3- METHODS IN PLANT HISTOLOGY THE UNIVERSITY OF CHICAGO PRESS CHICAGO, ILLINOIS THE BAKER & TAYLOR COMPAXY NEW VOHK THE CAMBRIDGE UNIVERSITY PRESS LONDON THE MARUZEN-KABUSHIKI-KAISHA TOKYO, OSAKA, KYOTO, FfKUOKA, SENDAI THE COMMERCIAL PRESS, LIMITED SHANGHAI GS7 METHODS IN PLANT HISTOLOGY ^f, 6' By CHARLES J. CHAMBERLAIN, Ph.D., Sc.D. Professor Emeritus of Morphology and Cytology in the University of Chicago FIFTH REVISED EDITION THE UNIVERSITY OF CHICAGO PRESS CHICAGO • ILLINOIS COPYRIGHT 1901, 1905, 1915, 1924, AND 1932 BY THE UNIVERSITY OF CHICAGO. ALL RIGHTS RESERVED. PUBLISHED JUNE 1901 SECOND EDITION OCTOBER 1905, THIRD EDITION MAY 1915 FOURTH EDITION OCTOBER 1924, FIFTH EDITION AUGUST 1932 SECOND IMPRESSION OCTOBER 1933 COMPOSED AND PRINTED BY THE UNIVERSITY OF CHICAGO PRESS CHICAGO, ILLINOIS, U.S.A. iu PREFACE TO THE FIRST EDITION This book has grown out of a course in histological technique con- ducted by the author at the University of Chicago. The course has also been taken by non-resident students through the Extension Divi- sion of the University. The Methods were published over a year ago as a series of articles in the Journal of Applied Microscopy, and have called out numerous letters of commendation, criticism, suggestion, and inquiry. The work has been thoroughly revised and enlarged by about one-half. It is hoped that the criticism and suggestion, and also the experience gained by contact with both resident and non-resident students, have made the directions so definite that they may be fol- lowed, not only by those who work in a class under the supervision of an instructor, but also by those who must work in their own homes without any such assistance. More space has been devoted to the paraffin method than to any other, because it has been proved to be better adapted to the needs of the botanist. The celloidin method, the glycerin method, and free- hand sectioning are also described, and their advantages and disad- vantages are pointed out. The first part of the book deals with the principles of fixing and staining, and the various other processes of microtechnique, while in the later chapters these principles are applied to specific cases. This occasions some repetition, but the mere presentation of general prin- ciples will not enable the beginner to make good mounts. The illustrations and notes in the later chapters are not intended to afford a study of general morphology, but they merely indicate to students with a limited knowledge of plant structures the principal features which the preparations should show. The photomicrographs were made from the author's preparations by Dr. W. H. Knap, and Figures 52, 57, and 59 (Figs. 61, 66, and 68 of second edition) were drawn by Miss Eleanor Tarrant; all other figures of plant structures were made from the author's drawings. Corrections and suggestions will be heartily appreciated. ^ Charles J. Chamberlain Chicago June 1, 1901 PREFACE TO THE SECOND EDITION It is gratifying to the author to learn that the kindly reception ac- corded to Methods in Plant Histology has exhausted the edition. Since the first edition appeared, a httle more than four years ago, laboratory methods have been greatly improved, and systematic experiments have made it possible to give much more definite directions for making preparations. In the present edition much more attention has been given to col- lecting material. Professor Kleb's methods for securing various repro- ductive phases in the. algae and fungi have been outlined in a practical way. Methods for growing other laboratory material are more com- plete than in the earlier edition. The paraffin method has been much improved, and the glycerin method has been almost entirely replaced by the Venetian turpentine method, to which a whole chapter is devoted. Other new chapters deal with microchemical tests, freehand sections, special methods, and the use of the microscope. The author is deeply indebted to his colleague, Dr. W. J. G. Land, for numerous suggestions and improvements in methods. Corrections and suggestions will be heartily appreciated. Charles J. Chamberlain Chicago July 1, 1905 VI PREFACE TO THE THIRD EDITION The continued appreciation accorded to Methods in Plant His- tologij has exhausted the second edition. Since that edition appeared, methods have become more and more exact, so that the present vol- ume is practically a new book. The general arrangement of the sub- ject matter, and directions for collecting material and for securing reproductive phases in the algae and fungi have been retained, and a chapter on ''Photomicrographs and Lantern Slides" (chap, xii) has been added. Great improvements have been made in the paraffin method, so that sections are easily cut which were impossible ten years ago, while ten years of added experience with the Venetian turpentine method have made it possible to describe it so definitely that even the beginner should find no serious difficulty. The author is deeply indebted to his colleague, Dr. W. J. G. Land, for numerous suggestions and improvements covering the whole field of microtechnique. He is also greatly indebted to Dr. S. Yamanouchi for many improvements in the methods applicable to algae and mi- totic figures. Corrections and suggestions will be heartily appreciated. Charles J. Chamberlain Chicago May, 1915 VI 1 PREFACE TO THE FOURTH EDITION It is gratifying to the author that the appreciation accorded to Methods in Plant Histology, when it first appeared as a series of articles in the Journal of Applied Microscopy, has continued through three editions of the book. While the chapter headings and general arrange- ment remain about the same as before, the book has been almost en- tirely rewritten. Directions for collecting material have been amplified and the preparation of the most familiar laboratory types has received par- ticular attention. While no radical changes have been made in the paraffin method, the process has been shortened and improved; the Venetian turpentine method, introduced in the second edition and im- proved in the third, has come into such general use that the experi- ence of many laboratories has been added to that of our own, and the directions have become so definite that there is little excuse for fail- ures. The cellulose acetate method, which may do as much for woody structures as the Venetian turpentine method has done for its class of mounts, is outlined in a tentative way, and the chapter on 'Thoto- micrographs and Lantern Slides" has been extended and improved. The introduction of American stains, which are becoming very ac- curately standardized, has occasioned some modifications throughout. The author is even more deeply indebted than before to his col- league, Dr. W. J. G. Land, for suggestions and improvements covering the whole field of microtechnique and photography. He is also indebted to Dr. S. Yamanouchi for improvements applicable to algae and mi- totic figures. Dr. Paul J. Sedgwick is responsible for much of the im- provement in photomicrography and for many of the photomicro- graphic illustrations. To Miss Ethel Thomas, who assisted me for many years, I am indebted for improvements, criticisms, and sugges- tions covering the whole range of the book. Besides, I must thank a host of colleagues and students all over the world for help in all phases of the subject. Corrections and suggestions will be heartily appreciated. p Charles J. Chamberlain October, 1924 viii luji A PREFACE TO THE FIFTH EDITION As one edition of this book has followed another, we have tried to keep the methods up to the highest possible standard. We have tried to keep our own technique up to date and have had the pleasure of seeing various phases of microtechnique as practiced in the leading universities of our own country and also in the laboratories of Eng- land, Europe, and the Orient. The directions for collecting material have been still further ampli- fied, and directions for fixing, dehydrating, and staining have been improved. "Paleobotanical Microtechnique" and "Laboratory Photography" have been amphfied and given chapter headings. There is a new chap- ter on "Illustrations for Pubhcation." The steam method and Jeffrey's vulcanizing method for sectioning hard woods, and Gourley's method for staining living vascular tissues will repay the time one must spend to gain a mastery of these phases of technique. The paraffin method has been improved for both dehcate and hard tissues. Improvements have been made throughout; in fact, the book has had a rewriting, not a mere revision. The author is even more deeply indebted than before to his friend and colleague. Dr. W. J. G. Land, for suggestions and improvements covering the entire field of microtechnique and photography. He is also indebted to Dr. S. Yamanouchi for refinements in the paraffin method — from fixing to the finished mount — which make 3 and 2 micron sec- tions easy, 1 micron sections not too difficult, and | micron sections a possibility. Dr. Paul J. Sedgwick has written the parts deahng with "Botanical Photomicrography" and "Movie Photomicrography." To Miss Ethel Thomas, who assisted me for many years, I am in- debted for improvements, criticism, and suggestions covering the entire range of the book. Besides, I must thank a host of colleagues and students all over the world for help in all phases of the subject. Corrections and suggestions will be heartily appreciated. ^ Charles J. Chamberlain Chicago April, 1932 IX CONTENTS PART I PAGE Introduction 3 Chapter I. Apparatus 7 Chaptkr II. Reagents 18 Killing and Fixing Agents 18 The Alcohols 19 The Chromic- Acid Group 21 Picric Acid 29 Corrosive Sublimate 30 Iodine 32 Formalin 32 General Hints on Fixing 33 Dehydrating Agents 34 Clearing Agents 37 Miscellaneous Reagents 40 Chapter III. Stains and Staining 42 The Haematoxylins 45 The Carmines 53 The Anilins 56 Double Stains and Triple Stains 65 Chapter IV. Genera:. Remarks on Staining 72 Choosing a Stain ' 72 Theories of Staining 73 Practical Hints on Staining 77 Chapter V. Temporary Mounts and Microchemical Tests . . 80 Temporary Mounts 80 Microchemical Tests 82 Chapter VI. Freehand Sections . . •. 87 Woody and Herbaceous Sections 88 Objects Mounted without Sectioning 97 Chapter VII. The Glycerin Method 100 Chapter VIII. The Venetian Turpentine Method 106 xi ^279G xii CONTENTS PAGE Chapter IX. The Paraffin Method 112 Killing and Fixing 112 Washing 114 Hardening and Dehydrating 115 Clearing 116 The Transfer from Clearing Agent to Paraffin 117 The Paraffin Bath 118 Imbedding 120 Cutting 122 Fixing Sections to the Slide 125 Removal of the Paraffin 129 Removal of Xylol or Turpentine 129 Transfer to the Stain 129 Dehydrating 129 Clearing 130 Mounting in Balsam 130 A Tentative Schedule for Paraffin Sections 131 Chapter X. The Celloidin Method 132 Chapter XL The Cellulose Acetate Method 138 Chapter XII. Special Methods 140 Very Large Sections 140 Stony Tissues 141 Steam Method for Hardwood Sections 141 Jeffrey's Vulcanizing Method 142 Clearing Thick Sections or Small Objects 143 Land's Gelatin Method 144 Schultze's Maceration Method 144 Jeffrey's Maceration Method 145 Protoplasmic Connections 145 Staining Cilia 149 Chondriosomes 149 Canaliculi 151 Gourley's Method for Vascular System 151 Staining Living Structures 152 Chapter XIII. Paleobotanical Microtechnique 154 Sections 154 Peels 157 CONTENTS xiii PAGE Chapter XIV. Botanical Photography 159 Lantern Slides 160 Maps and Graphs 166 Enlargements 167 Overexposures and Underexposures 170 Photomicrographs {By Dr. Paul J. Sedgwick) 171 Movie Photomicrographs (By Dr. Paul J. Sedgwick) 177 Photographic Formulas 181 Chapter XV. Illustrations for Publication 190 PART II Specific Directions 197 Chapter XVI. Myxomycetes and Schizophytes 199 Myxomycetes 199 Schizophytes 201 Cyanophyceae 205 Chapter XVII. Chlorophyceae 210 Chapter XVIII. Phaeophyceae 240 Chapter XIX Rhodophyceae 247 Chapter XX. Fungi 252 Phycomycetes 252 Hemiascomycetes 258 Ascomycetes 259 Lichens 265 Basidiomycetes 266 Chapter XXI Bryophytes— Hepaticae 274 Chapter XXII. Bryophytes— Musci 285 Chapter XXIII. Pteridophytes — Lycopodiales 292 Chapter XXIV. Pteridophytes — Equisetales 300 Chapter XXV. Pteridophytes— Filicales 304 Chapter XXVI. Spermatophytes— Gymnosperms 322 Cycadales 322 Ginkgoales 331 Coniferales 333 Gnetales 345 Chapter XXVII. Spermatophytes— Angiosperms 347 XIV CONTENTS PAGE Chapter XXVIII. Using the Microscope 371 Micrometry 371 Artificial Light 375 Chapter XXIX. Labeling and Cataloguing Preparations . . 377 Chapter XXX. A Class List of Preparations 379 Chapter XXXI. Formulas for Reagents 386 Fixing Agents 386 Stains 390 Miscellaneous 396 Bibliography 400 Index 409 PART I INTRODUCTION '^-CJ!^ From the days of Nehemiah Grew and Robert Hooke to the time of Matthias Schleiden and Herman Schacht, histological technique was in a very primitive condition, each investigator making his own micro- scope and other apparatus. Stems and similar objects were held in the hand and cut with sharp knives, while things which did not lend themselves to such technique were dissected with needles, even such difficult objects as embryo sacs and embryos being teased out so that fairly accurate views were obtained. Schleiden's cell theory (1838), the "theory" that a plant consists entirely of cells, was developed from such preparations. Carl Zeiss, one of Schleiden's students, dissatisfied with his micro- scope, quit the study of botany and proceeded to improve microscopes. His success gave the scientific world immensely better microscopes, and the better microscopes brought improvements in histological tech- nique. By 1850, stains were being used, and improvements came so fast that a history of technique, rather than the practice of it, would be needed if one were to trace the whole subject. Before the end of the century, there were numerous fixing agents, paraffin baths, and micro- tomes, and staining had reached a high degree of efficiency. Unfortunately, some of the skill of the older botanists in teasing material and studying it in the living condition had been lost; but this valuable phase of technique is being revived and, with the micro- manipulator, results are being obtained which surpass the wildest dreams of Schleiden and Strasburger. In the fourth edition we stated that the pollen grain of a hly, placed on a dark background, is barely visible to the naked eye; but with modern technique, such a pollen grain can be cut into fifty sections, the sections can be mounted and stained without getting them out of order, a photomicrograph can be made from the preparation and a lantern slide from the photomicrograph, and finally there appears on the screen a pollen grain 10 feet long, with nuclei a foot in diameter, nucleoli as large as baseballs, and starch grains as large as walnuts. While this is a striking illustration, modern technique shows its efficiency better in demonstrating the structure of chromatin and the 3 4 INTRODUCTION finer details of protoplasm and its derivatives. For general morpho- logical work, sections are not cut so thin. About 10 /x is a serviceable thickness, with 5 /x for oil immersion details. Algae and fungi, with their small nuclei, usually need 3 or 2 /x sections. With improvements in microtomes and in technique, the 1 ^ section is not so rare, and rib- bons of root-tips have been cut at | /x. A microscope, giving several times the magnification of any microscope now on the market, is in an advanced experimental stage. Should such microscopes get into use, it would be necessary to regard a 2 /x section as rather thick, and nothing thicker than 1 ^ could be regarded as thin. Such a micro- scope would bring great improvements in technique. Every investigator whose work demands a microscope should study technique until he gains such a grasp of fundamentals that he will be able to make the modifications which individual problems may re- quire. Those who think that such work is mere mechanical drudgery, which can be done by an assistant, are likely to become armchair in- vestigators, drawing false conclusions or becoming scholastic grafters, according as the assistant is mediocre or talented. Besides, there is always the danger that a talented assistant may "hold out" some- thing. A younger member of a faculty, doing research work for his superior, may make an important discovery but, knowing that his superior has little knowledge of the progress of the investigation, may "hold it out" and, later, pubhsh it himself, preaching the same sermon from a different text. Benjamin Franklin's advice, "If you want your business done, go; if not, send," applies very well to investi- gations involving histological technique. We strongly advise the student to collect his own material in the field, for such collecting is a valuable part of a botanical education. There are details of habitat and behavior which are never described in books. One learns gradually, by experience, that certain kinds of plants grow in certain kinds of places; and further, that not only the season, but even the weather, may be an important factor. A heavy rain may cause some algae to disappear; while the same rain, followed by a few days of sunshine, will bring ideal conditions for collecting Myxomycetes and many other fungi. One learns that while Lycogala is pink, it is in the free nuclear condition, and that Stemonitis is in that condition as long as it is white; and Volvox may be abundant at the bottom of a pond when there is scarcely any in suspension. The successful investigator learns how a flower bud should look, if it is to INTRODUCTION 5 yield floral development, how the flower looks when the embryo sac is mature; and how it looks after fertilization. Such studies add immensely to the value of a preparation. A con- siderable part of a botanical education can be gained by collecting material, making and studying preparations, reading what is available, and thinking. Some biologists regard cut and stained sections as a mere mess of artifacts, giving only distorted, misleading views of the actual struc- tures; and it must be admitted that some structures, notably the liquid albuminoids, are changed by the processes of fixing, imbedding, and staining. However, no one knows this better than the expert cytolo- gist. Every student of plant structures should compare the fixed and stained material with the living. Chromosomes in the pollen mother- cells of a lily can be counted and measured and photographed in the living condition; and such details which can be seen in the living con- dition are so like those in fixed and stained material that we believe that finer details, which can be seen dimly or not at all in living ma- terial, are equally well preserved. Chromosomes as small as those in the spore mother-cells of Osmunda cinnamomea can be seen in the living condition, the various stages in mitosis can be traced, and chon- driosomes can be seen moving vigorously in living cells. Protoplasmic connections can be seen easily in living cells of the endosperm of Diospyros discolor, and can be seen, dimly, in the endosperm of a date seed, as one gets it on the market. And so we believe that smaller protoplasmic strands, which cannot be seen in the living condition, but which are easily seen in thin, well-stained sections, are not arti- facts produced by fixing, imbedding, and staining, but are real struc- tures which have merely been made visible by these processes. Of all the structures with which the cytologist has to deal, protoplasm suffers most from fixing and imbedding. But even here, protoplasm, like that in the egg of a cycad, shows the large vacuoles and smaller and smaller vacuoles in the living condition, so that still smaller vacuoles, not visible in the living condition, are no more likely to be artifacts than those so easily seen in the living condition. However, all who make preparations for microscopic investigation should be constantly on guard, and should always compare the living material with that which has been subjected to the various processes of microtechnique. CHAPTER I APPARATUS The microscope is the most important piece of apparatus. It should have a rack and pinion coarse adjustment, a fine adjustment, two eyepieces magnifying about 5 and 10 diameters, a low-power objective of about 16-mm. focus, and a high-power objective of about 4-mm. focus, a double nosepiece, an iris diaphragm, and an Abbe condenser. A cheap and practical form is shown in Figure 1, and similar instru- ments are for sale by all the leading companies. The rest of the apparatus is for getting material ready for observa- tion with the microscope. The following list includes only the appa- ratus necessary for making preparations: a microtome; a razor; a hone and a good razor strop; a paraffin bath; a turntable; a scalpel; a pair of needles; a pair of scissors; a pair of forceps; staining-dishes ; soHd watch glasses; bottles; a graduate (50 or 100 c.c); pipettes; slides, 1X3 inches; round covers, 18 mm. or f inch in diameter; and square covers, | inch. Longer covers will be needed for some of the serial sections. Keep the apparatus clean, especially the microscope. Since the chemicals used in histological technique are likely to damage the stage and substage of the microscope, it is well to place upon the stage a piece of glass 3 or 4 inches square. A lantern-slide cover is just right for this purpose. It is not necessary to fasten it to the stage, since it is merely for protection while examining slides which are wet with reagents. In our own laboratory we use for examining wet slides a cheap microscope with only a single low-power objective and a single ocular. Some knowledge of the structure and optics of the microscope is necessary if one is to use it effectively. Why are there so many dia- phragms? Why is there an arrangement for raising and lowering the condenser? Why does the mirror bar swing? Why is one side of the mirror plane and the other concave? Everyone who uses even a cheap microscope should know the answers to questions like these. All the leading manufacturers furnish, free of charge, booklets, explaining 7 8 METHODS IN PLANT HISTOLOGY the construction of the microscope and giving practical directions for its care and use. Aside from the microscope itself, the microtome is the most impor- tant piece of apparatus in the laboratory. In recent years there has been immense improvement in microtomes, but we still have only two general types — the sliding and the rotary. If there is to be only one microtome, it should be of the sliding type; for all kinds of sectioning can be done with the sliding microtome, while only paraffin ribbons can be cut with the rotary. The ro- tary microtome is convenient, rapid, and, to a large extent, eliminates the necessity for skill; but a good sliding mi- crotome, in the hands of an expert, will yield paraffin sec- tions superior to any which can be cut with a rotary mi- crotome. Paraffin sections of root-tips have been cut as thin as I M with the medium-priced sliding microtome shown in Figure 2. It should be provid- ed with a clamp which will hold any kind of knife ; but we should strongly recommend, in addition, the clamp shown in Figure 3, which will hold any of the thin safety razor blades. For any sections not more than f inch square, the safety razor blade is long enough. Paraffin ribbons f inch wide can be cut with safety razor blades. Celloidin sections, up to f inch square, can be cut with the safety razor blade; but larger sections must be cut with a microtome knife. Fig. 1. — An efficient microscope of moderate price. The leading optical companies put the same objec- tives and oculars upon such instruments as upon their most expensive stands. APPARATUS 9 Clamps have been devised for holding safety razor blades for cutting with rotary microtomes (Fig. 4). Fig. 2. — A sliding microtome capable of the best paraffin sectioning, and also good for cutting sections of stems, roots, and other things. Fig. 3. — Clamp for sliding microtome for holding thin safety razor blades Success with safety razor blades depends, to a large extent, upon the blade. Many blades, which are fairly good for shaving, are worthless for microtome cutting, because they are too soft. After the leading domestic firm had refused to furnish blades much harder than those 10 METHODS IN PLANT HISTOLOGY on the market, John Watts, 24 Redcross Street, London, E.G. 1. England, furnished blades so hard that they broke when tightened in the ordinary handles used for shaving. These blades are ideal for his- -71 (D Fig. 4. — Clamp for holding safety razor blades in the Spencer rotary microtome tological work. In ordering, one should state that he wants blades specially hardened for microtome use. Many have trouble with safety razor blades, especially when used with the rotary microtome, where the best holder is of the type shown in Figure 4. The blade is bent into a curve, making the angle of the cutting edge look less than it really is. The holder must stand more nearly vertical than a regular microtome knife to give the cutting edge the same angle. Study Figure 5, which shows cor- rect position. If the blade is too nearly vertical, it will rub the paraffin instead of cutting it ; while there will be scraping instead of cutting, if the blade is too far from the vertical position. The position will vary slightly with hard and soft, and with thick and thin, sections. If the blade projects too far beyond the holder, it will vibrate; if it does not project enough, the paraffin will hit the holder. Figure 5 will make this clear. The stout razors our grandfathers used to shave with are excellent for freehand sectioning and even for cutting sections on the micro- tome. The blade should be sharpened flat on the under side and beveled on the upper, as shown in Figure 6. If sharpened without this chisel bevel, there will be a "wire" edge, and smooth cutting will be impossible. A satisfac- tory bevel can be secured by merely tilting the knife while finishing the sharpening. The heavier microtome knives have a back which is Fig. 5. — The correct relative positions of holder, blade, and par- affin, for both sliding and rotary micro- tomes. Fig. 6. — A grandfather's razor with chisel-edge sharpening. APPARATUS 11 put on during sharpening, thus getting a proper bevel. Modern razors, ground hollow on both sides, are worthless for histological work. There should be two good hones: a fine carborundum hone for the prehminary sharpening, and a yellow Belgian hone for finishing. About 10X2| inches is a good size. If the second hone be quite hard and the finishing skilfully done, little or no stropping may be neces- sary. The best strops used by barbers are satisfactory for microtome knives. Fig. 7. — Land's electric constant apparatus, showing diagram of the automatic switch, as de- scribed in the Botanical Gazette of November, 1911. Great improvements have been made in paraffin baths. A type de- vised by Dr. Land has never been surpassed in accuracy. The novice is likely to have trouble with it. This bath is not on the market, but, with the help of the diagrams, one can make it and having made it one can use it (Figs. 7 and 8). A detailed description of the thermostat and heater is given in the Botanical Gazette of November, 1911. Unless the coil in the heater is perfectly protected, there will be a short cir- cuit; but this danger can be obviated, in large measure, by using oil instead of water in the jacket. Another form of heater, using long electric bulbs instead of a coil, can be made with less skill. From a sheet of transite | inch thick, make a box just the size of the bottom of the bath and about 4 inches 12 METHODS IN PLANT HISTOLOGY deep. In this box put three long electric bulbs. Two of the bulbs, about 250 watt, giving a temperature of about 40° C, should be con- nected with the regular outlets and should be on all the time. The third bulb, which should be capable of raising the temperature 15° or 20° C, should be connected with a thermostat. An efficient thermo- FiG. 8. — Thermostat, heater, and switch of Land's electrical constant apparatus Stat, capable of keeping the variation in temperature within about 1° can be made in a short time. Take a piece of soft steel f inch wide and iV inch thick, and 17 inches long; lay upon it three strips of aluminum of the same width and length, but only gV inch thick. These four pieces are fastened together by drilling numerous holes, not more than j\ inch in diameter, and riveting with pieces of brass wire. After fasten- APPARATUS 13 ing the four pieces together for about 7 inches, begin to bend all four pieces, fastening them together as the bending continues, so that finally there will be a horseshoe shape with the parallel sides about 1| inches apart. One end of the horseshoe is fastened to a block of trans- ite or other non-conductor, while the other end moves freely. On this free end is fastened a good platinum contact, which can be bought at any automobile supply store. This contact should be on a screw, so that it may be adjusted. A similar contact is fastened to a post, so that the two contacts are about i inch apart. Anyone familiar with Fig. 9. — Paraffin bath electricity can make the connections. Various temperatures are se- cured by changing the distance between the platinum points. To pre- vent sparking, there must be a condenser in the circuit leading to the thermostat. A telephone condenser (0.500MF) is satisfactory. Pos- sibly, some of the cheap radio condensers would do. Anyone can make a bath which, if carefully watched, gives excellent results (Fig. '9). It is simply cut from brass, 2 or 3 mm. in thickness, with three legs screwed into it. There should be brass boxes, 10 or 12 cm. long, to contain the paraffin. It is neither necessary nor de- sirable that the boxes have covers. Boxes are easily made from square 14 METHODS IN PLANT HISTOLOGY brass tubing, 1 inch square. Cut the tubing into pieces 10 or 12 cm. long, saw off one side, and solder pieces into the ends with hard solder. The brass plate can be heated with any kind of flame at the pointed end. Since the Venetian turpentine method has almost entirely displaced the glycerin method, the turntable is disappearing from the botanical laboratory; but some objects, like Nemalion and moss protonema, are still mounted in glycerin or glycerin jelly; and so one still finds occa- sional use for this once necessary piece of apparatus. A serviceable form is shown in Figure 10. More expensive turntables, with devices Fig. 10.— Turntable for automatic centering, present no practical advantages, and the centering devices are often in the way. Much histological work usually done with scalpels can be done with safety razor blades, especially since holders of the Gits type have be- come common. For trimming paraffin blocks and handling paraffin ribbons, a more rigid type is necessary. A scalpel with a straight edge is preferable. Needles are used so constantly that it is well to have clamping holders. However, nothing is quite equal to a rather large handle whittled out from a piece of light pine. Scissors are seldom used in the botanical laboratory except for cutting out labels. Rather stout scissors, with blades about 2\ inches long, are best for general purposes. It is convenient to have two pairs of forceps, a strong pair for han- dling slides and a lighter pair, preferably with broad shovel-shaped points, for handling cover glasses. Curved forceps are not necessary; the cover-glass forceps, used by bacteriologists for staining on the cover, are of no use in botanical histology. APPARATUS 15 For staining on the slide, Stender dishes are very convenient. The form shown in Figure 11 A, about 60X90 mm., is in general use. Some prefer the Coplin jar, shown in Figure IIB; but it is troublesome to clean, and if sHdes are placed back to back, as shown in the figure, water is carried up in dehydrating and xylol is carried down. When a large number of slides of the same kind are to be stained at one time, the cheap and practical device, shown in Figure 12, is a time-saver. It is simply a coil of brass wire, 0.064 inch in diameter (No. 14, Band's gauge), wound so that the coil is about | inch across. Such a coil, carrying 15 slides, will go into an ordinary Stender dish, except that the coil projects enough to prevent the cover from fitting. Taller k. n 1 1 1 A B Fig. 11. — Staining-dishes: A, Stender dish; B, Coplin jar glasses, from the five-and-ten-cent store, can be used for the absolute alcohol and xylol, which must be kept well covered. We have been using a coil made from wire 0.051 inch in diameter (No. 16 Band's gauge), wound so that the coil is If inch across. It holds the slides and the ordinary Stender dish can be covered. Biological supply houses use rectangular staining dishes and holders carrying 50 slides. Solid watch glasses, or Minots, as they are often called, are always useful. Each student should have a dozen or more. Each student should have three bottles of about 1-liter capacity for 90 per cent alcohol, absolute alcohol, and xylol. In addition, a half-dozen bottles, holding about 100 c.c, will be useful. There should be two bottles, holding about 50 c.c, for clove oil. If one is doing much research work, it will be convenient to have many more bottles for graded series of alcohols and xylols. 16 METHODS IN PLANT HISTOLOGY There should be a graduate, preferably 50 c.c. or 100 c.c. If the bottles are of uniform size, 50 c.c, 100 c.c., 500 c.c. and 1,000 c.c, the student should soon be able to estimate with sufficient accuracy for making up reagents which do not require extreme accuracy. Three or four pipettes, or medicine droppers, will be useful. Occa- sionally, the glass of an ordinary pipette, thrust into a small camera bulb, will save time in drawing off reagents. Fig. 12. — -Coil of brass wire holding 15 slides SHdes and covers are a constant expense. Many shdes now upon the market are imperfect. Beware of slides which are not perfectly flat. Be skeptical in regard to any claim that slides are already clean enough to use. Of course, there should be no bubbles. "White" shdes are to be preferred to those which appear greenish in the box. For ordinary class work, slides of medium thickness are more serviceable, but for critical cytological work many investigators prefer very thin slides. Slides and covers, as you buy them, arc never clean, no matter how APPARATUS 17 nice they may look. Leave them overnight or 24 hours in cleaning fluid: Bichromate of potash 20 g. Sulphuric acid 30 c.c. Water 250 c.c. Rinse well in clean water and then leave them overnight or 24 hours in soapy water with plenty of soap. Rinse thoroughly in clean water and wipe dry. The cleaning fluid cleans. The soap is necessary on account of the acid in the cleaning fluid, and rinsing removes the soap. Unless the acid is completely removed, many stains will fade. There is never any objection to very thin covers, except that they require care in cleaning. For mounts which are to be used with an im- mersion lens, it is better to have the cover of the same width as the slide. The advantage is evident, since there is no danger of getting balsam on the cover when wiping off the immersion fluid ; besides, one can put sections to the very edge of the slide and still be sure that they will be covered. Since most mounts for research work are mounted under long covers and are intended for examination with immersion lenses, we should recommend covers of 25X50 mm., or even 25X60 mm. Round covers are desirable only when mounts are to be sealed on a turntable. Larger slides and correspondingly larger covers are needed for special purposes. By consulting a catalogue, which will be furnished by any dealer, the beginner can determine what he needs to buy, and what he can find substitutes for, if it is necessary to be very economical. \ CHAPTER II REAGENTS During the eight years since the fourth edition of this book ap- peared, practically no new ingredients of formulas have come into gen- eral use; but there have been new combinations of ingredients and considerable improvement in the use of reagents. The following ac- count presents not only those reagents which are in constant use but some of those which are used only occasionally. The Microtomist's Vade-Mecum, by Lee, is written from the standpoint of the zoologist, but it contains very complete formulas for stains and other reagents which are equally valuable to the botanist. For convenient reference, a list of reagents, including stains, is given in chapter xxxi. Stains are treated more fully in chapter iii. KILLING AND FIXING AGENTS No process in microtechnique is in more urgent need of improve- ment than this first step of killing and fixing. In nearly every investi- gation involving histological technique some fixing agent or other is recommended; but usually so little attention is paid to other factors which may be just as important, that it is doubtful whether the fixing agent is responsible for the excellence or mediocrity of the prepara- tions. If the fixing is bad, it is impossible to get good preparations; but insufficient washing after fixing, too rapid dehydration, too long an immersion in the paraffin bath, or too high a temperature in the bath may result in poor preparations even when the fixing has been good. If material is examined at every stage, mistakes can be corrected — in the next lot of material. Usually the same reagent is used for both killing and fixing. The purpose of a killing agent is to bring the life-processes to a sudden termination, while a fixing agent is used to fix the cells and their con- tents in as nearly the Uving condition as possible. The fixing consists in so hardening the material that the various elements may retain their natural condition during all the processes which are to follow. 18 REAGENTS 19 Zoologists often use chloroform or ether for killing an organism, and then use various fixing agents for various tissues. Most of our formulas are merely empirical, for very few botanists are expert chemists, and those who have some knowledge of chemistry are interested in physiological problems rather than in microtechnique. The principal ingredients of the usual killing and fixing agents are : alcohol, chloroform, chromic acid, dichromate of potash, potassium iocUde, copper acetate, acetic acid, osmic acid, formic acid, picric acid, sulphuric acid, platinum chloride, iridium chloride, corrosive subH- mate, and formalin. We shall consider first : THE ALCOHOLS a) Ninety-five per cent alcohol. — This is in quite general use for material wliich is needed only for rough work. It is extremely con- venient, since it kills, fixes, and preserves at the same time and needs no changing or washing. It really has nothing to recommend it for fine work. It causes protoplasm to shrink, but cell walls usually retain their position, so that 95 per cent alcohol will do for freehand sections of wood and many herbaceous stems, where it is not necessary to pre- serve cell contents; but even freehand sections of tender stems, like geraniums and begonias, will look better if better reagents are em- ployed. Alcohols weaker than 95 per cent are not to be recommended as fixing agents, although 70 per cent alcohol, or even 50 per cent, will preserve material for habit work. The time required for fixing in 95 per cent alcohol is about the same as for absolute alcohol. The subse- quent treatment is the same, except that material to be imbedded in paraffin or celloidin must be dehydrated in absolute alcohol. Material preserved in weaker alcohols and intended only for habit study may be kept in the reagent until needed for use. Unless some glycerin be added, material left in 95 per cent alcohol becomes very brittle. Stems, roots, and similar objects may be kept indefinitely in a mixture of equal parts of 95 per cent alcohol and glycerin. Methyl alcohol, or wood alcohol as it is commonly called, serves equally well. b) Absolute (100 per cent) alcohol. — This is a fair killing and fixing agent, it causes but little shrinldng of the protoplasm, and is a time- saver if material is to be imbedded in paraffin. The time required for fixing in alcohol is very short. For small fungi, hke Eurotium, 1 minute 20 METHODS IN PLANT HISTOLOGY is long enough. Root-tips of the onion, anthers of the hly, and similar objects require from 15 to 30 minutes. Larger objects may require an hour. No washing is necessary, but all plant tissues contain water; consequently, if material is to be imbedded in paraffin, the alcohol used for fixing should be poured off and fresh alcohol added before proceed- ing with the clearing. If material is to be mounted in Venetian turpen- tine, as is hkely to be the case in small filamentous fungi, the transfer to the stain may be made directly from the absolute alcohol to any stain dissolved in an alcohol not weaker than 85 per cent. Small forms with no vacuoles may be transferred to a weaker alcoholic stain or even to an aqueous stain ; but neither the fixing nor the rude transfer would be at all satisfactory with forms like Zijgnema or Saprolegnia. Acetic acid is used with alcohols to counteract the tendency to shrink. One of the most widely known of the alcohol combinations is c) Carney's fluid. — Absolute alcohol 6 parts Chloroform 3 parts Glacial acetic acid 1 part The penetration is very rapid. An object like an onion root-tip is doubtless killed in less than a minute and 15 or 20 minutes is long enough to fix an object of this size. Wash in absolute alcohol, changing frequently, until no odor of acetic acid or chloroform remains. For a root-tip, the entire process does not require more than an hour. It is better to imbed in paraffin at once, but when this is not convenient, the material may be washed in absolute alcohol until the odor of acetic acid and chloroform disappears, cleared in xylol, and, with a block of paraffin about half the bulk of the liquid added, may be left indefinite- ly. Cyanin and erythrosin, fuchsin and iodine green, and similar com- binations stain brilliantly after this reagent. d) Acetic alcohol. — Farmer and Shove recommend for fixing root- tips of Tradescantia virginica a mixture of 2 parts absolute alcohol and 1 part glacial acetic acid. The mixture is allowed to act for 15-20 minutes, after which the acid is washed out with absolute alcohol and the material is imbedded as soon as possible. e) Formalin alcohol. — This is one of the most satisfactory alcohol combinations. Various proportions are used by different workers. Professor Lynds Jones, who first brought the combination to my no- tice, added 2 c.c. of commercial formalin to 100 c.c. of 70 per cent al- REAGENTS 21 cohol. We have used a larger proportion of formalin, often as much as 10 c.c. to 100 c.c. of 70 per cent alcohol. Results which seem equally good have been secured by adding from 4 to 10 c.c. of formalin to 100 c.c. of 50 per cent alcohol. Material in this fixing agent may be left until needed for use. /) Formalin acetic alcohol. — This combination, for general anatomi- ical work, might almost be called a universal fixing agent. About 5 c.c. of glacial acetic acid and 5 c.c. of commercial formalin to 90 c.c. of 50 per cent or 70 per cent alcohol is generally satisfactory. If the proto- plasm shrinks away from the cell wall, increase the proportion of acetic acid to 7 per cent or even to 10 per cent. We should not recommend more than 10 per cent of acetic acid in any fixing agent. When one is on a long trip, moving frequently from place to place, with httle opportunity to make the numerous changes which are neces- sary when using the chromic formulas, this is the best fixing agent we have found. It will fix and preserve an amount of material equal to its own weight, and the material may be left in the solution for months. The reagent is good for almost any material, except the unicellular and filamentous algae and fungi, which are more satisfactory in media containing no alcohol. THE CHROMIC-ACID GROUP Chromic acid, or solutions with chromic acid as a foundation, are the most generally useful killing and fixing agents yet known to the botanist. A 1 per cent solution of chromic acid in water gives good results, but it is better to use the chromic acid in connection with other ingredients, such as acetic acid, formic acid, osmic acid, etc. Chromic acid does not penetrate well, and this is one reason why it is seldom used alone. Unfortunately it precipitates some liquid albuminoids in the form of filaments and networks, which may be mistaken for struc- tural elements. In botanical work, acetic acid is nearly always mixed with chromic acid. The pickles of the dinner table show that acetic acid is a good preservative, and that it causes little or no shrinking. It penetrates rapidly, and is hkely to cause swelling rather than shrink- ing, thus counteracting the tendency of chromic acid to cause plas- molysis. The swelhng is as bad as shrinking. If the proportion of acetic acid is too high, material may even break up; but 2 per cent, or even 6 per cent, may be used to show the topography of an embryo sac of an angiosperm, or the free nuclear stage of the endosperm of a gym- 22 METHODS IN PLANT HISTOLOGY nosperm; and for filamentous algae, which are to be mounted whole, 3 per cent is very effective. It is convenient to have in the laboratory the following stock solu- tion of chromo-acetic acid from which various solutions can be made as they are needed Chromic acid crystals 10 g. Glacial acetic acid 10 c.c. Water 1,000 c.c. Keep the stock solution in a glass-stoppered bottle because the acetic acid evaporates very rapidly. To make a solution containing 0.5 g. of chromic acid and 2 c.c. of glacial acetic acid to 100 c.c. of water, add 50 c.c. of water to 50 c.c. of the stock solution, and then add to the weakened solution 1.5 c.c. of glacial acetic acid. Any desired proportions can be secured in a similar way. Weighing the crystals for every new proportion is more tedious. The proportions of the various ingredients, for the present at least, must be determined by experiment. With favorable objects Uke fern prothallia, Spirogyra, and other things which can be watched while the fixing is taking place, suitable proportions are rather easily determined, because specimens, after being placed in the reagent, may be examined at frequent intervals, and combinations which cause plasmolysis may be rejected and different proportions tried until satisfactory results are secured. For example, fern prothallia might be placed in the following solution: chromic acid, 2 g.; acetic acid, 1 c.c; and water, 97 c.c. If plasmolysis takes place, as it probably will, weaken the chromic or strengthen the acetic. In general, it will be better to weaken the chromic, but not to less than | per cent. If there is still some shrinking after the chromic has been reduced to ^ per cent, strengthen the acetic. For most fern prothallia, the stock solution, with the addition of 2 c.c. of glacial acetic acid to 100 c.c. of the solution, is satisfactory for material to be mounted whole, and also for sections. A combina- tion may be quite satisfactory for fern prothallia and still fail to give good results with Spirogijra, and a combination which is excellent for Spirogyra may fail utterly with Vaucheria. For critical work the most favorable proportions must be determined for the particular object under observation. In observing the effect of the fixing one can deter- mine whether there is any noticeable plasmolysis or distortion, but whether the fixing is thorough can be determined only by noting how REAGENTS 23 the tissues endure the subsequent processes. When the effect of the reagent cannot be observed directly, it is well to make a freehand sec- tion and thus determine whether plasmolysis takes place. It is not safe to judge the action of a fixing agent by the appearance of sections cut from material which has been imbedded in paraffin, because shrinking of the cell contents often takes place during the transfer from absolute alcohol to the clearing agent or during infiltration with paraffin, and sometimes even during later processes. When there is doubt as to proportions, we should suggest 2 g. chromic acid, 3 c.c. acetic acid, and 300 c.c. water as a good formula for most purposes. A large quantity of the fixing agent is required and it cannot be used again. The volume of the fixing agent should be at least 25 times that of the material to be fixed. We use about 50 volumes of the fixing agents to one of the material. The time required for fixing undoubtedly varies with different ob- jects, but even a delicate object, like Spirogijra, which is penetrated immediately, should remain in the fixing fluid for from 18 to 24 hours. Most botanists leave material hke onion root-tips and lily ovaries in the chromo-acetic acid about 24 hours. Two days, or even 3 or 4 days, does no damage, and we should prefer 48 hours rather than to use less than 24 hours. Christman, in his work on rusts, left material for three days in Flemming's fluid, a much more vigorous agent than the chromo-acetic acid. We have often imbedded material which had been in chromo- acetic acid for several days, and it seemed to have suffered no injury. It is well known that zoologists allow fixing agents like Mliller's fluid and Erlicki's fluid to act for weeks before the material is passed on to the next stage, and it may well be questioned whether botanists have not made a mistake in allowing the chromic solutions to act for so short a time. More rapid penetration, and consequently more imme- diate killing, can be secured if the reagent is kept warm (30°-40° C). The warming also shortens the time required for fixing, but, for cyto- logical work, it is quite possible that the danger of producing artifacts may be increased by the heat. After fixing is complete, all reagents containing chromic acid as an ingredient should be washed out with water. Running water is desir- able, and where this is not convenient the water must be changed fre- quently. About 24 hours is long enough for complete washing in running 24 METHODS IN PLANT HISTOLOGY water. Shorter periods may be sufficient for some things, but 24 hours will not do any damage even to the most delicate objects, and a shorter time may be insufficient. Heavy objects which sink promptly may be placed in a Stender dish or wide-necked bottle under a gentle stream of water. There is little danger in this method if the material is heavy enough to remain at the bottom : the only ob- jection is that much of the water does not reach the bottom. Here is a better method: tie a piece of cheesecloth over the neck of a bottle ; slip over the water tap a rubber tube with a piece of glass tube tied in the end, and slip the glass tube through the cheesecloth nearly down to the bottom of the bottle. The washing will then be very thorough. A method devised by Dr. Dudgeon is simple and very efficient, especially for delicate materia or objects which have a tendency to float. A glass tube, 1 inch in diameter, is cut into pieces 2f inches in length. The glass is then heated to round off the sharp edges and, while the glass is still very hot, one end is flared a little, so that a piece of cloth can be tied over or fastened over it with a rubber band. Bolting cloth or bolting silk is best because water passes through it so readily. For flaring the ends of the tubes a triangular piece of copper ^V i^^ch thick is very convenient (Fig. 13). Heat the copper, cut the edges into a cake of beeswax, and turn the instrument around a little in the end of the hot glass tube. Any num- ber of these tubes can be placed in a jar without any danger of losing material. Another method which we have found satisfactory is to put the material into a tea filter, which can be got at the five-and-ten-cent store. This is good for everything except filamentous forms which stick in the little holes. Any number of the tea filters can be placed in a jar and washed from a single water tap. Here is still another method: take a box about 6 inches wide, 18 inches long, and 4 inches deep ; bore |-inch holes in the bottom, and into each hole put a piece of rubber tubing about 4 or 5 inches in length. Pipettes can be fastened in the ends of these rubber tubes. Place the Fig. 13. — Instrument for flar ing glass tubes. REAGENTS • 25 box under the tap. In the botanical laboratory at Woods Hole, Massachusetts, large quantities of material are washed at one time by using an ordinary washtub with the bottom arranged as just described for the box. If one is using such a large box or tub and does not need all the streams of water, the tubes not in use may be closed by means of clamps. Where running water is not available we should recommend the washtub, as just now described. The tub could be filled, and, with a single lot of material, would run for 10 or 12 hours. With delicate filamentous or branching algae and fungi, extreme care must be taken in the washing, for the material must not get tangled. Put some material — not too much — in a large Petri dish, propped up on an inverted dish and tilted just enough to let the water r^'yT^^ZZZZi -A\ .^ -^ V J Fig. 14. — Washing delicate filamentous and branched material run off gently. As a precaution, the supporting dish may be placed in a larger dish (Fig. 14). The stream of water should be from a pipette fastened in the end of the rubber which has been slipped over the water faucet. Drops of water are even better than a small stream. Although some objects might be washed in 10 or 12 hours, it is better to wash for 24 hours. Nothing would be damaged by the longer time and subsequent processes, especially staining, might be improved. Everyone has his own chromo-acetic acid formulas. Some of those in more general use are the following: a) Stock chromo-acetic solution. — Chromic acid 1 g. Glacial acetic acid 1 c.c. Water 100 c.c. This solution has been used quite extensively in embryological work upon the higher plants. It fixes thoroughly, but often causes plas- molysis in cells with large vacuoles. 26 METHODS IN PLANT HISTOLOGY h) Weak chromo-acetic solution (Shaffner's formula). — Chromic acid 0 . 3 g. Acetic acid 0.7 c.c. Water 99.0 c.c. This has also been used in embryological work. It causes little or no plasmolysis. Difficult material, like Aster heads and ripe Capsella pods, cuts more readily after this reagent than after the stronger solu- tions. c) Strong chromo-acetic solution. — Chromic acid 1 g- Glacial acetic acid 3 c.c. Water 100 c.c. For fern prothallia, most liverworts, moss capsules before they have begun to get reddish or brownish, and most filamentous algae and fungi, this is a good fixing agent. d) Licent's formula. — One per cent chromic acid 80 c.c. Glacial acetic acid 5 c.c. Formalin 15 c.c. This formula has been recommended for coenocytic algae and fungi and for embryo sacs. The most famous and, up to the present time, the most satisfactory of all the chromic mixtures, are the Flemming solutions or modifica- tions of them. In his work on mitosis and upon the structure of proto- plasm he used two solutions, commonly called the stronger and weaker solutions, which contained osmic acid in addition to the chromic and acetic acids. Various proportions of the three ingredients have been used by various investigators. While the chromic and acetic acids may be made up in a stock solu- tion, it should be remembered that the acetic acid will evaporate un- less kept in a very tightly stoppered bottle. The osmic acid should be kept in a tightly stoppered bottle, always using a glass stopper, and the bottle should be completely covered with black paper. The osmic acid must not he added to the other two ingredients until you are ready to drop the material into the fixing agent. REAGENTS 27 e) Flemming's fluid (stronger solution). — . f One per cent chromic acid 45 c.c. \ Glacial acetic acid 3 c.c. B. Two per cent osmic acid 12 c.c. This formula has been very popular for cy tological work, and has been highly recommended for chromosomes, centrosomes, achromatic struc- tures, and mitotic phenomena in general. The fluid should be allowed to act for from 24 to 48 hours, and the washing should be very thorough. Material should be in very small pieces | inch square, or in thin slices I inch or less in thickness, for the fluid penetrates poorly. The blackening due to the osmic acid may be removed by peroxide of hy- drogen juvst before the slide is passed from the alcohol into the stain. Harper and Holden, in their work on Coleosporium, recommended 4 hours on the slide in a 3 per cent solution of the peroxide of hydrogen. Some prefer a stronger solution of the peroxide of hydrogen, even 20 per cent. The peroxide should be in water, if one is following it by an aqueous stain, but may be in 50 per cent alcohol if it is to be followed by an alcoholic stain. Yamanouchi has used chlorine for bleaching, and the results are fully equal to those obtained with peroxide of hy- drogen, and the chlorine is cheaper. Make the bleacher as follows: Place some potassium chlorate crystals — a group about as large as a grain of wheat — in the bottom of a 100 c.c. Stender dish; add one drop of 25 per cent hydrochloric acid in water; immediately fill the Stender full of 30 per cent alcohol and thus dissolve the fumes in alcohol. This will bleach sections in 10 minutes, or even less. Wash in 30 per cent alcohol 2 or 3 hours before staining. Trondle uses 1 per cent chromic acid in water for bleaching; it is slow, requiring about 8 hours, but he maintains that material stains better than after bleaching with perox- ide of hydrogen. According to Miss Merriman, the linin in the nuclei of onion root-tips is not so well preserved in this solution, but the arrangements of the chromatin granules is brought out with greater distinctness. Flemming's safranin, gentian violet, orange combination gives excellent results after this reagent. /) Flemming's fluid (weaker solution). — (One per cent chromic acid 25 c.c. One per cent acetic acid 10 c.c. Water 55 c.c. B. One per cent osmic acid 10 c.c. 28 METHODS IN PLANT HISTOLOGY As in all solutions containing osmic acid, mix A and B only as needed for immediate use. g) Benda's fluid. — One per cent chromic acid 16 c.c. Two per cent osmic acid 4 c.c. Glacial acetic acid 2 drops This modification of Flemming's stronger solution has been used in various investigations upon chromatin. h) Merkel's fluid. — Equal volumes of a 1.4 per cent solution of chromic acid and a 1.4 per cent solution of platinic chloride. This is also an expensive re- agent. It is recommended for mitotic phenomena, but does not seem to equal Flemming's solution. i) Hermann's fluid. — One per cent platinic chloride 15 parts Glacial acetic acid 1 part Two per cent osmic acid 4 or 2 parts This is the most expensive fixing agent yet discovered, and for bo- tanical purposes it does not seem to be any better than the cheaper chromic mixtures. It is mentioned here with chromic mixtures because it originated as a variation of Flemming's fluid, the platinic chloride being substituted for the chromic acid. Recently, it has been resur- rected and highly recommended for the structure of the chromosome. Personally, I do not believe it is equal to Flemming's weaker solution; and, even in this weaker solution, the percentage of osmic acid may be too high. j) Chicago formula. — Chromic acid 1 g. Glacial acetic acid 2 c.c. One per cent osmic acid 6 to 8 c.c. Water 90 c.c. The osmic acid, of course, to be added immediately before using. REAGENTS 29 For a couple of years an extensive series of experiments has been carried on with root-tips of Vicia faha, Allium cepa, and especially with Trillium erectum. The stock chromo-acetic solution was tried with the addition of from 1 to 10 c.c. of 1 per cent osmic acid, fixing from 24 to 48 hours. While the solutions with the lower percentages of osmic acid fixed fairly well, they proved decidedly inferior in staining, especially with Haidenhain's iron-alum haematoxylin. Solutions with 9-10 c.c. of osmic acid are unnecessarily strong. The solution with 8 c.c. of osmic acid produces the best fixing, and the staining is brilliant, especially with Haidenhain's haematoxylin. For some things, espe- cially algae, the chromic acid may be reduced and the osmic acid in- creased. Some suggestions are made in Part II, in connection with various objects. PICRIC ACID Use a saturated solution in water or 70 per cent alcohol. One gram of picric acid crystals will saturate about 75 c.c. of water or alcohol. This reagent penetrates well and does not make the material brittle. It is to be recommended when difficulty is anticipated in the cutting. If used cold, the time varies from 1 to 24 hours, depending upon the character of the tissue and size of the specimen. If used hot (85° C), 5 or 10 minutes will be sufficient. This fixing agent is used rather ex- tensively by zoologists, especially for embryological work. Botanists have not given it fair trial. Since it seems worthless for mitotic figures; they have not made a thorough trial of it for other objects. It might be worth while to try it for embryo sacs, free nuclear stages in the female gametophytes of gymnosperms, and similar things for which satisfactory fixing has not yet been devised. Material should be washed in 70 or 50 per cent alcohol. Water is injurious, and some even go so far as to avoid aqueous stains, unless the material has been thoroughly washed. The washing should be continued until the material appears whitish and the alcohol no longer becomes tinged with yellow. Picro-carmine gives its best results after this reagent. Picric acid can be combined with various other fixing agents, and so we have picro-sulphuric acid, picro-nitric acid, picro- chromic acid, picro-chromic-sulphuric acid, picro-osmic acid, picro- alcohol, and picro-corrosive sublimate. The picric acid in all mixtures should be rather strong. 30 METHODS IN PLANT HISTOLOGY A picric-acid combination which has gained some popularity for cytological work is Bouin's fluid. — Formalin (commercial) 25 c.c. Picric acid (saturated solution in water) 75 c.c. Glacial acetic acid 5 c.c. Fix about 24 hours. Rinse in water for a few minutes to remove the more supei-ficial picric acid, and then complete the washing in 35 per cent or 50 per cent alcohol. There is likely to be some swelling, but spindles of mitotic figures stain well. The formula has given good re- sults with early stages in the female gametophyte of Pinus and would be worth a trial with the embryo sacs of angiosperms. CORROSIVE SUBLIMATE Corrosive sublimate, or bichloride of mercury, is soluble in water and in alcohol. About 5 g. will make a saturated solution in 100 c.c. of water. It is very much more soluble in alcohol, but for practical pur- poses 5 g. in 100 c.c. of 50 per cent alcohol may be regarded as a favorable solution. Corrosive sublimate used alone does not give as good results as when mixed with acetic acid, chloroform, or picric acid. Fixing is very rapid, the material being fixed almost as soon as it is penetrated by the fluid. Material which is at all transparent, like some ovules and the endosperm of gymnosperms before the formation of starch, becomes opaque as soon as fixed, and so the time needed for fixing is easily determined. From 10 minutes to 30 minutes should be sufficient for onion root-tips or lily ovaries. Smaller or larger objects require shorter or longer periods. When used hot (85° C), the fixing is much more rapid. While a few minutes' fixing may be sufficient, we let the reagent reach the boiling-point, then remove the flame, and just as soon as the bubbling ceases, put the material in and leave it until the liquid becomes cool. It may be left for 20 minutes, or even 30 minutes, without any damage. Wash out aqueous solutions with water and alcoholic solutions with alcohol. In either case, the washing must be very thorough, since preparations from incompletely washed material are sure to be dis- figured by crystals of corrosive sublimate. After material fixed in the aqueous solution has been washed in water for an hour, add a little of the iodine solution used in testing for starch. The liquid will turn brownish or amber-colored, and then clear up; add a little more, until the liquid fails to clear up completely, a very slight amber remaining REAGENTS 31 for an hour or even permanently. After material fixed in the alcoholic solution has been washed in 50 per cent alcohol for an hour or more, add the iodine solution used in testing for starch or even add, drop by drop, tincture of iodine, until the color fails to disappear. With uni- cellular forms, filamentous forms, and thin things, like fern prothallia, such washing is likely to be sufficient, but with more bulky material which is to be sectioned, the crystals may appear in the paraffin rib- bon. In such cases, the slide should be dipped for a minute in the iodine solution just before staining. Camphor may be used instead of iodine to hasten the washing, but it does not give any color reaction. Material should be imbedded as soon as possible, since it gets brittle if allowed to remain in alcohol. Kinoplasmic structures do not stain well with gentian violet, but safranin and the haematoxylins stain almost as well as after chromic- acid mixtures, and the carmines give their most brilliant stains, as a result of the formation of mercuric carminate. The following formulas are merely suggestive: a) Corrosive sublimate and acetic acid. — Corrosive sublimate 3 g. Glacial acetic acid 5 c.c. Alcohol (50 per cent) or water 100 c.c. h) Corrosive sublimate, acetic acid, and formalin. — Corrosive sublimate 4 g. Glacial acetic acid 5 c.c. Formalin 5 c.c. Alcohol (50 per cent) or water 100 c.c. This is our favorite formula. For material which is to be mounted in glycerin, glycerin jelly, or Venetian turpentine, use the aqueous so- lution; for material which is to be imbedded, use the alcoholic. Ciha are caught and preserved; and even delicate organisms, Hke Volvox, do not collapse. c) Corrosive sublimate, acetic acid, and picric acid. — Corrosive sublimate 5 g. Glacial acetic acid 5 c.c. Picric acid, saturated solution in 50 per cent alcohol 100 c.c. Miss Ethel Thomas recommends tliis formula for the female game- tophyte of Pinus. 32 METHODS IN PLANT HISTOLOGY d) Corrosive sublimate and picric acid (Jeffrey's solution). — Corrosive sublimate, saturated solution in 30 per cent alcohol 3 parts Picric acid, saturated solution in 30 per cent alcohol 1 part It would be worth while to try other combinations. IODINE Iodine is well known as an antiseptic. It is also a good fixing agent for unicellular, colonial, and filamentous forms. It penetrates rapidly. To a saturated solution of potassium iodide in distilled water, add iodine to saturation. Filter and dilute with distilled water until the solution has a rich brown color. For fixing, dilute still further to a light-brown color. The solution fixes in 10-24 hours, but material may be left in it for several days. Wash thoroughly in tap water which has stood long enough to give off all excess of air. If the staining of the starch does not disappear, a | per cent solution of tannic acid in water will remove any excess color. FORMALIN Formalin is an excellent preservative. It has been mentioned al- ready as an ingredient in several formulas. Commercial formalin has a strength of 40 per cent. Throughout this book, a 2, 4, or 6 per cent formahn is understood to mean 2, 4, or 6 c.c. of commercial formalin to 98, 96, or 94 c.c. of water, alcohol or any other ingredient. Com- mercial formalin is sure to contain some formic acid. For most pur- poses, it is neither necessary nor desirable to remove the acid. For studying the origin of vacuoles, it is necessary to have neutral forma- lin, which can be secured from commercial formalin by distillation. Place some sodium bicarbonate in a flask of formalin and distil by heating over a Bunsen flame. It is not worth while to distil more than is needed for immediate use, since the formic acid soon reappears. For filamentous algae and fungi a 3-6 per cent solution of the or- dinary commercial formalin in water is very good. Material is left in the solution until needed for use. For marine algae, sea water should be used instead of fresh water. Both marine and fresh-water material should be washed for an hour in fresh water before staining. Material of Polysiphonia, left in a 12 per cent solution of formahn for 10 years, REAGENTS 33 showed scarcely any shrinking of cell contents, the filaments were not breaking up, and even the color had scarcely faded. A 6 per cent solution will fix one-fourth its volume of material. With material like filamentous algae or leafy liverworts, a 10 per cent solution will fix all one can put into the bottle without crowding. For class use, material should be washed in water for several minutes, because the fumes are irritating to the eyes and mucous membranes. For a study of chondriosomes or the origin of vacuoles, the follow- ing combination is satisfactory: Bensley's formula. — 1. Formalin (neutral) 10.0 c.c. 2. Dichromate of potash 2 . 5 g. 3. Corrosive sublimate 5 . 0 g. 4. Water 90.0 c.c. Make the solution 2, 3, 4, and then add the neutral formalin. Fix about 24 hours. Wash in water, but use the iodine — necessary on account of the corrosive sublimate — just before staining sections on the slide. Yamanouchi's formula. — Formalin (neutral) 10 c.c. Water 100 c.c. This simpler formula brings out the chondriosomes very clearly. Fix overnight or even 24 hours. A thorough washing is easy and staining is brilliant, especially with Haidenhains iron-alum haema- toxylin. GENERAL mNTS ON FIXING Since it is desirable that a fixing agent penetrate quickly to ah parts of an object, the material should be in small pieces. The best fixing agents do their best work near the surface of the piece. Of course, filamentous algae and fungi, and delicate objects like fern prothallia and root-tips, are simply thrown into the fixing agent. Alcohol, formahn alcohol, or formalin alone may penetrate j-inch cubes; but the chromic-acid series, which gives the best results in cytological work, penetrates so poorly that cells more than jV i^^ch from the surface are not likely to be well fixed. Most objects should be trimmed with a razor so that no part shall be more than j\ inch from 34 METHODS IN PLANT HISTOLOGY the surface. Even then, it must be remembered that a waxy or cu- tinized or suberized surface presents an almost impassable barrier to the chromic series. Some objects, although small, cause trouble in various ways. Many buds are hairy and will not sink; if such things are dipped quickly in strong alcohol, they will usually sink. If rather large air bubbles pre- vent the material from sinking, as in case of perichaetical leaves of some mosses and involucral leaves of liverworts, a little dissection or a careful snip with the scissors will often obviate the difficulty. If an air-pump is available, some bubbles are easily removed, but air bub- bles in cells may resist even the air-pump. An aspirator fastened to a water tap is very efficient in removing bubbles. Heating followed by rapid cooling is recommended by Pfeiffer and Wellheim for removing air, but, for cytological work, the remedy is worse than the bubbles. It is often asked whether fixing agents really preserve the actual structure of cell contents. It must be admitted that some things — notably the liquid albuminoids — are much modified in appearance, but the most competent observers are now inclined to believe that such delicate objects as chromosomes, centrosomes, the achromatic figure, and even the structure of protoplasm, can be studied with confidence from material which has been fixed, imbedded, and stained. Extensive investigations upon various objects in the hving condition have strengthened this confidence. It is certain that we have not yet found the ideal fixing agent for cell contents. Such an agent must not be a solvent of any of the cell contents, must penetrate rapidly, must preserve structures perfectly, and must harden so thoroughly that every detail shall remain un- changed during the subsequent processes of dehydrating, clearing, imbedding, sectioning, and staining. DEHYDRATING AGENTS Objects which are to be imbedded in paraffin or celloidin, and also all other objects which are to be mounted in balsam or Venetian tur- pentine, must be dehydrated, i.e., they must be freed from water. The slightest trace of water is ruinous. Alcohol is used almost exclusively for dehydrating. The process must be gradual. If material has been fixed in an aqueous solution, it must pass through a series of alcohols of increasing strength, beginning with about 3 per cent alcohol. Twen- REAGENTS 35 ty years ago, most botanists were beginning with 35 per cent alcohol; in the second edition of this book (1905) we recommended 15, 35, 50, 70, 85, 95, and 100 per cent as a safe series, since it causes no obvious plasmolysis of the cell contents. As investigations have become more and more critical, especially investigations upon the structure of chromatin, it has been found that even 15 per cent alcohol is too strong for a beginning. It is maintained that, in addition to the damage done by transferring from water to so strong an alcohol, the final dehydra- tion is not so perfect as it is when the series begins with a weaker alco- hol. Yamanouchi, whose work upon delicate algae has been particular- ly successful, uses the following series: 2|, 5, 7| ,10, 15, 20, 30, 40, 50, 70, 85, 95, and 100 per cent. After such gradual early stages, there seems to be no objection to the less gradual stages which follow. Of course, there is no particular virtue in the fractions: it is convenient to make a 10 per cent alcohol, then dilute it one-half for the 5 per cent, and dilute the 5 per cent one-half for the 2| per cent. The 7| per cent is made with sufficient accuracy by adding a little water to the 10 per cent alcohol. It is not safe to suggest minimum times for each grade of alcohol. One might as well recognize that in histological technique speed and excellence seldom go together. For the first six grades, three grades a day, morning, noon, and evening, seem to be safe: for 30, 40, 50 and 70, two grades, morning and evening; 85, for 24 hours, changing the alcohol 2 or 3 times, since this is the best place for hardening; 95, for 24 hours; for the absolute alcohol, 24 hours, with 2 or 3 changes, should complete the dehydration. If pieces are larger than |-inch cubes, the times should be longer. In all cases, the absolute alcohol should be changed 2 or 3 times. The grades below 85 per cent can be used repeatedly. The absolute alcohol should not be used again for this purpose, but may be put back into the 95 per cent bottle. It is always well to filter the alcohols when pouring back into the bottle. Otherwise, there would soon be an accu- mulation of starch grains, pollen grains, spores, and various other things. Waste alcohol as strong as 85 or 95 per cent will be useful for rinsing one's hands when dealing with Venetian turpentine. If it is necessary to be very economical, the stronger alcohols may be filtered into a single large bottle and the strength of the mixture can then be determined by using an alcoholometer. Knowing the strength of the mixture, one can easily make any weaker grade. 36 METHODS IN PLANT HISTOLOGY Be very sure that bottles or Stenders for absolute alcohol are perfect- ly dry; also, keep the bottles well corked and keep the lids on the Stenders. The importance of excluding moisture cannot be exagger- ated. Tightly fitting corks and closely fitting covers are better than absorbents; prevention is better than cure. The lower grades are made up from 95 per cent alcohol. Formulas for alcohols — The following formulas will enable anyone to make the other grades of alcohol from 95 per cent alcohol and water. 95 95 95 95 95 95 95 95 95 10 15 20 30 40 50 60 70 85 85 80 75 65 55 45 35 25 10 The foregoing are the formulas for various alcohols from 10 to 85 per cent. The fh'st column shows the formula for making 10 per cent al- cohol. The percentage of alcohol secured in each case is indicated by the middle number in each column. In the first formula, subtract 10 from 95; the result, 85, is the number of cubic centimeters of water which must be added to 10 c.c. of 95 per cent alcohol in order to obtain 10 per cent alcohol. The mixture contains 95 c.c. of 10 per cent alco- hol. If more or less than 95 c.c. of the mixture is needed, take propor- tional parts of 10 and 85. This simple method is a time-saver, but if the bottles or Stender dishes are to be filled frequently, it will be a still further saving of time to use a long label (Fig. 15) and, after pouring in the 95 per cent alcohol, draw a line showing how high it reaches; and then, after pouring in the water, draw another line. The next time it is necessary to fill the bottles merely pour in 95 per cent alcohol until it reaches the first line, and then pour in water until it reaches the second line. It is not necessary to use distilled water if pure drinking-water is available. Synthol is used like alcohol, and many believe it to be a good substitute. Acetone has also been used with more or less success for all grades except absolute alcohol. Some investigators use more or less complicated dif- fusion apparatus and make the dehydration process ex- tremely gradual. Judging from the finished preparation, we find no ad- vantage in the method. In the diffusion process, the solution is con- stantly changing. This may not be an advantage. Fig. 15.— Label for stain- ing-dish. REAGENTS 37 Some very minute objects, like bacteria and the smaller Cyano- phyceae, may be dehydrated by heating them until all water is drawn off, but, of course, this shows merely the form, with little or nothing of the internal structure. CLEARING AGENTS Clearing agents are so named because they render objects transpar- ent. When clearing agents are used to precede infiltration with paraf- fin, the clearing is merely incidental, the real purpose being to replace the dehydrating agent with a solvent of paraffin. The clearing is use- ful, even in this case, because it indicates when the replacing has be- come complete. When the clearing agent is used to precede infiltration with paraffin, the material should always be most thoroughly dehydrated with abso- lute alcohol before beginning with the clearing agent. When the clear- ing agent is used to clear sections or small objects just before mounting in balsam, absolutely perfect dehydration is not necessary with all clearing agents. Bergamot oil, carbolic acid, and Eycleshymer's clear- ing fluid (ecjual parts of bergamot oil, carboHc acid, and cedar oil) will clear readily from 95 per cent alcohol. Sections to be cleared in xylol or clove oil should be dehydrated in "absolute" alcohol. If the abso- lute alcohol is below 99 per cent, xylol will not clear perfectly; but clove oil clears readily from 99 per cent. If the absolute alcohol is not up to 99 per cent it is a good practice to go from the alcohol to clove oil; and then, from clove oil to xylol. Water may be removed by distilling or by putting a "drier" into the alcohol. Put some calcined copper sulphate into the bottle of "abso- lute" alcohol, shake, and allow to stand for 24 hours. Then pour off and add fresh copper sulphate, shake, and repeat the operation until the fresh copper sulphate no longer gets conspicuously blue when put into the alcohol. When the alcohol can be mixed with xylol without becom- ing milky, it may be called absolute alcohol. Some put a httle calcium sulphate into the "absolute" alcohol and keep it there, pouring off the alcohol very gently as it is needed. By any of these methods, 99 per cent alcohol can be brought up to usable absolute alcohol. Distilling, followed by a drier, will bring 95 per cent alcohol up to a usable absolute alcohol. 38 METHODS IN PLANT HISTOLOGY Xylol. — In our opinion, xylol is the best clearing agent to precede infiltration with paraffin. After the material has been dehydrated, it should be brought gradually into xylol. Thirty years ago it was cus- tomary to bring material directly from absolute alcohol into xylol; twenty years ago, two or three mixtures of absolute alcohol and xylol were used before reaching the pure xylol; at present, those who are do- ing the most critical work are making this process still more gradual. As cytologists have been studying more and more minute structures, the methods have become more and more critical. As in the case of the alcohol series, the xylol series has its grades closer together at the be- ginning than at the end. The following series seems to be sufficiently gradual: -jV, h I, 2; f; P^re xylol. It is hardly necessary to use a graduate in making up the series. For the |, use equal parts of xylol and absolute alcohol; for the |, use equal parts of the | and absolute alco- hol; for the I, use equal parts of the | and absolute, and for the iV> equal parts of the | and absolute. The f can be guessed at with suffi- cient accuracy. We prefer a closer series of xylols, using 2|, 5, 7|, 10, 15, 20, 30, 40, 50, 75, and 100 per cent. Infiltration with paraffin is more thorough with this closer series. We use it and recommend it. Three grades a day, morning, noon, and night, will do for filamentous algae and fungi, fern prothallia, onion root-tips, and similar objects. For larger pieces the times should be longer. For |-inch cubes, change morning and evening. For still larger pieces, 24 hours in each grade is not too long. In all cases, the pure xylol should be changed 2 or 3 times. While the pure xylol must not be used again for this purpose, it is still good for dissolving paraffin ribbons when staining on the slide. Xylol is the best agent for clearing sections just before mounting in balsam. Preparations cleared in xylol harden more rapidly, and this is such a decided advantage that even when sections have been cleared in cedar oil or clove oil it is worth while to give them a minute or two in xylol before mounting. Besides, clove oil is a solvent of many of the most frequently used stains and, consequently, preparations in such stains would fade, if transferred directly from clove oil to balsam. Xylol evaporates so rapidly that one must take care not to let sec- tions become dry before applying the balsam. Thin sections perfectly dehydrated seem to clear in a few seconds; but, even with very thin sec- tions, it is better to let the xylol act for at least a minute. Sections 20 n in thickness should remain in the xylol 5 minutes before mounting in bal- REAGENTS 39 sam. If there is much moisture in the air, or if the absohite alcohol is not above suspicion, clear sections in clove oil before transferring to xylol. Chloroform. — Some botanists use chloroform to precede the infiltra- tion with paraffin. In the later stages of infiltration it is more easily removed than xylol. It seems to possess no other advantages, and for clearing sections just before mounting in balsam it is inferior to xylol or clove oil. Its value in hardening celloidin and as a fixing agent en- titles it to a place in the histological laboratory. Cedar oil. — It is not always easy to get good cedar oil. If the stuff offered for sale looks like turpentine and smells like it, it is worthless for histological purposes. Good cedar oil has a shghtly amber tint, the color resembling a weak clove oil. It should have the pleasant odor of cedar wood. The very expensive cedar oil used with immersion lenses is not needed for clearing or for preceding infiltration with paraffin. It is claimed that material cleared in cedar oil does not become so brittle as that cleared in xylol or chloroform. Dr. E. J. Kraus has used cedar oil extensively in clearing large ob- jects— strawberries and gooseberries either whole or cut in two, sec- tions of apple 2 to 4 mm. thick, and similar objects. This method is proving valuable in vascular anatomy, some material showing the course of bundles very clearly in pieces so large as centimeter cubes. Xylol can be used in the same way, but is so volatile that specimens often dry up. Dr. Land suggests equal parts of xylol and carbon disul- phide for clearing large objects which are to be examined without sec- tioning. Clove oil. — This is an excellent agent for clearing sections and small objects just before mounting in balsam. It clears more readily than xylol. When the absolute alcohol has deteriorated so that xylol no longer clears the sections, clove oil may still clear with ease. While clove oil will clear from 95 per cent alcohol, it is better to use absolute. Since preparations cleared in clove oil harden slowly, it is a good plan to treat them with xylol before mounting in balsam. Gentian violet is somewhat soluble in clove oil, and this fact makes it possible to secure a beautiful differentiation, because the stain is extracted from some elements more rapidly than from others. The stain may be extracted completely from the chromosomes during the metaphase and still remain bright in the achromatic structures. After the desired differen- tiation has been attained, the preparation should be placed in xylol to remove the clove oil, since the continued action of the clove oil would 40 METHODS IN PLANT HISTOLOGY cause the preparation to fade. Do not use a Stender dish for clove oil, but keep it in a 50 c.c. bottle. Put on a few drops, and immediately drain them off in such a way as to remove the alcohol as completely as possible. Then flood the slide and pour the clove oil back into the bottle, repeating the process until the proper differentiation has been reached. Replace the clove oil with xylol and mount in balsam. With stains not soluble in clove oil, the xylol is not necessary, except to facil- itate the hardening of the preparation. Clove oil may be used in removing the celloidin matrix from celloidin sections. It is useless as an agent to precede infiltration with paraffin. Eycleshymer's clearing fluid. — This is a mixture of equal parts of bergamot oil, cedar oil, and carboHc acid. It clears readily from 95 per cent alcohol, and consequently is useftd in clearing celloidin sections when it is desirable to preserve the celloidin matrix. In sections stained with haematoxyhn, or haematoxylin and eosin, the stain may be removed completely from the matrix by the use of acid alcohol, and the matrix may be preserved by clearing from 95 per cent alcohol. It is not intended that the mixture should be used to precede infiltra- tion with paraffin. Other clearing agents. — Bergamot oil, carbohc acid, turpentine, benzine, gasohne, and other reagents have been tried for clearing, but none seem to be worth more than a warning mention. MISCELLANEOUS REAGENTS Canada balsam is used almost exclusively for mounting. Very thick balsam is disagreeable to handle and makes unsatisfactory mounts. Very thin balsam, in drying out, allows bubbles to run under the cover. Xylol is cheaper than balsam, and consequently the balsam on the market is likely to be too thin for immediate use. The stopper may be left out until the balsam acquires the proper consistency. Balsam must not be acid. If there is the sUghtest acid reaction, most stains will fade. Paraffin should be of at least two grades, a soft paraffin melting at 40° to 45° C, and a hard paraffin melting at 52° to 54° C. Grubler's paraffin and most imported paraffins melt at the temperature indicated on the wrappers. The melting-point indicated on the wrappers of paraffins sold by some American dealers does not enable one to make even a guess as to the real melting-point. Paraffin marked 70° C. may melt at 60° C, and other grades are hkely to melt before the tempera- REAGENTS 41 tiire indicated on the labels is reached. The fact that the price rises with the melting-point may explain the discrepancy. Test every grade with a thermometer. If it is desired to get a paraffin melting at 52° C. and your sample melts at 50° C, add a little paraffin with a melting- point above 52° C. ; if the sample melts at 55° C, add a httle with a melting-point below 52° C. Grubler's paraffins need no modification, but paraffins only slightly inferior can be improved by the addition of bayberry wax. A piece of the wax, not larger than a grain of com, to a pound of paraffin, is hke- ly to improve the infiltration and cutting. Paraffin may be used repeatedly. Keeping it in the Hquid condi- tion in the bath month after month has an advantage, since it be- comes more and more tenacious and homogeneous. Glycerin, glycerin jelly, Venetian turpentine, and gold size are de- scribed in the chapter on "The Glycerin Method" (chap. vii). Cel- loidin is described in the chapter on "The Celloidin Method" (chap, x), and cellulose acetate in the chapter on "The Cellulose Acetate Method" (chap. xi). The reagents already described are noted further in connection with specific applications. Reagents used in making mi- crochemical tests are described in the chapter on "Temporary Mounts and Microchemical Tests" (chap. v). A list of reagents will be found in chapter xxxi. Cleaning fluid. — No matter how nice they may look and no matter what dealers may claim, slides and covers, as you buy them, are never clean. This is a good cleaning fluid: Potassium dichromate 2 g. Water 10 c.c. Sulphuric acid 23 cc. Dissolve the potassium dichromate in water and add the sulphuric acid. For cleaning slides and covers, this solution may be diluted 25 or even 50 times with water. Leave slides and covers in the solution for 24 hours, rinse thorough- ly in water, and then put them into soapy water and leave them over- night or 24 hours. Rinse thoroughly and wipe dry. If the least trace of acid is left, many stains will fade. For developing trays and most kinds of laboratory glassware, the solution can be used, full strength, for a few minutes or an hour. The solution can be used repeatedly. CHAPTER III STAINS AND STAINING As edition after edition of this book has appeared, there has been a decrease rather than an increase in the number of stains in general use; but there has been a notable improvement in the use of some stains which have long been popular. For cytological work Haidenhain's iron-haematoxylin holds more firmly than ever its place at the head of the list, with Flemming's triple stain an easy second. For anatomical work, safranin still holds first place for the lignified elements of the vascular system, but the claim of Delafield's haema- toxylin to first place for cellulose tissues is no longer undisputed, for anilin blue is giving excellent results and Hght green seems to give more accurate views of the phloem than we were securing with any of the other stains. However, it must be admitted that preparations of coniferous woods, stained in safranin and Delafield's haematoxylin by Thomson and his students at the Toronto laboratory, have not been surpassed. The fact that excellent preparations can be made, almost without trial, by using combinations already perfected doubtless deters in- vestigators from experimenting with other stains. There is still abun- dant room for experimenting with various stains, especially in the use of mordants and in the effect of the same stain or combination after various fixing agents. It is to be regretted that botanists who need microtechnique have so little knowledge of chemistry, and that chem- ists have no interest in developing methods of staining. During the past few years, American stains have been developed until many equal and some even surpass the famous Griibler products; and, besides, the American stains are becoming standardized. The Commission on Standardization of Biological Stains, and especially its able president, Dr. H. J. Conn, cannot be too highly commended for the great improve- ment in American stains. The first standardized stain, methylene blue, was put on the market in the summer of 1923 and, before the end of the year, safranin was added. By the end of 1929, forty-three stains had been certified. The certification means that the stain has passed 42 STAINS AND STAINING 43 spectrophotometric tests, has been tested chemically, and has been tried in actual practice. The advantage to the one who uses stains is that, when he finds a certified stain which is satisfactory, he can al- ways get exactly the same stain again. The earlier stains, even the Griibler stains, were merely textile dyes, usually more or less modified. A student once asked Professor W. J. G. Land what was the difference between gentian violet and crystal violet, and received the reply, "Gentian violet is crystal violet plus mud." Textile dyes were often weakened or adulterated. Biological Stains, an excellent book by Dr. Conn, gives not only an account of the work of the commission but also an interesting history of stains and staining. Stains may be classified in various ways: e.g., there are three great groups of stains — the carmines, the haematoxylins, and the anilins. Stains may be classified as basic and acid, or they may be regarded as general and specific. A general stain affects all the elements, while a specific stain affects only certain elements, or stains some elements more deeply than others. Stains which show a vigorous affinity for the nucleus have been called "nuclear stains," and those which affect the cytoplasm more than the nucleus have been termed "plasma stains." Of course, such stains are specific. We shall consider some of the more important haematoxylins, car- mines, and anilins, reserving general directions and theoretical ques- tions for another chapter. The formulas are largely empirical. Some of those given here are taken from The Microtomist's Vade-Mecum (Lee), which is easily the most complete compendium of stains and other reagents concerned in microtechnique. Biological Stains, al- though not covering so much ground, is, in many respects, superior, and the formulas are for the standardized stains. Other formulas are from Botanical Microtechnique (Zimmermann) and from Stirling's Histology, and still others are from current litera- ture and from our own laboratory. The directions for using a stain apply to stains made up according to the formulas which are given here, and may need modification if other formulas are employed. It is hoped, however, that the directions will give the student sufficient insight into the rationale of staining to enable him to make any neces- sary modifications. Since American stains have come into general use, the need for rationale is even greater, especially if the American stains are made up according to standard formulas, which are based 44 METHODS IN PLANT HISTOLOGY largely upon the Griibler products. In general, it would seem that the American stains are purer and that they act more rapidly. The current practice in staining paraffin sections on the slide differs from the practice in staining freehand sections or small objects which are to be mounted whole. In case of paraffin sections, the cell contents are usually as important and often more important than the cell walls; consecjuently, extreme care must be given to every detail. With free- hand sections the cell contents often drop out, but even when they re- main, the cell walls are usually the important features; and so the process is considerably shortened. For staining freehand sections, it is customary to use solid watch glasses, unless the sections are very large. The details of the method are given in chapter vi, on "Freehand Sections." Fig. 16. — Arrangement of staining-dishes For staining sections on the slide, nothing is better than the ordi- nary Stender dish. The arrangement of Stender dishes shown in Figure 16 is very convenient. The advantage is obvious. With two dishes each of xylol, xylol-alcohol, absolute alcohol, and 95 per cent alcohol, one set can be used in passing down to the stain, and the other, which is thus kept free from any paraffin in solution, can be used in passing back to the balsam. Even for paraffin sections, some use only three alcohols, 50, 95, and 100 per cent, and the first two may be simply poured over the slide; in this case, only one Stender dish — for the 100 per cent alcohol — is necessary in the alcohol series, the other two alcohols being kept in bottles. This short method gained great popularity because it was used in Strasburger's laboratory at Bonn. It was the influence of this school and its great master which led to the adoption of the short schedule in the second edition of this book. A few years' trial showed the weakness of the method, and we re- turned to the longer schedule. The crudeness of the short schedule is doubtless responsible for the tenacity with which the Bonn school has clung to the theory of linin and chromomeres. The young investigator STAINS AND STAINING 45 should be warned that during the last twenty years of his life, Stras- burger, who had been a leader in technique, cut very few sections and did practically no staining, but used preparations made by assistants. Let us now consider a few of the most important stains. THE HAEMATOXYLINS The most important haematoxyhns are Heidenhain's iron-alum haematoxylin, Delafield's haematoxylin, Mayer's haem-alum, and Boehmer's haematoxyhn. All the haematoxylins mentioned contain alum, and, according to Mayer, who has written the most important work on haematoxylin stains,^ "the active agent in them is a compound of haematin with alumina. This salt is precipitated in the tissues, chiefly in the nuclei, by organic and inorganic salts there present (e.g., by the phosphates), and perhaps also by other organic bodies belonging to the tissues." These salts are fixed in the tissues by the Idlhng and fixing agent, and when the stain is applied a chemical comljination results. The first American haematoxylin was not satisfactory. It was dark and did not stain well. Manufacturers bleached it, but that made the staining worse. They then made a darker, but better, product. If crys- tals are dark colored, feel sticky when rubbed between the fingers, and go into solution quickly, the stain is not likely to be satisfactory. Crystals of a light yellowish sand color, dissolving slowly and requiring about six weeks to ripen, are much better. Haematoxyhns stain well after any of the fixing agents described in the preceding chapter, but they are most effective when used after members of the chromic-acid series. Heidenhain's iron-alum haematoxylin. — This stain, introduced by Heidcnhain in 1892, immediately gained great popularity and now, after more than 40 years' constant use, still maintains first place in cytological investigations. Two solutions are used, and they are never mixed : A. Four per cent aqueous solution of ammonia sulphate of iron. B. One-half per cent aqueous solution of haematoxylin. In making solution A, use the violet ferric crystals, not the ferrous. The first solution acts as a mordant, i.e., it does not stain, but pre- 1 "Ueber das Farben mit Hamatoxylin," Miltheilungen aus der Zoologischen Station zu Neapel, 10:170-186, 1891, and "Ueber Hamatoxylin, Carmin und ver- wandte Materien," Zeitschriftfiir wissenschaftliche Mikroscopie, 16:196-220, 1899. 46 METHODS IN PLANT HISTOLOGY pares the tissue for the action of the second solution. It is better to make a 4 per cent solution and dilute it when a 2 per cent or weaker solution is needed. The bottles and also the Stender dishes containing the ammonia sulphate of iron nearly always become coated with a yellowish film of iron oxide. This film also forms on sections or on material to be mounted whole, but is so thin that it would be over- looked unless one compared the sections with others without any film, just as ordinary glass looks all right unless you compare it with fine cut glass. This film forms only when the temperature of the fluid rises above 18° C. So, keep the solution below 18° C. while using it. Solution A is at its best as soon as the crystals are completely dis- solved and it remains in practically perfect condition for about two months, after which it gradually deteriorates. The haematoxylin crystals for solution B should be dissolved in distilled water. This will require about 10 days, during which time the bottle should be shaken often and vigorously. The solution must then be allowed to ripen for a month before it is ready for use. During the ripening, which is an oxidation process, a cotton plug should be used instead of a cork, to facilitate the oxidation ; but as soon as the stain is ripe, a cork — preferably a closefitting glass stopper — will prolong the maximum efficiency. If kept in a cool place, away from strong light and kept quiet, the stain may retain its efficiency for six months. When needed, pour out very gently. The same solution, on a table in the laboratory, poured out as one pours other stains, would lose its efficiency in less than a month. As soon as the rich wine color begins to disappear, the solution is worthless. Some prefer to dissolve the haematoxylin crystals in alcohol — about 10 g. in 100 c.c. of absolute alcohol. This solution should stand until it has a deep wine-red color. This will require 4 or 5 months, and a year is not too long. From this stock solution, make up small quantities as needed. About 4 or 5 c.c. of this stock solution in 100 c.c. of water gives a practically aqueous solution, and it is already ripe. The general method is as follows: treat with A, stain in B, and then return to A to reduce and differentiate the stain. Never transfer directly from A to B, or from B to A; always wash in water before passing from one of the solutions to the other. While all follow the general method just indicated, no two investi- gators would prepare exactly the same schedule, even for staining the same object, e.g., root-tips; neither investigator would use the same STAINS AND STAINING 47 schedule for a root-tip and an embryo sac ; an alga might require differ- ent treatment, and all the preceding variations might fail miserably with the pollen tubes of cycads. This stain is so important that every worker must learn it, and the only way to learn it is to become ac- quainted with the general outline of the process and then adapt every step to the case in hand. For the sake of illustration, I asked a prominent cytologist, Dr. S. Yamanouchi, who has been notably successful in staining mitotic fig- ures, to write a schedule indicating his methods of using this stain. While he protested that the practice could not be written down, he kindly prepared the following schedule, not for the instruction of his colleagues, but to introduce the method to beginners. The schedule is for paraffin sections. Throughout the schedule, I have interpolated comments and suggestions. Yamanouchi's schedule. — 1. Xylol, 5 minutes, to dissolve the paraffin. Do not heat the slides to melt the paraffin. However, a gentle warming which does not approach the melting-point of the paraffin does no damage and makes the paraffin dissolve more readily. The xylol soon has consider- able paraffin in solution, but 100 c.c. of xylol should remove the paraffin from at least 100 slides with ribbons 25 mm. long and 10 fx thick. If the ribbons are only 5 /j. tliick, 200 slides can be treated. 2. Xylol and absolute alcohol, equal parts, 5 minutes. 3. Absolute alcohol, 5-7 minutes. 4. Ninety-five, 85, 70, 50, and 35 per cent alcohol, 5 minutes each. If material has been fixed in a reagent containing osmic acid, it should be bleached. For this purpose, 10-15 c.c. of hydrogen peroxide may be added to 100 c.c. of the 50 per cent alcohol, where the sUdes should re- main until the blackening disappears. 5. Water, 10-20 minutes. If any alcohol is left in the sections, the staining will not be brilliant. Change the water several times. 6. Iron-alum. Use the 4 per cent solution. For many objects, like the archegonia of gymnosperms and the embryo sacs of angiosperms, 1 hour is usually enough. For cliromosomes in root-tips and anthers, 2 hours may be long enough; but for algae, 2 hours is generally a minimum. 7. Wash in water, 5 minutes. The water should be changed several times. If the washing is not thorough, the differentiation will not be sharp. 48 METHODS IN PLANT HISTOLOGY 8. Haematoxylin. Many objects, like the archegonia of gymnosperms and the embryo sacs of angiosperins, will stain sufficiently in 5 or 6 hours; most algae re- quire at least 20 hours. 9. Wash in water, 5 minutes, changing as often as the water shows any color. 10. Iron-alum, 2 per cent solution. No time can be indicated here. The preparation must be watched under the microscope. After some experience, one can form some judg- ment from the color tone, as the slide stands in the Stender dish of iron- alum, but the finishing must always be done under the microscope. In general, if it requires more than 2 hours to secure good differentiation, use a stronger iron-alum; if the differentiation is reached in less than | hour, use a weaker iron-alum. A 3 /x section of a root-tip from material fixed in chromo-acetic-osmic acid, with 8 c.c. of 1 per cent osmic acid to 100 c.c. of the stock solution, should be perfectly differentiated in 2 hours. If the stain comes out too rapidly, use 2 per cent iron-alum for an hour and finish in a 1 per cent solution. The less the proportion of osmic acid, the faster will the stain be extracted. If the stain is coming out rather slowly, as it should, one can handle from 6 to 10 slides at one time. Put the slides on a 5X7 glass plate and put the plate on the stage of the micro- scope. The iron-alum can be added or removed with a pipette. As slide after slide reaches the proper differentiation, it is placed in water. 11. Water, 30 minutes. The water should be changed several times. If this washing is not thorough, the preparation wdll fade, on account of the continued action of the iron-alum. If an aqueous counter-stain is used, apply it at this point. 12. Thirty-five, 50, 70, 85, 95, and 100 per cent alcohol, 5 minutes in each. If an alcoholic counter-stain is used, apply it near the alcohol of the same strength as the stain. 13. Absolute alcohol and xylol, equal parts, 5 minutes. 14. Xylol, 2-5 minutes. 15. Balsam. While this schedule should enable the student to apply the method not only to sections but to objects to be mounted whole, like filamen- tous algae and fern prothallia, an additional schedule for such things is given in chapter viii on "The Venetian Turpentine Method." The times given above must not be accepted as final. Many prefer to wash 3 or 4 times as long after the first immersion in iron-alum. Some think that 4 hours is enough for the entire process. In staining: STAINS AND STAINING 49 with iron-alum haematoxylin for protoplasmic connections, 24 hours in 4 per cent iron-alum, with 1 or 2 minutes washing in water, 2 days in haematoxylin, 5 minutes washing in water, and 1 minute in 1 per cent iron-alum may be a good schedule to start with. The times must be determined for each case. Many put the slide into iron-alum in the morning and finish the process in the afternoon. These short schedules are not likely to prove satisfactory with mitotic figures. A plan which has proved convenient and very successful is to put the slide into the iron-alum in the morning, wash in water for an hour at some con- venient time in the afternoon, leave it in the | per cent haematoxylin overnight, and finish the preparation the next morning. It is a long process, requiring care, patience, and judgment, but it is worth the effort. Chromosomes, centrosomes, and pyrenoids take a brilliant black; or, if the second treatment with iron-alum be more prolonged, a blue black or purple. Achromatic structures stain purple, but the stain can be extracted while it is still bright in the chromosomes. Lignified, suberized, and cutinized structures stain lightly or not at all. Cellu- lose does not stain so deeply as with Delafield's haematoxylin. Arche- sporial cells and early stages in sporogenous tissue stain gray. Many details which are not so brilliantly colored often show good defini- tion. If a counter-stain is desired, anything which gives a serviceable contrast may be used. In any case, the haematoxylin stain must be complete and the washing thorough before the second stain is applied. An aqueous stain should be applied just after the final washing in water; an alcoholic stain should be applied during the process of pass- ing the slides through the alcohols, staining in a solution of safranin in 50 per cent alcohol from the alcohol of a concentration nearest that of the stain; and staining after the final absolute alcohol, if the stain is dissolved in clove oil. A stain of 3 or 4 minutes in safranin adds an excellent differentiation in case of many algae and does not obscure nuclear details. The exine of pollen grains may take a brilliant red with safranin in 5 or 10 min- utes, contrasting sharply with the mouse gray of the intine. Orange G, in clove oil, often gives a pleasing contrast. Delafield's haematoxylin. — "To 100 c.c. of a saturated solution of ammonia alum add, drop by drop, a solution of 1 g. of haematoxylin dissolved in 6 c.c. of absolute alcohol. Expose to air and light for one 50 METHODS IN PLANT HISTOLOGY week. Filter. Add 25 c.c. of glycerin and 25 c.c. of methyl alcohol. Al- low to stand until the color is sufficiently dark. Filter, and keep in a tightly stoppered bottle" (Stirling and Lee). The addition of the glyc- erin and methyl alcohol will precipitate some of the ammonia alum in the form of small crystals. The last filtering should take place 4 or 5 hours after the addition of the glycerin and methyl alcohol. The solution should stand for at least 2 months before it is ready for using. This "ripening" is brought about by the oxidation of haema- toxylin into haematin, a reaction which may be secured in a few min- utes by a judicious application of peroxide of hydrogen. However, we prefer to let the haematoxylin ripen naturally. There is no objection to making this stain in considerable quantity, since it does not deterio- rate. We have used Delafield's haematoxylin which had been in a cork- stoppered bottle for 20 years, and it still gave the rich characteristic stain. Transfer to the stain from 50 or 35 per cent alcohol or from water. The length of time required is exceedingly variable. Sometimes sec- tions will stain deeply in 3 minutes, but it is often necessary to stain for 30 minutes or even longer. This stain may be diluted with several times its own volume of water; when this is done, the time required is correspondingly long, but the staining is frequently more precise. The length of tune required will be fairly uniform for all material taken from the same bottle. This fact indicates that the washing process, which follows killing and fixing, is an important factor; if the washing has been thorough, the material will stain readily; but if the washing has been insufficient, the material may stain slowly or not at all. The washing is particularly important when the fixing agent contains an acid. Transfer from the stain to tap water. Distilled water is neither necessary nor desirable. Some writers recommend washing for 24 hours, but this is entirely unnecessary; for paraffin sections on the sHde, 5 or 10 minutes is long enough, and even for rather thick free- hand sections 20 or 30 minutes is sufficient. Use plenty of water and keep changing it as often as it becomes in the least discolored. Precipi- tates are often formed when shdes are transferred directly to alcohol from this stain, and sometimes even after washing in water. A few gentle dips in acid alcohol (2 drops of HCl to 100 c.c. of 70 per cent alcohol) will usually remove the precipitates. This extracts the stain more rapidly from other parts than from the nuclei, and hence gives a STAINS AND STAINING 51 good nuclear stain, while at the same time it removes any disfiguring precipitates. Some prefer to stain for a very short time and use no acid alcohol, but, as a rule, it is better to overstain and then diHerentiate in this way, because sharper contrasts are obtained. Transfer from acid alcohol to 70 per cent alcohol and leave here until a rich purple color replaces the red due to the acid. Since small quantities of the acid alcohol are carried over into the 70 per cent alcohol, it is well to add a drop of ammonia now and then to neutralize the effect of the acid. Too much ammonia is to be avoided, for it gives a disagreeable bluish color with poor differentiation, probably on account of the precipitation of alumina. The preparation is now dehydrated in 95 per cent and then in absolute alcohol, cleared in xylol or clove oil, and mounted in bal- sam. The following is a general schedule for staining paraffin sections on the shde in Delafields' haematoxylin : 1. Stain (from water or from 35 or 50 per cent alcohol) 10 minutes 2. Rinse in water 10 minutes 3. Thirty-five and 50 per cent alcohol 3 minutes each 4. Acid alcohol 5 seconds 5. Seventy per cent alcohol 3 minutes 6. Eighty-five per cent alcohol 3 minutes 7. Ninety-five per cent and 100 per cent alcohol . . 3 minutes each 8. Xylol and 100 per cent alcohol, equal parts 3 minutes 9. Xylol 3 minutes 10. Mount in balsam. If, after rinsing in water, the stain is evidently too weak, put the slide or section back into the stain until it appears overstained. Place the slide in acid alcohol. If an acid alcohol with 2 drops of HCl to 100 c.c. of 70 per cent alcohol reduces the stain too much in 10 or 15 seconds, use less acid or stain longer. Transfer to 70 per cent alcohol without any acid. As soon as the color changes from red to purple, examine under the microscope. If it is still overstained, return to the acid alcohol ; if the stain is too weak, return to the haematoxylin and try it again. After the haematoxylin is just right, apply a contrast stain, if you wish to double stain. Before transferring to the xylol wipe the alcohol from the back of the slide, or at least rest the corner of the slide upon blotting-paper for 2 or 3 seconds, in order that you may not carry over so much alcohol into the xylol. Add a drop of 52 METHODS IN PLANT HISTOLOGY balsam and a cover. Since the xylol is very volatile, this last step must be taken quickly. If blackish spots appear they are usually caused by the drying of sections before the balsam and cover are added; if there are whitish spots or an emulsion-like appearance, the clearing is not thorough; this may be caused by poor xylol (or other clearing agent); by absolute alcohol which is considerably weaker than its name implies (the absolute alcohol must test at least as high as 99 per cent, and ought to test as high as 99.5 per cent, if xylol is to be used for clearing) ; or by passing too quickly through the absolute alcohol and xylol, or even by moisture on the cover glass. The last danger is easily avoided by passing the cover quickly through a Bunsen or alcohol flame before laying it on the balsam. Delafield's haematoxylin is the most generally useful stain in the haematoxylin group. It brings out cellulose walls very sharply, and consequently is a good stain for embryos and the fundamental tissue system in general. With safranin it forms a good combination for the vascular system, the safranin giving the lignified elements a bright red color, while the haematoxylin stains the cellulose a rich purple. It is a good stain for chromatin, and the achromatic structures show up fairly well, but can be brought out much better by special methods. Arche- sporial cells and sporogenous tissue are very well defined if proper care be taken. Lignified and suberized walls and also starch and chromato- phores stain lightly or not at all. Whenever you are in doubt as to the selection of a stain for general purposes, we should advise the use of Delafield's haematoxylin. Mayer's haem-alum.— Haematoxylin, 1 g., dissolved with gentle heat in 50 c.c. of 95 per cent alcohol and added to a solution of 50 g. of alum in a hter of distilled water. Allow the mixture to cool and settle; filter; add a crystal of thymol to preserve from mold (Lee). It is ready for use as soon as made up. Unless attacked by mold, it keeps indefinitely. Transfer to the stain from water. It is seldom necessary to stain for more than 10 minutes, and 4 or 5 minutes is generally long enough. As a rule, better results are secured by diluting the stain (about 1 c.c.-lO c.c. of distilled water) and allowing it to act for 10 hours or overnight. This is a good stain for the nuclei of filamentous algae and fungi, since it has little or no effect upon cell walls or plastids. Wash thor- oughly in water, transfer to 10 per cent glycerin, and follow the Vene- tian turpentine method, as described in chapter viii. STAINS AND STAINING 53 Erlich's haematoxylin. — Distilled water 50 c.c. Absolute alcohol 50 c.c. Glycerin 50 c.c. Glacial acetic acid 5 c.c. Haematoxylin 1 g. Alum in excess. Keep it in a dark place until the color becomes a deep red. If well stoppered, it will keep indefinitely. Transfer to the stain from 50 per cent or 35 per cent alcohol. Stain from 5 to 30 minutes. Since there is no danger from precipitates and the solution does not overstain, it is not necessary to treat with water or with acid alcohol, but the slide may be transferred from the stain to 70 per cent alcohol. Eosin, eryth- rosin, or orange G are good contrast stains. Jeffrey uses safranin and ErHch's haematoxylin for woody tissues. Boehmer's haematoxylin. — j Haematoxylin 1 g. \ Absolute alcohol 12 c.c. f Alum 1 g. \ Distilled water 240 c.c. The solution A must ripen for two months. When wanted for use, add about 10 drops of A to 10 c.c. of B. Stain from 10 to 20 minutes. Wash in water and proceed as usual. Cellulose walls take a deep violet. The closing membrane (torus) of the bordered pits of conifers will usually stain deeply in about 15 min- utes. Lignified, suberized, and cutinized structures stain slightly or not at all. When they do stain, the color is not violet, but a light yellow or brown. THE CARMINES Botanists have never given the carmines a fair trial, doubtless be- cause the stains were not considered worth it; but the splendid prepa- rations by Professor Powers of various members of the Volvocaceae, and by Belling in staining pollen mother-cells whole, prove that we should pay more attention to this group. Only a few of the multitudi- nous formulas will be considered. The carmine solutions keep for several years, some of them even improving with age, if distilled water has been used in the formulas 54 METHODS IN PLANT HISTOLOGY and the stains have been kept in tightly stoppered bottles. If the solu- tion becomes turbid, it should be filtered. Greenacher's borax carmine. — Carmine 3 g. Borax 4 g. Distilled water 100 c.c. Dissolve the borax in water and add the carmine, which is quickly dissolved with the aid of gentle heat. Add 100 c.c. of 70 per cent alco- hol and filter (Stirling). The following is a slightly different method for making this stain from the ingredients mentioned above: Dissolve the borax in water, add the carmine, and heat gently for 10 minutes; after the solution cools, add the alcohol and filter; let the solution stand for 2 or 3 weeks, then decant and filter again. Stain the material in bulk from 50 per cent alcohol 1-3 days, then treat with acid alcohol (50 c.c. of 70 per cent alcohol-t-2 drops of hy- drochloric acid) until the color becomes a clear red; this may require only a few hours, but may take 2 or 3 days. The material may then be passed through the rest of the alcohols (6-24 hours each), cleared, im- bedded, and cut. After the sections are fastened to the slide, the paraf- fin should be dissolved off with xylol. The balsam and cover may be added immediately, or the xylol may be rinsed off with alcohol and a contrast stain may be added. Alum carmine. — A 4 per cent aqueous solution of ammonia alum is boiled 20 minutes with 1 per cent of powdered carmine. Filter after it cools (Lee). Stain from 12 to 24 hours and wash in water. No acid alcohol is needed, since the solution does not overstain. Carmalum (alum lake).— Use 1 g. of the powdered stain to 100 c.c. of very dilute ammonia water. Filter, if there is any precipitate. Mayer's carmalum. — Carminic acid 1 g. Alum 10 g. Distilled water 200 c.c. Dissolve with heat; decant or filter and add a crystal of thymol to avoid mold. With material of Volvocales fixed in weak aqueous potassium iodide. STAINS AND STAINING 55 SO weak that the solution has a hght brown color, or fixed in weak os- mic acid, only 4 or 5 drops of 1 per cent osmic acid to 50 c.c. of distilled water, this carmalum is good for material to be mounted whole. Di- lute the stain considerably, put in a crystal of thymol to prevent mold, and allow the stain to act for weeks, or even a couple of months. It is not likely to overstain. Alum cochineal. — Powdered cochineal 50 g. Alum 5 g. Distilled water 500 c.c. Dissolve the alum in water, add the cochineal, and boil; evaporate down to two-thirds of the original volume, and filter. Add a few drops of carbolic acid to prevent mold (Stirling). Stain as with alum carmine. It used to be a common practice to stain in bulk in alum cochineal and counter-stain on the shde with Bismarck brown. Balling's iron aceto-carmine 1. — For counting chromosomes in pollen mother-cells mounted whole, Belhng used a modified aceto-carmine method. The preparations are good for an immediate count, but do not last longer than a few days or a week. "Ordinary aceto-carmine is prepared by heating a 45 per cent solu- tion of glacial acetic acid to boihng with excess of powdered carmine, cooling, and filtering. The young anthers are teased out with steel blades or needles in a drop of this until it changes slightly toward blu- ish red. An excess of iron spoils the preparation." Balling's iron acato-carmine 2. — "To a quantity of aceto-carmine a trace of solution of ferric hydrate dissolved in 45 per cent acetic acid is added until the hquid becomes bluish red, but no visible precipitate forms. An equal amount of ordinary aceto-carmine is then added. The anthers are teased out with nickled instruments." A cover-glass is then added and sealed with vaseline. The preparation lasts only a few days, but is much superior to any obtained by the usual intra vitam processes. The method is not as easy as it might seem to be. Much time will be saved by reading the detailed account given in Belling's book, The Use of the Microscope. McClintock's iron acato-carmina. — Dr. Barbara McClintock's modification of the Belling method makes the preparations permanent. 56 METHODS IN PLANT HISTOLOGY 1. Fix anthers in 1 part glacial acetic acid and 3 parts absolute alcohol from 12 to 24 hours. 2. Squeeze contents of an anther into a drop of Belling's iron aceto-carmine. Remove all structures except the pollen mother-cells, which must come into contact with both cover and slide. Otherwise they will be washed off. Place a cover glass over the drop. 3. Heat over an alcohol flame for a second, repeating 4 or 5 times. The drop must not boil. 4. Place the slide in a Petri dish filled with 10 per cent acetic acid until the cover rises from the slide. Some of the pollen mother-cells will stick to the slide and some to the cover. 5. Place both slide and cover in equal parts of absolute alcohol and acetic acid. 6. Pass through 1 part acetic acid to 3 parts absolute alcohol, 1 part acetic to 9 parts absolute alcohol, absolute alcohol, equal parts absolute alcohol and xylol, pure xylol, a few minutes in each. 7. Mount in balsam. Dr. McClintock used certified carmine (NCa2). THE ANILINS Many of the most brilliant and beautiful stains yet discovered be- long to this group. These stains are very numerous, but not so numer- ous as their names; for different names have been given to the same stain, and the same name has been given to different stains. For- tunately, the Committee on Standardization of Biological Stains is doing a good work in standardizing the nomenclature as well as the stains themselves. A valuable list of synonyms, with the preferred designations, was published in Science, 57:743-746, 1923, and other references to the work of the commission are given in the Bibliography on page 341, and brought up to 1929 in Dr. Conn's book, Biological Stains. General formula. — Make a 10 per cent solution of anilin oil in 95 per cent alcohol, shaking frequently until the anilin oil is dissolved; then add enough water to make the whole mixture about 20 per cent alcohol; then add 1 g. of cyanin, erythrosin, safranin, gentian violet, etc., to 100 c.c. of the solution. Solutions containing anilin oil do not keep so well as aqueous or alcoholic solutions. Personally, we hardly ever use solutions containing anilin oil. The anilins keep well in balsam, but not so well in glycerin. Xylol is a good clearing agent for all of them; but clearing in clove oil im- STAINS AND STAINING 57 proves stains like gentian violet, which are more or less soluble in clove oil. Even in such cases, xylol should follow the clove oil, or the prepa- ration will fade. While the anilins are not as permanent as the haematoxyhns, most of them keep fairly well if the staining has been carefully done. Prepa- rations fade if exposed long to bright sunhght. Keep the slides in the box when not in use, and even when in use, do not leave them on the laboratory table, exposed to the sun. We have preparations, made more than 30 years ago, in which the safranin and gentian violet are still bright; and others made more than 15 years ago, in which Mag- dala red and anilin blue have not faded. Some of the anilins are acid, some basic, and some are neutral. The rapidity with which sections must be transferred from one fluid to another makes many of them more difficult to manage than the haematoxylins or the carmines, but the stains are so valuable that even the beginner should spend most of his time with the anilins. Many anilins stain quite deeply in from 1 to 20 minutes, but if the stain washes out during the dehydrating process, stain longer, even 10-24 hours if necessary. Often the brilliancy of the stain can be in- creased by leaving the slide for 5 minutes in a 1 per cent solution of permanganate of potassium before staining. The permanganate acts as a mordant. The following are the more important anilins now in use by botan- ists. The directions apply to solutions made up according to the for- mulas given with the different stains. Safranin. — For the botanist, safranin is the most useful of all the anihn stains, and safranin 0 is practically the only safranin he needs. This stain, although certified, still has a certain measure of variability, but is comparatively uniform. In the fourth edition of this book we advised making the stain by mixing equal parts of an alcohol-soluble and a water-soluble safranin. We thought we generally got a better stain and probably we did, sometimes, because the stains were not uniform, and, by this method, we had two chances, instead of only one, for getting a good stain. The certified safranin 0 is equally soluble in water or alcohol. A 1 per cent solution in 50 per cent alcohol, or in water is best for general work and can be diluted when desirable. Flemming, who developed the famous triple stain — safranin, gentian violet, orange — dissolved 0.5 g. of alcoholic safranin in 50 c.c. of abso- lute alcohol and, after 4 days, added 10 c.c. of distilled water. 58 METHODS IN PLANT HISTOLOGY The first American safranins were very unsatisfactory; but there have been great improvements, and one company, the National AniUn and Chemical Company, has produced a safranin with 90 per cent dye content, much stronger than any of the European stains. This is the safranin which has been certified by the Committee on Standardiza- tion of Stains. Other companies are also improving. Coleman and Bell's Safranin Y, for baciUi, is good for staining xylem. All safranins keep indefinitely, solutions 20 years old staining as well, or better, than when fresh. An anilin safranin may be made according to the general formula. The transfer to the stain depends upon the formula. If the stain is aqueous, transfer to the stain from water; if made up in 50 per cent alcohol, transfer to the stain from 50 per cent alcohol. Sections of woody tissues, cut from living material, should be put into 95 per cent alcohol for about 1 hour and then transferred to the alcoholic stain. If cut from formalin material, sections should be left in water for | hour, changing the water several times; then in 15, 35, and 50 per cent alcohol, about 5 minutes in each grade; and then be stained in alcoholic safranin. If cut from formalin alcohol acetic-acid material, the sections should be placed in 50 per cent alcohol for at least 10 minutes, changing once or twice before being placed in the alcoholic stain. These are minimum times: longer times are just as good and may be better. The time required for staining varies with the tissue, the fixing agent, and the quality of the stain. In general, it may be said that 2 hours is a minimum and 24 hours a maximum. If the staining be too prolonged, delicate structures, like starch grains, crystals, and various cell constituents, may wash out. The mere fact that the whole section does not wash off does not mean that everything is fastened to the shde. On the other hand, with a short period, it is difficult to get a sharp differentiation. In staining a vascular bundle, one should be able to wash the safranin from the cellulose walls and still leave a brilliant red in lignified structures. For paraffin sections, 3-6 hours will usually be sufficient. It is a good practice to put the shdes into the stain in the morning and finish the mounts any time in the after- noon. For freehand sections of woody tissues, 24 hours is not too long, and in a 50 per cent alcoholic safranin, the sections may be stained for a week. From the stain, transfer to 50 per cent alcohol. If the sections are STAINS AND STAINING 59 deeply stained, and sufficient differentiation is not secured within 5 or 10 minutes, a drop of hydrochloric acid added to 50 c.c. of the alcohol will hasten the extraction of the stain. If staining vascular tissue, draw the stain from the cellulose walls, but stop before the lignified walls begin to fade. If a contrast stain is to be added, like light green, which weakens the safranin; or anihn blue or Delafield's haematoxylin, which need to be followed by an acid; the safranin should be strong enough to allow the necessary reduction. If staining mitotic figures, draw the stain from the spindle, but stop before the chromosomes be- gin to weaken. When the desired differentiation has been reached, wash out the acid in 50 per cent alcohol, if acid has been used. About 5 minutes should be sufficient for the washing. If safranin is to be used alone, pass through 50, 70, 85, 95, and 100 per cent alcohol, through the xylol-alcohol, then through xylol to bal- sam. If clove oil is used, omit the xylol-alcohol, but follow the clove oil with xylol to hasten the hardening of the preparation. If a second stain is to be added, transfer from the 50 per cent alcohol to any alcoholic stain. If the second stain is an aqueous stain, rinse the slide or sections for a minute in water before applying the stain. Safranin is the most generally useful of all the red stains, and, for- tunately, it is quite permanent. Lignified, suberized, cutinized, and chitinized structures stain red, as do also the chromosomes, nucleoli, and centrosomes. Directions for using safranin in combination with other stains will be given in connection with various objects. Acid fuchsin. — Use a 1 per cent solution in water or in 70 per cent alcohol. The solution in alcohol is preferable if sections are to be mounted in balsam. This stain often acts with great rapidity, 2 or 3 minutes being sufficient. The method for using acid fuchsin with woody tissues is given in the chapter on "Freehand Sections" (chap, vi). In staining embryo sacs, pollen grains, and such structures, longer periods are better. Stain 1 or 2 hours, and then differentiate in a sat- urated solution of picric acid in 70 per cent alcohol. This may require 30 seconds, or even several minutes. Rinse in 70 per cent alcohol until a bright red replaces the yellowish color due to the acid, and then pro- ceed as usual. Basic fuchsin. — This stain has become valuable to botanists through the researches of Gourley, who used it to stain the vascular 60 METHODS IN PLANT HISTOLOGY system of living plants. It does not diffuse during dehydration or clearing. Detailed directions are given on page 151. Dissolve 0.5 g. basic fuchsin in 20 c.c. of 95 per cent alcohol and dilute with 1,000 c.c. of tap water. A smaller quantity can be made by dissolving 50 mg. in 2 c.c. of 95 per cent alcohol and diluting with 100 c.c. of tap water. Congo red. — This is an acid stain resembling acid fuchsin. For cy- tological work use a | per cent aqueous solution; for anatomical work use a saturated solution. It is a good stain to use after malachite green or anilin blue. Transfer to the Congo red from water, stain 15 minutes, wash in water, transfer — for wood sections — to 85 per cent alcohol, and wash until the green or blue color of the previous stain begins to show through the red. Then treat quickly with absolute al- cohol, clear in xylol, and mount in balsam. Eosin. — This has long been a favorite stain, but for most purposes it has been replaced by similar stains giving better differentiation. The dry stain is made in two forms, one for aqueous and the other for alcoholic solution. Each should be used with its intended solvent. Make a 1 per cent solution in alcohol or water. It is worth mentioning that the aqueous solution is an excellent red ink. For material to be mounted whole in glycerin, glycerin jelly, or Venetian turpentine, stain overnight or, better, 24 hours; pour off the stain, which may be used repeatedly; treat, without washing in water, with a 2 per cent aqueous solution of acetic acid for 5 or 10 minutes, changing 2 or 3 times; transfer to 10 per cent glycerin without washing in water, since the stain will be brighter if the whole solution is slightly acid. When the glycerin becomes thick, mount in glycerin jelly. If the Venetian turpentine method is to be used, wash the glycerin out in alcohol shghtly acidulated with acetic acid (a couple of drops of acetic acid to 50 c.c. of alcohol), and do not drain off the last alcohol too com- pletely before transferring to the 10 per cent Venetian turpentine. According to Lee, the glycerin should be slightly alkaline. The alka- linity can be brought about by adding half a gram of common salt to 100 c.c. of the 10 per cent glycerin. We have found that eosin keeps better when the media are slightly acid. For staining paraffin sections, the alcoholic solution is better and the time may not be more than a few minutes, especially if the eosin is being used as a contrast stain. We have found the Eosin Y, of Coleman and Bell, very satisfac- STAINS AND STAINING 61 tory, especially for fungi to be mounted whole. With the rapid im- provement in the manufacture of stains, it is very probable that other dealers will have equally good products. Investigators will save time and money by keeping track of the findings of the commission on Standardization of Biological Stains. Haematoxylin and eosin and methyl blue and eosin are good com- binations. The eosin should follow the other stain. Erythrosin. — This is really an eosin, but there is some difference in the method of manufacturing. It is more precise and a more transpa- rent stain than eosin and is to be preferred for nearly all staining of paraffin sections. IMake a 1 per cent solution in distilled water or in 70 per cent alcohol. It gives good results when made up according to the general formula. Erythrosin stains rapidly, from 30 seconds to 3 minutes being suffi- cient. When used in combination with other stains, erythrosin should come last. Magdala red.— The name, Magdala red, is too indefinite to mean anything. The Magdala red echt (genuine) , of Griibler, is worth nothing as botanical stain. Sometimes a bottle labeled simply Magdala red gave fine results. It would seem that the occasional stains which suc- ceeded are practically the same as the American stain, phloxine; at least, a stain called Phloxine B (color index number 778), made by the National Anilin and Chemical Company, behaves like the best "Mag- dala red" and seems to give uniform results. Phloxine B.— Probably the occasional lots of Magdala red which proved to be so satisfactory in the Magdala red and anilin blue com- bination were phloxine. Phloxine 1 g- Ninety per cent alcohol 100 c.c. This stain is particularly valuable for staining algae which are to be mounted whole. In this case it should be followed by anilin blue. Full directions are given in chapter viii. For staining sections to be mounted in balsam, dilute the phloxine one half with water. Stain for 24 hours, dehydrate in 95 and 100 per cent alcohol, clear in clove oil, transfer to xylol, and mount in balsam. Phloxine stains lignified, suberized, and cutinized structures, and also chromosomes, centrosomes, nucleoli, and pyrenoids. It is likely to overstain, but the differentiation is easily secured by placing the 62 METHODS IN PLANT HISTOLOGY finished mounts upon a white background in the direct sunHght. When the desired differentiation has been reached, it is better to avoid direct sunHght, although the mounts do not seem to fade in the or- dinary hght of a room. Except for special purposes, it is better to use this stain in combina- tion with blue, green, or violet. Gentian violet. — Dissolve in the anilin oil solution as directed in the general formula. Although it does not keep as well as the aqueous solution, it stains better, especially when dealing with the achromatic structure of the mitotic figure. Often the brilliancy of the stain can be increased by leaving the slide for about 5 minutes in a 1 per cent aque- ous solution of permanganate of potassium before applying the stain, or in Gram's iodine solution (1 g. iodine, 2 g. potassium iodide, 300 c.c. water) after staining in violet. The greatest objection to the aqueous and anilin-oil solutions of gentian violet is that the stain washes out so rapidly in alcohols that it is impossible to run the slide up through the series. The usual prac- tice is to dip the slide in water to remove most of the stain and thus avoid carrying it into the alcohol : then transfer directly from water to 95 per cent alcohol, allowing the alcohol to act for only 2 or 3 seconds, then allow the absolute alcohol to act for 5 or 6 seconds, and then, while the stain is still coming out in streams, begin the treatment with clove oil. Holding the slide in one hand, pour on a few drops of clove oil, and immediately drain off in such a way as to carry off the alcohol. This clove oil should not be used again. Then flood the slide repeat- edly with clove oil, pouring the clove oil back into the bottle. A 50-c.c. bottle of clove oil is large enough. About 100 mounts can be cleared with 50 c.c. of this oil. The clove oil is a solvent of gentian violet, but it dissolves the stain from some structures more rapidly than from others; e.g., the stain may be completely removed from the chromo- somes while it is still bright in the achromatic structures. As soon as the stain is just right, drain off the clove oil and put the slide into a Stender dish of xylol for a couple of minutes. The xylol will soon take on an amber color, but this will not reduce its efiiciency in clearing; on the contrary, its efficiency will improve. However, the least trace of clove oil, carried over into the balsam, will finally cause the stain to fade. Therefore, transfer the slide to fresh, clear xylol and let it re- main for 2 or 3 minutes before mounting in balsam. As may be inferred from what has preceded, alcohol would soon ex- STAINS AND STAINING 63 tract the stain, without any appHcation of clove oil. The clove oil is used, not only because it extracts the stain more slowly, but because it dissolves the stain from some structures more rapidly than from others; e.g., the stain may be completely removed from the chromo- somes while it is still bright in the achromatic structures, so that with safranin and gentian violet one can get red chromosomes on a violet spindle. Many use the gentian violet dissolved in clove oil. Dissolve 1 g. gentian violet in 200 c.c. absolute alcohol and pour into 200 c.c. of clove oil. Stir thoroughly or shake in a bottle, then pour into an open dish and keep warm until the mixture evaporates down to about 200 c.c. Then keep in a well-stoppered bottle. Put a little on the sHde with a pipette, allow it to stain for 2-10 minutes and then drain back into the bottle, for the stain can be used repeatedly. If gold orange or orange G, dissolved in clove oil, is to be used, put it on at this point and allow it to act for 10-20 seconds. Then drain it off into its bottle and put the slide into a Stender dish of xylol. Transfer to a Stender dish of clear xylol, and mount in balsam. Some still use cedar oil to follow the clove oil. This stops the action of the clove oil, but the preparations harden slowly. Gentian violet or, better, crystal violet, is an excellent stain for achromatic structures in all stages of development. Chromatin, in many of its stages, is also stained. In metaphase and anaphase one should be able to get red chromosomes and violet spindles with safra- nin and gentian violet. If the chromosomes also persist in retaining the violet, shorten the stain in gentian violet. Cilia stain well; starch grains stain deeply, chromatophores less deeply, and Hgnified walls may not stain at all. One should be able to get red lignified walls and violet cellulose walls with safranin and gentian violet. Cyanin. — This stain is also called Quinolein blue and Chinolin blue. Dissolve 1 g. of cyanin in 100 c.c. of 95 per cent alcohol and add 100 c.c. of water. The cyanin would not dissolve in 50 per cent alcohol. We have not found Griibler's cyanin at all satisfactory with the fore- going formula. With the general formula the Griibler's cyanin will not dissolve. We use a cyanin prepared by H. A. Metz and Company, 122 Hudson Street, New York. This cyanin dissolves completely when made up according to the general formula. It stains rapidly, 5-10 minutes usually being sufficient. Chromosomes take a deep blue, but the spindle is only slightly affected. Lignified structures stain blue. 64 METHODS IN PLANT HISTOLOGY while cellulose walls are scarcely affected and the stain is easily washed out. Iodine green. — Use a 1 per cent solution in 70 per cent alcohol. Stain for an hour, rinse in 70 per cent alcohol, dehydrate in 95 per cent alcohol and absolute alcohol, clear in xylol or clove oil, and mount in balsam. If the stain washes out too rapidly and does not give sufficient differentiation, stain longer, overnight or even 24 hours. Lignified structures stain green, but, after proper washing, cellulose is scarcely affected. A bright green may be left in the chromosomes after all the stain has been washed out from the spindle. Acid fuchsin, erythrosin, and eosin are good contrast stains for mi- totic figures. Acid fuchsin or Delafield's haematoxylin are good for cellulose walls. Light green (Licht Griin). — Light green is an acid stain, soluble in water, alcohol, or clove oil. It stains quickly and forms a sharp con- trast with safranin or phloxine. Stain in safranin and then, with little or no washing out, stain in a weak alcoholic solution of acid green (about 0.2 g. in 100 c.c. of 95 per cent alcohol). From 20 seconds to about 1 minute may be sufficient. The green rapidly reduces the safranin, and consequently the staining must not be too prolonged. A successful preparation should show red chromosomes and green spindle. Lignified walls should be red, and cellulose walls, green. Malachite green. — A 1-3 per cent aqueous solution is good for cellu- lose walls. The stain contrasts well with Congo red. Methyl green. — A 1 per cent solution in water is good for staining lignified structures. Lee recommends that the solution be acidulated with acetic acid. This is not necessary for staining lignified membranes nor for staining chromosomes. Methyl green has long been a favorite stain for hving tissues. It is more easily controlled than iodine green, especially in double staining to differentiate lignified and cellulose walls. Acid green. — Make a solution according to the general formula, or simply make a 1 per cent solution in water. This stains cellulose walls and achromatic structures, but scarcely affects hgnified walls or chromosomes. Anilin blue. — Strong alcoholic solutions are best for botanical work. Even though the dry stain may be intended for aqueous solution, make a 1 per cent solution in 85 or 95 per cent alcohol. STAINS AND STAINING 65 This stain can be recommended for cellulose walls, achromatic structures of mitotic figures, for cilia, and it is particularly valuable for algae. Directions for using it with algae are given in chapter viii. Orange G. — Make a 1 per cent solution in water, in 95 per cent al- cohol, or a j\ per cent solution in clove oil. We prefer the solution in clove oil. The orange dissolves very slowly if put directly into clove oil. It is better to dissolve | gram of orange in 50 c.c. of absolute alcohol. In a well-corked bottle in the paraffin oven at 52° C, this much orange may go into solution. Then remove the cork and allow about half of the alcohol to evaporate. Pour on 200 c.c. or more of clove oil and let it stand for several hours. If any of the stain has not gone into solution, pour off the clear fluid, which is now ready for use, and pour some more clove oil on the residue, allowing the residue to go slowly into solution. In staining, we use a small bottle of the orange, pouring it on the slide and draining it back into the bottle. The absolute alcohol, carried into the clove oil in this way, does no damage, except that it dilutes the stain a little. Transfer to the aqueous stain from water; to the alcoholic stain from 85 per cent alcohol, since the stain is always applied as a second or third stain ; use the solution in clove oil after the dehydration in abso- lute alcohol. Times are always short and are to be reckoned in sec- onds rather than in minutes. If the solution in clove oil has been used, the slide should be transferred to xylol before mounting in balsam. This is a plasma stain. It is distinctly a general rather than a selec- tive stain, but is valuable as a background for other structures which have been stained violet or blue or green. It first came into prominence as the third member of the triple stain — safranin, gentian violet, orange. Gold orange. — This stain, which many incorrectly suppose to be the same as orange G, is much more readily soluble in clove oil and stains with much greater rapidity. DOUBLE STAINS AND TRIPLE STAINS Occasionally one uses a single stain to bring out some particular structure, but in most cases two, or even three, stains are used. In staining a vascular bundle, one stain may be selected which stains the xylem, but not the phloem, while another of a different color stains the phloem, but not the xylem, thus affording a sharp contrast. In 66 METHODS IN PLANT HISTOLOGY staining mitotic figures, one stain may stain the chromosomes, while another of a different color may be used to stain the spindle. One stain may affect another, the second stain often weakening the first. There is not likely to be much success without patience and con- stant observation. Flemming's safranin, gentian violet, orange. — Safranin has long been a famous stain for mitosis. This triple combination was pubhshed in 1891, but its value in plant cytology was not thoroughly appreciated until five or six years later, when its application was developed to a high degree of perfection by various investigators of the Bonn (Ger- many) school. Three methods, which may be designated as A, B, and C, will be described. A. According to Flemming, stain 2 or 3 days in safranin (dissolve 0.5 g. safranin in 50 c.c. absolute alcohol, and after 4 days add 10 c.c. distilled water) ; rinse quickly in water; stain 1-3 hours in a 2 per cent aqueous solution of gentian violet; wash quickly in water, and then stain 1-3 minutes in a 1 per cent aqueous solution of orange G. Trans- fer from the stain to absolute alcohol, clear in clove oil, and mount in balsam. We are indebted to Flemming for the chromo-acetic-osmic fixing agent. No cytologist has made a greater or more permanent con- tribution to cytological technique, but his method of using the triple stain is mentioned only as a matter of history : no one uses it today. B. The following formulas and method seem to be better for mitotic phenomena in plants : Make a 1 per cent solution of safranin in 50 per cent alcohol, a 1 per cent aqueous solution of gentian violet, and a 1 per cent aqueous solution of orange G. Transfer paraffin sections to the stain from 50 per cent alcohol after the xylol or turpentine used in dissolving away the paraffin has been rinsed off. Stain from 3 to 24 hours. If the period be too short, the washing out is so rapid that it is difficult to stop the differentiation at the proper point, and, besides, the red is likely to be less brilliant. Rinse in 50 per cent alcohol until the stain is washed out from the spindle and cytoplasm, but stop the washing before the chromosomes begin to lose their bright red color. If the washing out takes place too slowly, treat with shghtly acidulated alcohol (one drop of HCl to 50 c.c. of 50 per cent alcohol) for a few seconds. The acid must be removed by washing for 15-30 seconds in alcohol which has not been acidulated. STAINS AND STAINING 67 Wash in water for a couple of minutes and then stain in gentian violet. The time required is so variable that definite instructions are im- possible. The gentian violet should stain the spindle, but not the chromosomes. If the stain be too prolonged, it may be impossible to get it out from the chromosomes and still leave it bright in the spindle. If the period be too short, the stain will wash out from the spindle. For mitotic figures in the germinating spores of the liverwort, Pellia, 30 minutes is not too long. In this case, the stain washes out easily from the chromosomes without the use of acid, and the spindle takes a rich violet which is not easily washed out. In embryo sacs of Lilimn try 10 minutes. In pollen mother-cells try 5-10 minutes. For root-tips try 2-10 minutes. Chromatin in the early prophases and in telophases will stain with the violet, and the violet will not wash out, but in phases in which fully formed chromosomes are visible the violet can be washed out if the period has not been too long. If the aqueous gentian violet or crystal violet fails to stain the spindle, try the anilin oil solutions. Remove the shde from the gentian violet and dip it 5 or 6 times in water and then stain from 30 seconds to 1 minute in orange G. The orange stains cytoplasm and at the same time washes out gentian violet. Transfer from the orange G to 95 per cent alcohol, dipping the shde a few times in this merely to save the absolute alcohol. Dehydrate in absolute alcohol 3-30 seconds. Clear in clove oil, as already described in the paragraph on gentian violet. Transfer to xylol and mount in balsam. Safranin and gentian violet are often used without the orange. In this case, transfer from the gentian violet directly to 95 per cent alco- hol, and proceed as before. An objection to both these methods is that the gradual series of al- cohols cannot be used, because the gentian violet washes out so rapid- ly. If one should try a filament of Spirogyra with either of these methods, it would hardly be recognizable when it reaches the balsam; but with thin sections, especially when well fastened to the shde, con- ditions are different and there does not seem to be much damage. With any aqueous solution of gentian violet or crystal violet, the violet is less likely to be lost, if permanganate of potash is used before the stain, or Gram's iodine after it. 68 METHODS IN PLANT HISTOLOGY We are using a third method which seems to be better than either of the two just described. Shdes can be run up, through the alcohols in the usual way. C. Use the safranin solution described in B, but use clove oil solu- tions of gentian violet and orange G, or gold orange. Make the orange solution as described on page 65. The solution of gentian violet is prepared in the same way, but it does not take as long to get a good solution. After staining in safranin and dehydrating in absolute alcohol, flood the slide with the clove oil solution of gentian violet or crystal violet and stain for 5-30 minutes, or even for hours if necessary. Drain the stain into the bottle, for it can be used repeatedly. Put pure clove oil on with a pipette and watch until the stain is satisfactory. In a mitotic figure, the chromosomes should be red, and the spindle, violet. Pour off the clove oil and put on the orange. About 10-30 seconds will usu- ally be long enough. Pour the orange back into its bottle, rinse with pure clove oil, and place the slide in a Stender dish of xylol. This xylol will soon take on an amber color. Transfer to clear xylol and mount in balsam. With freehand sections, which are likely to be much thicker, the process is the same but the times will be longer. Safranin and light green. — Stain in 50 per cent alcoholic safranin, wash in 50 per cent alcohol, but stop while the safranin is still some- what stronger than desired in the finished mount ; then stain in light green (1 g. in 100 c.c. of 90 per cent alcohol) for 10-30 seconds. The light green not only stains vigorously but reduces the safranin. Pass through 95 and 100 per cent alcohol, clear in xylol, and mount in balsam. The light green can be dissolved in clove oil. With a pipette, flood the slide with light green, stain for 30 seconds, pour the light green back into its bottle, rinse with pure clove oil, transfer to xylol, and mount in balsam. Used in either way, this combination is very good for anatomical work, especially for vascular anatomy. Cyanin and erythrosin. — Both solutions may be made according to the general formula for anilins, or 1 per cent aqueous solutions may be used. Miss Thomas recommends 1 gram dissolved in 100 c.c. of 95 per cent alcohol. After the solution is complete, add 100 c.c. of distilled water. Since the combination is used only for paraffin sections or for . STAINS AND STAINING 69 small organisms dried down on the slide, her formula is preferable. Transfer to the alcoholic cyanin from 50 per cent alcohol, stain 5-10 minutes or longer; rinse cjuickly in 50 per cent alcohol, transfer to eryth- rosin, and stain 30 seconds to one minute. Rinse quickly in 50 per cent alcohol, then in 95 per cent and absolute alcohol. Clear in xylol and mount in balsam. If aqueous stains are used, transfer to the cyanin from water, rinse in water, stain in erythrosin, rinse in water, and transfer directly to 95 per cent alcohol. If the cyanin washes out, stain for 1 hour, and if it still washes out, omit the rinsing and transfer directly from the cyanin to the erythrosin. The erythrosin may be used first ; in this case stain for 5 minutes in erythrosin, transfer directly to cyanin, and stain for about 10 seconds. Dehydrate in 95 per cent and in absolute alcohol, clear in xylol or in clove oil, and mount in balsam. The stains wash out so rapidly that the series of alcohols cannot be used. Chromosomes and nucleoli stain blue, and achromatic structures, red. Lignified structures stain blue, and cellulose walls, red. The various cell constituents are often sharply differentiated. It was this combination which suggested the now obsolete terms, "cyanophilous" and "erythrophilous." Phloxine and anilin blue. — In the fourth edition, this combination was described under the heading, Magdala red and anilin blue, but the occasional lots of the red stain which gave satisfactory results were probably phloxine. Make both solutions as directed in chapter viii on "The Venetian Turpentine Method." For paraffin sections, stain 3-24 hours in phloxine, dip in 95 per cent alcohol to rinse off the stain, and then stain 2-10 minutes in the anilin blue. Dip in 95 per cent alcohol to rinse off the stain, and treat for a few seconds with alcohol slightly acidulated with hydrochloric acid (one drop to 50 c.c. of 95 per cent alcohol). In the acid alcohol the blue will become more intense, but the red would soon be extracted. Wash in 95 per cent alcohol to remove the acid. If the acid has weakened the phloxine put a pinch of sodium carbonate into the 95 per cent alco- hol. The red may brighten. If the red is too weak, return to the phloxine and try again. From the 95 per cent alcohol transfer to abso- lute alcohol, to xylol, and then mount in balsam. 70 METHODS IN PLANT HISTOLOGY For staining algae, more complete directions are given in chapter viii. Acid fuchsin and iodine green mixtures. — Two solutions are kept separate, since they do not retain their efficiency long after they are mixed : J Fuchsin acid 0 . 1 g. \ Distilled water 50 . 0 c.c. -r, j Iodine green 0 . 1 g. \ Distilled water 50 . 0 c.c. [ Absolute alcohol 100 . 0 c.c. C ^ Glacial acetic acid 1.0 c.c. [ Iodine 0 . 1 g. Mix equal parts of A and B. Transfer to the stain from water. The proper time must be determined by experiment. For a trial, 24 hours might be recommended. Transfer from the stain directly to solution C and from C to xylol. A. Acid fuchsin 0 . 5 g. in 100 c.c. of water B. Iodine green 0. 5 g. in 100 c.c. of water Mix a pipette full of A with a pipette full of B; stain 2-8 minutes; transfer to 85 per cent or 95 per cent alcohol, dehydrate rapidly, clear in xylol, and mount in balsam. Both these formulas are good for mitosis. Acid fuchsin and methyl green. — Both may be used in 1 per cent aqueous solutions. For mitotic figures, stain in green for about an hour, wash in water or alcohol until the green is extracted from the spindle, and then stain for about 1 minute in the fuchsin. Dehydrate in 95 and 100 per cent alcohol, clear in xylol or clove oil, and mount in balsam. If the green washes out, stain longer; if it is not readily extracted from the spindle, shorten the period. If the fuchsin stains the chromosomes, shorten the period, and lengthen it if the fuchsin washes out from the spindle. The chromosomes should take a brilliant green, and the spindle, a bright red. Delafield's haematoxylin and erythrosin. — Stain first in the haema- toxylin, and after that stain is satisfactory, stain for 30 seconds or 1 minute in erythrosin. This is a good combination, and, for most plant structures, gives a far better differentiation than the traditional hae- matoxylin and eosin, since the erythrosin has all the advantages of the eosin and is more transparent. Orange G is also a good stain to use with Delafield's haematoxylin. STAINS AND STAINING 71 Directions for staining in safranin and Delafield's haematoxylin are given in the chapter on "Freehand Sections" (chap. vi). Heidenhain's iron-haematoxylin and orange G. — This haematoxy- hn is very satisfactory when used alone. A hght staining in orange G, however, sometimes improves the mount. After the last washing in water, stain for about 30 seconds in orange G ; or, if the orange is in clove oil, stain after dehydrating in absolute alcohol. Eosin, erythrosin, and most plasma stains fail to increase the effect of a good stain in iron-haematoxylin ; but staining overnight in safranin and reducing the stain until it becomes a faint pink in the protoplasm may still show a distinct red in the nucleoli. Then stain in iron-alum haematoxylin in the usual way. Combinations might be described almost without limit. Several more will be suggested in Part II in connection with various groups of plants. We have not tried to make the Ust of stains complete, but we have described more than any botanist will learn to use critically. Master two or three good combinations and don't spend much time with the rest. CHAPTER IV GENERAL REMARKS ON STAINING The function of a stain is to make structures visible which cannot be seen without staining, or to bring out clearly structures which are only faintly visible. A filament of Spirogijra shows the chromatophore clearly if merely mounted alive in a drop of water; the nucleus is visi- ble and the pyrenoids can be distinguished. Such a study is necessary if one is to understand anything about the plant and, for an elemen- tary class, this much is sufficient ; but a drop of iodine solution applied to the edge of the cover would emphasize certain details, e.g., the starch would appear blue, the nucleus a light brown, and the cyto- plasm a lighter brown. This illustrates at least one advantage to be gained by staining; it enables us to see structures which would other- wise be invisible, or almost invisible. Much of the recent progress in morphology and cytology has been due to the development of critical methods of staining. Some of the combinations and methods recom- mended by various workers are good in themselves, while others, not so good, have yielded results because they have been so skilfully used. CHOOSING A STAIN Some stains which are excellent in differentiating certain structures are worthless for others; but the worthless stain may be the best one for something else. Beautiful and instructive preparations sometimes result from some happy chance, as when a shde is passed through al- cohols which have become tinged with various stains. Such slides may show four or five stains well balanced ; but uniform success demands skill and judgment in manipulation, and also a knowledge of the struc- tures which are to be differentiated. Let us take a vascular bundle for illustration. Safranin stains the xylem a bright red, but, with judicious washing, is entirely removed from the cambium and cellulose elements of the phloem. A careful staining with Delafield's haematoxylin now gives a rich purple color to the cellulose elements which were left un- stained by the safranin, thus contrasting sharply with the lignified elements. If cyanin and erythrosin be used, the xylem takes the blue while the cambium and phloem take the red. 72 GENERAL REMARKS ON STAINING 73 The mere selection of two colors which contrast well is not sufficient. Green and red contrast well, but safranin and iodine green would be a poor combination, for both would stain chromosomes and neither would stain the spindle ; both would stain lignified structures and nei- ther would give satisfactory results with cellulose walls. Both stains are basic. Acid green would have given a contrast in both these cases, because it stains achromatic structures and cellulose walls. In general, an acid stain should be combined with a basic one, but there are so many exceptions that it is hardly worth while to learn a list of basic and acid stains. Stains which stain chromosomes are likely to be basic, and those which do not stain chromosomes are likely to be acid or neu- tral. If it were true that acid stains affect only basic structures, and basic stains affect only acid structures, a classification of stains would be of great value. Safranin and gentian violet are both basic but, with the safranin properly washed out and the gentian violet properly ap- plied, chromosomes stain red while the spindle stains violet. Lignified cell walls stain red, while cellulose walls stain violet. The exine of a pollen grain stains red while the intine stains violet. It is very evident that, to secure a contrast, it is not necessary that one stain be basic and the other acid. The only way to insure success is to become famihar with the action of each stain upon the various structures. THEORIES OF STAINING The history of staining does not go back to Aristotle, but a carmine was used for microscopic purposes as early as 1770. Even in 1838, when Schleiden announced his cell theory, staining had not become an important part of histological technique, although some use was being made of carmine and iodine. Beginning with 1850, stains were used more and more and, during the last thirty years of the century, de- mands for better stains became so insistent that in Germany com- panies were formed to furnish stains for microscopic use. The famous Griibler Company is still furnishing excellent stains, some of which, like their haematoxylin, have not been surpassed. During the World War, stains were produced in America and, when the Commission on Standardization of Biological Stains was organized, stains improved rapidly and many of them have become not only excellent but uni- form, so that, when a stain has become valuable for some particular purpose, one can get more exactly like it. 74 METHODS IN PLANT HISTOLOGY A short history of staining is given in Dr. Conn's book on Biological Stains, in which one can find references to more extensive works on the subject. As soon as staining became recognized as a necessary part of histo- logical technique, theories began to appear, some of them suggestive, some instructive, and some only amusing. In 1890 Auerbach, a zoologist, published the results of his studies upon spermatozoa and ova. He found that, if preparations containing both spermatozoa and ova were stained with cyanin and erythrosin, the nuclei of the spermatozoa took the cyanin, while the nuclei of ova preferred the erythrosin; hence he proposed the terms "cyanophilous" and "erythrophilous." Auerbach regarded these differences as an in- dication of sexual differences in the cells. Rosen (1892) supported this theory, and even went so far as to re- gard the tube nucleus of the pollen grain as female, on account of its erythrophilous staining. In connection with this theory it was sug- gested that the ordinary vegetative nuclei are hermaphrodite, and that in the formation of a female germ nucleus the male elements are extruded, leaving only the erythrophilous female elements; and, simi- larly, in the formation of a male nucleus the female elements are ex- truded, leaving only the cyanophilous male elements. As long ago as 1884 Strasburger discovered that with a mixture of fuchsin and iodine green the generative nucleus of a pollen grain stains green, while the tube nucleus stains red. In 1892, in his Verhalten des Pollens, he discussed quite thoroughly the staining reactions of the nuclei. The nuclei of the small prothalhal cells of gymnosperm micro- spores are cyanophilous Hke the male generative nuclei. The nuclei of a nucellus surrounding an embryo sac are also cyanophilous, while the nuclei of structures within the sac are erythrophilous. His conclusion is that the cyanophilous condition in both cases is due to poor nutri- tion, while the erythrophilous condition is due to abundant nutrition. A further fact in support of the theory is that the nuclei of the adventi- tious embryos which come from the nucellus of Funkia ovata are de- cidedly erythrophilous, while the nuclei of the nucellus to which they owe their food-supply are cyanophilous. In division stages nuclei are cyanophilous, but from anaphase to resting stage the cyanophilous condition becomes less and less pro- nounced, and may even gradually change to the erythrophilous. An additional fact in favor of this theory is that in Ephedra the tube GENERAL REMARKS ON STAINING 75 nucleus, which has very httle cytoplasm about it, is cyanophilous. Strasburger claimed that there is no essential difference between male and female generative nuclei, and subsequent observation soon showed that within the egg the sex nuclei rapidly become alike in their re- action to stains. Malfatti (1891) and Lilienfeld (1892-93) claim that these reactions are dependent upon the amount of nucleic acid present in the struc- tures. During mitosis the chromosomes consist of nearly pure nucleic acid and are intensely cyanophilous, but the protoplasm, which has little or no nucleic acid, is erythrophilous. There is a gradual transi- tion from the cyanophilous condition to the erythrophilous, and vice versa, the acid structures taking basic stains, and basic structures, the acid stains. The terms "erythrophilous" and ''cyanophilous" soon became obso- lete, and many claimed the affinity is for basic and acid dyes, rather than for blue or red colors. That the terms were misnomers became evident when a combination like safranin (basic) and acid green (acid) was used, for the cyanophilous structures stained red, and the eryth- rophilous, green. According to Fischer (1897 and 1900), stains indicate physical but not chemical composition. Fischer experimented with substances of known chemical composition. Egg albumin was shaken until small granules were secured. These were fixed with the usual fixing agents, and then stained with Delafield's haematoxylin. The extremely small granules stained red, while the larger ones became purple. Since the granules are all alike in chemical composition, Fischer concluded that the difference in staining must be due to physical differences. With safranin, followed by gentian violet, the larger granules stain red and the smaller, violet; if, however, the gentian violet be used first, then treated with acid alcohol, and followed by safranin, the larger granules take the gentian violet and the smaller, the safranin. In root-tips similar results were obtained. Safranin followed by gentian violet stained chromosomes red and spindle fibers violet, while gentian violet followed by safranin stained the chromosomes violet and the spindle red. One often reads that chromosomes owe their strong staining ca- pacity to nuclein, and especially to the phosphorus, but, according to Fischer, this is shown to be unfounded, since albumin gives similar results, yet contains no phosphorus, and is not chemically allied to nuclein. 76 METHODS IN PLANT HISTOLOGY Probably the most important reason which led Fischer to undertake this series of experiments was the claim that certain granules of the Cyanophyceae should be identified as chromatin because they behaved like chromatin when stained with haematoxylin. Fischer's experi- ments not only proved that chromatin cannot be identified in this way but raised the question whether staining reactions ever indicate chem- ical composition. At present, it would seem that, in most cases, the staining indicates only physical differences. However, in some cases there is a chemical reaction, e.g., when material fixed in bichloride of mercury is stained in carmine, mercuric carminate is formed. It would be very convenient if we knew just how much dependence should be placed upon staining reactions as a means of analysis. If two structures stain alike with Delafield's haematoxylin, does this mean that they have the same chemical composition; or if, on the other hand, they stain differently, must they necessarily be different in their chemical composition? Delafield's haematoxylin, when carefully used, gives a rich purple color, but a careful examination will often show that in the same preparation some structures stain purple, while others stain red. Does this mean that the purple and red structures must have a different chemical composition? Many people believe that structures which stain differently with a given stain must be chemically different, but they readily agree that structures which stain alike are not necessarily similar in chemical composition. Chromosomes of dividing nuclei and lignified cell walls stain ahke with safranin; chromosomes and cellulose cell walls stain much alike with Delafield's haematoxylin; but everyone recognizes that the chromo- some is very different in its chemical composition from either the cellu- lose or the lignified wall. However, in an indirect and somewhat uncertain way, one can in- fer the nature of certain structures from the staining. For instance, if sections of various objects have been stained with safranin, we may draw the following inferences with more or less confidence : if cells in the xylem region of a vascular bundle stain red, their walls are ligni- fied; if cortical cells, which may appear quite similar in transverse sec- tion, stain red, they are likely to be suberized; if the outer walls of epidermal cells stain red, they are cutinized; but if the outer boundary of the embryo sac of a gymnosperm stains red, it is chitinized. Of course, these inferences can be made only because the various struc- tures have been tested bv more accurate methods. GENERAL REMARKS ON STAINING 77 Whatever doubt or uncertainty there may be in regard to theories of staining or in regard to the value of stains as a means of analysis, there is no doubt that stains are of the highest importance in differen- tiating structures, and in bringing out details which would otherwise be invisible. PRACTICAL HINTS ON STAINING The number of stains in the catalogues is becoming so great that it is impossible to become proficient in the use of all of them. As we have already intimated, it is better to master a few of the most valuable stains than to do indifferent work with many. An experienced tech- nician knows that it is impossible to judge from a few trials whether a given stain or combination is really valuable or not. As a matter of fact, some of the most valuable combinations, hke Haidenhain's iron- alum haematoxylin and Flemming's safranin, gentian violet, orange, require patient study and long practice before they yield the magnifi- cent preparations of the trained cytologist. The beginner, especially if somewhat unacquainted with the details of plant structure, may be- lieve that he has an excellent preparation when it is really a bad, or at most an indifferent, one. To illustrate, let us suppose that sections of the pollen grain of a lily have been stained in safranin and gentian vio- let. If the preparation merely shows a couple of dense nuclei and a mass of uniform cell contents surrounded by a heavy wall, the mount is poor. If the two nuclei are quite different and starch grains are well differentiated in the tube cells and the wall shows a violet intine con- trasting sharply with a red exine, the mount is good. Anything inter- mediate is indifferent. If mitotic figures have been stained with cyanin and erythrosin, a first-class preparation should show blue chromo- somes and red spindles; if stained with safranin and gentian violet, the chromosomes should be red and the spindles, violet. In staining growing points, apical cells, young embryos, antheridia, archegonia, and many such things, the cell walls are the principal things to be differentiated, if the preparations are for morphological study. As a rule, it is better in such cases not to use double staining, but to select a stain which stains the cell walls deeply without obscur- ing them by staining starch, chlorophyll, and other cell contents. For example, try the growing point of Equisetum. The protoplasm of such growing points is very dense. If Delafield's haematoxyhn and eryth- rosin be used, the haematoxyhn will stain the walls and nuclei, and 78 METHODS IN PLANT HISTOLOGY will slightly affect the other cell contents, but the erythrosin will give the cytoplasm such a dense stain that the cell walls will be seriously obscured. It would be better to use haematoxylin alone. For counting chromosomes, it is better to stain in iron-alum haematoxylin alone, or in safranin alone. The same suggestion may well be observed in trac- ing the development of antheridia, archegonia, embryos, and similar structures. In using combinations, it must be remembered that the second stain often affects the first, e.g., if safranin is to be followed by Dela- field's haematoxylin, in staining a vascular bundle, it will not do to make the safranin just right and then apply the haematoxylin, for the acid which must be used to differentiate the haematoxylin and to avoid precipitates will also reduce the safranin, and the red will be too weak. You must overstain in safranin so that the reduction will finally leave it just right. The same hint will apply if safranin is to be followed by anilin blue, since, here, also, acid must be used; if light green is to follow the safranin, the stain itself is so acid that the safranin must be rather strong before the light green is applied. Orange, whether in water or in clove oil, reduces many stains and, consequently, such stains must be strong enough to allow the weakening. These hints are only samples: the student must observe the behavior of the various stains when used singly and when used in various combinations. With perfect confidence, we can give advice to the beginner and to the seasoned investigator: master a few stains. With Haidenhain's iron-alum haematoxylin, stain sections of root-tips until you can see the finest details of the chromatin. When you can make a perfect preparation of a root-tip with this stain, you will have made a good start toward a satisfactory histologial technique. Gentian violet is often used to stain the ciha of sperms; but Haidenhain's iron-alum haematoxylin will stain the ciha of cycad sperms so critically that the base of the cilium, as it passes through the Hautschicht (plasma mem- brane), will be differentiated from the part projecting beyond the Hautschicht. Master this haematoxylin stain and then, with sections of root-tips, practice the safranin, gentian violet, orange combination until you can stain the chromosomes red, the spindle violet, and the cytoplasm orange. For vascular anatomy, learn to stain xylem with safranin; and, for a contrast, stain cellulose walls with Delafield's haematoxylin, gentian violet, light green, or anilin blue. For filamen- tous algae and fungi, master the iron-alum haematoxylin method; and GENERAL REMARKS ON STAINING 79 then try the phloxine and aniUne-blue combination. Do not pass judg- ment against a standard method or even a new method just because you fail to get results at the first trial. After you have become pro- ficient with the iron-alum haematoxylin for mitotic figures in higher plants, you are sure to fail if you try the same procedure with Rhizo- pus; but, nevertheless, the stain is just as good for Rhizopus as for figures in pollen mother-cells or root-tips. Permanent preparations are such a necessary part of most advanced work that one is in danger of delaying the critical observation until he has made a permanent mount. It cannot be repeated too often that one should develop also the technique of studying the living structures. It is impossible to make a permanent mount of the rotation of proto- plasm. Study motile spores while they are moving before you make permanent preparations. The difficult and complicated histological technique loses much of its value unless it is accompanied by a thor- ough study of living material. CHAPTER V TEMPORARY MOUNTS AND MICROCHEMICAL TESTS Skill in making freehand sections, without any microtome, and in teasing with needles, and in making dehcate dissections under the simple microscope are absolutely necessary in any investigation deal- ing with the structure and development of plants. Preliminary study with the aid of such methods not only gives a broader view of struc- tures in all dimensions and helps the interpretation of stained micro- tome preparations but is necessary in determining whether material is worth all the labor of making permanent mounts. That particular class of temporary mounts intended only for chemical tests is con- sidered separately in the second part of this chapter. TEMPORARY MOUNTS A preliminary examination of almost any botanical material may be made without any fixing, imbedding, or staining. If a little starch be scraped from a potato, and a small drop of water and a cover-glass be added, a very good view will be obtained, and if a small drop of iodine solution be allowed to run under the cover, the preparation, while it lasts, is better than some permanent mounts. The unicellular and fila- mentous algae can be studied quite satisfactorily from such mounts. The protonema of mosses and the prothallia of ferns should be studied in this way, even if a later study from sections is intended. The addi- tion of a little iodine identifies the starch and makes the nucleus more plainly visible. If the top of a moss capsule be cut off at the level of the annulus, a beautiful view of the peristome may be obtained by simply mounting in a drop of water, or, in a case like this where no collapse is to be anticipated, the object may be mounted in a small drop of glycerin — just enough to come to the edge of the cover without oozing out beyond — and the preparation may be made permanent by sealing with balsam, gold size, or any good cement. The antheridia and archegonia of mosses may be examined if the surrounding leaves are carefully teased away with needles. Freehand sectioning with a sharp razor and judicious teasing with a pair of needles will give a fair 80 TEMPORARY MOUNTS AND MICROCHEMICAL TESTS 81 insight into the anatomy of the higher plants without demanding any further knowledge of technique. This rough work is a very desirable antecedent to the study of microtome sections, because most students see in a series of microtome sections only a series of sections when, in the mind's eye, they ought to see the object building itself up in length, breadth, and thickness as they pass from one section to another. The movements of protoplasm can, of course, be studied only in the living material. Every laboratory should keep Chara growing at every season of the year. Mount a small portion and note the movements in the internodal cells. Avoid any pressure and any lowering of the temperature. A gentle raising of the temperature will accelerate the movements. A leaf of Elodea shows the movements very clearly, especially in the midrib region. The stamen hairs of Tradescantia have long been used, their color, resembling a faint haematoxylin stain, making them particularly favorable. Stinging hairs show a brisk move- ment if they are mounted quickly and without injury. Fortunately, the common onion always furnishes favorable material for demon- strating the movements of protoplasm. Strip the epidermis from one of the inner scales of the bulb and mount in water. The granules may appear to better advantage in yellow light, like that of an ordinary kerosene lamp. In studying the movements of protoplasm, a drop of aqueous car- mine, allowed to run under the cover, will bring the protoplasm more clearly into view. The protoplasm is often so nearly colorless that one recognizes it only by the movement of plastids and various granules which are carried along in the current. The discharge of spores and gametes should be observed in the liv- ing material : the difference in the behavior of spores and gametes is very striking and can be appreciated only while they are alive. Most aspects of growth and movement can be studied best in the living con- dition. In short, it is well to make a preliminary study of everything. The development of the micromanipulator has brought such an immense improvement in the technique of studying living material that some investigators can make tiny glass needles which they can insert into a cell, hook a needle into each end of a chromosome, stretch it, and allow it to contract. This remarkable instrument, which can be used with any high-grade microscope, opens an attractive field for work in cytology and microchemistry. The germination of spores and the growth of pollen tubes can be 82 METHODS IN PLANT HISTOLOGY Studied in the hanging drop. For facilitating such cultures there are many devices, such as hollow-ground slides, glass rings, rubber rings, etc. (Fig. 17). A device which is better for most purposes, and which is easily made by any student, is to cut a square or round hole f inch in diameter in a piece of pasteboard | inch thick, 1 inch wide, and 1| inches long. The pasteboard is then boiled to sterilize it and to make it fit more closely to the slide. While the pasteboard is still wet, press _ it to the slide, make the II II culture in a drop of ^ ,, ^^ ^ . J , water or culture solu- riG. 17. — Ine hanging-drop culture tion on the cover, and invert the cover over the hole. A little water added at the edge of the pasteboard from time to time will keep it from warping and will at the same time provide a constant moist chamber. In collecting material for mitotic figures in anthers it is necessary to examine fresh anthers, if one wishes to avoid a tedious and uncertain search after the anthers have been imbedded. By teasing out a few cells from the apex and a few from the base of the anther the stage of development is readily determined, and anthers which do not show the desired stages can be rejected. By allowing a drop of eosin or methyl green to run under the cover, the figures are more easily detected. The actual progress of mitosis has been observed in the stamen hairs of TradescanUa. If care be taken not to injure the hairs or let the tem- perature drop, a mount in water, or in 1 per cent sugar solution, or in the juice of the plant, may live long enough for a study of a complete mitosis, which takes 2 or 3 hours. MICROCHEMICAL TESTS Botanical microchemistry has developed to such an extent that it has become an independent subject, like bacteriology. We shall con- sider only the commonest tests which are needed constantly by stu- dents of morphology. For a thorough presentation of the chemistry of the cell, we are still looking forward with great anticipation to a forth- coming book by Dr. Sophia Eckerson, whose critical tests and analyses we have observed for many years. In the meantime, Pflanzenmikro- chemie, by Dr. 0. Tunmann (Gebriider Borntrager, Berlin), is recom- mended to those who read German. Zimmerman's Botanical Micro- technique (Henry Holt & Co., New York) is still recommended to those who must rely upon English. We shall give only a few tests, but in TEMPORARY MOUNTS AND MICROCHEMICAL TESTS 83 considering the various stains we shall indicate the effect of each stain upon the various plant structures. Starch. — Mount the starch or starch-containing structures in water, and allow a drop of iodine solution to run under the cover. Starch assumes a characteristic blue color. The solution may be prepared by dissolving 1 g. of potassium iodide in 100 c.c. of water and adding 0.3 g. of sublimed iodine. A strong solution of iodine in alcohol (about 1 g. in 50 c.c. of absolute alcohol) keeps well. A drop of this solution added to 1 c.c. of water is good for testing. With too strong a solution, the starch first turns blue but rapidly becomes black. Grape-sugar. — In cells containing grape-sugar, bright-red granules of cuprous oxide are precipitated by Fehling's solution. It is better to keep the three ingredients in separate bottles, because the solution does not keep long after the ingredients are mixed. The solutions may be labeled A, B, and C. j Cupric sulphate 3 g. 1 Water 100 c.c. j Sodium potassium tartrate (Rochelle salt) ... 16 g. \ Water 100 c.c. p / Caustic soda 12 g. \ Water 100 c.c. When needed for use, add to 10 c.c. of water 5 c.c. from each of the three solutions. The sections, which should be two or three cells in thickness, are warmed in the solution until little bubbles are formed. Too much heat must be avoided. Mount and examine in a few drops of the solution. The twig or organ may be treated with the solution, and the sections may be cut afterward. Other substances pi-ecipitate copper, and may be mistaken for grape-sugar by the beginner. Cane-sugar. — Cuprous oxide is not precipitated from Fehling's so- lution by cane-sugar, but after continued boiling in this solution the cane-sugar is changed to invert-sugar and the copper is precipitated. The solution becomes blue. Proteins. — The proteins turn yellow or brown with the iodine solu- tion. It is better to use a stronger solution than when testing for starch. It must be remembered that many other substances also turn brown when treated with iodine. When proteins are warmed gently in concentrated nitric acid, the acid becomes yellow. The color may be deepened by the addition of a little ammonia or caustic potash. 84 METHODS IN PLANT HISTOLOGY When proteins are heated with Millon's reagent, the solution be- comes brick-red or rose-red. This reaction takes place slowly even in the cold. The following is one formula for this reagent: Mercury 1 c.c. Concentrated nitric acid 9 c.c. Water 10 c.c. Dissolve the mercury in the nitric acid and add the water. Fats and oils. — The fatty oils are not soluble in water and are only shghtly soluble in ordinary alcohol. They dissolve readily in chloro- form, ether, carbon disulphide, or methyl alcohol. Alcannin colors oils and fats deep red. The test is not decisive, be- cause ethereal oils and resins take the same red color. Dissolve com- mercial alcannin in absolute alcohol, add an equal volume of water, and filter. The fats and oils in sections left in this solution for 24 hours should be bright red. The reaction is hastened by gentle heating. Osmic acid, as used in fixing agents, colors fats and oils brown or black, and the black remains even after all the processes of the paraffin method. The black can be bleached out in hydrogen peroxide, or chlorine (see p. 27). In case of fats and oils, solubility and color reactions are useful, but must be regarded as corroborative evidence, not as decisive proof. For more critical and detailed methods, consult the book by Tunmann, which will also give the literature of the subject. The middle lamella. — Even the origin and development of the mid- dle lamella are none too well known; its microchemistry has pro- gressed but little beyond the color-reaction stage. The middle lamella consists largely of pectin or pectic compounds. The easy isolation of cells, when treated with Schultze's maceration, depends upon the ready solubility of pectins in this reagent. Many intercellular spaces arise through the natural solution or gelatinization of the lamella. In polarized light, with crossed Nicols, the middle lamella is resolved into three lamellae, the middle one appearing dark, and the two outer lamellae, hght. Ruthenium red is a good stain, since it gives as good results as any and has the advantage of keeping well in balsam or glycerin jelly. Make a very weak solution — 1 g. to 5,000 c.c. of water, or even weaker — and keep it in the dark. It stains many other things besides the lamella, but is, nevertheless, a good stain. TEMPORARY MOUNTS AND MICROCHEMICAL TESTS 85 Pectin is not at all confined to the middle lamella, but is found in other membranes, particularly in spore coats. Cellulose. — In concentrated sulphuric acid cellulose swells and finally dissolves. It is also soluble in cuprammonia. The cuprammonia can be prepared by pouring 15 per cent ammonia water upon copper turnings or filings. Let the solution stand in an open bottle. It does not keep well, but its efficiency is readily tested. Cotton dissolves al- most immediately as long as the solution is fit for use. With iodine and sulphuric acid, cellulose turns blue. Treat first with the undiluted iodine-potassium-iodide solution described in the test for starch, then add a mixture of two parts of concentrated sul- phuric acid and one part of water. With chloroTodide of zinc, cellulose turns violet. Dissolve commer- cial chloroiodide of zinc in about its own weight of water and add enough metallic iodine to give the solution a deep brown color. The cell walls of fungi consist oi fungus cellulose. When young, they give a typical cellulose reaction ; when older, they become insoluble in cuprammonia and, with iodine and sulphuric acid, show only a yellow or brown, instead of the typical blue. With chloroiodide of zinc, the wall stains yellow or brown, instead of violet. Reserve cellulose, which is common in thick- walled endosperm of seeds, shows the same microchemical reactions as ordinary cellulose. Callose. — The thickening on the sieve plate differs from cellulose in its staining reactions, and in its solubility. It is insoluble in cupram- monia, but will dissolve in a 1 per cent solution of caustic soda. Stain in a 4 per cent aqueous solution of soda (Na2C03) for 10 minutes, and transfer to glycerin. The callus should take a bright red. If stained very deeply and then transferred to a 4 per cent soda (with- out the corallin), the stain is extracted from the cellulose but remains in the callus. Unfortunately, the preparations are not permanent. If stained for about an hour in a dilute aqueous solution of anilin blue, the stain may be extracted with glycerin until it remains only in the callus. After the blue is satisfactory, a few minutes in aqueous eosin will afford a good contrast. The preparation may be mounted in balsam and is fairly permanent. Lignin. — Lignified walls are insoluble in cuprammonia. The iodine and sulphuric acid or the chloroiodide of zinc, used as in testing for cellulose, give the lignified walls a yellow or brown color. After a treat- 86 METHODS IN PLANT HISTOLOGY ment with Schultze's maceration fluid, lignified membranes react like cellulose. Phloroglucin in a 5 per cent aqueous or alcoholic solution applied simultaneously with hydrochloric acid gives lignified walls a reddish- violet color. The preparations do not keep. Cutinized and suberized walls. — These are insoluble in cupram- monia or concentrated sulphuric acid. They are colored yellow or brown by chloroiodide of zinc, or by iodine and sulphuric acid, when applied as in testing for cellulose or lignin. With alcannin, they take a red color, but the red is not as deep as in case of fats and oils. After soaking in an aqueous solution of caustic potash, suberized mem- branes take a red-violet color when treated with chloroiodide of zinc. If a strong, fresh alcoholic solution of chlorophyll be allowed to act upon suberized membranes for 15-30 minutes in the dark, they stain green, while lignified and cellulose walls do not take the stain. The preparations are not permanent. A solution of alcannin in 50 per cent alcohol stains suberized and cutinized walls red, but the color may not be very sharp. Cyanin can be recommended. First, treat with Eau de Javelle (po- tassium hypochlorite), which can be obtained ready for use at any drug-store. This destroys tannins, and the lignified walls lose their staining capacity. Make a 1 per cent solution of cyanin (Griibler's) in 50 per cent alcohol and add an equal volume of glycerin. This should show blue suberized walls, while the lignified walls remain unstained. Gum, mucilage, and gelatinized membranes. — These are all soluble in water and are further characterized by their strong power of swell- ing. They are insoluble in alcohol. A series of forms with various color reactions is included under this heading. Crystals. — Nearly all crystals which are found in plants consist of calcium oxalate. Crystals of calcium carbonate, calcium tartrate, and calcium sulphate also occur. Calcium oxalate is soluble in hydrochloric acid or nitric acid. It is better to use the concentrated acids. The crystals are insoluble in water and acetic acid. Sulphuric acid changes calcium oxalate into calcium sulphate. When treated with barium chloride, crystals of calcium sulphate become covered with a granular layer of barium sulphate, while crystals of calcium oxalate are not affected. Calcium carbonate, when treated with hydrochloric acid or acetic acid, dissolves with effervescence. The acetic acid should be rather dilute. CHAPTER VI FREEHAND SECTIONS^ The real freehand sections of pre-microtome days, cut by holding the object in one hand and the knife in the other, are becoming less and less frequent in well-equipped laboratories. Since the subsequent technique for such sections is the same as for those cut with a microtome without imbedding, both kinds of sections can be treated together. However, every student should learn to cut real freehand sections: the laboratory is no place for one who is awkward with his hands; manual dexterity must be acquired if there is to be any success in morpholog- ical studies, which demand critical preparations. Not only freehand sections, but other small thin objects which can be treated like sec- tions, will be considered in this chapter. The beginner should start with the freehand section because the processes are rapid and it is easy to find the causes of imperfections and failures. For cutting sections of small twigs, roots, rhizomes, and similar ob- jects, we use a safety razor blade, either held directly in the fingers or in the type of clamp shown in Figure 3. For those who use the old- fashioned razor, the grandfather type, shown in Figure 6, is the best. In cutting, brace the forearms against the sides, hold the object firmly in the left hand, and cut with a long, oblique stroke from left to right. The edge of the razor and the direction of the stroke should be toward the body, not away from it as in whittling. If the material is fresh, the object and the razor should be kept wet with water, the razor being dipped in water for every stroke. For hard objects, hke twigs of oak or maple, the grandfather razor will need sharpening after cutting a dozen sections. It is a waste of time to put off sharpening until the razor has become noticeably dull, for all sections except those cut when the razor is perfectly sharp are sure to be inferior. With softer material the razor may hold its edge for hundreds of sections. Those sections which seem to be worth further treatment should be 1 Before attempting the freehand sectioning, the beginner should read the paragraphs on killing, fixing, washing, hardening, dehydrating, and clearing, beginning on pp. IS and 112. 87 88 METHODS IN PLANT HISTOLOGY placed at once in water or in a fixing agent and, of course, the choice of a fixing agent should be determined before the sections are cut. With the advent of a cheap, efficient sliding microtome, the hand microtome began to fall into disuse and, today, it has almost dis- appeared. The sliding microtome (Fig. 2) reduces to a minimum the necessity for manual dexterity, but it is a more complicated machine. Study the various parts before you begin to cut sections. How is the knife ad- justed? How is the object clamp raised and lowered? How is the thickness of the section determined? In case of a simple microtome like the one shown in Figure 2, the student should soon answer such questions without any help from the instructor. In case of more com- plicated microtomes, a demonstration by the instructor will save both time and machine. In cutting sections of wood or herbaceous stems, the knife should be set obliquely so as to use as much as possible of the cutting edge. In most cases it is neither necessary nor desirable to cut very thin sections by this method; 10 ^ is very thin, and 20, 30, or even 40 /x is usually thin enough. Cut with a firm, even stroke, wetting both knife and object after every section. Use water, if the material is fresh; if preserved, use the preservative. Some use a brush in removing sections from the knife, but nothing is quite equal to one's finger; anyone who is in danger of a cut while performing this act is in need of this little practice in manual dexterity. WOODY AND HERBACEOUS SECTIONS Safranin and Delafield's haematoxylin. — In order to make the di- rections as explicit as possible, let us follow the processes from collect- ing the material to labeling the slide. The rhizome of Pier is aquilina is a good object to begin with. Dig down carefully until the rhizome is exposed ; then with a sharp knife cut off pieces a foot in length, wrap them in wet paper, and bring them into the laboratory. If the rhizome has been cut carelessly or pulled up, as is usually the case, the finished mount will show ruptures between the bundles and bundle sheaths, disfiguring what should have been a beautiful preparation. While the material is still fresh and moist, cut the sections, placing them in water as fast as they are cut. When through with the cutting rinse the sections in water and transfer to 95 per cent alcohol, where FREEHAND SECTIONS 89 they should remain for at least 30 minutes— an hour, or even over- night, does no damage. Pour off the alcohol and pour on a 50 per cent alcoholic solution of safranin (a 1 per cent solution of safranin in 50 per cent alcohol). Stain overnight, or even for 24 hours. Pour off the safranin (which may be used repeatedly) and pour on 50 per cent alcohol. The alcohol will gradually wash out the safranin, but this stain is washed out more rapidly from cellulose walls than from those which are lignified. The sections should remain in the al- cohol until the stain is nearly — but not quite — washed out from the cellulose walls, while still showing a brilliant red in the large lignified tracheids. If 5 or 10 minutes in the alcohol draws the safranin from the lignified walls as well as the cellulose, stain longer; if the differenti- ation is not secured in 5 or 10 minutes, a small drop of hydrochloric acid added to the alcohol will hasten the process. Some recommend staining for only 1 or 2 hours, but the washing-out process is likely to be rapid and uncertain. Pour off the alcohol and wash the sections thoroughly in ordinary drinking-water. The washing should be particularly thorough if acid has been used to hasten the previous process, for the preparations will fade if any acid remains. Stain in Delafield's haematoxylin 3-30 minutes. Usually 5 minutes will be about right. Delafield's haematoxylin will stain the cellulose walls, but will have little or no effect upon lignified structures. Transfer to drinking-water, not distilled water. The red color of the whole section, as it appears to the naked eye, will be rapidly replaced by a rich purple. Continue to wash in water for 2 or 3 minutes after the purple color appears. If the cellulose walls show only a faint pur- plish color, put the sections back into the stain and try a longer period. If the color is a deep purple or nearly black, add a little hydrochloric acid (1 drop to 50 c.c. is enough) to the water. It is better to put the drop into a bottle of water and shake thoroughly before letting the acidified water act upon the sections. As soon as the sections begin to appear reddish, which may be within 4 or 5 seconds, pour off the acidified water and wash in drinking-water, changing the water 3 or 4 times a minute, until the reddish color caused by the acid has been re- placed by the rich purple color so characteristic of haematoxylin. The acid not only secures differentiation by dissolving out the stain from lignified structures more rapidly than from cellulose walls but it also removes the disfiguring precipitates which almost invariably accom- 90 METHODS IN PLANT HISTOLOGY pany staining with Delafield's haematoxylin. The acid also washes out the safranin; it is for this reason that the washing after safranin should be stopped while there is still some red color in the cellulose walls. The acid should not only reduce the density of the haematoxylin and remove precipitates but should also remove the little safranin which may remain in the cellulose walls. After the purple color has ap- peared, the sections should be left in water for 20 or 30 minutes, changing frequently. They might be left in the water for several hours. The acid must be washed out thoroughly or the stains will fade. Now place the sections in 50 per cent alcohol for 1 minute, then in 95 per cent alcohol for 1 minute, 100 per cent alcohol for 5 minutes, and then transfer to xylol. As soon as the sections become clear — in about 1-5 minutes — they are ready for mounting in balsam. If the o Fig. 18.— The label sections do not clear readily, as may be the case if the air is damp, or if the alcohol or xylol is not quite pure, transfer from the absolute alcohol to clove oil, which will clear, even if the absolute alcohol is rather poor. Then transfer from clove oil to xylol; the objection to mounting directly from clove oil is that preparations harden more slowly than when mounted from xylol. With a section-lifter, or scal- pel, or brush, transfer 3 or 4 sections to a clean, dry slide, put on 1 or 2 drops of balsam, and add a cover, first heating it gently to remove moisture. If xylol has been used for clearing, it is necessary to work rapidly; for the sections must never be allowed to dry. Use square or oblong covers for such mounts, reserving round covers for glycerin mounts^ If material is abundant, use as many sections as you can cover conveniently. If you have used several stains with the same material, select for each mount sections from the different stains. In ordinary wood sections each mount should show the three most im- portant views, transverse, longitudinal radial, and longitudinal tan- FREEHAND SECTIONS 91 gential sections. It is wasteful to use three slides and three covers to show these three views, or to make a mount containing only a single section of the rhizome of Pteris. Put the label at the left. Write first the genus and species; then indicate what part of the plant has been mounted. The date on which the material was fixed is often valuable. In many cases, the date of collecting the material is desirable. The beginner is likely to write also the stains used, and other details, which he will find quite unnecessary after a little experience. Figure 18 illustrates a good style of labeling and mounting. The following is a convenient summary of the foregoing processes, beginning with the sections in 95 per cent alcohol : 1. Sections in 95 per cent alcohol. 2. Safranin, 12-24 hours. 3. Fifty per cent alcohol, with or without acid, until color is right, generally about 2-10 minutes. 4. Water, 5 minutes, changing frequently. 5. Delafield's haematoxylin, 3-30 minutes. 6. Water, 5-10 minutes, changing frequently. 7. Water slightly acidulated, 5-10 seconds. 8. Water, to wash out acid, 20-30 minutes. 9. Fifty per cent alcohol, 5 minutes. 10. Ninety-five per cent alcohol, 5 minutes. 11. One hundred per cent alcohol, 5 minutes. 12. Xjdol, 5 minutes. 13. Balsam. 14. Cover and label. If clove oil seems necessary, finish as follows: 12. Clove oil, 2-5 minutes. 13. Xylol, 5 minutes. 14. Balsam. 15. Cover and label. Another method has given excellent results, especially with old stems, pieces of dry boards,' etc. Cut pieces 5-10 mm. square and 2-3 cm. long, boil in water 10-24 hours and, when cool, transfer to equal parts of 95 per cent alcohol and glycerin. Material may be left in this mixture indefinitely. Hard woods should remain here for at least two weeks before cutting, since the mixture is a good preservative and hard woods left in it for a year are easier to cut. 92 METHODS IN PLANT HISTOLOGY After sections have been cut, wash them in tap water, then in dis- tilled water, and stain half an hour or more in weak Delafield's haema- toxylin — about 5 parts of the solution, as given in the formula, to 100 parts of water — and then wash in distilled water. Stain in weak saf- ranin — about 2 parts of the stock solution to 100 parts of water — overnight or even for several days. Wash in tap water, then wash in 95 per cent alcohol for 30 seconds or longer, according to the appear- ance of the stain. Dehydrate in absolute alcohol, clear in clove oil, transfer to xylol, and mount in balsam. This method is very good for gymnosperm woods. Since it usually happens that processes are commenced, but cannot be completed, at a single laboratory period, it is necessary to know where sections may be left for several hours or until the next day with- out suffering injury. At 1, 2, or the pure water of 8 in the schedule given above, sections may be left until the next day. If it is not de- sirable to mount all of the sections which have been prepared, they may be kept indefinitely in clove oil or xylol. If the sections are to re- main for a year or more in the clearing agent, xylol is to be preferred. Shells with good corks are best for keeping such material. For the study of vascular anatomy, this is the most permanent stain which has come into general use. More recently, safranin combined with anilin blue or with light green has been coming into favor. Both these methods will be described. Safranin and anilin blue. — Use the alcoholic safranin already de- scribed, and a 1 per cent solution of anilin blue in 90 per cent alcohol. With this combination we should recommend a long stain in saf- ranin, not less than 24 hours. Wash in 50 per cent alcohol, but do not extract all the safranin from the cellulose walls. Stain 2-10 minutes in anilin blue. Rinse a few seconds in 95 per cent alcohol, then treat for about 5 seconds with 95 per cent alcohol slightly acidulated with hy- drochloric acid— one drop in 50 c.c. of alcohol may be enough. The weak blue should at once change to a bright blue and, at the same time, the acid will remove some of the safranin. It is for this reason that we proceed while the sections are still somewhat overstained in safranin. Wash for 1 or 2 minutes in 95 per cent alcohol to remove the acid. A trace of sodium carbonate, just enough to make the alcohol alkahne, may be added to the 95 per cent alcohol. If any acid remains, the safranin will fade. Dehydrate in absolute alcohol 5 minutes, clear in xylol, or first in clove oil and then in xylol, and mount in balsam. FREEHAND SECTIONS 93 For convenient reference, the process may be summarized, but it must be remembered that all the schedules are intended merely to introduce the beginner to the method. 1. Sections in 95 per cent alcohol. 2. Stain in safranin, 24 hours. 3. Fifty per cent alcohol until the stain becomes weak in cellulose walls, but not until it is removed entirely. 4. Anilin blue, 2-10 minutes. 5. Ninety-five per cent alcohol, 2-5 seconds. 6. Ninety-five per cent alcohol, slightly acidulated with hydrochloric acid, 5 seconds. 7. Ninety-five per cent alcohol, with or without a trace of sodium carbonate, 1 or 2 minutes. 8. Absolute alcohol, 1-5 minutes. 9. Xylol, 5 minutes. The xylol may be preceded by clove oil. 10. Mount in balsam. Lignified and suberized walls should stain bright red, and cellulose walls, bright blue. To make this beautiful combination a success, it is necessary to be very careful. If too much safranin is extracted at stage 3, the acid at stage 6 will still further weaken the red stain and the contrast will not be sharp. Safranin and light green (Land's schedule). — This is another beauti- ful combination, and the student should be successful from the first, since the light green is simpler to apply than either Delafield's haema- toxylin or anilin blue. Land uses either aqueous, anilin, or alcoholic safranin, and uses the light green in clove oil, or in a mixture of clove oil and absolute alcohol. Make a saturated solution of light green in clove oil. Since the solu- tion takes place slowly, the mixture should stand several days before using. If a small quantity of absolute alcohol be added to the clove oil, the stain dissolves more readily. For some structures the stain is more brilliant than with the simple clove-oil solution. Sections from fresh material are fixed in 95 per cent alcohol; sections from preserved material are rinsed in alcohol or water before staining. The following schedule will summarize the method: 1. Safranin, 2-24 hours. 2. Fifty per cent alcohol, until differentiated. 3. Dehydrate in 95 and 100 per cent alcohol. 4. Light green (in clove oil), 3-30 minutes. 94 METHODS IN PLANT HISTOLOGY 5. Xylol: 2 or 3 c.c. of absolute alcohol may be added to each 100 c.c. of xylol, if the free light green shows a tendency to precipitate, 6. Mount in balsam. This stain is particularly good for phloem. Since the light green is not likely to overstain and only slightly weakens the safranin, the combination is a rather easy one, so that even the beginner can hardly fail to get a good preparation. Malachite green and Congo red. — I am indebted to Dr. Sharp for this method, which has been popular in Professor Gregoire's labora- tory at Louvain. Sections of fresh material should be treated with 95 per cent alco- hol and then transferred to water. 1. Three per cent aqueous solution of malachite green or methylene blue, G hours or more. 2. Wash in water. 3. Congo red, 1 per cent aqueous solution, 15 minutes. 4. Wash in water. 5. Rinse in 80 per cent alcohol. As soon as the malachite green or anilin blue color appears through the red, transfer quickly to 6. Absolute alcohol. 7. Xylol. 8. Balsam. Iodine green and acid fuchsin is another good combination for such sections. The stain will be particularly brilliant if sections from fresh material are fixed in 1 per cent chromo-acetic acid for 24 hours; and then washed for an hour in water. Beginning with the sections in water, the procedure is as follows : Stain in aqueous iodine green for 12-24 hours. Then wash in water until the stain is nearly all washed Out from the cellulose walls but is still brilliant in the lignified walls. If the stain acts for too short a time, the washing-out process necessary to remove the stain from the cellulose walls will leave only a pale green color in the lignified walls. Stain in aqueous acid fuchsin for 2-10 minutes. This should stain the cellulose walls sharply, but should not act long enough to affect the lignified tissues. Pour off the stain (which may be used repeatedly), and pour on 95 per cent alcohol, and immediately pour it off and add absolute alcohol. The 95 per cent alcohol should not act for more than 5 or 10 seconds, its only function being to save the more expensive absolute alcohol. From 10-30 seconds will usually be long enough for FREEHAND SECTIONS 95 the absolute alcohol. Too long a period in the alcohols will weaken the stain. Clear in xylol or clove oil, and mount in balsam. If a 50 or 70 per cent alcoholic solution of iodine green has been used, the stain should be washed out in 50 per cent alcohol; otherwise the treatment is the same. Methyl green (aqueous solution) and acid fuchsin is a good com- bination, and the student may find it easier to get a good differentia- tion than with iodine green. Follow the directions for the aqueous iodine green and acid fuchsin. It may be necessary to wash more rapidly, since the methyl green is easily extracted. Safranin and gentian violet. — This is a good combination for vascu- lar anatomy. Stain overnight in safranin, rinse in 50 per cent alcohol until the stain is reduced to a light pink color in the cellulose walls; then rinse in water and stain 5-10 minutes in aqueous gentian violet or crystal violet. Rinse in water, dehydrate in 95 and 100 per cent alcohol, differentiate and clear in clove oil, transfer to xylol, and mount in balsam. Orange may be added to this combination, making a triple stain. In this case, do not reduce the safranin at all, but rinse quickly in 50 per cent alcohol, then in water, stain in gentian violet, rinse in 95 per cent alcohol, and stain for 1 or 2 minutes in orange dissolved in clove oil. This will not only reduce and differentiate the gentian violet, but will reduce the safranin. Transfer to xylol and mount in balsam. If the safranin is drawn out too rapidly, stain for 15-30 seconds in the orange, transfer to clove oil without any orange until the gentian violet is satisfactory; then transfer to xylol and mount in balsam. Both the violet and the orange may be used in clove oil. In this case, transfer to the violet from absolute alcohol, and from the violet to the orange ; then to pure clove oil until the violet looks right. Trans- fer to xylol and mount in balsam. The bordered pits of conifers, the Bars of Sanio, and the middle lamella are beautifully stained by this method. For the Bars of Sanio, it may be necessary to stain in the clove oil violet over night, or even for 24 hours. Other combinations might be suggested, e.g., iodine green or methyl green with Bismarck brown, methyl green with Delafield's haematoxylin; orange G might be added after the safranin and Dela- field's haematoxylin, and various other stains might be tried. In dou- ble staining it is usually best to combine a basic with an acid stain. 96 METHODS IN PLANT HISTOLOGY Green and red make a good contrast, but a section stained with iodine green and safranin would be a failure, because both stains would stain the xylem and neither would stain the cellulose. Both stains are basic. Red lignin and green cellulose could be secured by using safranin and acid green. Green lignin and red cellulose as already indicated, can be got with iodine green and acid fuchsin. The time required for the different processes varies greatly, and the time required for a subsequent process is often more or less dependent upon the time given to processes which preceded it. Good mounts of sections of the petiole of A'uphar advena have been secured from ma- terial which had been cut, fixed, stained in safranin and Delafield's haematoxylin, and mounted in balsam, the entire time being less than 30 minutes. This is an extreme case, and nothing is gained, except time, and the saving of time is apparent rather than real, for the his- tologist always has something to do while the sections are in the stain. Preserved, fresh, and dry material. — If sections are to be cut from material preserved in formalin, the piece should be washed in water, since the odor is annoying and the fumes are injurious to the eyes, and the acid in the formalin interferes with most stains. The sections are placed in the stain from water. Sections from alco- holic material are transferred directly to the stain. If the material is in a mixture of alcohol and glycerin, the sections should be washed in water or 50 per cent alcohol until the glycerin has been removed before transferring to the stain. Some material cuts well when fresh, but cuts with difficulty when preserved. On the other hand, some material cuts well when pre- served, but hardly at all when fresh. Some material which is too soft to cut when fresh can be cut with ease after it has been in formalin alcohol for a week or more. Oak, hickory, maple, and other hard woods need special treatment because they are so hard to cut. It is a good practice to boil the ma- terial in water and treat with hydrofluoric acid before any sectioning is attempted. The following is the usual method when this acid is used: Cut the material into blocks 5 or 6 mm. square and 2 or 3 cm. long and boil in water for several minutes; then transfer to cold water and, after several minutes, repeat the boiling. The alternate boiling and cool- ing, which should be repeated several times, drives out the air. Trans- fer to equal parts of commercial hydrofluoric acid and water. From 1 to 3 weeks will be enough for most woods. Some oaks, ebony, apple. FREEHAND SECTIONS 97 etc., may require a longer time and the acid may be used pure. Three days in 10 per cent acid may be enough for corn stems. Wash thor- oughly in water for a day or two. Then leave in equal parts of 30 per cent alcohol and glycerin for several days before cutting. Material may be left indefinitely in the mixture of glycerin and alcohol. An article by Dr. La Dema M. Langdon, dealing with the prepara- tion and sectioning of hard, woody tissues, appeared in the Botanical Gazette of July, 1920. By her method, hard, woody tissues are softened so that they cut readily and can even be imbedded in paraffin and cut successfully. OBJECTS MOUNTED WITHOUT SECTIONING Fern prothallia, mounted without sectioning, make very useful preparations. Select desirable stages and fix in chromo-acetic acid for 24-48 hours (chromic acid, | g.; acetic acid, 3 c.c; water, 100 c.c). Wash in running water 6 hours or overnight; stain in Delafield's hae- matoxylin for 5-30 minutes; wash in water slightly acidulated with hydrochloric acid for a few seconds, and then wash thoroughly in tap water. The prothallia must now be brought through a graded series of alcohols— 2i, 5, 7i 10, 20, 35, 50, 70, 85, 95, and 100 per cent. About 2 hours should be the minimum in each grade, except 85, where 24 hours is better, since this is the best place to complete the harden- ing. About 2 hours will be long enough for the 95 and 100 per cent. Then use mixtures of alcohol and xylol. Then use a series of xylol- alcohol, beginning with 5 parts xylol to 95 parts absolute alcohol. The foUowing series seems to be close enough: 5, 10, 20, 35, 50, 75, pure xylol. An hour in each may be sufficient. Then bring the prothallia into a mixture of xylol and balsam, using at least 10 parts of xylol to 1 of balsam. If left in a shell, without cork- ing, the xylol will soon evaporate, so that in a few days the prothallia may be mounted. Use the balsam in which the material has been standing, because any other balsam may have a different concentra- tion. At every step in the process the prothallia should be examined under a microscope, so that any shrinking may be detected. If each succeeding step is tested with a single prothallium, a general disaster may be avoided. If plasmolysis takes place, weaken the reagent and try another prothallium. When a safe strength is found, bring on the bulk of the material, and use the same method with succeeding steps. The dangerous places are likely to be the transfer from alcohol to 98 METHODS IN PLANT HISTOLOGY xylol and the transfer from xylol to balsam. The process is tedious, but the mounts are very firm and durable. The Venetian turpentine method is less tedious and is likely to produce better results than the method just described. Fix in the chromo-acetic mixture just men- tioned. Stain some prothallia in iron-alum haematoxylin and some in phloxine and anilin blue. When both have reached the thick turpen- tine, fine preparations can be made by mounting prothallia from both lots under the same cover. Sori of ferns. — Instructive mounts of sori or of individual sporan- gia may be made without sectioning. It is better to choose ferns with thin leaves, since leaves thicker than those of As-plenium thelypteroides are likely to be unsatisfactory. If this fern is at hand, cut off several of the small lobes which bear from three to six pairs of sori. Fix in chromo-acetic acid; wash in water; stain in Delafield's haematoxyhn, or omit staining altogether; pass through a series of alcohols, allowing each grade to act for at least 10 minutes; clear in clove oil; and mount in balsam. If the sori have begun to turn brown, better views of the annulus will be obtained without staining. Mosses and liverworts. — Nearly all mounts are more successful by other methods, for which the student should consult the chapters on Bryophytes (chaps, xxi and xxii). Excellent mounts of the peristome of the moss can be made as follows : From fresh or preserved capsules cut ofif the peristome just below the annulus. Treat with 95 per cent alcohol 10 minutes, absolute alcohol 10 mmutes, clear in clove oil or xylol, and mount in balsam. It is a good plan to put at least three peristomes on a slide, one with the outside up, one with the inside up, and another dissected to show details of the teeth. Fairly good unstained mounts of the archegonia and antheridia of small mosses can be obtained by following the directions for mounting the sori of ferns. Beautiful and instructive mounts of the more delicate foliose Jungermanniaceae can be made by staining lightly in Delafield's hae- matoxylin whole plants, or pieces as long as can be covered conven- iently. The method is that just given for fern prothallia. The mount should show both dorsal and ventral views. The epidermis shows its best surface views without sectioning. Se- lect some form with large stomata, hke Lilium or Tulipa, strip pieces of epidermis from both sides of the leaf, and place them immediately in absolute alcohol for 10 minutes. Stain in Delafield's haematoxylin; FREEHAND SECTIONS 99 after this stain is satisfactory and all acid has been washed out, stain for 1 or 2 minutes in aqueous eosin, erythrosin, or acid fuchsin; place directly into 95 per cent alcohol for a few seconds (merely to save the absolute alcohol), then into absolute alcohol for about 30 seconds, and then into clove oil. Mount in balsam. The epidermis is likely to curl and, unfortunately, patience seems to be the only remedy. In mount- ing, be careful to get pieces from both sides of the leaf, and be sure that the pieces are outside up. The inside of the epidermis is usually more or less rough, on account of the mesophyll torn off with it. Sedum piirpurascens will show various stages in the development of stomata, even in epidermis stripped from mature leaves. The epidermis of the Sedums strips off very easily. If the large Sedum maximum is avail- able, it is not difficult to strip off pieces 2 or 3 centimeters wide and several centimeters long. There is not much tendency to curl. The pieces may be spread out flat in a Petri dish, fixed in the chromo- acetic-osmic solution, just recommended for fern prothallia, or in this solution without the osmic acid. Wash in water, stain in Delafield's haematoxylin and eosin, or in safranin and gentian violet. Then wash in water and run up through a series of alcohols — 5, 10, 20, 35, 50, 70, 85, 95, and 100 per cent — about 30 minutes in each grade, except 85, which should be allowed to act for a couple of hours or overnight. Then transfer to clove oil, xylol, and balsam. This is a more tedious method, but it is worth the trouble. We have had the best results with the haematoxylin and eosin combination. Other objects. — The cases just given will suggest other objects which might be mounted by such methods. Nearly all objects which used to be mounted in balsam without sectioning are now handled successfully by the \>netian turpentine method. CHAPTER VII THE GLYCERIN METHOD Mounting in glycerin, once a very popular method, has become al- most obsolete. In its day, it was very good for unicellular and fila- mentous forms and for various small objects; and we still use it for moss protonema, which keeps the natural green and brown colors for years. The transfer from glycerin to glycerin jelly is easy and safe, and glycerin jelly has considerable consistency, so that it is easy to seal. We ahnost never mount in glycerin, but transfer to glycerin jelly. The glycerin method, except the final mounting, also constitutes the first part of the Venetian turpentine method and, consequently, it is neces- sary to learn the capabilities and limitations of glycerin. The method, from fixing to mounting, as used in connection with staining and without staining, will now be described. Stained preparations. — The familiar Spirogyra is a good form to begin with. Fix in chromo-acetic-osmic solution (| g. chromic, 3 c.c. acetic acid, 6 c.c. 1 per cent osmic acid, and 90 c.c. water); or in a chromo-acetic solution without osmic acid (1 g. chromic, 3 c.c. acetic acid, and 96 c.c. water). Fix 24 or 48 hours and wash in running water for 24 hours. At this point it is the usual practice to stain and transfer to 10 per cent glycerin; but preparations are more nearly perfect if the material receives more hardening than the chromic fixing agents can give it, before it goes into the stain. Therefore, run it up, as if it were to be imbedded in paraffin, until it reaches 85 per cent alcohol. The following series is recommended: 2|, 5, 7|, 10, 15, 20, 30, 40, 50, 70, and 85 per cent, with ^ hour in each of the first five grades; 2-4 hours each in 20 and 30; 5 or 6 hours each in 40 and 50; all day or overnight in 70 ; 24 hours in 85. The 85 per cent completes the hardening so that, with reasonable care, the subsequent processes do little or no damage. After the hardening in 85 per cent alcohol, run back to water through the same series of alcohols, with one hour in each grade down to 30; below 30, 20 minutes in each grade; water, 20 minutes, and then stain. The most generally satisfactory stain is Haidenhain's iron-alum haematoxylin. If there was any osmic acid in the fixing agent the 100 THE GLYCERIN METHOD 101 material must be bleached in hydrogen peroxide or in chlorine before proceeding with the stain (see p. 27). After bleaching and washing in water, treat with 2 per cent iron-alum, 4 hours; wash in water, 30 minutes; stain in § per cent haematoxylin overnight or 24 hours; wash in water, 30 minutes; and transfer again to iron-alum. This time, the iron-alum will extract the stain. The rapidity of the action of the n-on- alum will now depend upon the fixing agent. If it contained 6 or 7 c.c. of 1 per cent osmic acid to 100 c.c. of the solution, it may require an hour, or even more, to make a satisfactory differentiation of the stain. If there was no osmic acid in a chromic fixing agent, the differentiation may be complete in 10 minutes. If the stain becomes too weak in 4 or 5 minutes, use 4 per cent iron-alum as a mordant, stain longer, and use 1 per cent iron-alum for the second treatment. Haidenhain's iron- alum haematoxylin gives its most brilliant results when chromic mix- tures with 5-7 c.c. of 1 per cent osmic acid to 100 c.c. of the chromo- acetic acid have been used. Different species of Spirogyra and even different collections, fixed in the same reagent, will differ in their reaction to stains; and different unicellular and filamentous forms in different fixing fluids, will present so much difference in times that only general suggestions can be given. When the stain is satisfactory, wash in running water for an hour. If this second iron-alum is not washed out thoroughly, its continued action will cause the preparation to fade. Put the material into 10 per cent glycerin (1 part glycerin and 9 parts water), and then allow the water to evaporate gradually in a place as free from dust as possible. Nothing is better than a Petri dish for this purpose, because it presents a large surface for the evaporation of the water in the mixture. If there is much dust, cut a piece of filter paper just the size of the dish and let it float on the 10 per cent glycer- in. The liquid will soak through the paper and evaporate without ex- posing the material itself to the dust. The process may be hastened, safely, by warming up to 35° C. The temperature of a paraffin bath — 45° to 52° C. — causes such rapid evaporation that the material is likely to shrink. The concentration from 10 per cent glycerin to pure glycerin should not require less than three days. This time is easily regulated by the amount of 10 per cent glycerin and the size of the exposed sur- face. When the glycerin has become about as thick as pure glycerin, the material is ready for mounting. Place a small drop of glycerin, with 102 METHODS IN PLANT HISTOLOGY the material, in the center of the slide, taking care not to put on so much that there will be a confusing tangle. Use scissors constantly so as not to injure filaments by trying to tease them out. Put on a round cover. There should he just enough glycerin to come to the edge of the cover-glass, but not any more, for it is impossible to seal a mount if glycerin has oozed out beyond the cover. The mount should now be sealed. Canada balsam, various asphalts, cements, fiat varnish, gold size, and other things have been used. Canada balsam is always at hand and seems to be as good as any. Preparations which had been sealed with gold size more than fifty years before have been exhibited in perfect condition, but they must have been hidden away in some museum, for a glycerin mount would never survive fifty years of laboratory use. The gold size, as painters L Fig. 19. — Slide, natural size, showing size and form of ring use it, is likely to be too thin for sealing mounts. Put some of it in a 1-ounce bottle with a wide neck and leave the cork out until the gold size thickens a little. Should it become too thick, thin it with turpen- tine. Nothing but practice will enable one to spin a good ring, but a good camel's-hair brush, a good turntable, and a balsam neither too thick nor too thin will facilitate matters. Gently touch the cover and slide at three or four points with the tip of the brush, so that a very small drop of balsam will bind the cover to the slide. In half an hour the tiny drops of balsam will have hardened sufficiently for the next step, the spinning of the ring. Clip the slide to the turntable so that the cover glass is perfectly concentric with the rings, give the turntable a gentle spin, and with the brush touch the shde as far out from the cover as you wish the ring to extend, then gradually approach the cover. Dip the brush in the balsam again, and gradually extend the ring until it is about iV inch wide on the cover. The touch must be extremely gentle or the cover will be moved. Do not try to put on a thick ring the first time, but let a thin ring harden for an hour (months would do no damage), and then a thicker ring can be added without any danger. Thin rings are too Hkely to be broken, and thick rings are in the way if the preparation is to be examined with high powers. A medium ring is THE GLYCERIN METHOD 103 best, and it should consist of two coats, for a crack would seldom appear at the same place in both coats. A good shape and thickness for a ring are shown in Figure 19. The following is a summarj^ of the foregoing processes: 1. Fix in chromo-acetic-osmic acid, 24 hours. 2. Wash in water, 24 hours. 3. Alcohols, 2^-85 per cent. 4. Alcohols, 85-2| per cent. 5. Wash in water, 30 minutes. 6. Iron-alum, 4 hours. 7. Wash in water, 30 minutes. 8. Haematoxylin, | per cent, 24 hours. 9. Wash in water, 30 minutes. 10. Iron-alum until the stain is satisfactory. 11. Wash in water, 30 minutes. 12. Ten per cent glycerin. 13. Mount and seal. 14. Label. The times just given may seem unnecessarily long, but one can al- ways do something between times. The following summary, taken from the fourth edition of this book, is much shorter and it gives good results, unless you compare the slides with those made by the longer schedule, just as ordinary glass looks good, unless you put it side by side with plate glass : 1. Fix in cln-omo-acetic-osmic acid, 24-48 hours. 2. Wash in water, 24 hours. Bleach and wash in water. 3. Iron solution, 2 hours. 4. Wash in water, 10 minutes. 5. One-half per cent haematoxylin, 3-24 hours. 6. Wash in water, 10 minutes. 7. Iron solution until stain is right. 8. Wash in water, 1 hour. 9. Ten per cent glycerin. 10. Mount and seal. If material has been fixed in formalin, it should be washed in water for 30 minutes before starting with stage 3 of the long schedule, or the first iron-alum of the short schedule. Material preserved in formalin- alcohol-acetic acid, with 70 per cent alcohol, should be run down to water before staining. If the alcohol is only 50 per cent, put the 104 METHODS IN PLANT HISTOLOGY material in 70 and 85 per cent, a day in each, and then run down to water. Mayer^s haem-alum is also a good stain for filamentous algae and fungi which are to be mounted in glycerin. The process, after fixing and washing in water, is as follows: 1. Transfer to the stain from water. It is seldom necessary to stain longer than 10 minutes. As a rule, it is better to dilute the stain (about 1 c.c. to 10 c.c. of distilled water) and allow it to act for 10 hours or overnight. 2. Wash in water, 20 minutes. 3. Ten per cent glycerin until sufficiently concentrated. 4. Mount and seal. Eosin is a good stain for many algae and fungi which are to be mounted whole, if sharp outlines rather than cell contents are to be brought out. After material has been fixed and washed in water, run it up to 85 per cent alcohol and back to water. Stain in an aqueous solu- tion of eosin for 24 hours; pour off the eosin, which can be used repeat- edly, and pour on a 1 or 2 per cent solution of acetic acid in water. Pour this off and pour on some more of the acid, until very little stain washes out. The process may require 2-5 minutes. Then place in 10 per cent glycerin containing about | per cent acetic acid, and allow the glycerin to concentrate. The acetic acid is to prevent the stain from washing out. When the glycerin has reached the proper concen- tration, mount and seal as before. The following is a rapid method for forms like Aspergillus and PenicilUum: Fix in 100 per cent alcohol about 2 minutes; stain in aqueous eosin 5 minutes; wash in water about 1 minute; fix in 1 per cent acetic acid 1 minute; then mount directly in 50 per cent glycerin to which about 1 per cent acetic acid has been added. It is hardly worth while to try this method with forms which have large cells; they are sure to collapse. If a form like Eurotium passes through the earher processes without danger, but collapses when put into the 50 per cent glycerin, put it into the 10 per cent glycerin and allow the glycerin to concentrate. Mounting without fixing or staining. — It is sometimes desirable to retain the natural color of an object. The chlorophyll green can usu- ally be preserved by mounting directly in glycerin without any previ- ous fixing. Other colors also are often preserved in this way. Moss protonema makes beautiful preparations by this method. If possible, THE GLYCERIN METHOD 105 select protonema showing the very young moss plants. The brown protonema and bi'own bulbils preserve their color perfectly. Wash the dirt away from the protonema, which is then placed in 50 per cent glycerin. Let the glycerin concentrate, transfer to glycerin jelly, and mount in the usual way. The method is very useful when one finds a single specimen of Pediastrimi, or any small form which would be lost in the more com- phcated processes. Place a large drop of 10 per cent glycerin on a shde; with a pipette, transfer the object to the drop, and allow the glycerin to concentrate. Then add a cover and seal the mount. GLYCERIN JELLY It is almost never necessary to mount anything in glycerin, because material can be transferred directly from glycerin to glycerin jelly. If the glycerin jelly is well made, it is quite firm and mounts will last for a year or two, without sealing; but it is better to seal them with balsam. A very good formula is known as Kaiser's gelatin. It is made as follows: One part by weight of the finest French gelatin is left for about 2 hours in 6 parts by weight of water; 7 parts of glycerin are added, and for every 100 grams of the mixture, 1 gram of concentrated carbolic acid. The whole is warmed for 15 minutes, stirring all the while until all the flakes produced by the carbolic acid have disap- peared. Filter while warm through a fine-mesh cheesecloth. To make a mount, take a small piece of the glycerin jelly, not more than half as large as a grain of wheat — the exact size will depend upon the material — warm it until it melts, and then transfer to it the mate- rial which has already been brought into thick glycerin. It is a good plan to touch the material to filter paper in order to remove as much glycerin as possible; for the less glycerin the firmer the mount will be. The mount may be sealed as soon as it is cool; but some prefer to let it stand for a week or two before sealing. In any case, it is a fairly firm mount, so that there is no danger of moving the cover. Everything which can be brought safely into pure glycerin can be mounted in glycerin jelly, and the preparation is much more stable than a glycerin mount. CHAPTER VIII THE VENETIAN TURPENTINE METHOD Venetian turpentine is made from the resin of Larix europea. It looks like Canada balsam and in many ways behaves like it; but it is readily soluble in absolute alcohol. Consequently, material can be transferred directly from absolute alcohol to Venetian turpentine, without passing through xylol or any similar reagent. The mounts are as hard and durable as balsam mounts and they become as trans- parent as if a clearing agent had been used. While the method was described by Pfeiffer and Wellheim in 1894, it received no recognition in the United States or even in Europe. I made a casual trial of it when preparing the first edition of this book more than thirty years ago ; but the preparations were such miserable failures that the process did not seem worth mentioning. The method was next brought to my attention during a demonstra- tion in Strasburger's laboratory at Bonn. He was using preparations of Zijgnema and Spirogyra, the staining of which surpassed anything I had ever seen. He remarked that it was not worth while to consult Pfeiffer and Wellheim 's lengthy article, because his preparations had been made by the authors and no one else had made a success of the method. However, when I returned, I made a careful study of the process, and finally learned to use it successfully. The details as given in that paper were too indefinite for practical use, but, after one has learned the method, the article can be read with profit. The practical advantages of the method are the elimination of the dangerous xylol stage, the hard durable mounts, and a greater variety of stains than can be used with glycerin. After fixing, washing in water, running up in alcohols from 2| to 85 per cent, running back to water, and staining in an aqueous stain, e.g., iron-alum haematoxylin, the process is as follows: 1. Ten per cent glycerin until concentrated. 2. Wash the glycerin out thoroughly in 95 per cent alcohol. 3. Complete the dehydration in 100 per cent alcohol. 106 THE VENETIAN TURPENTINE METHOD 107 4. Ten per cent Venetian turpentine in an exsiccator until the turpentine becomes thick enough for mounting. 5. Mount in the Venetian turpentine. Even after the method became established, there occurred a period of several years during which it was practically impossible to get a Venetian turpentine suitable for histological use. Consequently, it was necessary to resort to glycerin jelly or to try various schemes for bringing material into Canada balsam ; but good Venetian turpentines are again available and are even more satisfactory than those which established the method in popular favor. We have tested two brands which are giving uniform and excellent results: these are the "Venice Turpentine (True)," sold by the Fuller-Morrison Company, of Chica- go; and the ''Turpentine Venetian" (No. 2605), sold by the National Anilin and Chemical Company, of New York. There are probably other good turpentines and still others are likely to appear. The general outline, just given, is not sufficiently definite for a working introduction. The following concrete examples, describing the use of Venetian turpentine with an aqueous stain, with an alco- holic stain, and with a combination of aqueous and alcoholic stains, will be more practical than general directions. The steps from fixing to mounting, as used with an aqueous stain, will be described first, since this will introduce the method in its least complicated form. Heidenhain's iron-haematoxylin. — Using Spirogyra as a type, pro- ceed as follows: 1. Fix 24 hours in chromo-acetic acid. 1 per cent chromic acid 100 c.c. Glacial acetic acid 3 c.c. The volume of the fixing agent should be at least 50 times that of the material to be fixed. 2. Wash in running water 24 hours. (After this washing in water, the material may be run up to 85 per cent alcohol for hardening, and then back to water.) 3. Two per cent aqueous solution of iron-alum (ammonia sulphate of iron), 4 hours. ■» 4. Wash in running water, 20 minutes. 5. Stain overnight, or 24 hours, in ^ per cent aqueous solution haema- toxyUn. 6. Wash in water, 20 minutes. 108 METHODS IN PLANT HISTOLOGY 7. Two per cent aqueous solution of iron-alum, until the stain is satis- factory. This can be determined only by examining frequently under the microscope. 8. Wash in water, 2 hours. If this washing is not thorough, the continued action of the iron-alum will cause the preparations to fade. 9. Transfer to 10 per cent glycerin, and allow the glycerin to concentrate until it has the consistency of pure glycerin. It is not necessary to use an exsiccator. Merely put the glycerin into shallow dishes, and leave it exposed to the air, but protected from dust. If the material is in Petri dishes or other dishes with a large surface, 3 or 4 days will be sufficient. This process may be hastened by warming, if the temperature does not go above 35° C. If the reduction from 10 per cent glycerin to pure glycerin is accompUshed in less than 48 hours, the change in the con- centration is so rapid that material is likely to suffer. 10. Wash out the glycerin with 95 per cent alcohol. It will be necessary to change the alcohol several times. From 10 to 20 minutes will be sufficient if the alcohol is changed frequently. This alcohol cannot be used again for the same purpose, but it will be useful in cleaning one's hands and in cleaning dishes which have contained Venetian turpen- tine. 11. Complete the dehydration in 100 per cent alcohol: 10 minutes should be sufficient. 12. Most failures are now ready to occur. From the absolute alcohol the material is transferred to a 10 per cent solution of Venetian turpentine in absolute alcohol. The turpen- tine thickens as the alcohol evaporates, and when it reaches the con- sistency of pure glycerin the material is ready for mounting. The 10 per cent Venetian turpentine is very sensitive to moisture, and most failures are due to this characteristic ; consequently the concentration cannot be allowed to take place with the turpentine exposed to the air of the room. Use an exsiccator. This will not only absorb the moisture from the air, but will soon remove the alcohol from the turpentine mixture. Make an exsiccator as follows: Place a saucer full of soda lune (sodium hydroxide with Hme) on a plate of glass, and cover with a bell jar. This is a simple and effective exsiccator. Instead, you may simply scatter soda lime in the bottom of any low museum jar with tight-fitting cover. The tin cans, with tight covers, in which you get your pound of "Improved Vacuum Coffee" make good exsiccators for small amounts of material. You may improvise other forms ; the essen- THE VENETIAN TURPENTINE METHOD 109 tial thing is to provide a small, air-tight place in which the soda lime may work. Instead of soda lime you may use fused calcium chloride or the white sticks of sodium hydroxide. We are now ready for the transfer from absolute alcohol to the 10 per cent Venetian turpentine. Make the transfer quickly. Pour off the absolute alcohol and place the dish, with the material, in the exsicca- tor; then pour on the 10 per cent turpentine, and immediately put on the cover. This is better than to pour on the turpentine and then try to get the dish well placed in the exsiccator. The greater the surface of soda lime exposed, the more rapid will be the concentration of the Venetian turpentine. The concentration must not be too rapid. Not less than 2 days should be allowed for the concentration of 30 c.c. of the turpentine in an ordinary Minot watch glass. Great care must be taken not to let any of the soda lime, or other drier, get into the turpentine. When the lime has become saturated, it may be heated until dry, and then used again. If material is put into an exsiccator with nearly saturated lime, the turpentine becomes milky. If the material is very valuable, wash in absolute alcohol until entirely free from any milky appearance, and start again in 10 per cent turpentine. If the material can be replaced, throw away the milky stuff and start at the beginning. It may teach one not to put material into an exsiccator with half- saturated lime. In Tucson, Arizona, the Venetian turpentine method is easy, with no need for an exsiccator. Dr. J. G. Brown tells me that the air is so dry that the 10 per cent Venetian turpentine can be left exposed to the air until it concentrates, just as we leave the 10 per cent glycerin. As soon as the turpentine has attained the consistency of pure glycerin, it may be exposed to the air without any danger from mois- ture; but the turpentine would soon become too thick for mounting. If the turpentine has become too thick, thin it with a few drops of absolute alcohol or with 10 per cent or any thin solution of Venetian turpentine. Mount the material in a few drops of the Venetian turpentine and add a cover. Tapping on the cover with the handle of a needle or scalpel will often separate the filaments so that they are more con- venient for examination. Square covers may be used since it is entirely 110 METHODS IN PLANT HISTOLOGY unnecessary to seal the mounts, which are as hard and durable as those mounted in balsam. Material in the thickened Venetian turpentine, when not needed for immediate mounting, may be put into small bottles. The corks should be of the best quahty ; otherwise the turpentine will become too thick. While it can be thinned by adding thin turpentine, it is better, for easy mounting, not to let the turpentine become too thick. If the turpentine is only a little too thick, warming it gently will thin it enough for making mounts; but if any material is to be put away, a few drops of absolute alcohol or of a thin Venetian turpentine should be added. Material in Venetian turpentine, well corked and kept in the dark, does not fade or deteriorate in any way. Phloxine and anilin blue. — Fix in chromo-acetic acid and wash in water, as described in the previous schedule. Transfer from water to 10 per cent glycerin and allow the glycerin to concentrate. It is not necessary to use an exsiccator since there is no danger from moisture in the air. When the glycerin attains the consistency of pure glycerin, wash the glycerin out with 95 per cent alcohol. This washing must be very thorough; otherwise the staining will not be satisfactory. 1. Stain in phloxine. A double stain in Magdala red and anilin blue has sometimes given very satisfactory results; but, just as often, has been entirely worthless. The reason for the discrepancy seems to be that stains sold under the name of Magdala red are of various composition, some of them containing no Magdala red at all. The standardized stain, phloxine, seems to be identical with successful lots of Magdala red and results are rather uniformly successful. Make a 1 per cent solution of phloxine in 90 per cent alcohol and stain for 24 hours. 2. Rinse the material for a minute in 90 per cent alcohol. 3. Stain in anilin blue, using a 1 per cent solution in 90 per cent alcohol. We prefer to make a fresh solution every time we have anything to stain. It is not necessary to measure it. A little of the powder — about half the bulk of a grain of wheat— in 30 c.c. of 90 per cent alcohol, will give an efficient solu- tion. The time required for successful staining will vary from 3 to 30 minutes. Do not put all the material into the anilin blue at once, but, by trying a few filaments at a time, find out what the probable periods may be. 4. Rinse off the stain in 90 per cent alcohol, and then treat for a few seconds in acid alcohol (1 very small drop of HCl to 30 c.c. of 90 per cent alcohol). The acid alcohol fixes and brightens the anilin blue, but extracts the phloxine. If the anilin blue or the acid alcohol acts for too short a time, the blue will be weak; if they act too long, the red is lost entirely. If the blue overstains too THE VENETIAN TURPENTINE METHOD 111 much, wash it out in 95 per cent alcohol. The phloxine is not Hkely to over- stain. 5. Absolute alcohol, 5 or 6 seconds. 6. Transfer quicklij to 10 per cent Venetian turpentine and proceed as in the previous schedule. The surprising beauty of successful preparations will compensate for whatever failures may occur. Nuclei and pyrenoids should show a brilliant red, while the chromatophores and cytoplasm should be dark blue. The cell walls should show a faint bluish color. Heidenhain's iron-alum haematoxylin and eosin. — Follow the schedule for iron -haematoxylin until the glycerin has been washed out in 95 per cent alcohol. Then stain for an hour in a solution of eosin in 95 per cent alcohol. Wash for a minute in 95 per cent alcohol, then a minute in absolute alcohol, and then transfer to the 10 per cent Vene- tian turpentine. Heidenhain's iron-alum haematoxylin and safranin. — Follow the schedule for iron-haematoxylin until the glycerin has been washed out in alcohol, and then add to the 95 per cent alcohol several drops of a solution of safranin in 95 or 100 per cent alcohol and allow the stain to act for 30 minutes or an hour. Then dehydrate in absolute alcohol and transfer to 10 per cent Venetian turpentine. Other stains may be used. Aqueous stains should be used before starting with 10 per cent glycerin. Alcoholic stains should be in strong alcohol — about 90 per cent — and should be applied just after washing out the glycerin. This method is equally good for filamentous fungi and also for the prothallia of Equisetum and ferns, for delicate liverworts and mosses, and similar objects. If you have a good turpentine, good stains, arid avoid moisture, the Venetian turpentine method should not be difficult, and the results with filamentous and unicellular forms and other small objects surpass anything yet secured by other processes. CHAPTER IX THE PARAFFIN METHOD For studies which demand very thin, smooth sections, the paraffin method still holds the first place, with no near competitor. Some have added to the paraffin a little of this or that or the other, and these additions have corrected, somewhat, the imperfections of poor paraf- fins. A thoroughly good paraffin will yield smooth ribbons at 1 ^ and, in cold weather, ribbons f\ y. and even I n'm. thickness have been cut in our laboratory. Modern microtomes, while rather comphcated, give wonderful results and, to some extent, eliminate the element of skill. The microtome, shown in Figure 20, with the cooling attach- ment designed by Dr. Land, has cut even ribbons, Iju in thickness, from the antheridial receptacles of Marchantia. With the compara- tively inexpensive sliding microtome shown in Figure 2, page 9, smooth, even ribbons of root-tips have been cut as thin as | m- It is doubtful whether any other microtome, however expensive, has ever cut thinner or smoother sections. Both of these microtomes were de- signed by Mr, H. N. Ott, president of the Spencer Lens Company. With these microtomes, especially with the small sliding one, serial sections can be cut of pollen grains and spores too small to be seen by the naked eye. Many of the principles involved in this method are general in their application, and some of the processes are common to other methods. One who has mastered the paraffin method should have little trouble with any other method of preparing plant material for microscopic examination. The following are the stages from fixing material to the finished mount : KILLING AND FIXING As stated in the chapter on "Reagents" (chap, ii), the purpose of a kiUing agent is to bring the life-processes to a sudden termination, while a fixing agent is used to fix the cells and their contents in as near- ly the living condition as possible. The fixing consists in so hardening the material that the various elements may retain their natural condi- tion during all the processes which are to follow. Usually the same re- 112 THE PARAFFIN METHOD 113 agent is used for both killing and fixing. Zoologists, from humane mo- tives, may use chloroform for killing, while other reagents are used for fixing. In fixing root-tips, anthers, and other material for a study of mitotic figures, it is necessary that killing be very prompt. In a weak solution of chromo-acetic acid, nuclei which have begun to divide may complete the division, although the reagent might hinder nuclei from Fig. 20. — Spencer rotary microtome with electric motor, and Land's apparatus for temperature control. entering upon division. A strong chromo-acetic solution will increase the number of mitotic figures, and the chromo-acetic-osmic solution, given on page 28, will still further increase the number, and the pro- portion of anaphases w^ill be greater. Take the kilhng and fixmg fluids into the field. If one waits until the material is brought to the laboratory there may be some fixing, but it will, in many cases, be too late to do much killing. Material which has begun to wilt is not worth fixing. Material like Spirogijra, however, may be wrapped in several thicknesses of newspaper, placed 114 METHODS IN PLANT HISTOLOGY in a botany can and brought into the laboratory. Before fixing, it should be placed in water for half an hour. Such forms suffer more from lack of air when placed in a bottle or a can than from lack of water when wrapped in wet newspaper. Branches with developing buds may be brought in and kept in water. Cones of the cycad, Ceratozamia, sent from Jalapa, Mexico, have arrived in Chicago with cell division still going on at a rapid rate. But such cases are extremes; as a rule, take the killing and fixing fluids into the field. Always have the material in very small pieces, in order that the reagents may act quickly on all parts of the specimens. Pieces larger than cubes of 1 cm. should be avoided whenever possible. While one sometimes needs sections 2 or even 3 cm. long, it is not hkely to be necessary to fix pieces more than 4 or 5 mm. in thickness. For very fine work no part of the specimens should require the reagent to pene- trate more than 1 or 2 mm. For fixing agents of the chromic-acid series, the volume of the re- agent should be about 50 times that of the material. Fixing agents with alcohol as an ingredient will fix a larger propor- tion of material. It must be remembered that the water, which is al- ways present in living tissues, weakens the fixing agent. The time required for fixing varies with the reagent, the character of the tissue, and the size of the piece. About 24 hours is a commonly recommended period for chromic-acid solutions, but 2 or even 3 days will do no harm. Directions for making and using the various fixing agents are given in the chapters on "Reagents" (chaps, ii, xxxi). WASHING Nearly all fixing agents, except the alcohols, must be washed out from the material as completely as possible before any further steps are taken, because some reagents leave annoying precipitates which must be removed, and others interfere with subsequent processes. Aqueous fixing agents with chromic acid as their principal ingredient are washed out with water; aqueous solutions of corrosive subhmate are also washed out with water. Use running water whenever possible and, whenever running water is not available, change the water fre- quently. With both methods, the tubes with bolting silk on each end, or the tea filters from the five-and-ten-cent store will facilitate the washing. Very effective "bottles" for washing can be made of wire THE PARAFFIN METHOD 115 gauze. With shears, cut a piece about 8 cm. square; bend it into a cyhnder; solder along the edge; and solder in a circular piece for a bot- tom. Stand several of these in a dish about 6 cm. deep; let a gentle stream of water come in at the bottom, and wash for 24 hours. Alcoholic solutions should be washed out with alcohol of about the same strength as the fixing agent; picric acid, or fixing agents with picric acid as an ingredient, must not be washed out with water, but with alcohol, whether the picric acid be in aqueous or alcoholic solu- tion. HARDENING AND DEHYDRATING After the material has been washed, it is necessary to continue the hardening and also to remove the water. Alcohol is used almost en- tirely for these purposes. It completes the hardening and at the same time dehydrates, that is, it replaces the water in the material, an ex- tremely important consideration, for the least trace of moisture inter- feres seriously with the infiltration of the paraffin. The process of hardening and dehydrating must be gradual; if the material should be transferred directly from water to absolute alcohol, the hardening and dehydrating would be brought about in a very short time, but the violent osmosis would cause a ruinous contraction of the more delicate parts. In recent years, cytologists have been making the dehydration process more and more gradual. Twenty years ago most workers began the dehydration process with 35 per cent alcohol and used the series 35, 50, 70, 85, 95, and 100 per cent alcohol. Some placed an intermediate grade between water and 35 per cent alcohol. If plasmolysis — the tearing away of the protoplast from the cell wall — was avoided, the series was thought to be suffi- ciently gradual; but a series which may avoid plasmolysis may not be adequate if one is to study the finer details of cell structure. The following series is recommended: 2|, 5, 7|, 10, 15, 20, 30, 40, 50, 70, 85, 95, and 100 per cent. There is no particular virtue in the fractions: it is convenient to make 10 per cent alcohol, dilute with an equal volume of water for the 5 per cent, and dilute the 5 per cent with an equal volume for the 2| per cent. It will be noted that the series begins with very close grades and that the intervals are gradually in- creased. The claim is that, by beginning with very weak alcohols in close grades, more perfect dehydration can be secured at the end of the series. Various devices, like constant drip and osmotic apparatus, have been proposed to secure a more gradual transfer; but these have 116 METHODS IN PLANT HISTOLOGY no advantages, unless the mixture is very complete before it reaches the material. If the drops fall near the material, the liquid is in a con- stant turmoil. In passing through the graded series, it is not necessary to use a large amount of alcohol: 2 or 3 times the volume of the material is sufficient. The grades of alcohol may be used several times, but it must be remembered that pollen grains, fungus spores, starch grains, and vari- ous granules are likely to be left in the alcohol, so that it is wise to pour back through a filter each time, thus keeping the alcohols clean. As the alcohols absorb water from the material, they become weaker and weaker. If the various alcohols be poured in a large "waste alco- hol" bottle, when a couple of liters have been accumulated, the strength may be determined by testing with an alcoholometer. Then any grade of less strength can be made from this stock. The time necessary for each of the stages has not been determined with any certainty. We recommend three grades a day, morning, noon, and evening, for the first six grades; for the 30, 40, and 50, change twice a day, morning and evening; 85, at least 24 hours and better 48 hours, for this is the best grade in which to complete the hardening which will make the material able to withstand the subse- quent processes; 95, overnight or 24 hours; absolute, 24 hours, chang- ing two or three times. Material may be left in any of the grades over- night, or 24 hours. If it is to be kept in alcohol, leave it in 85 per cent but, where labor is no object, it is better to go on and imbed it in paraffin. CLEARING Let us suppose that the material has been thoroughly dehydrated, so that not the slightest trace of water remains. If the supposition chances to be contrary to fact, all the work which has preceded, as well as all which is to follow, is only an idle waste of time. The purpose of a clearing agent is to make the tissues transparent, but clearing agents also replace the alcohol. At this stage the latter process is the essential one, the clearing which accompanies it being incidental. The clearing, however, is very convenient, since it shows that the alcohol has been replaced and that the material is ready for the next step. Various clearing agents are in use. Xylol is the most generally em- ployed, and for most purposes it seems to be the best. Bergamot oil, cedar oil, clove oil, turpentine, and chloroform are used for the same THE PARAFFIN METHOD 117 purpose. Cedar oil and chloroform may, in some cases, be as good as xylol. Only a small quantity of the clearing agent is necessary, enough to cover the material being sufficient; but since the grades, except pure xylol, can be used repeatedly, it is better to use four or five times the bulk of the material. Filter as in case of the alcohols. The transfer from absolute alcohol to the clearing agent should be gradual, like the hardening and dehydrating processes. The most sue- * cessful workers have been making this transfer more and more gradual. Thirty years ago it was customary to transfer from absolute alcohol directly to xylol; then a mixture of equal parts of absolute alcohol and xylol was interpolated ; in the second edition of this book three grades were placed between the absolute alcohol and xylol. It is undoubtedly better to make the transfer still more gradual. The following series seems to be safe, 2|, 5, 10, 15, 25, 50, 75, and 100 per cent xylol. These mixtures of absolute alcohol and xylol can be made with sufficient accuracy without measuring in a graduate. The 50 per cent grade is made by mixing equal parts of absolute alcohol and xylol; the 25 per cent, by adding to the 50 per cent an equal volume of absolute alcohol ; make the 10 per cent grade from the 25 per cent by adding a little more than an equal volume of absolute alcohol ; in the same way, make the 5 per cent from the 10 per cent, and the 2| per cent from the 5 per cent. The different grades may be kept in bottles and may be used repeatedly. A couple of drops of safranin dissolved in absolute alcohol, added to the 50 or 75 per cent xylol, will color the material a little and will often be helpful in orienting after the imbedding in paraffin. Three grades a day, morning, noon, and night, will do for all the grades, except pure xylol. It will do no harm to leave the material overnight in any of the grades. The pure xylol should be allowed to act for 10-24 hours, with 3 or 4 changes. This xylol can be used to make up any of the lower grades. Throughout the dehydrating and clearing it is a good plan to keep the material in Number 4 shells, which are made from glass tubing about 25 mm. in diameter. Other clearing agents may be used, but the process must be just as gradual. THE TRANSFER FROM CLEARING AGENT TO PARAFFIN This should also be a gradual process. The most convenient method is to place a small block of paraffin in the pure clearing agent with the 118 METHODS IN PLANT HISTOLOGY material, but the block of paraffin should not rest directly upon the objects. Dr. Land uses coarse wire gauze, cut into strips about 15 mm. wide and tapered at both ends. The strip is then bent so that the pointed ends rest upon the bottom of the Number 4 shell, while the middle portion forms a flat table upon which the paraffin may rest. Dip the wire gauze table into xylol and then slip it carefully into the Number 4 shell. The table portion should be 10 or 15 mm. above the material, and there should be enough xylol to extend a few millimeters above the table. Place on the table a block of paraffin about equal to the volume of the xylol in the shell. The table not only prevents the paraffin from injuring the material by mechanical pressure but insures considerable diffusion before the mixture of paraffin and xylol reaches the specimens. After 24 hours (or several days, if time permits) at room temperature, place the shell on a pasteboard box — slide boxes are good — on top of the paraffin bath. Do not place the shell directly upon the metal of the bath, since it is better to minimize heat. As soon as the paraffin is dissolved, add some more, this time leaving the cork out, in order that the xylol may evaporate. About 24 hours on the top of the bath should be sufficient. THE PARAFFIN BATH This step is usually called infiltration, but when the transfer from the clearing fluid to paraffin is made gradually, as has just been indi- cated, the process of infiltration is already begun. It is now necessary to get rid of the xylol or other clearing agent. This is accomplished, to a considerable extent, by pouring off the mixture of xylol and paraf- fin and replacing it with pure melted paraffin. Pour off the pure paraffin immediately. This is important. You will notice that often, when the pure paraffin is poured on, a froth or scum will appear on the surface. Much of the xylol will be in this scum, and, if allowed to re- ma" n, it would diffuse into the mass and greatly prolong the time needed for infiltration. So, pour it off and add more pure paraffin, for some xylol remains in the tissues and must be removed. Dot not put the shell into the bath, but use a flat dish of some sort. The main object is to have a fairly large surface exposed, so that the remaining xylol may evaporate as rapidly as possible. Change the paraffin 3 or 4 times. A good 52° C. paraffin will yield smooth sections from | /x up to 20 n. Where thicker sections are needed, a 45° C. paraffin should be used. For many years we have used only a 52° C. paraffin. THE PARAFFIN METHOD 119 A good paraffin is an absolute necessity, if preparations are to be of the highest grade. Some brands of parowax are fairly good if sections do not need to be thinner than 10 m- For critical work, with sections from 5 m down to |/x, Griibler's 52° C. paraffin stands at the head of the list. It melts at the temperature indicated on the package. Since the price rises with the melting-point, many paraffins are marked higher than they really are. Some paraffins can be improved by heating almost to the boiling point for several hours. If any scum appears, skim it off; if anything settles to the bottom, pour the paraffin off gently and throw away the sediment. If, with prolonged heating, the paraffin takes on a slightly amber color, keep that paraffin for your best work, for it is likely to be good. The addition of bayberry wax — a piece about a centimeter square and 5 mm. thick — to a pound of paraffin is likely to improve any paraf- fin except the best. Dr. Land added asphalt and secured a paraffin which yielded In ribbons with a rotary microtome. Do not throw away the paraffin which you pour off, but put it in a waste jar or beaker, or, still better, in a small tin lard pail, in which you have made a lip to facilitate pouring. This can be placed in the bath, or, in winter, on the radiator, and the xylol will gradually evaporate. After long heating, the paraffin not only becomes as good as new but even better, since it becomes more homogeneous and tena- cious. If it contains dust or debris of any kind, it may be filtered with a hot filter. The time required varies with the character of the material and the thoroughness of the dehydrating and clearing. If this schedule has been followed up to this point, the time will be much shorter than most investigators now deem necessary. In dehydrating and clearing, material could be left overnight at any stage; but in the paraffin bath, the time must be reduced to the minimum. If 30 minutes is enough, an hour may be ruinous; if an hour is right, 2 hours may mean dis- aster. A few hints may be helpful. Fern prothallia of average size in- filtrate perfectly in 20-25 minutes; onion root-tips, in 30-45 minutes; ovaries of Liliimi philadelphicum or L. canadense at the fertilization stage, from 45 minutes to 1 hour; 5 or 6 mm. cubes of the endosperm of cycads, containing archegonia, 2-3 hours; median longitudinal sections of the ovulate cones of Pinus banksiana, 4 or 5 mm. thick, may re- 120 METHODS IN PLANT HISTOLOGY quire 6 or 8 hours; if serial sections through the entire cone are wanted, Dr. Hannah Aase found that the time must be prolonged to 24 or even 48 hours. Some particularly difficult material which will be mentioned in the chapter on "Special Methods," may require several days. When one is dealing with many lots of the same kind of material, as in research work, the time required for infiltration is easily determined. As a rule, minimize heat. It is, probably, never necessary to use paraf- fin with a melting-point higher than 52° C. With Land's cooling device sections 1 ^ in thickness can be cut from 52° C. paraffin. A few final hints may not be amiss. If the dehydration is not com- plete, it is practically impossible to replace the alcohol completely with xylol. Unless this replacement is complete, the infiltration with paraf- fin will be imperfect. Students are likely to prolong the time in the paraffin bath in a vain attempt to force paraffin into a tissue which still contains some xylol. When the alcohol series and xylol series have done their work perfectly, the time in the bath is likely to be shorter than most investigators allow for this stage. IMBEDDING Material may be imbedded in paper trays or any apparatus made for the purpose. A satisfactory imbedding-dish is a thin rectangular porce- lain dish glazed inside. This dish, called a Verbrennungsschale, is made by the Konigliche Porzellan-Manufactur, Berlin, Germany. The most convenient sizes are 40X50X10 mm., 68X45X10 mm., and 91 X 58 X 15 mm. As listed, these dishes are not glazed; care should be taken to indicate that the dishes must be glazed inside {innen glasiert). The paper tray, if well made, is as good as anything. Thick ledger linen or thin, smooth cardboard makes good trays. Smear the dish or tray with glycerin or soapy water to prevent sticking. Another way to prevent sticking is to put a piece of tissue paper in the dish, pour on water and make the tissue paper fit the inside of the dish, and then pour on the paraffin with the material to be imbedded. The paraffin will not stick to the paper. If several objects are to be imbedded in one dish, it is best to have the dish as near the temperature of melted paraffin as possible; otherwise, the objects may stick to the bottom, and it will be impossible to arrange them properly. Hot needles are good for arranging material. Great care should be taken not to have the dish too hot, since too high a temperature not THE PARAFFIN METHOD 121 irifffffffi lllllflMff ifiiMMin Fig. 21 only injures the material but also prevents a thorough imbedding. Pour the paraffin with the objects into the imbedding-dish and arrange them so as to facilitate the future cutting-out from the paraffin cake. Or, keeping the imbedding tray at the temperature of the paraffin bath — never hotter — the objects may be picked out gently and arranged as they are placed in the paraffin in the tray. Look at Figures 21 and 22, repre- senting the arrangement of root-tips in a paraffin cake. From a cake like that in Figure 21 it is easy to cut out tips for sectioning. The arrangement, or rather the lack of it, shown in Figure 22 should be remembered only as an exasperating example. After the objects have been arranged, cool the cake rapidly by allowing the bot- tom of the dish to rest upon cold water. As soon as a suf- ficiently firm film forms on the surface of the cake, let water flow gently over the top. After the cake has been under water for a few min- utes, the paraffin will either come out and float on the water or, at least, it will be easily removed from the dish. If paraffin cools slowly it crystallizes and does not cut well. The layer of par- affin should be just thick enough to cover the objects, not only as a matter of economy, but because a thick layer retards the cooling. Very small objects, like the megaspores of Marsilea, ovules of Silphiu?n, etc., may simply be poured out upon a cool piece of glass, which has been smeared with glycerin or soapy water. In this way, thin cakes are made which harden very rapidly. If one is doing much imbedding, it is worth while to have the par- affin cakes uniform in size and to have a convenient method of filing. Fig. 22 Figs. 21 and 22. — Paraffin cakes of root tips, the upper (Fig. 21) showing a good arrangement; the lower (Fig. 22) showing fewer tips and most of these not in position to be blocked without injury. 122 METHODS IN PLANT HISTOLOGY In our own collection, there are more than 6,000 paraffin cakes. They are filed in pasteboard boxes 28 cm. long, 10 cm. wide, and 2 cm. deep. With the generic name written on the box, and the boxes arranged al- phabetically or, preferably, taxonomically, it is easy to find anything in the large collection. CUTTING As soon as the paraffin is thoroughly cooled, it is ready for cutting. Trim the paraffin containing the object into a convenient shape, and fasten it upon a block of wood. Blocks of pine f inch long and f inch square are good for general purposes. Put paraffin on the end of the block so as to form a firm cap about | inch thick. Warm the cap and the bottom of the piece containing the object, and press them Hghtly together; then touch the joint with a hot needle, put the whole thing into cold water for a minute, and it is ready for cutting. Cutting can be learned only by experience, but a few hints may not come amiss: a) The knife must be sharp. This condition, which used to be the most difficult, has become the easiest; for any paraffin section, up to 2 cm. square, can be cut with a safety razor blade. The holder shown in Figure 3, with the shding microtome shown in Figure 2, with a hard safety razor blade, preferably the Watts blade described on page 10, will furnish relief from the tedious sharpening of microtome knives which, at their best, are not equal to a good safety razor blade. The "Gem" or "Star" blade is good for paraffin sections and is unequaled for wood sections, but the back must be broken off to make it fit the holder shown in Figure 3. Fortunately, The American Safety Razor Corporation, Brooklyn, N.Y., will furnish the Gem blade, specially tempered and tested, and with the back removed for microtome use, at 50 cents for a package of 10 blades. Some students have trouble with safety razor blades. There must be a good holder. The holders shown in Figures 3 and 4 eliminate any trouble from this factor. The angle must be right. A study of Figure 5 should eliminate any trouble from this source. In general, the safety razor blade should project farther beyond the holder, and the angle between the blade and paraffin should be greater for thin sections (1-5 m) than for thicker sections. The blade should project the least, and the angle should be the least, for hard sections and thick sections. Find the thickness at which the paraffin and object cut best. When in doubt as to the proper thickness, cut at 10 m- When the room temperature is at zero centigrade, onion root-tips in 52° C. paraffin THE PARAFFIN iMETHOD 123 should yield good ribbons at 2 and 3 yu, on a rotary microtome; and on a good sliding microtome, should yield good sections at 1^, or even thinner. The ideal temperature for 1-0.5 ^ sections, with 50° or 52° C. paraffin, is — 2° C. In warm weather, the microtome should be cooled on a block of ice and the knife and object must be kept cool by holding a small piece of ice against them every two or three minutes. A small piece of ice can be kept against the knife or holder by a rubber band. The ice is only a necessary evil. Try to arrange your time so as to do your cutting in cold weather. Let the room get cold, put on a warm coat, and go to work. If you are still in the microtome knife stage, get two good hones, one for use when the knife is rather dull and the other for finishing. For the first hone, nothing equals a fine carborundum hone. About 5.5X22.5 cm. is a good size. A hard Belgian hone, of the same size, may be a little better for finishing. Flood the stone with water, and rub it with the small slip which accompanies all high-grade hones; this not only makes a lather, which facilitates the sharpening, but it also keeps the surface of the hone flat. As soon as the edge of the knife appears smooth and even under a magnification of 30 or 40 di- ameters, the sharpening is completed with a good strop. It is better to sharpen the knife every time you use it. A first-class microtome knife, in perfect condition, will cut good sections, but it requires both time and skill to keep the edge perfect. Of course, for large sections, more than 18 mm. in diameter, a regular microtome knife is necessary. To get the right bevel, use a "back." For knives longer than 4 inches, we prefer to have a back only on the upper side. For blades 4 inches or less in length, the tube-like back, giving a bevel like that of a safety razor blade, is satisfactory. With the Watts safety razor blade in the holders shown in Figures 3 and 4, we have cut smooth ribbons of Selaginella strohili, sections through the sporangium region of the whole plant of Isoetes, sections of stems of Cucurbita, in fact, we have not used an ordinary micro- tome knife for cutting paraffin ribbons for more than 15 years. Many fail at the first attempt and go back to the continual drudgery of sharpening microtome knives. If the holder shown in Figure 4 is placed in the rotary microtome at the angle used for a microtome knife, failure is certain; for the blade, which is bent into a curve, will scrape rather than cut. A study of Figure 5 should enable anyone to secure the proper angle. 124 METHODS IN PLANT HISTOLOGY h) Keep the microtome well cleaned and oiled. Xylol is good for cleaning a microtome and the oil used for sewing machines is thin and efficient. Three-in-one oil is all right if the microtome is in constant use, but not so good if the microtome is to remain idle through a long vacation. c) Trim the block so that each section shall be a perfect rectangle. B Fig. 23.— Ribbons A ribbon of sections hke that shown in Figure 23 A is much better than one like B of the same figure, because sections will usually come off in neater ribbons if the knife strikes the longer edge of the rec- tangle, so that the sections are united by the longer sides rather than by the shorter. Crooked ribbons are caused by wedge-shaped sections, and are always to be avoided, because they make it difficult to economize space, and also because they present such a disorderly appearance. The knife, which should be placed at a right angle to the block and not obliquely, should strike the wJiole edge of the block at once, and should leave in the same manner. If sections stick to the knife, it may be that the knife is too nearly parallel with the surface of the block, as in Figure 24 A. By inclining the knife as in Figure 245, this difficulty is often obviated. A spUt or scratch in the paraffin ribbon may be caused by a nick in the knife. Use some more favorable position of the edge, or sharpen the whole knife. A spHt or a scratch in the ribbon is often caused by some hard granule which becomes fastened to the inner side of the edge of the knife. This is the most common cause of the difficulty. Simply wipe B Fig. 24. — Position of the knife THE PARAFFIN METHOD 125 the under side of the knife with a gentle stroke of the finger, shghtly moistened with xylol. For the best results, the knife should be wiped in this way after every section. The sliding microtome is more con- venient for wiping than the rotary. Ribbons 1 n and even 0.5/1 thick and nearly transparent can be cut on the sliding microtome shown in Figure 2. Such a thin ribbon is not hkely to be more than 10 cm. long; but at 10 n, ribbons 15 or 20 cm. long can be cut on a shding micro- tome. It is a good plan to put a piece of ledger linen paper on the holder or knife. The paper, long enough to hold a short ribbon, will probably stick for a minute or two if merely moistened. If it comes off, a little mucilage or a little piece of gummed paper, overlapping a small part of the holder and paper, will keep the paper in position. Sometimes hard paraffin does not ribbon well. This difficulty may be remedied by dipping a hot needle in soft paraffin and applying it to the opposite edges of the block to be cut. Often the mere warming of the opposite edges of the block with a hot needle is sufficient. Another method, suggested by Dr. Land to facilitate the cutting of difficult material, has come into general use, and is very effective. Paraffin absorbs a small amount of water, or water penetrates be- tween the crystals of paraffin. At any rate, water reaches cell walls and, perhaps, other structures which have not been completely infil- trated, and thus softens them. The paraffin cakes may be left for weeks in water. Cakes of class material may be put in water in a fruit can and kept until ready for use. After such treatment, smooth rib- bons may be cut from material which would hardly cut at all without it. If the failure to ribbon well is due to electricity, a very small drop of water on the knife will hold the sections and not prevent them from slipping along in a ribbon. After cutting, the ribbons may be kept for a few weeks, on filter paper, in a closed box; but no time is better for mounting than imme- diately after cutting. A ribbon carrier is very convenient. A good carrier can be made by mounting a couple of spools 20 or 30 cm. apart, with a strong piece of cloth for a band. More elaborate carriers may be made if one has tools. FIXING SECTIONS TO THE SLIDE Mayer's fixative. — Sections must be firmly fixed to the slide, or they wiU be washed off during the processes involved in staining. Mayer's albumen fixative is excellent for this purpose. Formula : 126 METHODS IN PLANT HISTOLOGY White of egg (active principle) 50 c.c. Glycerin (to keep it from drying up) 50 c.c. Salicylate of soda (antiseptic, to keep out bacteria, etc.) 1 g. Shake well and filter through cheesecloth. It will keep from 2 to 6 months, but, to say the least, it is never better than when first made up. Put a small drop of fixative on the slide, smear it evenly over the surface, and then wipe it off with a clean finger until only a scarcely perceptible film remains ; then add several drops of distilled water and float the sections or ribbons on the water. Warm gently until the par- affin becomes smooth and free from wrinkles. Wrinkled or curved ribbons may be straightened by touching with a needle at each end and pulling gently, just as the ribbon begins to smooth out in the warming. When the water is warmed, the ribbon almost always stretches. Theoretically, it should not stretch at all, if the cutting has been per- fect. If a ribbon cut at 10 /x does not lengthen more than 10 per cent, the cutting is fairly good; if it lengthens 20 per cent, cut thicker sec- tions or lower the temperature, using ice if necessary. If a 5/x ribbon does not lengthen more than 10 per cent, the cutting may be regarded as good; at 1 ^ or 0.5 /x, if it does not lengthen more than 20 per cent, the cutting has been good. Be very careful not to melt the paraffin, be- cause the albumen coagulates with less heat than is required to melt the paraffin. Consequently, there would be nothing to fix the ribbon to the slide. After the sections become smooth, remove as much of the water as possible. This precaution is usually neglected. Drain off what you can. Then, by touching filter paper to the edge of the sections, get rid of some more water. As the water is removed by the filter paper, the edge of the ribbon comes first into contact with the slide and thus seals in some water. Touch the edge of the ribbon with a hot needle and use the filter paper again. If care be taken at this point, there is no danger later, even when using stains requiring 24 or even 48 hours in liquids. This may seem slow and tedious, but when you compare a slide of mitosis in root-tips, or the reduction divisions in pollen mother- cells put up this way, with the usual hasty preparations, you will see a difference. If you should heat the ribbon so hot as to melt it, some things can be saved by cooling the ribbon and floating it off to another slide THE PARAFFIN METHOD 127 smeared with fixative. Of course, since the ribbon had not got into contact with the fixative, much would be lost. It is a good plan to put the slide on a metal bath with a piece of corrugated pasteboard under the shde. Very convenient warming plates, which can be kept at a con- stant temperature, are sold by most dealers in laboratory supplies. A very convenient warming plate is easily made. Simply take a box, or make one, about 2 feet long, 1 foot wide, and 6 inches deep, using a piece of glass or a piece of brass | inch thick for the top. Heat it by putting in such an electric bulb as will give the temperature desired. With the bulb in one end of the box, there will be various temperatures in different parts of the box. This is fine for straightening ribbons and for use during imbedding. It can even be used as a paraflfin bath where the times are so short that the temperature can be watched constantly. After the sections have become smooth and the surplus water has been removed, leave the slides where they will be warm, but well under the melting-point of the paraffin, overnight or for 24 hours. If free from dust, they may be kept for several days, or even weeks, before staining. If the sections are very thick, so that they do not need to be smoothed out on water, they may be laid carefully on the fixative, patted down with the finger and they are ready for staining. Sections may stick to the beginner's finger, but he should soon learn to avoid such troubles. Land's fixative. — Mayer's fixative is so easily prepared and it keeps so well that it is in universal use; but, in many cases, it will not hold the section to the slide. Moss archegonia and moss capsules are likely to wash off, especially if cut rather thick. Large sections of cones of conifers are almost sure to float off as soon as the slide comes into the xylol or alcohol. Sections of ovules of cycads, as soon as they attain a length of 1.5-2 cm., are likely to wash off. For handling these more difficult cases, Dr. Land devised a fixative which has proved satisfac- tory, even in such extreme cases as sections of ovulate cones of Pinus hanksiana 2 cm. long. Formula: Gum arabic 1 0 g. Dichromate of potash 0 . 2 g. Water 100.0 c.c. The mixture will not keep ; the formula is given merely to indicate its composition. Make a 1 per cent solution of gum arabic in water, which will keep as well as Mayer's fixative; but make the dichromate 128 METHODS IN PLANT HISTOLOGY solution immediately before using. Do not make the solution stronger than 1 per cent; usually 0.2 per cent is strong enough. Dr. Land does not measure, but simply adds enough dichromate crystals to make the water pale yellow. Smear a few drops of the 1 per cent solution of gum arable on the slide; flood with the dichromate solution; warm to straighten the rib- bons; drain off the excess water and let the preparation dry in the light. The exposure to light renders the gum insoluble in water. LePage's glue or Mayer's albumen fixative may be used instead of gum arable. The foregoing directions are taken from Dr. Land's notes. With the ordinary Mayer's albumen fixative, the dichromate of potash, without the gum arable, may be used in floating out ribbons, and makes a stronger fixative than the Mayer's formula. Szombathy's fixative. — Gelatin 1 g- Distilled water 100 c.c. Salicylate of soda (a 2 per cent solution) 1 c.c. Pure glycerin 15 c.c. Dissolve the gelatin in water at 30° C, add the salicylate of soda, shake well, cool, and filter through cheesecloth; then add the 15 c.c. of glycerin. The solution should be perfectly clear. A couple of drops of the fixative, with a couple of drops of 2 per cent formalin, is rubbed on the sHde. The sections are then added, and straightened out. The formalin makes the gelatin insoluble. The fixa- tive is much like Land's and is used for difficult material which is not held by Mayer's fixative. Haupt's fixative. — Gelatin 1 g. Phenol crystals 2. g Glycerin 15 c.c. Distilled water 100 c.c. This is a modification of Szombathy's formula. The gelatin is dis- solved at 30° C. Gelatin which will not dissolve at this temperature is not satisfactory. Place a small drop of the fixative on the slide and smear with the finger until the film is very thin. Flood the slide with a 2 per cent solu- tion of formalin in distilled water, put on the ribbon, warm it, and proceed in the usual way. THE PARAFFIN METHOD 129 REMOVAL OF THE PARAFFIN To remove the paraffin, place the shde in a Stender dish of xylol. About 5 minutes will be sufficient for sections 10 ii thick, but 10 or 20 minutes will do no harm. While a gentle heating will hasten the process and will do no harm, you should never heat the slide e7iough to melt the paraffin. Never attempt to warm the paraffin over a lamp. Over- heating is ruinous. REMOVAL OF XYLOL OR TURPENTINE To remove the xylol, place the slide in equal parts of xylol and abso- lute alcohol in a Stender dish. After 5 minutes, transfer to absolute alcohol, which should also be allowed to act for 5 minutes. A little of the paraffin will unavoidably be carried into the xylol-alcohol ; a smaller quantity may be carried into the absolute alcohol. The ar- rangement shown in Figure 15 will completely remove the paraffin before staining, TRANSFER TO THE STAIN After the paraffin has been removed with xylol or turpentine, and the xylol or turpentine has been rinsed off with alcohol, the next step is the staining. If the stain is a strong alcoholic one (85-100 per cent alcohol), transfer directly to the stain. If the stain is in 70 per cent alcohol, pass through 95 and 85 per cent alcohol, 5 minutes in each, before staining. If an aqueous stain is to be used, pass down the whole series — 95, 85, 70, 50, 35, and water — 5 minutes in each, before placing the slide in the stain. This is rather tedious, but, for cytological work, it seems to be necessary, and one might as well learn early that rapid work and good preparations seldom go together. DEHYDRATING After the sections have been stained, they must be dehydrated. If they have been stained in a strong alcoholic solution, transfer to 95 and then to 100 per cent alcohol, 2 minutes in each, if the stain does not wash out too rapidly. If stained in an aqueous solution, pass through the series — water, 5, 10, 20, 35, 50, 70, 85, 95, and 100 per cent alcohol — about 2 minutes in each. With stains which wash out rapidly, the times must be shortened and some of the alcohols must be omitted. With aqueous gentian vio- let, all must be omitted except the 95 and 100 per cent, and even in these the time must be shortened to a few seconds. 130 METHODS IN PLANT HISTOLOGY CLEARING After the sections have been dehydrated, they must be cleared, or made transparent by some clearing agent. The clearing agent must be a solvent of balsam, but it is not at all necessary that the balsam shall be dissolved in the particular clearing agent which has been used. Xylol balsam is used, not only when preparations have been cleared in xylol, but also when they have been cleared in clove oil, cedar oil, bergamot oil, or other clearing agents. Xylol is the most generally useful clearing agent. Place the sHde in equal parts of xylol and absolute alcohol and then in pure xylol, allow- ing each to act for about 2-5 minutes. Clove oil is also an excellent clearing agent. The clove oil should follow the absolute alcohol, without any mixtures. Pour on a few drops of clove oil, and drain them off at once in such a way as to carry with them whatever alcohol may still remain. Then flood the slide repeatedly with clove oil, draining the clove" oil back into the bottle. If judiciously used, 50 c.c. of clove oil is enough to clear 50 prepara- tions. Sections are usually cleared in a few seconds. The only objec- tion to clove oil is that mounts harden slowly. To overcome this diffi- culty, the shde should be dipped in xylol for a minute before mounting in balsam. Synthetic oil of wintergreen is much less expensive and some claim that it is just as good as clove oil. We prefer clove oil. For clearing sections on the slide, other clearing agents are hardly worth mentioning. MOUNTING IN BALSAM After the sections are cleared, wipe the slide on the side which does not bear the sections. Put on a drop of Canada balsam and add a clean,! thin cover. Before the cover is put on, pass it through, the flame of an alcohol lamp to remove moisture, for it would be a pity ' Slides and covers should be treated with hydrochloric acid, or equal parts of hydrochloric acid and water, for several hours. They should then be thoroughly rinsed in water and wiped with a cloth perfectly free from hnt. After rinsing in water, they may be kept in 95 per cent alcohol. When a cover is needed for use, it is Dr. Land's practice to rest the corner of the cover on a piece of filter paper to remove the drop of alcohol; then pass the cover through the flame of a Bunsen or alcohol lamp. The film of alcohol will burn and the cover may warp, but it will usually straighten, and it will be clean and dry. The mixture of sulphuric acid and dichromate of potash, used for cleaning labo- ratory glassware, is equally good for slides and covers. THE PARAFFIN METHOD 131 indeed to injure a preparation at this stage of the process. Add a label, and the mount is complete. A TENTATIVE SCHEDULE FOR PARAFFIN SECTIONS It will be useful to give several tentative schedules for the use of beginners. It cannot be too strenuously insisted that these schedules are only tentative, their sole object being to give the beginner a start. The following is a tentative schedule for the ovary of a lily at any period before fertilization. The pieces should not be more than 12 mm. in length. 1. Cliromo-acetic acid, 1 day. 2. Wash in water, 1 day. 3. Two and one-half, 5, 10, 15, 20, and 35 per cent alcohol, three grades a day, morning, noon, and night; 50 and 70 per cent, change morning and evening; 85 per cent, 24 hours; 95 per cent, all day or overnight; absolute alcohol, 24 hours, changing 2 or 3 times. 4. Mixtures of absolute alcohol and xylol; 2|, 5, 10, 15, 25, 50, and 75, 3 grades a day, morning, noon, and night; pure xylol, 24 hours, chang- ing 2 or 3 times. 5. Paraffin and xylol, 48 hours. 6. Melted paraffin in the bath, 30-40 minutes, changing 2 or 3 times. 7. Imbed. 8. Section; about 10 /x is a good thickness. 9. Fasten to the slide. 10. Dissolve off the paraffin in xylol, 5 minutes. 11. Xylol and absolute alcohol, equal parts, 5 minutes; 100, 95, 85, and 70 per cent alcohol, 5 minutes each. 12. Stain in safranin (alcohoUc), 6 hours or overnight. 13. Rinse in 50 per cent alcohol, using a trace of HCl if necessary; then in 70, 85, 95, and 100 per cent alcohol, 5 minutes each. 14. Stain in gentian violet dissolved in clove oil (or in clove oil with a httle absolute alcohol), 10 minutes. 15. Treat with pure clove oil until the gentian violet stain is satisfactory. 16. Rinse in xylol, 1 minute. 17. Mount in balsam. 18. Label. That the paraffin method is tedious and complicated is universally recognized. Many substitutes have been tried, but without enough success to justify even a reference. CHAPTER X THE CELLOIDIN METHOD The celloidin method has almost disappeared from botanical micro- technique, because material too hard for imbedding in paraffin can be cut without any imbedding at all, and material too delicate to be cut without a supporting medium can be imbedded in paraffin. But these two categories do not cover all the ground; celloidin still has its ad- vantages. Stems too hard for the paraffin method, which lose the cortex or, at least, suffer breaks with the steam method or when cut freehand and cold, can often be cut successfully in celloidin. Years ago, a piece of rotten wood from an ancient Egyptian mum- my case was brought to the writer for identification. It could be rubbed into powder in the fingers, and had to be handled gently to keep it from falhng to pieces. It was cut very successfully in celloidin. Stems too hard for paraffin may be cut in celloidin when it is desired to pre- serve cell contents. Celloidin is still very valuable for most of the sec- tions used in medical schools, because the sections can be prepared in great numbers and each student can take a section, add a drop of bal- sam and a cover, and have a preparation of his own ready to study. Where serial sections are necessary, as in most morphological and cy- tological work, the method is too tedious to be worth even a trial, un- less the sections cannot be cut in any other way. Besides, most of the more valuable stains color the celloidin matrix, and if the matrix be removed, the more delicate elements may be displaced or even lost. Celloidin and collodion are forms of nitro-cellulose. They are in- flammable, but do not explode. Schering's celloidin, which is only a collodion prepared by a patented process, is in general use for imbed- ding. Granulated and shredded forms of celloidin are on the market, but the tablets are more convenient. Directions for making the vari- ous solutions accompany the celloidin. To make a 2 per cent solution, add to 1 tablet enough ether-alcohol to make the whole weigh 2,000 g. To make a 4 per cent solution, add another tablet, and to make a 6 per cent solution, add an additional tablet, and so on. The collodion method was pubhshed by DuvaP in 1879. Celloidin 'Duval, Journal de Vanatomie, 1879, p. 185. 132 THE CELLOIDIN METHOD 133 was recommended by Merkel and Schiefferdeckei-i in 1882. The princi- pal features of the method are as follows : Material is dehydrated in absolute alcohol, treated with ether-alcohol, infiltrated with celloidin, imbedded in celloidin, hardened in chloroform or alcohol; after which, it is cut, stained, and mounted. Eycleshymer, who brought the celloidin method to a high degree of efficiency, pubhshed in 1892 a short account, which may be sum- marized as follows: Put the celloidin tablet, or fragments, into a wide- mouthed bottle, and pour on enough ether-alcohol (equal parts ether and absolute alcohol) to cover the celloidin. Occasionally shake and add a little more ether-alcohol until the celloidin is all dissolved. The process may require several days. The solution should have the con- sistency of a very thick oil. Label this solution Number 4. Solution Number 3 is made by mixing 2 parts of solution Number 4 with 1 part of ether-alcohol. Solution Number 2 is made by mixing 2 parts of Number 3 with 1 part of ether-alcohol. Solution Number 1 consists of equal parts of ether and absolute alcohol. After dehydrating, the material is placed successively in solutions 1, 2, 3, and 4. For an object 2 mm. square, 24 hours in each solution is sufficient; for the brain of a cat, a week is not too long. A paper tray may be used for imbedding. Pour the object, with the thick solution, into the tray and harden in chloroform for 24 hours; then cut away the paper and place the block in 70 per cent alcohol for a few hours. The material may be left indefinitely in a mixture of equal parts of 95 per cent alcohol and glycerin. Before cutting, the object is mounted upon a block of wood. A block, suited to the microtome clamp, is dipped in ether-alcohol, which removes the air and insures a firmer mounting. Dip the end of the block of wood in solution Number 3, and the piece of celloidin contain- ing the object in solution Number 1. Press the two firmly together, and place in chloroform until the joint becomes hardened. Set the blade of the microtome knife as obliquely as possible. Both the object and the knife should be kept flooded with 70 per cent alco- hol, and the sections, as they are cut, should be transferred to 70 per cent alcohol. Stain in Delafield's haematoxylin for 5-30 minutes. Wash in water for about 5 minutes, and then decolorize in acid alcohol (2-5 drops of hydrochloric acid to 100 c.c. of 70 per cent alcohol) until the stain is 1 Merkel and Schiefferdecker, Archiv fur Anatomie und Physiologie, 1882. 134 METHODS IN PLANT HISTOLOGY extracted from the celloidin, or at least until the celloidin retains only a faint pinkish color. Wash in 70 per cent alcohol (not acid) until the characteristic purple color of the haematoxylin replaces the red due to the acid. Stain in eosin (preferably a 1 per cent solution in 70 per cent alcohol) for 2-5 minutes. Dehydrate in 95 per cent alcohol for about 5 minutes. Absolute alcohol must not be used, unless it is de- sirable to remove the celloidin matrix. Eycleshymer's clearing fluid (equal parts of cedar oil, bergamot oil, and carbolic acid) clears readily from 95 per cent alcohol. Mount in balsam. If serial sections are necessary, arrange the sections upon a slide, using enough 70 per cent alcohol to keep the sections moist, but not enough to allow them to float. Cover the sections with a strip of thin toilet paper, which can be kept in place by winding with fine thread. After the sections have been stained and cleared, remove the excess of clearing fluid by pressing rather firmly with a piece of blotting-paper. Then remove the toilet paper and mount in balsam. With occasional slight modifications, we have used the method as presented by Eycleshymer in his classes. Instead of the graded series of celloidin solutions, we use a 2 per cent solution, which is allowed to concentrate slowly by removing the cork occasionally, or by using a cork which does not fit very tightly. The material is imbedded when the solution reaches the consistency of a very thick oil. If the material is to be cut immediately, we prefer to imbed it and fasten it to the block at the same time. The blocks should have surface enough to accommodate the objects, and should be about I inch thick. White pine makes good blocks; cork is much inferior. Tie a piece of ledger linen paper around the top of the block, letting it project above the top of the block far enough to make for the object a little tray with the end of the block for a bottom. Place the block for a moment in ether-alcohol and then dip into the 2 per cent celloidin the end of the block which was left rough by the saw. With the forceps remove a piece of the material from the thick celloidin and place it upon the block, taking care to keep it right side up. Dip the block with its object first in thick celloidin, then in thin, and after exposing to the air for a few minutes drop it into chloro- form, where it should remain for about 10-20 hours. It should then be placed in equal parts of glycerin and 95 per cent alcohol, where it may be kept indefinitely. If the material is hard, like many woody stems, it will cut better after remaining in this mixture for a couple of weeks. THE CELLOIDIN METHOD 135 The following schedules, beginning with the celloidin sections in 70 per cent alcohol, will give the student a start in the staining: Delafield's haematoxylin and eosin. — 1. Seventy per cent alcohol, 2-5 minutes. 2. Delafield's haematoxylin, 5-30 minutes. 3. Wash in water, 5 minutes. 4. Acid alcohol (1 c.c. hydrochloric acid+100 c.c. of 70 per cent alcohol) until the stain is extracted from the celloidin, or at least until only a faint pinkish color remains. 5. Wash in 70 per cent alcohol (not acid) until the purple color returns. 6. Stain in eosin (preferably a 1 per cent solution in 70 per cent alcohol), 2-5 minutes. 7. Dehydrate in 95 per cent alcohol, 2-5 minutes. Do not use absolute alcohol unless you wish to dissolve the celloidin, which is not necessary with this staining. 8. Clear in Eycleshymer's clearing fluid, usually 1-2 minutes, but some- times 5-10 minutes. 9. Mount in balsam. Safranin and Delafield's haematoxylin. — 1. Seventy per cent alcohol, 2-5 minutes. 2. Safranin (alcohohc), 6-24 hours. 3. Acid alcohol (a few drops of hydrochloric acid in 70 per cent alcohol) until the safranin is removed from the cellulose walls. 4. Wash in 50 per cent alcohol, 5-10 minutes to remove the acid. 5. Delafield's haematoxylin, 2-5 minutes. 6. Wash in water, 5 minutes. 7. Acid alcohol, a few seconds. 8. Dehydrate in 95 per cent alcohol, 2-5 minutes, then in absolute alcohol, 2-5 minutes, which will partially dissolve the celloidin. 9. Clear in clove oil, which will complete the removal of the celloidin. 10. Be sure that the sections are free from fragments of celloidin and then mount in balsam. Stains which can be used with celloidin are limited because they stain the matrix; but some material can be stained in bulk with an aqueous stain while the material is in water, or with an alcoholic stain while passing through the alcohols. Safranin is good for xylem, alum carmine is more generally useful, and borax carmine is good for some animal tissues. A second stain, like Delafield's haematoxylin, which can be extracted from the matrix, can be used after the sections have been cut. 136 METHODS IN PLANT HISTOLOGY Jeffrey's improvements in the celloidin method have been de- scribed in considerable detail by Plowman. ^ Sections of hard stems and roots cut by this method could hardly be surpassed, and they are perfectly adapted to the requirements of photomicrography. The fol- lowing is a brief abstract of Plowman's paper: 1. Preparation of material. — Dead and dry material should be re- peatedly boiled in water and cooled to remove air. An air-pump may be used in addition. Living material may be fixed in a mixture of pic- ric acid, mercuric chloride, and alcohol: Mercuric chloride, saturated solution, in 30 per cent alcohol. 3 parts Picric acid, saturated solution, in 30 per cent alcohol 1 part Fix 24 hours, and wash by passing through 40, 50, 60, 70, and 80 per cent alcohol, allowing each to act for 24 hours. 2. Desilification, etc. — Sihca and other mineral deposits are re- moved by treating with a 10 per cent aqueous solution of commercial hydrofluoric acid. The material is transferred to this solution from water or from the 80 per cent alcohol. The process may require 3 or 4 days, with one or two changes of the acid and frequent shaking of the bottle. An ordinary wide-mouthed bottle, coated internally with hard paraffin, should be prepared, since the acid is usually sold in bottles with narrow necks. The bottles are easily prepared by filling them with hot paraffin and simply pouring the paraffin out. Enough will stick to the bottle to protect the glass from the acid. Wash in running water 3 or 4 hours. 3. Dehydration.— Use 30, 50, 70, 90, and 100 per cent alcohol, allowing 12 hours in each grade. 4. Infiltration with celloidin. — There should be ten grades of celloi- din: 2, 4, 6, 8, 10, 12, 14, 16, 18, and 20 per cent. Transfer from abso- lute alcohol to the 2 per cent celloidin. (We should prefer a previous treatment with ether-alcohol.) The bottle should be nearly filled, and the stopper should be clamped or wired in place. Put the bottle on its side in a paraffin bath at 50°-60° C. for 12-18 hours. Cool the bottle quickly in cold water, taking care that the water does not get into the bottle. Pour out the 2 per cent solution (which, as well as all other so- lutions, may be used repeatedly), and replace it with the 4 per cent, and proceed in the same way with the other grades. When the 20 per cent solution is reached, a further thickening is gained by adding a 1 A. B. Plowman, "The Celloidin Method with Hard Tissues." Botanical Gazette, 37:456-461, 1904. THE CELLOIDIN METHOD 137 few chips of dry celloidin from time to time until the mixture is quite stiff and firm. Remove each block with the celloidin adhering to it and harden it in chloroform for 12 hours. Then transfer to a mixture of equal parts of glycerin and 95 per cent alcohol, where the material should remain for a few days before cutting. Cutting, staining, and mounting. — Although 10 m is usually thin enough, sections are readily cut as thin as 5 m by this method. Re- move the celloidin before staining by treating 10-15 minutes with ether; then wash in 95 per cent alcohol and transfer to water, and then to the stain. Stain to a fairly dense purple in an aqueous solution of Erlich's haematoxyhn; wash in dilute aqueous solution of calcium or sodium carbonate, and then in two changes of distilled water. Add a few drops of alcohohc solution of equal parts of Griibler's alcoholic and aqueous safranin, and stain to a rich red. A dilute stain acting 1-2 hours is better than a more concentrated stain acting for a shorter time. Transfer directly to absolute alcohol, clear in xylol, and mount in balsam. Haidenhain's iron-haematoxylin is a very satisfactory stain for photographic purposes. The celloidin method has its disadvantages as well as its advan- tages. It is extremely slow and tedious, and it is rarely possible to cut sections thinner than 10 /z while, on the other hand, it gives smoother sections. Succulent tissues, which are usually damaged by the paraffin meth- od, are easily handled without any injury in celloidin. The fact that the method may be used without heat is often a further advantage. Stems and roots, which cannot be handled at all in paraffin, cut well in celloidin, and much larger sections can be cut than in paraflan, but most material of this kind can be cut without any imbedding. When material is to be imbedded, use celloidin as a last resort. Use parafiin when you can, celloidin when you must. CHAPTER XI THE CELLULOSE ACETATE METHOD When the celkilose acetate method first appeared, more than ten years ago, we hoped that it was destined to be as successful with hard woody tissues as the Venetian turpentine method has been with uni- cellular and filamentous forms; but, up to date, American investiga- tors have found nothing to arouse any enthusiasm for this method, which seems to be at its best in the fogs of London. We have obtained the cellulose acetate from Cellon and tried it repeatedly with hard and soft woods, but have never secured sections which could be compared with those obtained by other methods. However, no one but the authors got good results with Venetian turpentine for many years after the method was published; so, let us hope that the method will yet yield as good sections on this side of the Atlantic as it does on the other. Hard woods like oak, and even harder material, have yielded smooth thin sections. Cellulose acetate does not injure the finer de- tails of structures and, on that account, is superior to hydrofluoric acid. We are quoting, in full, Mrs. Williamson's account in the Annals of Botany for January, 1921. A NEW METHOD OF PREPARING SECTIONS OF HARD VEGETABLE STRUCTURES In order to prepare sections of hard vegetable structures it is essential that some method should be devised by which the structure is not only embedded but softened, so that sections can be cut easily and smoothly. After various methods had been tried, the cellulose acetate method successfully used by Dr. Kernot for embedding and sectioning the fabric of aeroplane wings was used. It was discovered that this method not only embedded hard vegetable structures, but also softened them so that sections are easily obtained. It proved best to use cellulose acetate of French manufacture made from pure cellulose, as the viscosity is more uniform than that of English manufacture, which is obtained from the cellulose of wood. In the preliminary experiments pieces of oak and beech, cut into half-inch cubes, were passed through strengths of alcohol, then placed in pure acetone for two hours and finally into a 12 per cent solution of cellulose acetate in ace- 138 THE CELLULOSE ACETATE METHOD 139 tone. There they were left for two months, and excellent sections were ob- tained. Further experiments showed that the passage through alcohols was unnecessary. In the final experiments the pieces of wood were placed in water and the air removed from them, after which they were put into pure acetone for 1-2 hours and finally into the solution of cellulose acetate. It was found that the length of time of immersion in the solution of cellulose acetate neces- sary for softening the tissues varied with the hardness of the wood, the mini- mum time for soft woods being two days; for woods such as oak and beech, at least six days are required. Experiments were tried with sal {Shorea robusta) and Pyingadu (Xylia dolabriformis) , one of the Indian ironwoods, which is extremely hard. After fourteen days in the cellulose acetate solution it was possible to obtain transverse sections of these hard woods. The cellulose acetate solution is therefore capable of softening even the hardest wood in a relatively short time. In order to stain sections — either hand or microtome — obtained by this method, it is necessary to wash them in i)ure acetone for 1 to 2 minutes to remove the cellulose acetate, wash in alcohol 1 to 2 minutes, and pass on to the stains selected. Various staining methods for cell walls— such as aniUn chloride, methjdene blue, and Congo red, ammoniacal fuchsin and Kleinen- berg's haematoxylin, etc.— were tried with success. A comparison with stained sections of untreated wood revealed no differences. DeHcate tissues in the wood and hyphae of fungi infecting the wood also stain well and are unaffected by the treatment. A satisfactory method of preparing sections of hard vegetable structures is therefore supplied by the use of a 12 per cent solution of cellulose acetate in pure acetone for softening and embedding.— H. S. Williamson, Imperial College of Science and Technology. Correspondence with Mrs. Williamson indicates that the various brands of cellulose acetate behave differently. Cellulose acetate ob- tained from wood is unsatisfactory. We found that cellulose acetate made from photographic films was also unsatisfactory. Mrs. William- son used a cellulose acetate sold by Cellon (Richmond) Ltd., 22 Cork Street, London, England, and manufactured by the Societe Chimique des Usines du Rhone. The time may be shortened by keeping the temperature at 40° C. In making the solution, use 12 g. of cellulose acetate to 100 c.c. of pure acetone. CHAPTER XII SPECIAL METHODS It has been the object of the preceding chapters to give the student an introduction to the principal methods of preparing plant material for microscopic study, and to afford him such a grounding in funda- mentals that he will be able to develop methods which may be neces- sary in special cases. A few methods designed to meet special difficulties are given in this chapter and others are mentioned in the second part of the book, in connection with various laboratory types. VERY LARGE SECTIONS It is sometimes desirable to cut very large sections. Sections as large as a cornstalk may be cut freehand, but cut better when im- bedded in paraffin or celloidin. Even when cutting a paraffin section of a corn stem, have the knife somewhat oblique, and if the section shows a tendency to curl, as it probably will, a gentle touch with the finger will prevent the curling. If the knife, for the best cutting, is too oblique for ribbons, take each section off separately and put it in a box. A section of a stem of Zamia 5 or 6 cm. in diameter is difficult to handle by the usual methods. If a large microtome, such as is used in cutting complete sections of large brains, is available, the piece of stem is easily held for the cutting. Some of the medium-sized sliding microtomes now have a rigid clamp which will grip a block 3 cm. square. The lower part of the piece can then be trimmed to fit the clamp, leaving the upper part round, so that sections across a stem 6 or 7 cm. in diameter may be cut without much difficulty. With a rather soft stem, like Zamia, the surface must be flooded with 95 per cent alcohol after each section, if it is desirable to cut thin sections. From stems 3 cm. in diameter, sections can be cut at about 20 to 30 /x. If the section is not more than 3 or 4 cm. in diameter, it can be mount- ed on a 50X75 mm. sHde. Sections 6 or 7 cm. in diameter can be mounted on lantern slides; if large covers are not available, use another lantern slide for a cover. It will be easier to get neat mounts if the cover is cut down so as to leave a margin 2 or 3 mm. wide. It is not 140 SPECIAL METHODS 141 easy to mount a thick section between 2 lantern slides of the same size, on account of the balsam which oozes out at the edges. Such prepara- tions may be used directly as lantern slides. Large sections of the stem of a tree fern make good mounts without any staining. STONY TISSUES Sections of the stony tissues of hickory nuts, walnuts, peach stones, and similar refractory substances cannot be cut by ordinary methods. With a fine saw, saw sections about 1 mm. in thickness. Put some fine carborundum on a piece of plate glass, wet it, lay the section on it and, with the finger on the section, rub with a circular motion until one side of the section is quite smooth. Turn the section over and rub the other side. When the section has become quite thin (about half a millimeter) use a piece of plate glass, about 10 cm. square, instead of the finger. When the section is thin enough, wash it thoroughly, de- hydrate, clear, and mount in balsam. The long, narrow pores show better without any clearing. In this case, dry the section thoroughly, heat a few drops of balsam on the slide to drive off the solvent, put the section into the balsam, and add a cover. The air caught in the long, narrow pores will make them ap- pear as black lines. Sections of most nuts show excellent detail with- out any staining. Thin sections, however, may be stained in the usual way. STEAM METHOD FOR HARDWOOD SECTIONS In 1926 Dr. Josef Kisser published a useful method for cutting sec- tions of hard woods. The method consists, essentially, in letting steam play upon the block as the sections are being cut. The hot steam is easily secured by a simple apparatus which can be set up in a few min- utes in any laboratory. All that is needed is a flask, holding about 300 c.c, and some glass tubing (Fig. 25). The temperature of the steam should be about 90° C. If the steam is too hot, the material dries; if the temperature is much below 90° C, there is little advantage from the steam. The Spencer Lens Company's holder for thin safety razor blades, while excellent for thin paraffin sections, does not hold the thin blade so well for wood sections. However, the holder holds the Durham du- plex blade very well, and the Gem or Star blade, with the back broken off, is ideal for wood sections. Of course, if one likes to sharpen micro- tome knives, they are long and will cut while they are sharp. 142 METHODS IN PLANT HISTOLOGY It is a good plan to cut the wood into pieces suitable for cutting, so that the sections will be about 5X7 mm. Boil these pieces for 24 hours and then put them into equal parts of 95 per cent alcohol and glycerin for at least a week. After the boiling, very hard woods should be treated with 25 per cent hydrofluoric acid for a week and then washed Block to be cut u I Bunsen Fig. 25. — Apparatus for steam method thoroughly in water before being placed in glycerin and alcohol. A soft wood, like Pinus strohus, needs no steam or previous treatment, except a few hours' soaking in water. Kisser cut transverse sections, 5 mm. square, of ebony. Sections of cocoanut shell 2 mm. square and 6 /x thick were cut smoothly. JEFFREY'S VULCANIZING METHOD Jeffrey cut very thin sections of the hardest woods and even such refractory material as peach stones and the shells of various nuts. SPECIAL METHODS 143 Tissues are softened at a temperature of about 320° F., in an or- dinary dental vulcanizer. The time required varies. For a three- or four-year-old branch of oak, an hour is usually enough; but for a piece of seasoned oak wood, the time is likely to be 4 or 5 hours. Brass pipe f-1 inch in diameter is cut into lengths to fit the vulcan- izer, and the ends are threaded for brass caps. On one end, the cap is made tight by "sweating" lead solder into the thread. The other end is made tight by putting into the cap a piece of cardboard and, on top of the cardboard, a circular piece of lead. The tube is then placed in a vise, the water or alcohol, with the material, is put into the tube, and the cap is screwed on tight with a wrench. After vulcanizing, the material is allowed to cool slowly, and then is treated for a few days in a mixture of 2 parts water and 1 part hydro- fluoric acid. Wash well, run up through the alcohols, and preserve in equal parts glycerin and 95 per cent alcohol, until needed for cutting. By this method, Jeffrey cut transverse sections of cocoanut shells and hard woods as thin as 2 or 3 fi. CLEARING THICK SECTIONS OR SMALL OBJECTS It is sometimes desirable to make very thick sections to show gener- al topography rather than detail. A longitudinal section of the fully grown ovule of Ginkgo or a cycad may be cut as thick as 3-5 mm. so as to include the entire group of archegonia. A slab can be cut from each side of the ovule with a fine saw, and a razor can be used for smoothing. If the section is from fresh material it should be fixed, washed, etc., with about the same periods as if it were to be imbedded in paraffin. When thoroughly cleared in xylol, the section should be put into a flat museum jar of suitable size and kept in xylol. Before the stony coat of a cycad or Ginkgo ovule becomes too hard to cut readily with a safety razor blade, the ovule should be run up to 85 per cent alcohol before cutting the slabs off from the sides, because the turgidity of the endosperm would cause distortion. If the base of the living ovule be placed in basic fuchsin, the vascular system will be stained. Then fix in alcohol and follow Gourley's method, described later in this chapter. Such preparations, when cleared and placed in a smooth glass dish with a light beneath, are very instructive. Sections of Zamia or other cycad stems, 2 mm., or even 5 mm. thick, make instructive mounts, since thej^ show the peculiar course of the bundles, a feature which is largely lost in thin sections. 144 METHODS IN PLANT HISTOLOGY Kraus prepared large objects very effectively by dehydrating, clearing in xylol, and then transferring to cedar oil. Sections of an apple, either longitudinal or transverse, about 3 or 4 mm. thick, cleared in this way, are very instructive. Strawberries, gooseberries, and similar objects treated in this way afford a kind of study which is too often neglected. Dr. LaDema M. Langdon cleared 15 mm. cubes of mature wood of Dioon spmulosum in this way, but used equal parts of xylol and carbon disulphide for clearing. By placing a light under the dish containing the object, the bundles could be traced perfectly. LAND'S GELATIN METHOD It is sometimes desirable to get sections of partly disorganized material. A matrix is necessary to hold the parts in place, but dehy- dration may make the tissue unnecessarily hard to cut. Soak ordinary gelatin (which can be obtained at the grocery) in water until no more is taken up. Then drain off the excess water and liquefy the gelatin by heating. Place the material— previously soaked in water— in the melted gelatin and keep it there for several hours. Place also in the gelatin some small blocks of hard wood to serve as supports in the microtome. The material to be sectioned is oriented in a gelatin matrix on the supporting blocks, cooled until the gelatin sets, and then placed in strong formalin to harden the gelatin. In cutting, flood the knife with water. If the material is to be stained, stain it in bulk before putting it into the gelatin, since the gelatin stains very deeply. Of course, the gelatin could be dissolved with hot water, or hot water and acetic acid, but all the advantage of a matrix would be lost. It would be worth while to try this method thoroughly with soft, succulent tissues and with hard tissues which become still harder if dehydrated. SCHULTZE'S MACERATION METHOD Various solutions are used to separate a tissue into its individual cells. These solutions dissolve or weaken the middle lamella so that the cells are easily shaken or teased apart. Schultze used strong nitric acid and potassium chlorate. Put the material, which should be in very small pieces, into a test-tube; pour on just enough nitric acid to cover it, and then add a few crystals of potassium chlorate. Heat gently until bubbles are evolved, and let the reagent act until the SPECIAL METHODS 145 material becomes white. Four or five minutes should be sufficient. The fumes are disagreeable and are very injurious to microscopes. Pour the contents of the tube into a dish of water. After the material is thoroughly washed in water, it may be teased with needles and mounted, or it may be put into a bottle of water and shaken until many of the cells become dissociated. After a thorough washing in water, the material may be stained. The large tracheids of ferns, dissociated in this way and stained in safranin or methyl green, make beautiful preparations. JEFFREY'S MACERATION METHOD A method which I saw at Toronto and which gives much better re- sults was credited to Professor Jeffrey. Wood is cut or split into sec- tions about 300 fx thick, which are then boiled and cooled until free from air. The macerating fluid consists of equal parts of 10 per cent nitric acid and 10 per cent chromic acid. The time will vary with dif- ferent woods, but is likely to be about 24-48 hours if the temperature is about 35° C. When properly macerated the cells may be shaken apart or are very easily teased apart. Before staining, the material should be very thoroughly washed to remove the acid. A study of such material is very valuable in modern anatomical work. Maceration methods which act in a few minutes are likely to be so violent that fine details will not be preserved. PROTOPLASMIC CONNECTIONS As a rule, protoplasmic connections are not hkely to be seen in an ordinary preparation. It used to be thought that the rather large protoplasmic strands seen at the sieve plates of the pumpkin and other Cucurbitaceae were exaggerated examples of protoplasmic continuity ; but, as a matter of fact, the large strands do not extend entirely through the plate. The real continuity, through the middle lamella, is scanty and hard to demonstrate. Very satisfactory material for the demonstration of the connecting strands is furnished by the endosperm of the Japanese persimmon, Diospyros kaki. Usually, as you get this persimmon at the grocery store, it is seedless, but occasionally it has seeds. Fix in 10 per cent formalin, or in formalin-alcohol (10 c.c. formahn to 50 c.c. of 50 per cent alcohol) ; or in glycerin-alcohol (equal parts 95 per cent alcohol and glycerin) for a week. The endosperm of the American persimmon, 146 METHODS IN PLANT HISTOLOGY Fig. 26. — Diospyros discolor: section of endo- sperm fixed in formalin and stained in Haiden- hain's iron-alum haematoxylin. X1135. Diospyros virginiana, is good, but small and harder to hold. Best of all is the endosperm of the Philippine Diospyros discolor (Fig. 26). In Diospyros discolor the best view will be obtained in sections cut parallel with the flat surface of the seed. Imbedding is neither necessary nor desirable. Clamp the endosperm in the microtome directly and cut sections 8-10 m thick. For cutting pieces of endo- sperm too small to be clamped directly, fasten the piece to a con- venient block with cellulose ace- tate, which is easily made by dis- solving a photographic film (with the emulsion removed by keeping it a little while in hot water) in acetone. The solution should have the consistency of thick syrup. Celloidin or even glue will hold the piece to the block. Put the sec- tions in ether and leave them there for 24 hours, changing once or twice to remove any oily or fatty substances. If oils and fats are not removed completely, a good stain will he impossible. Staining is not difficult, if all oils and fats have been removed and the display of proto- plasmic connections is very striking (Fig. 26). 1. Fix in formalin, formalin-alcohol, or in glycerin-alcohol for a week. 2. Cut sections 8-12 fi thick. 3. Chloroform or ether, 24 hours. 4. Absolute alcohol, 95 per cent and 50 per cent alcohol, 20 minutes each. 5. Wash in water, 5 minutes. 6. Iron alum, 4 per cent, 8-24 hours. 7. Wash in water, 30 minutes. 8. Stain in ^ per cent haematoxyUn, 24 hours. 9. Wash in water. At this stage, examine carefully, because it may not be necessary to reduce the stain in iron-alum. If the connections are not too deeply stained, simply dehydrate, clear, and mount in balsam. After the first 5 stages, a strong stain in Delafield's haematoxylin, 10-24 hours, followed by a very weak hydrochloric acid, has given good results. A sharp stain in crystal violet, differentiated with orange SPECIAL METHODS 147 in clove oil, often fails, but sometimes succeeds; and, when successful, the connections stand out beautifully. The endosperm of Phytelephas (vegetable ivory), of dates, and many other pahns, and probably most hard endosperms, will show the connections by the methods just described; but in many cases it is necessary to resort to special methods in order to demonstrate the con- tinuity. In these special methods a reagent is used which causes the membranes to swell before the stain is applied. It is only by such an exaggeration that the more dehcate connections can be shown. Put thin sections of fresh material into a mixture of equal parts of sulphuric acid and water and allow the reagent to act for 2-10 sec- onds. Wash the acid out thoroughly in water and stain in anilin blue. According to Gardiner, this stain should be made by adding 1 g. of the dry stain to 100 c.c. of a saturated solution of picric acid in 50 per cent alcohol. The staining solution is then washed out in water, and the sections are mounted in glycerin. The sections may be dehydrated, cleared in clove oil, and mounted in balsam. The anilin blue may be used in 50 per cent alcohol acidulated with a few drops of acetic acid. Chloroiodide of zinc may be used instead of sulphuric acid. Treat the fresh sections for 2 hours with the iodine and potassium iodide solution used in testing for starch; then treat about 12 hours with chloroiodide of zinc. Wash in water and stain in anihn blue. Examine in glycerin. Try Meyer's pyoktanin method with seeds from dates as you get them on the market. Remove any oils and fats with ether, transfer to absolute alcohol, 95, 85, 70, 50, 35, and 20 per cent alcohol, about 5 minutes in each; then wash well in water and use the following re- agents : 1. Iodine, potassium iodide solution: iodine 1 part, potassium iodide 1 part, water 200 parts. 2. Sulphuric acid 1 part, water 3 parts; this mixture to be saturated with iodine. 3. Pyoktanin coeruleum 1 g., water 30 c.c. This pyoktanin is a very pure methyl violet obtained from E. Merck in Darmstadt. Put sections of the date seed into a watch glass full of the first solu- tion, and allow it to act for a few minutes; then mount in a drop of the solution. The connections will be only very faintly stained, showing a slightly yellowish color. At the edge of the cover, add a drop of the second solution. The preparation will darken a little. Then allow a 148 METHODS IN PLANT HISTOLOGY small drop of the third solution to run under the cover. Allow the stain to act for about 3 minutes. Then plunge the whole preparation into water. The action should be stopped before the entire section has be- come blue. Now wash the section quickly. If there are annoying, granular precipitates, remove them with a soft brush. Mount in glyc- erin. The membrane should be a clear blue, while the protoplast and connections should be a blue-black. The following is Strasburger's modification of Meyer's method, and shows the connections with great distinctness; and the preparations are permanent. 1. Treat the fresh sections with 1 per cent osmic acid, 5-7 minutes. 2. Wash in water 5-10 minutes. 3. Treat with a solution of iodine in potassium iodide (0.2 per cent iodine and 1.64 per cent potassium iodide), 20-30 minutes. 4. Transfer to 25 per cent sulphuric acid, which should act for at least half an hour; 24 hours may be necessary. 5. Bring the sections into 25 per cent sulphuric acid which has been satu- rated with iodine. Add a drop of Meyer's pyoktanin solution (1 g. pyok- tanin coeruleum as sold by E. Merck in Darmstadt in 30 c.c. of water). In about 5 minutes the sections will be stained sufficiently and can be examined in glycerin. If there are annoying precipitates, remove them with a soft brush. According to Meyer, the swelling is an advantage only when the walls are very thin. When the walls are thick, the connections show better without any previous swelling. Try the following method with the seeds of Diospyros, Latania, Chamerops, Phoenix, or Fhytelephas: Soak in water and cut thin sec- tions. Extract the oily and fatty substances with ether; wash in 95 per cent, or in absolute alcohol; stain in anilin blue (Hoffman's blue 1 g. dissolved in 150 c.c. of 50 per cent alcohol) for a few minutes. Examine in glycerin. This method succeeds very well with seeds of the date. Permanent preparations may be secured by the following method : 1. Fix in 1 per cent osmic acid, or in absolute alcohol, 5-10 minutes. 2. Stain for 24 hours in Delafield's haematoxylin. 3. Wash for a few minutes in acid alcohol (5 drops of hydrochloric acid in 50 c.c. of 70 per cent alcohol). 4. Wash for a few minutes in ammonia alcohol (5 drops of ammonia to 50 c.c. of 70 per cent alcohol). 5. Dehydrate in absolute alcohol, clear in xylol, and mount in balsam. SPECIAL METHODS 149 STAINING CILIA The cilia of the large spermatogoid of Gingko and the cycads stain beautifully in iron-alum haematoxylin, which not only stains the cilia but even differentiates the free portion from the part between the blepharoplast and the surface. The cilia of sperms of Bryophytes and Pteridophytes stain better with gentian violet or crystal violet. The periods are long; not less than 30 minutes, and often several hours will be required. The cilia of the motile spores of Thallophytes may often be demon- strated by allowing a drop of the iodine solution used in testing for starch to run under the cover. Zimmermann gives the following method: Bring the objects into a drop of water on the slide and invert the drop over the fumes of 1 per cent osmic acid for 5 minutes. Allow the drop to dry. Then add a drop of 20 per cent aqueous solution of tannin, and after 5 minutes wash it off with water. Stain in a strong aqueous solution of fuchsin (or carbol fuchsin) for 5 minutes. Allow the preparation to dry com- pletely, and then add a drop of balsam and a cover. The cilia should take a bright red. Zimmermann also found the following method satisfactory for the cilia of the zoospores of algae and fungi : Fix by adding a few drops of 1 per cent osmic acid to the water containing the zoospores; then add an equal amount of a mixture of fuchsin and methyl violet. The fuchsin and methyl violet should be 1 per cent solutions in 95 per cent alcohol. In a few seconds the ciha stain a bright red. The brilliant staining of the cilia of motile sperms of cycads with iron-alum haematoxylin would warrant a trial in any other form. Skilfully used, this stain will give good results with almost anything. CHONDRIOSOMES During the past twenty years the terms chondriosomes, mitochon- dria, Chondriokonten, and about fifty others, have become increasingly frequent in botanical literature. These chondriosomes, although ex- tremely small, can often be seen in living cells. They move as actively as bacteria and very effective moving picture photomicrographs have been made. While probably present in most cells, they are not differ- entiated by the methods usually employed for other purposes. Most of them bear a superficial resemblance to coccus, bacillus, and spirillum forms of bacteria (Fig. 27). 150 METHODS IN PLANT HISTOLOGY Many fixing agents either destroy the mitochondria or make it al- most impossible to demonstrate them. Fixing agents containing al- cohol or any considerable percentage of acid are to be avoided. We recommend neutral formalin, with 10 c.c. of formalin to 90 c.c. of distilled water. Get the neutral formalin from commercial formalin by distilling with sodium carbonate. It is not worth while to distil more than you need within the next 24 hours, because the formalin will not remain neutral. Formic acid appears in it and its value as a fixing agent for chondriosomes is at an end. Fix for 48 hours, wash in water, and follow the regular procedure for Haidenhain's iron-alum haematoxylin. The chondriosomes should stain black. B C Fig. 27. — Chondriosomes. A, periblem of root tip of Allium cepa. Fixed in 10 per cent neutral formalin and stained in iron-alum haematoxylin. Preparation by Yamanouchi. B, cells in young megasporangium of Asparagus officinalis. Fixed in formalin chromic acid and stained in iron-alum haematoxylin. Both X1135. C, canaliculi in root tip of Allium cepa, fixed in Bensley's solution and stained in iron-alum haematoxylin. X1200. Benda's solution, followed by Haidenhain's iron-alum haematoxy- lin, will give good results. A solution recommended by Bensley is good also for plant material. Bensley's solution. — Osmic acid, 2 per cent 1 part Corrosive sublimate (HgCl,), 2^ per cent 4 parts Add 1 drop of glacial acetic acid to 10 c.c. of this solution. Fix for 24-48 hours and wash thoroughly in water. On the slide, bleach with hydrogen peroxide; wash in water; treat with the iodine solution used in testing for starch; then wash in water. The shde is now ready for staining. We recommend the usual Haidenhain's iron-alum haema- toxylin. SPECIAL METHODS 151 Bensley recommends the following method which we have found rather uncertain, but which, when successful, yields magnificent prepa- rations: On the slide, bleach for 2 or 3 seconds in a 1 per cent aqueous solution of permanganate of potash ; then treat with a 5 per cent aque- ous solution of oxalic acid until the preparation becomes white (a few seconds); wash in water, and then stain as follows: 1. Copper acetate (neutral) saturated solution in water, 5-10 minutes. 2. Wash in water. 3. One-half per cent haematoxylin, 5-10 minutes. 4. Wash in water. 5. Potassium dichromate (neutral), 5 per cent solution in water until the preparation blackens, usually 30 seconds or less. 6. Differentiate in Weigert's ferricyanide solution. Borax 2 . 0 g. Ferricyanide of potassium 2 . 5 g. Water 200.0 c.c. 7. Wash in water and proceed as usual. Yamanouchi's method. — Fix in a 10 per cent solution of neutral formalin in water for 24 hours; wash in water, 24 hours; dehydrate, leave objects as small as onion root-tips at least 24 hours in the 85 per cent alcohol; clear in xylols; imbed; stain in iron-alum haematoxylin. CANALICULI By using special methods, Bensley has obtained views of the proto- plasm of plants, quite different from those seen in ordinary prepara- tions. In the cell of a root-tip a series of small canals, or vacuoles, appears, which is much more definite and extensive than the usual dis- play of vacuoles and which appears before any vacuoles can be recog- nized in preparations made in the usual way (Fig. 27C). Being a zoologist, he called these vacuoles canaliculi. GOURLEY'S METHOD FOR VASCULAR SYSTEM It is well known that if stems be cut under water in which there is a dilute solution of aqueous eosin, the stain will rise in the bundles, making them very prominent, but the material cannot be fixed and cleared because the stain diffuses. In the fourth edition of this book it was stated that such preparations would be still more valuable if they could be fixed and cleared. Dr. Gourley has developed such a method which is valuable, not only for stems, but for various other things. He 152 METHODS IN PLANT HISTOLOGY used basic fuchsin, prepared as follows: 50 mg. basic fuchsin is dis- solved in 2 c.c. of 95 per cent alcohol and diluted with 100 c.c. of tap water. Dr. Gourley used two lots of basic fuchsin, one from the Will Corporation and the other from Coleman and Bell. Neither lot has been certified by the Commission on the Standardization of Biological Stains. Young or old plants of Coleus — tomato, bean, etc. — are lifted from the soil and the roots are washed free of adhering material. The root system is then immersed in the stain and a part cut off beneath the surface of the solution. In 24-48 hours the bundles will be well stained. Gourley succeeded with plants 6 feet in height. Cut off the upper 6 inches of a Coleus plant, immerse the lower end in the stain and cut off 5 mm. of the stem under the surface of the stain. In 24 hours, rinse off any surface stain in water. Transfer to 50, 60, 70, 85, 95, and 100 per cent alcohol, at least 12 hours in each. Better change only once a day, especially with large pieces. Clear in f abso- lute alcohol and \ xylol, ^ xylol and | alcohol, j alcohol and f xylol, and pure xylol, 24 hours in each. The material is now ready for study but, since xylol evaporates so rapidly, add cedar oil or carbon disulphide. In a glass dish with a smooth bottom and an electric bulb underneath, the vascular system can be traced in great detail. An ovule of a cycad, cut from the sporophyll under the staining solu- tion, will have the vascular system well stained within 24 hours. When dehydrated and cleared, the simple outer, and much-branched inner, vascular supply are very striking. Small fruits and other objects can be studied in this way. In bottles with wide mouth and glass corks, such preparations should keep for years. After the stain has been taken up, material may be boiled in water or in a very dilute solution of potassium hydrate. After partial disinte- gration, pieces can be dissected so that the larger vascular units are easily followed. Gourley's method is at its best where transpiration is strong and the vascular system simple. STAINING LIVING STRUCTURES Some stains will stain living structures. Cyanin, methyl blue, and Bismarck brown have been recommended for this purpose. The solu- tions should be very dilute, not stronger than 1 : 10,000 or 1 : 500,000. The solutions should be very slightly alkaline, never acid. It is claimed SPECIAL METHODS 153 that such sohitions never stain the nucleus, and that if the nucleus stains at all, it is an indication that death is taking place. Campbell succeeded in staining the living nuclei in the stamen hairs of Tradescantia by using dilute solutions of dahlia and of methyl violet (0.001-0.002 per cent in water). Dividing nuclei were stained. For determining the stage of development of fresh material it is often necessary to use a stain. For this purpose stronger stains may be used, since it is unimportant whether the tissue is killed or not. An aqueous solution of methyl green or eosin can be recommended. With 1 per cent solutions, diluted one-half with water, mitotic figures can be recognized with ease. CHAPTER XIII PALEOBOTANICAL MICROTECHNIQUE During the past three or four years, new methods in paleobotanical microtechnique have greatly minimized the drudgery of making prepa- rations for microscopic examination. The subject of paleobotany has been making such rapid progress that scarcely any problem involving the anatomy of living vascular plants can be investigated intelligently without some knowledge of Mesozoic and Paleozoic forms. Material, especially that of Paleozoic pteridophytes and gymnosperms, is be- coming available in the United States, largely through the discoveries of Dr. Noe and his students. Consequently, it is increasingly necessary for laboratories to have apparatus and technique for making rock- sections. SECTIONS The outline of the process of cutting a rock-section is very simple: 1. Saw the rock into two pieces. 2. Polish the cut surface. 3. Fasten the cut surface to a piece of glass mth hot shellac. 4. With the saw, make another cut, as close to the glass as possible, so as to leave a thin section firmly fastened to the glass. 5. Grind and polish until the section is as thin as possible, or as thin as you want it. 6. Wash all polishing powder off \\ath water. 7. Dry completely and, either with or ^^^thout moistening in xylol, mount in balsam. A word of suggestion in regard to these various points may not be amiss. 1. Most rock-sections are cut with a rather expensive and quite compUcated instrument, called a "petrotome." The saw is of the cir- cular type, is made of tin or other soft metal, has no teeth, but has diamond dust driven into the margin. A rigid clamp holds the object, and the saw, constantly cooled by a stream of water, gradually cuts through the specimen. If the piece to be cut is more than 5 or 6 cm. in diameter, a band saw is better; and if the piece is 10 or 20 cm. in diameter, the band saw is necessary. The "saw" is not of the type 154 PALEOBOTANICAL MICROTECHNIQUE 155 used in sawing wood, but is a plain band of metal which must not be too hard. To be ideal, it should have diamond dust driven into the margin; but, since the expense would be considerable, carborundum powder and water can be used instead. A band saw is a dangerous piece of apparatus and the operator should be thoroughly protected from a broken, whipping saw. 2. The cut surface can be polished on a revolving brass plate, kept wet, and liberally powdered with fine carborundum. Or, the cut surface can be rubbed by hand on a piece of plate glass, with plenty of carborundum. This is a more rapid method and most investigators prefer it. When the surface has become even and smooth the specimen is ready for the next step. 3. Fasten the polished surface to the glass slide upon which the sec- tion is to be mounted. Plate glass 3 or 4 mm. thick is best for sections larger than 3 or 4 cm. square. Gradually heat the slide until it is quite hot. Melt upon the slide the thin flakes of white shellac used by painters; heat the object and press the polished surface very firmly into the melted shellac. Canada balsam, from which the xylol has been driven off by heating, can be used instead of shellac. Much of the Paleozoic material is in the form of coal balls. After the ball has been cut in two, it is often difficult to hold the hemispherical piece in a clamp, especially if the piece is small. In such cases, it is better to fasten the polished surface to a convenient .piece of marble, about 2.5 cm. thick, and 5 or 6 cm. square. The marble is easily held in the clamp. As soon as the slide, or marble, and object are cool, the next cut can be made. 4. Fasten the object in the clamp and saw as close to the glass, or marble, as possible, thus leaving a thin section cemented to the slide or marble. If marble has been used, the section is removed by heating or by dissolving it off with xylol. It can then be fastened to the glass slide for grinding and polishing. Anyone who can handle tools should soon be able to cut a section 1 mm. thick. A skilled technician can cut sections as thin as 0.5 mm. 5. The second grinding must be very careful and accurate. This can be done on the revolving brass disc; but here, again, a piece of plate glass, with plenty of carborundum, and water, is gaining favor over the more expensive revolving disc. The glass slide allows one to note how the process is progressing. 156 METHODS IN PLANT HISTOLOGY 6. When the section becomes thin enough, or even before if it be- gins to crack, wash off the powder. 7. It is usually a good plan to use rather thick balsam for mount- 1 ^aB^l 91S ffBfhfffli W^^r i ^S \ mt ^ iii' *-viJ3^t ^H i m H^Ki' ^^^HT l^V"'*-^ /^^53?^ JTSS*.^vTt3S W^A_A/V\fvt/TrSc }l^SM •TJbm-M ^^ ^"r'^^td ^r"^^ ...Twtf c*T \ Att-^/X. S xX- "^j^C^I^ ^^®BtJ 1 ^^W^^^§ ■^Si ^'^tajL.jl''^^ 1 i/-*, jf)S^\£j^^'\^ 1 MHB^rjj|n^Kfvfa^^^BS|K ^S #^ J^^K. jt''^^^*w^ */i ^/^ ig i^li'^staj^ji^"^'^ VJtS ^^^' K' :.^ ^^^^^^^^^^^^^ ^:; ^ Fig. 28. — Sphenophyllum pleurifoliolatum: transverse section of a stem cut from a coal ball of the Upper Carboniferous. Eastman Commercial Ortho film.Wratten E filter (orange), J. Swift and Son 1-inch lens; arc light; exposure, 1 second. Slide by Lomax, negative by Sedgwick. X 19. PALEOBOTANICAL MICROTECHNIQUE 157 ing, even if it should be necessary to heat it a Httle to make it flow well. By this method, sections of fossils 10X15 cm. have been cut thin enough for examination with a 4 mm. objective. Sections 3 or 4 mm. square have been cut thin enough for satisfactory examination with a 2 mm. oil immersion lens. Figure 28 shows that a rehable study can be made from sections cut from the solid rock. Of course, this method can be used for such objects as walnut and hickory shells. It is not really necessary to cover the section. Just polish the sur- face with fine carborundum and then polish still smoother with a fine powder of tin oxide. The section will look as if mounted in balsam. A fine view can be secured without making any section. Merely polish the cut surface as described in the preceding paragraph and ex- amine by reflected light. Excellent photomicrographs can be made from such surfaces. PEELS Peels seems to be as good a name as any for a type of "section" which is saving the paleobotanist a lot of time and is making possible a near approach to serial rock-sections. Peels are made with celloidin or with gelatin. Celloidin peels. — Probably every investigator has his own method of making a celloidin peel. The following method, written by Dr. Fredda Reed, applies to calcified petrifactions: 1. After cutting a coal ball in two, polish the surface of the petrifaction with fine carborundum powder. 2. Wash surface. 3. Treat with 5 per cent hydrochloric acid 2-5 minutes. The thickness of the plant tissue peeled depends upon the length of the acid treatment. 4. Wash gently in running water. 5. Dry. 6. When thoroughly dry support the rock in a horizontal position and pour over the surface a solution of equal parts of absolute alcohol and ether quickly (before the alcohol-ether solution has had time to evaporate) : 7. Pour on 25 per cent celloidin (dissolved in equal parts of absolute alcohol and ether). 8. Peel. When the absolute alcohol and ether have evaporated, the cel- loidin is left as a thin film which may be peeled off by inserting a scalpel under the edge. 158 METHODS IN PLANT HISTOLOGY 9. Wash the film in 5 per cent hydrochloric acid to remove any excess mineral, then wash gently in water. 10. Dry in blotting paper under pressure, otherwise the peel curls badly. 11. The entire film, or only desirable portions of it, may be cleared in xylol and mounted in balsam. After every peel, polish the surface before making another. - The following variations, used by various investigators, are worth trying: After the peel has been taken off, cleaned, and washed (stage 9 of Dr. Reed's schedule), instead of drying and using blotting pnper, put the peel into 50, 70, 85, and 95 per cent alcohol, about 15 minutes in each. If the celloidin appears to soften in 95 per cent alcohol, short- en the time. Transfer to Eycleshymer's clearing fluid (which does not cause such curling as the xylol), and mount in balsam. Silicified material is treated in the same way, except that hydro- fluoric acid (10 per cent in water) is used instead of hydrochloric and is allowed to act longer, about 3 or 6 minutes. Gelatin peels. — In Nature, of March 15, 1930, John Walton de- scribes peels of gelatin. The rock surface is prepared and etched with acid, as in case of celloidin peels, and allowed to dry. Then a hot solution of jelly, containing formalin and glycerin, is poured on the surface. The amount of the mixture poured on will depend upon the size of the object and the desired thickness of the peel. To cover a sur- face 1 square decimeter in area, use about 2 g. of the pure gelatin used in making bacteriological cultures, 50 c.c. water, 0.5 c.c. glycerin, and 0.5 c.c. formalin. The surface must be surrounded before the etching process with a rim of plasticene, or some other substance, and should be leveled with a spirit level. The water and glycerin are mixed, heat- ed, and the jelly stirred until dissolved. Heat to 60°-80° C, stir in the formalin quickly, and immediately pour the mixture over the sur- face of the petrifaction. Let the jelly set and remove to a warm well- aired place to dry. Avoid dust. When the jelly is dry, peel it off. It may be lizard to start the peel but, once started, it comes off rather easily. Clear in xylol and mount in balsam. It used to be claimed that in petrifactions all organic matter has been replaced by the mineral. The fact that a peel can be made proves that the old claim is incorrect. The mineral matter is etched down by the acid, the organic matter remaining and coming off in the peel. CHAPTER XIV BOTANICAL PHOTOGRAPHY The only field of photography with which histology is directly con- cerned is photomicrography, together with paper prints and lantern slides made from photomicrographic negatives; but this field is so difficult that time, temper, and money will be saved by beginning with pictures of trees, flowers, buildings, maps, graphs, lantern slides, and such experimental apparatus as one needs in illustrating investi- gations. It is assumed that the student knows something about an ordinary camera and that he knows the usual routine of making negatives and paper prints. The first step is to get a good negative. Begin with a tree or a build- ing. The most important factor in securing a good negative is correct exposure. The professional photographer does not need any meter, but the rest of us had better use one to determine the length of expo- sure. The Watkins Bee Exposure Meter is good and inexpensive. The sensitive paper is reliable for a year, even in the tropics. The Wynne Infallible Exposure Meter lives up to its name. The Harvey and the Voigtlander are good meters of the mechanical type. The Bewi and the Justaphot are more expensive, but they almost remove the neces- sity for judgment in making exposures. The American Photography Exposure Tables are very convenient and instructive, since the various factors, light, stop, plate, latitude, and time of day, are estimated separately. The tables give also practically all the formulas and direc- tions an amateur needs. An amber-colored filter, increasing the expo- sure about three times, will keep the clouds, which would otherwise be lost, and will give much better color values. Next, select a good plate or film. Professor Land, when asked his opinion of the comparative merits of film packs and cut films, said, "Packs cost twice as much as cut films and are only half as good: plates are better than either." But films are improving and the cut film now has an emulsion as good as that on a plate. Such an emulsion cannot be put on a roll film or film pack, because the film has to be 159 160 METHODS IN PLANT HISTOLOGY pulled over a small roller. The "commercial ortho" cut film is very satisfactory; but, as one learns to get the right exposure, the panchro- matic film can be used. Mr. Charles H. Carpenter, the veteran photographer of the Field Museum in Chicago, gave this advice to a beginner: "Pick out a good plate or film, and one developer, and stick to them for the first ten years." LANTERN SLIDES After securing a good negative, the method of making a lantern slide or paper print depends upon the size of the negative. Slides and paper prints of the same size as the negative are printed by contact. It is hardly worth while to make a lantern slide by enlargement from a tiny negative; the best lantern slides are made by reduction from larger negatives. On the other hand, excellent paper prints can be made by enlargement. Lantern slides by contact. — From a 3jX4| negative a lantern shde can be printed by contact, just as one would make a contact print on paper, because the lantern-slide plate (3jx4) is so nearly the size of the negative. If one wants a lantern slide of some feature of a larger negative, the lantern-slide plate can be placed over the desired portion and a contact print can be made. The negative can be placed in a printing frame and the lantern-slide plate placed over it, emulsion side against emulsion side. If the printing frame is larger than the nega- tive, put a piece of clear glass in the frame. If the negative is a glass plate, remember that you are dealing with three thicknesses of glass and do not use too much pressure. Hold the frame in the left hand, as far from the bulb as possible, and snap on the light for a second. If the plate is overexposed, use a weaker bulb or increase the distance. If the plate is completely de- veloped within a minute, the exposure was too long. The exposure should be such that full development will require about 1 minute and 45 seconds. The prominent features should show clearly when viewed from the back of the slide. If the negative is uneven, the distance from the hght may be in- creased so as to lengthen the exposure to several seconds, thus giving time to shade the weak parts. If a negative is harsh and shows too much contrast, hold it closer to the light and shorten the exposure; if weak and lacking in contrast, hold it farther away and lengthen the exposure. BOTANICAL PHOTOGRAPHY 161 Dealers in photographic supplies sell a box for making paper prints. It is equally good for making lantern slides, but rather expensive. A box which will give just as good results can be made in a few hours (Fig. 29). Find or make a box about 18 inches high and large enough to have a glass top 9X11 inches fitted into it. Fit a ground glass into it, about 8 inches from the clear glass in the top. On the bottom, put a red bulb in the middle, with four white bulbs (50 or 60 watt) around it. The red light can be plugged in separately and the four white bulbs can be wired so as to glow only when the switch is closed ; or the lights can be wired so that, with only one plug, the red light will be on as long as the plug is in, but the white lights will come on only when the switch is closed (Fig. 29). For holding the lantern-slide plate firmly against the negative — the dull emulsion side of the lantern-slide plate and the negative should always be in contact with each other — the device shown in Figure 30 is better than a hinged lid. With this box, the exposure can be estimated very accurately, since the distance between the negative and the light is constant. With only a ground glass to diffuse the hght, the exposure is likely to be too short. Put one or two sheets of white paper on top of the ground glass. With both ground glass and paper to diffuse the light, the exposure should be about 2 seconds for a good negative. The number of sheets of white paper should be increased or diminished until 2 seconds gives an exposure which will develop fully in If minutes. With a dense negative, the time will be longer; with a thin negative, it is better to add one or more sheets of paper, so that 2 seconds will be about right for the exposure. After the plate, film, or paper has been developed and rinsed for about 10 seconds in the acid stop, place it in "hypo." The hypo removes the silver which was not acted upon by the ex- posure to light. Lantern slides by reducing and enlarging. — If a slide is to be made from a 4X5 or larger negative, there must be a reduction. A camera is necessary. A 3jX4| camera is large enough. If any larger size is used, the plateholder must be "kitted" down to 3jX4, the standard size of lantern slides in America. In using the larger cameras, mark upon the ground glass the exact size and location of the lantern- slide plate. Fasten the negative in some convenient place where the 162 METHODS IN PLANT HISTOLOGY £u ^ 9 inches— Sinches ■^ J JJoor Ground cflas^ j^ i\ L 10 inches f=^ f=\ . f r — \ lb plug Fig. 29.— Box for making lantern slides and paper prints. .4bove, view of front: below, view of bottom. BOTANICAL PHOTOGRAPHY 163 light may shine through it : diffuse daylight is good. Then arrange the camera just as in taking any ordinary picture. The board shown in Fig. 30. — Block to press lantern slide plate against the negative Figure 31 will be just as useful in making lantern slides as in making photomicrographs. At one end of the board fasten a frame which will hold an 8 X 10 negative and also hold kits for smaller negatives (Fig. 315 and C). The long slot in the board will allow the camera to be fastened at the proper dis- tance. If buildings, trees, or shadows are in the way, tilt the board so as to have a clear sky for a background. Be very careful in focusing; it is best to examine, with a pocket lens, the image on the ground glass. In general, use a rather small stop, F16 is good. If reducing from an average A Fig. 31. — A, board for photomicrographic and lantern slide work; B, end view with clips to hold negatives; C, side view of block to be used on board when making lantern slides. 164 METHODS IN PLANT HISTOLOGY 5X7 negative, in good daylight, with an F16 stop, try 2 or 3 seconds. If enlarging from a negative somewhat smaller than a lantern slide, try 8 or 10 seconds. Other things being equal, the best lantern slides are made by reduction from larger negatives and the poorest, by en- largement from smaller negatives. The superiority of the larger negative is easily demonstrated. With a 5X7 camera, make a negative of some elaborately ornamented building, making the building just cover the plate. Then with a vest- pocket camera, or even a 2jX3|, make a similar negative, so that the building just covers the plate. Make lantern slides from both nega- tives, so that the building is of the same size on both lantern slides. While the building, as it appears on the screen, is of the same size, no matter which slide is used, the one from the larger negative will show finer detail. The same is true for all kinds of plant subjects. Small cameras are easy to carry and their small films are cheap, but they have no other recommendation. A 3iX4|, with a high grade 4.5 anastigmatic lens, is good, even for scientific work; but a 5X7 is better. If you are strong and ambitious or have some one to carry the heavy load, take an 8X 10. Staining lantern slides. — Some of the stains used in staining mi- croscope slides will give a pleasant tone to lantern slides. Light green gives a clear, moonlight effect. Phloxine gives a transparent, rosy tint. Sepia and other tones could doubtless be imitated by this easy method. Clearing lantern slides. — Sometimes a slide will seem perfectly clear, just as it comes from the fixing bath, especially from an acid fixing bath; usually, however, it will be better to transfer the slide from the fixing bath to a weak solution of acetic acid — just enough acid to give the solution the taste of weak vinegar — and then rock for a minute before washing. The following clearing fluid may be used in the same way : Metric Apothecaries' Alum 20 g. (1.3 gr.) Iron sulphate 20 g. (1.3 gr.) Citric acid 20 g. (1.3 gr.) Water 500 c.c. (17 oz.) Coating lantern slides. — After the slide has become thoroughly dry, a coat of balsam or shellac will add much to its brilliancy. Dilute the Canada balsam with xylol until it becomes almost as thin as water; balance the slide on the thumb and first, second, and third fingers. BOTANICAL PHOTOGRAPHY 165 holding it as level as possible ; pour the balsam over it, letting the bal- sam flow evenly over the whole surface ; then tilt the slide and pour the balsam back into the bottle. Put the slide in the rack to dry. Mounting. — Add a suitable mat and a clean lantern-slide cover. Re- member that the effect of a first-class lantern slide may be impaired or even ruined by an inartistic mat. Bind the slide and cover together with a lantern-slide binding strip. Paste on the label, or, if you prefer, paste the label on the mat before binding, so as to have it protected by the cover. Lay the slide down so that the positions are just as they were in the original, and then paste the "thumb mark" in the lower left-hand corner. The Gevaert lantern slide plates are used by many botanists. They are good for general work and the formulas for sepias, purples, and reds by direct development make them attractive for lantern slides of scenery. Developers. — For Lantern Slides from Strong Negatives Distilled water 1,000 c.c. Metol Hydrochinon Sodium sulphite (crystal) . Sodium carbonate (crys- tal) Potassium bromide 3g. Ig- 40 g. 50 g. Ig. For Lantern Slides from Weak Negatives Distilled water 1,000 c.c. Metol 1§ g. Hydrochinon 6 g. Sodium sulphite (crystal) 50 g. Sodium carbonate (crys- tal) 100 Potassium bromide 1 g- g- The time of development will be about 2-2| minutes for black tones, and 1-li minutes for warm tones. For sepia, purple and red tones. — Use the Gevaert warm tone lan- tern-slide plates and expose as for ordinary tones. Use the following developer. A B Distilled water 1,000 c.c. Distilled water 250 c.c. Metol H g- Ammonium carbonate 30 g. Hydrochinon 6 g. Ammonium bromide 30 g. Sodium sulphite (crystal) . 50 g. Sodium carbonate (crys- tal) 100 g. Potassium bromide 1 g. 166 METHODS IN PLANT HISTOLOGY The ammonium carbonate evaporates so rapidly that it must be kept in closely stoppered bottles, preferably in emery stoppered bot- tles. The different tones are obtained by mixing A and B in different proportions. Cold sepia, 8 parts A to 1 part B. Develop, 5-8 minutes. Warm sepia, 10 parts A to 3 parts B. Develop, 12-15 minutes. Purple-red, 8 parts A to 5 parts B. Develop, 20-30 minutes. Do not try to shorten the time of development by giving a longer exposure. Rinse thoroughly and fix in an acid fixing bath. The following is recommended for all plates: Hypo 150 g. Sodium bisulphite 15 g. Water 1,000 c.c. MAPS AND GRAPHS One often needs a lantern slide of a map. An easy but very unsatis- factory method is to copy from an atlas or, not quite so bad, from a base map. The atlas will have so many places named that what you want to show will be obscured. The best way is to trace a rather large map of the desired region, rub the underside of the tracing paper with a soft pencil and then, by following the lines with a hard pencil, trans- fer the outline to pure white cardboard. Ink it with dead black India ink making the lines very bold. If the map is 38 cm. wide, hnes 1 mm. wide are not too coarse for the outlines of coasts and islands. When they appear on the lantern slide, they will not be more than 0.2 mm. wide. Lettering should be correspondingly bold. Letters less than 1 cm. high on a map 38 cm. wide will not be very conspicuous when they appear on the screen. In copying a map 38 cm. wide, using a contrast lantern-slide plate (red label plate, if Cramer's is used), with F16 in fair diffuse light, try 20 seconds. If such work is always done in the same place, you will soon know whether to use 15, 20, or 30 seconds. If artificial light is used, there should never be a mistake after you have once determined the proper exposure. Develop in a contrast or process developer. Use hydrochinon : there should not be any metol in the formula. Maps are more satisfactory when colored, if there are both land and BOTANICAL PHOTOGRAPHY 167 water areas. After washing out the hypo, stain the shde in an aqueous anilin bkie or light green for 10 or 15 minutes. When dry, use a brush to color the land areas orange or any desired color. The second color is likely to be more satisfactory if very dilute. With all slides which are to be colored, it is better to omit alum from the hypo solution. For graphs, use the same method, except that hypo solutions with alum and acid may be used. Lantern slides can be made from typewritten tables in the same way. The paper should be pure white and the letters, dead black. Illustrations in books can be copied in the same way. While nothing surpasses a process plate for negatives from which you wish to get dead black and clear glass in the lantern slide, or dead black and pure white in a paper print, process films are very good and convenient for filing and are in no danger from breaking. ENLARGEMENTS Lantern-slide plates are often used in making photomicrographs of histological preparations, but even when a 5X7 plate is used, an 8 X 10 enlargement, or even an 11X14, is better for reproduction, assuming, of course, that the negative is good enough to stand enlargement. If the negative is sharp and has no defects, a glossy paper can be used; but if the negative lacks a little in sharpness or has defects, a paper with a velvet surface is better, for the large print can then be touched up with a pencil or pen and, when reduced to the size used in scientific journals, the reproduction will be better than the engraver could have obtained from a small print. Any prints as small as 3jX4j or even 5X7, should be on glossy paper and should be squeegeed, if they are to be only 4 inches wide when reproduced. The engraver recommends glossy paper for all prints. While the enlargement from photomicrographs is the kind which comes within the range of histology, it is better to practice with nega- tives of trees, flowers, and people. Select your best negatives and use a paper with a velvet surface to begin with. The negative can be placed in your camera where you place a plate for exposure. You can cut the partition out from the plate holder — except a small border to hold the plate — and then put the negative in as you would a plate; or you can make a frame, just the size of a plate holder to hold the glass negative. For fihns, use two clear glass plates 168 METHODS IN PLANT HISTOLOGY orounef f/ass bound together along one edge with a piece of lantern-sUde binding tape. The relative positions of light, ground glass, negative, and paper are shown in Figure 32. A 200-watt light or, better still, four or five 60-watt lights, will be sufficient for most work. There should be a reflector behind the Hght. The ground glass, about 2 inches from the negative, is to diffuse the Hght. Almost any camera which takes ordinary plate holders will do, but we should advise a strong 5X7 camera of the general Primo pat- tern. The vertical position is bet- ter, because the paper is easier to manipulate. If you prepare your own apparatus, it will save time to look at enlarging cameras on the market, or at least to study the illustrations of them in cata- logues. The time of exposure will vary with the light, the negative, the paper, and the amount of enlarge- ment. With a 200-watt bulb, or a group of 60-watt bulbs, a good negative, an average paper, and an enlargement of three diame- ters, try 20 seconds. It will save time and money if you use a full sheet of paper and expose for 5 seconds; then cover a strip about an inch wide with a piece of black paper or a metal squeegee plate and expose the rest for 5 seconds more; then cover another inch and expose for 5 seconds; and so on across the paper. When you develop, you will see which exposure was best and, after a few trials, you will be able to estimate the exposure without any trial or, at least, will be able to estimate by trying a small piece of paper. There are various developers, but the one given below seems to be as good as any. foble Fig. 32. — Relative positions of reflector, light, ground glass, negative, lens, and table. At top, above light, there should be holes for ventilation. BOTANICAL PHOTOGRAPHY 169 H2O 1,250 c.c. Metol 1 g. Sodium sulphite 15 g. Hydrochinon 4 g. Sodium carbonate 15 g. Potassium bromide 1 8 g. There is a relation between the length of exposure and the time of development. Suppose that with a good negative an exposure of 10 seconds with 2 minutes of development gives the best results. Then an exposure of 5 seconds, with 4 minutes of development, or an exposure of 20 seconds with 1 minute of development, should yield prints nearly as good. Too long an exposure gives dark, unsatisfactory prints; while, with too short an exposure, the paper becomes gray or spotted in the effort to get pictures by prolonging the development. With uneven negatives, the exposure can be controlled, more or less, by holding a suitably shaped piece of cardboard between the lens and the paper, keeping the cardboard in motion .to avoid sharp contrasts. Transfer from the developer into the stop. Here again, there can be some control, because — with the trays close together — part of the print can be slipped into the stop, while the rest develops a little longer, so that a sky with clouds can be developed after the develop- ment of the foreground has been stopped. By applying the stop with a tuft of cotton, good prints can be secured from negatives which would yield only flat prints without such treatment. Then transfer to hypo. Many prefer a plain fixing bath: Water 1 liter Hyposulphite of soda 250 g. It does not keep well and must not be used if it shows the least brownish color. Make it fresh every day. A single print would be fixed in 15 or 20 minutes; but prints are al- most always piled, one on top of another, in a tray of hypo. They should be moved frequently, taking the one from the bottom and put- ting it on top. With such movement, half an hour, or even an hour, is not too long. Then wash in running water for half an hour, or even an hour. If the prints are in a sink, a rubber tube can be fastened to the faucet, and one can pinch the end of the tube so as to spray the prints. In this way, prints may be washed thoroughly in 15 minutes. The most ex- 170 METHODS IN PLANT HISTOLOGY pensive rotary washers have no advantage over this method, except that they save your time. The prints are then spread out to dry. Let the print drain until the water comes off only in drops; then lay the prints down, face up, on blotting paper or newspaper and wipe off surplus water with a large tuft of cotton. When almost dry — not quite "bone dry" — they can be piled up, one on top of another, or with a thin piece of white blotting paper between, and put under gentle pressure. A board with two or three bricks will be enough. If put under pressure too soon, prints are likely to wrinkle. If glossy paper is used, place the prints face down upon a clean squeegee plate, and press them with a rubber roller or with a rubber like a window cleaner. When dry, they should come off easily. OVEREXPOSURES AND UNDEREXPOSURES Negatives, lantern slides, and paper prints which have been over- exposed or underexposed can often be reduced or strengthened until they are as good as if the exposures had been correct. Reducing overexposures. — The reducing solution should be applied as soon as the negative, lantern slide, or paper print comes from the fixing bath. If they have been washed and dried, they should be soaked in water for five or ten minutes before using the reducing solu- tion. The following is a good reducing solution for most purposes : Metric Apothecaries' / Water 473 c.c. (16 oz.) \ Hyposulphite of soda 31 g. (1 oz.) f Water 473 c.c. (16 oz.) \ Red prussiate of potassium 31 g. (1 oz.) Solution B must be protected from the light. Cover the bottle with black paper and keep it in the dark when not in use. Mix only for immediate use 8 parts of A to 1 of B and use in rather subdued light. A darkroom is not necessary, but avoid bright light. When the negative or lantern slide becomes satisfactory, wash it in water as thoroughly as if it had just come from the ordinary hypo fixing solution. The reduction can be done locally with a tuft of cotton. When possible, it is better to make a new, correct exposure than to reduce or intensify an incorrect one. BOTANICAL PHOTOGRAPHY 171 Intensifying underexposures. — Even if a negative or lantern slide has been considerably overexposed, it can be reduced quite satis- factorily; if much underexposed, httle can be done for it; if only slight- ly underexposed, it may be greatly improved by the following solution : Metric Apothecaries' (Bichloride of mercury 2 g. (31 gr.) Water 100 c.c. ( 4 oz.) Bromide of potassium 2 g. ( 31 gr.) J Sulphite of soda crystals 10 g. (154 gr.) \ Water 100 c.c. ( 4 oz.) The solutions keep indefinitely and may be used three or four times. Apply the intensifier after fixing in hypo and washing in water. If the negative or slide has been allowed to dry, soak it in water for half an hour before intensifying. Place the negative or slide in A, rocking the tray as in developing, until it becomes gray or even white. Wash in water for 5 minutes and then transfer to B and leave until the dark color can be seen on the back of the negative or slide. Wash in water as thoroughly as after fixing in hypo. Some use a saturated aqueous solution of the bichloride of mercury, without the bromide of potassium; and, instead of solution B, use water to which ammonia has been added — about 1 part ammonia to 40 parts water. Excellent sepia tones may be secured in this way. Wash well in water. After the plate has been thoroughly washed in water, wipe it gently with a tuft of cotton. The cotton must, of course, be thoroughly wet; it is better to hold the plate under a stream of water while wiping. This should always he done before placing a negative or slide in the rack to dry, after a washing in water. PHOTOMICROGRAPHS By Dr. Paul J. Sedgwick For the making of photomicrographs at the highest magnifications a regular photomicrographic camera, consisting of a heavy, rigid, optical bed to which are attached the camera proper, the microscope, the condensing lenses, the filters, and the light source, is almost a necessity. Such an outfit certainly facilitates the making of photomi- crographs at any magnification, and the worker who is required to make any considerable number of photomii'rographs should be so 172 METHODS IN PLANT HISTOLOGY equipped. In the absence of such equipment very satisfactory work can be done with any ordinary camera, microscope, and suitable light source if the worker will exercise sufficient care in their arrangement and alignment. If the camera has a long bellows draw, it should be possible to make pictures with magnifications as great as 1,000 di- ameters. Without regular photomicrographic equipment it is scarcely worth attempting higher magnifications. In arranging the equipment for taking a photomicrograph it is ab- solutely essential that all the equipment be in perfect alignment. This is taken care of in the expensive photomicrographic cameras by pro- viding a heavy metal bed or track to which all of the equipment can be attached and along which the various parts can be shifted as necessary in arriving at the correct optical adjustment. A satisfactory substitute for this rigid optical bed can be constructed from a board of sufficient length to accommodate all of the equipment. A guideway for the camera can be made of wooden strips and screwed to the board. The microscope can be bolted to the board by long bolts with buttei-fly nuts. The microscope is inclined at 90°. The guideway for the camera must be of a sufficient height to raise the camera to a level which will center it with the microscope. If a table can be spared, it might be found to be more convenient to have one table set apart for photomi- crographic work (Fig. 33). A guideway could be fastened to the table, and holes could be bored in the table for bolting down the microscope. In the discussion that follows it will be assumed that a photomicro- graph of a part of a vascular bundle is desired and that the photograph is to have a magnification of approximately 400 or 500 diameters. Furthermore, it will l^e assumed that the section has been stained in safranin and anilin blue. The apparatus is arranged as follows. The microscope is inclined at the inclination joint. The lens of the camera is removed, and the tube of the microscope is inserted through the hole in the lens board. Some sort of light-tight arrangement must be made where the tube of the microscope enters the camera. A piece of black velvet will serve if carefully wrapped around the tube of the microscope. The microscope is lined up with the camera as accurately as possible and bolted in place. The mirror of the microscope is removed and the light source is placed in line with the camera and the microscope. The light source may be an arc light, a gas lamp with incandescent mantle, a concen- trated filament mazda projection bulb, or a lamp designed especially BOTANICAL PHOTOGRAPHY 173 03 a a > W a C3 a o o .a a c8 a a < CO 174 METHODS IN PLANT HISTOLOGY for photomicrographic work. The light should be lined up as accurate- ly as possible. One or more condensing lenses should be inserted be- tween the light and the condenser of the microscope in order to con- centrate the rays. For this, one may use a spherical flask filled with water, or a simple hand reading glass will serve. The condensing lenses of a small stereopticon can be adapted. If a round flask filled with water is used, it will serve both as condenser and heat-absorbing unit. If lenses are used for the auxiliary condensing system, it will be necessary to provide a flat-sided flask or small battery jar with water to absorb the heat and thus protect the slide that is to be photo- graphed. The auxiliary condensing system should be placed in such a position as to project a magnified image of the light source on the iris diaphragm of the condenser of the microscope. The slide which is to be photographed is next brought into focus on the ground glass of the camera with the coarse and fine adjustments of the microscope. The substage condenser is focused to obtain critical illumination. This will bring the light to a focus on the section to be photographed. An image of the source of light will now be seen on the ground glass superimposed upon the image of the object. By examin- ing the ground glass of the camera, any further adjustments that may be necessary can be made in order to bring the light source, the auxili- ary condensing system, the substage condenser, the microscope tube, and the camera into perfect alignment. After all of the adjustments have been correctly made, move the substage condenser a very short distance either toward or away from the slide so as to remove the image of the light source from the ground glass. This adjustment should be very slight and will not appreciably affect the quality of the image of the object to be photographed. It will now be necessary to decide upon the plate or film to be used for the photograph and it will also be necessary to choose an appropri- ate light filter. The yellow filters which are used in ordinary photog- raphy will be found to be useful, but, if a great amount of photomicro- graphic work is to be done, the worker should have a set of light filters such as the set of 9 Wratten filters for photomicrography sold by the Eastman Kodak Company. This set includes the filters that will be found to be most useful. In choosing the plate or film and the light filter it is necessary to come to a decision in regard to the effect that is desired in the picture. The choice of plate, or film, and filter will de- pend upon whether it is desired to obtain the maximum contrast be- BOTANICAL PHOTOGRAPHY 175 tween the object being photographed and the background or whether it is desired to obtain the maximum detail within the object. For the beginner, perhaps the most confusing part of the procedure comes in choosing the proper fiUer. The yellow and orange filters will probably be the ones most frequently used, for when they are used with a good orthochromatic or panchromatic plate or film the result will be an approximately correct rendering of the color values in black and white. It is not possible to state just which filters will serve for each set of conditions but certain rules will aid in determining. To obtain the greatest amount of contrast between the specimen and the back- ground use a filter or a combination of filters which transmits only that part of the spectrum completely absorbed by the specimen. This will cause the specimen to appear black in the picture. If the result is unsatisfactory because detail within the specimen has been sacrificed in favor of contrast between the background and the specimen, try a filter or a combination of filters whose spectral transmission is not exactly limited to the part of the spectrum absorbed by the object. To eliminate the contrast between the object and the background use a filter or combination of filters transmitting that portion of the spec- trum which is also transmitted by the object. After a little experience one will be able to estimate the probable photographic result by mak- ing a visual examination with first one and then another of the filters in place. We started with the assumption that a photograph was to be made of a part of a vascular bundle in a section that had been stained with safranin and anilin blue. If the photograph were to be made without a light filter and on an ordinary uncorrected plate or film which is oversensitive to the blue portion of the spectrum and completely in- sensitive to the red part, the picture would not be satisfactory. The red-stained part of the specimen would appear black and the parts stained in anilin blue would hardly appear in the photograph. This effect would result from the insensitiveness of the ordinary plate or film to red and the oversensitiveness to blue which gives blue almost the value of white light. Instead, a good color-corrected plate or film should be used and a combination of filters such as the Wratten "B" and "E" filters. This combination of filters will increase the contrast of the part of the specimen stained in blue since the spectral trans- mission of the combination is included within the absorption band of anilin blue. While these filters will not give the maximum contrast for 176 METHODS IN PLANT HISTOLOGY the safranin-stained portion of the specimen, the contrast will be sufficient and the result will be more satisfactory than if the safranin stain had photographed as pure black, because all detail in the vessel walls would then have been lost. A booklet, Photomicrography, published by the Eastman Kodak Company, includes a table showing the spectral absorption bands of some of the stains used in botanical microtechnique. It also gives data on the transmission spectra of the Wratten filmers ordinarily used in photomicrographic work. Three other booklets published by the East- man Kodak Company will be helpful though they do not pertain di- rectly to photomicrography. These are, Wratten Light Filters, The Photographij of Colored Objects, and Color Films, Plates and Filters for Commercial Photography. The final focusing of the microscope should be accomplished after the Hght filter is in place in the path of the light and close to the sub- stage condenser. The plate or film holder should be inserted, the light turned off, the dark slide removed from the plate or film holder, and the exposure made by turning on the light and turning it off. Making the exposure in this way obviates the necessity for using a shutter in the set-up of equipment. During the time of the exposure the table on which the camera is resting should not be touched and no one should be permitted to walk across the floor. These precautions are necessary to prevent a blurring of the image. The time of the exposure will have to be determined by trial. After a correct exposure has been obtained it will be possible to compute further exposures with different filters from the factors given in the booklet, Photomicrography. In the preceding discussion it was assumed that a photomicrograph was to be made at fairly high magnification. For photographs at lower magnifications the ocular is removed from the microscope, and it will probably be best to remove also the draw tube. Care must be exer- cised to avoid reflections within the microscope. A tube of dull, black paper may have to be inserted in the microscope. A microscope with a large barrel is useful for low-power work. When using 48 mm., 32 mm., and possibly 16 mm. objectives it will be necessary to remove the substage condenser of the microscope in order to get the complete field illuminated. It may also be necessary to insert a piece of ground glass in the path of the light to give even illumination. Critical illu- mination will be lost in this way, but for low-power work it will not matter. BOTANICAL PHOTOGRAPHY 177 The relative positions of the various parts of the equipment as set up for high-power work are given below: Ground glass Camera bellows Microscope Slide Substage condenser Light filter Auxiliary diaphragm Auxiliary condenser Water-cooling cell Auxiliary condenser Light MOVIE PHOTOMICROGRAPHS By Dr. Paul J. Sedgwick Before the advent of the now popular 16 mm. amateur motion pic- ture film, the cost of making motion pictures was prohibitive for most workers. In screen time 100 feet of the amateur size film is equivalent to 250 feet of the standard 35 mm. film. A further economy is brought about by the fact that the 16 mm. film is a reversal film. There is no extra cost for the making of a positive. The original film is developed as a negative and then immediately converted into a positive by a process of reversal. The entire charge for processing is included in the original purchase price for the film. The 16 mm. film is being widely used in the preparation of teaching material and is finding some use in the recording of the results of re- search. Photomicrography with the motion picture camera is not very different from ordinary photomicrography. For movie photomicro- graphs at normal speed any good amateur motion picture camera will serve. Most of the amateur cameras are equipped with spring motors which operate the shutter so as to take 16 pictures or frames per sec- ond. Many of the cameras have half-speed attachments and some have slow motion attachments. With the half-speed attachment 8 frames are exposed each second, and when the finished pictures are later pro- jected, the speed of the action is then seen to be doubled. The slow motion attachments found on some cameras expose either 64 or 128 frames per second which permits the slowing down of fast action. Where time-lapse pictures are desired of such subjects as the germina- 178 METHODS IN PLANT HISTOLOGY tion of a spore, or the conjugation in Rhizopus, it is necessary to ar- range a motor and a gear system to operate the camera so as to make exposures of individual frames at intervals of several minutes (Fig. 34). For this type of work the most adaptable camera is the original Eastman Model A amateur motion picture camera which is a hand- crank camera and does not have a spring motor. Indeed, the Bausch and Lomb time-lapse mechanism is designed to be used with the East- man Model A camera. In making movie photomicrographs, the microscope is generally used in its normal vertical position. The lens of the camera is removed and the camera is connected to the microscope with some sort of an optical connector or double ocular. The Zeiss Company makes a special optical attachment for this purpose in which most of the light from the object is reflected into the camera by a silvered prism and a small amount of light passes through to the inspection ocular. The Bausch and Lomb Company also provides such an attachment with the equipment that they sell for movie photomicrography. In the absence of a special attachment any double ocular such as the dem- onstration oculars used in elementary teaching may be substituted. Some sort of double ocular is necessary so that it will be possible to watch the action during the time that the picture is being made. It will be necessary to adjust accurately the inspection ocular so that the image seen through it will be in focus when the image formed on the film by the other ocular is also in focus. To ac- complish this adjustment, remove the film gate in the camera and place a piece of onion-skin paper which is 16 mm. in width against the aperture plate. If it is preferred, a piece of ground glass cut to a width of 16 mm. may be used instead. Be sure to have the ground surface forward. Focus the microscope very carefully on some object until a sharp image is seen on the onion-skin paper or ground glass which is against the aperture plate. Examine this image with a magnifying lens to verify the focus. After this image is sharp, examine the object through the inspection ocular and bring the inspection ocular into focus by adjusting the ring or collar provided for this purpose. Be very careful not to alter the position of the objective while doing this. After adjusting the inspection ocular so that it is in focus, re-examine the image formed in the camera to make certain that it is still in focus. When both of the images are in focus, the gate may be replaced in the camera and the camera threaded with film. Further focusing as BOTANICAL PHOTOGRAPHY 179 .5 a CO m . c3 -a M 2- » eg .^ t^ .a a^ o >> Qi CD t-r o c a t- > o- a ^< u to <*- u fe o ft > 0) © •-MS 5- § "^ "o i go « 3 1, C o ^ O •>, **-< aj - & o 3 « *i J3 -Q C 03 _. 0) C ^ a ^ ^ M =3 Cow c3 m '^ o :« a m ~ "^ a^8 ■^ G o g M a, CO OJ OJ S ^ o a g c3 <; m g I 'g <= CO o ^ 2 S o ft ft 180 METHODS IN PLANT HISTOLOGY the object is being photographed can be accomphshed by watching the action through the inspection ocular and using the fine adjustment of the microscope as necessary. Because of the running of the mechanism in the automatic cameras or the turning of the crank in the hand-cranked model, it is necessary that the support for the camera be very rigid. In one set up used by the writer the camera is braced with heavy two-by-fours and held in place by large bolts. The Ughting system must be arranged with care. Strong illumina- tion is needed for normal speed work as in the photographing of zoospores and gametes of the algae, or in photographing the movement of the cytoplasm in a cell of Elodea. It is necessary to have sufficient light to give ample exposure in the brief period of approximately ^\ of a second that the camera shutter is open when making 16 frames per second. If too much light is used the living cells may be damaged. A water-cooling cell should always be used between the light and the mirror of the microscope. It may be advisable to use the superspeed panchromatic film as this is approximately three times as sensitive to artificial light as is the regular panchromatic film. In making tune- lapse pictures as in photographing the growth of the hyphae of a fungus with perhaps one frame exposed every three or four minutes the amount of light that is used may be very small. Each frame is in reality a time-exposure in any time-lapse mechanism in which the shaft of the camera is turned continuously at a very slow rate. No exact information can be given in regard to the amount of light that will be necessary for a correct exposure. This must be determined by experiment. Upon the basis of numerous experiments one can build up in time an experience that will make it possible to estimate the amount of light that will be needed for any subject. At the beginning of each experiment it will be well to expose a foot or two of film on the subject and then remove the film and develop it as a negative. If the strip shows good quality as a negative, it is safe to assume that the exposure is correct and that the reversal positive will have good qual- ity when it is returned from the processing station. If this procedure is followed, it will be necessary that the camera and microscope be setup in a dark room, so that the strip of film can be removed from the roll without fogging the rest of the film. Motion picture photomicrographs find one of their most valuable applications in making possible the slowing down of action that is too BOTANICAL PHOTOGRAPHY 181 fast to be studied at normal speed and in the speeding up of action that is too slow to be studied at normal speed. The following table will indi- cate the apparent rate of action as seen on the screen according to the rate at which the frames were exposed in the camera. Rate of Exposure of Frames Rate of Action on Screen 128 frames per second | normal 64 frames per second j normal 32 frames per second | normal 16 frames per second normal 8 frames per second 2 times normal 1 frame per second 16 times normal 1 frame per minute 960 times normal 1 frame every 4 minutes 3,740 times normal 1 frame every 6 minutes 5,760 times normal 1 frame every 10 minutes 9,600 times normal 1 frame every 15 minutes 14,400 times normal PHOTOGRAPHIC FORMULAS The makers of photographic plates, films, and papers know what they have put into their emulsions and, consequently, it is better to use their formulas in developing their products. In making solutions, dissolve the ingredients in the order in which they appear in the formulas, making sure that each one is dissolved before another is added. Remember that metol should be dissolved in warm water. A vigorous use of the stirring rod is always worth while and, with sodium sulphite or sodium carbonate, a hard cake will form unless the salts are added slowly with constant stirring. If metol is not completely dissolved before the sulphite is added, they combine and form an inert mass which makes the developer just that much weaker. Use distilled water whenever possible. When distilled water is not available, boil the water — but not in an iron kettle — and then cool and filter it. Ingredients of formulas. — Many of the formulas in common use have the same ingredients, but in different proportions. Those in most common use are metol, hydrochinon, pyrogallic acid, pyrocatechin, sodium sulphite, sodium carbonate, and potassium bromide, with potassium metabisulphite, carbonate of potassium, sodium sulphide, acetone, sulphuric acid, and citric acid appearing less often. Hyposulphite of soda, commonly called "hypo," with or without a 182 METHODS IN PLANT HISTOLOGY hardener to harden the emulsion and an acid to counteract the effect of the alkahne developer is used to dissolve the silver which has not been affected by light. Acetic acid is often used as a "short-stop" between the developer and the "hypo." Metol.—Metol should be completely dissolved in warm water before adding any sulphite. It is a vigorous developer, bringing the entire image up so quickly on the surface of the plate or film that the be- ginner might stop development too soon. Turn the plate over and look at the other side to make sure that development has taken place throughout the entire thickness of the film. Potassium bromide re- strains the rapidity of the action, allowing the metol to soak through the emulsion before the surface development is too extreme. It is al- most always used in combination with hydrochinon. Elon, pictol, and rhodinol are about the same as metol and may be substituted for it in formulas. Hydrochinon. — This developer comes in needle-like crystals which can be dissolved in water at room temperature. If the temperature is below 50° F., it does not act. It develops best at temperatures from 65° F. to 70° F. It is not used alone except for maps, graphs, and such things, where dead black lines and dots with a clear glass background are desired. It is generally used in combination with metol, forming the famous M.Q. developer, in which the metol softens the harshness and gives detail even in the shadows, so that the result is an artistic picture, whether you want a print on paper or a lantern slide. A much larger proportion of hydrochinon is needed for paper prints than for plates or films. P7/ro.— This is often called pyrogallol or pyrogalhc acid. Profession- al photographers generally use pyro. Combined with metol it is widely used in tank development of films. Properly used, it produces splendid negatives; but it has been replaced to a great extent, by newer devel- opers. Sodium sulphite.— In a tight can or a well-stoppered bottle, this reagent will keep for years. It takes no part in the developing but acts as a preservative, its function being to prevent too rapid oxidation of the metol or other developing agent. Weigh carefully, because too much or too little may cause fog, especially with hydrochinon. Potassium metahisulphite.— This acid salt is probably the best pre- servative when pyro is the developing agent, for pyro keeps only in an BOTANICAL PHOTOGRAPHY 183 acid solution. Citric acid, sulphuric acid, and oxalic acid are also used as preservatives with pyro. Sodium carbonate. — This makes the emulsion swell and thus allows the metol and other developing agents to penetrate more rapidly. The dry granular form is better than the crystal or anhydrous forms. Weigh carefully because too much carbonate will cause fog. Stir vigor- ously until dissolved. Potassium carboriate. — Hydrochinon works faster with potassium carbonate than with sodium carbonate, but the potassium carbonate is more likely to cause fog, and it may cause blistering and frilling, especially in warm weather. Sodium carbonate is to be preferred. Potassium bromide.- — The bromide retards the action and keeps down fog. It restrains the developing agent from acting upon the sil- ver which has not been affected by light. Too much bromide retards the action so much that details are not likely to be brought out in the shadows. The right amount checks chemical fog and produces con- trast and clearness by retarding the development of the shadows, a desirable result, especially in case of overexposed negatives. Hyposulphite of soda. — There is great difference of opinion in regard to the composition of the "hypo" bath. Many prefer plain hypo, about 100 g. hypo to 400 c.c. of water. Professor W. J. G. Land, whose negatives, lantern slides, and paper prints could hardly be surpassed, uses this plain hypo bath, which is also preferred by many of the best English photographers. With this bath, there should be an acid "short- stop" between the developer and the bath. Pour acetic acid into water until it tastes about like a weak vinegar. If too strong, it will cause pimples. If no short-stop is used, the alkaline developer is carried over into the hypo and soon spoils it. About 10 or 15 seconds in the short- stop will usually be long enough. The addition of alum to the hypo is desirable for negatives because it hardens the emulsions ; but it is undesirable for lantern slides or pa- per prints if you wish to color them, because the alum makes them hard to color, so that various "sizing" solutions have to be used. FORMULAS For convenient reference some well-known photographic formulas have been brought together here. There will be a still further saving of time if the formula is pasted on the bottle and coated with shellac to keep it from getting wet. While the scientist has no use for antiquated 184 METHODS IN PLANT HISTOLOGY weights and measures, these are so often the only ones given by makers of plates and films that we have added them, often approximately, in parentheses. Grains are in apothecaries' weight and ounces are in avoirdupois. Developers for plates, films, lantern slides, and papers.— The fol- lowing standard formulas can be recommended. Many others, some of which may give better results for special cases, will be found in the references at the end of this chapter. Metol-hydrochinon for lantern slides (Cramer). — A i\/r„t-;„ Apothecaries'- Metnc Avoirdupois Water 750 c.c. (25 oz.) Metol (or Pictol, Elon or other substitutes) 2 g. (30 gr.) Hydrochinon 6 g. (90 gr.) Sulphite of soda 30 g. (1 oz.) B Water 750 c.c. (25 oz.) Carbonate of soda 15 g. ( 5 oz.) Mix A and B in equal parts. This developer keeps well even when mixed and can be used repeatedly. When fresh, add one drop of a 10 per cent bromide of potassium solution to each 30 c.c. of the de- veloper. This is a good developer for lantern slides. Metol-hydrochinon for negatives and papers (Wall).— ■j^ , . Apothecaries'- Metnc Avoirdupois Water 1,0(^0 c.c. (32 oz.) Metol (or substitutes) 2 . 25 g. (34 gr.) Sodium sulphite (dry) 45 g. (H oz.) Sodium carbonate (dry) 35 g. ( Ig oz.) Hydrochinon 4.7 g. (70 gr.) For negatives, dilute with an equal volume of water. For paper, use 1 part to 3 parts of water and add 0.02 g. (about I gr.) of potassium bromide to each 30 c.c. of the chluted developer. Cramer's Pyro-Soda for negatives. — A ■««■ i. • Apothecaries'- Metno Avoirdupois Water 640 c.c. (16 oz.) Sodium bisulphite 4.5 g. (75 gr.) Pyrogallol 30 g. (1 oz.) BOTANICAL PHOTOGRAPHY 185 B Water 640 c.c. (16 oz.) Sodium sulphite (dry) 60 g. (2 oz.) C Water 640 c.c. (16 oz.) Sodium carbonate (dry) •SO g. (1 oz.) Mix 1 part of each of the three solutions and 8 parts of water. Developer for line work (Cramer). — A Metric Apothecaries'- Avoirdupois Water 1,000 c.c. ( 32 oz.) Hydrochinon 45 g. ( 1^ oz.) Sodium sulphite 30 g. ( 1 oz.) Sulphuric acid 4 c.c. ( 60 minims) B Water 1,000 c.c. ( 32 oz.) Sodium carbonate 30 g. (1 oz.) Potassium carbonate 90 g. (3 oz.) Potassium bromide 8 g. (120 gr.) Sodium sulphite 90 g. (30 oz.) Use equal parts of A and B. This is good for maps and graphs and also for lantern slides where considerable contrast is desired. Land's Contrast Developer for negatives and lantern slides. — Hydrocliinon 20 g. Sodium sulphite (dry) 60 g. Sodium carbonate (dry) 140 g. Potassium bromide 12 g. Water 1,000 c.c. If kept tightly stoppered, with no air space between the liquid and the cork, this developer keeps almost indefinitely. When some is taken out for use, the rest should be put into a smaller bottle, so that there shall be no air between the liquid and the cork. Another formula, developed by Dr. Land to meet the trying condi- tions of the tropics, is also useful for lantern slides. 186 METHODS IN PLANT HISTOLOGY Land's Tropical Developer for negatives and lantern slides. — Hydrochinon 8 g. Metol 3g. Sodium sulphite (dry) 30 g. (60 g. if crystals are used) Sodium carbonate (dry) 30 g. (90 g. if crystals are used) Potassium bromide 2 g. Water 1,000 c.c. This formula will develop an underexposed plate when the usual developers fail. With this developer, the image flashes into sight with surprising suddenness, but do not become startled and remove the slide too soon, lest you fail to secure details. In the tropics, where it is often impossible to get reasonably cool water, this developer is a boon to the scientist who cannot wait until he gets into a favorable place for developing. Process Developer for negatives, films, la7itern slides, and paper. — A ■., . ■ Apothecaries'- Metnc Avoirdupois Water 500 c.c. ( 16 oz.) Hydrochinon 22 g. (176 gr.) Sulphuric acid 2 c.c. ( 30 gr.) Sodium sulphite 15 g. ( § oz.) B Water 500 c.c. (16 oz.) Sodium carbonate log. ( | oz.) Potassium carbonate 45 g. ( U oz.) Potassium bromide 4 g. (32 gr.) Sodium sulphite 45 g. ( U oz.) With a process plate, a process film, or a "red-label" Cramer lan- tern-shde plate, or similar plate by other makers, this developer is ideal for maps, graphs, typewritten tables, and similar subjects. In making copy for the negative, use smooth, pure white paper and dead black ink. The wretched lantern slides, seen so often at scientific meet- ings, result from using the same plates and developers which are used for landscapes. BOTANICAL PHOTOGRAPHY 187 Metric Apotbeoaries'- Avuirdupois 50 c.c. (40 oz.) 1 g. (15 gr.) 15 g. ( ^oz.) 4 g. (60 gr.) 15 g. ( ioz.) l.Sg. (26 gr.) Developer for bromide enlargements (Agfa). Ml Water 1,250 Metol Sodium sulphite Hydrochinon Sodium carbonate Potassium bromide The time required for exposure and development depends upon several factors, but, assuming that, with a good negative and Agfa Velvet Cyco paper, 10 seconds is about right for the exposure, two minutes will probably be the maximum time required for complete development. Some papers will require much less exposure and some will require more. Plain hypo hath. — Many prefer a plain hypo bath, because the addi- tion of a hardener makes lantern slides and prints more difficult to color. With this bath, we strongly recommend an acetic acid "short- stop"— water with enough acetic acid to make it taste like a weak vinegar — to be used between the developer and the hypo. About 10- 20 seconds is long enough. As developer is carried into the short-stop, add a few drops of acetic acid occasionally to maintain the acidity. If a lantern slide should not look bright and snappy as it comes from the hypo, rock it in the acetic acid solution until it brightens. This bath is good for plates, films, lantern slides, and papers. Metric Avoirdupois Water 1,000 c.c. (32 oz.) Hyposulphite of soda 250 g. ( 8 oz.) Acid-fixing a.nd hardening bath. — Metric Avoirdupois Water 1,000 c.c. (32 oz.) Hyposulphite of soda 250 g. (8 oz.) B Water 250 c.c. (8 oz.) Sulphite of soda 22 g. ( f oz.) Sulphuric acid 4 c.c. ( | oz.) Powdered chrome alum 15 g. ( | oz.) Pour B into A, stirring vigorously. This is then a one solution bath, with a greenish color, which can be used repeatedly. It hardens the 188 METHODS IN PLANT HISTOLOGY emulsion and does not stain. Plates should be left in the bath for 15 minutes after the silver seems to have been dissolved. Short-stops. — After development, a plate, film, or paper should be rinsed in water or, preferably, in a short-stop before going into the hypo. Enough acetic acid added to the rinsing water to give it the taste of weak vinegar will stop the development immediately and will save the hypo. If the acetic acid is too strong, it will cause pimples on the emulsion. Small blisters are often caused by a direct transfer from the developer to the hypo. Hardener for plates and films. — In the tropics, and in warm weather anywhere, it is desirable to harden the emulsion. Metric Avoirdupois Water 300 c.c. (10 oz.) Chrome alum (granular) 30 g. (1 oz.) After development, transfer to the hardener without previous rinsing in water and rock for 15 seconds; then transfer directly to the hypo. The hardener may be used repeatedly. Even if washing must be done in water at 80° or 85° F., the emulsion will not be damaged after treat- ment with this hardener. Sepia tones. — Hypo must be washed out thoroughly before bleach- ing. Stock Bleaching Solution Stock Toning Solution Metric Avoirdupois Metric Avoirdupois Potassium bromide ... . 30 g. (loz.) Sodium sulphide 30 g. (1 oz.) Potassium ferricyanide . 90 g. (3 oz.) Water 300 c.c. (10 oz.) Water '. 300 c.c. (10 oz.) Bleach in 1 part stock bleaching solution to 9 parts water until the print is light brown. Rinse in water 1 minute. Tone in 1 part stock toning solution to 20 parts water. There is no danger in prolonging either bleaching or toning. Wash as thoroughly as after hypo. Sepia tones may be obtained by bleaching in a saturated solution of bichloride of mercury with 3 drops HCl to 30 c.c. (after hypo has been washed out) ; washing in water and toning in 1 part ammonia to 10 parts water. Wash thoroughly. MISCELLANEOUS HINTS 1. Keep all chemicals in a dark, cool, dry place. 2. Keep bottles tightly stoppered and packages tightly closed. 3. Store ammonia, sodium sulphide, and ammonia sulphide away from the rest of the chemicals. BOTANICAL PHOTOGRAPHY 189 4. Do not keep plates, films, or papers in the same room with the chemicals. 5. Keep everything clean. Dust may be removed from a plate by tapping it gently on the table. If a brush is used at all, keep it clean, for it is as likely to distribute dust as to remove it. 6. If hypo is spilled or even rocks over a little, wipe it up, preferably with a rag wet with potassium permanganate. If allowed to dry, the hypo dust may cause spots on negatives and prints. 7. A temperature of 65°-70° F. is about right for most photographic work. 8. Developers keep longer if made up in two solutions, with the developing agents in one and the carbonate of soda in the other. 9. Be skeptical about "safe lights," even when dealing with lantern slides, slow plates, or even with papers. 10. Most spots are due to dirt, in the dark room, in the camera, on the lens, or on the emulsion. REFERENCES Some of the references are advertising pamphlets, but since they are intend- ed to bring the best results with their products, such directions are worth studying. Cramer, Manual on negative-making and formulas. St. Louis, Mo., or 30 E. Randolph St., Chicago, 111. : Cramer Dry Plate Co. (Furnished free.) Fraprie, Frank R., American photography exposure tables and manual. Boston, Mass.: American Photographic Publishing Co. (About Sl.OO.) Mallinckrodt Chemical Works, The chemistry of photography. St. Louis, Mo. : Mallinckrodt Chemical Works. (50 cents.) Eastman Kodak Company, Lantern slides; how to make and color them. Color plates and filters. Walmsley, W. H., The A.B.C. of photomicrography. New York City, N.Y.: Tennant & Ward. Duncan, F. Martin, First steps in photomicrography, a handbook for novices. London, E.C. 4., England: Iliffe & Sons, Ltd., 20 Tudor St. Glover, B. T. J., Print perfection, how to attain it. London, E.G., 4., Eng- land: British Periodicals, Ltd. 19, Cursitor St. (About 50 cents.) Wall, E. J., Photographic facts and formulas. Boston, Mass.: American Photography Publishing Co. ., Practical color photography. Boston, Mass.: American Photog- raphy Publishing Co. Hampton, M. Monroe, Photo coloring and tinting. Chicago, 111.: Central Camera Co., 230 S. Wabash Ave. ($L00.) Bailey, R. Child, Photographic enlarging. Dorset House, Tudor St., Lon- don, E.C. 4, England: Iliffe & Sons. CHAPTER XV ILLUSTRATIONS FOR PUBLICATION Illustrations will be made from some of the preparations used in the course of any histological or cytological investigation. Sometimes— and probably too often — photomicrographs are made. These have been considered in the preceding chapter. Thirty years' experience with the illustrations of a prominent bo- tanical periodical makes me bold enough to venture some suggestions not only to beginners but even to my colleagues. Before you begin to make a drawing for publication, get pure, smooth, white cardboard and dead black waterproof India ink. Get good steel pens. Remember that for drawings which are to be reduced one-half, "crow quill" pens are dangerous, because their lines and dots are likely to be too small for successful reproduction, especially if the drawings are to appear as text figures. Freehand drawings, for most botanists, are difficult and they gener- ally look crude. Drawings of apparatus, where much can be done with a ruler and a compass, are not so bad. Make the hnes bold, so that they may be reduced to one-half the size of the drawing. Maps 3 feet long to be reduced to page width (usually about 4 inches) or "the long way of the page," which always irritates the reader, are often sent to journals. Letters \ inch in height, when such a map is reduced to page width, would be only 3V inch in height — unreadable; and "the long way of the page," only 2^^ inch in height — still unreadable. To appear I inch high in the periodical, the letters must be f inch high in the drawing. The author should think how he wants letters, lines, and other things to look in the reproduction and make his drawing of the map accordingly. For very coarse lines, unless they can be made with a ruhng pen or a compass, use a "speedball" pen, which makes a line of uniform weight. In making graphs, never use the yellow- or pink- or blue-lined paper which is so common in class work. If a piece of research is worth publishing, it is worth doing right. Take a piece of smooth white Bristol board and, with a ruling pen set for a heavy line, make the vertical line at the left of the sheet and join it at the bottom with a 190 ILLUSTRATIONS FOR PUBLICATION 191 heavy horizontal line. Some use only these two lines ; but it looks better to rule, in lighter lines, the space between. Figures and letters can be pasted on, or may be indicated in pencil and the printer can do the rest. If you choose to do your own lettering. Exercises in Lettering, "Slant Gothic," by George G. Greene, published by the Bruce Publish- ing Company, Milwaukee, Wisconsin, will be helpful. Where several curves are to be shown, the ideal method would be to use different colors; but, in practically all scientific journals the expense makes this method prohibitive. So, use a solid heavy line for one curve, a broken line for another, a dotted line for another, etc. rndoa'armis \Per/cyc/e ,Ph/oem Endodermis - Pericucle Ph/oem Xu/em Fig. 35. — Two drawings of a part of a vascular bundle of Pteris aquiUna, A showing a poor style, looking as if the layer of cells just outside the endodermis might be the outer border of the structure; B, a better style, showing that the cells just outside the endodermis are not the outer border. X150. The camera lucida minimizes the amount of skill required in mak- ing highly magnified drawings. Draw first with a pencil, making very light lines which will easily rub out; then ink while you still have the preparation under the microscope for constant reference. Such draw- ings are almost always reproduced by the zinc-etching process. Pen- cil drawings can be reproduced by the more expensive photolitho- graphic method, or by the still more expensive lithographic method. They can also be reproduced, although not so satisfactorily, by the halftone process. In making a detail from a tissue, do not make a border of entire cells so that it will look like an outer margin ; but leave it so that it will look like a detail which had more surrounding it (Fig. 35). 192 METHODS IN PLANT HISTOLOGY If one should need a line drawing of a tree, or flower, or mushroom, a kind of drawing that, ordinarily, requires real artistic ability, there is a way to get an outline with as good proportion and perspective as even the best artist could not surpass. Photograph the object, make a lantern slide, and then throw the object on cardboard and trace with a pencil. It is more convenient to catch the object on a mirror placed at 45° and thus throw the object on the cardboard resting on the table. Drawings can be made from photomicrographs in the same way. Instruments can be bought which will throw the object down di- rectly, saving a lot of time in many cases. However, the projection method is not as slow as it seems and it is not expensive, since lantern slide plates can be used. By projecting upon durable cloth, very effi- cient charts can be made in this way. The lithograph is so expensive that it has almost disappeared from botanical journals, except in countries where labor is cheap. This type of illustration is still furnished to those who are fortunate enough to get grants from Foundations. The copy may be in pencil or in ink or in a combination of pencil, ink, and washes. If your copy is poor, ex- plain to the lithographer how you want it and he can fix it for you. A photolithograph is excellent, better than a lithograph, if you can draw better than the lithographer; but not so good as a lithograph, if the lithographer is your superior. The photolithograph is rather ex- pensive; consequently, journals which are having a hard time to live within the budget look with disfavor upon anything more expensive than the zinc etching and the halftone. The copy for a photolitho- graph should not be a mixture of pen and pencil work, but should be all in pencil or all in ink. To get different shades with ink, dilute the ink for lighter tones: do not use a pencil. The best reproductions by the photolithographic method are made from copy entirely in dead black ink on pure white cardboard, different shades from light to dark being obtained by dots and lines. Such a photolithograph can be printed in a lithograph colored ink, and the finished product looks like an expensive genuine lithograph. Photographs of trees, flowers, and landscapes are nearly always re- produced by the halftone process. Remember that there is a screen which makes black lines through light parts and light lines, through black parts, thus reducing the contrasts so that a good print from a good negative will be disappointing. This may be remedied, to some extent, by using a contrasty plate or film, and a contrasty developer ILLUSTRATIONS FOR PUBLICATION 193 with the negative; and a contrasty paper for the print. For good re- production, the print should look too hard for an artistic picture. Overdo it just enough to offset the flattening which the halftone proc- ess cannot avoid. If the page width of the journal in which the illus- tration is to appear is 4 inches, do not use a negative smaller than 5X7. If you have no camera larger than a 3|X4j, get an 8X10 en- largement on glossy paper. If your camera is smaller than 3jX4j, try to describe the situation. Good English will be better than poor illustrations. There are many other methods of reproducing drawings and photos, but the practical investigator had better learn to make copy for repro- duction by the zinc etching and the halftone, and work on reproduc- tion by the photolithographic process to show details of chromosomes and the structure of cytoplasm and similar difficult features. PART II SPECIFIC DIRECTIONS In the preceding chapters the principles and methods of technique have been described in a general way. It is difficult, especially for a beginner, to apply general principles to specific cases, and, besides, the material which he might select for the preparations might not yield the most valuable collection. We have tried to select types in- volving all kinds of botanical microtechnique and which, at the same time, illustrate fundamentals upon which the theories of evolution, phylogeny, genetics, and physiology are based. An ambitious student, by making preparations, studying them, and reading about them, can become well grounded in comparative morphology without attending any classes. Hofmeister, the father of morphology, got his botanical training in this way while clerking in a store. We shall not discuss morphology, but shall try to answer, by means of sketches and specific directions, the multitudinous questions which confront the instructor in the laboratory. For those who have had a thorough training in general morphology the following suggestions will be in some degree superfluous ; but we are asked so often to recommend books which will help the student to interpret the preparations which he has made that a few references may save the student the trouble of asking for this kind of advice. Nearly all of the books are in English. Algae. — Oltmanns, F., Morphologie und Biologic der Algen. Jena : Gustav Fischer, 1927. West, G. S., and Fritsch, F. E., British fresh water algae. Cambridge University Press. 1927. Yamanouchi, Shigeo, Morphology and cytology of the algae. University of Chicago Press (to be published in 1932). Smith, Gilbert M., Algae (to be published in 1932). Fvingi. — Gwynne-Vaughan, H. C. L., and Barnes, B., The structure and de- velopment of the fungi. Cambridge University Press. 1927. Gaumann, E. a. (trans, by W. D. Dodge), Comparative morphology of fungi. McGraw-Hill Book Co., 1928. Stevens, Frank L., The fungi which cause plant disease. Macmillan, 1919. 197 198 METHODS IN PLANT HISTOLOGY Lister, Arthur, Mycetozoa. 3d ed. by Gulielma Lister. Published by the British Museum. 1925. Bryophytes. — Campbell, D. H., Mosses and ferns. Macmillan. 1918. Land, W. J. G., Morphology of Bryophytes (in preparation). Pteridophjrtes . — Campbell, D. H., Mosses and ferns. Macmillan. 1918. Bower, F. 0., The ferns (3 vols.). Cambridge University Press. 1926. Gymnosperms. — • Coulter, J. M., and Chamberlain, C. J., Morphology of gymnosperms, University of Chicago Press, 1917. Angiospenns. — Coulter, J. M., and Chamberlain, C. J., Morphology of angiosperms (out of print). Appleton. 1903. ScHURHOFF, p. N., Die Cytologie der Bliithenpflanzen. Stuttgart: Ferdi- nand von Enke. 1926. Chamberlain, Elements of Plant Science. McGraw-Hill Book Co. 1930. Wettstein, Richard, Handbuch der Systematischen Botanik. Leipzig: Franz Deutike. 1926. Jeffrey, E. C, The anatomy of woody plants. University of Chicago Press. 1917. Eames, a. J., and McDaniels, L. H., An introduction to plant anatomy. McGraw-Hill Book Co. 1925. Strasburger, E., Text-book of botany. 6th English ed. Macmillan, 1930. Sharp, L. W., An introduction to cytology. McGraw-Hill Book Co. 1926. Scott, D. H., Studies in fossil botany. London: A. & C. Black. 1923. The directions for collecting and growing laboratory material con- stitute an important feature of this part of the book. With a few exceptions, the order in which the forms are presented is that given in Engler's Syllab^is der PflanzenJamUien. CHAPTER XVI MYXOMYCETES AND SCHIZOPHYTES MYXOMYCETES An organism large enough to be seen with the naked eye and so pe- cuhar that biologists do not know whether it is a plant or an animal, should be studied by both botanists and zoologists. In the sporangium stage of its life-history, the organ- ism looks and behaves like a plant; in the Plasmodium stage, it looks and behaves like an animal. Writers who think these organisms are plants, call them myxomycetes; those who think they are animals, call them mycetozoa. With the exception of a few forms like Fuligo (often found on oak, stumps and on oak bark in tan- yards), the myxomycetes are small, and are usually overlooked by col- lectors (Fig. 36). A careful exami- nation of rotting logs in moist woods will usually reveal an abun- dance of these delicate and beauti- ful organisms. Various species may be found in spring, summer, and autumn. The plasmodia are most abundant just after a warm shower. A couple of days of dry weather will then bring sporangia in abun- dance. The specimens should be pinned to the bottom of the box for safe carrying. An excellent collecting-box can be made from an ordinary paper shoe-box. On the bottom of the box place a thin piece of soft pine, or a piece of the corrugated paper so commonly used 199 Fig. 36. — Trichia, a myxomycete. A, hab- it of a group of sporangia growing on rotten wood. Natural size. B, single sporangium with some of the rotten wood. The dots repre- sent fairly well the size and distribution of the nuclei at this stage. X70. C, D, and E, succes- sive stages, much later than B, showing con- densation of the wall, origin of elaters.(Z)), and mature sporangium (E). Preparation stained in safranin, gentian violet, orange. X320. 200 METHODS IN PLANT HISTOLOGY in packing; or, better still, a sheet of cork. At each end nail in a piece of pine half an inch thick and an inch high. Upon these end pieces place a thin piece of pine, thus making a second bottom, which, of course, should not be fastened. A second pair of ends with a third pine bottom nailed to them may rest upon the second bottom. The three bottoms will give a considerable surface upon which the material may be pinned. For most purposes, the specimens are simply allowed to dry, and are then fastened with glue or paste to the bottom of a small box. Plasmodia and young sporangia may be fixed in chromo-acetic acid, but staining with iron-alum haematoxylin is likely to be more satis- factory if some osmic acid is added. The solution — 0.7 g. chromic acid, 3 cc. glacial acetic acid, and 7 c.c. 1 per cent osmic acid to 90 c.c. distilled water — will be good for the younger stages of any myxo- mycete. Sections cut easily in paraffin. For general structure, 5 /x is thin enough; but for nuclear detail 3, 2, or even 1 ju will be better. Since the material cuts so easily, this is a good place to practice cutting thin sections. Remember that a good sliding microtome is better than a rotary for very thin sections. The safranin, gentian violet, orange combination is good for a study of the general development and for some cytological features, but iron- alum haematoxylin is better for nuclear details. Spores of most myxomycetes will germinate as soon as they are thoroughly ripe, and, during the first year, germination is more prompt than in case of older spores. Fresh spores may germinate in half an hour; the time may extend to several hours; spores two or three years old may germinate in three or four days, or may not germinate at all. We have never succeeded in germinating spores which were more than three years old. The longevity is doubtless different in different species. In most cases, spores will germinate in water, if they will germinate at all. For small cultures, the hanging-drop method, described on page 82, may be used. Plasmodia may be raised by sowing spores on moist, rotten bark or wood and placing the culture under a bell jar, where the moist, sultry condition favorable to their growth is easily imitated. Plasmodia may be got upon the slide by inclining the slide at an angle of about 15°, with one end of the slide at the edge of the plasmodium, and allowing water to flow very gently down from the upper end of the slide to the lower. The proper flow of water could be secured by dropping water MYXOMYCETES AND SCHIZOPHYTES 201 from a pipette, but a less tedious plan is to arrange a siphon so as to secure a similar current. The Plasmodium will creep up the slide against the current, furnishing an excellent illustration of rheotropism. Enough Plasmodium for an illustration may be formed in two or three hours. Examined under the microscope, the preparation should give an excellent view of the streaming movements of protoplasm. The following is another method for getting the plasmodia upon the slide : Place the slides upon a pane of glass and upon each slide place a small piece of plasmodium-bearing wood. Cover with a bell jar. Wet blotting paper or a small dish of water included under the jar will help to create the warm, sultry atmosphere necessary. The slides may be covered with the Plasmodium in a few hours. Permanent prepara- tions may be made by immersing the slide in chromo-acetic acid, then washing and staining without removing the Plasmodium from the slide. Acid fuchsin is a good stain for bringing out the delicate strands of the Plasmodium. Iron-alum haematoxylin, followed by acid fuchsin or erythrosin, brings out both nuclei and cytoplasmic strands. Some of the foregoing methods are taken from an article by Pro- fessor Howard Ayers in the January and February (1898) numbers of the Journal of Applied Microscopy. Other methods, with directions for various experiments, are given in the same article. In 1931, in the American Journal of Botany, Howard published a very effective method of cultivating myxomycete plasmodia. He used oat agar. Cook 30 g. rolled oats, 15 g. agar, and 1 liter of water for 15 minutes in a double boiler. Pour into a flask and autoclave at 15 pounds for 15 minutes. When nearly cool, pour into Petri dishes. Temperatures from 20° to 26° C. are good for the growth of most plasmodia. If the Plasmodia are allowed to dry slowly by exposing them to air, they pass into the sclerotium condition, where they may remain for a year or more. To revive them, place them under moist conditions, but never cover them with water. Within 24 hours they may become active again. SCHIZOPHYTES (Fission Plants) BACTERIA {Schizomycetes. Fission Fungi) Bacteria are studied almost exclusively from the standpoint of health and disease, and bacteriological methods are, largely, those which have been developed for determining species, structure and phylogeny being incidental. The methods of the medical school will 202 METHODS IN PLANT HISTOLOGY not contribute much to our knowledge of the internal structure of bacteria. The methods given here will merely enable the student to study the form and size of those bacteria which are more easily demonstrated. Foul water at the outlets of sewers and such places will usually afford an abundance of Coccus, Bacillus, Spirillum, and Beggiatoa forms. Place a drop of water on a slide, heat it gently until the water evaporates, then stain with fuchsin or methyl violet, dehydrate, clear in xylol, and mount in balsam. The hay infusion is a time-honored method for securing bacteria for study. Pour hot water on a handful of hay, and filter the fluid through blotting paper. Place the fluid in a glass dish, and cover with a piece of glass to keep out the dust. When the fluid begins to appear turbid, bacteria will be abundant. The active movements are easily observed in a mount from the turbid water. As the bacteria pass into the rest- ing condition, they form a scum on the surface of the water. Usually, the first to appear is a somewhat rod-shaped form, the Bacterium termo of the older texts. Spirillum and Coccus forms often appear later. Scrape the inside of your cheek with your finger nail and you are almost sure to find some bacteria. If you let your teeth go without brushing for 24 hours you can get bacteria by scraping your teeth with your finger nail. Throw flies or other insects into water from a pond or ditch and bacteria will soon appear. From such material or from vigorous cultures smear a little on the sUde and allow it to dry for 24 hours in a warm place, free from dust; or, if you are in a hurry, dry it by passing it several times over the flame of a Bunsen burner. Then try the following rapid method: 1. Place on a clean cover a drop of water containing the bacteria and dry completely in a flame or on a hot plate. 2. Stain 2-5 minutes in gentian violet or methyl violet. 3. Rinse quickly in water. 4. Dip into 95 per cent alcohol to reduce the stain. 5. Remove most of the alcohol by touching a corner of the cover with filter paper and then dry completely by passing through a flame. 6. Mount in balsam. Ciha of bacteria can be stained in various ways. In the hay infusion, the first form to appear is likely to be one which goes under the name of Bacterium termo. It is a harmless ciliated form. Bacillus tijphosis, the bacillus of typhoid, is a good ciliated form; but students had MYXOMYCETES AND SCHIZOPHYTES 203 better leave dangerous bacteria alone. Try Bacterium termo with the following solution : Ferric chloride, aqueous (1 part ferric chloride to 20 parts water) 25 c.c. Alum, saturated aqueous solution 75 c.c. Shake and add a saturated aqueous solution of basic fuchsin 10 c.c. Filter and allow it to stand for some time; then stain for 5 minutes, gently warming but not to the boiling point. Rinse in water and stain lightly in carbol fuchsin. If the ciha are not stained, repeat the process. Mount in balsam. Here is another method. After drying the smear, treat with a 10 per cent aqueous solution of tannin for 2 minutes. Wash in water, 10 sec- onds. Put a few drops of acid fuchsin on the smear and heat until bub- bles begin to appear. Let it cool, wash in water, 1 minute; then to 95 per cent alcohol, 20 seconds; absolute alcohol, 5 seconds; xylol. Mount in balsam. The cilia should be red. The following method is much better if the bacteria are vigorous and the haematoxylin is at its best: make smears from the hay infusion, invert over fumes of 1 per cent osmic acid, 10 seconds; dry in a warm, dry room; heat gently; 10 per cent aqueous solution of tannin, 10 sec- onds; water, 10 seconds; iron-alum 4 per cent, 4 hours; water, 1 min- ute; i per cent haematoxylin, overnight; water, 5 minutes; 1 per cent iron-alum, until stain is right; water, 2 minutes; 30, 50, 70, 85, 95, and 100 per cent alcohol; xylol. Mount in balsam. The ciUa should be black. Still another method : After drying the smear, treating with tannin, and rinsing slightly with water, put on a few drops of acid fuchsin and heat until bubbles appear. Cool off; wash in water, 1 minute; 95 per cent alcohol, 20 seconds; 100 per cent, 5 seconds; xylol. Mount in balsam. Fine preparations may be obtained by inoculating a mouse with Anthrax, and then cutting paraffin sections of favorable organs. For making mounts of a dangerous form like Anthrax, secure properly fixed material from a bacteriologist. Let him cut the liver and spleen (and any other parts) into small pieces, about 5 mm. square and 2 or 3 mm. thick, and put them into the chromo-acetic-osmic solution (0.7 g. chromic acid, 3 c.c. glacial acetic acid, 7 c.c. 1 per cent osmic 204 METHODS IN PLANT HISTOLOGY acid, and 90 c.c. water). Fix 24-48 hours and then follow the usual schedule for imbedding in paraffin. Cut 2-5 fx thick and stain in Haidenhain's iron-alum haematoxylin, or try the following schedule, which is good for Anthrax and many other bacteria: 1. Gentian violet, 5 minutes. 2. Rinse in water a few seconds. 3. Gram's solution (iodine 1 g., potassium iodide 2 g., water 300 c.c.) until the color is almost or quite black ; tliis will generally require 1 or 2 min- utes. 4. Ninety-five per cent alcohol until the color has nearly disappeared. 5. Rinse in water and examine. If the bacteria are well stained, a counter- stain may be added. 6. Light green or erji^hrosin, 5 seconds; or Bismarck brown, 5 or 10 seconds. 7. Ninety-five and 100 per cent alcohol, dehydrating as rapidly as possible. Not more than 5 or 10 seconds can usually be allowed. 8. Xylol, 1-5 minutes. 9. Balsam. After the rinsing in water of stage 5, the preparation may be dehydrated rapidly in 95 per cent and 100 per cent alcohol, and then stained for 5 or 10 '^y '^Xv' 6 seconds in orange dissolved in clove oil. _ ^ %>4, " ''"'^ From the clove oil, transfer to xylol and mount in balsam (Fig. 37). The bacteria are the only plants in which a nucleus has not been conclu- sively demonstrated, and some claim that a nucleus is present even in bac- teria. In determining the presence or absence of a nucleus in bacteria, the crude method, just given, would be of no value, and even the most critical methods of the bacteriologist, who mounts the organisms whole, would be entitled to only scant consideration. The presence or absence of a nucleus will have to be determined by a study of thin, well-stained sections of perfectly fixed material. r Fig. 37. — Bacillus anthracis, from a paraffin section cut from the liver of a mouse and stained in crystal violet; w, white blood corpuscle; r, red blood cor- puscle. X580. MYXOMYCETES AND SCHIZOPHYTES 205 CYANOPHYCEAE. BLUE-GREEN ALGAE (Schizophyceae. Fission Algae) The blue-green algae include unicellular, colonial, and filamentous forms. They occur everywhere in damp or wet places. On the vertical faces of rocks where there is a constant dripping of water, brilliant blue-green forms are abundant. In the Yellowstone National Park the brilliant coloring of the rocks is due in large measure to members of this group. Many forms occur as brownish or greenish gelatinous layers on damp ground or upon rocks, or even upon damp wooden structures in greenhouses. Other forms float freely in water or on the surface of the water.. Oscillatoria. — For most purposes it is best to study Oscillatoria in the living condition. It is readily found in watering-troughs, in stag- nant water, on damp earth, and in other habitats. The commonest forms have a deep blue-green or brownish color. It is very easy to keep Oscillatoria all the year in the laboratory. Simply put a little of a de- sirable form into a gallon glass jar half filled with water. By adding water occasionally to compensate for evaporation, the culture should keep indefinitely. In a jar with a tightly fitting cover we have kept such a culture for years without renewing the water. For the purposes of identification and herbarium specimens the ma- terial may simply be placed on a slip of mica and allowed to dry. When wanted for use, add a drop of water and a cover, and the mount is ready for examination. After the examination has been made, re- move the cover, allow the preparation to dry, and then return it to the herbarium. Good mounts may be made by the Venetian turpentine method. Species of medium size are more satisfactory for a study of the nucleus than the very large species. Fix in a chromo-acetic-osmic solution (1 g. chromic acid, 3 c.c. acetic acid, and 7 c.c. 1 per cent osmic acid). Stain in iron-alum haematoxylin, and follow the Venetian turpentine meth- od. While the nuclei are easily seen in such preparations, still better views can be secured from sections of paraffin material fixed in the same solution or in Flemming's weaker solution. The section should be 1-3 M thick. After staining with haematoxyhn, stain lightly with orange dissolved in clove oil. In paraffin sections the scattered condi- tion of the material as it appears in thin sections is very annoying. As soon as the material is thoroughly washed in water, arrange it so that the filaments will all have the same general direction. This will enable you to get longitudinal and transverse sections. As you begin with the 206 METHODS IN PLANT HISTOLOGY alcohols, use a Petri dish and lay a slide over the material, and keep it there until you imbed in paraffin. This will keep the filaments from Fig. 38. — Oscillatoria: photomicrograph from a paraffin section 3 /i in thickness and stained in iron-alum haematoxylin. X373. spreading out too much, and you will be able to get as much on one slide as you would be likely to get on a dozen slides without such precaution. Oscillatoria, as it appears in section, is shown in Figure 38. Tolypothrix. — This form occurs as small tufts, either floating in stagnant water or attached to plants and stones. Some species grow upon damp ground. It furnishes an excellent example of false branching (Fig. 39) . Like all small filamen- tous algae, it may be dried on mica for herbarium purposes. Venetian turpentine mounts and par- affin sections are prepared as in Oscillatoria. Tolypothrix is even better than Oscillatoria for a study of the nucleus. Sc5rtonema is a similar form which is fairly common. It is often found as a feltlike covering on wet rocks. In staining forms like Tolypothrix and Scyto- nema, which have a thick sheath, take care not to obscure the cell contents by staining the sheath too deeply. If the sheath is not stained at all, you may not be able to see the nature of the false branching. Iron-alum haematoxylin, with orange in clove oil for the sheath, is good for sections. Phloxine, with light green or Fig. 39. — Tolypothrix, showing "false branch- ing": h, heterocyst; c, concave cell; b, end of false branch with begin- ning of new sheath. X620. MYXOMYCETES AND SCHIZOPHYTES 207 Fig. 40. — Scytonema: filament showing thick sheath and characteristic branching. X640. anilin blue for the sheath, is good for Venetian turpentine mounts (Fig. 40). Nostoc. — Nostoc is a cosmopoHtan form. It occurs on damp earth or floating freely in water. In a fruit can or a battery jar, Nostoc is easily kept year after year in the lab- oratory. Young specimens are gener- ally in the form of gelatinous nodules, but in older specimens the form may be quite various. It is very easy to make sections, since the gelatinous ma- trix cuts well and holds the filaments together. Chromo-acetic acid is a good fixing agent. Stains which stain the gelatinous matrix make the prepara- tions look untidy, but they show that each filament of the nodule has its own gelatinous sheath. Small nodules may be stained in bulk and be got into Venetian turpentine. Crushed under the cover, they make instructive preparations. Rivularia. — This form is readily found on the underside of the leaves of water-lilies {Nuphar, Nymphaea, etc.), but is also abundant on submerged leaves and stems of other plants. It occurs in the form of translucent, gelatinous nodules of various sizes. Chromo-acetic acid gives beautiful preparations, but good results can also be secured from formalin or picric acid material. The most instructive preparations for morphological study can be obtained by the Venetian turpentine method. Stain in iron-haema- toxylin and very lightly in erythrosin, the latter stain being used merely to outhne the sheath. When ready for mounting, crush a small nodule under a cover glass. The paraffin method is easily ap- plied, since the gelatinous matrix keeps the filaments in place. Any form of similar habit may be prepared in the same way. Gloeotrichia. — Gloeotrichia (Fig. 41), in its later stages, is a free- floating form. In earlier stages it is attached to various submersed aquatic plants. The nodules, when young, are firm like Nostoc, but as they grow older and larger they become hollow and soft. The older forms become so much dissociated that they lose their characteristic form and merely make the fixing fluid look turbid. Allow a drop of such material to spread out and dry upon a slide which has been 208 METHODS IN PLANT HISTOLOGY slightly smeared with albumen fixative. Leave the sHde in 95 per cent alcohol 2 or 3 minutes to coagulate the albumen fixative, and then stain in safranin. If the background appears untidy, stain for 24 hours, or longer; you can then extract the stain from the back- ground, and still leave the long spore and some of the other fea- tures of the filament well stained. A touch of light green will bring out the sheath. Iron- alum haematoxylin, followed by orange in clove oil, gives a beau- tiful differentiation. The firmer young nod- ules can be treated like Nostoc. "Wasserbliithe."— Fig. 41. — Gloeotrichia: photomicrograph from a preparation gome of the CyaUO- stained in cyanin and erythrosin; negative by Dr. W. J. G. Land. ^^^^^^^ ^^^^^ ^^ ^^^^^ on the surface of quiet or stagnant water. The name, Wasserbliithe, "water flowers," was given because the scums are often iridescent. Some of the commonest forms are Coelo- sphaerium and Anabaena (Fig. 42). Some of the Chlorophyceae also oc- cur as Wasserbliithe. Where the ma- terial is very abundant, it may be collected by simply skimming it off with a wide-mouthed bottle, but where it is rather scarce, it is better to filter the water through bolting silk and finally rinse the algae off into a bottle, adding enough formalin to the water in the bottle to make a 5 per %» .n? A B Fig. 42. — Anabaena: A, hormogonium showing well-defined nuclei; B, older filament showing a spore and a heterocyst. MYXOMYCETES AND SCHIZOPHYTES 209 cent solution. The material may be kept here indefinitely, but after 24 hours it is ready for use. If the forms are small, like Anabaena, smear a sHde lightly with Mayer's albumen fixative, as if for paraffin sections, add a drop of the material and allow it to dry overnight or for 24 hours; then immerse the slide in strong alcohol for a few minutes, and pro- ceed with the staining. Cyanin and erythrosin form a good combina- tion for differentiating the granules. Delafield's haematoxylin, used alone, stains some granules purple and others red. Iron-alum haema- toxylin is excellent for heterocysts. With patience, these Wasserhliithe forms may be stained in iron-haematoxylin and brought into Venetian turpentine, from which they will yield much better preparations than can be secured by the drying-down method. Sometimes Anabaena, mixed with Gloeothece or Gloeocapsa, occur floating in gelatinous masses which hold together fairly well so that it is easy to fix in the chromo-acetic-osmic solution recommended for Oscillatoria, stain in iron-alum haematoxylin, and follow the Venetian turpentine method. With such material we have tried a more expeditious method with excellent results. After staining in haematoxyhn, we have used a series of alcohols, 21, 5, 10, 15, 20, 30, 40, 50, 70, 85, 95, and 100 per cent, allowing only 3 or 4 hours for the entire series. Then use mixtures of clove oil and absolute alcohol, beginning with 1 part clove oil to 4 parts alcohol, followed by equal parts clove oil and alcohol, then 3 parts clove oil to 1 of alcohol. At this point, stain in orange dissolved in clove oil. Drain off the stain and transfer to pure clove oil. Then place the material in thin balsam, about 1 part of the balsam used for mounting to 3 parts of xylol. Here the material may be kept 'indefi- nitely. Mounts may be made in balsam from this stock. Figure 42 was drawn from material prepared in this way. CHAPTER XVII CHLOROPHYCEAE. GREEN ALGAE For experiments in most phases of botanical microtechnique, no group of plants affords better material than the green algae, since the killing, fixing, and staining can be watched directly; the effect of the change from one solution to another can be observed; and even the be- havior during infiltration with paraffin can be determined with con- siderable accuracy. Since the Chlorophyceae furnish our best illustrations of the evolu- tion of the plant body, the origin and development of sex, and also the beginning of alternation of generations, they occupy a prominent place in any well-planned course in the morphology of plants; and, if they were better known, the ease with which the reactions of the indi- vidual cell may be observed would make them valuable to the physi- ologist. They are found in both fresh and salt water, but are most abundant in fresh water. The ponds, ditches, and rivers of any locality will yield an abundance and variety both of the unicellular and the multicellular members of this group. Most of the algae are independent, but there are epiphytic, endophytic, and saprophytic species. The larger forms and those which grow in tufts or mats are readily recognized in the field. Many of the smaller forms are attached to other water plants. Drain the water plants and then squeeze them over a bottle. The sediment is Hkely to contain a variety of unicellular and other small algae. Many of the genera are easily kept in the laboratory. It is not necessary to have very large aquaria. Ordinary glass battery jars holding about a gallon are good for most forms. Jars holding 2 gallons wiU be as good or better. For some cultures which are to be kept for a long time, like Scenedesmus, small glass jars, or dishes, with ground- glass tops are desirable. For a limited amount of material, quart or 2-quart fruit cans are very efficient. Put about an inch of pond dirt in some, clean sand in others, and in still others use a gravel bottom. Many forms grow well without any soil or sand in the bottom of the jar. When possible, use the water in which the algae were growing, since 210 CHLOROPHYCEAE 211 very few take kindly to a sudden change of water. If the material has been brought to the laboratory in a very small quantity of water, fill the jar about two-thirds full with tap water. Let the water run for 2 or 3 minutes before you fill the jar, since the water standing in the pipes is injurious, or even fatal, to most algae. Add water occasion- ally, only a little at a time, to compensate for evaporation. If the water has evaporated until the jar is about one-third full and you fill it nearly to the top with tap water, you are likely to kill some of the most desirable forms. It is a mistake to put too much material into a jar. A wad of Spirogyra half as large as one's finger is as much as should be put into a gallon jar. As it grows to ten or twenty times that amount, it is not necessary to keep throwing it out, since it will gradually accommodate itself to conditions; but if the larger amount should be put into the jar when brought in from the field, it would die in a day or two. Cultures may be started even in the winter. A surprising number of the green algae live through the winter under the ice of ponds and rivers. Oedogonium commonly passes the winter in the sporeling stage. Clado'phora may be found the year around. Coleochaete, on stems of plants like Typha, can be taken from under the ice and, in a few days, will be fruiting. Many pass the winter in the spore stage. Bring in some mud over which algae were growing the previous summer or autumn; put it into a jar and fill it two-thirds full of tap water. Also bring in sticks, leaves, and stones from good alga localities and put them into jars of tap water. Cultures may be started either by taking mud and sticks from under the ice or by taking them from places which have entirely dried up during the summer or autumn. A few such jars will be likely to yield a variety of material. If you have a good jar of Oedogonium, or some other desirable form, do not throw it out if the alga should disappear. Remember that tem- porary disappearances occur in nature. Allow the culture to become dry and then set it aside where it will be protected from dust. After a few months, pour on tap water and it is very likely that you will soon have a good jar of Oedogonium. Many algae behave similarly; some, like Volvox, appear for a short time and then disappear for a long time; some, like Cladophora, may last the whole year, and grow so luxuri- antly that the excess material must be removed; and some, like Ulothrix, we have not been able to cultivate at all in the laboratory. Some very useful hints on collecting and growing fresh-water algae 212 METHODS IN PLANT HISTOLOGY for class work will be found in an article by Dr. J. A. Nieuwland in the Midland Naturalist, 1:85, 1909. Professor Klebs has shown that various phases in the life-histories of many algae and fungi may be produced at will. By utilizing his re- sults, the fruiting condition may be induced in many of the common laboratory types. Knop's solution will be needed in most cases. A stock solution which can be diluted as required may be made as follows : Potassium nitrate, KNO3 1 g- Magnesium sulphate, Mg804 1 g. Calcium nitrate, Ca(N03)2 3 g. Potassium phosphate, K2HPO4 1 g- Dissolve the first, second, and fourth ingredients in 1 liter of dis- tilled water, and then add the calcium nitrate. A precipitate of cal- cium phosphate will be formed. For practical purposes this may be called a 0.6 per cent solution. Whenever a dilute solution is made from the stock solution, the bottle must be shaken thoroughly in order that a proper amount of the precipitate may be included in the diluted solution. To make a 0.1 per cent solution, add 5 hters of distilled water to 1 liter of the stock solution; for a 0.3 per cent solution, add 1 liter of distilled water to 1 liter of the stock solution, etc. The addition of a liter of a 0.2 per cent solution to 4 or 5 Hters of water will often produce a more thrifty growth. Directions for inducing reproductive phases are given in connection with the various types. With a good supply of glass jars, plenty of Knop's solution, a reason- able control over temperature, and the teacher's usual amount of pa- tience, most laboratory types can be studied in the living condition at all seasons of the year. Collecting algae need not be so laborious as most botanists make it. Forms hke Spirogyra, Zygnema, Cladophora, Vaucheria, and Hydro- didyon may be rolled up in wet newspaper and carried in a botany can. They suffer less from lack of water than from lack of air. Large quan- tities of material can be brought in and transferred to water after reach- ing the laboratory. Even after 24 hours in the wet paper, such forms seem to suffer no damage. Permanent preparations are needed to show details which are not so evident in the fresh material. The unicellular and filamentous mem- bers, together with such forms as Volvox, are best prepared by the Venetian turpentine method. The structure is so much more compli- cated than in the Cyanophyceae that it demands far more care and skill to make good preparations. CHLOROPHYCEAE 213 In many, probably in most of the green algae, nuclear and cell di- vision takes place at night. This is definitely known to be the case in Spirogyra, Zygnema, Closterium, Ulothrix, and others. Mitosis is most abundant about midnight, or an hour before midnight, and continues up to three or four o'clock in the morning. The most extensive work on the time of day at which nuclear division occurs is a paper by G. Karsten, "Ueber embryonales Wachstum und seine Tagesperiode," Zeitschrift filr Botanik, 7:1-34, 1915. Although the paper is in Ger- man, the numerous tables can be understood by those who are un- familiar with the language. The paper contains a bibliography of the subject. Chromo-acetic acid, with or without osmic acid, is a good killing and fixing agent for the entire group. We prefer the following formula : Chromic acid, 1 g.; glacial acetic acid, 3 c.c; 1 per cent osmic acid, 5 c.c. ; water, 90 c.c. If material is to be imbedded, it is better to increase the amount of osmic acid up to 7 c.c, since the staining of thin sections is likely to be more brilliant. With any fixing agent, it is worth while to place a few filaments in the mixture and watch the effect under the microscope. If plasmolysis occurs with the chromo-acetic mixture, weaken the chromic or strengthen the acetic until the suitable proportions are determined. In the previous edition, the usual method was to weaken the chromic acid. While this avoided any shrinking of the cell contents, the fixing was not very thorough, and material often suffered during staining or other subsequent processes. An extensive series of experunents, espe- cially with coenocytic forms which are notoriously difficult to prepare, proved that it is better not to let the chromic acid drop below 0.7 g. to 100 c.c. of water. Generally 3 c.c. of acetic will be enough to avoid any shrinking. If there is still a tendency to shrink with 4 c.c. of acetic, weaken the chromic down to 0.5 and let the solution act for 48 hours. One function of the osmic acid is to make the killing almost instan- taneous. If you put the little crustacean, Cyclops, into a 1 per cent chromo-acetic solution, it will keep up its characteristic movements for several minutes; but if you add one drop of 1 per cent osmic acid to 25 c.c. of the chromo-acetic solution, the movements stop in a few seconds. Besides, the osmic acid acts as a mordant for some stains, especially haematoxylin. About 24 hours in any of the chromic series and a 24 hours' washing in water will be sufficient for members of this group. Only a few of the most commonly studied will be mentioned. With marine forms use sea water in making up the fixing agents and 214 METHODS IN PLANT HISTOLOGY for most of the washing; but finish the washing in fresh water and use fresh water in making up alcohols and for the 10 per cent glycerin. For collecting, use "bolting cloth" or "bolting silk" of the finest mesh available. With a piece of thin cloth about 15 cm. square, laid over an ordinary coffee strainer, you can pour through about 4 hters of water in a minute. In this way you will secure all the Volvox in about a barrel of water in half an hour. Eudorina may be collected in the same way. Smaller members of the Volvox family like Pandorina, Gonium, and Chlamydomonas are too small to be held by the cloth; but if material is very abundant, the water goes through faster than the organisms and you will soon have many times as much material in a bottle as you could get by dipping. Many small organisms are effectively collected in this way, even when they are so small that most of them pass through the cloth. Material of Volvox and all the Volvocaceae may be fixed in the cor- rosive sublimate-acetic mixture, used hot — 85° C. If material is to be stained and mounted whole, use the aqueous mixture; if it is to be im- bedded and cut, use the alcoholic. For mounting whole, stain in iron- alum haematoxylin, or in phloxine and aniUn blue, following the Vene- tian turpentine method. Chlamydomonas is such an important type from the standpoint of evolution and phylogeny, that there should always be a supply of liv- ing material as well as some well-prepared slides. It often appears in the greenhouse. A small quantity in a Petri dish on white sand, moist but not flooded with water, may be kept for months. Add a pipette full of water occasionally to keep it from drying up. If there are zygotes, the material may dry up without any damage. If stained in iron-alum haematoxylin, stain one lot heavily to show cilia; another lot, lightly, to show internal structure. Some of each lot, with some in phloxine and anilin blue, and some in iron-haematoxylin, on each slide, make an instructive preparation. Of course, it is assumed that there has been a thorough study of living material. The principal stages in the life-history are shown in Figure 43. The Powers' methods yield beautiful, transparent preparations. Volvox.— Volvox is found in ponds and ditches, and even in shal- low puddles. The most favorable place to look for it is in the deeper ponds, lagoons, and ditches which receive an abundance of rain water. It has been claimed that where you find Lemna, you are likely to find Volvox; and it is true that such water is favorable, but the shading is CHLOROPHYCEAE 215 unfavorable. Look where you find Sphagnum, Vaucheria, Alisma, Equisetum fluviatile, Utricularia, Typha, and Chara. Dr. Nieuwland reports that Pandorina, Eudorina and Gonium are commonly found in summer as constituents of the green scum on wallows in fields where pigs are kept. The flagellate, Euglcna, is often associated with these forms. If you have a culture in the laboratory, do not throw it out Contractile Vacuo/e Nucleus \ Eye spot Pyrenoid Nucleus ■Pyrenoid Diagram of Vegetative Reproduction Diagram Gametic Reproduction Fig. 43. — Chlamydomonas: A, vegetative cell of the zoospore; B, the zoospore loses its cilia and rounds off; C, four new zoospores are formed within the parent zoospore; D, when eight new indi- viduals are formed within the parent zoospore, the eight are gametes. X 1,000. The second and third rows are diagrams of vegetative and gametic reproduction. Vegetative reproduction: 1, zoospore; 2, zoospore has lost its cilia and rounded off; 3, the rounded cell is dividing; 4, the rounded cell has divided, producing four zoospores; 5, the rounded cell instead of dividing as in 3 and 4 has produced four zo5spores within itself; 6, the four zoospores have escaped from the rounded cell. Gametic reproduction: 1, zoospore; 2, the zoospore has lost its cilia and rounded off; 3, eight gam- etes are formed inside the rounded cell; 4. gametes escaping; 5, two gametes uniting; 6, zygote formed by the two uniting gametes; 7, four zoospores escaping from the germinating zygote. From Chamberlain's Elements of Plant Science (McGraw-Hill Book Co., New York). when the culture disappears, because new coenobia are likely to de- velop from the oospores. Many fixing agents cause the protoplasmic connections between the cells to be withdrawn. Figure 44 ^, showing the protoplasmic connec- tions in nearly the living condition, was drawn from material fixed in the hot aqueous corrosive sublimate-acetic acid solution; B, fixed in a 1 per cent chromo-acetic solution, does not show the connections; C to E show details drawn from sections. For paraffin sections, the material, preferably in sufficient abund- ance to make a layer 5 mm. deep may be placed in a shell with a bot- 216 METHODS IN PLANT HISTOLOGY torn rounded like a test tube. The tube should be on top of the bath, with the cork out, to evaporate as much of the xylol as possible. Put it in the bath, standing it in some dish to keep it from tipping over. As soon as the paraffin melts, draw it off with a pipette just a little warmer than the paraffin. A hot pipette works well, but ruins the material. Change the paraffin four times. Half an hour in the bath should be enough for any of the Volvocales or any forms of similar consistency. Imbed by pouring out into a small tray so that there Fig. 44. — Volvox: A, surface view of several cells, fixed in tiie hot aqueous corrosive sublimate- acetic acid mixture, stained in iron-alum haematoxylin, and mounted in Venetian turpentine; B, fixed in chromo-acetic acid, but otherwise treated like A ; C-G, fixed in 1 per cent chromo-acetic acid, imbedded in paraffin, and cut 5^; C, E, and F stained in iron-alum haematoxylin; D and G stained in safranin, gentian violet, orange; C, new colony showing pyrenoids, p, and nucleus, n; D,a, nearly mature antheridium; E, young egg; F, egg before fertilization; G, egg after fertilization, showing oil globules, o; pyrenoids, p; starch cut off from pyrenoid, s. All X780. shall be a layer of material about 2 mm. thick. If you have trouble in pouring it out, just let it cool in the shell, putting it into luke-warm water for 15 or 20 seconds, then into cold water. Break the shell. There will be some loss of material in getting it ready for cutting. Sections should not be thicker than 5 ju) 3 or 2 /x will be better for de- tails. Safranin, gentian violet, orange, is a good combination for older stages, especially for pyrenoids and starch. Iron-almii haematoxylin is better for nuclei and the younger stages of oogonia, antheridia, and new colonies. CHLOROPHYCEAE 217 Figure 45 shows that even such dehcate forms as Volvox can be im- bedded in paraffin without shrinking. The Powers' methods. — Professor J. H. Powers' mounts of Volvox and other members of the Volvocaceae have been the deHght and despair of both botanists and zoologists for many years. They have never been surpassed and, probably, never equaled. Professor Powers has kindly given me an outline of his methods; but, as in other phases of technique, judgment, skill, and patience must be furnished by the stu- dent. For fixing. Professor Powers uses the aqueous potassium iodide solution used in testing for starch. This solution may be weaker than the usual formula so that it has a light brown color. From 10 to 24 hours is sufficient for fixing, but material may be left here for sever- al days. Wash thoroughly in tap water which has stood long enough to give off all of its excess of air; otherwise bubbles will form on the colonies, causing them to float and hindering subsequent processes. Change after change of water should be made rapidly, using large amounts of water and decanting just as soon as the colonies have settled. From 1 to 3 hours' washing should be sufficient to remove the brown color of the iodine. Stain in Mayer's carmalum. Use a pure carminic acid in making the stain, 1 g. carminic acid, 10 g. alum, and 200 c.c. distilled water. Dis- solve with heat, filter, and add a crystal of thymol to keep out fungi. After staining, follow the Venetian turpentine method, taking care to wash the glycerin out completely. The 10 per cent turpentine should not be allowed to concentrate too rapidly. Fig. 45. — Volvo.r: photomicrograph of a section from material fixed in chromo-acetic acid and stained in Dela- field's haematoxylin; from a preparation and negative by Dr. W. J. G. Land. 218 METHODS IN PLANT HISTOLOGY Material fixed in weak osmic acid is even better for protoplasmic connections and cilia. About 4 or 5 drops in 50 c.c. of distilled water is sufficient. From 6 to 24 hours is long enough for fixing. After either of these fixing agents, following the washing in water, material may be preserved in a nearly saturated solution of alum ; or in a dilute aqueous carmalum, with a crystal of thymol to prevent mold. Staining for 3 weeks in a weak carmalum stains the cells but not the matrix. About 3 months will be necessary to stain the matrix enough to make it a background for the cells. An aqueous solution of nigrosin gives an effect like that of iron-alum haematoxylin. Rose benzol and Lee's pyrogallic acid method were also useful. When forms so large and so delicate as Volvox are to be mounted whole, put a few small pieces of cover glass into the balsam to keep the cover from crushing the specimens. Diatoms. — Living diatoms are often found clinging in great numbers to filamentous algae, or forming gelatinous masses on various sub- merged plants. Cladophora is frequently covered with Cocconeis, an ehiptically shaped diatom; Vaucheria is often covered with small forms. Other algae will pay for examination, especially if they look brown. If stones in the water have a brown, slippery coating, you can be sure of diatoms. Sometimes the brown coating on sticks and stones is so abundant that it streams out with the current. If rushes and stems of water plants have a brown, gelatinous coating, you are likely to find millions of specimens of the same diatom. The surface mud of a pond, ditch, or lagoon will always yield some diatoms. They can be made to come out from the mud by putting a black paper around the jar and letting direct sunlight fall upon the surface of the water. The diatoms, in a day or even less, will come to the top in a scum which can be easily secured. Since diatoms form an important part of the food of molluscs, tunicates, and fishes, the alimentary tracts of these animals often yield deep-water forms which are not easily secured in any other way. Fresh water diatoms appear in greatest abundance in spring, are comparatively scarce in summer, and reappear in autumn, though not so abundantly as in the spring. Marine forms can be secured by scraping barnacles, oyster shells, and other shells. The big Stromhus shell from the West Indies, which we use to keep the door open, will yield a good collection if you get it before it is cleaned. CHLOROPHYCEAE 219 The silicious shells of diatoms are among the most beautiful objects which could be examined with the microscope (Fig. 46). To obtain perfectly clean mounts requires considerable time and patience, but when the material is once cleaned, preparations may be made at any time with very little trouble. Diatom enthusiasts have devised numer- '-' ^^'^ M Fig. 46. — Diatoms: diatomaceous earth from Cherryfield, Maine, a Pleistocene deposit, show- ing the great variety of forms usually found in such material; photomicrograph by Miss Ethel Thomas from a preparation by Rev. E. L. Little. X400. ous methods for cleaning them, and separating the various forms from one another, but we shall give here only a few simple, practical meth- ods. Dr. Wood's method of mounting frustules of living forms is easy and effective: Material may be obtained by skimming off the brownish scum found on ponds, by squeezing out water weeds, by scraping sticks and stones which are 220 METHODS IN PLANT HISTOLOGY covered at high water, or from the mud of filter beds and pumping-worls:s, or in other places. The material is put in a dish of water, and after it has settled the water is decanted. This is repeated until the water will clear in about half an hour. The sediment is then treated with an equal bulk of sulphuric acid, after which dichromate of potash is added until all action ceases. After a couple of hours the acid is washed out. To separate the diatoms, place the sediment in a glass dish with water, and when the water becomes clear give the dish a slight rotary motion. This will bring the diatoms to the top, when they may be removed with a pipette and placed in alcohol. To mount, place a number in distilled water, evaporate a few drops of the mixture on a cover- glass, which is then mounted on a slide in balsam. It is better to use a very slight smear of Mayer's albumen fixative to prevent the diatoms from floating to the edge of the cover. Many scouring soaps and silver polishes contain large quantities of fossil diatoms, and the diatomaceous earths are particularly rich. Diatomaceous earths from Cherryfield, Maine, and from Beddington, in the same region, are the richest we have seen (Fig. 46). The de- posits at Richmond, Virginia, have long been famous. In some of our western states there are deposits 300 feet thick, with 80 per cent of silica, the silica being the valves of diatoms. Break up a small lump of such material and boil it in hydrochloric acid. An evaporating dish or a test-tube is convenient for this purpose. Let the diatoms settle, pour off the acid, and then wash in water. As soon as the diatoms settle, the water should be poured off. The wash- ing should be continued until the hydrochloric acid has been removed. When the washing is complete, pour on a little absolute alcohol, and after a few minutes pour off the alcohol and add equal parts of turpen- tine and carbolic acid. The material will keep indefinitely in this con- dition, and may be mounted in balsam at any time. In making a mount, put a little of the material on a slide and allow it to become dry, or nearly dry, and then add the balsam and cover. If the balsam should be added too soon, the diatoms are likely to move to the edge of the cover. We have had excellent results with the following method: After washing in water, keep the diatoms in 5 per cent formahn. To make a mount, smear the slide very slightly with Mayer's albumen fixative, add a little of the material, and heat just enough to coagulate the albu- men. When perfectly dry, add a drop of balsam and a cover. Or, after coagulating the fixative, dip in absolute alcohol and then in xylol be- fore mounting in balsam. Without the alcohol and xylol, some air is CHLOROPHYCEAE 221 sure to be caught and it may accentuate some markings; but, in gener- al, we prefer to use the alcohol and xylol. To show the cell contents, diatoms must be fixed and stained. If they are clinging to filamentous algae, the algae with the diatoms attached should be put into chromo-acetic acid (24 hours) and then washed in water for 24 hours. Stain in iron-haematoxyhn and proceed by the Venetian turpentine method. When ready for mounting, the diatoms can be scraped off from the algae or other substratum. Saf- ranin gives a beautiful stain, bringing out the radiations and the pecu- liar centrosomes. This stain has been used very effectively in studying auxospores. When material is in gelatinous masses or is clinging firmly to some easily cut substratum, it may be fixed and imbedded in paraffin. The knife often breaks the frustules as cleanly as if they were cut. Desmids. — The desmids are unicellular, free-floating, or suspended algae. They are not found in salt water and are more abundant in soft water than in hard. Deep pools, quiet ponds, and quiet margins of small lakes are good collecting-grounds. Collections of other fresh- water algae often contain some desmids. It frequently happens that a single desirable desmid appears during examination of field collec- tions. In such a case, remove it with a fine pipette, and get it into a drop of water on a clean slide, invert it over a bottle of 1 per cent osmic acid for 8 seconds, leave the slide exposed to the air until almost all the water has evaporated, and then add a drop of 10 per cent glycerin. In a few hours (6-24) put on a cover and seal. It requires more time, care, and patience than it is worth to attempt staining in such a case. Sometimes desmids occur in great abundance. We have found Mi- crasterias so loosely attached to Chara that a quart bottle full could be squeezed off in a few minutes. A watering-trough yielded Cosma- rium in almost equal abundance. They may then be treated like the filamentous algae, except that more care must be taken not to lose them when changing fluids. Four or 5 drops of 1 per cent osmic acid to 50 c.c. of water fixes well, and material from this solution may be placed directly into 10 per cent glycerin and mounted by the Venetian turpentine method. It looks almost as if stained in iron-alum haema- toxyhn. The iodine solution used in testing for starch gives good re- sults and may be followed by any stains. The larger desmids stain beautifully in iron-alum haematoxylin. The Venetian turpentine method, with phloxine and anihn blue, 222 METHODS IN PLANT HISTOLOGY will give beautiful preparations. A deep stain with phloxine and a rather light stain with anilin blue is better for the pyrenoids and nu- cleus, while a light stain in the red and a deep stain in blue is better for the chromatophores. Material may be fixed and stained as directed for Volvox. Formalin 8 C.C., acetic acid 2 c.c, and water 90 c.c. fixes well, and material may stain in the solution indefinitely. Fig. 47. — Some common desmids. A and B, Clostcrium conjugating; C-F, Cosmarium; C and D, adult cells; E and F, dividing; the inner half-cells will grow until they reach the size of the outer half-cells; G, Micrasterias. A and B X640; C-F X770; G X640. Lutman found that nuclear division in Closterium takes place at night. This is probably true to a greater or less extent, for most of the Chlorophyceae. The division is peculiar. When the cell divides, each of the resulting cells gets a half-cell already of adult size; the other half-cell — the inner one — is very small, but it grows rapidly and, in a day or so, becomes as large as the other half. A few of the most common desmids are shown in Figure 47. Zygnema. — Zygnema is one of the most common algae of the ponds, swamps, and ditches. The mats are very slippery to the touch. In the field it resembles Spirogyra, but is distinguished by the two character- CHLOROPHYCEAE 223 istic chromatophores which are readily seen with a good pocket lens. Sometimes conjugation can be induced by bringing the material into the laboratory and placing it in open jars with plenty of water and not too much hght. The chronio-acetic-osmic acid solution is good for fixing. Stain in iron-alum haematoxylin and also in phloxine and anilin blue. Follow the Venetian turpentine method, and in mounting put material from both stains on each slide. Also, have both conjugating and vegetative material on each slide. There will probably be vegetative filaments mixed with those which are conjugating; but these will not be so vigorous and are not so likely to show dividing cells and plastids as material which has not begun to conjugate. Don't forget to run the material up to 85 per cent alcohol and then run it back before staining. To many, this may seem unnecessary, but the results are worth the time and labor. Textbooks describe ''stellate chromatophores" in Zijgnema. A good preparation should show that the chromatophores have an even out- line, with no trace of a stellate form. The boundary between the chromatophore and the protoplasm — often of a stellate form — in which the chromatophore is imbedded, should be seen in well-fixed material with either of the foregoing stains. The large starch grains, extending from the pyrenoid almost to the border of the chromato- phore, are better differentiated by the phloxine and anilin blue; the nucleus and pyrenoid are better stained by the iron-alum haematoxy- lin. Careful staining should bring out the features shown in Figure 48. The chromatophores do not stain as readily as those of Spirogyra, and consequently it is necessary to use stronger stains or more prolonged periods. Use the Venetian turpentine method. For a detailed study, imbed in paraffin and cut thin sections. After washing in water, arrange the filaments so that most of them will have the same general direction; then, in running up through the alcohols, keep the filaments from spreading too much by placing a slide on the material. After imbedding, the material can be cut into blocks about a centimeter square. If sections thinner than 5 fx are wanted, cut out smaller paraffin blocks. Spirogyra. — Probably no alga has been more studied by pupils, teachers, and investigators than Spirogyra (Fig. 49). Nearly all of the numerous species belong to the low, quiet waters of ponds and ditches, where they often form large, flocculent green mats nearly covering the 224 METHODS IN PLANT HISTOLOGY surface of the water. A few species occur in running water. The mats are very sUppery to the touch — a character which assists in recogniz- ing the genus in the field. In the larger species the characteristic spiral chromatophores can be seen with a good pocket lens, thus completing the identification, as far as the genus is concerned. Mats in which zygospores have been formed are likely to show a pale, or even a brownish, color, due to the brownish walls of the zygospores. This C Fig. 48. — Zygnema: fixed in the Chicago chromo-acetic-osmic solution: A and B stained in Magdala red and aniUn blue; C and D stained in iron-alum haematoxylin. All show the nuclei, the chromatophores differentiated from the cytoplasm, and the large starch grains arranged radially about the pyrenoids. In C, cell division has just taken place and the pyrenoids and chromatophore in each new cell are dividing. In D, a young zygospore, the two nuclei have not yet fused. X790. color, however, is not always, or even usually, due to zygospores, but is more often due to the death and degeneration of the plants. Mats in early stages of conjugation and those with young zygospores show as bright a green as vigorously growing material. Spirogyra is not easy to keep in the laboratory. The small species keep better than the larger ones. Put only a small amount of the material in a jar and use rain water. If it is necessary to use tap water, let the water run for a minute before taking the water for the culture. Most metals are poisonous to Spirogyra, even the small amount taken up by the water while standing in the water pipe being detrimental. CHLOROPHYCEAE 225 The species found in running water will usually conjugate within a week, when brought into the laboratory and placed in rain water or tap water. Species belonging to quiet waters, when brought into the Fig. 49. — Spirogyra: A, vegetative cell of a large species, with four spiral chromatophores, con- taining many large and small pyrenoids. B, a smaller species with only one spiral chromatophore. C, union of gametes: these large, non-motile gametes, consisting of the entire contents of the cell, are characteristic of the group. D, mature zygotes: the one on the right focused on the surface; the one on the left focused for the center, showing the nuclei of the two gametes. All X300. From Chamberlain's Elements of Plant Science (McGraw-Hill Book Co., New York). laboratory and placed in a 0.2 per cent Knop's solution, are likely to undergo rapid cell division and growth. After the alga has remained in such a culture for a few days or for a week, conjugation may be in- duced by transferring to rain water or tap water, and keeping the cul- ture in bright sunlight. Conjugation may begin within 3 or 4 days. 226 METHODS IN PLANT HISTOLOGY Variations in temperature between 1° and 15° C. have little influence upon conjugation. The Chicago chromo-acetic-osmic solution fixes well. Stain some material in iron-alum haematoxylin and some in phloxine and an- ilin blue. Use the Venetian turpentine method, and on each slide mount material stained in both ways. With phloxine and anilin blue the spiral chromatophore takes the blue and its pyrenoids the red. Fig. 50. — Scenedesmus If the material contains figures, stain in iron-haematoxylin. This will stain the figures, but will hardly touch the chromatophore or cell wall, thus allowing an unobstructed view of the figures. While figures occur occasionally in the daytime, collect your material at night, preferably near midnight. Spirogyra is easily imbedded and cut. Scenedesmus. — Scenedesmus (Fig. 50) is found everywhere as a con- stituent of the fresh water plankton. It is more abundant in stagnant water. It often appears in considerable quantity in laboratory cultures, where it may be kept for years in a tightly closed glass jar without renewing the water, the lid being removed only when material is needed. CHLOROPHYCEAE 227 The form is so small that in living material little more than the general form can be distinguished. Excellent mounts are easily and quickly made. Smear a very thin layer of albumen fixative upon the slide, and add a drop of water containing the Scenedesmus. The drop may be inverted for 4 or 5 seconds over the fumes of 1 per cent osmic acid. No washing is necessary, and good mounts may be made without any fixing whatever. Allow the drop to dry completely. It is better to leave it for 24 hours before proceed- ing. The usual difficulty with this form, and with many others, is that the back- ground stains and so makes the mounts untidy. The following method by Yam- anouchi will produce beautiful prepara- tions (Fig. 50) : 1. Dry on the slide, 24 hours. 2. Ten per cent alcohol overnight to re- move chlorophyll. 3. Safranin (alcoholic), 4 days. 4. Water, 5 minutes. 5. Aqueous gentian violet, 2 days. 6. Water, a few seconds. 7. Orange G, aqueous, 3 minutes. 8. Ninety-five per cent alcohol, a few seconds. 9. Absolute alcohol, 1 minute. 10. Clove oil, until the stain is satis- factory. Different collections of Scenedesmus stain very differently, but the time in clove oil is likely to be long, even as long as 6 hours. 11. Xylol, 5 minutes. 12. Mount in balsam. Fig. 51. — Pediastrum: A, colony with 16 cells; B, colony with 8 cells. Both are beginning to form new colo- nies inside each cell. X770. Pediastrum. — This is a beautifully regular little alga (Fig. 51). Scattered specimens often occur mixed with other algae and, occasion- ally, one finds it in abundance. Look in small ponds, ditches, and bog pools, where it is associated with other water plants. If it is at all abundant, centrifuging very gently for a few minutes may help you get a hundred specimens in a single drop. If material is scarce, put it into formahn-acetic acid — 5 c.c. formalin and 5 c.c. acetic acid to 228 METHODS IN PLANT HISTOLOGY 90 c.c. water. Stain with safranin, fuchsin, or carmine. Avoid stains which need much washing out. However, small scanty material like this can be handled in the following away: strip epidermis from the inner scales of an onion or from a Sedum leaf; fix in the above forma- lin-acetic acid solution; get the material into a drop or two of water on the epidermis; roll it carefully so as to make a little tube; tie the ends of the tube and treat like any macroscopic object. When ready to mount, the ends with the threads can be cut off and the material can be mounted in balsam. Such pieces can be treated like a root-tip, im- bedded in paraffin, and cut. The epidermis makes a ring around each section, thus making the objects easier to find. Hydrodictyon. — This is popularly known as the "water-net." Hy- drodidyon is found floating or suspended in ponds, lakes, or slow streams. The young nets are formed within the segments of the older nets. Examine segments 4 or 5 mm. in length for the formation of young nets. The old nets may reach a length of 10 cm. Cultures are easily kept in the laboratory. If material which has been growing in a 0.5-1 per cent Knop's solution be brought into tap water or pond water, zoospore formation may begin within 24 hours. Nets brought from the nutrient solution into a 1^ per cent cane-sugar solution produce zoospores for a few days. Nets of all sizes should be selected for study. The segments are coenocytic, and the nuclei of the older segments are hard to differen- tiate, except in stained preparations. Only one nucleus will be found in the young segments, but in the older segments the nuclei become very numerous. For fixing try 0.7 g. chromic acid, 3 c.c. acetic acid, 6 c.c. of 1 per cent osmic acid, and 100 c.c. water. If the cell contents of the vegeta- tive cells shrink away from the wall, reduce the chromic acid to 0.5 g. If there is still some tendency to shrink, increase the acetic acid to 4 c.c, leaving the chromic acid at 0.5 g. If the chromic acid is as weak as 0.5 g., fix for at least 24 hours, using a large amount of the fixing agent. About 100 times the weight of the material is not too much. For fixing, use the Chicago chromo-acetic-osmic formula. This should not produce plasmolysis in nets of any age. Iron-alum haema- toxylin will differentiate the nuclei and pyrenoids, which may look ahke with less precise stains. Use the Venetian turpentine method for mounting whole young nets and parts of older nets. Fine scissors should be used freely, because any attempt to arrange the material CHLOROPHYCEAE 229 with needles will make it look as if the whole method of preparation were wrong. Parts of nets mounted whole are shown in Figure 52. For details of the formation of starch and for the finer details of the development of zoospores and gametes, Hydrodictyon should be im- bedded and cut. Pleurococcus. — This form, which is used everywhere as a labora- tory type of the unicellular green algae, is found on the bark of trees, where it is more abundant on • i®^. f&l m:^ i "H ' 9 '■<9)' * M' © « ■(i)&' .9 ■ -^ ■ D ^^ the north side and near the ground. It is also found on stones and fences, and in moist situations generally. It is easily secured in nearly all locahties and at all seasons. The life-history of Pleuro- coccus is variously described in textbooks, but it is very doubtful whether there is any mode of reproduction except by cell division. The zoospores and gametes which are sometimes described probably belong to other forms which are occasion- ally associated with Pleurococ- cus, especially when growing in very moist situations. The life- history was examined very critically by the great algolo- gist, Wille, who not only con- cluded that cell division is the sole mode of reproduction but showed how investigators, even those relying upon cultures, had made their mistakes. Wille's paper was published in 1913 in Nyt Magazin for Naturvidenskaberne. A study of the living material is sufficient for any general course. The bright-green cells, scraped off and mounted in a drop of water, show the rather thick wall, the chromatophores, and usually the nu- cleus. A drop of iodine will bring out the nucleus, if it does not show already, and will also stain the pyrenoid, if the cell contains one. A Fig. 52. — Hydrodictyon: A, part of young net with segments showing one pyrenoid and one or two nuclei; B, parts of three segments with nucleus, n, and pyrenoid, p, well differentiated; C, part of a still older segment with nuclei more numerous than the pyrenoids; D, part of a nearly mature segment with nuclei much more numerous than the pyre- noids. Fixed in the Chicago chromo-acetic-osmic solution and stained in iron-alum haematoxylin. X600. 230 METHODS IN PLANT HISTOLOGY mount in Venetian turpentine, stained in phloxine and anilin blue, shows the nucleus very clearly. Vaucheria. — This form can always be obtained in greenhouses, es- pecially in the fernery, where it forms a green felt on the pots. The greenhouse form is likely to be Vaucheria sessilis. Another species, V. geminata, is very common in the spring, when it may be found in ponds and ditches (Fig. 53). Vaucheria is also found in running water, but in this situation is almost certain to be sterile. In the vicinity of Chicago, V. geminata appears late in March or early in April and within a few weeks begins to fruit abundantly. The fruiting contin- ues for from 4 to 8 weeks, and then the alga may disappear until later in the season, when some of the oospores germinate. Vaucheria sessilis is found at all sea- sons in the greenhouses, but it is usu- ally in the vegetative condition. Klebs found that the formation of oogonia and antheridia can be induced in V. repens (a variety of V. sessilis) within 4 or 5 days by putting the material into a 2-4 per cent cane-sugar solution in bright sunlight. The sex organs will not be formed in weak light or in darkness. The formation of zoospores may be induced in the following way : Cultivate in an 0.1 to 0.2 per cent Knop's solu- tion for a week, then bring the ma- terial into tap water, and keep the cul- ture in the dark. Zoospores may appear within 2 days. Bright light or a temperature higher than 15° C. will check the production of zoospores. A 2 per cent cane-sugar solution kept in the dark is also likely to furnish zoosporic material. If no zoospores are formed when the solution is kept in the dark, the nutrition has been too weak: strengthen the nutrient solution and keep the culture in the light for a few days; then put the culture in the dark, and zoospores should Fig. 53. — • Vaucheria: A, Vaucheria geminata, showing antheridium and five oogonia containing fertilized eggs; from a preparation fixed in formalin, acetic acid, and stained in iron-alum haema- toxylin. B, V. sessilis: from a prepara- tion fixed in chromo-acetic acid and stained in eosin and gentian violet. X150. CHLOROPHYCEAE 231 appear. The formation of zoospores may continue for a couple of weeks. Aplanospores of V. geminata are formed in nature when the plant is growing upon damp ground. The aplanospores may also appear in a 4 per cent cane-sugar solution. In fresh 0.5 per cent Knop's solution in bright light, cultures remain in the vegetative condition, and the result is the same in weak light if the nutrient solutions are seldom changed. Such cultures may be kept indefinitely by chang- ing the nutrient solution whenever a whitish scum appears on the surface. Vaucheria is not easy to fix. Solutions which give fine results with Spirogyra and Zygnema may be ruinous to Vaucheria. We have secured the best results with a formalin-acetic solution (10 c.c. formalin, 5 c.c. glacial acetic acid, and 90 c.c. water). Chromic-acid solutions, even with 4 or 5 per cent acetic acid, cause some plasmolysis. If the chromic acid is weakened enough to prevent plasmolysis, the solution should be allowed to act for 48 hours. The ad- dition of 1 per cent osmic acid up to 6 c.c. to 100 c.c. of the solution does not seem to cause any more shrinking, and nuclei are easier to stain. Iron-alum haematoxylin is the best stain. Phloxine and anilin blue give beautiful results, occasionally, but preparations are almost sure to fade. Eosin is good for topography, but will not show the nuclei. Use the Venetian turpentine method. In mounting, use small scis- sors freely. You cannot untangle a mat of Vaucheria so as to give good views. For the development of the oogonium and antheridium, for fertili- zation and for the structure and development of the various spores, thin sections are necessary. Imbed in paraffin. For nuclear details, use iron-haematoxylin ; for cytoplasm, use safranin, gentian violet, orange. Cladophora. — This genus is found in both salt and fresh water (Fig. 54). The fresh-water forms are usually attached to sticks or Fig. 54. — Cladophora; fixed in chromo-acetic acid and stained in iron-alum haematoxylin. 232 METHODS IN PLANT HISTOLOGY stones in quiet or running water. The mats feel rough and crisp and, even under a pocket lens, show the characteristic branching by which the form is easily recognized. The absence of a mucous coat makes Cladophora a convenient host for numerous parasitic algae, among which diatoms belonging to the genera Cocconeis and Gomphonema are particularly abundant. For laboratory cultures, select the forms found in quiet water, but for preparations, forms growing where the waves dash hard are better, since you can get a fine display of branches under a small cover. Forms growing in still water or in gently flowing water may look like un- branched filaments, under an ordinary cover. For fixing, use chromo-acetic-osmic acid, watching the effect of the solution and modifying the constituents until you find just what you need for that lot of material. If it looks all right at the end of 10 min- utes, it is likely to remain all right. Cladophora is one of the most difficult of all algae to fix well. Iron-alum haematoxylin, followed by the Venetian turpentine methods, gives the best results for nuclei and pyrenoids. Phloxine and anilin blue are better for the cell wall and chromatophores (Fig. 54). Ulothrix. — Where the problem of the origin and evolution of sex is studied, Ulothrix is an indispensable type (Fig. 55). Ulothrix zonata is found in springs, brooks, and rivers, occurring in bright green masses attached to stones in riffles, especially in sunny places. It is abundant on stones and piles along the beaches of lakes. Nuclear division takes place at night, most abundantly about midnight, and is followed by a rapid development of zoospores and gametes, which continue to be discharged throughout the forenoon. In the afternoon the material is largely vegetative. Another species is found in stagnant ponds, ditches, and even in watering-troughs and rain-barrels. It is difficult to keep in the laboratory the forms which are found in rapidly flowing water. However, if they are brought in still attached to stones and placed under a stream of tap water, they may live for a couple of weeks and may produce zoospores every morning. The production of zoospores may continue for a few days, if the material is merely put into a jar of water; in a 2-4 per cent cane-sugar solution the produc- tion of zoospores continues a little longer. No form is better than Ulothrix for illustrating to a class the differ- ence between zoospores and gametes. Even when gametes are not con- jugating, their more rapid movement is noticeable; and when conju- CHLOROPHYCEAE 233 gating, the awkward, jerky movements of the pair contrast sharply with the graceful movements of the zoospores. Fix in chromo-acetic acid— 1 g. chromic acid and 2 c.c. acetic acid to 100 c.c. water ; or in 0.7 g. chromic acid— 3 c.c. acetic acid, and 6 c.c. 1 per cent osmic acid to 100 c.c. water. Formalin-acetic acid— 10 c.c. for- malin and 5 c.c. acetic acid to 100 c.c. water— is a good fixing agent, especially when followed by phloxine and aniUn blue. The anilin blue m ^ 1 M A B C D F G Fig. 5.5. — Ulothrix: A, part of vegetative filament. B, a vegetative cell at the top; the cell be- low has divided and one more division would make 4 zoospores; the next cell shows 4 young zoo- spores, and the bottom cell has 4 zoospores almost ready to escape; e, eye spot; n, nucleus; p, py- renoid. C, each cell with 8 nearly mature zoospores. D, mature zoospore. £, filament with gametes. F, two gametes uniting. G, zygote formed by the two uniting gametes. X535. From Chamberlain's Elements of Plant Science (McGraw-Hill Book Co., New York). should stain the chromatophore and the phloxine the pyrenoids. Iron- alum haematoxylin should stain the chromatophore gray and the pyrenoids black. Mount on each slide material from both lots. Oedogonium. — Most species are found in quiet waters, especially in ponds and ditches. The best fruiting material is often attached to sub- merged twigs, rushes, and various plants, where, to the naked eye, it forms only a fuzzy covering. Some species form floating masses, bear- ing some resemblance to Spirogyra, but they are not so slippery. The fixing agents mentioned for Ulothrix are good for Oedogonium. The iodine solution used in testing for starch, or a weak aqueous solu- 234 METHODS IN PLANT HISTOLOGY tion of osmic acid (4 or 5 drops of 1 per cent osmic acid to 50 c.c. of water) is good for zoospores and androspores. For cell division and the peculiar method of forming the new cell wall, stain in phloxine and anilin blue. Iron-alum haematoxylin is better for most of the other phases; but the fertilized eggs stain very deeply. Consequently, stain some material lightly, for the fertilized eggs; and some more deeply for young eggs, chromatophores, and other phases. Mount in Venetian turpentine. For details of blepharoplasts and the development of the various motile forms, material should be imbedded and sectioned. Nanandrous species have antheridia only in the dwarf males; and species with antheridia in the ordinary filaments have no androspores in the hfe-history. A species with no dwarf males in the life-history (macandrous) is shown in Figure 56 A and B; a species with dwarf males in the life-history (nanandrous) is shown in C of the same figure. In studying Oedogoniuvi diplandrum, Klebs found that a change from a lower to a higher temperature would induce the production of zoospores. A culture which had been kept in a cold room with a tem- perature varying from 6° to 0° C, when brought into a warmer room with a temperature varying from 12° to 16° C, produced an abundance of zoospores within 2 days. Light does not seem to have any influence upon the formation of zoospores in this species, but hght is necessary for the formation of antheridia and oogonia. We have secured an abundance of oogonia and antheridia by keep- ing the material for 4 or 5 days in a very weak Knop's solution and then transferring to distilled water. The oogonia appeared in 3 or 4 days. The method seems to succeed with some species, especially those which occur floating or suspended in the water, but we have not suc- ceeded with species which form a fuzzy covering on grasses and twigs under water. Sterile material sometimes fruits when brought into the laboratory and placed in open jars with plenty of water and not too much light. Co\eochsiete.— C oleochaete is epiphytic upon the stems and leaves of submerged plants. Sagittaria is a good host plant. Look on petioles from the surface of the water down to 6 inches below, where the alga begins to get scarce. The well-lighted part of the host is better than the shaded part. Three or four species may be found growing so close together that they all come in the field of the microscope with a 16 mm. objective. C oleochaete scutata is the best-known species, but C. soluta, CHLOROPHYCEAE 235 C. orbicularis, and C. irregularis are equally common. C. scutata is often found on the floating leaves of Polygonum amphibium. Chromo-acetic acid (1 g. chromic acid and 3 c.c. acetic acid to 100 c.c. water) is a good fixing agent. For finer details and for sections, add 6 c.c. of 1 per cent osmic acid. d..J Fig. 56. — Ocdogonium. A, oogonial, and B, antheridial filament of a dioecious species; n, nucleus; p, pyrenoid; s, starch. D shows two groups of antheridia, with 16 antlieridia in the upper group and 8 in the lower. Most of the antheridia contain two nearly mature sperms. C, Ocdo- gonium nehraskense, a nanandrous species collected by Dr. Elda Walker; d, dwarf male. X300. From Chamberlain's Elements of Plant Science (McGraw-Hill Book Co., New York). The epidermis of the host plants is hard to peel off. With a fleshy plant, like Sagittaria, cut parallel to the surface pieces 8 or 10 mm. wide, lay the outer side on a piece of glass, and gently scrape off the hypodermal cells until practically nothing but the epidermis, with the Coleochaete, is left. Then fix and mount the epidermis with the Coleo- chaete. Stained in iron-alum haematoxylin and mounted whole, such 236 METHODS IN PLANT HISTOLOGY preparations are very effective. All the species mentioned are flat: C. pulvinata is hemispherical and might be mistaken for a small Rivularia. If you overstain with Delafield's haematoxylin and then reduce the stain with hydrochloric acid (about 3 or 4 drops to 100 c.c. water), the alga will stand out sharply against the host. A slight tinge of orange in clove oil will increase the contrast. Sections are easily cut and, especially in forms with a flat thallus, show features which might escape if one depended entirely upon plants mounted whole. Cut out small pieces of leaf or stem abundantly covered with Coleochaete, imbed in paraffin, and cut host and guest together. Chara. — Chara is found in ponds, lagoons, and ditches. Once seen, it is always readily recognized. In the ponds and lagoons along the southern shores of Lake Michigan it fruits so abundantly that the whole pond shows an orange color due to the immense numbers of antheridia. In the lagoons of the Chicago parks Chara is so abundant that it must be dredged out every summer. Chara is easily kept alive throughout the year in the laboratory. A 2-gallon glass jar with an inch of pond dirt, sand, and gravel at the bottom, and nearly filled with tap water, is all that is needed for a successful culture. If the jar is to be covered, it should not be more than two-thirds full of water. Not more than a dozen plants should be put into such a jar. A rather strong solution should be used for fixing. The following will give good results : Chromic acid 1 g. Glacial acetic acid 1 c.c. Water 100 c.c. In about 24 hours this not only fixes but dissolves the lime with which most species are coated. For paraffin sections select the tip of the plant, a piece about a centimeter in length. Sections of this may show, not only the large apical cell, but also various stages in the development of antheridia and oogonia (Fig. 57). For the development of the plant body from the apical cell and also for early stages in the development of oogonia and antheridia, the safranin, gentian violet, orange combination is excellent; for later stages, especially in the development of the an- theridia, iron-haematoxylin is much better. The antheridium of Chara stains so rapidly that the beginner uni- CHLOROPHYCEAE 237 formly makes poor preparations. In order to get good preparations of the antheridium it is necessary to disregard other structures, which will be stained lightly or not at all when the stain is just right in the antheridial filaments. B Fig. 57. — Chara: fixed in chromo-acetic acid and stained in safranin, gentian violet, orange. A, longitudinal section of apex showing five nodes; c, corticating filaments; j;, part of the frag- mented nucleus. B, note the different condition of the nodes and first branches. C, young an- theridium, a, and young oogonium, o. X230. If it is desired to mount whole branches showing the antheridium and oogonium in position, use the Venetian turpentine method, staining in phloxine alone, or in phloxine and anilin blue. Good mounts showing shield, manubrium, capitula, and filaments may be obtained by crush- 238 METHODS IN PLANT HISTOLOGY CHLOROPHYCEAE S/PHONEAE Coenocuf/c Phijllosiphonaceae CONFERVOIDEAE F/hmenfoud, uninucleate ColeochoefQceae Voucher/QceQe DerhesfQCSQe C/odopk oraceoe CQu/erpa ceae Bruops/dQceae \ Dosucladaceae l/a/aniaceae ^ 'oraceae Choetopk I OedoQoniaceae I U/ofrichQceae Botrudiaceae Uli/aceae /Pediastrum^ ^ /Hudrocfictuon ocenecfesmus •Fucfor/na , /"^Dialomaceae ^. -.■ >■■;,;.- „;, ,^^- a^, . * . ;>.i 1." '••i ■'•■ ; ",'. •>•■■;■•. ^' ■^■■■■•^ ■ V ■"■■ '.'-1- •;■.,'„."* ■^J^ "•'■'. ''K'-*- ■ V 'fV'V*:■'.•:% !WV.- .-''^^■.'-* . ' V.'"".- ••' ^'' *\^' ' -"*'"*'i".'-i ;■-•.■ ■^ V,*'T K-'- f- ■"j*;> V"iv >.V'*'*'-" •■■-...<.>;{ % ■:v;->y'*^':/" :''5^i'^ :•*:''] \\ .''*■-.:. ■ -■^-i' ,'*-'■ ■ ••.■.•■ >v r'i\* 'ivj; '. '/ '.'•'''. *iv'V'"' •\:;.-"i« v.-f -■-■ y i**'."' ;>•';• v.^'. -V ■•-• ,> :*;-■? .^. . . 't'^ .,v*: -^ : -- ^^ -.- i 11 V ■ '-:'r ^^-^^:j=?^:;' ■v:,;§ •■''*i ^*:-^ ^-.'t.- -V;;- ,.■;!-- ■;' '? . '.'..* »>.•••: Sf • ■:'. '•'■ -■•'■. . . '■-' .,■■'/.'":•.* %=: iV'.T;- ■:f:-%i^S ■'•'-'-■<■? i V ■?'s^- • fi '*:i".' * ' .-" .'. i^^ C. ■•'^i'^"''^' '*^C*'^ *:-• '■■■>* 1 i;^^CKjv^' .■■->^\ '•',* j!;,'-'^**--.J,'V V P _,.,^ )|g|^ % '^^J^'^ '/*«^^ ' :.i' ^^'t /^ z? Fig. 119. — Pinus laricio: A, top of prothallium, with an archegonium just before the cutting off of the ventral canal cell; fixed in Flemming's weaker solution and stained in Haidenhain's iron- alum haematoxylin; collected June IS, 1897. B, C, D, early stages in the development of the em- bryo; fixed in chromo-aoetic acid and stained in safranin, gentian violet, orange; collected July 2, 1897. X104. the stages shown in Figures 119 ^ and 120 the pollen tubes with their contents are rapidly working their way through the nucellus toward the archegonia, and consequently, in some of the material, it is better to retain enough of the tissues of the ovule to keep the nucellus in place. In later stages, after fertilization has taken place, the develop- ing testa should be removed with great care, for a very slight pressure is sufficient to injure the delicate parts within. With any fixing agent of the chromic-acid series, the free nuclear stages of the female gametophyte and, later, the archegonia and pro- SPERMATOPHYTES— GYMNOSPERMS 343 embryo stages, like those shown in Figure 119, are Hkely to show plasmolysis; but, by reducing the chromic acid to 0.5 c.c. and increas- ing the acetic acid to 3 c.c, and fixing for 48 hours, there is less danger. Fig. 120. — Pinus laricio: photomicrograph showing the formation of the ventral canal cell; iLsually this cell is not so large in proportion to the egg; fixed in Flemming's weaker solution and stained in safranin, gentian violet, orange; the preparation was made in 1897, the photomicrograph in 1915; Cramer contrast plate; 4-mm. objective; ocular X4; Abbe condenser; yellowish-green filter and also a strong filter used in outdoor photography; camera bellows, 75 cm.; arc light; exposure, 7 minutes. Negative by Miss Ethel Thomas. X587. Stages, like those shown in Figure 1195-D, without any shrinking, were secured by Miss Ethel Thomas by using hot alcoholic corrosive sublimate-acetic acid with formalin (4 g. corrosive sublimate, 5 c.c. 344 METHODS IN PLANT HISTOLOGY acetic acid, 5 c.c. formalin, 100 c.c. 70 per cent alcohol). Figures like that shown in Figure 120 are better in chromo-acetic-osmic acid. The period at which the various stages may be found varies with the species, the locality, and the season. In Pinus laricio the mega- spore mother-cells appear as soon as the young strobili break through the bud scales. At Chicago, in the season of 1897, material collected May 27 did not yet show archegonia; the ventral canal cell was cut off about June 21 (see Fig. 120), the fusion of the egg and sperm nuclei occurred about a week later, and stages like Figure 1192?, C, and D, were common in material collected July 2. In the season 189G all the stages appeared about 2 weeks earher. In Pinus sylvestris the stages appeared a little earlier than in Pinus laricio. After the stage shown in Figure 119 A has appeared, it is necessary to collect every day until the stage shown in Figure 119D is reached. If collections are made at intervals of 3 or 4 days, the most interesting stages, like the cutting off of the ventral canal cell, fertilization, and the first divisions of the nucleus of the fertilized egg, may be missed altogether. It should be mentioned that all the ovules of a cone will be in very nearly the same stage of development; consequently, it is worth while to keep the ovules from each cone separate. Stages like that shown in Figure 120 are rare in miscellaneous collections, but if ovules from each cone are kept separate and this figure is found, the rest of the ovules from that cone will be likely to show some phase of this interesting mitosis. Thuja and Juniperus are good types to illustrate the archegonium complex and the large, highly organized sperms. In Thuja occidentalis, in the Chicago region, a series from the appearance of archegonium initials to young embryos may be collected between June 10 and June 20. In Juniperus virginiana, in the same locality, polHnation occurs late in May and fertihzation takes place 12| months later. The mega- spores are formed late in April, and the development of the female gametophyte occupies about 6 weeks. The embryo.— The early stages of the sporophyte, usually desig- nated as the proembryo, have been mentioned already. From the time when the suspensors begin to elongate up to the appearance of cotyledons, instructive preparations can be made by mounting the embryo whole. Dr. J. T. Buchholz has developed a method for handhng these small objects. Remove the testa and then, under water, hold the endosperm gently with forceps and press the SPERM ATOPHYTES— GYM NOSPERMS 345 neck and upper part of the archegonium with a needle, pressing, and at the same time drawing, the needle away, so as to pull the young embryo out. Some of the embryos will be broken, but by care- ful manipulation more than half should be en- tirely uninjured. Fix in formalin (5 per cent in water) , stain in Delafield's haematoxylin, transfer to 10 per cent glycerin, and continue with the Vene- tian turpentine method. A preparation made in this way is shown in the photomicrograph (Fig. 121). These stages, and all subsequent stages, are easily cut in paraffin with- out removing the embryo from the endosperm. Cut a thin slab from opposite sides of the endosperm, fix in chromo-acetic acid, with or without osmic acid, imbed in paraffin, and stain in safranin and gentian violet. This will give a good view of the abundant starch and other food stuff, and at the same time will bring out sharply the cell walls of the embryo. Fig. 121. — Piiius banksiana: photomicrograph of young embryos teased out by the method described in the text; from a preparation by Dr. J. T. Buchholz; Cramer contrast plate; 16-mm. objective; no ocular or Abbe condenser; camera bellows, 75 cm.; safranin filter; arc light; exposure, 17 seconds. Negative by Miss Ethel Thomas. X54. GYMNOSPERMS— GNETALES Of the three peculiar genera belonging to this order only one. Ephedra, occurs in the United States. Welwitschia is found only in Damaraland, South Africa, and Gnetum is tropical and subtropical. 346 METHODS IN PLANT HISTOLOGY Gnetum thrives in the greenhouse, but the other two have not been cultivated successfully. The wood of Ephedra and Gnetum is extreme- ly hard, but can be cut by the hot steam method. The wood of Wel- witschia consists, principally, of a delicate parenchyma tissue, with numerous, large, rigid, spicular cells. Dr. Langdon cut beautiful sec- tions by treating with 50 per cent hydrofluoric acid for 3-6 weeks and imbedding in paraffin. There should be no haste. Allow 24 hours for each grade of alcohol and for each grade of the paraffin mixtures. Allow at least 24 hours for the xylol-paraffin mixtures, half of the time at room temperature and half, on the top of the bath. Put the mate- rial in an open dish in the bath. About 2 or 3 days will be required, and the paraffin should be changed 3 or 4 times. All of the Gnetales show vessels in the secondary wood, an angio- sperm character. The strobili can be fixed in formalin-acetic-alcohol, but in Ephedra the dry chaffy scales must be dissected away before completing the dehydration and infiltration with paraffin. If you can secure material of Ephedra, Dr. Land's researches present a very complete life-history, with dates of various stages and suggestions for fixing and staining. CHAPTER XXVII SPERMATOPHYTES ANGIOSPERMS This immense group demands knowledge and skill in the whole field of histological technique, for embryo sacs are so delicate that they are as difficult as the free nuclear stage in the female gametophyte of a gymnosperm, while the peach stone needs a petrotome rather than a microtome. Between these extremes there is everything imaginable in structure, chemical composition, and consistency. Some hints will be given, but the student will gradually learn what should be cut freehand and what should be imbedded, what stages in floral development, what stages in the development of the embryo sac, or what stages in spermatogenesis are likely to be correlated with easily recognized field characters ; and what fixing agents are likely to give the best results with various kinds of material. The vegetative structures. — In stems, roots, and leaves the more delicate structures should be imbedded in paraffin and the more rigid structures should be cut without imbedding at all; but it should be re- membered that the range of structures which can be imbedded in paraffin can be increased by the use of hydrofluoric acid and improve- ments in the paraffin method. The stem.— The vascular cylinder of the angiosperms is either an endarch siphonostele, or a polystele derived from it. For a study of the development of the stem, the common geranium {Pelargonium) may be recommended. Near the base of a fresh stem, about 1 cm. in di- ameter, cut freehand sections and fix them in formalin acetic alcohol for 24 hours. The sections may be left here for months. Transfer to 50 per cent alcohol and leave them here until all chlorophyll is ex- tracted. Probably 24 hours will be sufficient. Then stain in safranin and light green, or safranin and Delafield's haematoxylin. Such sec- tions will show both primary and secondary structures in both stele and cortex. Higher up, there will still be secondary structures in the stele, but none in the cortex; and still higher up will be found the origin of interfascicular cambium. All of these can be cut without im- bedding, but the earlier stages showing the differentiation of protoxy- 347 348 METHODS IN PLANT HISTOLOGY lem, metaxylem, and the origin of secondary xylem are too soft for successful freehand sections. Cut into pieces about 5 mm. long and fix in formalin acetic alcohol (10 c.c. formahn, 5 c.c. acetic acid, and 100 c.c. of 50 per cent alcohol). Imbed in paraffin. The vascular system can be traced very successfully by Gourley's basic fuchsin method. Take a small plant of Pelargonium {Geranium), Coleus, or Tropaeolum; use only 6 or 8 inches of the tip. Cut off the base — under the stain — and when the stain has reached the tips of the highest leaves, fix and clear. For a study of woody stems, Tilia americana (bass wood) is good, and shoots from 5-10 mm. in diameter are easy to cut. Very hard stems like Hicoria (hickory) and Quercus (oak) must be boiled and treated with hydrofluoric acid, if you expect to cut shoots more than 5-7 mm. in diameter. Tilia stems, up to 5 or 6 mm. in diameter, can be cut in paraffin. Fix for 3 or 4 days in formalin acetic alcohol; treat with 10 or 20 per cent hydrofluoric acid for 4 or 5 days, wash thorough- ly, and proceed as usual. About 24^8 hours in the bath should be sufficient. Harder stems, up to 2 cm. in diameter, should be fixed in the same way, treated with 20 per cent hydrofluoric acid, washed thoroughly, and put into equal parts of 95 per cent alcohol and glycerin, where they may remain indefinitely, but should remain at least a week. Cut by the hot steam method. Of course, veneer machines with very sharp knives cut large sections of the most refractory woods. While a random selection of stems would furnish material for prac- tice in technique we suggest that the stem of Clintonia shows a good siphonostele in a monocotyl; the rhizome of Acorus calamus is a good type for the amphivasal bundle and, although a monocotyl, still shows a differentiation into stele and cortex; Zea mays, universally used but not characteristic of monocotyls, shows scattered bundles, but not the amphivasal condition. Aloe, Dracaena, or Yucca will illustrate second- ary wood in monocotyls. Iris has a highly developed endodermis in the rhizome; and Nymphea or Nuphar will show scattered bundles in a dicotyl. Lenticels and tyloses are abundant and typical in Menispermum-, and very thin sections can be cut without imbedding; but both these structures are well developed while the stem can still be cut in paraffin without previous treatment in hydrofluoric acid. SPERMATOPHYTES— ANGIOSPERMS 349 The sieve tubes of the phloem are easily demonstrated in Cucurhita pepo, the common pumpkin; other members of the family furnish good material. Take pieces of stem about 1 cm. long and not too hard to cut in paraffin, fix in formalin-acetic-alcohol, and stain in safranin, gentian violet, orange. Beautiful sections of the sieve tubes and sieve plates can be obtained by cutting out, very carefully with a thin safety razor, a single vascular bundle with a little of the surrounding paren- chyma, and imbedding it in paraffin. The tropical Tetracera, one of the Dilleniaceae, has sieve plates so large that they are easily seen with a pocket lens. The phloem area is so large in the larger stems that it can be cut out, practically free from the xylem, and imbedded in paraffin. It was once thought that these large sieve tubes afforded an obvious illustration of the continuity of protoplasm; but, as a matter of fact, the actual protoplasmic connections are scanty and hard to demon- strate. Iron-alum haematoxyUn and orange will differentiate the strands if you are careful. Roots.— It has long been known that the root-tip furnishes con- stantly available material for a study of mitosis (Fig. 122). An onion thrown into a pan of water will soon send out numerous roots. Soak beans in water for several hours and then plant them in loose, moist sawdust. In a greenhouse, with "bottom heat," the primary root will be long enough in 2 or 3 days. The large, flat beans, especially Vicia faba, are very favorable. The root-tips of various species of Trillium, Tradescant'ia virginica, Podophyllum peltatum, Arisaerna triphyllum, and Cypripedium puhescens may be mentioned as known to be favor- able; but it is very possible that the best root-tip has not yet been tried. Cell division does not proceed with equal rapidity at all hours of the day. Kellicott has shown that in the root-tips of Alliu77i there are in each 24 hours two periods at which cell division is at the maximum, and two at which it is at the minimum. The maximum periods are shortly before midnight (11:00 p.m.), and shortly after noon (1:00 P.M.). The minima, when cell division is at the lowest ebb, occur about 7:00 A.M. and 3:00 p.m. When cell division is most vigorous, there is little elongation, and when cell division is at the minimum, cell elonga- tion is at the maximum. Consequently, root-tips of Allium should be collected about 1 :00 p.m. or 11 :00 p.m. Lutman, later, made observa- tions upon periodicity of mitosis in the desmid, Closterium; and in 350 METHODS IN PLANT HISTOLOGY 1915, Karsten made a comparatively extended study of periodicity in various stems and roots, together with notes on algae. It is safe to say that the maximum number of mitoses in root-tips will be found shortly after noon (1 :00 p.m.) and shortly before mid- night (11 :00 P.M.). It is certain, however, that abundant mitoses may be found at other times — even at 3:00 p.m. — in sporangia of ferns, in B D Fig. 122. — Nuclear and cell division in root-tip of onion. .4, resting nucleus; B, spirem stage; C, spirem divided into chromosomes; D, metaphase, with chromosomes split; E, late anaphase; F, early telopha.se; G, late telophase; H, resting nucleus. X 1,200. From Chamberlain's Elements of Plant Science (McGraw-Hill Book Co., New York). anthers of angiosperms, in endosperm, and in free nuclear stages of the embryo of gymnosperms. Mitotic figures play such an important part in the development of the plant and in modern theories of heredity that it is worth while to acquire a critical technique in fixing and staining these structures (Fig. 122). During the past two years we have made an extensive series of SPERMATOPHYTES— ANGIOSPERMS 351 experiments with root-tips of Trillium sessile, obligingly furnished by my friend, Dr. R. C. Spangler, who fixed the tips, shortly after noon, in chromo-acetic-osmic solutions. With a stock solution of chromic acid 1 g., and acetic acid, 1 c.c, osmic acid was added in various pro- portions—! c.c. up to 10 c.c. to 100 c.c. of the stock solution. Fixing was excellent in all; but staining was best with 6, 7, or 8 c.c. of osmic to 100 c.c. of the solution. A long series of trials on onion root-tips indicated that, with the acetic acid raised to 2 c.c, the results were better. We recommend chromic acid 1 g., acetic acid 2 c.c, 1 per cent osmic acid 6-8 c.c, and water 90 c.c For convenience, this may be called the Chicago formula. Try the various fixing agents. Remember that no matter how good the fixing may be, material can be rumed in dehydrating, in clearing, or in the bath. Make yourself master of Haidenhain's iron-alum haematoxylin ; then add the safranin, gentian violet, orange combination; then safranin and anilin blue; and then experiment for yourself, but remember that the triumphs of modern cytology have been won with iron-haematoxyhn and that you cannot read intelligently the literature of the past twenty-five years until you have gained at least an approximate mastery of this stain. Of course, dehydration, clearing, and infiltration must be very gradual. The schedule by Yamanouchi on page 47 will repay careful study. When chromo-acetic-osmic solutions have been used for fixing, there is a relation between the time needed in the second iron-alum and the amount of 1 per cent osmic acid. If the time required for differentiation in 2 per cent iron alum is less than 30 minutes, the per- centage of osmic acid was too low; if more than 2 hours, it was too high. About 1 hour in 2 per cent iron-alum, followed by 20-30 minutes in 1 per cent (or weaker) is about right. It is somewhat like an expo- sure in photography, except that an overexposure develops too fast, while an overfixing in osmic acid comes out too slowly. If the iron- alum haematoxylin is preceded by an overnight stain in safranin, and the safranin is drawn until it has almost disappeared from the chromo- somes, the figures will look as if stained only in iron-alum haematoxy- lin, but the nucleoli will show the red and the cytoplasm will have a slight tinge of pink. In staining with safranin, gentian violet, orange, allow the alcoholic safranin to act for 16-24 hours; then extract it with 50 per cent alcohol, slightly acidulated with hydrochloric acid, if necessary, until the stain has almost disappeared from the spindle; then pass through 70, 85, 95, 352 METHODS IN PLANT HISTOLOGY and 100 per cent alcohol; stain in gentian violet dissolved in clove oil, or in a mixture of clove oil and absolute alcohol, for 5-20 minutes; fol- low with orange dissolved in clove oil, remembering that this will weak- en the safranin and sometimes the gentian violet ; finally use pure clove oil to differentiate the gentian violet. Leave the slide in xylol for 2-5 minutes to remove the clove oil and to hasten the hardening of the balsam; If you use aqueous gentian violet or crystal violet, use the anilin oil formula. When the safranin is satisfactory, transfer to water and then to the violet. After staining in violet, dip in water to remove the ex- cess of stain, and then dehydrate rapidly in 95 per cent and absolute alcohol, differentiate in clove oil, and then transfer to xylol. The structure and development of the young root will be shown, to some extent, in preparations made for mitotic figures. The origin of dermatogen, periblem, plerome, and also of protoxylem, is well shown in Zea mays. An ear of sweet corn, as young and tender as you can find on the market, will furnish material. Cut out carefully pieces 2 or 3 cm. long, with two rows of grains and fix in formalin acetic alcohol. When needed for paraffin sections, cut out from the grain a rectangu- lar piece about 2X3 mm. and 4 or 5 mm. long; if you want to show also the structure of the entire grain, take a section the entire length of the grain, perpendicular to the flat side of the grain, and about 2 mm. wide. Cut the latter longitudinally; the rectangular pieces are sufficient for transverse sections. This is better than to try to cut out the grains before fixing. The roots of various cereals will repay study. The roots of Ranunculus repens and Sambucus nigra furnish good illustrations of the radial arrangement of xylem and phloem. Smilax shows the radial arrangement, with a large number of poles and a very highly differentiated endodermis. Cut it in paraffin. The mature root will need 2 or 3 days' treatment with 10 or 20 per cent hydrofluoric acid. The origin of secondary xylem and phloem is well shown in Sambucus 7iigra. Vicia faba is good for the origin of secondary roots. Pistia stratiotes, sometimes found on lily ponds in greenhouses, and common in Cuba and Southern Mexico, is unexcelled for showing the origin of secondary roots. On account of the hard root cap, it needs 48 hours in 5 or 10 per cent hydrofluoric acid. The arrangement of cells in the young roots of aquatic or semi-aquatic plants often shows a geometric regularity (Fig. 123). SPERMATOPHYTES— ANGIOSPERMS 353 The leaf. — Young and tender leaves should be fixed in formalin alcohol and out in paraffin. Cut sections freehand whenever there is sufficient rigidity. Resort to pith only when necessary. In cutting sec- tions of a leaf like that of Lilium, lay one leaf on another until you have a bundle of them which will be nearly square in transverse sec- tion. Wrap the bundle with string for about 15 mm.; cut the bundle transversely so that about 5 mm. of the bundle will project beyond the Fig. 123. — Sparganium eurycarpum: photomicrograph of transverse section of young root; fixed in chromo-acetic acid and stained in Bismarck brown; Cramer contrast plate; 16-mm. objec- tive; ocular X4; no Abbe condenser; yellowish-green filter; camera bellows, 1 meter; exposure, S seconds. X90. tied portion. Dip in melted paraffin, as already suggested for Pinus, fasten the tied portion in the shding microtome, and cut with the knife placed obliquely. About 15-20 n'lSSi good thickness for general leaf structure. In case of large leaves, cut out pieces 1 cm. wide and 3 cm. long and tie them together to make a good bundle for cutting. For the finest preparations, imbed in paraffin. The common Hlac, Syringa, has a good leaf to illustrate palisade and spongy parenchy- ma : the privet, Ligustrum, is even better. Ligustrum japonicum has 354 METHODS IN PLANT HISTOLOGY SO few cells between the upper and lower epidermis that it is a good type for students to draw. Buds will furnish beautiful preparations of young leaves and, at the same time, will show the vernation. Cut the bud transversely, a Httle above the middle; remove the bud scales, if they promise to cause trouble; retain only enough tissue at the base of the bud to hold the parts in place. Fix in formalin acetic alcohol; imbed in paraffin; and stain in safranin and light green. Epidermis, stripped from the leaf, fixed in 10 per cent formaUn in water for a day or two, and then stained in safranin and light green, will give excellent views of stomata. The development of stomata is particularly well shown in Sedum purpurascens, even in leaves which have reached the adult size. The epidermis is very easily stripped from a leaf of Sedum. If the big Sedum maximum is available, pieces of epidermis 6 or 7 cm. long and 2 or 3 cm. wide are easily stripped off, almost free from any underlying tissue. The epidermis of Lilium and Tradescantia shows fine, large stomata, but it is not so easy to strip off. In these two genera the stomata, as in nearly all leaves, show only the adult structure. Floral development. — For a study of floral development very young buds are necessary, and it is best to select those forms which have rather dense clusters of flowers, in order that a complete series may be obtained with as httle trouble as possible. The usual order of appearance of floral parts is (1) calyx, (2) corolla, (3) stamens, and (4) carpels; but if any of these organs is reduced or metamorphosed, their order of appearance may be affected. Floral development is easily studied in the common Capsella hursa- pastoris (Fig. 124). The best time to collect material is late in March or early in April. Dig up the plant, carefully remove the leaves, and in the center of the rosette a tiny white axis will be found. A series of these axes from 3 to 9 mm. in length, and from 1.5 to 3.5 mm. in di- ameter will give a very complete series of stages in the development of the floral organs. Preparations from the apex of the shoot taken after the inflorescence appears above ground are not to be compared with those taken early in the season, because the pedicels begin to diverge so early that median longitudinal sections of the flowers are com- paratively rare. Fix in chromo-acetic acid and stain in Delafield's haematoxylin. The sections should be longitudinal and about 5 m thick. Capsella shows the hypogynous type of development. The or- SPERMATOPHYTES— ANGIOSPERMS 355 der of appearance of floral parts is (1) calyx, (2) stamens, (3) carpels, and (4) petals. The ovary is compound (syncarpous). With its 6 stamens (2 of them shorter than the other 4), 2 carpels, and 4 nectaries marking the place of the missing parts, Capsella shows an interesting transition from the pentacyclic to the tetracyclic type of flower. Transverse sections of single flowers, just before the small petals expand, are best for this study. Ranunculus, which is also hypogynous, will illustrate the develop- ment of the simple (apocarpous) ovary. The ovules appear quite early, so that the archesporial cell, or even the megaspores, may be Sepal Microsporophyf/ ^ (Stamen) ■Wiegasporophyl! (Carpel) '-Petal 'Microsporophyll (Stamen) Fig. 124. — Capsella bursa-pastoris: floral development. X85. From Chamberlain's Elements of Plant Science (McGraw-Hill Book Co., New York). seen while the carpel is still as open as in any gymnosperm. The whole structure is a simple strobilus (Fig. 125). Rumex crispus (yellow dock) is also a good hypogynous type, and the densely clustered flowers afford a fine series of stages. Besides, in transverse sections, the early stages in spermatogenesis are very clear. In the willows, Salix, the bud scales must be removed and the copi- ous hairs should be trimmed off as much as possible with scissors, after which the catkin should be slabbed a little on opposite sides to facilitate penetration. This is a fine illustration of a compound stro- bilus. The cat-tail, Typha, presents a simple type of floral development. The leaves should be dissected away long before the flowers can be seen from the outside. The cylindrical clusters, varying in diameter from 2 or 3 mm. up to the size of one's finger, will afford a complete series of stages. Until the spike reaches the diameter of a lead pencil, 356 METHODS IN PLANT HISTOLOGY transverse sections are easily cut. For later stages, the outer part of the spike should be sliced off so that only enough spike is retained to hold the florets in place. Prunus and many other members of the Rosaceae furnish examples of the perigynous type of development. In many of them the floral parts do not occur in the usual succession. Sepal-\ Bracf- --Pefal ■-"Sepal Megasporophylls ' (Carpels) ^JVIicrosporopliylls ■'' fSlamens) -Petal •Sepal '—Brvicf jasporophyll XCarpel) Megasporangium (Ovule) Microsporophyll (Stamen) Microsporangiiim \-Petal Sepal Petal Sepal'' Fig. 125. — Ranunculus acris. floral development. Plant Science (JMcGraw-Hill Book Co., New York). X70. From Chamberlain's Elements of The epigynous type is well shown in the Compositae. The order of appearance is (1) corolla, (2) stamens, (3) carpels, and (4) calyx (pappus). The common dandelion, Taraxacum officinale, affords an excellent series with little labor. Examine vigorous plants which have, as yet, no flowers or buds in sight. Dig up the plant and dissect away the leaves. If there is a white cluster of flower buds, the largest not more SPERMATOPHYTES— ANGIOSPERMS 357 than 4 mm. in diameter, cut out the cluster, leaving only enough tissue at the base to hold the buds in place. Larger heads should be cut separately. Our most common thistle, Cirsimn lanceolatum, shows the floral de- velopment with unusual clearness, but the preparation of the material is somewhat tedious. The involucre, which is too hard to cut, must be carefully dissected away. Retain only enough of the receptacle to hold the developing florets in place. A series of sizes with disks vary- ing from 3 to 10 mm. in diameter will show the development from the undifferentiated papilla up to the appearance of the archesporial cell in the nucellus of the ovule. The Canada thistle, Cirshwi arvense, is equally good, but it is more difficult to dissect out the desirable parts. In the common sunflower, Helianthus annuus, the young floral parts, like the mature head, are so very large that a satisfactory study may be made with a low-power objective. As in the case of the thistle, the involucre must be trimmed away and only enough of the receptacle retained to hold the florets together. Erigeron philadelphicus furnishes a beautiful example of epigynous floral development, and the heads are so densely clustered that, in a single section, one may find various stages from heads with undiffer- entiated disk up to heads with florets showing pappus, corolla, sta- mens, and carpels (Fig. 126). In the Chicago region, the last two weeks in May are best for these stages. Spermatogenesis. — The earlier stages in spermatogenesis will be found in the preparations of floral development. The origin of the archesporium, the origin of sporogenous tissue, and the formation of the tapetum are beautifully shown in longitudinal and in transverse sections of the anthers of Taraxacum and many other Compositae. Transverse sections of the head of Taraxacum, or any similar head at the time when pollen mother-cells are rounding off in the center of the head, will show various stages from the mother-cells in the center to the tetrads of spores at the periphery. Transverse sections of the anther of Polygala give exceptionally well-defined views of the arche- sporial cells and sporogenous areas. Lilium, Trillium, Galtonia, Iris, Tradescanlia, Vicia, and Podo- phijllum can be recommended for demonstrating the nuclear changes involved in the formation of spores from the mother-cell (Fig. 127). Several species of Lilium are common in greenhouses, and these may be used where wild material is not available. In early stages, where 358 METHODS IN PLANT HISTOLOGY the sporogenous cells have not yet begun to round off into spore mother-cells, it is sufficient to remove the perianth, retaining just enough of the receptacle to hold the stamens in place. Transverse sec- tions show the six stamens and also the young ovary. After the spore mother-cells have begun to round off, each stamen should be removed so as to be cut separately. In securing the desirable stages showing the division of the mother-cell into microspores, much time and patience will be saved by determining the stage of development before fixing Bract Corolla •Corolla Corolla Stamen - Bract ^;; Youn^ flowers Corolla ,'Stamen (Microsporophyll) ^^ Carpel '(Megaspomphyll) Calyx (Pappus) Fig. 126. — Erigeron philaddpliicus: floral development, .-l and B X3.5; C, D, E, and F X 150. From Chamberlain's Elements of Plant Science (McGraw-Hill Book Co., New York). the material. Mitosis is more or less simultaneous throughout an an- ther. Long anthers are particularly favorable, since they may show a very closely graded series of the various phases of mitosis. An anther of Lilium may show mother-cells with nuclei in synapsis at the top, while the mother-cells at the bottom have reached the equatorial plate stage of the first division; or, the mother-cells at the top may show the first division, while those at the bottom show the second. Deter- mine the stage by examining a few mother-cells before fixing. From what has been said, it is evident that longitudinal sections should be cut to show mitosis. Transverse sections should be cut to show the general structure of the anther. It is not necessary to cut the SPERMATOPHYTES— ANGIOSPERMS 359 stamens into pieces before fixing, since they are easily penetrated and infiltrated; in later stages the stamens 77iust not be cut into pieces, since the pollen grains and even the pollen mother-cells are easily washed out. The problem of fixing spore mother-cells has received much atten- tion. In fixing mother-cells and the two mitoses by which spores are Fig. 127. — Lilium marlagon: reduction of chromosomes in the microspore mother-cells; A, bird's-eye view of metaphase showing 12 pairs of chromosomes; B, one chromosome of each pair is starting for one pole and the other for the opposite pole; C, anaphase, each of the 12 chromosomes starting for the pole in B is seen to be made up of 2 chromosomes, so that 24 chromosomes are going to each pole; D, each nucleus of this two-cell stage contains 24 chromosomes. E, the 24 chromosomes of each nucleus of D are being distributed, 12 to each pole, so that each nucleus of F contains 12 chromosomes; the 4 cells of F are young microspores. X530. From Chamberlain's Elements of Plant Science (McGraw-Hill Book Co., New York). formed, investigators have used almost exclusively the chromo-osmo- acetic acid solutions of Flemming, some preferring the weaker solution and some the stronger. These solutions were used in nearly all of the work done under Strasburger at the Bonn (Germany) school. 360 METHODS IN PLANT HISTOLOGY Professor Gregoire and his students have made this their principal fixing agent. In spite of the weight of authority, we beheve that the Chicago chromo-acetic-osmic formula is better. In the Flemming's weaker for- mula we believe the chromic acid is too weak; the acetic acid, with its tendency to cause swelling, is too strong ; and the osmic acid, unneces- sarily strong. The osmic acid undoubtedly accelerates the killing of the proto- plasm. This is seen more readily in animals. If Cyclops be brought into 30 c.c. of the solution A, the animals will swim for a while; if 5 or 6 drops of 1 per cent osmic acid be added to the solution, the animals cease their movements almost instantly. Doubtless the osmic acid has the same effect upon plant protoplasm. Where fixing is slow, very few mitotic figures are found with the chromosomes midway between the equator and the poles. Farmer and Shove, in studying these mitoses .and also vegetative mitoses in Tradescantia, claimed better results with a mixture of 2 parts of absolute alcohol and 1 part glacial acetic acid. They allowed the fixing agent to act 15-20 minutes, then washed in absolute alcohol, and imbedded by the usual methods. This proportion of acetic acid seems entirely too large for any accurate work with chromatin, and we doubt whether the structure of the cytoplasm is normal when so much acetic acid is used. The entire pollen mother-cell may be stained and mounted without sectioning. Two descriptions of technique appeared in 1912, one by Mann and the other by Pickett. Mann removes the pollen mother- cells before fixing and staining; Pickett fixes and stains the anther in toto and teases out the pollen mother-cells just before mounting. In Mann's method, the anther is placed in a drop of water and the tip is cut off; a gentle tapping with a needle will then cause the pollen mother-cells to float out into the drop. Fix in Bouin's fluid, 4-8 hours, wash in 50 per cent alcohol until no color remains, and then stain in iron-haematoxylin. At this stage we should put the material into 10 per cent glycerin and follow the Venetian turpentine method. Pickett fixed entire anthers in chromo-acetic acid for 30 hours, washed in water for 24 hours, and then passed up to 80 per cent alco- hol. At this point, he stained in strong cochineal or Kleinenberg's haematoxylin for 5 days, then completed the dehydration, cleared in cedar oil, teased out the mother-cells, and mounted in balsam. SPERMATOPHYTES— ANGIOSPERMS 361 The Belling method, as modified by Dr. McClintock, is more rapid and gives good permanent preparations. The pollen grain at the time of shedding generally consists of two cells, the tube cell and the generative cell, which afterward divides and forms two male cells or two male nuclei. Lilium and Erythronium furnish good illustrations of pol- len shed in the two-cell stage (Fig. 128). In Silphium, Sambu- cus, and Sagittaria the genera- tive nucleus divides before the pollen is shed. The division of the generative cell to form the two sperms takes place just be- fore fertilization; consequently, in forms like Silphium, fertiliza- tion is Hkely to occur within less than 72 hours after the division of the generative cell. Sections should not be more than 5 m thick, if they are to show a clear differentiation of exine, intine, starch, and other structures. If sections have been stained in iron-haematoxyhn, staining in safranin for from 3 to 7 minutes will give the exine a bright red color and will not obscure the haematoxylin, A rather sharp stain in gentian violet will stain the starch and also the intine. In Asclepias and many orchids, in which a common exine surrounds the entire mass of pollen grains, care must be taken not to overstain. In many cases the pollen grains will put out their tubes in a 2-5 per cent solution of cane-sugar in water. Where the interval between pol- lination and fertilization is known (about 72 hours in Lilium phila- delphicum and 96-100 hours in L. canadense) , pieces of the stigma and style showing pollen tubes can be selected with some certainty. Fig. 128. — Erythronium americanum: photo- micrograph of mature pollen grains; the one at the top, which is cut longitudinally, shows both the tube nucleus and the conspicuous generative cell; the other is cut transversely and shows the gen- erative cell, but not the tube nucleus. Stained in safranin and gentian violet; from a preparation by Dr. Lula Pace. Cramer contrast plate; 4-mm. ob- jective; ocular X4; yellowish -green filter; bellows, 85 cm.; exposure, 3 minutes. X615. 362 METHODS IN PLANT HISTOLOGY Oogenesis. — As in spermatogenesis, the early stages will be found in preparations of floral development. The preparations of Capsella will show the origin and development of the nucelliis (megasporan- gium) and also the megaspore mother-cell. The division of the mega- spore mother-cell to form four megaspores takes place shortly before the bud begins to unfold. A massive megasporangium with several ; , y •/ "^f.^'^A# ■^-^n"-^"* ' fT-"¥ I^W *^'? '4^1^ .i-L'.. :^^-^ *^' .*S v^t' /^ -< Fig. 129. — Lilium canadense: photomicrograph of part of transverse section of ovary, showing longitudinal section of ovule with megaspore mother-cell, just before the formation of integuments. Fixed in chromo-acetio acid and stained in iron-alum haematoxylin. Eastman Commercial Ortho film, Wratten H filter (blue); Spencer 4-mm. objective, N.A. 0.66; ocular X6; arc light; exposure, 2 seconds. Negative by Dr. P. J. Sedgwick. X400. megaspore mother-cells may be found in Ranunculus; a megasporan- gium with only one megaspore mother-cell and only one layer of cells surrounding it may be found in any of the Compositae. Erigeron philadelphicus is very good. Head from 4-6 mm. in diameter will show the stages from the megaspore mother-cell to the four megaspores. E. strigosus and E. annuus are equally good. In Trillium and in Cypri- pedium the embryo sac is formed from two megaspores, which are not SPERMATOPHYTES— ANGIOSPERMS 363 separated by walls. In Lilium, Tulipa, Fritillaria, Erythronium, and many others, the embryo sac is formed by all four megaspores, which are not separated by walls. In Peperomia, the Peneaceae, and some species of Ewphorhia, the sac is formed by the four megaspores, not separated by walls, and the sac has 16 free nuclei. In Plumbagella the four megaspores, not separated by walls, constitute the mature sac. '■l*. V . ?^>; Fig. 130. — Lilium philadelphicum: photomicrograph of transverse section of ovary showing, in one of the ovules on the left, the first mitosis in the megaspore mother-cell; and, in one of the ovules on the right, the second mitosis which gives rise to the four megaspore nuclei. Chromo-acetic acid; safranin, gentian violet, orange. Cramer contrast plate; 16-mm. objective; ocular X4; yellowish- green filter and also a strong filter such as is used in outdoor work; camera bellows, 30 cm. ; exposure, 2 minutes. Negative by Miss Ethel Thomas. X64. one of the megaspores functioning as the egg, two more fusing to form the endosperm nucleus, while the fourth megaspore aborts; so that the embryo sac, ready for fertilization, contains only two nuclei. The reduction of chromosomes takes place during the two mitoses by which the mother-cell gives rise to four megaspores. The figures are much larger than in the corresponding mitoses in spermatogenesis but so much more tedious to secure that most studies in reduction have been based upon divisions in the pollen mother-cell. Lilium is quite favorable for a study of oogenesis, but it must be remembered that it is exceptional in having an embryo sac formed from four megaspores. 364 METHODS IN PLANT HISTOLOGY In very young stages, before the appearance of the integument, the ovary may be removed from the flower and placed directly in the fixing agent, but at fertilization and later stages, strips should be cut off from the sides of the ovary in order to secure more rapid fixing and more Fig. 131. — Lilium philadelphicum: photomicrograph of second mitosis in megaspore mother- cell. Chromo-acetic acid; safranin, gentian violet, orange. Cramer contrast plate; 4-mm. objective; ocular X4; Abbe condenser; camera bellows, 1 m. ; yellowish-green filter and also a strong filter such as is used in outdoor work; exposure, 7 minutes. Negative by Miss Ethel Thomas. X626. perfect infiltration with paraffin. The dotted lines in Figure 132 C show about how much should be cut off. This is a much better plan than to secure rapid fixing and infiltration by cutting the ovary into short pieces, because the ovules will be in about the same stage of de- velopment throughout the ovary, and when one finds desirable stages like those from which these photomicrographs were taken, it is gratify- ing to have these pieces as long as possible. SPERMATOPHYTES— ANGIOSPERMS 365 I |iSi^:4?S-3^ B The Chicago chromo-acetic-osmic formula is good for the entire series. Iron-alum haematoxylin, with a light touch of orange in clove oil, is best for the chromatin. For general beauty and for the achro- matic structures, the safranin, gentian violet, orange combination has not been excelled. The photomicrographs (Figs. 129-131) illustrating the series from the archespo- rial cell (which, in this case, is also the primary sporoge- nous cell and the megaspore mother-cell) to the four meg- aspore nuclei will repay a careful study. One more mi- tosis produces the 8-nucleate embryo sac, but Lilium is not a good type for illustra- tive purposes, since the egg apparatus is not very defi- nitely organized. For the embryo sac at the fertilization stage, many of the Compositae are good. Senecio aureus is qujte favor- able, because it is easy to cut and the akenes do not spread. Aster gives an ex- ceptional view of the antip- odal region, but is rather hard to cut. Before fixing, trim the head as indicated in Figure 132. Silphium, especially S. ladniatum, furnishes an ideal view of the embryo sac. With thumbs and fingers grasp the two wings of the akene and carefully split it, exposing the single white ovule inside. This is rather tedious, but every ovule will yield a perfectly median longitudinal section of the embryo sac, and there is not the slightest difficulty in cutting. When the rays look their best, the embryo sac is ready for fertihzation, or the pollen tubes may be entering; as the rays begin to wither, you will find fertilization or early stages in the embryo and endosperm. Sections should be about 10 /i thick. Fig. 132. — A, head of Aster; B, pod of Capsella; C, transverse section of ovary of Lilium. The dotted lines show how the material should be trimmed before fixing. 366 METHODS IN PLANT HISTOLOGY The Ranunculaceae, especially Anemone patens var. wolfgangiana, show a rather large, broad embryo sac, with highly organized egg apparatus and antipodals. Sections should be 10-20 fx thick. For general views of the embryo sac, the safranin, gentian violet, orange combination is recommended. Fig. 133. — Plumbagella micrantha: longitudinal section of ovule showing the embryo sac, with the egg and endosperm nucleus ready for fertilization. Stained in iron-alum haematoxylin. Eastman Commercial Ortho film, Wratten E filter (orange); Bausch and Lomb 8-mm. objective, N.A. 0.50; Spencer ocular X6; arc light; exposure, 1 second. Preparation by Dr. K. von O. Dahlgren and nega- tive by Dr. P. J. Sedgwick. X208. The peculiar embryo sac of Plumbagella, with only two nuclei — the egg nucleus and the endosperm nucleus — when ready for fertilization, is shown in Figure 133. Fertilization. — The later stages cut to show the mature embryo sac will often show fertilization. The male and female nuclei almost in- SPERMATOPHYTES— ANGIOSPERMS 367 variably show a difference in staining capacity when the male nuclei are just discharged from the pollen tube. With cyanin and erythrosin, the male nucleus stains blue and the female, red; hence the obsolete terms "cyanophilous" and "erythrophilous." With safranin, gentian violet, orange, the egg nucleus stains more with safranin and the male with gentian violet; but as the two nuclei come into contact within the egg, they begin to stain alike, the male nucleus staining more and more like the fe- male. As fertilization pro- gresses, it becomes difficult or, at present, impossible to distinguish any difference be- tween the two nuclei. The male nucleus which takes part in the "triple fusion" to form the endosperm nucle- us behaves in the same way. Lilium is a very good and always available type for illus- trating fertiHzation (Fig. 134). Take ovaries from flowers whose petals have withered but have not yet fallen off. Though their embryo sacs and nuclei are smaller, Sil- phium and Helianthus are good types, because their curved or twisted male nuclei are easily distinguished from the spherical nuclei in the embryo sac. The embryo sacs of orchids are very small, but ovules are extremely numerous and the chances for securing the fusion of nuclei are correspondingly good. In Cijpripedi- um the nuclei do not fuse in the resting condition, but the chromo- somes of the two parents are perfectly distinct in the egg. The general statement that nuclei fuse in the "resting condition" is not correct, and probably the chromosomes of the two gametes never fuse. The endosperm. — Some of the preparations intended for fertiliza- tion will be likely to show early stages in the development of endo- sperm. Fig. 134. — Lilium philadelphicum: photomicro- graph of section showing fertiUzation and also the triple fusion; from a preparation and negative by Dr. W.J. G. Land. Xo85. 368 METHODS IN PLANT HISTOLOGY ^.-Endosperm -Embryo In rather long, narrow embryo sacs, a cell wall is likely to follow even the first division of the endosperm nucleus, so that the endosperm is cellular from the beginning. Ceratophyllum, Monotropa, and Ver- bena will furnish material of this type. In large, broad embryo sacs, the formation of en- dosperm is almost sure to be initiated by a series of simultaneous free nuclear divisions. This is a difficult condition to fix well, since the layer of protoplasm, with its free nuclei, sur- rounding a big vacuole, is likely to shrink away from the surrounding cells. As the free nuclear period comes to a close, walls appear at the periphery, and wall formation gradu- ally advances toward the center until the entire sac isfilled with tissue. Lilium, Capsella, and Ranunculus furnish examples of this type (Fig. 135). An intermediate condi- tion is seen in somewhat elongated embryo sacs of medium size, like those of Compositae. After a few free nuclear divisions, walls appear simultaneously throughout the entire sac. Silphium laciniatum is particularly good. Akenes from which the corolla has just fallen will furnish material. The embryo. — In most angiosperms the endosperm divides earher than the fertiUzed egg and in some cases, Uke Asclepias and Casuari- na, the free nuclear stage of the endosperm is completed and the cellu- —Micropyte Stalk (Funiculus) Fig. 135. — Capsella bursa-pastoris: ovule (megasporan- gium) with embryo and endosperm, the embryo wath plerome, periblem, and dermatogen differentiated. Cell walls are appearing in the endosperm. X60. From Cham- berlain's Elements of Plant Science (McGraw-Hill Book Co., New York). SPERMATOPHYTES— ANGIOSPERMS 369 lar stage is well advanced before the first division of the egg. In some forms, like the aroids, the embryo is massive, and differentiation into dermatogen, periblem, and plerome comes comparatively late; while in others, like the Cruciferae, the differentiation occurs very early. Cap- sella is a standard example of the latter type (Fig. 136). The stages shown in Figure 136 A-F will be found in pods about 3 mm. in length. These may be put directly into the fixing agent, but later stages, which are found in pods 5 mm. or more in length, should be trimmed, as indicated in Figure 132 B, before fixing. Formalin-alcohol-acetic acid is a good fixing agent; the Chicago chromo-acetic-osmic acid /0\ B D Fig. 136. — Capsclla bursa-pastoris: development of embryo. In D, dermatogen is shaded; in E, both dermatogen and the plerome of the root are shaded, and the periblem of the root is com- pleted; in F, dermatogen of root is completed. X520. From Chamberlain's Elements of Plant Science (McGraw-Hill Book Co., New York). with 3 c.c. of acetic acid instead of 2 c.c, is also very good, and Dela- field's haematoxylin stains better after the chromic series. Cut 5-10 n thick and parallel to the flat face of the pod. For a study of the monocotyl embryo. Iris, and especially I. pseiida- conis, can be recommended. The embryo is straight, and cotyledon, stem-tip, and root are clearly differentiated before the endosperm be- comes too hard to cut in paraffin. Fix pieces about 3 mm. wide cut perpendicular to the face of the cheese-shaped seed. Do not try to cut the whole pod. Sagittaria has been used quite extensively. It is easily obtained, the whole head can be cut with ease, even after the cotyledon and stem-tip are clearly differentiated, and the endosperm is instructive; but, in later stages, the embryo is curved, like that of Capsella, so that good views of the stem-tip are rare. 370 METHODS IN PLANT HISTOLOGY Zea mays, especially the sweet corn, is a good type to illustrate the peculiar embryo of the grasses. Directions have been given on page 352. In many forms good preparations of late stages may be secured by soaking the seeds in water until the embryo bursts the seed coat. Young seedlings furnish valuable material for a study of vascular anatomy. Parthenogenesis. — Many embryos are developed without fertiliza- tion. Taraxacum, the common dandelion, is an omnipresent example. Other widely distributed illustrations are Hieracium and practically all species of the Eualchemilla section of the genus Alchemilla. Par- thenogenetic forms show various irregularities in the mitoses leading up to the formation of the egg. CHAPTER XXVIII USING THE MICROSCOPE To use any instrument effectively, one should know something about its structure. The optical principles of the microscope are pre- sented in any textbook of physics. Excellent practical hints are given in two booklets published by the leading American optical companies. These booklets tell the beginner how to set up the microscope, how to keep it in order, and give directions concerning illumination, dry and immersion objectives, mirror, condenser, diaphragm, and various other things (Fig. 137). They were written for advertising purposes, but since they advertise by giving directions for securing the best re- sults with the microscope, the information is very practical. The Spencer Lens Company, Buffalo, New York; and the Bausch and Lomb Optical Company, Rochester, New York, furnish these booklets free of charge. A cheap microscope with a 16 mm. objective and one ocular can be used for examining preparations while they are wet with alcohols, oils, or other reagents. If it is necessary to use a better instrument for such work, cover the stage with a piece of glass — a lantern slide is of about the right size — and be extremely careful not to get reagents upon the brass portions. MICROMETRY One should learn to make some estimate of the size of a microscopic object just as one can make an estimate of the size of larger objects; but, in addition, everyone who uses a microscope should become able to determine just how much it magnifies and should learn how to measure microscopic objects. In any measurement one should note the tube length, which is usually 160 mm. Some companies still make the tube so short that it must be pulled out to reach the desired length of 160 mm., even when the nosepiece is in place. Where there is no nosepiece, the draw tube is simply pulled out until the length is 160 mm. (Fig. 138). Where a nosepiece is used, its height should be measured, and the draw tube should be pushed in a distance equal to the height of the nosepiece. There are in general use two practical methods of measuring micro- 371 372 METHODS IN PLANT HISTOLOGY scopic objects; one by means of the ocular micrometer; and the other, by means of camera lucida sketches. Measuring with the ocular micrometer. — A stage micrometer and an ocular micrometer are necessary. A stage micrometer should be ruled in tenths and one-hundredths of a millimeter. It does not matter SECULARS iNctmES SiNOCULftR BODY* NOSE PIECE OBJECTIVES R/\CK K PIKION COARSE /ADJUSTMENT BUTTONS INTERMEriflTE SLIDE HORSE SHO BASE Slide crrriers of MECHflNICHl. STAGE SCREW FOU CENTERINq STAGE ^^^-^ o3li(?ue light" P|/\PHRA oj' the Oculatf" Diaphragm! ^^•p— Field lens ' .— -DrawTube .--Body Tube objective, a Leitz 3. In the oc- ular micrometer, ninety-eight spaces covered just fifteen of the larger spaces of the stage micrometer. Since the stage micrometer is ruled in tenths and one-hundredths of a milli- meter, the fifteen spaces equal 1.5 mm., or 1,500 ju-^ Then ninety-eight spaces of the ocu- lar micrometer equal 1,500 fx; and one space in the ocular equals -g'g of 1,500 m, or 15.3 m- This value being determined, there is no further use for the stage micrometer. To measure the diameter of a pollen grain put the preparation on the stage, using the same objective and ocular micrometer, and note how many spaces a pollen grain covers. If the pollen grain covers five spaces, its diameter is five times 15.3 ix, or 76.5 n. In the same way, the value of a space in the ocular when used with the other objectives should be determined. The values for three or four objec- tives may be written upon an ordinary slide label and pasted upon the base of the microscope for convenient reference. This method is the best one for measuring spores and for most measurements in taxonomy. iQne millimeter= 1,000 At. The Greek letter ^ is an abbreviation for niKpov, or micron. _D raw Tube Diafi'ira grfl wilh Society Screw .SocUly Sera* ..--•Mount Baoxlens v^, ^^e ■front lens \ObjecLlVe ..WorkinqDistancej Slid&i Objact Fig. 138.— Tube length 374 METHODS IN PLANT HISTOLOGY Measuring by means of camera lucida sketches. — This method is of great importance in research work, because various details can be measured with far greater rapidity than by the other method. Upon a piece of cardboard, about as thick as a postal card, draw a series of scales like those shown in Figure 139. ' . im ^ lOVfJ 20O/. h-t§ 300h toof) I SOOtJ spencer /6mm. Oc./6 Diamefer of field /joo/jjjmm. U 7L 3^0^ 9\)0u""^7- to Co fi I 5 03 Droiun at ki^el of fable ^^ c^^f §-.^^ .^ > I /Mirror bar af /OO flirrorat^'' ^ P>c;^^ 09/ /y 0//00 '^^(f ofuof)ij JGOued^ f h.-t' Ml A ^nM'-^ Fig. 139. — Card scale for practical use Make a scale for each objective. It is not necessary to make scales for all the oculars, but only for the one in most constant use. It is absolutely necessary to note the tube length, length of the bar of the camera mirror and inclination of the camera mirror, and the level at which the scale is made. A variation in any of these details will change the scale. USING THE MICROSCOPE 375 In using the stage micrometer, place the cardboard on the table, and with the- aid of the camera lucida sketch the rulings of the mi- crometer. In Figure 139 note, for example, the scale drawn with Spencer 16 mm. objective, ocular X6. The spaces are drawn from the tenths of a millimeter rulings of the stage micrometer. Therefore, each space on the card represents one-tenth of a millimeter or 100 n, and the ten spaces shown on the card represent 1 mm., or 1,000 /x- By measur- ing with a metric rule the ten spaces upon the card, it is found that the scale is 102 mm. in length. The magnification of any drawing made with the same ocular and objective, under the same conditions, will therefore be 102 diameters. This does not mean that the magnifying power is 102 diameters, for the magnification of this combination is much less. A scale drawn at the level of the stage would show more nearly the magnifying power of the combination, but would still give too large a figure. The exact size of any object which has been sketched with this combination can now be measured by applying the cardboard scale, just as one would measure gross objects with a rule. The diameter of the field with this combination is 1,700 m- By knowing the diameter of the field with the various combinations, one can guess approximately the size of objects. Other combinations are made in the same way. An excellent check on the accuracy of the computations is to measure the same object by means of the ocular micrometer and by the scale card. If the results are the same, the computations are correct. In making sketches, it is a good plan to add the data which would beneeded at any time in making measurements, e.g., Spencer objec- tive 16 mm., ocular X6, table, 110, 45°, would show that the sketch was made at the level of the table, with the mirror bar at 110, and the camera mirror at an angle of 45°. ARTIFICIAL LIGHT During a considerable part of the year daylight is often insufficient for successful work with the microscope. Numerous contrivances for artificial illumination have been devised, some of them fairly good, but most of them thoroughly unsatisfactory. The best illuminator is the one which will give the most light with the least heat. More than two hundred years ago Hooke used a device for artificial illumination which probably suggested the apparatus used by the late Professor Strasburger at Bonn. The apparatus consists, essentially, 376 METHODS IN PLANT HISTOLOGY of a hollow sphere filled with liquid. A fairly good and practical light can be got with an ordinary lamp by allowing the light to pass through a wash bottle filled with a weak solution of ammonia copper sulphate. A piece of dark paper with a circular hole in it serves as a diaphragm and at the same time protects the eyes from the direct light of the lamp. With a good electric bulb, this not only furnishes a good hght, without any glare, and with no appreciable heat, but throws a good light on the pencil, which is an important consideration in drawing with a camera lucida. Optical companies are now making excellent lights for microscopes. These lights furnish good illumination and most of them have the effect of good daylight; but there is still too much heat. If laboratory tables are small, seating only one student, there should be a plug to attach the table to some convenient outlet; and also another outlet on the table for the microscope lamp. If the table is large, seating four or more students, there should be one or two outlets on the table for each student, and a single plug by which the whole table may be connected with a convenient outlet. For elementary classes, which are not likely to use higher powers than a 4 mm. objective with an ocular magnifying five or six times, individual lamps are not necessary in a well-lighted laboratory. Splen- did lights are now available for elementary work, almost as good as day- hght, and strong enough for ordinary dry lens microscopic work. CHAPTER XXIX LABELING AND CATALOGUING PREPARATIONS THE LABEL The first thing to write upon a label is the genus and species of the plant; the next thing would be the name of the organ or tissue, and then might be added the date of collection, e.g., Marchantia poly- viorpha, young archegonia, January 10, 1915. The date of making the preparation is of no value unless the student is testing the permanence of stains or something of that sort. It is hardly worth while to write upon the label the names of the stains used, for the student will soon learn to recognize the principal stains. A hasty sketch on the label will often indicate any exceptionally interesting feature in the preparation. To facilitate finding such a feature, it is a good plan to mark the par- ticular section or sections with ink, or better, with a diamond or car- borundum point, the marking being always on the underside of the slide so as not to cause any inconvenience if an immersion lens should be used. Where there are 2, 3, or more rows' of sections on a slide, a desirable feature may be found very quickly by marking on the label, 2d row, 4th etc. ' ' CATALOGUING PREPARATIONS As a collection grows, the student will need some device for locating readily any particular preparation. Some have their slides numbered and catalogued, but all devices of this sort are too cumbrous and slow for the practical worker in the laboratory. After forty years' experi- ence with a collection which now numbers more than thirty thousand preparations, we recommend the following system : Four wooden slide boxes of the usual type will do for a beginning; they should be labeled: Thallophytes. Bryophytes, Pterido- PHYTES, and Spermatophytes. As the collection grows and new boxes are needed, the classification can be made more definite, e.g., there should be a box labeled Bryophytes Hepaticae and one labeled Bryo- phytes Musci. As the liverwort collection grows, three boxes will be necessary, and should be labeled Bryophytes Hepaticae Marchanti- ales, Bryophytes Hepaticae Jungermanniales, and Bryophytes He- 377 378 METHODS IN PLANT HISTOLOGY paticae Anthocerotales. It will readily be seen that the process can be continued almost indefinitely, and that new slides may be at any time dropped into their proper places. A rather complete label gradually built up in this way is shown in Figure 140. BRYO PHYTES HEPATICAE Jungermanniales Porella platyphyllum Archegonia Fig. 140. — Label for slide box Since there should be some system in the classification, we should recommend the Engler-Gilg Syllabus der Pflanzenfamilien. The beginner will often find that the mere placing of a slide in the proper box and the box in its proper place on the shelf will refresh or increase his knowledge of classification. CHAPTER XXX A CLASS LIST OF PREPARATIONS Where a regular course in histology is conducted, it is a good plan to give each student at the outset a complete list of the preparations which he is expected to make. In a three months' course a fairly repre- sentative collection of preparations can be made. The availability of material determines what a list shall be. Besides gaining an introduc- tion to the use of the microscope and its accessories, a class meeting ten hours a week for ten weeks should be able to do as much work as is outhned below. In making the mounts, the order indicated in the list should not be followed. Begin with temporary mounts, and then study, in succession, freehand sections, the glycerin method, the Venetian turpentine meth- od, the paraffin method, the celloidin method, and special methods. A large proportion of the time should be devoted to the paraffin method. It is neither possible nor desirable that each student should in every case go through all the processes from collecting material to labeling. Some of the material may be in 85 per cent alcohol, some in formalin, some in glycerin, some in Venetian turpentine, and some in paraffin. One student may imbed in paraffin enough of the Anemone for the whole class; another may imbed the Lilium stamens; and by such a division of labor a great variety of preparations may be secured with- out a corresponding demand upon the time of the individual. LIST OF PREPARATIONS THALLOPHYTES MYXOMYCETES 1. Trichia varia. — Paraffin sections 5 /j.. Safranin, gentian violet, orange. SCHIZOPHYTES SCHIZOMYCETES 2. Bacteria. — Coccus, Bacillus, and Spirillum forms. Stain on cover glass or slide. 3. Bacillus anthracis. — In liver of mouse. Paraffin sections, 5 m- Stain in gentian violet, Gram's method. 379 380 METHODS IN PLANT HISTOLOGY SCHIZOPHYCEAE 4. Oscillatoria. — Fix in special chromo-acetic-osmic acid and stain in iron- alum haematoxylin to show nuclei. Venetian turpentine method. 5. Tolypothrix. — Use the Venetian turpentine method. Should show hetero- cysts, hormogonia, and false branching. 6. Nostoc. — Venetian turpentine method. 7. Wasserbluthe. — -The principal forms in this material are: a) Coelosphaermm kutzingianum. — Colonies in the form of hollow spheres. b) Anabaena gigantea. — ^Filaments straight. Preparations should show vegetative cells, heterocysts, hormogonia, and spores. c) Anabaena fios-aquae. — Filaments curved. Stain on the slide and mount in balsam. If material is abundant, stain in iron-alum haematoxylin and mount in Venetian turpentine. 8. Gloeotrichia. — Smear on the slide, stain in safranin and gentian violet, and mount in balsam; or use the Venetian turpentine method, staining in phloxine and anilin blue and crushing under the cover glass. ALGAE CHLOROPHYCEAE 9. Volvox. — Use the Venetian turpentine method. If paraffin material is available, cut 2-5 /x in thickness and stain in safranin, gentian violet, orange. 10. Scenedesmus. — Let a drop containing the material dry upon the slide, stain, and mount in balsam. 11. Hijdrodidyon. — ^Use the Venetian turpentine method. Each preparation should contain pieces of old and of young nets, and also at least one young net developing within an older segment. The greatest care must be taken not to injure the older segments while arrang- ing the mount. 12. Ulothrix. — Use the Venetian turpentine method. Each mount should show various stages in the development of spores and gametes. 13. Oedogonium. — Stain in phloxine and anihn blue and mount in Venetian turpentine. 14. Coleochaete. — Stain in Delafield's haematoxylin and mount in balsam. 15. Cladophora. — Stain some in iron-haematoxylin and some in phloxine and anilin blue. Mount both together in Venetian turpentine. 16. Diatoms. — Make mounts of the frustules and also stained preparations showing the cell contents. 17. Desmids. — Make mounts of available forms. Use the Venetian turpen- tine method if material is sufficiently abundant. 18. Zygnema. — Stain in iron-haematoxylin and mount in Venetian turpen- tine. 19. Spirogyra. — Stain in phloxine and aniUn blue and mount in Venetian turpentine. A CLASS LIST OF PREPARATIONS 381 20. Vaucheria. — Stain in iron-alum haematoxylin and mount in Venetian turpentine. 21. Chara.— Cut paraffin sections of the apical cell, ooginia, and antheridia. PHAEOPHYCEAE 22. Edocarpxis.— Stain some in iron-haematoxylin and some in phloxine and anilin blue. Mount both together in Venetian turpentine. 23. Cii > > Sporodinia, 254, 255 Sporogenous cell, 52 Sporophyte, of Hepaticae, 279 Staining dishes, arrangement, 44; stain- ing living tissues, 152 Stains and staining, 42; practical hints, 77 Star blade, 122 Starch, 83 Steam method, 141, I42 Stemonitis, 4 Stender dish, 15 Sticta, 265 Stony tissues, 141 Strasbiu-ger, 45 Slrombus, 218 Stypocaulon, 242 Suberin, 52, 86 Syringa (lilac), leaf, 353 Tapetal Plasmodium, 312 Taraxacum, floral development, 356; parthenogenesis, 370 Tea filter, 24 Teliospore, 268 Temporary mounts, 80 Tetracera, 349 Thallus, of Hepaticae, 276 416 METHODS IN PLANT HISTOLOGY Thamnidium, 261 Thermostat, Land's, 12 Thuja, 344 Tilia americana, 348 Tolypothrix, 206 Tradescantia virginica, root-tips, 349; reduction of chromosomes, 357 Trichia, 199 Trillium, erectum, 29; T. sessile, mitosis, 351, 357 Tropaeolum, 348 Tube length, 373 Turntable, U, 102 Turpentine, 40 TyVoses, 348 Typha, 211, 262; floral development, 355 U Ulothrix, 211, 232, 233 Uncinula, 262 Uredineae, 266 Urediniospore, 268 Uromyces, 273 Usnea, 265 Ustilagineae, 266 Utricularia, 215 Verbena, 368 Viciafaba, 29; root-tip, 349; secondary- roots, 352; reduction of chromo- somes, 357 Voluox, 4, 211, 212, 213, 214, 216, 217 W Washing, 23, 114 Washing delicate material, 25 Washing tubes (Dudgeon's), 24 Wash tub, 25 Wasserbliithe, 208 Watt's blade, 122 Welwitschia, 346 Wire coil, 16 X Xylaria, 264 Xylol, 38 Yamanouchi, schedule for root-tips, 21, 27; formula for mitochondria, 33, 151; schedule for Haidenhain's haematoxylin, 47 Yucca, 348 Vacuoles, 5, 20 Vade-Mecum (Lee), 43 Vaucheria, 22, 212, 215, 230 Venetian turpentine, 106 Zamia, 140, 322, 323, 329, 330 Zea mays, 348, 352, 370 Zeiss, 3 Zygnema, 20, 212, 222, 2^4 Zygorhyncus, 255 [PRINTED II IN USaJ I