isolates Trichinae by macerating for one or two days in 2 per cent, acetic acid, staining with aceto -carmine, and teasing. 966. Nemertina. — My best results have always been obtained by fixing with cold saturated sublimate solution, acidified with acetic acid. The other usual fixing agents, such as the osmic and chromic mixtures, seern to act as irritants, and provoke such violent muscular contractions that the whole of the tissues are crushed out of shape by them. CHAPTER XXXVI. 515 Prof. DU PLESSIS has suggested to me fixing with hot (almost boiling) water. I have tried it and found the animals die in extension, without vomiting their proboscides. So also JOUBIN, Bull. Mus. Hist. Nat., 1905, p. 326. I have tried FOETTINGER'S chloral hydrate method (§ 20). My specimens died fairly extended, but vomited their proboscides. According to Lo BIANCO narcotisation with a solution of 0-1 to 0 -2 per cent, in sea water for six to twelve hours is useful. OESTERGREN (§ 18) recommends his ether water. DENDY (see Journ. Roy. Mic. Soc., 1893, p. 116) has succeeded with Geonemertes by exposing it for half a minute to the vapour of chloroform. For staining fixed specimens in toto I have found that it is well-nigh necessary to employ alcoholic stains. Borax-carmine or Mayer's alcoholic carmine may be recommended ; not so cochineal or haematoxylin stains, on account of the energy with which they are held by the mucin in the skin. Sections by the paraffin method, after penetration with oil of cedar (chloroform will fail to penetrate sometimes after a lapse of weeks). BURGER (Fauna u. Flora Golf. Neapel, xxii, 1895, p. 443) studies the nervous system, nephridia, skin, muscle and intestine by the intra vitam methylen-blue method. He injects the animals with 0 -5 per cent, solution in distilled water, or 0 -5 per cent, salt water, and allows them to lie for six -to twelve hours or more in moist blotting-paper. See also MONTGOMERY (ZooL Jahrb., Abth. Morph., x, 1897, p. 6) ; and BOHMIG (Zeit. wiss. Zool., Ixiv, 1898, p. 484). 967. Cestodes. — Wash gently in 1 per cent, saline, and then fix in hot corrosive sublimate acetic (at circa 50° C.) and allow the tape- worms to remain in the dish till the fluid becomes cold. Wash in running water for twelve hours and transfer to 70 per cent, alcohol. Stain as in general methods. As pointed out by VOGT and YUNG (Traite d'Anat. Comp. Prat., p. 204), the observation of the living animal may be of service, especially in the study of the excretory system. And, as shown by PINTNER, Tsenise may be preserved alive for several days in common water to which a little white of egg has been added. TOWER (ZooL Jahrb., xiii, 1899, p. 363) has kept Moniezia expansa alive for several days in a mixture of 100 c.c. of tap water, 10 grs. of white of egg, 2 of pepsin, 2 of sugar, and 5 of prepared beef (" Bovox "). Chloride of sodium, he says, should be avoided. 33—2 516 METHODS FOE INVERTEBRATES. LONNBERG (Centmlb. Bakteriol., xi, 1892, p. 89 ; Journ. Roy. Mic. Soc., 1892, p. 281) has kept Tricenophorus nodulosus alive for a month in a slightly acid pepsin-peptone solution containing from 3 to 4 per cent, of nutritive matter and less than 1 per cent, of NaCL For the nervous system, TOWER (Zoo?. Am., xix, 1896, p. 323) fixes in a picro-platin-osmic mixture (stronger than that of 0. VOM RATH, § 101) for ten hours, then treats for several hours with crude pyroligneous acid, and lastly with alcohol, and embeds in paraffin. ZERNECKE (Zool. Jahrb., Abth. Anat., ix, 1895, p. 92) kills Ligula in the osmio-bichromic mixture of GOLGI (4 : 1), impregnates as usual, makes sections in liver, and treats them by the hydroquinon process of KALLIUS. Besides the peripheral and central nervous system, muscle-fibres, parenchyma cells, and the excretory vascular system are impregnated. He has also obtained good results by the methyl en- blue method. BLOCHMANN (Biol. Centralb., xv, 1895, p. 14) recommends the bichromate and sublimate method of GOLGI. See also KOHLER, Zeit. wiss. Zool., Ivii, 1894, p. 386 (stretches Tsenise round a glass plate or on cork, and fixes with 5 per cent, sublimate) ; LUHE, Centralb. Bakt., xxx, 1901, p. 166, and RANSOM, U. 8. Nation, Mus. Bull, Ixix, 1909, p. 8. 968. Trematodes. — If necessary, clean by shaking up in 1 per cent, saline (parasites). Decant off dirty liquid, one-third of the tube is filled again with 1 per cent, saline, in which the worms are shaken vigorously, and an equal quantity of HgCl2 solution is added quickly, the vigorous shaking being continued for several minutes thereafter. This treatment should kill the flukes in an extended condition. Leave in the fixer as indicated (corrosive one or two days, wash in water twelve hours if 10 per cent, formalin be substi- tuted for the HgCl2 ; leave about same time and store in 3 per cent, formalin). FISCHER (Zeit. wiss. Zool., 1884, p. 1). — Opisthotrema cochleare may be mounted entire in balsam. For sectioning, he recommends a mass made by dissolving 15 parts of soap in 17-5 parts of 96 per cent, alcohol. The sections should be studied in glycerin. Lo BIANCO fixes Trematodes with hot saturated sublimate. Looss (Arch. mik. Anat., 1895, p. 7) takes for Bilharzia warm (50° to 60° C.) 1 per cent, sublimate in 70 per cent, alcohol. BETTENDORP (Zool. Jahrb., Abth. Morph., x, 1897, p. 308) has had good results with the rapid Golgi method only on Distoma hepaticum, and prefers methylen blue. CHAPTER XXXVI. 517 HAVET (La Cellule, xvii, 1900, p. 353) has also had results with the Grolgi method on this form, and also with thionin (after fixing with sublimate), which demonstrates tigroid substance. Cercarice. — SCHWARZE (Zeit. wiss. ZooL, xliii, 1886, p. 45) found that the only fixing agent that would preserve the histological detail of these forms was cold saturated sublimate solution warmed to 35° to 40° C. For an " indifferent liquid, HOFMANN (ZooL Jahrb., xii, 1899, p. 176) takes 1 part of white of egg in 9 of normal salt solution. 969. Turbellaria.— BRAUN (Zeit. wiss. MiL, iii, 1886, p. 398) gets entire animals (Rhabdocoela) on to a slide, lightly flattens out with a cover, and kills by running in a mixture of 3 parts of liquid of Lang with 1 of 1 per cent, osmic acid solution. (BoHMiG [ibid.], commenting on this, says that for some of the tissues, such as muscle and body parenchyma, nitric acid and picro-sulphuric acid are very useful.) Sections may be made by the paraffin method. DELAGE (Arch, de Zool. exp., iv, 2, 1886) recommends fixation (of Khabdocoela Acoela) by an osmium -carmine mixture, for which see loc. cit., or by concentrated solution of sulphate of iron. Liquid of Lang was not successful. For staining, he recommends either the osmium -carmine or impreg- nation with gold (^ formic acid, two minutes ; 1 per cent, gold chloride, ten minutes ; 2 per cent, formic acid, two or three days in the dark). BOIIMIG (Zeit. wiss. Mik., iii, 1886, p. 239) has obtained instructive images with Plagiostomidae fixed 'with sublimate and stained with the osmium -carmine. GRAFF (Turbellaria Acvela, Leipzig, 1891 ; Zeit. wiss. Mik., ix, 1892, p. 76) says that chromo-aceto-osmic acid, followed by hsema- toxylin, is good for the skin, but not for the Rhabdites, which in Accela and Alloioccela seem to be destroyed by swelling. The same method is also good for the parenchyma of Amphichoerus cinereus, Convoluta paradox^ and C. sordida. Sublimate is good for -Convolute Roscoffensis. The nervous system may be investigated by the methods of DELAGE. For Dendroccela sublimate solutions, sometimes hot, appear indicated for fixing ; see the mixture of LANG, § 64, also CHICHKOFF (Arch, de BioL, xii, 1892, p. 438). ARNOLD (Arch. Zellforsch., iii, 1909, p. 433) kills Dendrocoelum in extension (?) with strong liquid of Flemming. OESTERGREN narcotises Dendrocoelum with his ether- water, § 18. JAENICHEN (Zeit. wiss. Zool., Ixii, 1896, p. 256) advises for Planaria, eyes especially, picro-sulphuric acid for an hour or two ; 518 METHODS FOR INVERTEBRATES. osmic acid is not good, and liquid of Miiller macerates. He stains with borax-carmine, makes sections, and puts them for ten minutes into osmic acid, then for five minutes into pyroligneous acid, on the top of the stove. He macerates the visual rods in a mixture of 1 part common salt, 1 of acetic acid, and 100 of water. He bleaches the pigment of the eyes with peroxide of hydrogen. WILHELMI (ibid., Ixxx, 1906, p. 548) throws Triclads into almost boiling mixture of Zenker, and after ten to thirty minutes removes to water for some hours, and then passes into iodine alcohol. Echinodermata . 970. Holothurioidea.- -These are difficult to fix on account of their contracting with such violence under the influence of irritating reagents as to expel their viscera through the oral or cloacal aperture. VOGT and YUNG (Anat. Comp. Prat., p. 641) say that Cucuniaria Planci (C. doliolum, Marenzeller) is free from this vice ; but they recommend that it be killed with fresh water, or by slow intoxication, §25. Synapta may be allowed to die in a mixture of equal parts of sea water and ether or chloroform (S. Lo BIANCO). OESTERGREN (§ 18) puts Synapta into his ether water, but Dendro- chirota first into magnesium sulphate of 1 to 2 per cent., for some hours. GEROULD (Bull. Mus. Harvard Coll., xxix, 1896, p. 125) paralyses Caudina with sulphate of magnesia, § 24, and fixes with liquid of Perenyi (or sublimate for the ovaries). Holothurids, Dr. WEBER informs nie, are admirably preserved in formaldehyde ; a weak solution is sufficient. For the staining of muscles with methylen blue, see IWANZOFF, Arch. mile. Anat., xlix, 1897, p. 103 ; and for the study of calcareous plates, see WOODLAND, Quart. Journ. Micr. Sci., xlix, 1906, p. 534 (fixation with osmic acid, staining with picro -carmine, followed by Lichtgriin). 971. Echinoidea. — I advise that they be killed by injection of some fixing liquid. For preservation, formaldehyde has proved admirable in all respects, and greatly superior to alcohol (WEBER). Lo BIANCO kills by pouring over them (mouth upwards) a mixture of 10 parts acetic acid and 1 of 1 per cent, chromic acid, and brings at once into weak alcohol. Or he makes two holes in the shell, lets the water run out and alcohol run in. Sections of spines may be made by grinding, see § 177. CHAPTER XXXVI. 519 Spicula and the skeleton of pedicellarise may be cleaned by eau de Javelle, see DODERLEIN (Wiss. Ergeb. Tiefsee-Eioped., v, 1906, p. 67). 972. Asteroidea.— HAMANN (Beitr. Hist. Echinodermen, ii, 1885, p. 2) injects the living animal with a fixing liquid through the tip of a ray. The ambulacral feet and the branchiae are soon distended by the fluid, and the animal is then thrown into a quantity of the same reagent. In order to study the eyes, with the pigment preserved in situ, they should be removed by dissection, should be hardened in a mixture of equal parts of 1 per cent, osmic acid and 1 per cent, acetic acid, and sectioned in a glycerin gum mass, or some other mass that does not necessitate treatment with alcohol (which dissolves out the pigment, leaving the pigmented cells perfectly hyaline). For maceration use one-third alcohol, the aceto-osmic mixture failing to preserve the rods of the pigmented cells. Specimens for externals only preserve in 70 per cent, -alcohol or formalin. They retain their shape better if they are put for two or three minutes into fresh water before being placed in the fixer. If the internal anatomy is to be studied, cut along the length of each arm so as to allow fluid to enter, and preserve in 2 per cent, chromic acid, etc. Wash in running water, transfer to 70 per cent, alcohol ; or the specimen may be preserved in formalin spirit or 5 per cent. formalin. Formaldehyde is not to be recommended for the cell preservation of Asteroidea (WEBER). See also *Lo BIANCO, op. cit. (he kills Brisinga with absolute alcohol), also §§ 17, 20. 973. Ophiuridea should in general be killed in fresh water if it be desired to avoid rupture of the rays (DE CASTELLARNAU, La Est. Zool. du Napoles, p. 135). Lo BIANCO kills small forms with weak alcohol, Ophiopsila with absolute alcohol, and Ophiomyxa with 0 -5 per cent, chromic acid. Russo (Richerche Lab. Anat. Roma, iv, 1895, p. 157) fixes Ophiothrix for an hour or two in 0-5 per cent, osmic acid and then decalcifies in solution of Muller for six to ten days. Or he fixes for three minutes in a mixture of 2 parts concentrated sublimate solution, 1 part 70 per cent, alcohol, and 1 part acetic acid (sp. gr. 1-06), and decalcifies in Muller or in 70 per cent, alcohol with 10 per cent, of acetic acid. He stains with paracarmine. 974. Crinoidea. — Lo BIANCO (loc. cit., p. 458) fixes Antedon rosacea with 70 per cent, alcohol, A. phalangium with 90 per cent. 520 METHODS FOR INVERTEBRATES. 975. Larvae of Echinodermata (from instructions written down for me by Dr. BARROIS). — For the study of the metamorphoses of the Echinoidea and Ophiuridea it is necessary to obtain preparations that show the calcareous skeleton preserved intact (a point of con- siderable importance, since this skeleton frequently affords land- marks of the greatest value), and that give clear views of the region of formation of the young Echinoderm (which is generally opaque in the living larva). They should also possess sufficient stiffness to allow of the larva being turned about in any desired way, and placed in any position under the microscope. Pluteus larvse should be fixed in a cold saturated solution of corrosive sublimate, for not more than two or three minutes, then washed with water, and brought into dilute Mayer's cochineal (§ 235). This should be so dilute as to possess a barely perceptible tinge of colour. They should remain in it for from twelve to twenty- four hours, being carefully watched the while, and removed from it at the right moment and mounted in balsam, or, which is frequently better, in oil of cloves or cedar-wood. Auricularia and Bipinnaria. — As above, but the earlier stages of the metamorphosis of Auricularia are better studied by fixing with osmic acid, staining with Beale's carmine, and mounting in glycerin. Larvce of Comatula are best fixed with liquid of Lang, and stained with dilute borax-carmine. It is important (for preparations that are not destined to be sectioned) to use only dilute borax-carmine, as the strong solution produces an over-stain that cannot easily be reduced. Narcotisation by chloral hydrate before fixing is useful, especially for the study of Pentacrinus larvse and of the young Synaptce formed from Auricularia. Without this precaution you generally get preparations of larvse either shut up (Pentacrinus), or entirely deformed by contraction (young Synaptce). See also MACBRIDE on the development of Amphiura squamata, Quart. Jo urn. Micr. Sci., xxxiv, 1892, p. 131 (osmic acid followed by liquid of Muller and alcohol ; decalcification with nitric acid in alcohol ; staining with Mayer's paracarmine or hsemalum) ; and SEELIGER on the development of Antedon, Zool. Jahrb., Abth. Anat., vi, 1892, p. 161. MACBRIDE (Quart. Journ. Micr. Sci., xxxviii, 1896, p. 340) fixes larvse of Asterina in osmic acid, brings into liquid of Muller for twelve to four- teen hours, imbeds in celloidin followed by paraffin (see § 171), and stains sections with carmalum or Delafi eld's haematoxylin, best after a foregoing stain of twenty -four hours in borax carmine. MAYER (Ghrundzuge, LEE and MAYER, 1910, p. 486) arranges a number of fixed and stained Plutei on a sheet of gelatin foil gummed CHAPTER XXXVI. 521 to a slide with euparal, dehydrates by adding alcohol by drops, and adds euparal and a cover. See also WOODLAND, Quart. Journ. Micr. Sri., xlix, 1905, p. 307. Ccelentemta. 976. Thread-Cells. — IWANZOFF (Bull. Soc. Nat. Moscow, x, 1896, p. 97) advises for the Nematocysts of Actiniae maceration by HERT- WIGS' method, § 534, or better, fixation for two to five minutes with vapour of osmium followed by a short washing with sea water or distilled water. For Medusne he also advises HERTWIGS' method, § 526, or treat- ment with a solution containing methyl green and gentian violet with a little osmic acid. 977. LITTLE (Journ. App. Mic., vi, 1903, p. 2116 ; Journ. Roy. Mic. Soc., 1903, p. 237) kills Hydra in hot saturated sublimate in 70 per cent, alcohol, washes with alcohol, stains for five minutes in strong solution of methylen blue, dehydrates rapidly, clears with cedar or bergamot oil, and mounts in balsam. Nematocysts blue, the rest unstained. 978. Actinida. — Ana3sthetise in menthol (§ 15), which will take some twelve hours or more. For ordinary sea anemones, formalin (5 to 10 per cent.) followed by formalin spirit is to be preferred. For corals, such as Caryophyllia, Alcyonium or Gorgonia, ansesthe- tise, and then add hot corrosive sublimate or 5 per cent, formalin, followed by cold saturated corrosive sublimate. Ninety per cent, spirit, not allowed to get weaker than 70 per cent., gives good results for anatomical work (ALLEN and BROWNE, loc. cit.). For other narcotisation methods see §§ 15 to 26. 979. Fixation. — In Le Attinie, Fauna u. Flora d. Golfes v. Neapel, ANDRES says that hot corrosive sublimate often gives good results. In the case of the larger forms the solution should be injected into the gastric cavity. Freezing sometimes gives good results. A vessel containing Actinise is put into a recipient containing an ice and salt freezing mixture and surrounded by cotton-wool. After freezing, the block of ice containing the animals is thawed in alcohol or some other fixing liquid. DUERDEN (Journ. Inst. Jamaica, ii, 1898, p. 449) narcotises with magnesium sulphate, § 24, and fixes with formol of 3 to 5 per cent. 980. Maceration. — For the HERTWIGS' method (Jen. Zeit., 1879, p. 457) see § 526. The tissues should be left to macerate in the acetic acid for at least a day, and may then be teased in glycerin. 522 METHODS FOR INVERTEBRATES. LIST (Zeit. wiss. Mik., iv, 1887, p. 211) treats tentacles of Anthea cereus and Sagartia parasitica for ten minutes with a mixture of 100 c.c. of sea water with 30 c.c. of Flemming's strong liquid, then washes out for two or three hours in 0 -2 per cent, acetic acid, and teases in dilute glycerin. Picro-carmine may be used for staining. 981. Nervous system.- -This group is generally held to be refractory to the Golgi impregnation. HAVET, however (La Cellule, xviii, 1901, p. 388), has obtained good results by the rapid method on young specimens of Metridium dianthus. Besides nerve-cells, there are impregnated neuro-muscular cells, gland-cells, and nematocysts. He leaves for five to eight days in the osmic mixture. He has also had good results by the intra vitam methylen blue method (this is also good for nematocysts). So also has GEOSELJ (Arb. Zool. Inst. Univ.- Wien, xvii, 1909, p. 269), adding the dye to the water with the animals till it gives a steel-blue tint. 982. Zoantharia with Calcareous Skeletons are difficult to deal with on account of the great contractility of the polyps. Sublimate solution, which ought very often to be taken boiling, sometimes gives good results. See also Lo BIANCO, loc. cit., p. 446. Sections. — See §§ 177 and 178, for undecalcified specimens. 983. The Alcyonaria have also extremely contractile polyps. In a former edition I suggested for their fixation either hot sublimate solution or glacial acetic acid (§ 84). S. Lo BIANCO has since recom- mended essentially similar processes. GARBINI (Manuale, p. 151) drenches them with ether, and brings into strong alcohol. WILSON (Mitth. Zool. Stat. Neapel, 1884, p. 3) kills Alcyonaria with a mixture of 1 part of strong acetic acid and 2 parts of concen- trated solution of corrosive sublimate, the animals being removed as soon as dead and hardened for two or three hours in concentrated sublimate solution. 984. Zoantharia and Alcyonaria. — BRAUN (Zoo/. Anz., 1886, p. 458) inundates Alcyonium palmatum, Sympodium coralloides, Gorgonia verrucosa, Caryophyllia cyathus, and Palythoa axinellce with a mixture of 20 to 25 c.c. of concentrated solution of sublimate in sea water with 4 to 5 drops of 1 per cent, osmic acid, and after five minutes passes into successive alcohols. (This method also gives good results with Hydra and some Bryozoa and Rotifers.) See also S 14. CHAPTER XXXVI, 523 BUJOR (Arch. Zool. exper., ix, 1901, p. 50) kills Veretillum in sea water containing 10 per cent, each of formol and ether, and after a minute passes into 2 per cent, solution of formol in sea water. 985. Hydroidea in General. — Directly the tow-net comes on board, the Plankton must be poured into a glass jar and jelly fishes at once picked out by means of a lifter or pipette, and placed in another very clean jar of sea water. Leave in this jar for half an hour to allow organisms to recover from shock. Note that the slightest trace of chemicals in the jar will prevent their expanding. The secret of successful preservation depends on keeping the animals in motion while you pour in the fixer. First stir the organisms very slowly and gently, and when all are in motion begin to pour the formalin slowly down the side of the vessel. About 10 c.c. of 10 per cent, formalin should go to 100 c.c. of sea water, but better more than this quantity. Keep stirring for at least two minutes after addition of fixer. Leave for a few hours and then transfer to 5 per cent, formalin ; finally store in 10 per cent. To obtain medusae in a nice state of expansion it is necessary to use an anaesthetic (see especially § 15). Hydrochloride of cocaine is possibly the best ; use a 1 or 2 per cent, solution. Place the medusae in a small glass vessel with just enough sea water to allow them to swim. After they have expanded add a little cocaine (3 c.c. of 1 per cent, solution for every 100 c.c. of sea water). If the medusae at the end of ten to fifteen minutes do not contract when touched with a glass rod no more cocaine is needed ; if they are still active add more narcotiser and stir ; an over-dose will cause prolonged contraction. After anaesthetisation add the formalin and keep stirring, and continue for a minute, or longer. Do not leave speci- mens in solutions of cocaine longer than necessary. (ALLEN and BROWNE in Science of the Sea. London. John Murray. 1912.) For further description of narcotisation methods see §§ 15 et seq. For killing by heat see § 13. Fixation. — In general polyps may be very well killed in saturated sublimate solution, in which they should be plunged for an instant merely, and be brought into alcohol. The solution should be employed cold in general for Gymnoblastea, hot for most Calyptoblastea. Ether attentively administered gives good results with Campanu- laridae. Hydra is very easily killed by a drop of osmic acid on a slide. For the methylen blue intra vitam method, see Chapter XVI ; also HADZI, Arb. Zool. Inat. Wien, xvii, 1909, p. 225. 986. Medusae : Fixation. — For narcotisation see § 17 and above. Trachymedusae and Acalephae may be fixed in the usual way in 524 METHODS FOE INVERTEBRATES. chromic or osmic mixtures. Osmic acid may be added to the sea water containing the animals, which should be removed to spring water as soon as they begin to turn brown. 987. BIGELOW (Mem. Boston 8oc. nat. Hist., v, 1900, p. 193) fixes the scyphistomes of Cassiopeia in Lo Bianco 's mixture of 10 parts of 10 per cent, solution of cupric sulphate with 1 of saturated sublimate, and hardens them in 5 per cent, bichromate of potash. 988. Medusae : Sections. — Paraffin and collodion are certainly not satisfactory as all-round methods for these watery organisms. The HERTWIGS (Nervensystem der Medusen, 1878, p. 5) embedded in liver with the aid of glycerin gum, and hardened the objects and the mass in alcohol. See also JOLIET'S glycerin gum method, and the gelatin methods in Chapter VIII. 989. Medusae : Maceration. — See, especially for the study of the nervous system, § 526. Doubtless in many cases the pyrogallic acid reaction, § 374, would give enhanced differentiation. 990. Siphonophora. — For the cupric sulphate method of BEDOT (Arch. Sci. phys. et nat., xxi, 1889, p. 556), which is admirable for the preparation of museum specimens, but not necessary for histological work, as well as for those of Lo BIANCO (op. cit., p. 454), FRIED- LANDER (Biol. CentrbL, x, 1890, p. 483), and DAVIDOFF (Anat. Anz., xi, 1896, p. 505) see previous editions. Lo BIANCO fixes most forms with the mixture given in 8 1021. ^j o For preserving, according to WEBER, formaldehyde is better than alcohol. DAVIDOFF (loc. cit.) fixes in it. 991. Ctenophora : Fixation. — Never store in formalin, always in 70 per cent, alcohol. Pleurobrachia are best killed in 5 per cent, formalin in sea water. Fill large measuring jar with this fluid, drop in the animals and leave till they sink to the bottom ; transfer to 5 per cent, formalin in pure water. After a week or so (not longer) transfer to very dilute alcohol, and up-grade to 70 per cent, strength. Beroe : Bring into small quantity of sea water, and when expanded add large quantity of corrosive sublimate saturated solution in sea water. When specimens become white, decant and add fresh water ; wash in several changes to remove corrosive, up-grade to 70 per cent, alcohol. Bolina dissolves at once in formalin ; kill in Flemming, selecting small specimens : leave half an hour, wash slightly, up-grade to 70 per cent, alcohol. (ALLEN and BROWNE in Science of the Sea. London. John Murray. 1912.) CHAPTER XXXVI. 525 Small forms are very easily prepared by means of osmic acid. For the large forms see Lo BIANCO, loc. tit., p. 457. He uses his copper sulphate mixture, § 987. SAMASSA makes sections by the double-embedding method. See Arch. mik. Anat., xl, 1892, p. 157. 992. Plankton, Preservation of, without Sorting (E. J. ALLEN and E. T. BROWNE in Science of the Sea. John Murray. 1912). — Pre- servation of whole catch of a tow-net is performed by stirring around the Plankton with a rod and adding a little 5 to 10 per cent, formalin. Keep on stirring for about a minute, then allow the organisms to settle to the bottom ; as soon as this occurs pour off as much of the liquid as possible and transfer the Plankton to a bottle ; again allow to settle and reduce the fluid to a minimum ; then fill the bottle with 5 or 10 per cent, formalin. A bottle should be not more than half full of Plankton. After a few days, or on the appearance of opalescence of the fluid, change the liquid. Another method is first to fill the Plankton by pouring some saturated solu- tion of picric acid into the jar containing the organisms, then add some formalin 5 or 10 per cent, and leave for an hour or two, occasionally stirring. Finally decant and add 5 or 10 per cent, formalin as before described ; the yellow colour of the fluid can be neglected. Never use corrosive sublimate with formalin, as crystals form, which adhere to the organisms. Porifera. 993. Spongiae : Fixation. — The smaller forms can be fairly well fixed by the usual reagents, osmic acid being one of the best. For the larger forms absolute alcohol is apparently the best. If any watery fluid be preferred, care should at all events be taken to get the sponges into strong alcohol as soon as possible after fixation, on account of the rapidity with which maceration sets in in watery fluids. FIEDLER (Zeit. iviss. ZooL, xlvii, 1888, p. 87) has been using (for Spongilla), besides absolute alcohol, an alcoholic sublimate solution and the liquids of Kleinenberg and Flemming. Staining. — To avoid maceration, I hold that alcoholic stains should be alone employed, and I recommend Mayer's tincture of cochineal, § 235. VON LENDENFELD (Zeit. wiss. Mik., xi, 1894, p. 22) uses aqueous solutions of Congo red and anilin blue for the coloration of collar-cells. MINCHIN (Quart. Journ. Mic. Sci., xl, 1898, p. 569) stains spicula sheaths with Freeborn's picro-nigrosin, § 742. 526 METHODS FOR INVERTEBRATES. ROUSSEAU (Ann. Soc. Belg. Mic., xxiv, 1899, p. 51) stains in nigrosin, picro-nigrosin, or indulin, or MAYER'S picro-magnesia carmine. Prof. Dendy informs me that he uses Hickson's brazilin (§ 378) a great deal in his work on sponges. For intra vitam staining, see LOISEL, § 207, ante, p. 130. For silvering, see § 356. Sectioning. — Calcereous sponges may be decalcified in alcohol, acidified with hydrochloric or nitric acid, and then embedded in the usual way. Siliceous sponges may be desilicified, § 566. For ROUSSEAU'S methods, see § 566. VOSMAER and PEKELHARING decalcify with a solution of picric acid in absolute alcohol (see Zeit. wiss. Mik., xv, 1899, p. 462). See also Johnstone-Lavis and Vosmaer, § 179. Preparation of Hard Parts. — Siliceous spicules are easily cleaned by treating them on a slide with hot concentrated nitric or hydro- chloric acid, or solution of potash or soda. The acids mentioned are very efficient, but may attack the silex of some delicate spicules. Potash solution is, therefore, frequently to be preferred, notwith- standing that, in my experience, it does not give such clean pre- parations. According to NOLL, eau de Javelle is preferable to any of these reagents, see § 544. Embryos and Larvae.— MAAS (ZooL Jahrb., Abth. Morph., vii, 1894, p. 334) fixes larvse in liquid of Flemming or Hermann, one to three minutes, and stains with borax-carmine, or with gentian violet and Orange G (Flemming). He also (Zeit. wiss. ZooL, Ixvii, 1900, p. 218) fixes young Sycons in absolute alcohol and stains with ammonia carmine (spicules in situ). DELAGE (Arch. Zool. Exper., x, 1892, p. 421) fixes larvae of Spon- gilla that have settled down on cover-glasses for three minutes in absolute alcohol, stains in alcoholic carmine, and brings through alcohol into oil of bergamot, then either mounts direct in balsam, or detaches the larvse from the cover and imbeds in paraffin (three minutes). GATENBY (Journ. Linnean Soc., 1920) uses methods for mito- chondria, especially Champy-Kull and Kopsch. Protozoa.* 994. Introduction. — In the special study of the protozoa the various reagents and methods of general cytology are in great part * By Dr. A. Drew, Imperial Cancer Research Fund. CHAPTER XXXVI. 527 applicable, but numerous modifications have been introduced to meet special cases. Speaking generally, the main lines of study applied to the protozoa may be summed up under the following headings : — Culture, collection, determination of life cycles, general morphology, physiology. It will perhaps be appropriate to say a few words by way of introduction under each of these headings. 995. Culture. — No general -method is applicable to all cases ; for many of the free living protozoa, such as infusoria, amoebae and flagellates, a simple 1 or 2 per cent, hay infusion is suitable, which may be conveniently placed in small petri dishes ; these give the additional advantage of providing a large surface. Amoebae are in many cases best grown on a solid agar medium, to be described later. Special media have been described for the culture of many patho- genic forms, such as trypanosomes, and quite lately Sr. Monica Taylor has described a useful method for cultivating Amoebae proteus (§ 1013). 996. Pure Mixed Cultures of Amoebae. — When working with protozoa it is extremely desirable to have pure cultures whenever possible. In the case of amoebae, bacteria are necessary as food, so that we must grow our amoebae with bacteria. A culture containing one species of amoeba, together with a pure culture of any particular bacterium, is spoken of as a pure mixed culture, and it is necessary to obtain such pure mixed cultures in order properly to study the biology of the amoebae. The preparation of a pure mixed culture involves two distinct processes : (1) The separation from its fellows of a single amoeba ; and (2) its subsequent cultivation with a pure culture of some selected bacterium. (1) Separation of a Single Cyst.- -The easiest method of accom- plishing this is by means of the following method. Take a glass capillary tube about 5 to 10 cm. in length and 1 mm. in diameter. Flame the centre, and draw out quickly to the fineness of a hair. Break into two equal portions and reduce each of these to a length of 5 cm., so that one obtains two short tubes consisting of a wider portion and a very fine capillary portion. Next select a culture of the amoebae rich in cysts, add a drop of sterile water, and rub a portion of the growth into this with a sterile platinum wire. Allow a minute portion of this emulsion to run into the capillary end of the prepared tube, and then run in sterile water till about 0 -5 cm. of the broad portion of the tube is filled. Mix the contents of the tube by vigorous rotation. Now prepare an agar film on a micro- scope slide, by melting one of the stock tubes of the agar and pouring 528 METHODS FOE INVERTEBRATES. a few drops on a slide. Allow this to set. Place on the microscope and focus the upper surface with an inch objective. Tap out on to filter paper some of the liquid in the capillary tube, and then, whilst looking through the microscope, gently touch the film with the fine end of the tube. A small volume of the suspension of cysts will run on to the jelly and will spread out in an area which is quite visible, and which occupies only a small portion of the field. If no cyst is present, or if there should be more than one, place another drop on a fresh film and repeat till a single cyst is obtained on the film. The method is simple, and with practice one can make half a dozen such cultures in an hour. Place the slide film surface downwards above water in a petri dish (this is conveniently done by resting on two corks), and cover the dish. Examine day by day, till numerous amoebae are found, and then allow them to encyst. From this culture, prepare cultures in test tubes containing the special agar sloped as for bacteriological work. Allow these to grow for a week till covered with cysts. This may be observed with a low-powered lens through the wall of the test tube. Prepare a 3 per cent, solution of hydrochloric acid in sterile distilled water and cover the jelly film in the test tube with this solution and replace the plug. Allow this to act for twenty-four hours. Pour off the acid, and fill up with sterile water, and again pour off, using aseptic precautions through- out. With a platunim loop scrape some of the emulsion of cysts off the tube on to the surface of a fresh sterile tube of agar. Then add a loopful of a very dilute emulsion of the selected bacterium and rub gently over the tube. In the course of twenty-four to forty- eight hours the amoeba will excyst, and multiply, and will generally be found to be in pure culture with the bacterium added. Should they not be so on bacteriological analysis, allow to encyst and again treat the cysts with the acid and proceed as already described. It will be found that the best organisms wherweith to grow amceba3 are those naturally occurring in water, one of the best being Bacillus fluorescens non-liquefaciens. When pure mixed cultures have been obtained, the stock cultures should always be kept in test tubes, as the petri dish method over water, although excellent for impure stock cultures, is very liable to allow contaminations to occur. By means of the method described, amoebse have been obtained in pure mixed culture with the following bacteria, and with the results detailed below. NAME OF BACTERIUM. REMARKS. B. fluorescens liquefaciens . . Excystation rapid, good growth. B. fliioresf-ens non-liguefaeiens . Excystation rapid ^ood growth. CHAPTER XXXVI. 529 B. pyocyaneus . Excystation fairly rapid, growth good but amoebae encyst rapidly. B. prodigiosus . . . \ B. megatherium . . . . Excystation fairly rapid, growth B. siibtilis . . . . j S°od- B. proteus vulgaris . . . j Excystation fairly rapid, growth B. coli . . . . .1 poor, amoebae encyst very B. typhosus ... . . 1 soon. B. phlei . . . . . \ B. Eabinowitcli Excystation extremely slow, B. Ndbarro 3 Pneumococcus growth very poor. Excyst fairly rapidly, growth Meningococcus . . . ( slow, encystment very slow. Staphylococcus aureus . . Excyst fairly rapidly, growth fair. 997. Collection.- -The great majority of the free living protozoa are inhabitants of either fresh or salt water, and to obtain satis- factory specimens some method of collection is essential. There are several pieces of apparatus which are more or less essential, viz., a collecting stick with ring and net, collecting bottle and cutting knife, a good pocket magnifier (about x 10), and small specimen bottles. The collecting stick can be obtained from all opticians, and it is usually fitted with the necessary appliances. The net is perhaps the most essential part of the apparatus. It consists of a bag of soft mull muslin fastened to a brass ring about 9 inches in diameter at one end, whilst the other carries a rimmed glass bottle about 3 inches long by 1 inch wide. The cutting knife is a strong curved hook with a sharp knife edge, which is screwed to the end of the stick, and is occasionally useful for cutting pieces of water weeds, which would otherwise be out of reach. A flat bottle, which can be obtained from most opticians, is extremely useful for examining specimens secured by the net. In a round bottle it is often extremely difficult to see minute organisms clearly when examined with the magnifier, but with the flat bottle one can usually quickly ascertain whether a sample is worth keeping for study with the microscope. The magnifier should be a good one and should be aplanatic. The most generally useful will be found to be either Watson's aplanatic loops; or one of Zeiss's hand lenses. The most generally useful powers will be either the : 6 or 10, preferably both. The various forms of pond life can be divided, for collecting purposes, into the free, and attached, and these groups will require different treatment in the methods of collection. The free swimming forms are often designated by the term " plankton," and these will be best ob- tained by means of the net. In order to collect material with the net it is passed through the water half a dozen times or so and then with- drawn ; the pond water runs out, but the various infusoria, rotifera, etc., are retained and are finally condensed in the bottle. They should then be emptied either into the flat bottle for preliminary examination, M. 34 530 METHODS FOR INVERTEBRATES. or into one of the specimen tubes to carry home. The attached forms, such as hydra, polyzoa, vorticella, are generally found on water weeds or bits of stick or submerged roots. In order to secure such organisms the weeds, etc., are severed with the cutting knife and are dragged on shore, placed in water in the flat bottle and examined. Polyzoa seem to have a preference for submerged rootlets, whilst forms such as vorticella are more frequently found on the roots and stems of duckweed. 998. Determination of Life Cycles. — The only really satisfactory method of determining life cycles is to follow a single organism con- tinuously throughout its various changes in the manner originally used by the late Dr. Dallinger, but such observations should be controlled by examination of suitably stained specimens in the different stages, under critical illumination. In the case of many of the parasitic pro. tozoa such methods are impossible, as cultures cannot be obtained. In such cases we are only able to examine fixed and stained preparations and endeavour to piece out a life cycle from the appearances observed. This must be checked by observations of the living material wherever possible. For free living protozoa some means of keeping a drop of the culture fluid from drying is necessary, but any means adopted must permit of the continuous examination of the organisms by high -power lenses. One of the best is that used by Dallinger and Drysdale, and described in The Microscope and its Revelations, edited by DALLINGER, 8th ed.5 Part I, pp. 341 to 344. For the majority of flagellates this is an excellent arrangement. Its chief drawback seems to be due to the fact that it does not permit the aeration of the culture fluid, and it is, there- fore, found that organisms such as large ciliates, many amoebae, vorti- cellse, etc., soon die out from lack of oxygen. The simplest method of examining such forms is the well-known hanging drop arranged on a hollow ground slide. This, however, does not permit of critical illumina- tion and completely upsets the corrections of the condenser. To get over this difficulty a slide with a small table ground out is used. The table is surrounded by a trench, and a ring of vaseline is painted round the outside of the trench, and a drop of the fluid containing the organisms to be studied is placed on the table. A cover -glass is then lowered on to the drop and adjusted for pressure by gently pressing on to the vaseline. It is advisable in most cases to arrange the drop so that it does not spread entirely to the edge of the table ; this ensures an air supply. For amoebae and many ciliates and flagellates the live slide described by DREW and GRIFFIN (Journal of Eoyal Microscopical Society, February 21st, 1917) maybe used. This form consists of a glass plate cut to fit the mechanical stage of the microscope, and with a glass arm cemented along one side. A piece of linen has a hole slightly larger than the cover- glasses to be used cut in it, and this is then damped with water and laid upon the slide. A small glass vessel filled with water is attached to the arm and is put in communication with the cloth by means of a piece of linen or soft wick. The linen is thus kept moist by capillarity. A drop of the culture is placed on the slide and a cover-glass placed upon it, and adjusted so that the circular opening in the linen touches it at the margins, pressure being regulated, if necessary, CHAPTER XXXVL 531 by the insertion of cigarette paper between slide and cover -glass. So long as water remains in the vessel attached to the arrn, the linen and the fluid under the cover will remain moist. For many purposes the live slide devised by Botterill and described in The Microscope and its Revelations by DALLINGER, p. 340, will be found extremely useful. 999. General Morphology. — Here great attention must be paid to modern cytologic methods. The most perfect fixing and staining technique should be used in any detailed study of the protozoa, and yet this is seldom found. Methods of fixation will necessarily differ according to whether one wishes to study nucleus, cytoplasm or such cytoplasmic inclusions as mitochondria or Golgi apparatus. Different methods are also frequently necessary according to the organisms studied. For purposes of convenience it is proposed to treat the fixing and staining of protozoa under the following head- ings i—Amoebce, Coccidia, Ciliata, Flagellata, Hcemamoebce. 1000. Amoabae. — For temporary purposes many amoebae may be stained and fixed by running a drop of 1 per cent, chromic acid under the cover-glass and then running in a little alum carmine followed by water. Staining intra vitam is very conveniently carried out by means of an agar jelly. A 2 per cent, solution of agar in distilled water is made ; this is cleared with egg white and filtered hot and distributed in 5 c.c. quantities in test tubes. For use a tube is melted by steeping in boiling water in a beaker and a few drops of the selected stain is added and well mixed. A little of the molten agar is then poured on to a slide and allowed to set. The amoebse are distributed in a drop of fluid on a cover-glass, which is then inverted on the jelly. The preparation is at once examined with the microscope. Staining takes place progressively till the nucleus is tinted, when ' death ' occurs. One of the best stains for such a method is Unna's polychrome methylene blue. For permanent preparations one of the most satisfactory methods is that used by the writer. Slides are coated with a very thin film of molten agar. This is conveniently done by pouring a very small quantity of the agar on one end of a perfectly clean slide and spread- ing it out quickly into a thin film over the slide by means of a warmed glass rod. As soon as the agar has set the slides are stored in a moist chamber till required. A drop or two of the fluid culture containing the amoebae is spread gently over the surface of the agar with either a platinum loop or a glass rod, great care being taken not to break the thin agar film. The slide is then placed film side up in a moist chamber and allowed to remain for from ten minutes to half an hour or more. In this time the amoebae have generally 34—2 532 METHODS FOR INVERTEBRATES. spread themselves out on the agar and many are in states of division. The slide is quickly removed and placed in a larger tube containing wool saturated with 2 per cent, osmic acid. The vapour fixes the organisms and the slide is removed in two to five minutes, and at once carefully placed in 50 per cent, alcohol. It should remain in this for fifteen minutes and is then transferred to 70 per cent., 90 per cent, and absolute aicohol fifteen minutes in each. It is then brought down to distilled water by passage through the alcohols, and is stained by Heidenhain's iron hsematoxylin method. The secret of success in this method is to prepare the agar coated slides with as thin a film as possible, otherwise it will peel off in the alcohol. Amoebae may be mixed with 1 per cent, serum water, and spread in a thin layer over slides, which are then fixed in Schaudinn's fluid, passed, as before, through the upgraded alcohols and back again to water, and stained with iron alum. Dobell's alcoholic iron hsematin method may be used for staining the free forms of amoebae, but is not so good for the cysts. A modification of Mann's stain by Dobell is also an excellent stain for amosbfe and cysts, and also sections of intestinal ulcers and tissues. The stain consists of Mann's methyl blue-eosin, which is made up in the usual way. Differentiation is carried out in dilute orange G in 70 per cent, alcohol. A simple staining in Lugol's iodine solution is also of use, especiaUy in the routine examination of faeces. 1001. Coccidia.- -These parasites stain rather badly and conse- quently may be examined by adding a drop or two of 1 per cent, aqueous eosin to the material containing them. The coccidia stand out as unstained bodies on a pink ground. Tissues may be fixed in the following solution :- 10 per cent, cobalt chloride in distilled water 20 c.c. 2 per cent, chromic acid in distilled water . 5 „ Formic acid ...... 1 drop. Schaudinn's alcoholic sublimate is also a good fixative. Bertarelli's method consists in fixing in saturated perchloride of mercury and staining in Grenadier's hsematoxylin and differentiating in acetic alcohol. B oriel' s method is of considerable utility. Tissues are fixed in the following solution for twenty -four hours : — Osmic acid . . . . . . . 2 gms. Platinum chloride . . . . 2 „ Chromic acid . . . . . 3 ,, Acetic acid ..... 20 c.c. Distilled water ... . 350 „ They are then washed in water for an hour or two, and passed through CHAPTER XXXVI. 533 the upgraded alcohols to paraffin. Sections are stained for one hour in saturated magenta, and are placed for five to ten minutes in a saturated solution of picric acid in water, 1 part, and saturated aqueous indigo carmine, 2 parts. They are washed in water and are decolourised in absolute alcohol and then in clove oil, and are mounted in balsam. 1002. Ciliates. — Osmic acid fixation is amongst those best suited for the ciliates, generally being employed either as vapour or wet on the slide. Many forms may be mixed with serum water (serum 1 part, water, 20 parts), and then spread on slides and fixed either in the osmic vapour or by means of Schaudinn's solution. Bouin's fluid is also very suitable for fixation of many forms such as Opalina and Lophomonas. Tissues are best fixed in either 10 per cent, formol saline or Flemming's solution. Staining may be by means of methyl green or any of the stains used for amoebae. In many cases Hollande's chloro-carmine gives very satisfactory results. 1003. Flagellata. — For the majority of these forms the technique employed for the study of the trypanosomes may be used. Slides may be coated with either serum water or a very thin layer of glycerin albumen. A drop of the fluid containing the flagellates such as blood may be spread quickly over it before drying occurs, and the slide at once placed in Schaudinn's sublimate or Flemming's solution for varying periods. Slides are then treated with upgraded alcohols, the weaker ones containing a little iodine. They are then brought down to water and stained with Heidenhain's iron hsema- toxylin. Dried blood films prepared in the usual manner may be fixed in alcohol-ether or absolute methvl alcohol and stained with »/ Giemsa's method. 1004. Hsemamcebse.- -These forms, the principal of which are the well-known malarial parasites, will be found in the blood, and heematological methods must be used. Blood is spread in thin layers on slides by placing a drop at one end of the slide and touching it with the end of another. • The blood will spread out in a thin layer and may then be drawn across the slide when a thin, usually one-cell, layer is obtained. Films made by streaking a drop of the blood over a slide by means of cigarette paper are also good. Such films are best fixed either in alcohol-ether (equal parts of each) for half an hour to one hour, or pure methyl alcohol free from acetone one hour. For a single method of staining such films, probably Eees' thionin is the best. It is prepared as follows. Thionin 1-5 grms., absolute alcohol 10 c.c., 5 per cent, carbolic acid solution. 100 c.c. Dissolve the thionin in the alcohol and add the carbolic solution. It is 534 METHODS FOR INVERTEBRATES. best diluted for use about one in five, and films are stained for from five to thirty minutes, washed, dried and mounted in balsam or euparal. The hsemamoebae are stained purple, nuclei blue, red cells faint blue or grey. Films may be differentially stained by means of eosin-methylene blue, Borrel's blue, or still better, by one of the Romano wsky methods. 1005. Eosin-Methylene Blue. — The film is prepared and fixed either by alcohol-ether or methyl alcohol, and is then stained for thirty seconds in a 0-5 per cent, solution of eosin, and is then washed and stained in a saturated solution of methylene blue for thirty seconds. In a successful preparation the red ceUs are stained pink and the nuclei of leucocytes and parasites blue. 1006. Ronald Ross's Thick Film Method.— Frequently the parasites in these cases are only present in very small numbers, and in the examina- tion of the ordinary thin films their presence may be overlooked. To meet such cases Ross recommends about 10 to 20 cubic mm. of the blood to be spread in a thick film on a slide, which is dried by waving gently over a flame and is then washed in water. By such treatment the haemoglobin is dissolved out from the erythrocytes. The film may now be stained for a minute in 0-5 per cent, to 1 per cent, aqueous eosin, followed by a 1 per cent, aqueous solution of methylene blue, made alkaline by the addition of 0-5 per cent, sodium carbonate and heated. For this may, with advantage, be substituted a solution of Unna's poly chrome methylene blue diluted 1 in 2 or 3 with water. Films after staining are washed, dried, and mounted in balsam. Only the leucocytes and the parasites are stained by this method. 1007. Borrel's Blue. — This method depends upon the formation of an oxidation product of methylene blue. Silver oxide is prepared by dissolving the nitrate in distilled water and precipitating the oxide with a 10 per cent, solution of sodium hydroxide. The precipitate obtained is washed thoroughly in distilled water and a saturated solution of methylene blue is added. The mixture is well shaken and allowed to stand for about a fortnight. The super- natant fluid is then pipetted ofT and constitutes Borrel's blue. Films are prepared and fixed for half an hour in either alcohol, ether- alcohol or absolute alcohol, and are then stained with Laveran's solution prepared as follows :- Borrel's blue ...... 1 c.c. 0-1 per cent, aqueous eosin . . .5 Distilled water . . . . . .4 The slides are placed film side downwards in this fluid and allowed to stain for from five to fifteen minutes, and should then be washed in distilled water treated with a 5 per cent, aqueous solution of tannic acid for one to two minutes, washed and dried. Red cells are stained pink, nuclei of leucocytes purple violet ; whilst the 55 55 CHAPTER XXXVI. 535 cytoplasm of the parasites is stained faintly blue their nuclei is reddish purple. 1008. Romanowsky Methods. — The Romanowsky methods depend essentially upon the formation of Azur and other oxidation products of methyleue blue, either alone or in combination with eosin. The most generally used are Leishman's stain, Wright's stain and the well-known Giemsa's stain. 1009. Leishman's Stain. — This stain is an extremely useful oue ; it is best purchased, but may be prepared as described in § 784, p. 385. Films are prepared in the ordinary manner, but are merely dried, not fixed. Five to 10 drops of the stain are poured on from a pipette and allowed to act for thirty seconds. An equal quantity of distilled water is then added and the diluted stain allowed to act for a further period of from five to ten minutes. The preparation is then well washed in distilled water, and is dried and mounted in balsam. Red cells are stained pale pinkish, the nuclei of the leucocytes red, parasites blue and their nuclei reddish purple. See §§ 784 et seq. 1010. Wright's Stain. — This stain is best prepared by dissolving Grubler's methyleue blue, 1 grin, in 0-5 per cent, sodium bicarbonate 100 c.c. The mixture is then heated at 70° to 80° C. for an hour. Cool and add 500 c.c. of a 0-1 per cent, solution of eosin (the yellowish shade water soluble variety). The solution of eosin should be added rather slowly, with constant stirring, till the blue colour disappears and the mixture is purple ; at this stage the fluid should have a metallic appear- ance on the surface, when a finely granular blackish precipitate is formed. This is collected and dried at 37° C. and a 0-3 per cent, solution of this dye is then made in pure absolute methyl alcohol. When required for use it is diluted with methyl alcohol (4 stain, 1 methyl alcohol). Stain as described for Leishman's method. 1011. Wilson's Stain. — This stain also depends upon the production of polychrome derivatives of methylene blue. It gives very satisfactory results when carefully used, closely resembling those obtained in good Giernsa preparations. Two grins, of silver nitrate are dissolved in 15 c.c. of distilled water, and to the solution so obtained is added 250 c.c. of a freshly-prepared lime water. The mixture should be shaken well and the precipitate of silver oxide collected on a filter and well washed with distilled water. The precipitate is then dried in an oven at a temperature not above 70° to 80° C. The moist silver oxide pre- pared from AgN03 and NaOH solution may be used in place of the dry product. To the oxide so obtained 2 grms. of methylene blue dissolved in 200 c.c. of 0-5 per cent, sodium bicarbonate solution is added. The mixture is then gently boiled in a porcelain dish for twenty to thirty minutes, stirring from time to time. Pour off one-third of the contents into a cylinder and then add to the remaining solution in the dish an amount of distilled water equal to that poured off. Boil the mixture in the dish again for twenty to thirty minutes. Again pour one-third of the contents of the dish into the cylinder, and boil the remainder for a 536 METHODS FOR INVERTEBRATES. further twenty to thirty minutes. Pour the contents of the dish into the mixture in the cylinder and make up the total volume to 200 c.c. Filter into a cylinder and add at once solution No. II., which is prepared as follows : 1 grm. water soluble eosiii (yellow shade) in 200 c.c. dis- tilled water. Mix and allow to stand for half an hour, and collect the resulting precipitate on a filter. Dry the precipitate at 60° C. In order to prepare the stain dissolve 0-2 grm. of the dried precipitate in 50 c.c. pure acetone -free methyl alcohol. The method of staining is identical with that of Leishman and Wright. 1012. G-IEMSA'S Method. — In many ways this is the easiest and best of the Romanowsky stains ; it is best bought ready prepared, either as a solution or the solid products of Burroughs, Wellcome & Co. For use, films are fixed in absolute methyl alcohol, alcohol-ether or absolute alcohol. They are then placed film side down in the staining fluid, which is prepared as follows :- Giemsa's stain . . . . .1 drop. Neutral distilled water . . .1 c.c. They should be stained for about one hour in this stain, washed, dried and examined. It is highly important that the water used should be neutral. This is best secured by boiling fresh distilled water for fifteen minutes, and then rapidly cooling and storing in a syphon vessel over soda-lime. 1018. M. TAYLOR'S Method for Amoeba Cultures (Nature, April, 1920).- -Water from such places as the drainage cuttings in birch, alder, and willow woods, or from the margins of ordinary pools and ponds, together with the filamentous algae and the brown scum, and included diatoms overlying the dead leaves and the other decaying organic matter forming the floor of such places, is gathered in autumn or in early spring. This is allowed to stand in tap-water for some time, until a rich brown scum appears on the top. The surface water, with the scum, is poured off into another glass vessel, and wheat is added (1 gram to a litre of water). In February, minute amoebae begin to make their appearance ; these become fully grown in May and June, and will then divide rapidly, forming a luxuriant culture until the late autumn, when encystment of most individuals again takes place. Once started, amoeba cultures require no further attention than a supply of water to compensate for evaporation, and the addition of wheat from time to time. Dr. J. Bronte Gatenby informs me that using Sr. Monica Taylor's method he has made successful cultures in boiled and unboiled London tap-water, in London rain-water and in spring-water. Sub-culturing is easily managed. Simply take about a pint of fresh rain-water or boiled tap -water ; about eight wheat seeds are just brought to the boil in a te&t tube of water, the latter poured away and the seeds shaken into a flat dish containing the rain-water. The dish is then inoculated CHAPTER XXX VI. 537 from an old culture and securely covered. In from two to three weeks the cultures may be going strongly. It is best to make half a dozen different cultures, placing them at different distances from an oven or thermostat. Not all of them " take." Small flagellates always seem to accompany successful cultures, but the appearance of rotifers and small annelids usually heralds the end of the amoebae. Sr. Monica, in a paper in press (Journ. Roy. Mic. 8oc. 1921), makes a further contribution to this subject : there are periods of depression in the cultures, new cultures may not " take " immediately, the cultures are best kept in the light near a window, and to avoid extinction of a strain by means of rotifers or small worms, one can only subculture. 1014. The Growth of Paramcecium in Sterile Culture Medium (by R. A. PETERS, Phys. Proceed., 1920). —Culture of a race of paramcecia about 50 /x in length, isolated from a single individual, have been obtained upon the following medium, the cultures being considered sterile for the reasons given below. Sodium chloride .... 0-06 per cent. Potassium chloride . . . .0-0014 „ Calcium chloride .... 0-0012 „ Basic sodium phosphate (NagHPOJ . 0-0001 „ Acid potassium phosphate (KH.2P04) . 0-0001 „ Magnesium sulphate .... 0-001 „ Sodium bicarbonate .... 0-002 ,, Glucose ...... 0-03 ., Histidine ...... 0-01 ,, Arginin ...... 0-01 „ Leucin ...... 0-01 „ Ammonium, lactate .... 0-003 ,, Ferric chloride ..... Trace Potassium iodide .... Trace Manganous chloride .... Trace The substances are made up with glass distilled water. The con- stituents are autoclaved separately, and the final mixture sterilised by heating to 80° C. on three successive days. The organisms were cultivated first in sterile media in depression slides, experiment showing the most suitable concentration for division. The first divisions were apt to be slow. After a number of individuals have been obtained in this way, they are sub-cultured in test tubes, using all sterile precautions, and when a satisfactory test tube culture has been obtained, it can be used for culture pur- poses as required. A culture has now been kept going by weekly sub-culture upon this medium for three months. (Temperature from 15° to 20° C.) From successful cultures grown in this way with the paramrecium in question, it was impossible to obtain any bacterial growth (or other 538 METHODS FOR INVERTEBRATES. growth) by sowing a drop of the parauicecial culture with a platinum loop, either upon (1) nutrient broth, (2) nutrient agar, (3) glucose agar (anaerobic) and (4) litmus milk at room temperature or at 36° C. It was also not possible to obtain growth upon the medium itself stiffened with a trace of agar. Examination of a growing culture under the ^2 oil immersion lens, however, showed the presence of peculiar rod- shaped bodies. These were about 10 p. long and 2 p. broad. They were motile, but appeared as a rule to be anchored at one end to the slide. They were never observed to divide. After a varying time they would cease to move in the moist drop preparation. A\7lien stained they were found to lie in rows, varying in shape from curved to straight. The absence of any sign of parasite or symbiotic organism in the paramoe- cium and the general resemblance of these bodies to split off cilia has led to the belief that the cultures in question really contain no other organism than the paramoecium. 1015. Method for Examination of Faeces for Protozoa (H. M. WOODCOCK, B. M. J., November, 1915). — -A very small quantity of the faeces is taken up on a platinum loop and well mixed with a drop of 0 -5 per cent, salt solution sufficient in amount to run under a coverslip. The faeces must be well diluted, otherwise cysts are apt to be overlooked. The faeces should be examined as freshly as possible, as after four or five hours most of the active flagellates become motionless and die. A convenient and rapid way of making a permanent preparation is as follows : — A thin smear of the diluted feeces is made on a slide in the same manner as a blood film, and the slide is immediately placed in a stain tube containing at the bottom a small quantity of 4 per cent, osmic acid plus 1 drop of glacial acetic acid for fixation, and is left in for about ten seconds. Allow the slide to dry in air and then place in absolute alcohol for fifteen minutes. Wash with tap-water and stain in Giemsa, 1 drop to 1 c.c. neutral distilled water for twenty minutes or so. Rinse with tap-water. 1016. DONALDSON'S Method of Detecting Protozoal Cysts in Faeces by Means of Wet Stained Preparations (Lancet, 1917). — Donaldson recommends the use of two solutions, A and B. A. (1) Five per cent, aqueous potassium iodide saturated with iodine to which is added an equal volume of ether. B. (1) A saturated aqueous solution of Rubin S. ; or (2) A saturated aqueous solution of eosin ; or (3) Stephen's scarlet writing fluid. Equal parts of stains A and B are mixed just before use. A few loopfuls of one of the above stain combinations are placed on a clean slide, a loopful of faeces is taken and rubbed up with the stain CHAPTER XXXVI. 539 to form a fairly smooth emulsion, and a clean coverslip gently lowered on to the drop. In order to get the best possible definition, it cannot be emphasised too strongly that the film so made should be spread out under the cover-glass by capillarity so as to form the thinnest layer possible, preferably a layer which is no thicker than the diameter of an Entamoeba coli cyst. The exact amount of stain to be used will soon be learned after one or two attempts, and depends upon the size of the loop employed and the size of the coverslip. It is a matter of considerable importance the way in which the film is made, especially where Kubin S., or, to a less extent, eosin has been used in the staining combination, for if the layer of fluid between the slide and coverslip be too thick the super- fluous film of fluid overlying the cysts tends to render the latter less bright. In the case of cysts of the size and shape of Entarnreba coli or histolytica, this makes little difference, but if one is hunting specially for the Lamblia, or, still more, Tetramitus mesnili, the colour contrast is not so marked, owing to the smaller size of the cysts and the conse quent thicker red layer of fluid overlying them. Where the worker has not acquired sufficient dexterity in making such a film, the difficulty may be got over by using Rubin S. or eosin of only half saturation in the stain combination, or by employing the scarlet writing fluid mentioned. In this way the effect of the super-imposed deeper red is to some extent obviated. In a wet preparation stained by this method there is a more or less homogeneous red background, from which the cysts stand out as brilliant yellow or greenish- yellow spheres which even the tyro cannot miss seeing. 1017. Method for the Tsetse Flies (M. EOBERTSON, Trans. Eoy. Soc., Series B , vol. 203, p. 1 6 1 ). — The newly hatched flies are starved for twenty- four to thirty-six hours and are then fed on the infecting monkey once, or in some cases twice. The infecting feed is the first blood ingested by the flies. After the infecting feed the cage is starved for one or two days and thereafter fed on clean monkey's blood every second or third day. Daily feeding is not essential to the welfare of glossina, and does not appear to occur in nature. Dissections are made in a drop of physio- logical salt solution. The trypanosomes are studied both in the live state and in fixed and stained preparations. Preserved material is fixed while wet by dropping the coverslip film side downwards into Schaudinn's solution ; the preparations are subsequently stained by Heidenhain's iron hsematoxylin. 1018. Immobilisation. — See the narcotisation methods §§ 20 to 25. According to SCHURMAYER (Jen. Zeit., xxiv, 1890, p. 402), nitrate of strychnin, of 0-01 per cent, or less, gives good results with some forms, amongst which are Stentor and Carchesium. Antipyrin (0 -1 per cent.), or cocaine of 0 -01 per cent., seems only to have given good results as regards the extension of the stalk in stalked forms. EISMOND (Zool. Am., xiii, 1890, p. 723) slows the movements of 540 METHODS FOE INVERTEBRATES. small organisms (small worms and Crustacea as well as Ciliata) by means of a drop of thick aqueous solution of cherry-tree gum added to the water containing them (gum arabic and the like, it is stated, will not do). The objects remain fixed in their places, with cilia actively moving, and all vital processes retaining their full activity. CERTES (Bull. Soc. Zool. France, xvi, 1891, p. 93) has found that an intra vitam stain may be obtained by adding methyl blue or " violet dahlia, No. 170. " to the gum solution. JENSEN (after STAHL ; see Biol. CentralbL, xii, 1892, p. 558) makes a solution of 3 grins, of gelatin in 100 c.c. of ordinary water by the aid of heat. This makes a jelly at the normal temperature. It is slightly warmed, and a drop of it is mixed in a watch-glass with a drop of water containing the organisms. See also VOLK, ante, § 886 ; STATKEWITSCH, Arch. Protistenk., v, 1904, p. 17 ; LYON, Amer. Journ. Phys., xiv, 1905, p. 427 (neutralised gum). 1019. Staining intra vitam. — See hereon BRANDT (Verh. physiol. Ges. Berlin, 1878) ; CERTES (Bull. Soc. Zool, 25 janv., 1881) ; and HENNEGUY (Soc. Philom., 12 fev., 1881). See also § 208. BRANDT recommends a 1 : 3000 solution of Bismarck brown ; also (Biol. Centralb., i, 1881, p. 202) " a dilute solution of heemato- xylin." "CERTES (op. cit,, pp. 21, 226, 264, and Zool. Anz., iv, 1881, pp. 208, 287) found that living Infusoria stain in weak solutions of cyanin, Bismarck brown, dahlia, violet 5 B, chrysoidin, nigrosin, methylen blue, malachite green, iodine green, and other tar colours, and hsematoxylin. The solutions should be made with the liquid that constitutes the natural habitat of the organisms. They should be very weak, that is, of strengths varying between 1 : 10,000 and 1 : 100, OCO. For cyanin, 1 : 500,000 is strong enough. As to the staining of the Nucleus, see PRZESMYCKI, Biol. Centralb., vii, 1897, p. 321 ; and as to that of the Granula, the same author, Zeit. wiss. Mik., xiii, 1896, p. 478. Also LOISEL, § 208. Examination in a coloured medium in which the organisms do not stain, but show up on a coloured background, is sometimes helpful. CERTES (Bull. Soc. Zool. de France, xiii, 1888, p. 230) recommends solution of anilin black — Infusoria will live in it for weeks ; FABRE- DOMERGUE (Ann. de Microgr., ii, 1889, p. 545) concentrated solution of diphenylamin blue. For the mitochondria and other granules, see FAURE-FREMIET (Arch. d'Anat. micr., xi, p. 457). Dahlia in salt solution, Pictet's fluid. Janus green, or crystal violet being useful. CHAPTER XXXVI. 541 For mitochondria and Golgi apparatus of a sporozoon, J. HTRSCHLER (Anat. Am., xlvii, 1914 — 15) used the Mann-Kopsch method, § 693. 1020. Demonstration of Cilia (WADDINGTON, Journ. Roy. Mic. Soc., 1883, p. 185). — A drop of solution of tannin, or a trace of alcoholic solution of sulphurous acid, added to the water containing the living organisms is efficacious. 1021. Fixing and Preserving. — Protozoa may be killed by heat, by toxic vapours or by toxic liquids. Almost instantaneous fixation can sometimes be obtained by steam or by iodine (Kent) or iodine vapour (Overton) : see § 83. E. S. GOODRICH (Quart. Journ. Micr. Science, Ixiv, 1919) modifies Kent's method in a way which we have found useful for amoebae. A strong solution of iodine in potassium iodide is diluted to about the colour of sherry with normal saline for terrestrial and fresh water animals, and with sea water for marine organisms. Such a solution is run under the coverslip and followed by the definitive fixing agent, e.g., Bouin's fluid, etc., and the preparation proceeded with in the usual way. The iodine does not appear to destroy any of the cell contents. With regard to fixation, read §§ 29, 30, § 655, § 663, and especially §§ 673 to 713. See also the important section from §§ 768 to 772, dealing with fats. WOODCOCK and WILSON'S Modification o£ Schaudinn's Fixative (Phil. Trans. Roy. Soc. B., ccvii, 1916, p. 379 ; and Univ. Calif. Pub., xvi, 1916, p. 244). — Woodcock gives saturated aqueous sublimate, 2 parts ; absolute alcohol, 1 part ; and acetic acid, 5 per cent. Wilson uses alcoholic sublimate -f- 5 per cent, acetic. See also Gilson and Petrunkewitsch fluids, § 69, and acetic alcohol, § 86. For Schaudinn's original fixative, refer to § 1031. Lucidol or Peroxide of Benzol. See §§ 107 and 783. For killing by heat, see § 13. PFITZNER (Morph. Jahrb., xi, 1885, p. 454) used concentrated solution of picric acid run in under the cover. ENTZ (Zool. Anz., iv, 1881, p. 575) adds liquid of Kleinenberg to the water containing the organisms in a watch glass. KORSCHELT (ibid., v, 1882, p. 217) employs in the same way 1 per cent, osmic acid, or, for Amoebae, 2 per cent, chromic acid. LANSBERG (ibid., p. 336) advises the same reagents, but recom- mends bringing the organisms into the fixing liquid with a pipette. For sulphurous acid, § 62. CATTANEO (Bollettino Scientific^, iii and iv ; Journ. Roy. Mic. 542 METHODS FOR INVERTEBRATES. Soc., 1885, p. 538) fixes for a few minutes with J per cent, solution of chloride of palladium. BRASS (Zeit. iviss. Mik., 1884, p. 39) employs a mixture of 1 part each of chromic acid, platinum chloride, and acetic acid with 400 to 1,000 parts of water. CERTES (Comptes rend., Ixxxviii, 1879, p. 433) fixes with 2 per cent, osmic acid, or its vapours (ten to thirty minutes). For details, see previous editions. LONGHI (Bull. Mm. Zool. Univ. Genova, 1892, No. 4) kills in 10 c.c. of 1 per cent, sulphate of eserin with 1 drop of 1 per cent, sublimate. SCALA (Rev. Mus. La Plata, xv, 1908, p. 57) fixes for five or ten minutes in a mixture of 2 mg. of atropin, 10 drops of formol, 10 grms. of glycerin and 50 c.c. of water. See also PUSCHKAREW, Zeit. wiss. Mik., xxviii, 1911, p. 145 (agar process for fixing and staining Amoebae). FOL (Lehrb., p. 102) fixes delicate marine Infusoria (Tintinnodea) with the perchloride of iron solution (§ 80), added to the water containing them, and stains with gallic acid. Lo BIANCO (loc. cit., p. 444) fixes Gregarinee with picro-sulphuric acid (one hour), Vorticellae with hot sublimate, Acinetse with subli- mate in sea water, or with osmic acid, Thalassicola with 0-5 per cent, chromic acid (one hour), Acanthometrse and Aulacanthae with 50 per cent, alcohol or with concentrated sublimate, or by adding a little osmic acid to the water. For Sphaarozoa he proceeds as BRANDT, § 1019. ZOGRAF fixes Rhizopoda and Infusoria as Rotatoria, § 886, but without narcotisation. See also FABRE-DOMERGUE, Ann. de Microgr., ii, 1889, p. 545, and 1890, p. 50 ; SCHEWIAKOFF, Biblioth. Zool., v, 1889, p. 5 ; Journ. Roy. Mic. Soc., 1889, pp. 832, 833 ; ZOJA, Boll. Sci. Pavia, 1892 ; Zeit. wiss. Mik., ix, 1893, p. 485 ; LAUTERBORN, Zeit. wiss. Zool., lix, 1895, p. 170 ; SCHAUDINN, ibid., p. 193; BALBIANI, Zool. Ans., xiii, 1890, p. 133; KARAWAIEW, ibid., xviii, 1895, p. 286. 1022. Embedding of Protozoa and other Small Objects (MINCHIN, Q. J. M. S., Ix, 1915, p. 508). — A thin slice of a block of amyloid liver preserved in alcohol is floated into a shallow glass vessel with a flat bottom, containing alcohol. The dish is placed on the stage of a dissecting microscope. The objects to be embedded are taken up in a pipette and placed on the slice of liver and orientated as desired. A tiny drop of glycerine and albumen solution is taken up on the CHAPTER XXXVI. 543 point of a needle and caused to touch the surface of the alcohol immediately above the small objects. The dense albumen solution falls at once through the alcohol and spreads out over the objects on the liver ; at the same time the glycerine is extracted and the albumen coagulated by the alcohol, with the result that the objects are stuck on to the liver. The liver is now trimmed with a scalpel into a rectangular shape and embedded in the usual way. Minchin used this method for the stomachs of fleas. I have found it most successful for Echinoderm and other small eggs. Or the f ollowing method may be used :- -The organisms should be strongly fixed, then dehydrated and cleared, and brought into melted paraffin in a small watch glass. After a few minutes therein they are brought on a cataract needle on to a small block of paraffin, and arranged there with a heated needle and sectioned. They may be stained after fixation, or the sections may be stained on the slide, § 186 or 187. ENTZ (Arch. Protistenk., xv, 1909, p. 98) brings the objects from clove oil into clove oil collodion of the consistency of honey, then brings them in this into a funnel made of paraffin, and when they have collected at the bottom of this puts it into chloroform, which dissolves the paraffin and hardens the collodion. Some Current Stains for Protozoa. — Below are given a number of special staining methods suitable for routine protozoological work. 1023. HEIDENHAIN'S Alcoholic Iron Hsematoxylin. — (1) Fix in Schaudinn's solution for fifteen minutes. (2) Place at once in 70 per cent, alcohol for at least one hour. (3) Mordant for five hours or more in 50 per cent, alcohol . . . . .10 parts. 4 per cent, aqueous iron alum solution . 1 part. (4) Stain for twelve to twenty-four hours in Heidenhain's hsematoxylin ... 1 part. 70 per cent, alcohol . . . . .10 parts. Heidenhain's hsematoxylin- Hsematoxylin ..... 1 grm. Absolute alcohol . . . . .10 c.c. Distilled water . . . . . 90 „ Thymol 1 crystal. Dissolve the heematoxylin in the absolute alcohol and then add the distilled water. (5) Differentiate in the same solution as was used in (3) for mordanting. 544 METHODS FOR INVERTEBRATES. (6) Wash thoroughly in several changes of 70 per cent, alcohol to remove mordant. (7) Dehydrate and mount in Canada balsam. 1024-. DOBELL'S Haematein Method (see Arch. f. Protistinkunde, 1914, p. 144.) — Films or sections are transferred from 70 per cent, alcohol into 1 per cent, solution of iron alum in 70 per cent, alcohol. This is most easily made in the way described by HICKSON (Quart. Journ. Micr. Sci. 44, 1901, p. 470), 1 grrn. of salt dissolved in 23 c.c. of warm water, then add 77 c.c. of 90 per cent, alcohol after cooling. They are mordanted in this for ten minutes, rinsed in 70 per cent, alcohol and transferred to a 1 per cent, solution of hsematein in 70 per cent, alcohol. They are left in this for ten minutes and then differentiated, either in the iron alum or in acidified 70 per cent, alcohol (0-6 per cent. HC1 in 70 per cent, alcohol). After differentiation wash in several changes of 70 per cent, alcohol and then pass up through the alcohols into any of the usual mounting media. Any alcoholic counterstain can be used, light green in 90 per cent, alcohol being very good. By this method the organism is stained a purple grey, and hard black and white contrasts are not obtained. The various nuclear and cytoplasmic constituents may be stained with individual intensity. (The hsRematein method is good, when it works, but often it is a complete failure ; certain amoebae, for instance, are not stained at all by it.) 1025. DOBELL'S Modification of Mann's Methyl Blue, Eosin Stain. - (1) Stain with Mann's methyl blue eosin mixture (see Lancet, p. 196) in distilled water till everything is overstained (two to eight hours). (2) Rinse in distilled water and differentiate in 70 per cent, alcohol dried from water, containing a small quantity of Orange G — just enough to colour it. It is best to keep Orange G- in concentrated solu- tion in 90 per cent, alcohol ; add sufficient of this with a glass rod to clear 70 per cent, before differentiating. This weak Orange G cannot be used often as it gets discoloured very quickly. (3). Dehydrate in absolute alcohol (not too long) and transfer to xylol (also not too long, as eosin comes out in time if left). (4) Mount in balsam. By this method permanent and pretty results are obtained, often quite as good or better than those got with Giemsa. The method is especially good after Bonin fixation, but it is important that all the picric acid is removed before staining. 1026. Giemsa Stain (MLNCHIN'S Method, Q. J. M. £., Lx, 1915, p. 510). — Slides are washed in tap water and put in dilute Lugol solution (1 c.c. Lugol to 25 c.c. distilled H20) for ten minutes. After this, rinsed in tap water and put into a 0 -5 per cent, watery solution of hyposulphite of soda for ten minutes. Next wash in a current of water for five minutes and then put into the stain. The distilled water used to dilute the Giemsa stain has to be neutralised in the way prescribed by Giemsa. CHAPTER XXXVL 545 A measured volume of the distilled water is taken and to it are added a few drops of hsematoxylin solution (5 per cent, in dist. H20) sufficient to tint it. Then a weak solution (1 per cent, in dist. water) of potassium carbonate is added drop by drop until the colour of the tinted water changes from yellow-red to reddish purple. In this way the number of drops of the carbonate solution required to neutralise a given volume of the distilled water is found. The slides are now placed in the stain, 1 (^rop C4iemsa to 1 c.c. neutralised distilled water for one hour. Transfer to a weaker solution (1 drop stain plus 4 or 5 c.c. water) and leave overnight. Remove excess of stain by washing in water. Differentiate stain by carrying slides through different strengths of acetone mixed with xylol, beginning with 95 per cent, acetone and ending with pure xylol. Mount in dammar or Canada balsam. (If these instructions are carefully followed the results are excellent, but care must be used.) 1027. Giemsa Method for Staining Gut Flagellates (J. G. THOMSON ).- ( 1 ) Fix wet sat. perchloride, 2 parts ; alcohol, 1 part. (2) Wash in weak alcohol (circa, 50 per cent.). (3) Wash in water (aq. dist.). (4) Wash in water, to which is added a few drops of Gram's iodin. (5) Wash in 1 per cent, solution of hypo. (6) Wash in running water. (7) Stain in Giemsa (1 drop, 1 c.c.) twelve hours. (8) Differentiate in (a) acetone, 95 per cent. ; xylol, 5 per cent., for about five minutes ; then (&) acetone, 70 per cent. ; xylol, 30 per cent. ; (c) pure xylol to clear. (9) Mount in Canada balsam. 1028. Iron Brazilin Stain (HICKSON). — (1) Fix in Schaudinn's solution for fifteen minutes. (2) Seventy per cent, alcohol for one hour. (3) Mordant for four hours in a 1 per cent, solution of iron alum in 70 per cent, alcohol. (4) Stain twelve to sixteen hours in a 1 per cent, solution of brazilin in 70 per cent, alcohol. (5) Wash thoroughly in several changes of 70 per cent, alcohol. (6) Dehydrate and mount in balsam. 1029. FONTANA'S Stain. — (1) Fix in Hugo's fluid for one minute. Acetic acid (B.P.) 1 c.c. Formalin ... 20 ,, Distilled water . . . 100 ,, (2) Wash thoroughly in tap water. (3) Mordant. Slide should be gently warmed. Tannic acid . . 5 grms. Carbolic acid . . 1 c-c- Distilled water . . . 100 c.c. M. 35 546 METHODS FOR INVERTEBRATES. (4) Wash thoroughly in tap-water. (5) Stain and warm gently : (a) 5 per cent, solution of silver nitrate in water ; (b) ammonia. Add the ammonia until the precipitate redis- solves in excess of ammonia. Then add more silver nitrate drop by drop until the precipitate reappears and remains constant. (6) Wash thoroughly in tap -water. (7) Dry rapidly in air* 1030. Sphserozoa* — BRANDT (Fauna u. Flora Golf. Neapel, xiii. 1885, -p. 7) fixes with chromic acid of 0-5 per cent, to 1 per cent, (half an hour to an hour), or with a mixture of equal volumes of sea Water and 70 per cent, alcohol with a little tincture of iodine for a quarter to half an hour, or with a 5 to 15 per cent, solution of sublimate in sea water. KARAWAIEW (ZooL Anz., xviii, 1895, p. 286) fixes Aulacantha for twenty-four hours in equal parts of strong liquid of Flemming and acetic acid, and hardens for several days in pure liquid of Flemming. See also Lo BIANCO, § 1021. 1031. Sporozoa. — WASIELEWSKI (Sporozoenkunde, Jena, 1896, p. 153) studies them living in their natural medium, or in normal salt solution, or in a medium composed of 20 parts white of egg, 200 of water, and 1 of common salt. He fixes Gregarinse and Coccidia with osmic acid, sublimate, or picro-sulphuric acid, and Myxosporidia with liquid of Flemming. SCHAUDINN (ZooL Jahrb., Abth. Anat., xiii, 1900, p. 197) fixes Coccidia with a mixture of 2 parts of saturated aqueous sublimate and 1 of absolute alcohol, with, if desired, a trace of acetic acid. STEMPELL (Arch. Protistenk., xvi, 1909, p. 389) fixes caterpillars infected with Nosema in 2 parts of saturated sublimate with 1 of alcohol and a little acetic acid, and stains sections for as much as four days in GIEMSA'S mixture, rinses with alcohol and passes through xylol into balsam. LEGER (ibid., iii, 1904, p. 311) fixes cysts for a minute in " acetic sublimate," puts for a minute into absolute alcohol, and stains as a smear with heemalum or iron hsematoxylin. BRASIL (Arch.. ZooL Exper., 4, iv, 1905, p. 74) fixes them for twenty-four hours in a mixture of 1 grm. picric acid, 15 c.c. acetic acid, 60 c.c. formol and 150 c.c. alcohol of 80 per cent., and stains paraffin sections in iron hsematoxylin followed by eosin and orange G. , or Lichtgriin and picric acid. 1032. Haematozoa. — GRASSI (Alt. Accad. Lincei, iii, 1900, p. 357) demonstrates the Malaria-parasites in the intestine, body-cavity and salivary glands of Anopheles by treating them with normal salt CHAPTER XXXVI. 547 solution containing 2 per cent, of formol (pure formol produces swellings), or in a mixture of 1 -5 grm. of salt and 250 c.c. of water with the white of an egg. He fixes with sublimate, makes paraffin sections, and stains with hsemalum or iron hsematoxylin. He stains the Sporozoites by making cover-glass preparations, which are allowed to dry, put for twenty-five minutes into absolute alcohol, and stained by the process of ROMANO WSKY, §§ 784, 1008. For minute instructions for the application of this process to sections, see GIEMSA, Deutsch. med. Wochenschr., xxxvi, No. 12, 1910 ; and SCHUBERG, ibid., xxxv, No. 40, 1909 (Zeit. wiss. Mik., xxvii, 1910, pp. 160, 161 and 513). For clinical methods, see COLES, The Diseases of the Blood. London, J. and A. Churchill, 1905. BRADFORD and PLIMMER (Quart. Journ. Micr. Sci., xlv, 1902, p. 452) fix Trypanosomes in vapour of equal parts of acetic acid and 2 per cent, osmic acid, or with GULLAND'S formol and absolute alcohol, and stain with methylen blue and eosin, and mount in turpentine colophonium. HINDLE (Univ. Calif. Pub. Zool., vi, 1909, p. 129) makes smears on cover-glasses coated with albumen, fixes for five minutes in liquid of Flemming, passes through water up to absolute alcohol, then for ten minutes into alcohol of 80 per cent, with a good propor- tion of iodine in potassic iodide, then into 30 per cent, alcohol, and stains with iron hsematoxylin or safranin, then with polychrome methylen blue, and lastly with UNNA'S orange with tannin, and gets quickly through alcohol into xylol and balsam. MINCHIN (Quart. Journ. Micr. Sci., liii, 1909, p. 762) makes cover- glass smears, fixes them with vapours of osmic acid (with or without acetic acid), and mounts them dry, or in balsam after fixing in liquids and various stains, amongst these that of TWORT. Half- saturated solutions of neutral red and Lichtgriin are mixed, the precipitate dried and dissolved to about 0-1 per cent, in methyl alcohol with 5 per cent, of glycerin. Three parts of this are diluted with 1 of water, the smears stained for an hour, differentiated with UNNA'S glycerin-ether, and mounted in balsam. This stain works best after fixation with sublimate. POLICARD (C. R. Soc. Biol., Ixviii, 1910, p. 505) stains Trypano- somes intra vitam by adding a drop of concentrated solution of neutral red to the edge of a drop of blood spread between slide and cover. 1033. Flagellata. — LAUTERBORN (Zeit. wiss. Zool., lix, 1895 p. 170) fixes Ceratium for about ten minutes in liquid of Flemming, puts into alcohol for twenty-four hours, brings back into water, 35 — 2 548 METHODS FOR INVERTEBRATES. bleaches if necessary with hydrogen peroxide, and stains with picrocarmine or Delafield's heematoxylin. He also embeds in paraffin, § 1022, and stains sections with iron hsematoxylin. ZACHARIAS (Zool. Anz., xxii, 1899, p. 72) fixes Uroglena, etc., with a mixture of 2 volumes saturated aqueous solution of boracic acid and 3 of saturated sublimate. 1034. Stains for Flagella. — The ROMANOWSKY stain will give a red stain of the flagella of some forms. The method of LOFFLER (Centralbl. Bakteriol., vi, 1889, p. 209 ; vii, 1890, p. 625 ; Zeit. wiss. MiL, vi, 1889, p. 359 ; vii, 3, 1890, p. 368 ; Journ. Roy. Mic. Soc., 1889, p. 711 ; 1890, p. 678) is as follows. To 10 c.c. of a 20 per cent, solution of tannin are added 5 c.c. of cold saturated solution of ferrous sulphate and 1 c.c. of (either aqueous or alcoholic) solution of fuchsin, methyl violet, or Wollschwarz." Cover-glass preparations are made and fixed in a name in the usual way, special care being taken not to over-heat. Whilst still warm the preparation is treated with mordant (i.e. the above-described mixture), and is heated for half a minute, until the liquid begins to vaporise, after which it is washed in distilled water and then in alcohol. It is then treated in a similar manner with the stain, which consists of a saturated solution of fuchsin in anilin water (p. 177), the solution being preferably neutralised to the point of precipitation by cautious addition of 0-1 per cent, soda solution. See also LIEBETANZ, Arch. Protistenk., xix, 1910, p. 23. BUNGE (Journ. Roy. Mic. Soc., 1894, p. 640 ; Zeit. wiss. Mik., xiii, 1896, p. 96) makes the mordant by mixing 3 parts of the tannin solution with 1 of liquor ferri sesquichlorati diluted twentyfold with water, and lets the mixture ripen for some days exposed to the air, or (Journ., 1895, pp. 129, 248) adds to it a few drops of hydrogen peroxide, until it becomes red-brown, when it is shaken up and filtered on to the cover-glass and allowed to act for a minute. The cover-glass is then mopped up and dried, and stained with carbol gentian. KOERNER and FISCHER (quoted from Encycl. mik. Techn., p. 514) make the mordant with 2 parts of tannin, 20 of water, 4 of ferrous sulphate solution of 1 : 2 strength, and 1 of saturated alcoholic solution of fuchsin. Warm, let it act for a minute, rinse and stain with anilin-water-fuchsin, or carbol fuchsin. Similarly ELLIS (Centralb. Bakt., xxi, 1903, p. 241 ; Journ. Roy. Mic. Soc., 1904, p. 249), but staining with Saureviolett, 1 part to 75 of alcohol and 75 of water. PEPPLER (Centralb. Bakt., xxix, 1901, p. 376 ; Zeit. wiss. Mik., CHAPTER XXXVI. 549 xviii, 1901, p. 222) makes the mordant with 20 parts of tannin in 80 of water, and 15 parts of 2-5 per cent, chromic acid added gradually. This mordant will keep for months. Rossi (Arch, per le Sc. med., xxiv, 1900, p. 297 ; Zeit. wiss. Mik., xviii, 1901, p. 226) takes for the mordant a solution of 25 grms. of tannic acid in 100 of caustic potash of 0-1 per cent., which will keep indefinitely. The stain is Ziehl's carbol fuchsin, § 289. Cover- glasses are prepared with a drop of culture, dried, and treated with 1 drop of the mordant and at the same time 4 to 5 of the stain, allowed to remain for fifteen to twenty minutes, washed, and mounted. See also Centralb. Bakt., xxxiii, 1903, p. 572 (Zeit. wiss. Mik., xix, 1903, p. 517). CTEMELLI (Centralb., xxxiii, 1903, p. 316 ; Zeit. wiss. Mik., xix, 1903, p. 516) mordants for ten to twenty minutes in 0-025 per cent, permanganate of potash, rinses and stains for fifteen to thirty minutes in a mixture of 20 parts 0 -75 per cent, aqueous solution of calcium chloride and 1 part of 1 per cent, neutral red solution. A method of PITFIELD is described by KENDALL, Journ. app. Mic. , v, 1902, p. 1836 (Journ. Roy. Mic. Soc., 1902, p. 502). The mordant consists of 10 parts of 10 per cent, tannin solution, 5 parts of saturated sublimate solution, 5 of saturated solution of alum, and 5 of carbol fuchsin. Mordant for a minute with heat, and stain with a mixture of 2 parts saturated aqueous solution of gentian violet with 10 of saturated solution of alum. VAN ERMENGEM (Journ., 1894, p. 405) fixes for a few minutes with a mixture of 1 part 2 per cent, osmic acid, and 2 parts 10 to 25 per cent, solution of tannin, washes, treats with 0-25 to 0-5 per cent, solution of nitrate of silver, then for a few seconds with a mixture of 5 parts gallic acid, 3 of tannin, 10 of acetate of soda, and 350 of water, then puts back again into the silver for a short time, then washes and mounts. See also STEPHENS, ibid., 1898, p. 685, and GORDON, ibid., 1899, p. 235, and the methods of TRENKMANN (Centralb.. vi, 1889, p. 433 ; Zeit. wiss. Mik., vii, 1890, p. 79) ; BROWN (Journ. Roy. Mic. Soc., 1893, p. 268) ; JULIEN (ibid., 1894, p. 403) ; SCLAVO (Zeit. wiss. Mik., xiii, 1896, p. 96) ; HESSERT (ibid., p. 96) ; MUIR (Journ. Roy. Mic. Soc., 1899, p. 235) ; McCRORiE (ibid., 1897, p. 251 ; he stains for two minutes in a mixture of equal parts of concentrated solution of night -blue, 10 per cent, solution of alum, and 10 per cent, solution of tannic acid) ; ZETTNOW (ibid., 1899, pp. 662, 664); MORTON (ibid., 1900, p. 131); WELCKE (ibid., p. 132) ; LEVADITI, C. R. Soc. Biol, lix, 1905, p. 326 (for Spiro- chcete pallida, EAMON'S neurofibril stain) ; MEIROWSKY, Munch, med. Wochenschr., Ivii, 1910, No. 27 ; KALB, ibid., No. 26 (Zeit. wiss. Mik., xxix, 1912, pp. 123, 124 ; both for Spirochcete). CHAPTER XXXVII.* THE CULTIVATION OF TISSUE " IN VITRO " AND ITS TECHNIQUE. 1035. A culture of tissue consists of a special medium, natural or artificial, such as lymph or plasma, iifoculated with small fragments of living tissues, and is characterised by an active growth of the cells of the fragment into the nutrient plasmatic or lymph medium. Cells wander out into the latter, and may live up to twenty days without any signs of necrobiosis. The cultivation of tissues outside the body was first accomplished successfully by Eoss Harrison, of Johns Hopkins University in the year 1907. This brilliant observer has demonstrated by a series of experi- ments that fragments of nervous tissue of the frog embryo, covered with fluid from the lymph sac of an adult frog, show growth of long nerve fibres (HARRISON, Proc. Soc. Exper. Biol. and Med., iv, 1907, p. 140). Alexis Carrel, at about the same time, had been studying the laws of redintegration of tissues, and adapted Harrison's technique to mam- malian tissues. M. Burrows, a pupil of Ross Harrison, at this period began to work on tissue- culture, and first used blood plasma instead of lymph. Subsequently Burrows adapted the technique of Harrison to the cultivation of tissues of the chick. In -September, 1910, Carrel and Burrows, working in conjunction at the Rockefeller Institute, succeeded in cultivating, in vitro, the adult tissues of mammals, and thus began a series of contributions which have taught us many valuable facts regarding senesence and rejuvenescence and the pathology of tissues (CARREL and BURROWS, Jour. Exper. Med., xiii, No. 3, 1911). Two methods of tissue-culture may be distinguished :- (1) Hanging-drop or smear cultures (Harrison). (2) Large plate cultures (Carrel and Burrows). The former are useful for direct observation of living growing cells, the latter can be studied when fixed and cut into sections. There has been a great deal of work done on tissue-culture, but most of it has been carried out by vertebrate pathologists and histologists. There seems little doubt that this field is a most promising one for zoologists as well as histologists. Many problems of gametogenesis and general cytology might be settled by recourse to tissue-culture, especially by the application of such methods to the cells of invertebrate animals, whose cytology had previously been examined in detail with the aid of modern techniques. * By J. B. G. CHAPTER XXXVII. 551 1036. Precautions to Insure Complete Sterilisation oJ Apparatus.- It cannot be too strongly emphasised that the utmost precautions must be taken to insure complete sterility of all apparatus. Bacterial infections of the cultures are fatal. The worker who is not familiar with the minute precautions taken by surgeons and bacteriologists in sterilising instruments, apparatus, etc., is advised to become so before undertaking tissue-culture work. A rigid asepsis is necessary for the preparation of any tissue-culture. These words are addressed especially to the zoologist who may undertake tissue-culture work ; dirty floors and benches, dirty walls and garments, and casual methods will all contribute towards failure. A clean, warm room should be set aside for making the cultures, another for making the various plasmas, and, if possible, another for incubators and incubator microscopes. The ordinary zoological or botanical laboratory is generally unsuitable for such delicate work. Dr. Drew informs me that whether in vitro culture be carried out in a special laboratory or not, it is advantageous to use a specially constructed glass chamber to shield the cultures from chance con- tamination. Such a chamber is supplied by Hearson's (§ 11), or can be made by any carpenter. The apparatus consists essentially of a glass box in a wooden or metal framework measuring about 2 feet 6 inches in length, 2 feet in width, and about 1 foot in height. The top is hinged so as to allow ready access to the interior for cleaning, etc. The side facing the worker is made of wood, with either two small wooden doors or a piece of thick sheet rubber pierced so as to allow the easy entrance of the hands. The box should have glass racks to contain the hollow ground slides and a glass table for the cover-glasses. The slides are cleaned and stored in absolute alcohol ; they are removed from this by means of forceps and are flamed in a Bunsen and quickly transferred to the glass racks ; cover-glasses are cleaned in acid bichromate, washed in water till free from all trace of the acid, rinsed in distilled water, then in absolute alcohol, and stored in ether. They are removed by means of forceps and flamed and placed on the previously sterilised glass table. Here both slides and cover-glasses are protected from all falling dust, and can be manipulated easily with the hands through the openings in the case. The majority of failures occur through infection taking place, and the glass chamber reduces such a possibility very greatly. 1037. Simple Culture Technique by means of Frog Lymph (HARRI- SON, op. cit., 1907). — Pieces of embryonic tissues of frog embryos 552 THE CULTIVATION OF TISSUE ''IN VITRO." about 3 mm. long are dissected out with clean instruments, removed to a coverslip, and covered by a drop of lymph freshly drawn from one of the lymph sacs of an adult frog. The coverslip is inverted over a hollow slide, and the rim sealed with paraffin wax. When reasonable aseptic precautions are taken, tissues will live under these conditions for from a week up to four weeks. GOLDSCHMIDT (Arch. f. Zellf., 1916) has investigated the sperm cells of Lepidoptera by tissue culture methods. 1038. Technique for Culture of Mammalian Tissues. — Preparation of the Animal for Procuring Plasma. — The animal is aneesthetised with ether, and must be kept just at the correct depth of anaesthesia. A. J. WALTON (Journ. Path, and Bact., xviii, 1914), from whose article the following paragraphs are partly culled, recommends for this purpose a wide-necked bottle, with a closely-fitting cork pierced with two tubes of wide bore, both of which pass down to within J-inch of the surface of the ether placed in the bottle ; one tube communicates with a tin funnel having a mackintosh flange ; this tube also has a side tube, and the other tube is open to the air. The animal's head is placed in the funnel, and, when the side tube is clamped, breathes air and ether vapour. By clamping either the side or the short tube the amount of air or ether can be suitably controlled. The hair of the throat, is either shaved off, or removed by the application of a solution of sodium sulphide ^ii ad Oi, which rapidly dissolves it. The skin is then sterilised by painting with a 2 per cent, solution of iodine in spirit. 1039. Preparation of Instruments, etc. — Previous to the operation the following apparatus is sterilised : — Short test tubes, 2| inches in length ; corks kept in stoppered bottles, to fit these tubes ; small glass cannulse in olive oil ; three glass tubes, 3 inches by 1 inch ; several narrow-bore pipettes which are kept corked in the last- mentioned tubes, and which just before the operation on the animal, are removed from the tube by means of sterile forceps, dipped in a deep tin of molten paraffin, everted to allow the paraffin to run out, and when cool placed in another sterile tube. Two small sterile test tubes, as mentioned above, are similarly treated in paraffin, and should be corked with sterile corks as soon as cool. These two tubes are placed in two other larger tubes made to fit the centrifuge, and ice is packed between. Just before the operation, the instruments and some rubber teats to fit the pipettes are boiled in water for ten minutes. CHAPTER XXXVII. 553 1040. Removal of Plasma.— When dogs, rabbits, cats, chickens, guinea pigs and rats are used, the carotid artery is ordinarily selected ; an incision is made in the mid-line in the neck, and as soon as the skin is divided the edges are clipped to sterile towels. The carotid is exposed, its distal end ligatured and its proximal end clamped. A little sterile oil is placed on the artery, which is opened, and one of the cannule from the sterile oil is taken, inserted and tied in position ; on releasing the clamp the blood flows freely. This is collected in the paraffined test tubes for centrifuging. The tubes should be in their ice- jackets ; they are corked at once and im- mediately centrifuged for about five minutes ; they are then removed and placed in an ice box at 0° C. For human plasma one may remove blood from a vein by means of a needle pipette sterilised in olive oil. ( After the centrifugilisation the supernatant plasma may be removed with pipettes coated in paraffin (§ 1039). It should be used immediately for making the cultures, but can be preserved for some time in a fluid condition if kept very cool. Chicken plasma can be so preserved for more than a week, human and dog plasma for a few days, while rat plasma always coagulates after a few hours (CARREL and BURROWS, Journ. Exp. Med., 1911) ; when coagulation takes place the plasma is no longer of use. CARREL and BURROWS (Jour. Exper. Med., xiii, 1911) found that dilution of the plasma had a marked influence on the rate of growth of splenic tissue ; normal plasma is not the optimum medium for growth of tissue ; the most favourable plasma for spleen culture contains two- fifths distilled water, and slightly less for liver and heart, and generally for skin, too. 1041. Preparation of Tissues. — The tissues for cultures should be in normal condition, and are best when taken from the living animal or immediately after death. Positive results can still be obtained, however, when the tissues have been deprived of circulation for more than thirty minutes. With a cataract knife and a fine needle, a small fragment of tissue is dissected from the animal and placed on a glass plate ; the piece is rapidly cut into smaller pieces about the size of a millet seed and transferred to a perfectly clean sterile coverslip. This process must be carried out rapidly because some tissues die in even as short a time as ten seconds when exposed to the air (e.g., thyroid). To prevent this the tissue may be dissected in serum or Ringer. 1042. Preparation of Cultures. — For cultures of the hanging-drop type one uses a hollow ground slide of a sufficient depth to prevent 554 THE CULTIVATION OF TISSUE " IN VITRO." the drop of plasma from touching the bottom. The tissue is quickly placed on a coverslip, 2 drops of plasma from the paraffined pipette (§ 1039) are added and evenly and thinly spread around the tissue ; this must be done before coagulation occurs. If the plasma is not spread evenly the tissue-culture will grow in many planes, and will be less easy to observe, manipulate, and to fix and stain. When the plasma is spread out the cover glass is inverted over a hollow slide of suitable depth ; a little sterile vaseline may be placed at each side of the cell to assist adhesion preparatory to waxing down. The latter process is done by brushing molten paraffin around the edge of the coverslip, and on the slide, to prevent drying. Im- mediately this has been done the preparation is transferred to an incubator. Carrell and Burrows use a small portable electric incubator which is used for carrying the finished cultures to a bigger incubator in the observation room. For the large plate cultures the same technique is used. Tissue may be rapidly removed, cut into very small fragments, suspended in Ringer, and then spread on the cover of a flat glass -covered (Gabrits- chewski) box, and covered with plasma. The Gabritschewski boxes after several days' incubation (three to five days), are opened, and the plasmatic jelly cut out into blocks and preserved. Or, instead of using Gabritschewski boxes, one may make the culture on large black plates, which must then be placed in glass boxes with cotton sponges soaked in water, in order to preserve the proper hu- midity. The boxes are then carefully sealed with paraffin and kept in such a position that the fluid products of the culture may drain to the bottom. 1043. Subculturing is generally difficult, the technique of culti- vating of tissue cells in series being far from perfect. One extirpates a piece of the primary culture at its most active period, and transfers it to a fresh medium, growth often, but not always, beginning anew. Tertiary cultures are made in the same way. CARREL and BURROWS (Journ. Exper. Med., xiii, 1911) find that a very good way is to cut out the middle of the old culture around the original piece of tissue, and then fill up the space with new medium. The old cells grow into the new plasma. 1044. Fixation and Staining of Cultures. — CARREL and BURROWS (Journ. Exper. Med., 1911) remove the cover-glass to which the culture is adherent, and immerse in corrosive sublimate, acetic acid, or formalin, or the various preparations of chrome salts. After- wards the culture is stained in heematoxylin of Benda, Heidengain or Weigert. Dr. A. Drew informs me that he has found that the best fixatives CHAPTER XXXVII. 555 for in vitro cultures are, 70 per cent, alcohol and 5 per cent, acetic, Flemming, and Bouin. In all cases the cultures on the slip should be first detached from the slide and placed in warm Ringer's solu- tion 37° C. for five minutes. They are then placed in the fixative. Alcohol-acetic gives the cleanest pictures. Staining is best done by Ehrlich's hsematoxylin, Delafield's hsematoxylin, iron hsema- toxylin, carmine or Giemsa. As counterstain either eosin or orange G-. may be used. 1045. Artificial Culture Media, — MARGARET E. LEWIS and W. H. LEWIS (Anat. Record, v, 1911, p. 277) have investigated tissue cultures of chick embryo cells made in artificial media. Eighty combinations of NaCl, CaCl2, KC1 and NaHC03 and water, to form culture media have been proposed. It was possible to obtain growth in such media, in which either the CaCl2 or the KC1 or the NaHC03 was omitted, but not when the NaCl was left out. Such growths continue only for several days, and are never as extensive as those grown in plasma media. More recently MARGARET K. LEWIS (Gontrib. to Embryology, ix, 1920, Nos. 27-46) for tissues of chick embryos of four to twelve days' incubation uses "Locke-Lewis' solution (90 c.c. of NaCl 0-9 per cent. + KC1 0-042 per cent., + CaCl2 0-025 per cent., + NaHC03 0-02 per cent., + 10 c.c. of chicken bouillon + 0-25 per cent, dextrose). The embryo was removed from the egg and placed in a petri dish con- taining 20 c.c. of the warmed solution. Pieces of tissue to be explanted were removed, washed through one or more changes of warm medium, and cut with sharp scissors into pieces about 0-5 mm. in diameter ; each piece was then placed in the centre of a coverslip, part of the drop drawn off, and the coverslip sealed on to a vaseline ring around the well of a hollow slide. Cultures thus prepared were kept in an incu- bator at 39° C.^ and observations made in a warm box at 39° C. CHAPTER XXXVIII. A GUIDE FOR STUDENTS OF MICROTOMY. 1046. Three Examples for Beginners : — (1) The preparation of whole at dined mounts of some small object (Daphnids). (2) The preparation of sections of the muscle or an organ of a vertebrate. (3) The preparation of an embryo (or tadpole) for the making of serial sections. Example I. — From a pond or ditch obtain some water-fleas (Daphnia or Simocephalus) ; allow the jar to stand for several hours till the suspended material has settled. Capture some of the organisms as follows : — Take a piece of glass tubing some 8 inches in length ; place a finger over one end, dip the other end under the water and by taking away the finger, suck up some of the Daphnids into the tube ; put your finger over the end of the tube, remove the latter and transfer the organisms to a capsule or watch-glass about 2 inches in diameter. With a clean pipette carefully suck up most of the water, hardly allowing the animals enough to swim in ; now add a fixative to kill the organisms (see § 2), and to coagulate their protoplasm (§ 29) as rapidly as possible so as to leave the groups of cells forming the organs intact and in situ. Use corrosive acetic acid (§ 63), 2 per cent, acetic acid in saturated aqueous corrosive. Pour the fixative into the watch-glass or capsule, till it is full (the watch-glass or capsule contains about 15 to 20 c.c.). Place a glass square or plate over the capsule, and leave it for thirty minutes. The organisms become opaque, indicating the coagulation of the proteids of their cells. With a pipette carefully remove as much of the fixing fluid as possible. Now that the organisms are killed, the mercury salt must be removed ; unless the fixative is thoroughly removed, it will form masses of pin-shaped crystals at a later stage when the animals are being mounted in balsam. To remove the corrosive sublimate, it is necessary to convert it into another substance which may be more easily washed away ; this is effected by immersing the animals in some 70 per cent, alcohol which has been coloured light port-wine shade with tincture of iodine (§ 63), whereupon the mercury bichloride becomes mercury iodide, which is very soluble in 70 per cent, alcohol. The iodine and alcohol mixture CHAPTER XXXVIII. 557 should be used until it no longer loses its colour, which indicates excess of iodine. The whole process should last several hours and may be carried on overnight. The iodine and 70 per cent, alcohol are poured away, and the animals washed for several hours (a minimum of two) in at least two changes of 70 per cent, alcohol to remove as much of the iodine as possible. The objects are then transferred to 50 per cent, alcohol for one half- hour, then into 30 per cent., for the same time. They are brought down these grades in order that shrinkage may not occur when they are being transferred to stains containing little alcohol, or none at all. Two stains may be tried, Mayer's acid haemalum (§§ 248 and 249), and Grenadier's alcoholic borax carmine (§§ 213 and 233). The time that both these stains should be used depends almost entirely upon the accessibility of the cells of the object to the stain. Daphnids are covered by a chitinous shell, which though delicate tends to prevent instant penetration. It is a good thing to leave the animals in the stain for about five hours at least, and overnight preferably. Take two clean capsules, pour into one about 10 c.c. of borax carmine, into the other a similar quantity of the hsemalum. With a camel-hair brush or a pipette transfer some of the organisms to the stains and leave as directed above. See that the capsules are securely covered. After some hours in the stain, the latter is poured away, and the process of differentiation (§ 203) is begun. The object of differentiation is to wash away superfluous stain from certain organs or parts of organs, in order that a contrast in depth of colour may be obtained in the various other organs and tissues. Both borax carmine and Mayer's acid hsemalum may be differentiated in acid alcohol (4 to 6 drops of HC1 to 100 c.c. of 70 per cent, alcohol), which should generally be allowed to act at least for as long as the stain has been used, and, if necessary, longer. In both cases when differentiation has reached the right stage, the objects examined under a microscope have a trans- parent appearance, and such parts as the viscera and muscles should be well contrasted. The borax carmine specimens are wrashed out for several hours in neutral 70 per cent, alcohol. They are then upgraded to 90 per cent, and absolute alcohol, two hours in each, or overnight in absolute alcohol, and cleared in cedar wood or clove oil for at least two hours, and then mounted in xyjlol balsam. The hsernalum specimens have to be brought to an alkaline solution in order to " blue " the stain, and to get rid of all acid. Some workers " blue ' the stain in 70 per cent, alcohol made slightly alkaline with ammonia or bicarbonate of soda, but the best results are obtained by downgrading the objects to tap-water, which is allowed to run over them gently till they go quite blue, which should occur for small objects within an hour. The animals are then gradually upgraded through 30, 50, 70 and 90 per cent., to absolute alcohol, and cleared as above described for borax carmine specimens. 558 A GUIDE FOR STUDENTS OF MICROTOMY. In order to obviate the differentiation stage, one may dilute both the borax carmine and the acid haemalum till they are about one-third or one -half as strong ; dilution of the borax carmine may be carried out with 50 per cent, alcohol (not methylated spirit) and with distilled water in the case of hsemalum, In these solutions the animals remain till sufficiently stained. But the best results are got by the overstaining and differentiation method. 1047. Example II. — From a frog remove a large leg or thigh muscle, and cut it into two pieces about as big as the nail of the little finger. If desired, the liver, a halved testis, or a kidney may also be used. Transfer the material to a capsule containing at least 20 c.c. of Zenker's or Helly's fluids (§§ 73, 684). Leave till next morning, and wash in running water under the tap for at least three hours, preferably over- night, then transfer to 50 per cent, alcohol for an hour ; then to 70 per cent, alcohol containing enough tincture of iodine to give the solution a light port-wine shade. Add more iodine as the colour disappears, prolonging the treatment overnight for large pieces. Pour away the alcohol, and add pure 70 per cent., in which the material is washed at least three hours. Transfer to 90 per cent, for several hours and leave in absolute alcohol overnight. Next morning it is safest to give the material another hour in a fresh change of alcohol absolute. Pour away a good deal of the alcohol and add about the same quantity of xylol or cedar oil. Shake, leave half an hour, and then transfer the material to pure xylol or cedar oil ; leave half an hour. Pour away some of the xylol, either add chips of hard wax to cover the tissue, or add some of the stock xylol and wax mixture. Leave an hour in thermostat on the upper shelf, pour off, and add molten pure wax ; leave one or two hours on the bottom shelf. Embed blocks (§§ 142, 143). 1048. Example III. Preparation of an Embryo for Serial Sections.- Fix in Bouin's fluid , corrosive acetic or picro-nitric, overnight (§§ 110, 63, 97). In the case of the first and last mentioned fixatives, the embryo is afterwards transferred to 30 per cent, alcohol (half-hour), 50 per cent, (two hours), and then washed for a day in several changes of 70 per cent. The corrosive acetic fixed specimens are similarly treated except that at this stage iodine solution is added to the 70 per cent, (or this may be done in 90 per cent.) alcohol till the corrosive sublimate is removed. Leave overnight in 90 per cent, alcohol (or at least three or four hours), and at least six hours in two changes of absolute alcohol (preferably overnight). De-alcoholisation and clearing must be done carefully as directed in § 591, p. 269. It is a good plan to bring embryos from absolute alcohol, through several gradually strengthening mixtures of alcohol and cedarwood oil — to pure cedar-wood oil, and then wash out in benzole. Embed in wax as described in § 591, generally about one hour in benzol and wax, and two hours in pure wax. Embed blocks (§§ 142, 143). Now read §§ 144 to 151. CHAPTER XXXVIII. 559 1049. General Plan of Procedure Applicable to Histological Specimens. Anaesthetise animal, kill it, quickly take out organ, cut pieces 1 cm x 1 era x £ cm. ( 2£ % K2 Cr-2 O7 5~ % Hg Cl? ( 5 % Gl. Acetic acid. Wash in running water 24 hours. Fix for 24 hours in Zenker's fluid Freezing method. Paraffin method. Preserve in 5% Formalin f Wash in water Pass through increasing strengths of ale. f 70% alcohol Impregnate with gum f Cut sections with freezing microtome Float in water on to slide f Stain with Picro-carmine 15 minutes Drain off and wipe away the stain around Remove Hg deposit with iodine in 80% alcohol f (Preserve in 80% alcohol) Stain t Pass through 90% alcohol .................. 1 day Dehydrate in abs. ale ................. 1 night Mount in Farrant's medium. Clear in xylol or chloroform... From 1 to 2 hours f Pass through xylol satd. with paraffin wax 1 hour Impregnate with paraffin at 52° C 2 hours and Embed and make blocks f Cut sections with microtome Celloidin method. Ale-ether aa 1 day [1° Apply fixative to slide or glycerine and albumen water] f Thin celloidin 1 week (15% in ale-ether) Float section on drop of water on slide Warm gently to open out the section Wipe away excess of water and dry in air rhick celloidin ......................... ..1 week (30%- in ale-ether) 3° Remove paraffin with xylol After evaporation, mount on block of vulcanised fibre Harden celloidin in chloroform ......... 1-2 hours and then in 80 % ale ...................... 1-6 hours f Cut with razor (oblique) wetted with 80 % ale. 4° Remove xylol with abs. ale. I 5° Pass through 90 and 70 % alcohol to water t 6° Stain in Hsematoxylin, etc., 5-15 minutes, etc. Stain without removing celloidin I (Remove celloidin with oil of cloves) Mount in balsam. 7° Wash in water, 5 minutes — 1 hour t 8° Counter-stain in Eosin — 1 minute, etc. t 9° Remove excess with 90% alcohol i 10° Dehydrate with abs. ale. 11° Clear in clove oil or xylol 12° Mount in balsam. (Modified from D. T. Harris' "Practical Histology.") 560 A GUIDE FOR STUDENTS OF MICROTOMY. 1050. General Rules and Hints for Students. — (1) Keep all your bottles and capsules as clean as possible. (2) Try to keep your bench in order (it is difficult, J. B. G.). (3) Keep notes of the time necessary for changing reagents. (4) Thoroughly clean your slides and coverslips in acid alcohol before using. See addendum. (5) Note that corrosive sublimate tends to harden material. (6) Corrosive sublimate is difficult to remove from tissue unless you use iodine. If not properly removed you will find numerous pin-shaped crystals in the finished sections. § 63. (7) Corrosive sublimate attacks the surface of steel and other metals. Use quills, or wooden needles for manipulating tissue in sublimate. (8) Watery stains after picric acid fixation will cause maceration if prolonged. § 93. (9) Unless very well washed out, picric acid should not be used in conjunction with thionin or toluidin blue. Precipitates form. Certain other dyes do likewise. (10) Osrnic acid crystals should be dissolved in the purest distilled water. Wash the tube with distilled water before you break it, removing label. Wash out capsules and bottles for osmic acid solutions in distilled water. Keep solutions in shade or dark. § 27. (11) Osmic acid tends to harden yolk and certain other cell materials. The vapour of osmic acid is injurious to the eyes and nose. (12) Osmic acid and fixatives containing it inhibit staining, but if necessary you can induce osmicated material to stain in delicate dyes by bringing sections down to distilled water and treating in a -25 per cent, solution of permanganate of potash for a short time. Permanganate also decolorises sections. See page 31. (13) Nitric acid tends to soften chitin and yolk, but it may inhibit staining a little. § 97. (14) Imbed material in paraffin in the shortest time possible, for materials left in the thermostat longer than necessary go hard, especially from xylol ; this refers especially to vertebrate material and yolky embryos. (15) Alcohol and chloroform dissolve fats and lipoids, acetic acid dissolves away lipins. Vegetable oils dissolve fats less readily than xylol or chloroform. Bead §§ 120 et seq. (16) Strong alcohol is bad for the finger nails and skin. (17) When diluting stains with alcohol, use solutions made up by breaking down pure absolute alcohol. Do not use methylated spirit, as this generally precipitates the stain. (18) You can soon learn to tell roughly the strength of alcohols by the smell. (19) Don't use the dregs of the absolute alcohol bottle for dehydrating anything. The dregs are no longer absolute. Keep a waste alcohol bottle for used liquid. (20) Some workers add a little bag of fused copper sulphate to their store bottles of absolute. This keeps the alcohol dehydrated. (21 ) After fixation, when dehydrating and embedding a piece of tissue, an egg or an embryo, it is at its softest when in weak alcohol, and its CHAPTER XXXVIII 561 hardest when in xylol or a clearing oil. FJatten or otherwise manipu- late a fixed object, while it is still in weak alcohol, or it will break up ; but some objects may be dissected successfully in clove oil. § 122. (22) Cells alter soon after death: formalin fixation is the best for corpse material. Carefully note § 31. (23) The organs of animals over-anaesthetised by chloroform or ether are often spoilt (especially in the vicinity of large blood vessels) and are sometimes useless even for general purposes. § 12. (24) Keep balsam or colophonium jar in the dark, or paint it black outside. Acid balsam soon removes stains from tissue ; acid balsam is the micrologists' bete noire. § 443a. (25) After Zenker fixation sections may overstain in eosin. (26) If finished sections have crystals in them this is due to improper washing out of fixative, or stain. (27) Formaldehyde gas dissolves in water up to 40 per cent. The commercial formalin is acid and must be neutralised with magnesium or sodium carbonate kept in a little bag in the stock bottle. § 108, (28) Formaldehyde gas is injurious to the skin and mucous membrane of nose. (29) If after staining in delicate dyes (e.g., methyl green), all the colour keeps coming out of the sections during passage through alcohols, try the following method : — Wipe superfluous water from around the sections, and dehydrate by dropping acetone on sections : then plunge into a jar of half acetone, half xylol, then pure xylol. (30) For clearing embryos or pieces of tissue for whole mounts, cedar- wood oil is better than xylol. § 120. (31) If bubbles get under the coverslip they can often be removed by gently warming, or by placing slide under bell jar of an exhaust pump. (32) If after mounting an object in balsam white or black lines and blotchy areas appear, this means that dehydration was not complete. Bring back through xylol to absolute alcohol. (33) When, after embedding, the block is set aside for a time and it is found that the object is surrounded by a halo of white wax, this means that all the clearing oil was not removed and is now exuding from the object. Ee-imbed in pure wax. (34) When, after embedding, the material seems soft and tends to fall out of the wax, this indicates that dehydration was not complete, and possibly also that the time in pure wax was not long enough. Without efficient dehydration it is impossible to make good sections. (35) If when cutting the sections curl up, it means that either the knife is blunt or the material has been overhardened during imbedding. Occasionally an incorrect slope of the knife may be the cause of curling. (36) When the sections will not form a ribbon, this means that either the wax is too hard or the slope of the knife is not correct. If the wax is hard, place 1 drop of soft wax on each side of the block and flatten it out with a warm knife. Read carefully pp. 83 to 90, (37) The broad side of a block sho'uld be parallel to the knife. (38) Some people use miniature drums for rolling up the wax ribbon. Laying them on a piece of foolscap does quite well. Avoid sticky M. 36 562 A GUIDE FOR STUDENTS OF MICROTOMY. paper. If sections accidentally adhere you can often release them by cautiously wetting the paper with absolute alcohol. ( 39) Before placing sections on a slide, write with a diamond pencil the number of the slide and the material used. At a pinch, a glass wax- pencil may be used in stead - (40) If you have not used a diamond, it is always possible to tell on which side of the slide the section lies, simply by slightly tilting the slide and observing the shadow thrown on the other side of the glass. (41) Finally, if your first attempts are failures, do not be discouraged — even the most skilful microtomists generally produce atrocities at their first attempts. Try again ! APPENDIX 1051. Chemicals, Stains, and Apparatus. — Addresses of British firms from which it is recommended that these be obtained are given in § 11. 1052. Cleaning Slides and Covers. — New ones should first be soaked in one of the following liquids : strong sulphuric, hydro- chloric or nitric acid, or aqua regia, or a mixture of an ounce each of sulphuric acid and bichromate of potash with from 8 to 12 ounces of water, then washed first with water and lastly with alcohol, and dried with a clean cloth. For used ones, if a balsam mount, warm, push the cover into a vessel with xylol or other solvent of the mount, and put the slide into another vessel with the same, leave for a few days, and then put into strong alcohol. If this is not sufficient, treat as for new ones. Some persons boil in lysol, which I do not find efficacious. For the final treatment, see p. 121. 1053. Gum for Labels. — Labels stuck on glass often strip off. This may be avoided (MARPMANN, Zeit. Angew. Mik., ii, 1896, p. 151 ; Journ. Roy. Mic. Soc., 1897. p. 84) by means of the following adhesive : 120 grms. of gum arabic are dissolved in a quarter of a litre of water, and 30 grms. of gum tragacanth in a similar quantity. After a few hours the tragacanth solution is shaken until it froths, and mixed with the gum arabic solution. Strain through linen and add 150 grms. of glycerin previously mixed with 2^ grms. of oil of thvme. j PEIRCE (Joain. app. Mic., ii, 1899, p. 627 ; Journ. Roy. Mic. Soc., 1900, p. 404) finds that if the end of the slide be painted with a thin solution of balsam, it may be written on with ink when dry, and the record preserved by a second coat painted over it. For other receipts see early editions. 36—2 INDEX. Names like Lo Bianco are given under the latter half of the name. Abderhalden, 357 Acalephse, 523 Acanthocephali, 513 Acephala, injection, 503 Acetate of lead, brain, 404 of potash, medium, 219 Acetic acid, fixation, 51 alcohol and sublimate, 53 and alcohol, 52 bichromate, 41 decalcification, 252 alum, carmine, 137 Aceto-carmine, 138 Acetone, 168 for dehydration, 4 fixation (Lucidol), 47, 59 and formol, 64, 494 and sublimate, 47 -chloroform, for narcotisation, 14 Achucarro, neuroglia, 490 Acid luchsm, 171 for mitochondria, 320, 321 and malachite green (Pianese), 175 and methyl green. 324 myelin, 452 and orange G, 172 Acid hsemalum, 153 haematoxylin, 155 magenta, 171 rubin, 171 Acidic dyes, 120 Acidophilous tissue, 128 Actinians, narcotisation, 13, 521 Adamkiewicz, myelin, 452 Adenoid tissue, 249 Adsorption, 120, 122 Adurol, 469 Agar, sections between coverslips, 307 Agar-Agar, growing amoebae on, 527 Agassiz and Whitman, 281 Agduhe, Bielschowsky, 431 Aguerre, 482 De Albertis, neuroglia, 485 Albumen, method of mounting sections, 113 mercurial mounting medium, 220 removal of from eggs, 260 et seq., 283, 284 Alcohol, for dehydration, preservation, 4 narcotisation, 13 fixation, 56, 58 for maceration, 244 Alcohol — contd. absolute, 58 table lor dilutions, 57 and nitric acid for decalcifying, 253 Alcoholic, mercury bichloride, 46 hsematoxylin, Apathy and de Groot, 156 Heidenhain, 543 Dobell, 544 cochineal, 144 Alcyonaria, 522 Alcyonidium, 500 Alcyonium, 12, 521, 522 Alexander, 262 Alfieri, bleaching, 256 Alizarin, 184 and crystal violet, 322 nervous system, 408 Alkanna, 368 Allen, methylene blue, 192 Allen, Ezra, 304 chromic Bourn's fluid with urea, 306 on clearing, 307 Allen, E. J., menthol, 12 Allen and Browne, 27, 523, 524, 525 Allerhand, iron myelin method, 451 Alt, 408 Altmann, 20 et seq., 38 acid fuchsin picric acid method, 320 corrosion, 249 fat, 366 fluid, 37 Alum, aniline, 355 carmine, 136 and picric acid, 138 -nitric acid for decalcifying, 253 hsematoxylin, generalities, 151 Aluminium haematein, 151 Alzheimer, 416, 480 Amann, lactophenol, 222 Amato, 425 Ammonia, carmine, 140 chrornate, 43 Ammonio-chloride of tin for mvelin, Besta, 448 Ammonium, bichromate, brains, 403 sulphocyanide for maceration, 245 vanadate, Golgi method, 476 Amoebae, cultures, 527 Amphibia, 275 et seq. brains, 405 Champy's fluid, 37, 318 INDEX. 565 Amphioxus, 281 Amphipoda, embryology, 288 Amyl nitrite, 233 Amyloid, 133 Andeer, 254 Andres, actinida, 12, 13, 521 Andrews, avian embryology, 272 Andriezen, Golgi method, 465 Anemones, narcotisation, 12 See Actinians. Anglade and Morel, Victoria blue neuro- glia stain, 483, 485 Anilin dyes, 1 59 et seq. blue, 182 blue-black, 183, 408 blue and carmine, 213 red, 169 oil, 69 anilin oil water, 166 Anitschkow, 110 Annelids, 39 blood vessels, 510 Champy-Kull, 322 killing, 12 nerves, 510 Antedon, 519 Apdthy, 97, 98, 511 alcoholic corrosive, 46 hsematoxylin, 156 bergamot oil method for celloidin sections, 117 embedding in oil of cedar, 78 Canada balsam, 226 cement for glycerine mounts, 231 gold, 198, 206, 207 glycerine gelatin embedding, 93 gum syrup medium, 221 ha3inatin mixture, 155 methylen blue, 189, 192, 193 on knife tilt, 85 neurofibril methods, 416 nitric acetic for maceration, 247 picro-saurefuchsin, 177 series-on-knife method for celloidin sections, 118 theory of gold impregnation, 203 Apel, 512 Aqueous humour, 219 Araneida, 287 Arctiscoida, 509 Argentamin, 415, 452 Argyroneta ova, 287 Arndt, bone saw, 370 Arnold, 178, 297, 517 chondriosomes, 323 staining kidney, 394 Arnstein, 190, 193, 342 method for corpuscles, 342 Aronson, myelin, 448 Arsenic acid, decalcifier, 254 Arthropoda, 504 et seq. embryology, 284 et seq. fixation, 44, 47, 55 mounting whole, 504 Artifacts, 20, 304 Artificial fecundation, 258 et seq. Artificial iodised serum, 219 Artom, ascaris ova, 290 Ascaris ova, 52, 289 Aschoff-Kiyono, 388 Ascidia, narcotisation, 12 buds, 282 general, 499 Ascoli, Cajal's method, 426 Asphalt varnish, 230 Asphyxiation, by boiled water, carbonic acid gas, 16 Assmann, blood stain, 384 Astacus, eye, 508 methylen blue, 192 nerve-endings, 344 Asteroidea, 519 Astrocytes, 479 Athanasiu and Dragoiu, &52 Atheson, 511 Athias, 467 Atta, ova, 287 Auerbach, 415 buds, 425 stain, 312 Augstein, 514 Auricularia, 520 Aves, embryology, 271 et seq. Gerlach's window method, 271 Axis cylinder, 452 (methylene blue) other stains, 454 et seq. and dendrite, advice on special forms, 459 processes similar to Golgi methods, 475 rapid process, 458 Azoeosin, 313 Azoulay, 209 ammonium, vanadate process, 476 osmic acid methods (myelin), 451 Babcock, 102 Babes (safranin), 166, 167 Babkin, 396 Bacteria, in amoeba cultures, 528 in tissue, 326, 354 Ballowitz, 348 electric organ, 346 mammals, 264 reptiles, 275 Balsams, 225 et seq. cedar wood, alcoholic, 226 method, section-grinding, 109 neutral, 226 Barnes, 514 Barrois, echinoderm larvae, 520 Bartel, 482 Baryta-water, for maceration, 245 Basic dyes, 120 Basophil granules, nerve tissue, 410, 414 ' Basophilous " tissue, 128 Bastian, gold, 205 566 INDEX. Bataillon and Koehler, 308 ascaris ova, 289 Batchelor, 390 Bath (paraffin), 77 Baumgarten, 183, 213 Bayeri, decalcifier, 254 ossifying cartilage, 377 Bayliss, 125, 309 on dyes, 120 on specificity of stains, 134 vaso -dilators, 233 Beale, 232 digestion by pepsin, 248 Beard, raja embryos, 280 Beauchamp, 513 Beckwith, 309 Bedot, 524 Bees, brain, 508 Behrens, 182,^221, 344 mounting medium, 224 salmon embryos, 280 Bell, cement, 229, 366 fat, 367, 369 Benario, blood, 381 Benda, 495 alizarin method, 322 copper hseinatoxylin, 157 crystal violet, 184 fatty acids, 368 " Flemming fluid," 319 iron hsematoxylin, 148 neuroglia stain, 482 picro-saurefuchsin, 177 rapid myelin method, 448 safranin. and light green, 181 secretion, granules, 315 Benecke, fibrils, 351, 388 Van Beneden, acetic acid, fixation, 51 method against contraction, 12 acetic alcohol, 52 live mammal embryo in serum, 267 mammals, 265 tsenia eggs, 289 and Neyt, ascaris ova, 290 Bengal rose, 180 Bengtsson, eggs of diptera, 285 Bensley, brazilin and water blue, (thyroid), 394 pancreas, 395 intestine, 393 Bensley-Cowdry, acid fuchsin methyl green stain, 324 Benzidine dyes, 388 et seq. teeth, 373 Benzoazurin, 170, 184 cartilage, 376 Benzol, 70 embedding, 78 peroxide, 59, 382 Benzopurpurin, 179, 390 Benzoyl, green, 181 Bergamot oil, 68, 323 for celloidin sections, 117 Bergh, annelids, 510 Bergonzini, 356 Berkeley, C4olgi method, 466 liver, 393 rapid myelin method, 447 Berlese, Acarina, 504 Berlin blue, aqueous masses, 240 of Mayer, 240 injecting teeth, 372 mass, Briicke, 236 Bernard, mollusc, 504 Berner, 368 Bernheim. 206 Beroe, 524 Bertarellis, protozoa, 532 Best's carmine stain for glycogen, 295 Besta, amrnonio-chloride of tin, 448 Cajal's method, 425 Golgi apparatus, method, 438 Bethe, 507 chitin, 506 rnethylen blue, 194 molybdenum-toluidiii blue, neurofi- brils, 417 Bettendorf, 516 Betz, hardening nerve, 403 Bevan Lewis, 183, 404 Lo Bianco, 14, 15, 37, 510, 514, 515, 516, 518, 519, 524 acetic fixation, 51 ascidia, 499 mollusc oida, 500 chromo-sublimate, 48 corrosive acetic, 44 gephyrea, 512 mixture for narcotisation, 13 protozoa, 542 Bichloride of mercury. See, under Mercury. Bichoff, mammals, 264 Bichromate of ammonia, 43 of calcium, 43 of potash, 41 decoloration of, 41 maceration, 245 Muller's fluid, 42 and alcohol, 43 fixation, nervous system, 403 et seq. -osrnic, 37 -platinic, 37 chromic -osmic, 37 and mercury, 48 -sublimate, Golgi method, 470 Bickfalvi, digestion, 249 Biebrich scarlet, 180, 313 Biedermann, methylen blue, 344 Bielaszewics, 150 Bielschowsky methods, 426 et seq. Da Fano modifications, 432 et seq. other modifications, 430 et seq. silver method, for connective tissue, 352 and Bruehl, ear, 497 and Plien, cresyl violet, 414 Bigelow, Medusae, 524 INDEX. 567 Bile capillaries, 393 Bilharzia, 516 Binet, 31 Bing, 64, 449 Biniodide of mercury mounting liquid, 224 Binnennetz, 316 Biondi, blood, 380 Bipinnaria, 520 Bismarck brown, 161, 169 for cartilaginous skeletons, 377 Bizzozero, 391 blood-platelets, 386 and Torre, blood, 382 Bjeloussow, gum arabic mass, 241 Bladder, frog, nerves, 349 Blastoderms, general, 260 of mammals, 267, 268 Bleaching, Mayer's chlorine method, 255 sulphurous acid, hydrogen peroxide, chlorine, 31 Bles, frog embiyology, 278 Bleu de Lyon, 183 Block ma nn. 115, 500 cestodes, 516 frog embryology, 276 Blood, 379 et seq. cells, mitochondria, 333 elective stain for reds, 388 fixation, 379 et seq. fixing in bulk, 380 new Golgi body, 387 and iron salts, 300 platelets, 386 -serum media, 219 stains like methyl green, etc., 380 Blue gelatine mass, 236 lumiere, 183 Blum, 401 Bobretzky, 286 Boccardi, 206 erythrosin and toluidin blue, 415 Bodecker, decalcification, 251, 373 Boeke, Bielschowsky, 430 — 431 Bohm, 206 and Oppel, 41, 219 reptile blastoderms, 274 Bohmer, hsematoxylin, 154 Bohmig, 515, 517 Bolina, 524 Bolton, 469, 447 Golgi method, 464 Bombyx mori eggs, 285 Bone, 369 et seq. decalcification, 251 dry sections, 369 et seq mounting, 370 non-decalcified, 369 soft parts, 371 et seq. saw, 370 Bonnet, 269 Bonney, 178 Bonome, 480 Bonvicini, hardening human brain, 404 Borax carmine, 141 Bordeaux R., 178 Borgert, 75 Boring, ascaris ova, 290 Born, 262, 278 Borrel, 213 -method, 532 Borrel 's blue, 534 Bouffard, Benzidine dyes, 388 Bouin, picro-formol, 62 frog larvae, 278 salmon embryos, 280 Boule, Cajal's method for 425 Bouma, 376 Boveri, ascaris ova, 290 embedding of echinoderm ova, 260 picro -acetic, 55 Boyce and Herdman. copper, 300 Boycott, 507 Brachiopoda, 500 Bradford and Plimmer, 547 Braem, Bryozoa, 282 Brain, cat, dog, neurofibrils, 421. See under Nervous System. cat, man, preliminary fixation, 402 insect, 508 preliminary treatment, 397 — 399 Branca, 168 sublimate formol, 63 Brandt, 546 glycerine jelly, 223 protozoa, 540 Brasil, 546 Brass, on embedding, 76 protozoa, 542 Braun, 514, 517 Braus, 26, 281 bile capillaries, 393 Brazilin, 211 for sponges, 526 Breglia, 449 Bremer, 181, 473 Bresslau, mesostoniid ova, 288 Brilliant kresylblau, blood, 383 Bristol, 511 Brittle object, cutting of, 88 Brock, maceration, 245 Brodmann, 398 Bromide of soda or potash, Simarro. 41 ii Brookover, 442, 468 Golgi method, 465 Brown, 549 Briicke, digestion, 249 Bruel, dipterous eggs, 285 Briihl, corrosion, 249 Bruno, mucin, 391 Brunotti, gold gelatin, 93 gelatin embedding, 108 Brunswick black, 230 Bryozoa, 282, 500 heat-killing, 12 Buchner, glycogen, 296 nucleoli, 314 568 INDEX. Budge, injection, 241 Bugula, 500 Bujor, 523 Bulb, 424 Bullard, 367 Bumpus, 103 Bunge, fluid for iron stain, 299 flagella stain, 548 Burchardt, 139 paraffin, 92 chrome fixation, 40 protopterus brain, 405 Burzynski, 94 Burger, nemertina, 515 Burrows, 550 Busch, 29, 32, 214, 252 decalcification, 251 March! , 450 Butschli, 154 Buzzi, eleidin, 341 Cade, gastric glands, 393 Cajal, 435, 465 double-impregnation, Golgi process, 461 avoidance of silver precipitates, 467 gold chloride-sublimate method of neuroglia, 487 Gt>lgi apparatus method, 436 methylene blue diffusion process, 478 myelin, 450 nucleolini, 311 retina, 495 spirals and funnels, 440 Cajeput, oil of, 68, 103, 411 Calberla, 161, 181, 182 Bismarck brown, 161 liquid, 222 Calcium chloride medium, 219 Calyptoblastea, 523 Camsal balsam. 227 Canada balsam, 225 et seq. Caoutchouc, cement of Miller, 230 Capitellidse, narcotisation, 13, 510 Capsicum berries, 368 Carazzi, 256 Carbol-pyronin -methyl green, 172, 355 Carbolic acid (clearing), 69 fuchsin, 169 thionin (King), 168 Carbon, bisulphide for embedding, 77 Carter on, 372 insects, 507 tetrachloride, 77 Carleton, Golgi apparatus, 438 nucleolini, 311 Carmalum, 137 and indigo -carmine, 212 Carmine -generalities, 136 alcoholic stains, 141 aluminium chloride solution, 138 and anilin blue, 213 ammonia, soda, lithium, magnesia, 140 Carmine-generalities — contd. and cochineal stains, 135 -gelatine masses, 234 Hoyer, 235 Fol, 235 Krause, 235 glycerine mass (cold), Beale, 238 Robin, 233 Grenacher's alum-carmine, 136 and malachite green, 213 for nervous system, 407 neutral alkaline, 140 blue, 183 Carminic acid, 135 Carnoy, 151, 178 acetic alcohol, 52 and Lebrun, frog eggs, 276 iron, 300 Carothers, Bouin and urea for insect chromosomes, 306 Carrel, 550 et seq. Carter, J. Thornton, bone, 369 et seq. on post-mortem changes, 25 teeth sections, 371 Cartilage and bone, 376 silver, 200 skeletons, 377 Caryophyllia, 521, 522 Cassiopeia, 524 Castellarnau, 519 Castle, Ciona, 281 Castor oil, mounting medium, 228 Catois, methylene blue method for fishes, 478 Cattaneo, 541 Cattani, funnels and spiral filaments, 410 Caullery, 499 Causard, 509 Caustic soda bleacher, 257 potash or soda, corrosion, 250 maceration with, 244 Cavalie, electric organs, 346 Cavazzani, 215 Cedar wood oil, 66 et seq., 77 for minute dissections, 7 Cell inclusions, 316 et seq. granules, lymph and blood cells, 315 Celloidin embedding, 95 et seq. See also under Collodion, knife smeared in vaseline, 102 for injection, 241 sections, Apathy's method, 118 Bolles Lee's method, 116 Summer's method, 116 staining, 102 Celloidinum inelasticum, 96 Cements and varnishes, 229 et seq. Centrifuge, for oogenesis studies, 332 Centrosomes, 315 Cepede, 307 Cephalopoda, 282 eyes, 502 Ceratium, 547 Cercaria, 517 INDEX. 569 Cerebellum, 424. See under Nervous System. preliminary treatment, 399 Cerebrum, hardening, 399 Cajal's methods, 424 Cerfontaine, amphioxus, 281 Ascaris ova, 290 worm, 509 Certes, protozoa, 540 Cesaris-Demel, 383 Cestoda, 515 ova, 289 Chaetopoda, 509 marine, 510 Chalicodoma, eggs, 286 Chambers, intra vitam stain, 310 Champy, 314 fluid, 37 iodide of osmium method, 331 trichloracetic, 53 Champy-Kull,mitochondrial method, 321 fixation, Gatenby, 322 Chenzinsky, 181 blood stain, 383 Chick, embryology, 271 et seq. axis cylinder and dendrite, 459 Child, fish embryos, 279 Chilesotti, 407, 477 Chilopoda, blood, 381 China blue, spirals, funnels, 440 Chitin, 506 et seq. bleaching, 256 tests for, 507 Chiton, eggs, 284 Chitonidse, 503 Chloral hydrate, for narcotisation, 14 jelly, 224 maceration, 248 mounting medium, 220 Chlorcarmine, 139 Chloreton, for narcotisation, 14 Chloroform, for killing, injurious in cytology, 11 clearing, 70 embedding, 76, 78 vapour, collodion embedding, 98 Cholesterin, 356 et seq. Chondriokonts, 316 Chondriome, 316 Chondriosomes, 316 Chorion, removal, 284 et seq. Chromates, 40 Chromate masses, 237 Chromatin, microchemistry, 294, 308 et seq. and enzymes, 308, 309 digestive fluids, 294 methyl green test, 293 Chrome salts, Burckhardt on, 40 Chromic acid, decalcification, 252, 254 fixation, 32, 33 washing out, 33 formol, 63 for hardening, 34 Chromic acid — contd. and hydrochloric acid, decalcifier, 254 maceration, 246 nerve tissue, 404 and platinum chloride (Merkel), 39 Chromidia, 308, 309, 310, 316 Chromo-acetic acid, 35 Chromo-aceto-osmic decalcifier, 254 Chromo-fonnic, 35 Chromo-nitric acid (Perenyi), 39 decalcifier, 254 Chromophility tests, 309, 310 Chromosomes, techniques for, C4atenby, 303 et seq. Chromo-sublimate, 48 Chrysoidin, 364, 369 Ciacco, 206, 343 method, 369 (fat) tendon, 347 Ciaglinski, myelin, 452 Ciechanowski, bile capillaries, 393 Cilia, mollusc, 503 protozoa, 541 Ciliated epithelium, 339 Ciliates, general staining, 533 Cilimbaris, 353 Cinnamon (cassia) oil, 67 Cladocera, Haker, 288 Clasmatocytes, 356 Cleaning slides, 112, Appendix Clearing, 5, 65 Clove oil, for minute dissections, 7, 67 Coal gas, for killing, 11 tar dyes (plasma), 171 Cobb, differentiator, 3 Cocaine, for narcotisation, 14 Coccidia, staining, 532 Cochineal, 135 alum carmine, 137 Cochlea, 375, 496 et seq. Coe, Distomum, 289 Ccelenterata, 521 maceration, 524 nervous system, 522 Ccerulein S.,' 182 Cohnheim, gold, 204 Cold-blooded animals, injection, 241 Cole, gum mass, 110 Coleoptera, eggs, 286, 287 Collargol, 434 Collin, 510 and Lucien, 438 Collinge, 281 Collodion, embedding, 95 et seq. alcohol hardening, 100 bone sections, 372 bath, 96 for celloidin sections (Weigert), 118 blocks, clearing in cedar oil, 104 clearing and mounting, 102 cutting, 101 dry cutting, 104 hardening, 98 newer method, 103 570 INDEX. Collodion — contd. Obregia's syrup method of mounting sections, 115 older method, 96 preservation of blocks, 100 rapid process, 104 section mounting with albumen, 116 method of mounting paraffin sections, 115 and paraffin, 105 solution of, 97 Collodionisation, brittle objects, 88 Colloidal gold, 133 Colloidal-complex, 120 Colloids, 123 Colloxylin, 96 Colophonium, 226 and wax method, section-grinding, 109 Colucci, 226* Comatula larva, 520 Congelation masses, 109 Congo-Corinth, 179 Congo red, 121, 122, 124, 178 et seq. nervous system, 408 myelin, 453 Conklin, Crepidula, 284 Connective tissue, 350 et seq. . Bielschowsky, 434 Conser, 500 Contraction, prevention of, 1 1 acetic acid in, 12 corrosive sublimate in, 12 Cooling paraffin, 82 Copal method, 108 Copepoda, 288, 505 Copper, in tissue, 300 bichromate, 64 chloride and acetate fixation, 53 mounting fluids, 220 ferrocyanide injection mass, 234 formol, 64 nitrate, 54 sulphate and corrosive for nerve tissue, 405 fluid for frog eggs, 279 Corallin, 169 Corals, decalcification, 251 Cori, narcotisation mixture, 14, 15 osniic solutions, 29 Cornea, 342 et seq. maceration, 247 fibres, maceration, 246 silver method, 198 Corning, 475 neurokeratin, 441 Corpuscles, Golgi, 347 Meissner, Krause, 342 Nissl, 180 tactile, 341 Herbst and Grandry, 342 Corrosion, 249 et seq. Corrosive sublimate. See also under Mercury bichloride. preventative of contraction, 12 Corrosive sublimate— contd washing out, 26 Cowdry, 304, 415 acid fuchsin stain, 324 Janus green, 332 Mitochondria, 338 Cox, 415, 434 Golgi process, 472 et seq. neurokeratin, 441 Cramer, 30 fatty substances, 356 osmic vapour method, 330 Feiss and Bullock, 366 Creases (paraffin sections), 88 Creighton, 297 Creosote (clearing), 69 Crepidula, 284 Cresyl violet, nerve, 414 Crinoidea, 519 Cristatella, 282, 500 Crustacea, 505 et seq. Crystal violet, 184 for mitochondria, 322 Crystalline lens, 247, 343 Csokor, 230 bone saw, 371 Ctenophora, 524 Cucumaria, 518 Cultures, manipulation of amoebae, 527 Cunningham, micro-injection, 270 Curare, 292 for narcotisation, 16 Curling of sections, 86 Curreri, 469 Cutting, paraffin, 86 tissue, 319 Cyclas ova, 284 Cytological methods, 292 et seq. Cytoplasmic inclusions. 316 et seq. Czokor, 137 Daddi, fat, 367 Dahlia, 162, 169, 333, 340 Dakin, 503 Dallinger, 530 Damar, 226 Davidoff, 524 Tunicata, 281 Dead cells, 131 Dealcoholisation, 65 et seq. or clearing, 5 Death, 25, 131 Decalcification, 251 et seq. teeth, 251 et seq. and 372 et seq. Decapod, eyes, 508 Decapoda, ova, 288 Deecke, 404, 407 Deetjen, 387 Deflandre, fat, 369 Degenerate nerve, Marchi, 449 Dehydration, 2 by alcohol, acetone, methylal, aniline oil, 4 Dejerine, 407 INDEX. 571 Dekhuyzen, 218, 386 liquids, 43 osmacet, 380 Delafield. hsematoxylin, 154 Delage, Turbellaria, 517 sponge, 526 Delamare, 351 Dell'Isola, Golgi method, 463 Delia Rosa, Indian ink mass, 240 Delia Valle, Orchestia, 288 Deltapurpurin, 179 Demoor, 214 Dendrite stains, 454 et seq. Dendrocoelum, 517 Dendy, Geoneniertes, 515 sponges, 526 Denne, orientation of objects, 81 embedding method, 78 et seq. Dentine, 370 et seq. Dependorf, 374 Depigmentation, 256. See Bleaching. Descemet's membrane, 343 Desiccation method for paraffin sections, 111 Desilification, 251 et seq., 255 Dewey, 373 Dewitz, 503 Dictyosome, 316 Dietrich, 366, 509 Digestion, 248 et seq., 308 and dissociation, 243 et seq. Dimmer, 115, 119 Diomidoff, 405 Diptera eggs, 285 Disse, 377 Dissections-minute, cedar wood oil for, clove oil for, 7 glycerine for, 8 Distomum ova, 289 Dobell, modification of Heidenhain stain for protozoa, 544 Mann's stain, 544 Doderlein, 519 Dog brain, 402 Dogiel, 342 corpuscles of Herbst, 342 Meisser and Krause, 342 Grandry's corpuscles, 421 iris, 348 methylen blue, 189, 190 et seq. for epithelia, 195 tendon organs, 347 Donacia eggs, 286 Donaggio, 158 neurofibril methods, 417 et seq. Donaldson, 405 faeces, 538 Doncaster, chromosome fixation, 305 Double-embedding collodion paraffin, 105 Double-staining in hsematoxylin and acid fuchsin, 326 Downey, benzidine dyes, 388 Drasch, 204 Dreuw, 341 Drew, formol -chrome method. 325 manipulation of amoeba cultures, 527 protozoa, 526 et seq. staining amoebae, etc., 531 tissue culture, 551, 554 Drew-Griffin live-slide, 530 Drew-Murray, connective tissue stain, 354 Driessen, 297 Drost, 504 Di'iiner, 26 Duboscq, blood, 381, 465 Dubreuil, connective tissue, 350 Duerden, ccelenterates, 521 Duerk, 354 Dunham's mixture (celloidin sections), 103 Durig, Golgi method, 464 Duval, 404 carmine and anilin blue, 213 collodion embedding, 95 orientation method for blastoderms, 273 silver, 200 Dyes, nature of, 120 electric charges, 122 Ear, inner, 496 et seq. Eau de Javelle and Eau de Labarraque bleachers, 256 corrosion, 250 for eggs, 284, 285 for frog embryology, 276 Eberth and Runge, 469 Echinodermata, 518 et seq. larva?, 520 Echinoderms, decalcification, 251 Echinoidea, 518 spines, 518 Edinger, 34 Edington, blood, 381 Egg-capsules, removal, 284 orthoptera, 286 -Egg, white of, injection mass, 240 Eggs, Unio, 284 Ehrenbaum, grinding, 109 Ehrlich, 155 blood, 379 hsematoxylin and eosin, etc., 214 Indulin-Aurantia-Eosin, 180 mast cell method, 356 methylen blue, 188" et seq. neutral red, 179 triacid mixture, 175 Ehrlich- Biondi, 161, 173 and Lazarus, 297 Ehrmann, 340 Eichler, 498 Eisath, neuroglia granules, 486 Eisenberg, Nile blue, 368 Eisig, 13, 510 fluid, 40 maceration, 245 572 INDEX. Eismond, 539 Ekman, 500 Elasthsematein, 353 Elastic tissue, 352 et seq. fibres, of spleen, 394 Elderberries, Kappers, 409 Electric organs, 345 Eleidin, 341 Ellis, 548 Elschnig, 97 Embedding, gelatine masses, 92 et seq. collodion, 95 paraffin, 76 et seq. lead-gum, 106 boxes, 73 brass squares, 74 thimbles, 73 trays, 72 in vacuo, 80 Embryological methods, 258 et seq. Embryonic cartilage, 376 et seq. Embryos, Bielschowsky methods, 429 Cajal's methods, 424 fixation, 260, 268, 278, etc. Emery, aqueous carmine mass, 240 Encephala, 401, 404 Endothelium, silver, 200 Engelmann, 218 Enriques, 501 Entamceba, 538 Entire objects, preparation of, fixing agents for, best stains for, .7 Entz, 81, 543 protozoa, 541 Enzymes and chromatin, 308 Eosinophilous cells, 180, 383, 386 Eosins, 180 et seq. methyl green, 181 methylen blue, 383 et seq. Epeira eggs, 287 Ependynia cells, 479 Epidermis, digestion, 249 neurofibrils, 421 Epiploon, silver method, 199 Epithelia, silver method for, 198 Epithelium, maceration, 244, 245 Eppinger, 393 Erhard, 297 Erlanger, ascaris ova, 290 Erlicki's fluid, 42 Van Ermengem, 549 Ernst, 341 Erytnrosin, 180 with toluidin or methylen blue for nerve tissue, 415 Eternod, 83 Ether method, for celloidin sections, 116 for narcotisation, 13 Eucaine, for narcotisation, 15 Euler, 309 Eaparal, 227 Evans, benzidine dyes, 388 micro -injection, 270 Everard, 214 Ewald, blood, 380 section washing apparatus, 3 Examination media, 216 et seq. Eycleshymer, 98, 103 Eye, 493 et seq. arthropod, 508 et seq. bleaching, 255, 257 mollusca, 502 Fabre-Domergue, 220 protozoa, 540 Faded sections, treatment of, 6 Faeces, examination for protozoa, 538 smears, lucidol, 382 Fairchild, 3 Fajerstain, 342 hsernatoxylin, Golgi method, 476 Da Fano, 428, 434, 435 nervous system, 397 et seq. axis cylinder stains, 454 et seq. Bielschowsky method, generalities, 426—427 modifications, 432 et seq. Cajal's methods, 419 et seq. advice on Cajal's methods, 424 modification of CajaVs method, 425 cobalt nitrate, Golgi apparatus method, 437 formaldehyde, Golgi methods, 463 Golgi preparations, on cutting, 467 on mounting, 468 Golgi-Cox method, 473 special treatment for Golgi-Cox preps., 474 neurofibrils, 416, 417, 418 neuroglia methods, 479, 484 Golgi's sublimate method, 470 Fanz, grinding bone, 370 Farrant's medium, 221 Fat and glycogen stain, 296 Fatty substances, Cramer and Gatenby, 356 et seq. Faure-Fremiet, centrifuge, 332 protozoa, 540 Faussek, cephalopoda, 283 Feist, 407 Felizat, 168 Ferreri, decalcifying, 255 Ferria, 352 Ferric and ferrous salts, 297 et seq. Fettponceau, 367 Fibres of Sharpey, 375 Fibrils, connective tissue, 350 et seq. Fibrin, Weigert stain, 388 Fick, 184 kerato-hyalin, 340 Siredon, 277 ' Fieandt, neuroglia granules, 486 Fiedler, 525 Field and Martin, 105 Fiessinger, 297 Films, blood, 379 fixation, 381 et seq. INDEX. 573 Pinotti, 408 March! method, 450 myelin, 452 Fischel, 505 chick embryos, 273 Fischer, 223, 344, 353 on coagulation, 20 et seq. nucleoli, 314 trematodes, 516 Fischler, 368 Fish, 405 embryos, Bielschowsky (Paton), 430 eggs, 279 et seq. methylen blue nerve method, 478 V Fish, on clearing celloidin sections, 103 brain of Desmognathus, 405 decalcification, 253 Golgi method, 464 Fixation, in embryology, 259 et seq. by immersion, 25 by injection, 26, 397 by salts, 40 Fixing agents, cytological, 301 theory of, 18, 19, 131 Flagella. stains for, 548 Flagellata, general stains, 533 Flatau, 402 Golgi, corrosive method, 472 Flattening sections, 90 Flechsig, 206, 449, 472 Flemming, 339 chromo -acetic, 35 chromo-aceto-osmic, 35 Dahlia, 169 decalcified bone sections, 375 orange method, 171, 177 picro-osmic, 56 safranin, 166 Flesch, 444, 498 blood, 380 Floyd, 508 Flustra, 500 Foa, 214 sublimate and bichromate, 49 Foettinger, 14, 515 Fol, 16, 542 mixture, 39 mounting watery sections, 116 carmine mass, 235 picro-chromic, 56 Fontana, tannin-silver protozoon stain, 545 Foot and Strobell, smears, 308 Formaldehyde, 60 et seq. and alcohol, 62 modifications of Golgi method, 463 mordant, 165 Formalin, blood, 381 for nervous system, 401 and picric, 62 chrome techniques, 323 and sodium chloride, for maceration, 244 Formalin — -could Formol, chromic, 63 Formol-Miiller, 63 nitric acid for decalcifying, 253 sublimate, 63 vapour, blood, etc., 381 Formic acid, gold method, 204 Foster and Balfour, embryology, 271 Fraenkel, 297, 449 Francotte, 150, 289 Freeborn, 407 picro-nigrosin, 350 Freezing methods, 109 Frenkel, palladium and osmic, 50 Frenzel's mercuro-nitric, 47 Fresh cells, 293 Frey, 219 Friedenthal, hardening mass, 237 Friedlander, 524 Frog embryology, 275 eggs, removal of mucin, 276 et -seq. Gatenby's fluid for eggs, 277 methylen blue, 191 skin, 391 Frohlich, 176 Frozen sections, 109 brain, 406 Fuchsin (basic), 169 See Acid Fuchsin. carbolic, 169 Ftirst, bleaching, 256 Fusari, 377 Gage, 101, 297 albumen and mercury mounting medium, 220 alum, nitric acid decalcifier, 253 celloidin sections, 117 clearing mixture, 69 maceration, 248 with formalin, 244 Galesescu, 482 Gallein, myelin, 448 Galli, China blue, 440 Garbini, 522 Gardiner, ova of polychoerus, 288 Garlic water, 115 Gaskell, 94 Gastric glands, 392 et seq. Gastropoda, 283, 500 embryology, 283 eyes, 502 Gatenby, 308, 435 amoeba culture, 536 benzidine dyes, 388 et seq. centrifuge, 332 Champy-Kull fixation, 322 Cramer's osmic vapour method, 330 differentiation between cytoplasmic inclusions, 334 et seq. Donacia eggs, 287 double stain for mitochondria, 326 (and Cramer) fatty substances, 356 on Fischer's theory, 20, 21 574 INDEX. Gatenby — contd. on fixing agents, 22, 23, 24, 25 Flemming modification, 36 fluid for frog eggs, 277 on killing, 1 1 Limnsea embryology, 284 Mann-Kopsch method, 328 Maim-Kopsch-Altmann combination stain, 329, on methods of mammalian embryo- logy, 263 mitochondria, etc., 316 et seq. mitochondrial fluid, 319 new advances in embryological tech- nique, 258 plan for cytological research, 337, 338 sponges, 526 technique for chromosomes, 303 et seq. tissue culture methods, 550 et seq. on Unna's ox}^polarity theory, 21 and Woodger, 365 Gaule, desiccation method for paraffin sections, 111 solution, 46 Gaultheria, oil of, 68 Gavazzeni, 341 Geberg, 206 corpuscles, 342 Gedoelst, 249 neurokeratin, 441 Gee and Harrison, 124 Gehardt, reptile blastoderms, 274 lens, 343 Van Gehuchten, 413, 463 axis cylinder and dendrite, 459 fixing fluid, 415 ear, 498 Gelatin, embedding masses, 92 injection masses, 232 blue, 236 green, etc., 237 red, 234 yellow, 237 cement, 229 freezing mass, 110 and glycerine, 93 Gemelli, flagella, 549 Gentian blue, 183 violet, 162, 167 et seq. neutral, 396 for fibrin, 388 carbol, for flagella, 548 Geoffrey, mounting medium, 224 Gephyrea, 511 Gerlach (gold), 207 Gerota, 202, 398 brains, 402 Golgi method, 464 Gerould, 518 Giemsa, blood stain, 384 et seq. for neuroglia, 487 for protozoa, 536, 544, 545 Gierke, 197, 214 maceration, 245 Giesbrecht, 505 embedding, 78 Van Gieson (picro-saurefuschin), 176, 215 Giglio-Tos, blood, 382 Gilbert, 442 Gilson, bleaching, 256 chloral hydrate jelly, 224 copper formol, 64 mercurial mounting medium, 220 mercuro-nitric, 47 Sandarac mounting media, 227 rapid celloidin method, 104 Glands, 391 et seq. Glaue, 514 Glia, 479 et seq. Glochidia, 284 Glucose mounting medium, 221 Glycerine, gelatin and glycerine injec- tion media, 233 gelatin, 93 and HC1. decalcifier, 254 jelly, 223 for maceration, 247 for minute dissections, 8 mounting media, 222 et seq. Glycerised blood-serum, 219 Glychseaialum, Mayer's, 153 Glycogen, 294 et seq. and cell inclusions, 338 Goadby, 220 Goblet cells, 392 Gold, colloidal forms, 133 gelatin embedding, 93 gelatin mass, Tandler, Pearl, Mayer, Mozejko, 238 impregnation, generalities, 202 et seq. preservation of specimens, 208 Gold-size, 230 Gold-sublimate, Golgi method, 475 Goldmann, 131 benzidine dyes, 388 et seq. Goldscheider and Flatau, 414 Goldschmidt, 552 Golgi, 26, 435 axis cylinder and dendrite stains, 454 et seq. sublimate method, 470 et seq. mixed process, 460 avoidance of precipitates, 466 cutting and manipulation, 467 mounting, 468 body in red cells, 387 Golgi method, modifications, 461 formaldehyde process, 463 theory of impregnation, 460 methods for funnels and spiral fila- ments, 439 bichromate-sublimate modifications, 472 gold, 207 osmio-bichromate mixture, 458, 516 INDEX. 575 Golgi — contd. processes for rejuvenation of over- hardened tissue, 462 apparatus, generalities, 316 bone and teeth, 376 Kopsch techniques, 327 et seq. Mann-Kopsch-Altmann method, 329 other methods (silver), 438 plants, 325 silver methods of Golgi, Veratti, Da Fano, Cajal, 435 et seq. Sjovall method, 331 corpuscles of, 347 Golgi-Cox, 472 Da Fano's special mounting method, 474 making permanent preparations, 473 Golgi-Kopsch apparatus, 316 Golgi-Veratti, Golgi apparatus method, 435 Golodetz, 8, 210, 366 ana Unna, cholesterin, 341 Golovine, 179 Goodrich, 308, 377 modification of Kent's method, 541 Goodsiria, 282 Gordon, 180, 549 Gorgonia, 521, 522 Goronowitsch, 280 Gothard, methylen blue, 414 Graeffe, paraffin solvents, 76 Graff, Hirudinea, 511 Turbellaria, 517 Graham, 390, 514 Gram, gentian violet, 167 Grandis and Mainini, 300 Grand-Moursel and Tribondeau, pan- creas, 395 Grandry, corpuscles, 342 Grassi, haematozoa, 546 Gray, 498 Greef, 493 Green, B., 14 Green leucocytosis, oysters, 300 Gregory, micro -injection apparatus, 270 Grenacher, borax carmine, 136, 141 bleaching mixture, 257 eyes of mollusca, 502 Greppia, 469 Griesbach, 178, 182, 352 blood, 380 Grinding sections, 108 de Groot, alcoholic hsemalum, 156 iron carmalum, 139 Groselj, 522 Grosser, partially aqueous ink mass, 241 Griinpulver, 159 Griinstein, bladder, nerves, 349 Grynf eltt and Mestrezat, 256 Guanin, 301 Gudden, 465 niyelin, 446 Gudger, salmon embryology, 280 Gulick, Ascaris ova, 290 Gulland, 379 blood fixing fluid, 381 Gum, and chloral hydrate mount, 221 glycerine, Allen, Langerhans, 221 mounting medium, 221 syrup masses, 110 Thus, 228 Gurwitsch, 150 Guyeisse, 392 Gymnoblastea, 522 Gymnotus, electric organ, 346 Haber and Guild, Cajal's method, 426 Hadzi, 523 Haecker, 301 Hsemacalcium, Mayer, 155 Heemalum, 152 de Groot, 156 Hsemastrontium, Mayer, 156 Heematein, 145 et seq. neurofibrils, 416 Hsematoxylin, 145 et seq. Benda and Heidenhain, 148, 149 Bohmer, 154 Delafield, 154 chemical nature of, 145 chrome, Hansen, 157 Heidenhain, 156 Schultze, 157 combination stains, 213 copper, Benda, 157 elastin, 353 Golgi-method, 476 iron, 148 Kleinenberg, 155 for iron in tissue, 298 mounting in glycerine, 152 osmium, Schultze, 158 phospho-tungstic, Mallory, 158 and picro-Saurefuchsin, 215 ripening, 146 and safranin, 214 and Saurefuchsin, 214 stock solutions, 146 tin, Donnaggio, 158 vanadium, 157 Heematoxyline noire, 151 Haemoglobin, 300 Heemosiderin, 300 Hair, 341 Halle and Born, orientation method, 98 Bela Haller's mixture, maceration, 247 Halliburton, 410 Hamann, 506, 513 Asteroidea, 519 Hamburger, 218 Argyroneta ova, 287 Hamilton, 404 congelation method, 1 10 Hammarsten, 357 Hanazawa, dentine, 371 Hance, fixation of mammalian chromo- somes, 305 576 INDEX. Hansen, 140, 151, 176, 180 chrome-hsematoxylin, 157 Hantsch, 223 Hardening, 27 injection mass, 237 nerve tissue, 398 Hardy, 123, 512 Hari, 391 Harmer, 202 Harris, 15, 154 hsernatoxylin, for elastin, 353 myelin, 449 methylen blue, 195 Harrison, Ross, tissue culture, 550 et seq. Hart, 353 Harting, 219 gamboge glycerine mass, 239 Hartmann, 269 mammalian embryology, 265 opossum embryology, 267 Haswell, Temnocephala ova, 289 Hatschek, Amphioxus, 281 Haug, 251, 449 decalcifier, 254 Havet, 517 ccelenterates, 522 Hayem, blood fluid, 380 Heat, for killing, 12 Heckert, eggs of Distomum, 289 Heidenhain, 163, 173 alcoholic haematoxvlin for protozoa, 543 carbon bisulphide embedding, 507 centrosomes, 315 chrome-haematoxylin, 156 theory of dyeing, 125 Ehrlich-Biondi, 173 gelatin glycerine jelly, 223 iron hsematoxylin, 147 et seq. vanadium hsematoxylin, 157 Heinke and Ehrenbaum, 281 Heinrich, 352 Held, 150, 326 formol Miiller, 63 method for marginal neuroglia, 484 methylen blue and erythrosin method, 415 Helix, 501 ova, 283 Heller, 209 and Gumpertz myelin, 451 Helly, sublimate and bichromate, 49 Henchman, gastropoda, 283 Henking, examination medium, 293 methods for arthropod eggs, 285 ova of Phalangida, 287 Henneguy, 38 acetic alum carmine, 137 albumen water method for section mounting, 114 chick, 273 fish embryology, 279 gastropod embryology, 283 Henneguy — conid . mammal blastoderm, 268 permanganate method, 165 treatment of faded sections, 6 Hennings, 505 eyes of arthropoda, 509 Insecta, 505 Henocque, gold, 205 Herbst, 506 corpuscles, 342 Herdlicka, brain, 402 Hermann, 401 fluid, 38 pyrogallol method, 209 safranin and gentian, 168 L'Hermitte and Guccione, 485 Herrick, Astacus ova, 288 Hertwig, silver for marine animals, 202 frog eggs, 278 maceration mixture, 246 Triton eggs, 277 Van Herwerden, nuclease, 309 Herxheimer, 184, 340 Scharlach R., 367 Hesse, eyes of heteropoda, 502 - Hessert, 549 Heteropoda, 500 eyes, 502 Heyder, Arion embryos, 284 Heymons, 501 eggs of Orthoptera, 286 Hickson, 508 Brazilin, 211 eosin and hsematoxylin, 214 maceration, 248 High refractive mounting liquids, 224 Hill, J. P., 274 clearing and embedding, 262, 269 manipulation of ova, 266 ' marsupial mixture,1' 265 paper for reconstructions, 262 modification of picro-nitric, 55, 264 treatment and isolation of eggs of mammals, 264 Hill, 461, 466 myelin method, 448 Kindle, 547 Hippel, 495 Hirota, orientation, 274 HirschJelder, 512, 513 Hirschler, 328 Donacia eggs, 286 protozoa, 541 Hirudinea, 511 killing, 12 methylen blue, 192 nervous system, 511 His, 38, 197 Histiocytes, 388 Histriobdella, 511 Hochstetter, injection, 241 Hoehl, 37, 249 Hofer, 15 INDEX. 577 Hoffmann, 81, 390, 508, 517 chick embryos, 273 green, 182 Hogben, chromosomes, 303 Hoggans, histological rings, 199 perchloride of iron, 209 Hollands's chlorcarmine, 139 Holmes, Planorbis ova, 284 Holmgren, trophospongium, 439 Holothuria narcotisation, 12 Holothurioidea, 518 Homans, pancreas, 390 Homarus, 288 Honing, 84 Hopewell-Smith, teeth, 373 Hopkins, maceration, 246 Horn, hair, nails, 341 Hornowski, 351 Horny structures, maceration, 247 Hoskins, chick, 273 Hot water, for sudden killing, 12 Houser, 442 Hoyer, 75, 401 carmine mass, 235 gold, 205 Golgi method, 463 mounting medium, 221 mucin, 391 shellac mass, 242 silver method, 200 nitrate yellow gelatin mass, 237 Huber, 468 ' Hudson, 512 Human brain, 402, 404 neurofibrils, 421 embryos, 42 Hyatt, 108 Hydatina, 512, 513 ova, 288 Hydra, 521 methylen blue. 189 Hydrochloric acid, carmine, 143 for decalcification, 251 et scq. for maceration, 247 Hydrogen peroxide bleacher, 256 Hydroidea, general, 523 heat killing, 12 Hydroxylamin, for narcotisation, 15 Hymenoptera, eggs, 286 Hypochlorite of potash, corrosion, 250 of soda, corrosion, 250 Ide, 105 Idiozome, 316 Igacuschi, 393 Ilberg, 415 Imbedding, 71 et seq. See aho Embedding, for amphibia, 276 in paraffin or collodion, 5 Iinms, 504 Impregnation methods, 197 et seq. M. Impregnations other than gold, silver, or osmium, see page 210. iron sulphate, 210 palladium chloride, 210 perchloride of iron, 209 mass of gelatin, 237 Indian ink, injection of insects, 509 embryos, 270 mass, 240 India-rubber and paraffin, 92 Indifferent liquid^, 217 media, 292 Indigo, 212 Indigo-carmine, 182, 212 with oxalic acid, 212 Indophenol, 367 Indulin, 409 aurantia-eosin, ISO and nigrosin, 182 Injection masses, purely aqueous, 240 partially aqueous, 240 et seq. celloidin, etc., 241 Fol, Tandler, Beale, 238 gamboge glycerine, 239 gum arabic, 241 indigo carmine, Thoma, 239 milk, 241 warm, 232 cold, 238 methods, 232 tt *<'/. arthropods, 508 Hirudinea, 511 mammalian and other embryos, 270 mollusca, 503 " natural,'' 242 Innervation, bladder. )>49 Insabato, 352 Insects, 504 et set/. double embedding, ."(Mi carbon bisulphide, 507 , mounting whole, 504 — 5i>.~> Instruments, microscopes, microtomes. 8 Intercellular bridges, 339 Intestine, 393 Intranuclear rodlet of Roncoroni, 421 Intra vitam, methods, 292 stains, mitochondrial, 332 staining, alizarin, 130 Bismarck brown, 130, 162 Bolles Lee on, 129, 130 Congo red, 130 Fischel, 130 kidney, 394 Loisel, 130 methylen blue, ISli <-l w/. neutral red, 130, 179 protozoa, 540 sulphorhodamin, 130 theoretical, 129 et seq. In vacuo, embedding, 80 Inversion plasma stains, 184 Invertebrates, general methods, 499 ct seq. dendrites and axis cylinders, 454 37 578 INDEX. Invertebrates — contd. ganglia, Cajal, 425 nervous .system, 416, 417 Iodide of osmium, 331 of potash and biniodide, liquid, 224 for maceration, 244 Iodine, extraction of corrosive, 45 faeces examination, 538 fixation, 51 green, 182 method for glycogen, 295 vapour fixation, 51 Iodised serum, 219 for maceration, 243 Indium chloride, 50 Iris, 348 Iron, 297 et seq. alum fixation, 50 Brazilin, 211 for protozoa, 545 carmalum, 139 carmine, 139 cochineal, 140 hcema'oxyUn, 147 et seq. Benda, 148 Biitschli, 150 rapid method, 150 Weigert, 150 perchloride fixation, 50 sesquichlorate, myelin, 451 Isamin blue, 390 Islets of Langerhans, 395, 396 Isolation of single cyst, etc., 527 Israel, 180, 212 Iwanzoff, 518 electric organ, 346 Hatchett Jackson, 66 Jacoby, 183 Jacquet, leeches, 511 Jadassohn, 340 Jaderholm, 419 Jaenichen, 517 Jager, 223 Jakimovitch, 201 Jander, 502 bleaching, 257 Janssens, 151, 183 Janus green, 181, 332, 333 pancreas, 395 4 Japanese " method of section mount- ing, 114 Jaquet, 510 Jelinek, 101 Jenner, blood stain, 383 Jensen, protozoa, 540 Joest, 509 Lindsay Johnson, 206 aceto-osmic, 51 collodion method, 101 eye, 494 fluid, 37 on metallic stains, 198 Johnston, 262, 415 nerves of Petromyzon, 405 Johnstone-Lavis and Vosmaer, section grinding, 109 Joliet, gum glycerine embedding, 107 Jones, 300 Jonescu, 508 Jordan, 66 Jorgensen, 309 nucleoli, 314 Joris, 434 Joseph, 197, 341 white-of-egg mass, 240 Juliusburger, Nissl, 413 Kadyi, 407 brain, 402 Kaes, myelin, 447 Kaiser, 170, 223, 513 glycerine gelatin, embedding, 93 myelin, 447 solution, 44 spinal cord, 408 Kalb, 549 Kallius, 455, 469 embryonic cartilage, 376 Golgi modification, 461 Kaplan, 477 myelin, 452 neurokeratin, 441 Kappers, elderberries, 409 Karawaiew, 546 anobium eggs, 287 Karger, 493 Karyosome, 310 et seq. Kastschenko, 262 Selachian embryos, 280 Kat6, Cajal's method, 425 Kattwinkel, 400 Katz, ear, 497 Kawamura, 366 Keibel, 270 Kent, iodine fixation, 51 Kenyon, 508 Keratohyalin, 340 Kernschwarz, 211 Graham Kerr, 119 reconstruction method, 26. Kerschner, 205 Kidney, 394 Killing, amphibians, pithing, II birds, lizards, newts, 1 1 by sudden heat, 12 by hot water, 12 large mammals, ether and chloro- form for, coal gas for, 11 King, brain hardening, 405 Bufo eggs, 278 carbolic thionin, 168 Kingsbury, 368 Kingsley, Limulus ova, 288 Kionka, orientation method, 274 Kishinouye, spider eggs, 287 INDEX. 579 Kizer, blood, 381 Klein, cornea, 343 Kleinenberg, haematoxylin, 155 picro-sulphuric, 55 Knife position, paraffin cutting, 83 slope, 85 Knowen, 81 Koch, 449 Von Koch, copal method, section grind- ing, 108 Kockel, 388 Kodis, 404 myelin, 442 Koe;ner and Fischer, tannin fuchsin ilagella stain, 548 KoJoid, gastropod embryology, 283 Kohler, 516 Kolliker, mammal blastoderm, 2(38 embryology of mammals, 264 indigo-carmine for bone, 376 Kollman's fixative for fish eggs, 280 Kolmer, 26, 495 Kolossow, 32, 206, 209 modification of Cajal's axis cylinder stain, 462 prickle cells, 339 Kolster, stomach, 392 Kopsch, 386 osmic method, 327 Golgi method, 464 mollusc eyes, 502 teleost embryology, 280 Korotneff, 13 Korschelt, 541 cephalopoda, 283 Kostanecki, mercuro-nitric, 48 and Siedlecki, Ascaris ova, 290 and Wierzejski, mollusc eggs, 284 Kotlarewski, 404 Kowalewsky, teleost eggs, 280 Kowalski, 425 Kozowsky, myelin, 440 Krause, 173, 182, 393, 482, 496 carmine mass, 235 salivary glands, 392 Krauss, 201 Krecker, 14 Kresofuchsin, 169 Kresyl blue, 383 Kresyl-echtviolett, 184 Kresyl violet, 184 Krogh, 415 Krohnthal, lead sulphide impregnation, 475 Kroraayer, 184, 388 plasma fibrils, 339 Kronecker's serum, 218, 264 Krtiger, 354 Harpactida ova, 288 Kiihne, maceration, 247 Kuhnt, 496 Kiikenthal, 14, 510 Lumbricus, 509 Kull, 392 Kultschizky, 341 bichromate and sublimate, 43 double embedding, 105 mucin, 391 myelin method, 447 preservation of tissue in ether or xvlol, 4 */ * rubin for neuroglia, 486 solution, 42 spleen, 393 Kupffer, 393 Kuskow, digestion, 249 Lachi, 401 < lolgi method, 463 Lactic acid, 233, 254 Lactophenol, 222 Laevulose, for myelin preparations, 448 Laffont, 341 Lahille, 499 Lake, 132 Lakmoid, 393 Lambert, Epeira eggs, 287 Lamellibranchiata, 284, 500 et seq. cilia, 503 eyes, 502 maceration, 247 pigment, 257 Lams, 265 Landois, maceration solution, 245 Landolt, 496 Lane, pancreas, 396 Lang, corrosive liquid, 46 mollusca, 501 Langdon, 510 De Lange, 450 Langerhans, 341 gum glycerine, 22 1 islets of, 395 Langeron, 59 Lanis, fixation of mammal eggs, 265 Lankester and Bourne, 508 Lausberg, 541 Lanthanin, 302 Larvse, cchinoderm, 52<> Laslett, 447 Lattice fibres, 393, 394 Laurent, 181 Lauterborn, 75, 547 Lavdowsky, 192, 228, 498 maceration, 248 Laveran's solution, 534 Law, nerve-endings, 374 Lawrence, 223 Lead-gum embedding, 10('» Lead sulphide, Golgi method, Krohn- thal, 475 Leber, 495 Lebrun, Anurau embryology, 276 Lecithin. See under Fatty Substances, 356 ef seq. Bolles Lee, 407 decalciiication, 251 37—2 580 INDEX. Bolles Lee — contd. celloidin section mounting, 116 chick embryos, 272 chromosome stain, 303 on choice of stain, 134 dry cutting process, celloidin, 104 ear and eye, 493—498 fish embryos, 279 fixation of ground cytoplasm, 301, 302 gold, 206 iron carmine, 139 Kernschwarz, 211 mounting fluid, 222 mounting Golgi preparations, 468 myelin stain, 442 osmic-pyrogallol method, 208 paper cell mounting method, 230 on staining nucleus infra vitam, 310 Tunicates, 499 sponges, 525 Leeches, 511 maceration, 247 nervous system, 511 Legal, alum carmine, picric, 138 Legendre, Golgi apparatus, 438 Leger, sporozoa, 546 Legros, 201 Amphioxus, 281 Leiper, 514 Leishman, blood stain, 385 protozoa, 535 Lemon-juice gold method, 204 Von Lendenfeld, 525 Von Lenhossek, 342, 455 Nissl bodies, 413 mollusc eyes, 502 platinum chloride, 50 Lennhoff, methylen blue, nerve, 414 processes for nerve cells, etc., 476 Lennox, 496 Lens, eye, 343 Lenssen, 513 Hydatina, 288 Leontowitsch, 195 Lepidoptera ova, 286 Lepkowsky, 375 vessels in teeth, 373 Leuckhart, embedding boxes, 73 Leutert, 300 Levaditi, 549 blood smears, 383 Levi, fixation of mammal eggs, 26.3 mitochondria! method, 325 Levulose, 221 Levy, 389 Bevan Lewis, 408 Lewis, 389, 511 tissue culture, 555 Lewy, 450 Lichtgriin, 159 Liebetanz, 548 Liesegang, 407 Cajal's method, 426 Ligaraentum nuchac, digestion, 249 Light green, 181 Lillie, 332 Unio eggs, 284 Limax, 283, 501 Lime salts, 300 Limnsea ova, 284 Limulus, 288 Linville, mollusc ova, 283 Lipin, 357 Lipoids, 357 List, 214, 314 Coccids, 506 Mytihis, 500 Sagartia, 522 Lithia, for picric fixed material, 54 Lithium carmine, 140 Little, Hydra, 521 Live slide, Drew and Griffin, 530 Liver, 393 mollusc, 501 Lizard blastoderms, 275 Locke's solution, 218 Locy, spider eggs, 287 Loele, 390 Loewy, method for integument, 339 Loftier, stain for flagella, 548 Loisel, 367 fat, 369 sponge, 526 Long, constant temperature box for mammal eggs, 266 and Mark, fixing fluid for mammals, 266 mouse embryology, 263 Longhi, 542 Longworth, 342 Lonnberg, 516 Looss, 514, 516 corrosion of chit in, 250 Nematodes, 513 Lophomonas, 533 Lord, methylen blue, nerve, -.414 Lb'wit, blood fluid, 380 gold, 204 Loyez, 442 Lucidol, 308 in acetone, 59 blood, 382 Ludford, 308 Lugaro, 417 collargol, 434 Lugol's solution. 45 Liihe, 516 Luithlen and Sorgo, 414 Lumbricus epidermis, nerve, 421 nerve, 425 sections, 509 Lundvall, 377 Lustgarten, 169 Luxenburg, Nissl bodies, 413 Lymphatics, in teeth, 373 glands, 394 Lymph-spaces, methylen blue, 195 Lysol, for maceration, 248 INDEX. 581 Maas, 213, 249 sponges, 526 Macallum, 300, 301 iron methods, 297 Mac Bride, larvse of Echinoderms, 520 Maceration, 243 et seq. Bela Haller's mixture, 247 epithelium of molluscs, 503 Ranvier, 244 Moleschott, 244 Landois, 245 Macklin, 271, 389 Maclean, 357 Macrophages, 388 Von Maehrenthal, 209 Magdala red, 169 for spleen fibres, 394 Magenta, 169, 171 Magini, 477 Magnesia carmine, 140 Magnesium chloride or sulphate, for narcotisation, 15 Malachite green, 181 Malaria, 546 pigment, 300 Malassey, 218 Mall, 378 Mallory, 354 eosin and methylen blue, 181 neuroglia, 483 phospho-molybdic acid haematoxylin, 157 phospho-tungstic, 158 saure-fuchsin and phospho-molybdic acid, 351 Weigert stain, 481 Mammalia, Cajal's methods, 424 eggs 2,66. See a/so under Ova. Mammals, chromosomes, 305 et seq. clearing and embedding, 269 embryological methods, 263 fixation of whole tubes, 268 isolation of eggs, 263 et seq. Manchester brown, 161 Manfredi, 206 Mann, 26, 31, 114, 155, 398, 468 chromo-sublimate, 48 fat, 366 extraction of corrosive sublimate. 45 Golgi-Cox, 473 Golgi method precipitates, 466 methyl blue eosin, 183 osmio -sublimate, 48 stain, for protozoa, 544 sublimate formol, 63 Mann-Kopsch method, 328 Marcacci, maceration, 247 Marcano, blood, 381 Marchi, degenerate nerve, 449 et seq. mollusca, 501 tendon organs, 347 Marcus, 446 Maresch, 352, 394 Marfori, 300 Marie, 400 Marina, 405 Marine animals, fixation of, 27 cell inclusions, 332 Mark, collodionisation, 88 reconstruction, 262 Marsh, 229, 230 Martin, 392 trachese, 508 Martinotti, 167, 196, 354, 369, 386, 467, 408 Golgi method, 465 elastic tissue, 352 Mason, brains, 405 Massart, 214 Masson, connective tissue, 351 Mast cells, 354 et seq. Unna's method, 356 Matschinsky, bone sections, 371 Matuszewski, 450 Maurice and Schulgin, 213 Mawas, 496 Maximow, 356, 389 Altmann method, 322 May-Griinwald, 384 Mayer, 15, 33, 155, 184, 297, 350 acetate of potash, 151 albumen water method for sections, 113 alcoholic cochineal, 144 aluminium chloride carmine, 138 benzol embedding, 78 bleaching, 256 carmalum, 137, 212 cartilage, 377 chitin, 508 cochineal stain, 135 decalcification, 253 desilification, 255 eye, 339 glychsemalum, 153 hsemacalcium, 155 hsemalum, 152 et seq. hsemastroiitium, 156 on hsematoxylin staining, 145 iron staining, 298 methylen blue, 193, 196 mucin stains, 392 paracarmine, 142 picro-hydrochloric, 56 picro -magnesia carmine, 141 picro-nitric, 55 Plutei, 520 section stretcher, 87 triacid, 175 Maysel, 161 McClung, urea in fixing, 305 McClure, 502 McCrorie, 549 Medium of Farrant, 221 Medulla, hardening, 399 Medusae, 523, 524 killing, 12 maceration, 246 582 INDEX. Medusae — c^ntd. narcotisation, 13 sections, 524 Mehnert, 275 Meirowsky, 54!) Meisenheimer, gastropoda, 283 Melanin, 300 Melting point of paraffin, 91 Membrana. nictitans, 339 limitans, marginal neuroglia, 4S.~> Membranes of eggs, 284, 285 Mendel and Bradley, 300 Menthol, for narcotisation, 12 Mercier, 449 Mercury, bichloride, 44 alcoholic liquids, 46 ft seq. and acetic acid, 44 extraction, 45 sodium thiosulphate, extraction by, 46 washing out, 45 and bichromate, 48 and formol, 63 mounting media, 220 nitric mixtures, 47 sublimate and salt, 4(5 Merkel, indigo-carmine, 212 MerkePs fluid, 39 Merton, 502 Merzbacker, 485 Mesostomidse ova, 288 Messner, picrocarmme, 414 Metachromasy, 133 Metachromatic dyes, 133 Metagelatin vehicle, Fol, 238 Metallic stains, 197 et seq. theory of, 197 et seq. osmic acid, pyrogallol, 208 Metcalf, Chiton ova, 284 Methyl, aniline green, 159 blue, 183 with eosin (Mann), 183 nephridia, 511 i/rcen, 159 et seq. for ehromatin, 293 with eosin, 181 mixture for maceration, 247 salicylate oil, 68 violet, 162 for fibrin, 388 fiuid for blood, 382 plasma fibrils, 340 B., 184 Methylen blue, 170, 186 et seq. blood, 383 Cajal's diffusion process, 478 Distomum, 516 and eosin (Mallory), 181 eosin stains for blood, 383 et seq. and erythrosin, Nissl granules, 415 fixation of stain, 192 et seq. frog bladder, 349 intra vitam staining, 187, 344, 477 Methylen — could. for epithelia, lymph-spaces, 195 insect eyes, 508 method, cornea, 343 methods for sections, 194 modes of staining, 190 myelin, 449, 452 nerve endings, 344 methods, 477 et seq. Nissl bodies, 411 polychrome, 186 Saurefuchsin, 351 for skeletons, 377 Methylenazur, 186, 187 Meves-Flemming modification, 36 Victoria green stain, 333 Meyer, Berlin blue neurofibril method, 434 methylen blue nerve method, 477 myelin, 445 new celloidin method, 103 Mibelli, 352 Mica sheets, for mounting sections, 1 19 Michaelis, 133, 181 blood stain, 383 fat, 367 polychrome methylen blue, 187 Triton eggs, 277 Michailow, 190, 195 Microcbemistry, 293 et seq. Microglia, 493 Micro-injection, 270 Microscopes, 8 Microtome, 8 sliding, freezing, rotary, Jung, Minot, Tetrander, rocking, 9 Migula, 219 Miller, cement, 229 Minchin, albumen method for mammal eggs, 266 embedding protozoa, 542 hsematozoa, 547 sponges, 525 Minervini, 353 Mitochondria, fresh examination, 332, 333, 334 Catenby on, 316 protozoa, 540 teeth and bone, 375 vital stains, 332 Mitrophanow, 183, 342 chick embryos, 273 integument, 339 myelin, 447 Mitsukuri, reptile blastoderms, 275 Mixed process of C4olgi, 460 Mobius, 348 maceration medium, 246 Moerner, 377 Moleschott and Piso Bonne, salt alcohol for maceration, 244 Molge, embryology, 277 Moll, cartilage, 376 Mollison, Alkanna, 367 INDEX. 583 Mollusca, 282, 500 et embryology, 282 et seq. to kill extended, 500 maceration of epithelium, 503 mucus glands, 504 narcotisation, 12 nervous system maceration, 247 Molluscoida, 500 Molybdenum-toluidin blue, neurofibrils, 417 Mbnckeberg and Bethe, 31 bleacher, 256 Mondino, 472 Montanari, 419 Montgomery, 515 nucleoli, 314 ova of Theridium, 287 Monti, 477 gastric glands, 393 Mordant, 131 Mordanting, for coal tar dyes, 165 Moreaux, formol - picric - trichloracetic, 62 Morel, 175 Morgan, Ascidia, 282 cockroach eggs, 285, 286 frog embryology, 276, 278 Morphia, for narcotisation, 16 Moseley, 503 Mosse, 415 argentamin, 452 Mosso, 380 Mott, 129 Mounting media, glycerine, 222 resinous, 225 Farrant, Allen, Brun, 221 sections, collodion paper method, 116 Mayer's albumen method, 113 watery, 116 Mozejko, 503 vaso-dilators, 233 Muchsematein, 392 Mucicarmine, 392 Mucilage syrup mass, 110 Mucus, 391 et seq. glands, mollusca, 504 Muir, 549 blood, 381 Mullenix, 497 Muller, 392 Berlin blue mass, 240 fluid, 42 with formol, 63 for maceration, 245 silver method, 201 Mummery, embedding tooth germs, 372 nerve tissue of teeth, 373 J. A. Murray, Altmann's method, 321, 365 on glycogen stain, 296 connective tissue stain, 354 chrome-osmic method, 326 lead gum, 107 Muscle-libres, maceration, 246 and tendon, 344, 347 spindles, 345 Myelin, ammonio -chloride of tin, Besta, 448 and axis cylinder stains, 452 Berkley rapid method, 447 bulk stain, 448 iron method, 451 Kultschitzky, 447 osmic acid, 450 et seq stains, 442 silver nitrate, 451 Weigert stains, 442 Myers, 415 Mytilus, 500 Myxosporidia, o46 Nabias, gold chloride CJolgi method, 47»; mollusca, 501 Nageotte, 399, 448 Nails, 341 Nakahara, urea fixation, 305 Nakanishi, blood, 383 Nansen, 245 Naphtha, clearing, 77 Naphthalin, monobromide, 224 rose, 169 Naphthol, 390 Naphthylamin brown, 408 Naples water bath, 79 Narcotisation, anemones, Holothuria, Ascidia, Mollusca (menthol), 12 Medusae, starfishes (chloroform), Actinia?, Capitellidae (ether alcohol), 13 . Cristatella, 14 larvae (chloretone), 14 Alcyonella, Bryozoa, Annellida, Mol- lusca, Xemertians, Actiniae (chloral), 14 hydroxylamin, 15 magnesium chloride or sulphate, 15 morphia, curare, strychnin, prussic acid, 16 asphyxiation : tobacco, carbonic acid gas, soda water, 16 hydrogen peroxide, 17 Hydra (cocaine), 14 Vorticellida?, Rotatoria, Vermes (cii- caine), 15 Ascidia, Rhopalaea, 15 Ciona, 16 Medusae, 16 snails, 16 menthol, nicotine, 12 chloroform, cocaine, eucaine, ether, alcohol, 13 methyl alcohol, chloreton, chloral hy- drate, 14 Nasal mucosa, maceration, 247 Nathusius, 341 Nealey, bone sections, 372 581 INDEX. Nebenkern, 316 Nelis, brain fixation, 405 Nematodes, 513 et seq. corrosion of chit in, 250 ova, 289 Nemertina, 514 et seq. heat killing, 12 Nephridia, Histriobdella, 511 Nerve endings, beetle, 344 Bielschowsky, 426 et seq. frog, 344 gold method, 344 insect, 345 methylen blue method, 344 silver method, 345 bichromate of silver, 345 of teeth, 373 et seq. staining, methylen blue, 189, 191 Nervous system, Achucarro's tannin neuroglia method, 490 Apathy's neurofibril method, 416 axis cylinder, 452 axis cylinder at/d dendrite stain* (Golgi and othei's), 454 Cajal's double-impregnation process, 461 Golgi bichromate - sublimate method, 470 processes similar to Golgi methods, 475 CajaTs method, advice as to choice of formula, 424 (Golgi), axis cylinder and dendrite, treatment of larvse, 459 — 460 axis cylinder and dendrite, form- aldehyde modifications, 463 Berkley rapid myelin method, 447 Bielsclwicsky wrtliod*, introduc- tory, 426 for sections, 427 for peripheral nerve fibres, 428 for pieces, 429 modifications, 430 et seq. Cajal's gold chloride neuroglia method, 487 methods, special objects, 424 neurofibril methods, 419 et seq. collodionising sections, 406 Da Fano's Bielschowsky methods, 432 Donaggio's neurofibril methods, 417 Fish's fluid, 402 f ration, 397 alcohol, 400 chromic salts, 403 et seq. formalin, 401 general methods, 397 stains, 407 Golgi method, avoidance of preci- pitates, 466 cutting and mounting, 467— 469 Golgi internal apparatus, 435 et seq. Nervous system — conid. Golgi-Cox modification, 472 hardening, 398 et seq. Hold's method for marginal neuro- glia, 484 injection fixation, 397 Kultschitzky, myelin, 447 maceration, 245, 246, 247 Marchi method, 449 nzurofibrils, 416 et seq. other methods, 434 neuroglia, 479 et seq. Nissl substance, 410 et *c(/. Pal method, 446 phosphomolybdic acid hsemato- xylin, 157, 158 polarised light, 398 preliminary fixation for Sauro- psida, 405 Sand's neurofibril method, 434 sections, 405 et seq. special rt/fnJfigicaJ method*, 410 et seq. sustaining apparatus of medullary sheaths, neurokeratin, 439 Simarro's process, 419 Weigert's myelin stains, 442 ct seq. Nesteroffsky, 206' Nettovitch, 505 Neuberger, decalcifier, 254 Neubert, 297 Neukirch, 297 Neumayer, 262, 270 Neurofibril methods, 416 et seq. advice on, 424 Neuroglia, 479 Achucarro's tannin method, 496 Cajal's gold chloride sublimate method, 487 granules, 486 protoplasmic, 486 marginal, H eld's method, 484 methods, 479 et seq. stains, Anglade and Morel, 483 Da Fano, 484 Held, 484 Oppenheim, 486 Weigert, 480 Benda, 482 Mallory, 483 Neurokeratin, 439, 441 Neurosomes, 415 Neutral, balsam, 226 dye, 120 gentian, 396 red, 179 et seq. blood, 382 granules, 389 kidney, 394 mucin, 392 pancreas, 395 Neutralisation of carmine mass, 235 Neuville, silver impregnation mass, 237 INDEX. 585 Nicolas, 32 embedding in gelatin, 93 reptiles, 275 Nicolle, 168 Nicotine, for narcotisation, 12 Nigrosin, 170, 182, 408 Nikiforow, 102 Nile blue, connective tissue, 354 fat, 362, 368 Nissl, 410, 411, 415, 450, 480 brain hardening, 403 bodies, 410 et seq. stains other than Nissl' s, 412 granules, nature of, 130 methylen blue and erythrosin, 415 method, precautions against fading, 413 modifications, 413 Nitric acid, and acetic, for maceration, 247 bleacher, 257 and chlorate of potash, for macera- tion, 247 corrosion, 250 decalcification, 252, 253 for fixation, 38 fixative for neurofibrils, 417 formol, 64 maceration, 247 nerve tissue, 404 Noack, 83 Noll, corrosion, 250 Nordmann, mast cells, 355 Nosema, 546 Nowak, sublimate formol, 63 tactile corpuscles, 342 Nuclear stains, coal tar, 159 ft *cy. Nuclease, 308 Nucleoli, Gatenby on, 308, 310 malachite green for, 181 Nucleolini, 311 Nucleus, '' vital " stains, 310 Nudibranchs, 502 Nuttall, Cooper and Robinson, 506 Obersteiner, brain, 403 Obregia, 469 method for paraffin and celloidin sections, 115, 119 Obst, nucleoli, 314 Octopus, 501 Odenius, maceration, 247 Odier, Golgi method, 465 Oestergren, 13, 515, 517 Ohlmacher, 167 alcoholic corrosive, 47 tiuid, brain, 405 formaldehyde process, 165 myelin, 452 picric acid, 176 Oils, 65 et seq. See also under definitive name, e.g., Cedarwood. winter green, 27 1 Okajima, elective stain for reds, 388 fat, 368 Olmacher, 404 Olt, manipulation of frozen sections, 109 Opalina, 533 Ophiothrix, 519 Ophiuridea, 519 Oppel, lattice fibres, 393 Oppenheim, neuroglia granules, 486 Oppitz, 201 Orange G., 172, 173, 177 Orcein, 212 % embryonic cartilage, 376 elastin, 353 method, Unna, 351 water blue, 340 Orchella, 211 Orcin, 212 Organic acids, fixation by, 51 Orientation methods, 273 el so/. for blastoderms, 273 in embedding, 80 et seq. Origanum oil, 68 for celloidin sections, 102 Orr, 451 Marchi method, 450 Orth's fluid, 260 Orthoptera eggs, 286 Orton, 503, 508 Osmacet, 380 Osmium chloride, 50 'Osmium tetroxide, reduction, 29, 32, 124 acetic acid maceration, 246 bleaching, 31 restoration of staining, 31 for blood, 380 gold method (Viallane), 205 Kopsch method, 327 nerve tissue, 404 and fat, 356 et seq. and picric, 56 pyrogallol method, 208 regeneration of, 30 fixation by vapour, 30 after-treatment, 30 sublimate, 48 vapour method, Cramer, 330 Ossifying cartilage, 254, 377 Osteoblastic areas, 378 Ostracoda, 505 Wo. Ostwald, 125 Ova, of Echinoderms, fixation, 260 fixation of mammalian, 265 frog's, 275 et seq. Insecta, Pisces, 259 treatment of uterine eggs of mam- mals, 266 Ovary as index to pregnancy, 263 Ovens, paraffin, 79 Overhardened tissue, rejuvenation of, 462 Overton, 31, 34 iodine vapour, 51 Oviatt and Sargent, 233 586 INDEX. Oxalic acid, for maceration, 247 Oxidised hsematoxylin (Unna), 155 Oxidisers as fixers, 21 Oxydase reaction, 390 Oxygen, place in tissue, 390 Pacini, 220 blood, 380 Pal, 472 Paladino, 453 Palladium chloiade, 50 decalcification, 252 • Palythoa, 522 Pansch, injection, 242 Pancreas, 395, 396 inclusions, 338 Pancreatin, digestion, 248 Paneth, cells, 392 Paper trays, embedding, 73 Papillae foliata?, 342 tiee also under Corpuscles. Pappenheini, 356 hsemopoietic tissue, 386 mast cells, 355 panoptic stain, 383 pyronin and methyl green, 172 Paracarmine, 142 Paraffin, pure, 91 , overheated, 92 sections, flattening, 90 clearing and mounting, 91, 111 et •seq. embedding, 76 et seq. shaping block, 83 et seq. cooling, 82 bath, 77 Paramoecium, culture, 537 Paravicini, 418 Parietal cells, 393 Paris green, 160 Parker, 508 bleaching, 257 methylen blue, 194 and Floyd, sheep brain, 402 Parlodion, 95 Parmenter, Amblystoma, 306 Parolein, 228 Parthenogenesis, 259 Partington and Huntingford, 32, 364 Partsch, cochineal alum carmine, 137 decalcification, 254 Passarge and Krosing, 354 Paton, 272 Bielschowsky modification, 430 Patten, 286, 502 mollusc a, 504 orthopterous eggs, 286 orientation during embedding, 81 Patterson, chick, 273 Pauropoda, 505 Pavlov, myelin, 446 Peabody, corrosion, 249 Pearl, worms, 509 Pedicellina, 500 Pekelharing, 297 Pelagic fish ova, 281 Penfield, Golgi apparatus, 438 Holmgren's trophospongium, 439 Pensa, reconstruction method, 261 Peppier, 549 Pepsin, digestion, 248 Peptic cells, 392 Peptonum siccum, 233 Perchloride of iron impregnation, 209 Perdrau, Bielschowsky method, 434 Perenyi's fluid, 39 Perez, fly pupae, 286 Periplaneta eggs, 285 Permanganate of potash, bleacher, 31, 256 Henneguy's mordant, 165 for osmic solution, 29 Perophora, 282 Peroxide, of benzol, blood, 382 of hydrogen, bleaching, 256 for killing, 17 of magnesium, 256 of sodium, 256 Perrier, worms, 509 Pen-in, 122 Perusini, 453 Peter, 140 reconstruction method, 262 yolk stain, 260 Peters, R. A., culture of Paramoecium, 537 Petromyzon, nerves, 405 Petronne, 387 Petrunkewitsch, Hymenoptera, 286 rnercuro-nitric, 48 Pfitzner, 182 protozoa, 541 safranin, 166 Phalacrocera, 286 Phalangida ova, 287 Phenol, solution for fixation, 47 Phenylen brown, 161 Philippson, 339 Phloroglucin decalcifying mixtures, 254 Phloxin, 180 Phoronis, 512 Phosphatides, 356 et seq. Phospho-molybdic acid hsematoxylin, Mallory, 157 Phospho-tungstic acid fixative, 38 Phosphoric acid decalcification, 252, 254 Photographic negative varnish mount, ing medium, 228 Photoxylin, 95 Physa eggs, 284 Pianese, 175 methylen blue eosin, 181 Picraininic acid, 176 Picric acid, 54 decalcification, 252 plasma stain, 176 INDEX. 587 Picric acid — conld. and alcohol, 55 for maceration, 248 Picro-acetic, 55 Picro-carmine, 140 general nerve stain, 414 Picro-chromic, 56 Picro-hydrochloric, 56 decalcifier, 254 Picro-indigo-carmine, 212 Piero-nigrosin, 182, 213, 350, lux Picro-nitric, 55 decalcifier, 254 Picro-osmic, 56 Picro-platinic, 56 with formol, 63 Picro-Saurefuchsin, 176, 350 Picro-sublimate, 48 Picro-sulphuric, 55 Pictet's liquid, 219 Pink salt, 418 Pintner, 29, 515 Pisces, embryology, 279 d seq. Pitfield, flagella, 549 Pittock, reconstruction method, 262 Pizon, Ascidia, 282 Placenta, glycogen, 295 Planaria, 44, 517 Plankton, general preservative, 525 Planorbis ova, 284 Plants, Golgi apparatus, 325 Plasma eel's, 354 et seq. fibrils, 339 stains, 171 et seq. Bolles Lee, 314 Plasrnodium, 546 Plasmosome, 310 et seq. Plastic reconstruction, 261 Plastochondria, 315 Platino-aceto-osmic, 38 Platinum chloride, 49 Plainer, neurokeratin network, 441 Kernschwarz, 211 Pleschko, 194 Du Plessis, 515 Pleurobrachia, 524 Pluteus, 520 Podwyssozki, 167 fluid, 36 Polaillon, perchloride of iron impregna- tion, 209 Polariscope, 332 for fat, 361 et .seq. in nervous system, 398 Policard, 547 Politzer, 496 Polumordwinow, 413 Polychoerus ova, 288 Polychrome methylen blue, mast cells, 355 nerve cells, 476 for nerve tissue, 414 Polychrome toluidin blue, 196 Polyclads ova, 289 Porifera, 525 et seq. decalcification, 251 Post-chroming, 326 Post-impregnation for gold, 207 et seq. Post-mortem changes, 25 Post-osmicating, 326 Potash method, for skeletons. 37 S Potassium, 301 See under Bichromate. bichromate, maceration with, 245 chlorate and nitric acid, for macera- tion, 247 iodide, brain, 404 permanganate, for maceration, 246 sulphocyanide, for maceration, 245 Potter, 446 Pouchet, bleaching, 256 Pranter, 353 Precipitates, Golgi method, 466 Prenant, 181, 301, 497 Preservation media, 216 by alcohol, ether, xylol, or toluol glycerine mixtures, acetic acid, cedarwood oil, 4 Preyer, 13 Priapulus, 512 Prickle cells, 339 Primerose soluble, 180 Pritchard, 498 Progressive stains, coal-ta/, 159 Propylic alcohol balsam, 227 Protease, 308 Protozoa, 526 et seq. Borrel's blue, 534 Borrel method, 532 cell inclusions, see pages 316 — 338 collection, 529 culture, 527 et seq. of paramoecium, 537 current stains, 543 et seq. determination of life cycle, 530 Drew-Griffin live slide, 530 embedding, 542 examination in a coloured medium, 540 of faeces, 538 et seq. /i.ciug and preserving, 541 > t >*9 Salamandra, embryology, 277 Salicylic acid, fixation, 53 Saling, Tenebrio, 506 eggs, 287 Salivary glands, 392 Salkind, lead gum embedding, 106 Salmonidse, embryology, 280 Rabl-Ruckhard method, 280 Salts, for fixing, 40 solutions, 218 for maceration, 244 sublimate, 46 Samassa, 468, 525 Samter, 75, 288 Sanchez, 425 Sand, 482 neurofibril method, 434 Sandal-wood oil, 68 Sandarac, 228 media, Gilson, 227 Sanders, 473 Sankey, 408 Sansom, Carnoy modification, 53 Sanzo, fixing apparatus, 260 Sarcolemma, 344 Sata, fat, 367 Sattler, 201 Saurefuchsin. See under Acid Fuchsin. Saurerubin and Orange G, 393 Sauer, kidney, 394 Saureviolett, 181 Savini, 177, 415 Sazepin, 506 Scala, 542 Scarlet R. See Scharlach. Scarpatetti, 408 Schafer, 232 Schaffer, 251, 261, 300, 350, 356 bone, 370 decalcification, 252, 254 thionin for bone and cartilage sec- tions, 376 Schaper, 262 Scharlach R, 356 d ««/. fat, 367 Herxheimer, 367, MS Schaudinn, fluid, 546 Woodcock's modification, 541 Schaxel, 309 Schiefferdecker, 67, 68, 444 celloidin masses, 241 eye, 496 maceration mixture, 247 and Kossel, 356 Schlemmer, Bielschowsky, 431 Schmaus, 407 590 INDEX. Schmidt, gastropod embryology, 283 Schmorl, thionin for bone and cartilage sections, 376 Schneider, 300 aceto-carmine, 138 Schonemann, 262 Schreiber, Golgi method, 464 Schridde, 356 blood, 381 mitochondrial method, 325 Schrotter, 408, 449 Schuberg, malaria, 547 Schulemann, 389 Schultze, 219, 496 chrome hsematoxylin, 157 frog embryology, 278 iodised serum, 219 muscle, 348 osmium hsematoxylin, 158 oxydase reaction, 39) potash method, 378 " tendon, 347 Schumacher, 354 Schiirmayer, 539 Schiitz, 425 Bielschowsky, 430 Schwalbe, 407 cochlea, 497 Schwarze, 517 Sclavo, 549 S. G. Scott, 119, 180,498 blood, 381 Romanowsky stain, 386 neutral balsam, 226 standard hsematoxylin stain, 312 Scott and Osborn,triton embryology, 277 Scyphistoma, 524 Secretion granules, 315 Section-grinding, 108 Sections, crumpling, 88 flattening, 86, 90 mounting, Henneguy's method, 114 Seeliger, 520 Segregation granules, 389 Sehrwald, 468 Golgi methods and precipitates, 466 Seidenmann, 192 Seiler, alcoholic balsam, 226 carmine and indigo, 212 decalcifier, 254 Selachia embryology, 280 et seq. Selenka, 269 Seligmann, 493 Sensory ganglia, 424 Serial section mounting, 1 1 1 Serum, for maceration, 243, 244 media, 219 Severeanu, injection, 242 Shearer, 51 1 Sheep, brain, 402 Sheldon, 446 Shell, mollusc, 503 Shellac, for brittle sections, 89 embedding, 108 Shipley, 271, 310 benzidine dyes, 389 and Macklin, trypan blue, 378 Shun Ichi Ono, glycogen, 338 Siebenmann, 498 Silver, carbonate, neuroglia method, 492 impregnation, double-staining, 202 for elastin, 354 fixation, 201 marine animals, 202 metallic stain, 198 et seq. for nerve. See under Golgi, Ramon y Cajal, and Bielschowsky. nitrate, reduction, 201 picrate, lactate, acetate, for staining, 201 Simarro, neurofibrils, 419 Siphonophora, 524 Siphunculus, 511 Siredon, embryology, 276 Sjovall, 435 formol and osmic acid, 331 Skeletons, cartilage, 377 Skin nerves, 341 Slides, cleaning of, 112 Slow-worm eggs, 275 Small objects, embedding, 75 Smears, blood, 379 of gonads, 307 et seq. lucidol fixation, 59 Smirnow, 341 Golgi method, 465 Smith, eyes of gastropods, 502 and Mair, 366 Lorrain Smith, Nile blue, 368 Smooth muscle, 348 Snails, asphyxiation, 16 Snake blastoderms, 274 Snessarew, Bielschowsky, 352 Soap masses, 92 Sobotta, 269, 280 Amphioxus, 281 mammals, 263 Soda carmine, 140 Sodium chloride and alcohol, for macera- tion, 244 nitrite, 233 sulphalizarinate, myelin, 449 Solferino, 169 Solger, bleaching, 256 muscle, 344 salivary glands, 392 Solvents, for paraffin, 76 Soulier, maceration, 245 De Souza, pyridine, 59 Spalteholz, method of clearing, 270 Spec, 92, 270 Sphaerozoa, 546 Spicules, sponge, 526 Spielmeyer, 442 Spinal cord, 424 axis cylinder and dendrite, 459 hardening, 399 See under Nervous System. INDEX. 591 Spindles, fixation, 301 Spiral filaments, 439, 440 Spleen, 393 Sponges, 525 desilicification, 255 embryos and larvae, 520 sections, spicules, 526 Sporozoa, 546 Spuler, 140 sublimate formol, 63 Squire, 173 blueing sections, 151 glycerine jelly, 223 Staining, adjective, 131 in bulk, 6 with carmine, theory of, 135 effect of heat, 124 electrolytes in, 131 hindrances to, aids to, 25 nature of, 121 progressive, 132 regressive, 132 removal of dyes, 125 substantive, 131 two kinds distinguishable, 128 unsafe criterion of chemical consti- tution, 133, 134 vessels, 6 Stappers, 505 Starfishes, narcotisation, 13 Statoblasts, 282 Stauffacher, cyclas eggs, 284 Steensland, 450 Stein, 497 decalcification, 251 Stempell, 546 Stephens, 549 Stephenson's high ref active mounting medium, 224 Stirling, maceration, 245 Stoeltzner, 300, 442 Stohr, 214 Storch, 482 Strahuber, 477 Zur Strassen, 514 Ascaris ova, 290 Strasser, 262, 407 Stratum granulosum, 340, 341 Strecker, brain, 402 Streeter, myelin, 448 Van der Stricht, decalcification, 254 thysanozoon eggs, 289 Strieker, gum embedding, 108 Strong, 398, 449 brain of acanthias, 405 copper bichromate, 64 Golgi method, 463 iron alum fixation, 50 Stropeni, 355 Strychnin, for narcotisation, 16 Students, guide for, 556 Studnicka, 352 Stylaria, asphyxiation, 16 Styrax, 370 Styrax and Liquidambar, 228 Sublamin, 49 Sublimate, bichromate, 43 Golgi method, 470 See under Mercury and Corrosive substitution stain-;, 164 Suchannek, 3 anilin oil, 69 bergamot oil, 68 mounting medium, 227 Suchanow, Golgi apparatus, 438 Sudan III., for blood, 383 Daddi, 367 fat, 356 et seq. Sulima, 14 Sulphonic acid, 121 Sulphuric acid, maceration, 247 Sulphurous acid, 43 bleacher, 256 decalcification, 253 for teeth, 372 Sumita, 300 Summers, method for celloidin sections, 116 Sumner, 279 Suschkin, chick embryos, 273 Suspensoids, 123 Sussdorf, 391 Sustaining apparatus, of medullary sheaths, 439 Sympathetic ganglia, 424 Synapta, 518 Syrup media, 219 Szecsi, lucidol, 59 for blood, 382 Szent-Gyorgi, eye, 494 Sziitz, 185, 208 Tachiol, 425 Tactile corpuscles, 341 et seq. hair, nerve endings, maceration, 247 Tadpoles, infra vitam staining, 179 Taenzer-Unna, orcein method, 353 Tafani, 498 Taguchi, Indian ink mass, 240 Tannin, for mounting, 220 Tannin-fuchsin, for flagella, 548 Tap water substitute, 151, 313 Tartuferi, 343 eye, 495 Taylor, Sister Monica, amoeba culture method, 536- — 537 chromosome methods, 30:5 Teeth, Carter, 369 et seq. decalcification, 253 embedding through carbon bisul- phide, 372 lymphatics, 373 soft parts, 371 et seq. vessels, 373 Tegumentary organs, 339 et Teleost eggs, 279, 280 embryology, 259 592 INDEX. Teljatnik, 451 Tellyesniczky, acetic bichromate, 41 Temnocephala ova, 289 Tendon, 344, 347 silver, 200 Tenebrio eggs, 287 Terpinol, 69 as mounting medium, 228 Test-cells of Ascidia, 282 Theory of dyeing, electrical, 124 of fixation, 18 of staining, 120 Theridium, 287 Thiersch, 212 Thin, 496 Thionin, 162, 168 cartilage, 376 for Golgi apparatus, 438 for infra tit am staining of nerve, 196 mucin, 391 pancreas, 396 for skeletons of cartilage, 377 Thionine pheniquee (Nicolle), 168 Thiophen green, 182 Thoma, decalcification, 253 indigo-carmine mass, 239 and Fromherz, corrosion, 249 Thome, 173 Thompson, Arthur, reconstruction method, 262 Thomson, J. G., Giemsa stain, 545 Thread cells, 521 Thyme, oil of, 68 Thymus, 394 Thyroid, 394, 395 Thysanozoon ova, 289 Tigroid substance, 410 et seq. Timofejew, 196, 393, 405 Tirmann, 300 Tischatkin, 3 Tissue culture, 550 et seq. Toison, staining fluid for blood, 382 Tolu balsam cement, 231 Toluidin blue, 169 for cartilaginous skeletons, 377 and erythrosin, 415 for intra vitam staining of nerve, 196 myelin, 449 Nissl granules, 412- — 413 Toluol, 70 Tomaselli, neurofibrils, 418 Tonkoff, 183 Torpedo, electric organ, 346 Tortoise embryos, 275 Tower, Moniezia, 515, 516 Tozer, 513 Tracheae, 508 Tracheata, 505 Trachymedusse, 523 Trematodes, 516 et seq. ova, 289 Trenkmann, 549 Triacid mixture, 175 Trichina, 514 Trichlor-acetic, 53 decalcifier, 254 Trimming blocks, Eternod, 83 Triple stain, Bonney, 178 Triton (Molge), embryology, 277 Trophospongium, 439 True fats, 356 Trypan blue, 389, 390 cartilaginous skeletons, 377 dental pulp, 373 violet, 390 Trypanosomes, 533, 539, 547 Trypsin, digestion, 248 Trzebinski, 405 Tschaskin, 389 Tschernyschew, 446 Tsetse flies, 539 Tullberg, 15 Tunicata, 499 killing, 12 ova, 281 et seq. Turbellaria, 517 et seq. embryology, 288 Turpentine, clearing, 69 for mounting, 227 dissolving fat. 322, 32'.) Uexkull, 16 Underwood, 375 Unio, methylen blue, 192 Unmasking iron, 299 Unna, 32, 34, 350, 388, 391 carbol-pyronin-methyl green, 172 collodion mass, 96 half -ripe haematoxylin stock, 153 on hsematoxylin staining, 145 keratin, 341 mast cells, 355 methylen blue and Saurefiichsin, 351 orcein method, 351 oxidised haematoxylin, 155 polychrome methylen blue, 186 Rongalit white, oxygen test, 390 safranin and wasserblau, 351 smooth muscle, 348 water-blue orcein, 339, 340 Upson, 407, 477 Uranium acetate, 54 nitrate silver method, for Golgi apparatus, 436 Urea and Bouin's fluid, 306 and chromosome fixation, 305 Urodele, embryology, 276 Ussow, cephalopoda, 282 Vanadium chloride process, 475 hsematoxylin, 157 Varnishes and cements, 229 et seq. Vaso-dilators, 232 et seq. Vassale, 450 Weigert method, 444 and Donnagio, Golgi method, 465 INDEX. 593 Vastarini-Cresi, embryonic cartilage, 377 silver, myelin, 451 Vejas, 408 " Vejdovsky, 514 Venderovic, 407, 450 Venetian soap, 411 Venice turpentine, 227 for cementing, 230 Ventral cord, insect, 508 Veratti, 468 Veretillum, 523 killing, 12 Verhoeff, 354 Vermes, embryology, 288 e t seq. general, 509 et seq. Vernon, 309 Vert d'Alcali, 160 Lumiere, 159 Verworn, 14 Vesuvin, 161 mast cells, 355 Viallane, 509 osmic gold method, 205 Vialleton, cephalopoda, 282 silver impregnation of chick, 274 Victoria blue, 169 mucin, 392 neuroglia stain, 483 green, 181 Violet B, 184 of Lauth, 168 Virchow, 33, 280 Vital stains, benzidine, 390 mitochondrial, 332 See under Intra Vitarn. Scharlach VIII, myelin, 453 Vivante, bone, 375 Vivisection Acts, caution against, 265 Vogt and Tung, 511 Cestodes, 515 Cucumaria, 518 worms, 509 Volk, 17 Vosmaer, reconstruction method, 261 and Pekelharing, sponges, 526 Vosseler, wax feet, 243 Waddington, 13, 541 Waite, 288 Waldeyer, 498 decalcification, 252 Walsem, 449 Walton, tissue culture, 552 Ward, 16 Gephyrea, 511 Warnke, 407 Washburn, mollusc ova, 284 Washing out, after fixation in alcohol, formol, acetic acid, picric acid, nitric acid, corrosive, osmic, chrome, 26 liquids for, 26 Wasielewski, sporozoa, 546 M Wasserblau, 183 blood-platelets, 386 and orcein, Unna, 340 and safranin, 351 spleen, 394 Wassermann, 300 Watase, cephalopoda, 282 Watch-glass, embedding in, 74 Water-baths, paraffin, 79 Water-blue. See mtfor Wasserblau. Watery media, 217 Wax feet, 243 et seq. Webb, gum mass, 110 Weber, 512 Siphonophora, 524 Wedl, 211 Weed, 271 Weidenreich, 341 blood, 380 Weigert, 177, 450, 461 elastin stain, 353 fibrin stain, 388 hsematoxylin, 150 method for celloidin sections, 118 myelin, formol material, 444 stains, 442 et seq. neuroglia stain, 480 picro-Siiurefuchsin, 215 Weigert-Pal, myelin method, 446 Weigl, Mann-Kopsch method, 328 Weil, 109 bone, 370 teeth sections, 373 Wellings, intra vitam staining of teeth, 373 Wermel, 381 Werner, smooth muscle, 348 Wester, 507 Weysse, 269 Wharton's jelly, 391 Wheeler, eggs of Orthoptera, 286 White, bone sections, 370 Whitman, brains, 405 frog embryology, 276 pelagic fish ova, 281 Hirudinea, 511 Wickersheimer, 220 Widakowich, 270 Widmann, lens of Arachnida, 509 Van Wijhe, ammonia carmine, 141 cartilaginous skeletons, 377 Wilhelmi, 518 Will, reptile embryology, 275 Willebrand, blood stain, 383 Wilson, J. T., and Hill, J. P., 269 Wilson, Alcyonaria, 522 stain, 535 Wimmer, 482 Winiwarter, 177, 270 Wintergreen oil, for clearing, 271 Wislocki, 390 Von Wistinghausen, 155 De Witt, 3 4 Witte, pancreatin, 249 38 594 INDEX. Wittmaack, 497 inyelin, 451 Wolff, 388, 428 bladder, 349 Wolf rum, elastin stain, 353 Wollschwaiz, 548 Wolter, 453 chloride of vanadium process, 475 Wolters, 376 myelin, 447 Woodcock, faeces, 538 and Wilson. Schaudinn fixation, 541 Woodger, 365 Woodland, 518, 521 Woodworth, reconstruction, 261 Woolf, 168 Worcester's liquid, 280 Worms, 509 Wright, blood platelets, 386 Romanowsky stain, 535 Wynn, 447 Xylol, 70 Yellow gelatin mass, 237 Yolk stain, Peter, 260 See, under Fat and Cytoplasmic Inclusions, 316 et seq. 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