3aS^E=iF=i^F^F=^E^F=?SSI=i^B3 1 I Marine Biological Laboratory Library E E Woods Hole, Mass II I I I I I I I J . . lll&m R, Amberson J Universi " Maryland E B July 1, 1955 ID I EE »/^v?«^N* Presented ty JEaSB^^^^B^B^^^^ESE The Microtomist's Formulary and Guide The Microtomist's Formulary and Guide by Peter Gray, Ph.D., D.I.C., F.R.M.S. Head, Department of Biological Sciences University of Pittsburgh THE BLAKISTON COMPANY, INC. New York Toronto Copyright 1954, by The Blakiston Company, Inc. This book is fully protected by copyright, and no part of it, with the exception of short quotations for review, may be reproduced without the written permission of the publisher Library of Congress Catalog Card Number: 53-11567 PRINTED IN THE UNITED STATES OF AMERICA BY THE MAPLE PRESS COMPANY, YORK, PA. To Freda ^/<^IC/^ ^i^CAl Preface The preface of a book affords the author an opportunity of speaking to his reader in a comparatively direct and personal manner, and of acquaiiiting the prospective user of the book with the considerations which impelled the author to write it. (From The Bookman's Glossary. 3d ed. New York, Bowker [c. 1951]. Reprinted by permission of the R. R. Bowker Co.) A few generations ago the English periodical Punch offered to its readers a "letter of advice to those about to be married": the applicants received the single word "Don't." The advice is pertinent for those about to write a source reference work. You may well, in reading this book, become incensed at what you beheve to be its inaccuracies, errors, and faulty arrangements. This is exactly how I felt, twenty years ago, when I struggled with the reference books on micro- technique which I was then using. You may decide, as I did, to try to write a better book. You will find it a wearisome and disillusioning task. The research will, of course, be wholly dehghtful, but it will be followed by a period of brutal hard labor. Not only will you have to write, but then, if you are to produce a pub- Hshable book, it will have to be condensed and rewritten. Add to this the fact that the finished work has then to be reread four separate times as it goes through press, and you will join me in hoping that your activities do not too strongly resemble those of the dog mentioned in the Book of Proverbs. This book would never have been completed without the help of the librarians of the University of Edinburgh, the Wood's Hole Marine Biological Labora- tory, the University of Rochester, the Carnegie Library of Pittsburgh, and the University of Pittsburgh. I am especially indebted to Miss Lorena Garloch, and her assistants in the Reference Department of the University of Pitts- burgh Library, for their extraordinary skill in tracking down obscure journals and securing them for me on inter-library loan. The illustrations for this work, as for my Handbook of Basic Microtechnique, were prepared from my phot()grai)hs and sketches by Mrs. Gloria Green Hirsch. 1 am glad thai r('\ icwcrs of the published book share my enthusiasm for her work. The number of those, including the author and his wife, who have had a hand in typing this book is legion. It should be recorded, however, that Mrs. Mary Roman single-handed produced the first complete (1500 page) typescript vii Vlll PREFACE and that Mrs. Dolores Johnson, and Miss Kristine Pallesen, have stood by the author durmg the distressing hurly-burly known as "getting the book into press." Dr. James Lackey, then scientific editor of the Blakiston Company, encour- aged me over long periods to persevere in producing a publishable book. His successor, Mr. WiUiam Keller, approved what had been done and, with Mr. Willard Shoener converted my efforts to their present form. My debt to these gentlemen, and to their editorial assistants, is immense. Acknowledgement is made with thanks to the American Optical Company for figures 56, 57, and 84, to the Fisher Scientific Company for figures 34 and 38, and to the Carbide and Chemical Corporation for some of the data in Chapter 25. Permission to reproduce copyright material of the R. R. Bowker Company and the Oxford University Press is specifically acknowledged at the places where these reproductions occur. Peter Gray Edinburgh 1933 Pittsburgh 1953 List of Contents Preface vii Introduction , . 1 PART I— THE ART OF MAKING MICROSCOPE SLIDES Foreword to Part 1 7 1. Dry Wholemounts 10 Slides for dry mounts — coverslips — cells — background — cell cements — • coverslip cements for dry mounts — typical preparation: Strewn slide of foraminifera or radiolaria. 2. Fluid Wholemounts — Aqueous Type 21 Cells for aqueous mounts — cell cements — preservation media — coverslip cements — sealing the coverslip — typical preparations: Wholemount of Microcystis, Wholemount of a rotifer. 3. Fluid Wholemounts — Non-aqueous Type 32 Choice of mounting media — dichromate-gelatin seals — hot resin seals — typical preparations: Nematode in glycerine, Diatom in monobromnaphtha- lene. 4. Wholemounts in Gum Media 42 Choice of mounting medium — types of object to be mounted — finishing slides — typical preparation: Mite in Berlese's medium. 5. Wholemounts in Jelly Media 46 Process of mounting — finishing jelly mounts — typical preparation: Wholemount of a small crustacean. 6. Wholemounts in Resinous Media 51 Narcotizing and fixation — choice of stains — dehydration — clearing — mounting in balsam — finishing balsam mounts — typical preparations: Carmine stained wholemount of Pectinatella, Skeleton of an insect, Double stained alga in venice turpentine, Minute fresh-water organisms. 7. Smear Preparations from Fluid Material 69 Preparation of smears — fixing smears — drying smears — typical prepa- ration : Monocystis from the seminal vesicle of the earthworm. ix O^t^t^^ x list of contents 8. Smeae Preparations from Cut Surfaces 74 Preparation of smears — typical preparation : Diagnostic smear of Negri bodies. 9. Squash Preparations from Solid Bodies 76 The process of maceration — staining and mounting squash prepara- tions — typical preparations: Macrosporocytes of Crocus, Dissociated Hydra. 10. Ground Sections 80 Preparation of the crude section — grinding and polishing agents — typical preparations: Transverse section of bone, Section of coral with polyp in situ. 11. Sections of Free Material 88 Nature of sections — microtomes for free sections — methods of holding material — hardening and fixing material — staining and mounting sec- tions — typical preparations: Transverse section of leaf of Ligustrum, Section of wood. 12. Paraffin Sections 94 Selection of a fixative — dehydrating agents — clearing agents — embed- ding media — technique of dehydrating, clearing and embedding — micro- tomes — knives and knife sharpening — block mounting — cutting paraffin ribbons — staining and mounting sections — cleaning and labelling slides — typical preparations: Transverse section of frog's intestine, Section of amphibian embryo. Sagittal section of whole mouse. 13. Nitrocellulose Sections 142 Nitrocellulose — preparation of nitrocellulose solutions — infiltration — casting celloidin blocks — cutting sections — staining and mounting — typical preparation : Transverse section of lily bud. 14. Sections from Double Embedded Material 153 Explanation of process — typical preparation : Sections, intended for recon- struction, of pluteus larva. 15. Frozen Sections 157 Choice of a supporting medium — refrigerants — cutting frozen sections — staining and mounting — typical preparation: Section of fatty tissue. 16. Injections 162 Selection of injection mass — methods of injection — typical preparations: India ink injection of chicken embryo. Lead chromate injection of kidney glom- eruli, Carmine-gelatin injection of intestinal capillaries. PART II— METHODS AND FORMULAS USED IN MAKING MICROSCOPE SLIDES Foreword to Part II 173 LIST OF CONTENTS ■ XI 17. Preservatives (Referenced AS P) 175 P 00 General observations — P 10 Preservatives miscible with water — 11 inorganic reagents, 12 organic reagents, 13 other preservatives — P 20 Preservatives not miscible with water. 18. Fixatives (Referenced as F) 182 General observations — Standard fixative solutions — Formulas arranged by classes — ^1 osmic, 2 platinic, 3 mercuric, 4 cupric, 5 picric, 6 chromic, 7 dichromate, S other inorganic salts, 9 other organic reagents: alone, combined together, or with 0.1 formaldehj-de, 0.2 acetaldehyde, 0.3 acetone, 0.4 other mod'i&er : with or icithout 0.001 acetic, 0.002 trichloracetic, 0.003 formic, 0.004 nitric, 0.005 sulphuric, 0.006 hydrochloric, 0.007 oxahc, 0.008 other inorganic acids, 0.009 other organic acids — Basal fixative solutions — Formulas arranged alphabetically. 19. Accessory Fixative Formulas (Referenced as AF) 254 AF 00 General observations — AF 10 fixative removers — AF 20 De- calcifying AGENTS and AGENTS FOR SOFTENING CHITIN AF 30 BLEACHING AGENTS — AF 40 Macerating agents — AF 50 Narcotizing agents. 20. Formulas and Techniques for Dye Stains of General Applica- tions (Referenced as DS) 267 DS 00 General observations — DS 10 Dye staining techniques of GENERAL APPLICATION — DS 11 NucLEAR STAINS, 11.1 hematoxylin (typical preparations: Kat testis with iron-hematoxylin, Chicken embryo wholemount with alum hematoxylin, Chicken embryo sections with acid-alum hematoxylin), 11.2 carmine (typical preparations: Liver fluke with carmalum, Medusa with alcoholic borax-carmine, Chromosomes with iron aceto-carmine) , 11.3 other natural dyes, 11.4 synthetic dyes (typical prepsirations: Pollen grains with safranin, chromosomes with magenta) — DS 12 Plasma stains, 12.1 single con- trast formulas, 12.2 double contrasts from one solution (typical prepara- tions : Squalus embryo with 'picro-indigocarmine, Rat tongue with celestin blue — picro-acid fuchsin) , 12.3 complex contrast formulas (typical preparations : Section of earthworm with hematoxylin-acid fuchsin-anilin blue, Section of mouse head with hematox^jlin- ponceau 2R-light green) — DS 13 Complex TECHNIQUES INVOLVING BOTH NUCLEAR AND PLASMA STAINING, 13.1 thiazin eosinates (typical preparation: Blood smear with methylene blue-azur A- methylene violet-eosin Y), 13.2 thiazin eosinates with other dyes, 13.3 methyl green techniques (typical preparation: Suprarenal body with methyl green- acid fuchsin-orange G), 13.4 acid fuchsin techniques (typical preparation: Section of Amphioxus with acid fuchsin-anilin blue-orange G), 13.5 safranin techniques, 13.6 hematoxylin techniques, 13.7 other complex techniques. 21. Formulas and Techniques for Dye Stains of Special Applica- tions (Referenced as DS) 374 DS 20 Dye staining techniques op special application — DS 21 Sk- LECTivE stains FOR HISTOLOGICAL ELEMENTS, 21.1 skeletal tissues (typical preparations: Bones in wholemount of small salamander mth alizarin, Carti- lage in embryo with methylene blue. Root skeleton with acid fuchsin-iodine XU LIST OF CONTENTS green), 21,2 nervous tissues (t3T3ical preparations: Section of brain with methylene blue, Section of spinal cord with hematoxylin. Neuroglia with crystal violet), 21.3 blood, 21.4 other histological elements — DS 22 Stains foe CYTOLOGiCAL ELEMENTS, 22.1 nuclei (typical preparation: Mitosis with rose bengal-orange G-ioluidin blue), 22.2 mitochondria and Golgi (typical prepa- ration: Mitochondria in pancreas with acid fuchsin-toluidin blue-aurantia) , 22.3 Nissl granules, 22.4 yolk and fat granules, 22.5 plastids, 22.6 starch, glycogen and amyloid granules, 22.7 mucin, 22.8 other cell inclusions and extrusions — DS 23 Selective stains for specific organisms, 23.1 virus, Rickettsiae and Negri bodies (typical preparations : Rickettsiae in guinea pig scrotum with magentathionin, Negri bodies in guinea pig brain with ethyl eosin-methylene blue), 23.2 bacteria (typical preparations: Bacterial smear with crystal violet, Demonstration of Gram positive bacteria. Demonstration of tubercle bacilli, Flagella of Proteus vulgaris, Diploccocci in liver of rabbit), 23.3 other parasites and commensals (typical preparations •.Pencillium mycelia in orange rind with thionin-light green-orange G-erythrosin, Fungi in tissue scrapings), 23.4 other animals, 23.5 other plants — DS 24 Miscellaneous TECHNIQUES. 22. Accessory Dye Staining Formulas (Referenced as ADS) .... 514 ADS 10 Mordants and tissue revivers, 11 miscellaneous formulas, 12 mordants — ADS 20 Differentiating solutions, 21 for hematoxylin, 22 for other stains. 23. Formulas and Techniques for ]\Ietal Stains (Referenced as MS) 522 MS 00 General observations — MS 10 Osmic acid, 11.0 typical prepa- rations: (Golgi network in earthworm ovary), 11.1 staining solutions, 11.2 neurological techniques, 11.3 histological techniques, 11.4 techniques for cell inclusions — MS 20 Gold — MS 21 Gold used alone, 21.0 typical preparations: {Nerve termination in muscle), 21.1 staining solutions, 21.2 techniques — MS 22 Gold in combination with mercury, 22.0 typical preparation (Protoplasmic neuroglia in the cerebral cortex), 22.1 staining solutions, 22.2 neurological techniques — MS 23 Gold in other combina- tions, 23.0 typical preparation (Spinal cord with ammonium dichromate — gold chloride), 23.1 staining solutions, 23.2 neurological techniques, 23.3 cytological techniques, 23.4 other techniques — MS 30 Silver — MS 31 Silver nitrate, 31.0 typical preparations (Nervous elements of retina. Neuroblasts and axons in chicken embryo, Spirochaetes in sections), 31.1 staining solutions, 31.2 neurological methods, 31.3 cytological methods, 31.4 histological methods, 31.5 bacteriological methods — MS 32 Protein silver, 32.0 typical preparation (Sciatic nerve of cat to show axis cylinders), 32.1 neurological methods, other methods — MS 33 Silver diammine, 33.0 typical preparations (Nerve endings in taste buds, Oligigodendria and mi- croglia, microglia), 33.1 staining solutions, 33.2 neurological methods, 33.3 cytological methods, 33.4 histological methods, 33.5 bacteriological methods, 33.6 other silver diammine methods — MS 34 Silver in combination with other metals, 34.0 typical preparations (Purkinje cells in the cerebellar cortex. Structure of superior cervical ganglion. Neurons and dendrites in brain LIST OF CONTENTS Xlll of rabbit embryo), 34.1 staining solutions, 34.2 neurological methods, 34.3 histological methods, 34.4 cytological methods, 34.5 bacteriological methods — MS 35 Other Silver methods — MS 40 Other metals, 41.1 staining solutions, 41.2 neurological methods, 41.3 histological methods. 24. Accessory Metal Staining Formulas (Referenced as AMS) . . 612 AMS 10 Accelerators and mordants, 11 formaldehyde mixtures, 12 alcohol mixtures, 13 other mixtures — ADS 20 Solutions used after stain- ing, 21 developers, 22 toners, 23 differentiators, 24 fixers. 25. Solvents and Oils (Referenced as S) 622 S 10 Dehydrating agents — S 20 Clearing agents, 21 essential oils, 22 synthetic clearing agents — S 30 "Universal" solvents — S 40 Mix- tures. 26. Mounting Media (Referenced as M) 630 M 10 Mountants miscible with water, 11 gum arable media, 12 gelatin media, 13 other media — M 20 Mountants miscible with alcohol, 21 mastic media, 22 Venice turpentine media, 23 sandarac media — M 30 Mountants not miscible with water or alcohol, 31 canada balsam media, 32 damar media, 33 other natural resins, 34 synthetic resins. 27. Embedding Media (Referenced as E) 642 E 10 Media miscible with water — E 20 Media not miscible with water, 21 wax media, 22 nitrocellulose media, 23 resinous media, 24 other media. 28. Various Formulas (Referenced as V) 650 V 10 Cements, lutes and varnishes, 11 fluid, 12 solid, 13 other mix- tures — ^V 20 Adhesives, 21 for attaching sections to slides, 22 for attaching whole objects to slides, 23 for other purposes — ^V 30 Injection media — V 40 Cleaning formulas — V 50 Miscellaneous formulas. List of Abbreviations Used 669 List of Books and Journals Cited 670 Books — Journals not listed in "World List" — Journals hsted in "World List." Index 681 Introduction jOiC/^^ Scope of the Book This work consists of two parts. Part I (Chapters 1-16) is a treatise on the art of making microscope sUdes from biological specimens. Part II (Chapters 17-28) is a classified list of the formulas and tech- niques used in this art. Arrangement of Part I Each chapter deals with a specific type of microscope shde and is divided into two parts. The first part discusses problems in- volved in the preparation of such a sUde and the general methods by which these problems have been overcome. The second part is devoted to one or more specific ex- amples which describe in detail the appli- cation of the general methods to the production of an actual slide. The few literature references in Part I are confined to places where the author is describing a method of which he lacks personal experi- ence, or where he is giving opinions at variance with his own. Arrangement of Part II The chapters in Part II are devoted to specific types of formulas and give, where necessary, the techniques by which these formulas are used. Each chapter is sub- divided decimally in accordance with a scheme given in full at the beginning of the chapter and explained in the first para- graph of the chapter. Every formula or technique is thus identified by a number which is used, together with two or three letters identifying the chapter, in all cross references. Thus: DS 11.122 Mayer 1891 identifies a specific alum-hematoxylin of Mayer (he published five other alum- hematoxylin formulas) in any of the fifty places that reference is made to it. The formula is given only once and then in as- sociation with all the other alum-hema- toxylin (DS 11.122) formulas in the book. These decimal reference numbers are added to the page numbers all through, thus making it easy to run down a given type of formula or technique. Pet Names Some biologists have a pernicious habit of omitting literature references and using what Conn 1938 (20540b, 13:121) in a well-organized attack on them, calls "pet names." In cases where these pet names — such as paracarmine or B 15 — have be- come embedded in the folklore of micro- technique, the present author puts them in italics, immediately after the decimal reference, thus: DS 11.22 Mayer 1892 paracarmine — compl. script. The appended conipl. script, indicates that the word has occurred in a "great many writings." When only the originator of the technique appears to have used the pet name it is referred to with auct. Pet names should never be used in scientific writing but such sloppy scholarship as is inherent in a reference to "Bouin's Fluid" is almost worse. Bouin is the originator of many fixatives of which one happens to be popular at the moment; quite another was popular twenty years ago, when a casual reference to Bouin's Fluid meant a mer- curic formaldehyde mixture. Method of Giving Literature References The author indicates, after every for- mula or technique, the source from which he is quoting. The form used varies accord- ing to the type of source; and an example of each will be given. INTRODUCTION Direct Quotations from Books. The author's name, date, and page only are used, thus: DS 11.123 Anderson Anderson 1929, 129 At the end of Part II there is a list of books cited where "Anderson 1929" is ex- panded to a full bibliographic reference. Indirect Quotations from Books. It too frequently happens that a book quotes a formula by name, either without giving a reference at all or with an incorrect reference. When the present author has been unable to find the original he uses the abbreviation test. Thus: DS 11.123. Conklin test. 1930 Guyer Guyer 1930, 232. This indicates that the volume in ques- tion contains, on page 232, a formula for Conklin's picro-hematoxylin but offers no information as to where the original can be checked. Where the author of a book cited is quoting at second hand, the abbreviation cit. is used. Thus DS 11.24 Vignal test. 1907 Bohm and Oppel cit. Henneguy Bohm and Oppel 1907, 118 indicates that, on page 118 of the volume in question, there is a statement to the effect that Henneguy proposed, following the method of Vignal, to prepare a picro- carmine by this particular method. Where the author of a book cites him- self, or the authors cite themselves, with- out reference, the abbreviation used is test. ips. (standing for teste ipso or testihus ipsis). Thus: MS 31.22 Cajal 1925 test. 1933 ips. Cajal and de Castro 1933, 262 The present author would plead in self defense that he has tracked more than a thousand such references to the originals and that these test, and cit. references are used only where he has failed to find the original or where the original is incorrectly quoted. Direct Quotations from Journals. The author has used, in place of the name of the journal, the number assigned to that journal in the World List of Scientific Periodicals (Oxford, The University Press, 1927). Thus: DS 11.122 Carazzi 1911 23632, 28:273 indicates that Carazzi's formula is given on page 273 of volume 28 of the Zeitschrift far wissenschdftUche Mikroskopie und fiir mikroskopische Technik. The full titles of the two-hundred-odd journals cited will be found immediately preceding the index. The use of this number not only saves space, an important consideration in a volume of this magnitude, but also permits exact identification of the journal. The author decided to use these numbers quite shortly after he started checking refer- ences in the /. Anat. (either of two jour- nals) and the /. Bot. (any one of three journals). Indirect Quotations from Journals. The abbreviations test., test. cit. and test, ips. are used with journal references ex- actly as described for book references. Unpublished Information. The ab- breviations in verb, and in litt. indicate that the author has received unpubhshed information either verbally or in a letter. Thus: V 12.2 Fant 1932 in verb. indicates that Mr. Fant told the author the unpubhshed composition of his seahng medium for glycerol mounts in 1932. .Slavonic Names Where the author has cited Slavonic names from a Cyrillic alphabet original, he has transliterated according to the rules of the Library of Congress (Beetle, Clara, ed. A. L. A. Cataloging Rules for Author and Title Entries. Chicago, Ameri- can Library Association, 1949, p. 246) without regard for the writer's preference as indicated in, say, a German summary of his paper. Thus Yasvoyn, not Jasswoin, is cited from the original. Slavonic names cited from a Latin alphabet original are transcribed directly even though this in- volves referring to the same individual by several names. Slavonic names cited at second hand are also transcribed directly, no matter how obviously they may have been mistransliterated. INTRODUCTION Citing Latin alphabet names from Cyrillic alphabet originals has involved even more uncertainty. Russian writers not only omit references but also follow varying rules of transliteration, some tak- ing a phonetic and others a Hteral ap- proach. The name Huygens, for example, can be transliterated into a Cyrillic form which can then be phonetically trans- literated in German as Geugantz. Thus Roskin 1946 attributes to an individual whose name may be transliterated Shteve, Sleeve, Stieve, or Stive a fixative which re- sembles, but is not identical with, the formula attributed, also without refer- ence, to Stieve by Romeis 1948. The author has given the one as Stieve test. 1946 Roskin — and the other as Stieve test. 1948 Romeis. Faulty scholarship can certainly increase the confusion originated by the architects of the Tower of Babel. Names of Dyes The author has, with one exception, changed the names of all dyes to accord with the synonym preferred by Conn 1946 (Conn, H. J. Biological Stains 5th ed. Geneva, N. Y., Biotech, 1946). The author prefers, however, to use the name magenta, rather than basic fuchsin, to describe the mixture of magenta 0, magenta I* ma- genta II now sold as basic fuchsin. There is no discussion of the chemistry and synonymy of dyes in the present work; reference should be made to Conn (op. cit.) . The author has not given certification numbers or dye content in formulas, since they are available for so few. Names of Reagents Other than Dyes The author has in almost all cases fol- lowed the usage preferred by The Merck Index 6th ed. Rahway, N. J., Merck, 1952. The terms chromic acid, osmic acid, and picric acid, though technicallj^ incorrect, are so universal in biological literature that they have been retained. Similarly the terms alcohol and absolute alcohol (ab- breviated in the formulas to ale. and abs. ale.) have been used in place of ethanol. Chemical names used are those custom- arily found on the label of the reagent bottle and are not accompanied by chem- ical formulas, or otherwise qualified, unless the reagent is found equally commonly in several forms. Thus copper suZ/aie indicates the usual reagent CUSO4.5H2O. On the rare occasions when reference is made to the anhydrous salt, it is referred to as copper sulfate, anhydr. In any case of doubt, reference should be made to the Merck Index. Proprietary Compounds Proprietary compounds of known com- position, such as amidol and salvarsan have been referred to by the name preferred in the Merck Index. Proprietary compounds of secret composition have no place in con- temporary science and have been ignored. It is fantastic that purveyors of reagents should be permitted to sell nostrums of secret composition, thus indicating a con- tempt for technicians equal to that shown by medicine men for the yokels they gypped with snake oil. The author would make it very plain that he does not extend this attitude to "brands" of mixtures or reagents selected for technicians' use. Every maker of microscope slides is in- debted to those firms which select and blend materials specially for his use. Quantities and Measures The abbreviations ml. and Gm. have been omitted. It is to be presumed that all liquids will be measured in millihters and all sohds in grams. Formulas have been adjusted to give a rational total (usually 100) in terms of standard ingredients, no matter how the original was presented. This has been wearisome labor applied to thousands of formulas. It is doubtless con- venient to make up a solution by adding fifteen drops of a 2.5% solution of this to 30 drops of a 1.25% solution of that and then to dilute to 15 milliliters wdth 30% alcohol. As a published instruction, how- ever, it does not commend itself to writers of textbooks struggUng to avoid duplication. Index The last section of the book is a single- alphabet, fully expanded, index, alphabet- ized according to the rules of the American Library Association (Beetle, 1949, op. cit.). These terms may require explanation. INTRODUCTION A single-alphabet index is one in which all entries are placed in the same index. There are not separate indexes for authors, stains, etc. Fully expanded means that more than one entry leads to the informa- tion sought. For example, "Grenadier's alcoholic-borax-carmine" may be found whether the reader consults the word "Grenacher," "borax-carmine," alcoholic- borax-carmine," or "carmine." A con- densed index, which saves the author work and the publisher money without regard to the reader's feehngs, has "borax carmine see Grenacher," " carmine staining solutions, see author's name," etc. etc. Those who do not think it necessary to have rules of alphabetization might try indexing del Rio-Hortega, 2BD fixative, CS-IS mountant, and van't Hooft. The rules of the A.L.A. {op. cit.) may not be perfect but they are at least clearly ex- pressed and easily available. Part I The Art of Making Microscope Slides Foreword to Part I The preparation of objects for micro- scopic examination — more colloquially known as "making microscope slides" — has a twofold purpose. On the one hand it may be desired to preserve in permanent form objects too small or too dehcate to be handled by the ordinary methods of mu- seum preparation. Second, and far more important, it may be necessary to make permanent preparations of objects and tissues in such a manner that their struc- ture may be more clearly seen under the microscope. In both cases, the object is mounted on a slide, which is nowadays a standardized 3" XI" strip of thin glass. Originally microscope slides were very different and were usually made by taking a sHp of ivory, about 2" X }i", and drill- ing through it a hole of about %" in diam- eter. This hole was then enlarged from each side, about a third of the way through, to a }'i" diameter, thus leaving a ridge of ivory in the center. The depres- sion on each side of the slide was fitted ^^ith a ring of spring steel and several disks of mica of a half-inch diameter were fur- nished with each sUde. To make a mount, a piece of mica was inserted from one side and held in place by the slip ring, the ob- ject was placed on it and another disk of mica was then inserted from the other side and, in its turn, held in by a slip ring. This was the only type of shde available until about the middle of the 18th century when glass shdes first made their appearance. These glass slides were, however, of very little use, with their talc covers which re- mained the greatest bar to the progress of microtomy. Toward the close of the first half of the 19th century Messrs. Chance, Birming- ham, England discovered how to make thin glass coverslips. They were for many years (Queckett 1855, 287) the only manu- facturers, and until the discovery of oil immersion objectives microscopists were entirely dependent upon the increasing thinness with which these glasses could be supplied. Microscopists were still seeking for magnification rather than resolution, and by 1880 (Beale 1880, 351) a coversHp had been made sufficiently thin to permit the use of a ^^o-inch "high dry" objec- tive. Coverslips are now taken so much for granted that the contribution made to the development of biology through the intro- duction of thin glass is often overlooked. The earliest method of using these thin coverslips with glass shdes was by holding the cover in place with the aid of a paper label which covered all the slide except the area immediately over the object. These labels were often fancifully engraved to the design of the individual technician and an excellent and well-illustrated descrip- tion of their use is to be found in Martin 1872, pp. 46-52. Microscope mounts, as made today, consist of three types. These are, first, wholemounts, in which organisms or pieces of organisms are mounted under a cover- slip on a slide; second, smear -preparations, in which either a cut surface or a viscous fluid is smeared on a shde to form a thin layer which is subsequently preserved under a cover glass; third, sections, in which thin shces of objects are mounted under a coverslip. Where objects are cut into a series of sections, each of which is mounted in consecutive order on a slide, the preparation is known as a serial section. The simplest slide to prepare is that in which the object is mounted dry. An ex- ample of this is shown in Fig. 1 where a series of diatoms have been spread on a slide, and a covershp placed over 'them. This coversHp is held in place by a ring of cement which is prevented from running under the edges of the covershp by some 8 THE ART OF MAKING MICROSCOPE SLIDES Figs. 1 to 6. Types of microscopical preparation. 1. Dry wholemount of diatoms. 2. Freshwater hryzoan in deep cell of formaldehyde. 3. Crustacean in oval cavity in glycerol jelly. 4. Smear preparation. 5. Single section of plant stem. 6. Serial section of embryo. FOREWORD TO PART I 9 form of thin cell, which may be either of cement or paper, and which servos the additional purpose of preventing the crushing of the object by the coverslip. Most wholemounts are, however, pre- pared in a preservative medium, wliicli may be either aqueous, colloidal, or resin- ous. Many of these whole objects are rela- tively thick so that some method must be adopted of ])roviding space for them under the coversUp. Fig. 2 shows an object mounted in a deep cell of glass, while Fig. 3 shows an alternative method in which a relatively thick shde has had an oval cavity ground into it. As will be seen from the figure these mounts are heavily var- nished at the edges to prevent the evap- oration of fluid or the A\ithdrawal of water from the colloidal medium. Wholemounts prepared with | 'resinous media, w'hich harden and thus hold the coverslip in place, are frequently not varnished at the edges though some case can be made out (Gray 193G, Microsc. Rec, 38) for the ap- phcation of a ring of varnish around the edge of balsam mounts. Smear preparations (Fig. 4) are almost invarialily prepared in resinous media and equally invariably the edges of the covcr- shps are not varnished. Sections (Figs. 5 and 6), either single or serial, are univer- sally mounted in resinous media and the edges of the covershp are practically never varnished. Dry Wholemounts General Principles A dry wholemount consists essentially of an object or objects enclosed within a small, usually cylindrical, box attached to the center of a microscope slide. The floor of this box is almost invariably the surface of the slide itself while the roof is formed by the coversUp. The sides of the box are produced by the attachment of a cell, which may be a thin ring of cement, a washerlike piece of paper or plastic, or a squat cylinder of the same materials. The object may be attached directly to the glass surface of the sUde, if one desires to make a transparent mount, or the surface of the shde may be rendered opaque and the object then attached to whatever sub- stance is used to blacken the surface. There are a number of decisions to be made before preparing a dry wholemount. The considerations governing these deci- sions will be discussed in the order in which they present themselves to the technician. Selection of a Slide If the object is to be prepared as a trans- parent wholemount one has no choice but glass. None of the transparent plastics at present available have a sufficiently hard surface to be worth using. They are un- breakable and easy to handle when first made, but will become so scratched after even a few months of use as to be worth- less. The manufacturers of these slides point out that they may be repohshed at intervals, but there seems little point in preferring them to glass which does not become scratched. If the wholemount is to be prepared as an opaque object, which is the case in probably 90% of all dry wholemounts, there is little justification for using glass. It has two great disadvantages: first, it^is very easily broken; second, it is one of the most difficult materials to which to cause adhesives to stick. In the early days of microscope mounting it was customary to employ slides of well-seasoned mahogany and, though this practice is today confined to the mounters of Foraminifera, a brief description of the preparation of these shdes will be given. A piece of seasoned mahogany, 3" by 1" in section, is secured with the grain running parallel to the three-inch face. This block is set up on end in a vertical drill and a hole of the required diameter drilled as deeply into it as is possible with tools available. This hole should be about Ke of an inch smaller than the size of the coversUp which will be used; that is, if ^^-inch coversUps are customary an i He-inch drill is used to make the hole. The actual size of the covershps should be checked before drill- ing, since many coverslips which are sold as %-inch have a diameter of eighteen millimeters, or a trifle less than '*%4. When the hole has been drilled, the block of wood is transferred to a circular saw and slices about J^e of an inch in thickness cut from it. These slices are, in effect, 3" XI" microscope shdes with a hole of the required size in the center. A sheet of strong, thin card is then cut into 3" X 1" pieces, each of which is glued to the under side of one of the shdes. The best way to do this without warping the wooden strip is to use shellac as an adhesive, either em- ploying a very thick solution in alcohol and permitting it to dry under pressure, or 10 Coverslips DRY WHOLEMOUNTS 11 coating the sheet before cutting with the thick solution which is allowed to dry and then pressed under heat onto the lower surface of the slide. Sheets of photog- rapher's dry mounting tissue can be used for the same purpose, if a photographer's dry mounting press is available. This com- pletes a wooden microscope slide with a built-in, white-bottomed, cell. If a black bottom is required a disk of black paper is punched, coated with ordinary starch frame, the flanges of which are sufficiently deep to allow a thin slide to be slipped in as a cover. Selection of a Coverslip The chief difficulty in selecting a cover- slip for a dry wholemount using a deep cell is to find one which is thick enough. The only value attaching to very thin coverslips is that they permit the use of high power objectives. Most dry whole- Fig. 7. Turning a ring on a slide. paste on the underside, and pressed into position at the bottom of the hole. Numerous variations on slides of this type are possible. To mount a number of small objects on the same slide it is only necessary to drill a number of small holes at the end of the wooden slab and thus secure a slide with as many built-in cells as is required. Special shdes for Foraminifera are made where large collections are to be mounted. These consist of 3" X 1" slips of black card on the central two-thirds of which are printed 60 numbered divisions. At each end a section of thick card is glued on, so as to leave the black portion in the form of a shallow rectangular cell. The card so prepared slides into an aluminum mounts are used with low power objec- tives. The thickness commercially sold as No. 3 is the thinnest which should be con- sidered, unless high powers are certain to be used. Selection of a Cell A cell on a dry mount serves mainly to support the covershp, and the thickness required is therefore the primary con- sideration governing selection. Where the object is only a few hundredths of an inch in thickness, as in the case of diatoms or the smaller Radiolaria, a cement cell is the simplest. For a dry mount it is difficult to find a better cement than "gold size" and the preparation of a cell with this medium 12 THE ART OF MAKING MICROSCOPE SLIDES Cells will therefore be described. Cement cells are made with a turntable in the manner shown in Fig. 7. Turntables are of many- patterns but consist essentially of a rest for the hand and of a rotating circular plate bearing chps to hold the slide. These plates have the center marked, usually with a series of concentric rings engraved round it. The center of the shde must first be marked, and this is readily done by placing the slide on a sheet of paper, running a pencil around its edges, and then drawing the diagonals of the rec- tangle so formed. The shde is replaced on the rectangle and a dot made with India ink at the point immediately above the intersection of the diagonals. The shde is transferred to the circular plate of the turntable with the dot over the central point of the plate. The cement ring should be of the same diameter as the covershp and one of the circles on the plate may be used as a guide; if there are no guide hues, a ring should be marked on the underside of the slide of the same diameter as the covershp to be used. The brush is charged with the cement and the table spun quite rapidly by means of the milled ring shown in Fig. 7. It is safer to use this milled ring than to use the edge of the turntable be- cause the shde frequently projects slightly beyond the edge and may be tapped off center with the finger used for spinning. The charged brush is brought slowly down over the marked ring and held in contact with the spinning shde so that a circle of cement is drawn on the glass face. Re- member that you are not painting a thin ring of varnish on the slide; you are en- deavoring to build up a relatively thick layer of cement by allowing it to flow from the brush to the shde. The hairs of the brush should never touch the glass itself ; only the cement should touch the glass and thus be drawn off. In making a dry wholemount it is not very important how wide the ring is, but a %6-inch-wide ring for a %-inch covershp will be about cor- rect. As many shdes as are likely to be required are prepared at one time and may be left to dry indefinitely. Cells prepared with an ordinary sample of good gold size are safe to use after about 24 hrs. Building up a thick ring of cement by the applica- tion of successive coats is rarely satisfac- tory. A gold-size ring prepared in the manner described will have a thickness between one- and three-thousandths of an inch. If thicker rings are required, it is better to use cells made of paper, card- board, tin, or plastic. Paper rings are stamped from a sheet of the required thickness (a good quality bond paper runs from three- to five-thou- sandths of an inch) or from Bristol board (6- to 12-thousandths of an inch) or from cardboard (up to a thickness of about Ke of an inch). The best board to use is the dense black bookbinder's board (once known as millboard) since cheap yellow strawboards have such a rough surface that they can be made to adhere only with difficulty to a glass slide. SheUac is a good adhesive for attaching paper, or thin card, to a glass slide. A sheet of bond paper is coated on one side with com- mercial sheUac varnish and then dried. Rings of the appropriate size are stamped from this shellac-coated sheet, either by using two punches successively, or with a double punch. The outer diameter of the cell should be larger than that of the covershp, while the inner diameter should be less, so that when the cover is laid in place there wiU be an appreciable overlap of paper both inside a-nd outside. A large number of these stamped rings may be cut at one time. When required for use, one is centered on the shde and then pressed into place with a hot iron, raised to a temperature which will melt the sheUac. When the shde has cooled, it is turned upside down and observed with light reflected from it at an angle. If the cell is perfectly attached no adjustment of the angle of observation will produce mirrorhke reflections from the underside of the cell; if any considerable area of the underside of the cell shows mirrorhke re- flections, it is not properly attached and had better be rejected. Cardboard cells more than He of an inch thick are not satisfactory, for they are so porous that they admit moisture in humid weather and allow fungus growth on the specimen. The outside of the ceh may, of course, be covered with some waterproof cement, but this makes a clumsy looking mount and it Backgrounds DRY WIIOLEMOUNTS 13 is better to substitute either a plastic or a tin cell. Most plastic cells seem to be stamped out of vulcanite, though there is no reason why the numerous other plastics available today sliould not be used. It is almost im- possible to punch cells from sheets of plastic more than He of an inch thick without special machinery, so that it is better to buy them than to prepare them one's self. Excellent cells may, however, be prepared by anybody in possession of a lathe by buying extruded tubing, readily available in many types of plastic, and cutting from it lengths of the appropriate thickness. Opaque plastic should never be used to prepare cells more than i^g of an inch high, since the opaque wall interferes with the illumination of the contained object. Homemade cells are better pre- pared from tin than plastic, since an ordinary hammer and punch may be used to cut sheet tin, or sheet pewter, up to a thickness of nearly }i of an inch. When cells have been punched from sheet, rather than turned from a tube on a lathe, they will be found to have a turned-down edge where the punch came through. This edge must be removed before they are cemented by placing the cell on a sheet of fine sand- paper — sandpaper blocks sold for sharpen- ing draftsmen's pencils are excellent — and rubbing it backward and forward until a visual inspection shows that all the under- surface has come in contact with the abrasive. Cementing of cells to glass depends far more for its success on the cleanhness of the glass than on the cement selected. Gold size has the tremendous advantage that it will adhere firmly to slightly dirty glass, but it has the disadvantage that three or four days are required before it is sufficiently firm to continue mounting. If one is prepared to take the trouble to clean the glass thoroughly, almost any of the cements given in Chapter 28 under V 11.2 may be employed. To make a firm bond with liquid cements it is best to turn a ring of the cement in the appropriate place on the slide with a turntable, and to apply a thin coat of cement to the under- side of the cell. When both these coats are dry another thin coat is applied either to the slide or the cell. The two are then pressed together and dried under pressure. An easy way to apply this pressure is to take a 500-gram brass weight, which is usually to be found somewhere about the laboratory, and lay it carefully on top of the cell; or one can place another slide on top of the cell and clip the two slides to- gether with a strong spring paper clip. Whatever method, or cement, is em- ploj^ed, each slide must be inspected after it is dry to make sure that it is adhering perfectly over all, or almost all, of the base ; minute air holes will admit moisture and lead to molding of the contained speci- men. If the cell has been fixed with a cement under pressure, a small quantity of surplus cement will almost always have been extruded both outside and inside at the point of contact of the ring. That which has been extruded outside should be left in position, unless there is a great deal too much of it, but the material inside should be carefully scraped off with the edge of a scalpel. It is most unwise to endeavor to remove this cement with a solvent which is hkely to loosen the cell. Selection of a Background No background other than the glass it- self is necessary when the object is to be prepared as a transparent wholemount; but many objects prepared as a dry whole- mount are better displayed against a black or colored background. This background may either be a varnish apphed to the bottom surface of the cell, or a disk of the appropriately colored paper cemented in position. The best black paper is that used to wrap photographic plates, but colored papers of all tj^pes are available. The paper is punched into disks which are at- tached to the bottom of the cell with any adhesive. They will not be subjected to any strain, and office mucilage, or any of the formulas given in Chapter 28 under V 11.1 will be found satisfactory. If a black background is to be painted in place, the most satisfactory paint is the optical dead black, listed by some scien- tific suppHers or available occasionally from scientific instrument makers. The only one of these which may be prepared at home is the formula of Martin 1872 14 THE ART OF MAKING MICROSCOPE SLIDES Cements given in Chapter 28 under V 13.1. This is an excellent dead-black cement but, since it has a gold-size base, is very slow drjdng. The best colored backgrounds are the old wax and resin formulas, particularly those of Martin 1872, Oschatz 1842, and Mende- leef (1942); the formulas for these are in Chapter 28 under the heading V 12.2. These media have the advantage that they are thermoplastic so that they may be used both to provide a smooth back- ground and to secure the adhesion of the object to the bottom of the cell. They must be applied molten, and this is readily done if the circular stage of the turntable is heated while a small quantity of the cement is melted in a capsule. The cement is then apphed with a brush exactly as though it were a varnish and permitted to cool. These colored cement back- grounds were widely used in the old days and should receive more attention than is at present the case. Selection of a Cement to Hold the Object in a Cell This is the most difficult, as well as the most important, of the decisions which have to be made. A well-prepared dry wholemount should not have any visible cement obscuring the object, but the ob- ject must at the same time be so firmly held that it will stand the relatively rough handling to which most slides are sub- jected. If the sUde is to be mounted as a transparent wholemount, there is nothing, in the author's opinion, which can com- pare with gum tragacanth; and a simple dispersion of this gum in water, with the addition of some preservative such as thymol, is better than any of the more complex formulas. Tragacanth has the useful property of being transparent in thin films, but these thin films are not strong enough to hold objects larger than diatoms or butterfly scales. To use this gum one takes the slide on which the selected cell has already been prepared and turns, with the aid of a turntable, a very thin uniform layer on the bottom of the cell. The preparation of this thin uni- form layer requires experience and skill for which no description can substitute. The adhesive is then allowed to dry and the objects are arranged on it in the re- quired positions. As soon as all the objects have been laid on the dry film, it is placed in a moist, warm atmosphere wliich is usu- ally secured by bending open-mouthed over the shde and breathing very slowly and carefully. This makes the layer of tragacanth sticky so that the objects ad- here; it will dry again in a few moments. This was the method used to prepare those pictures, made with the scales of butterflies, or selected diatoms, which used to be a feature of the catalogs of old- time microscope preparers. There is, how- ever, no reason why the method should not be employed for scientific purposes, for it is often desirable, particularly when dealing with diatoms, to arrange them in a selected pattern. Small objects of this type may readily be placed in position if they are picked up on the end of a hair attached to a needle-holder; if they do not stick to the hair at the first trial, it is only necessary to moisten it with the hps. Gum tragacanth may also be used to attach larger objects (such as Foraminif- era and Radiolaria) to a paper background but it will not stick satisfactorily to either a resinous or wax surface. For these larger objects it is necessary to take a very small sable brush and place a drop of tragacanth on the surface of the paper background which has previously been moistened. The individual object is then picked up and pressed into the surface of the drop, which is then allowed to dry. If the appearance of the preparation is not very important a fairly thick smear of the mucilage may be placed all over the paper and the objects sprinkled on; this gives, however, a clumsy and unfinished appearance. The principal objection to the use of optical-dead-black varnish as a back- ground is that aqueous adhesives will not adhere to it. Both gum arable and gum tragacanth will stick for a certain length of time, but the author has never known a mount made with these adhesives in which the object did not loosen within a period of two or three months. Nothing is more annoying than to take the trouble to make a mount of ^elected foraminiferans and then, a few months later, to find one or two specimens rolling about inside the Cements DRY WHOLEMOUNTS 15 cell. If you are using an optical-dead-black based on gold size, clear gold size itself is th*e best cement. A series of fine drops of gold size are placed in the positions which the objects are subsequently to occupy, and the objects added one after another. This will result in perfect adhesion, but the sUde will have to be left uncovered and flat for at least two or three days, to harden the gold size before the cover is attached. When using an optical-dead- at a relatively high temperature and all too often this high temperature causes the dead-black cement to break away from the glass. When using these cements, a piece about one third of the size of the object which it is desired to attach is broken from the mass. These pieces of cement are placed on the bottom of the cell in the positions which the objects will occupy and the slide placed on a hot table to melt the cement. The author prefers Fig. 8. Using a warm table to attach cells with marine glue. black cement secured from a scientific supply house, it is necessary to secure some of the varnish medium in which the black has been suspended. The writer has seen hundreds of commercially prepared strewn sHdes of Radiolaria and Forami- nifera in which gum arable had obviously been used on black varnish backgrounds and in which from a third to a half of all the specimens were loose. As a second choice to the varnishes, there may be em- ployed one of the Canada-balsam-resin cements of the type of Fant 1932 (Chapter 28, V 12.2), or one of the Venice-turpen- tine cements of the type of Gage 1896. Un- fortunately these cements have to be used the type of table shown in Fig. 8 which consists of a strip of heavy metal, prefer- ably copper, bent back twice on itself and mounted on four legs. The top of the upper strip projects beyond the bent por- tions. A burner is placed under this pro- jection and adjusted to keep the end of the strip well above the boiUng point of water. It will be seen that the temperature steadily diminishes from shelf to shelf, that of the upper shelf being highest, that of the second shelf being lower, while the bottom shelf is scarcely warm. To deter- mine whereabouts on the shelf to place the slide it is only necessary to place a few chips of cement at about one-inch inter- 16 THE ART OF MAKING MICROSCOPE SLIDES Cements vals along the first and second shelves. After a few moments it will be apparent which point is just at the melting point of the cement in question. The slide bear- ing the small pieces in the position where the objects are to be mounted is then placed at this point and each object is individually placed in its own little pool of molten cement. It must be left at this temperature long enough for the object to reach the temperature of the cement or it will not stick. It is best not to cool these preparations suddenly, so that it is the author's practice to take them from the hot shelf on which the cement is molten, remove them to the shelf under- neath, and after they have cooled to that temperature to place them on the bench for their final cooling. For attaching opaque objects, particu- larly those of relatively large size, nothing is simpler than the wax-resin backgrounds which have been mentioned. In using these, a fairly thick coating is applied to the warmed slide and the object dropped into place. It is left until it has reached the temperature of the cement, or the cement is seen to be "creeping," and is then cooled. These media are more fre- quently employed by botanists for mount- ing dried-spore cases of mosses, and the like, but they also work admirably for zoological specimens. Selection of Cement for Attaching the Cover Glass Before discussing the selection of a cement for the attachment of a cover glass, which is the last step in the preparation of a dry wholemount, it is necessary to insert a warning that the word dry as applied to dry wholemounts must be interpreted literally. If wholemounts are being made in an American laboratory in winter at an inside temperature of 70°F. and an out- side temperature around 0°F., no diffi- culty will be encountered since the atmos- pheric humidity is practically nil. If, how- ever, the humidity is relatively high some method of drying the specimen must be used. The writer has in his possession several imperfectly sealed dry whole- mounts of ground sections of bone, made in Europe about forty years ago, which are entirely covered with fungus hyphae. The object may, it is true, be treated with some fungicide but this is rarely as effec- tive as, and usually more trouble than, making sure that the mount is dry before sealing. If the object has been attached by one of the techniques which involves heat- ing the slide and cement, it will probably have dried sufficiently, but it is desirable to make sure by leaving the uncovered mount overnight in a desiccator over some standard desiccant. When a coversUp is to be attached to a gold-size or other cement cell, it is best to use the same cement as was used in the preparation of the cell. A thin coat of this cement is applied to the top of the cell and left until it becomes tacky. A clean cover- slip is then placed on top and firmly pressed into position with a needle. It is easy to see whether adhesion is perfect and, if necessary, a small quantity of cement may be added from outside. It must be emphasized that only a very thin coat should be used because a thick layer will inevitably run in by capillary attrac- tion and thus ruin the specimens which have been mounted. It is really not important what cement is used when a coverslip is to be attached to the top of a paper, cardboard, or plastic cell. The author invariably uses gold size, largely from force of habit, but any liquid cement or varnish is adequate. A thin layer is painted on the upper surface of the cell and the coverslip pressed into place. The preparation should now be placed on one side luitil the adhesive is dry, and then finished with a coat of some black cement. Asphalt varnish [Benoit- Bazelle (1942) is an excellent formula] or Brunswick black (Beale 1880) both have the required characteristics of providing a waterproof seal while retaining a certain amount of flexibility. These formulas, and those of other suitable cements, are given under V 12.2 in Chapter 28. The old seal- ing-wax varnishes, and the modern cellu- lose-ester varnishes have the disadvantage that they tend to become brittle and break ofT after some years. Foraminifera DRY WIIOLEMOUNTS 17 Specific Example Preparation of a Strewn Slide of Foraminifera or Radiolaria Wholemoiints of the dried tests of Foraminifera, either fossil or I'cccut, are customarily referred to as strewn slides even though the individual tests may be arranged in place. Tests of Foraminifera may be obtained either from sand, from marine sludge, or from fossil deposits, and the method by which the shells are sepa- rated is in each instance different. Radio- laria are almost invariably obtained from fossil deposits. Forminiferal sands, which may be pur- chased or collected, are the best source of material. Large numbers of shells are thrown onto beaches in many parts of the world where they form whitish ridges. If such a ridge is observed it is only necessary to scoop off the surface with a spoon and to preserve it for further examination. Many scientific supply houses sell these sands. The separation of the dried shells from the sand is relatively simple. The whole may either be sprinkled onto the surface of a large vessel of cold water, in which case the majority of the shells will float, since they are filled with air; or carbon tetrachloride, the high specific gravity of which ensures that Foraminif- eral shells with only a small quantity of air enclosed will rise to the surface, may be substituted for water. If only a few shells are required they may be picked from the surface of the flotation medium with a brush and laid to dry on a disk of fine filter paper. If all the shells are re- quired, the surface layer containing the floating shells should be poured off through a fine sieve. Bolting silk is the best mate- rial from which to prepare this sieve, though fine brass screen wire may also be used. The separation of tests from marine deposits dredged from the bottom is not so easy since they are, in this case, mixed not only with particles of sand but also with considerable quantities of fine sludge which may include some organic matter. If the sludge is free from organic matter, the mass may be passed through a series of sieves under a jet of running water; but even under those circumst:iiices the tests will usually be discolored. Laporte 1946, p. 194 recommends that such tests be boiled in an alkaline solution of calcium hypochlorite {eau de Javelle) which serves the double purpose of bleaching the stains and removing any trace of organic matter which may remain. If the sludge is heavily contaminated with organic matter, the mass should be boiled for some time in a weak (2%) solution of sodium or potas- sium hydroxide before being sieved. Cush- man 1940, p. 26 suggests also that tests may be separated as the old gold miners separated gold by rotating the mass in a flattened dish. The Foraminifera, being somewhat lighter than the rest of the material present, will collect round the edges of the dish, from which they may be jerked with a circular motion. Tests of fossil Foraminifera may^be ob- tained from sandy deposits. They are also found embedded in clay deposits, or chalk. Foraminifera concreted in limestone can- not, in most cases, be removed and made into wholemounts. Foraminiferal tests ob- tained from sandy deposits may be sepa- rated by flotation in the manner already described, but these shells are usually dirty either from chalk or clay, and must be thoroughly cleaned if a satisfactory mount is to be prepared. It is best to boil them after they have been separated in a 5% solution of sodium carbonate. After they have boiled for some time the beaker containing them is removed from the flame and the Foraminifera are al- lowed to settle to the bottom. The cloudy alkahne solution is then poured off and re- placed with fresh solution and this is re- peated until the tests are sufficiently clean. If the dirt is particularly tenacious, it may often be loosened by boiling the tests in a relatively small quantity of the alkaline solution which is then poured while boiling over a mass of cracked ice. This sudden temperature change will often loosen dirt which cannot be removed by jg THE ART OF MAKING MICROSCOPE SLIDES Radiolaria any other method. When tests are cleaned blown off to diminish the pressure as in this manner it is essential that they rapidly as possible. The repetition of this should be thoroughly soaked, and prefer- process resu ts m the disintegration of ably also boiled, in a large quantity of materials which resist every other method, distilled water to remove the alkah. The separation of f oramini eral tests Methods for the separation of forami- from chalk is a relatively simple process, niferal tests from shale and clay deposits If one is only collecting the tests at ran- vary according to the degree of hardness dom, so that i does not matter if many of of the deposit The first exploratory step the more fragile forms are broken, the old should always be to boil the mass in a 5 % method of brushang under water has much solution of sodium carbonate. If the to recommend it. A piece of chalk is held solution speedily turns cloudy, it is in one hand under he surface water and a evident that the material is being dis- brush (an old tooth brush is excellent) is integrated satisfactorily and it is only scrubbed over the surface. L^^ge numbers necessary to continue boiUng long enough of tests, which fall to the bottom of the for the tests to separate. The cloudy solu- container, are removed by this method tion should be stirred up and poured off while the chalk remains m suspension and from time to time into a large cyhnder of can be poured off. Tests prepared by this distilled water. This should be allowed to method are never clean and must sub- stand for about 10 minutes, to permit all sequently be boiled in alkah to remove the foraminiferal tests to fall to the the adherent chalk. If it is desired to col- bottom and the cloudy supernatant lect the greatest possible number of shells, hauid then poured off. This may be re- chalk can often be disintegrated by boihng neated as often as experience shows to be either in 5% potassium hydroxide or m necessary to collect a mixture of forami- 5% sodium carbonate; this is, however, a niferal tests and fragments of the shale prolonged and messy business Chalk may mass at the bottom of the cyhnder. Sepa- also be disintegrated by the freezing and ration of the tests from the shale frag- thawing process, or by the autoclave proc- ments may either be by hand under a ess already mentioned. ^ ,. . . binocular microscope, or the mass may be The sihceous skeletons of radiolarians dried in an oven and sprinkled on cold are cleaned altogether differently and it water, or carbon tetrachloride, for the is difficult to improve on the method of flotation method previously described. If Roudabush 1938 {Ward's Bui 9). No the preUminary boiUng in sodium carbon- prehminary treatment is needed for ate does not result in a sufficiently rapid the easily disintegrated Barbados earths disintegration, two possible methods re- though other material may have to be dis- main The old method used to be to soak integrated by one of the methods already the mass thoroughly in water and then to described. The disintegrated pieces are reez^Tt; a household freezer giving tem- then boiled in 10% potassium hydroxide oeratures of -10° or -20°C. is excellent, for about 20 minutes. Throw the whole The frozen piece is then removed and mass into 10 times its own volume of thrown into boiUng water which almost water, stir vigorously, and allow to settle invariably breaks the mass into smaller for 10 minutes. Pour off the milky solu- Dieces This process of alternately freezing tion; refill the beaker with water, btir and boiUng is continued until the pieces vigorously, allow to settle for 15 seconds have become sufficiently smaU to enable and save the supernatant hquid. Repeat one to complete the separation of the tests the process; the two batches of decanted with boiUng alkaU. water will be found to have most of the An interesting alternative method for hberated radiolarians. , , ^, . shale has been suggested by Driver 1928 The pieces remaining at the bottom ot (J Pal 1 -253) who subjects the pieces to the beaker can be again boiled witti iu /o the action of high pressure steam in a potassium hydroxide and further batches laboratory autoclave. The pressure is run of liberated radiolarians poured off and up to about 20 lbs., maintained at this for accumulated, a few minutes, and the autoclave then The cleaned radiolarians in the ac- Foraminifera DRY WHOLEMOUNTS 19 cumulated decantations are allowed to settle for about 20 miinitos after the last batcli has been added and the suj)ernatant water poured off. Add carefully about twice as much nitric acid as there is sludge and boil for 20 minutes. Again wash with water, allow to settle antl decant. Now carefully add twice as much 10% potas- sium hydroxide as there is sludge, boil for 20 miiuites and again wash by decanta- tion. The material is now nearly clean — if it is not, repeat the nitric acid-potassium hydroxide cycle. Concentrate the nearly clean skeletons and then cover them with their own vol- ume of ammonium hydroxide. Stir at intervals for about 10 minutes, then slowlj^ add an equal volume of nitric acid. Wash thoroughly by decantation and ex- amine the skeletons; if they are not now clean, repeat the ammonium hj-droxide- nitric acid cycle. Concentrate the sludge as much as possible, wash it thoroughly with 95% alcohol, and then either allow to dry for strewn slides or store in alcohol and make balsam mounts in the manner described in Chapter 6. After the foraminiferan tests or radio- larian skeletons are accumulated in a small watch glass, they should be dry and quite free from any of the reagents used to clean them. It is also necessary to have ready on the bench a binocular dissecting micro- scope, two fine sable brushes, sHdes on which cells have already been cemented, and a container of mucilage of gum tragacanth. The author prefers to make all forami- niferal mounts in cells prepared from rings of vulcanite with bottoms of black paper. These should have been prepared the day before in the following manner. First take the required number of slides and clean them thoroughly. It is not nec- essary for the slides to be chemically clean — it is necessary only that the slide should be grease-free. A simple method of de- greasing slides is to take a commercial scouring powder, of the type used for household purposes, and make it into a paste with water. This paste is smeared Hberally on all surfaces of the required number of slides which are then dried. When the shde is dry, the scouring powder is removed by vigorous rubbing with a soft cloth. While the slides are drying, the retpiired number of vulcanite cells are laid out and a piece of fine sandpaper secured. Each cell is held on the sandpaper with the ball of the first finger and rubbed, with a circular motion, until all the lower surface has been abraded. The cell is then turned over and the process repeated. One side of the cell is then given a thin coat of gold size and placed with firm pressure on the center of a glass shde, which should then be left for three or four days. Any gold size which has been pressed out of the inner surface should be removed with the edge of a sharp pointed scalpel. A number of disks of black paper of the required size are then taken, coated on one side with any satisfactory adhesive, and pressed onto the bottom of the cell. It must be remembered that the cell should be of such a size that the coverslip, when laid on top, does not reach to the outer edge of the cell but only halfway across it. This may conveniently be done by using a %-inch cell with an 18-milhmeter covershp. The thickness of the cell selected is not of major importance but the writer usuall}^ prefers about }i2 of an inch when mount- ing Foraminifera. Let us suppose first that it is desired to prepare, from the materials at hand, an ordinary strewn shde hke those sold by biological supply houses. It is only neces- sary to moisten slightly the paper at the bottom of one of the prepared cells and to smear mucilage of tragacanth liberally over the surface. Plenty of tests are thrown onto the mucilage and the shde is placed on one side for about 10 minutes to dry. As soon as the mucilage is dry, the slide is inverted over the watch glass con- taining the tests and tapped sharply with the forefinger. This will cause all those tests which have not become attached to the mucilage to fall back into the stock, and will usually leave a continuous coat of foraminiferal tests over the black paper. It is generally, however, more satis- factory to mount selected tests in the re- quired position. In this case, the black paper on one of the prepared slides is thoroughly moistened and a fine sable brush is used to place small portions of mucilage of tragacanth in the positions which the selected tests are to occupy. 20 THE ART OF MAKING MICROSCOPE SLIDES Foraminifera Each drop of tragacanth should be slightly smaller, both in breadth and in thickness, than the test which is going to be placed on it. These drops of mucilage are most conveniently placed in the correct position with the aid of a binocular dissecting microscope. As soon as the drops have been placed the slide is pushed out of the field of the dissecting microscope and the watch glass, containing the specimens to be mounted, pushed into the field. A clean sable brush is then moistened with the lips. It should be sufficiently wet to cause the tip of the brush to come to a point but not suf- ficiently impregnated with saliva for any liquid to be showing. The tip of this brush is touched down onto the required speci- men and held in the field of the dissecting microscope with the right hand, while the left hand pushes the watch glass of speci- mens out of place and replaces the glass slide. It cannot be emphasized too much that the paper must be liberally moistened or the drops of gum tragacanth will dry in the period of time that it takes to select tests. If, when the cell is replaced under the binocular microscope, it is ob- served that the mucilage is dry, do not at- tempt to remoisten it; place another drop of mucilage on top of the dry portion. The shell, on the tip of the fine brush, is now pressed down in the selected position. If it is not exactly as required, it may be adjusted with the tip of a needle. After aU the tests required on any one slide have been placed in position, a fine sable brush is used to place a drop of water on top of each shell. This makes certain that there will be a perfect adhesion of the shell to the underlying mucilage. On a very dry day (that is, one on which the relative humidity is below 20%) the preparations may be sealed immediately; on humid days it is best to place the shdes in a desiccator overnight. In either case, the next step is simple. The top of each cell is spread with a very thin coat of gold size and a clean coverslip dropped into position. It is best to place one edge of the coverslip down first, supporting the other edge with a needle, and then to lower it by withdrawing the needle. As soon as it is in contact with the cell it is pressed down all round its circumference with a needle. It is not important that it should be in contact all over since further coats of cement will be placed on top. The initial coat of gold size is intended only to hold the coverslip in position through the next stages and it is better to have a very thin coat with an imperfect adhesion than to have a coat so thick that cement spreads onto the inner surface of the covershp. The slides, with their attached covers, are then placed on one side, preferably in a desiccator, for a period of about 24 hours to set the gold size. The shde is finished on a turntable (Fig. 7) by turning onto the upper surface of the cell a coat of any selected cement. The author prefers either asphalt varnish, or Brunswick black, though any tough and flexible cement may be used. It is quite important that the cell should be accurately centered on the turntable, and though this may be roughly done with the aid of the concentric circles engraved on the table, it is usually neces- sary to spin it once or twice and to make necessary adjustments manually. If lack of experience renders this difficult, it is suggested that a needle should be held stationary above the edge of the cell and the turntable rotated slowly. The table should be stopped when the edge of the cell (presuming it to be eccentrically placed) is at the maximum possible dis- tance from the needle. The cell is then pushed one half of this distance towards the needle, the needle replaced over the edge of the shde, turned as before, and readjusted. By this method even the most inexperienced can center a cell perfectly within a few moments. Only one coat of varnish is really necessary, though some people prefer to put on four or five, using the last coats to fill up the edge of the cell which is thus doubly protected. In the author's opinion this is not necessary and makes a clumsy mount. If, through accident, a test becomes detached in one of these slides it may be repaired easily. The coverslip should be broken by a sharp blow with the handle of a scalpel and the pieces removed with a pair of forceps. The top of the cell is then scraped clean with a scalpel and the test recemented in place. Fluid Wholemounts — Aqueous Type General Principles A fluid wholemount in an aqueous me- dium is essentially a miniature museum mount in which the glass jar has been re- placed by a cell mounted on a microscope slide. With the exception of the selection of a slide — for none other than glass is suitable — the choices confronting the technician are very much those discussed in the preparation of dry wholemounts in the last chapter, though the selection is in each instance different. It is necessary to select successively a type of cell, a cement for attaching the cell to the sUde, a cement for attaching the coverslip to the cell, and finally the mounting medium itself. Selection of a Cell The author prefers to use concave- ground glass slides instead of cells. At the present time these concave glass shdes are both difficult to obtain, and unsatis- factory when obtained, from American sources. It is, however, possible to secure in Great Britain slides into which have been ground circular concavities from 9 to 20 milhmeters in diameter, or oval con- ca\'ities in many sizes. The use of cavity sUdes avoids the difficulties of attaching a cell, and the slides are more waterproof than any cell. It is to be hoped that Ameri- can suppliers will make cavity slides avail- able to technicians who wish to make wholemounts in fluid media. If, however, cells must be used, the choice is very hmited. It is a waste of time to take paper and cardboard cells and to endeavor, by soaking them in various resins, to make them take the place of a plastic or metal cell. Cells of vulcanite anb tin are obtainable or may be pre- pared with the aid of a punch. These should, before use, be flattened on both sides in the manner described in the last chapter. Where a very deep mount is re- quired, it is better to use a glass cell which can be cut from thick-walled glass tube and ground flat on both faces. These cells are usually only obtainable in a %-inch size and care should be taken, as with other cells, to make sure that the edges of the coverslip selected will he on the sur- face of the cell. It is difficult to seal a dry wholemount, and impossible to seal an aqueous wholemount, in which the edge of the coverslip and the edge of the cell coincide. An almost perfect relationship is that of an 18-millimeter covershp to a fi-ineh cell, but unfortunately both 18 mm. and 3-^-inch appear to be used interchangeably by scientific supphers so that the measurements must be checked before mounting. When very thin objects are to be mounted, a cell can be made from gold size in the manner described in the last chapter. Selection of a Cell Cement The selection of a cement to attach the cell to the shde is of far more impor- tance in aqueous fluid mounts than in dry mounts. The cement must not only lje capable of holding the cell firmly to the glass, but must also make a waterproof seal which must remain waterproof for many years. In the author's experience no varnish is satisfactory, and one is forced to turn to the thermoplastic cements. Among these marine glue (Chapter 28, V 12.2 Beale 1880) or, if this is not obtain- 21 22 THE ART OF MAKING MICROSCOPE SLIDES Cements able, the very similar cement of Harting 1880 are the best. The marine glue here specified bears no relation to the so-called marine glue commonly sold today. The old-style marine glue, which is essentially a mixture of rubber (or gutta-percha) with shellac is one of the most water-resistant cements ever invented. This style of marine glue can still be obtained from suppliers of microscope-mounting acces- sories in Europe but does not appear at present to be on the market in the United States. If it is unobtainable, and the technician is unwilUng to make his own supply, gold size is the next best sub- stitute. This gold size must, however, be of the old-fashioned kind specified for microscope mounting and not one of the new varnishes which are placed on the market for the benefit of gilders. It should perhaps be explained at this point that gold size was the material used by the early gilders to apply sheets of gold leaf on large areas. It was partially polymer- ized and partially oxidized linseed oil mixed with small quantities of resin and diluted with turpentine. To the old gilders it had the advantage that it took a long time to harden so that it retained a tacky surface, to which the gold leaf could be applied, over a long period. The advan- tage of this material to the maker of microscope slides is that both boiled linseed oil and turpentine will selectively "wet" glass — that is, they will displace a fine film of water from the surface of the glass. They can therefore be applied to damp glass to which they will remain adherent. Modern gilder's varnishes — sometimes called gold size — have the advantage to the modern gilder that they remain tacky for any specified period; to the microscope mounter they have the disadvantage that they are made in the interests of the gilder, not of the tech- nician, and rarely contain ingredients which will adhere to moist glass surfaces. The attachment of a cell with gold size was described in Chapter 1 and need only be briefly reiterated. The slide is placed on a turntable (Fig. 7) and a ring of gold size of about the width of the cell turned on the center of the slide. The undersur- face of the cell is given a thin coat of gold size and both the slide and the cell are placed on one side until the varnished surfaces have become tacky. An additional thin coat of gold size is then applied either to the cell, or to the slide, and the two pressed together. Since, however, a water- proof seal is required, the cell must be pressed firmly against the slide until the cement has hardened, either by laying a heavy weight on top of the cell, or by placing another slide on top and clamping the two together. The attachment of a cell with marine glue is an altogether different proposition. If a solution of marine glue is used, a thick ring is turned on the sUde and a thick coat is applied to the underside of the cell. Both cell and slide are then warmed (the lowest step of the hot table shown in Fig. 8 may be employed) until all the solvent has been driven off. The slide is then laid on the upper shelf, which should be heated above the melting point of marine glue. The cell is placed on the now molten ring of cement and maintained in constant contact with it until its own coat of cement has melted and fused with the cement on the slide. The slide should next be transferred to the second or third shelf (which should be just below the melting point of the cement) and a heavy weight placed on top while the cement slowly solidifies. After a few minutes at this solidification temperature, the slide is re- moved, still with the weight or clips in position, and laid on one side to cool. It is then turned upside down and in- spected to make sure that no air bubbles have been caught in the cement. If solid marine glue is used, chips must be scraped from the block with a knife. A layer of these chips is then placed on one surface of the cell (which in this case must be of tin or some other metal) and the cell laid on the upper shelf of the hot table at a temperature which will melt the cement. A heated needle is used to remove as many air bubbles as possible from the molten cement and the glass slide is laid alongside it on the hot table. The hot slide is then pressed firmly to the molten cement on the upper surface of the ring. As soon as the ring is firmly pressed into place the slide is inverted, placed on the second Preservatives FLUID WITOLEMOUNTS — AQUEOUS TYPE 23 shelf, and a heavy weight placed on top until the cement is cooled. Whether gold size or marine glue be em- ployed, care must be taken to remove those portions which have been extruded into the interior of the cell. These cements swell up and become white in the presence of water, and even a trace of remaining cement will give an unfinished appearance to the slide. The excess cement may be removed by scraping with a scalpel, and a final cleaning may be given with a 10% solution of potassium, or sodium, hydrox- ide, which is wiped over the inside of the cell with a piece of cotton held in a pair of forceps. The cell is then thoroughly washed and laid on one side to dry. It is best to cement cells onto slides in ad- vance of requirements and thus secure an adequate reserve. Selection of a Preservative Medium The introduction of formaldehyde to microscopic technique was welcomed as the beginning of the millennium, and almost all of the older aqueous media were thrown overboard by mounters. This is to be regretted since formaldehyde is by no means a perfect medium, particu- larly for the preservation of small inverte- brates and single-celled plants, so that attention should be given to the list of aqueous preservative media in Chapter 17 (P 11.1). These media are mostly variations on the fluid of Goadby which was a mixture of sodium chloride and am- monium alum — designed to approximate an isotonic solution — containing a very small quantity of mercuric chloride as a preservative. Some of these solutions (cf. Kronecker 1907) had an alkaU added to preserve the green color of small algae. Another excellent preservative of green material is the solution of Ripart and Petit which is given in Chapter 18 (F 3000.0010 Ripart and Petit 1884) because it serves the dual purpose of fixation and preservation. A very similar formula was pubhshed by Woods in 1929 (Chapter 17, P 11.1) as a preservative for green algae. Simple solutions of various reagents may also be employed. The best of these is formaldehyde, which for purposes of mounting should never be neutraUzed since, once subjected to this treatment, it is liable to develop precipitates. The ordinary 1 to 10 dilution of 40% formalde- hyde, which is commonly employed for the preservation of gross biological speci- niens, is far too strong for microscope mounting of the type being discussed. It must be remembered that these strong solutions become greatly diluted from the water contained in the specimens placed in them, whereas in the case of a microscope mount the material will have already been impregnated with formaldehyde before being mounted. A dilution of 1 to 100 of the commercial 40% formaldehyde is adequate as a mounting fluid. Camphor water and chloroform water, which are merely saturated solutions of these rea- gents in distilled water, are also excellent preservatives for the more delicate Pro- tozoa and Algae. It must be emphasized that if glycerol is added to these media, the material will have to be handled by the special methods necessary for making glycerol mounts, which are described in the next chapter. Selection of a Coverslip Cement Though the cell is best attached to the slide with a thermoplastic cement, it must be obvious that a liquid cement must be used to attach the coversHp to a cell containing a fluid mounting medium. Numerous formulas have been developed for this purpose, and the author most warmly recommends either gold size, or the cements given under Behrens 1883 or Carany 1937 in Chapter 28 (V 12.1). These last two cements are quick drying, which is desirable, since at least three successive coats must be used properly to seal an aqueous wholemount. The first of these coats is designed to block off the water, and to provide a temporary support for a second layer of waterproof cement which would not adhere to the moist glass. This second coat of waterproof cement should always be an asphalt varnish (formulas are given in Chapter 28, V 11.2) and, if the black color is ob- jected to, any colored varnish may be coated over the asphalt to provide a more finished appearance to the mount. 21 THE ART OF MAKING MICRORCOrE SLIDES Sealing Sealing the Coverslip in Place It was pointed out in the last cliapter, and must be reiterated here, that before any sealing cement can be applied a pro- tective barrier must be erected to prevent this cement from running in by capillary attraction and mixing with the contents of the cell. More wholemounts are spoiled by this running in of cement than by any other method. The procedure, when mounting on a flat slide, or one containing a concavity, is somewhat different from that which is used in sealing a cell. The former will be described first. The process of attaching a coverslip and sealing it in place on an aqueous fluid mount in a concave shde is shown in sec- tion in Figs. 9-14. Fig. 9 shows a longi- tudinal section of the concave slide with the protective ring of cement in place. This ring should be the narrowest and the thickest which can be made with the aid of the finest sable-hair brush. It should certainly not be wider than >^4 of an inch, and if it can be built up to twice this depth, it will be all the better. This ring also assists in attaching the coverslip, so that the gold size should be given time to become tacky before the mount is made. It is a matter of convenience to run such rings on the turntable on the night before the mount is to be made. A ring made from a good specimen of gold size will remain tacky for at least 48 hours after it is turned. The diameter of this ring is quite critical. It should be at the outer edge about J-^4 of an inch less than the diameter of the coverslip. If it is smaller than this, too big a space will be left between the edge of the cell and the coverslip; if it is larger than tliis, perfect sealing is impossible. Fig. 10 shows the same slide after the object has been placed in the cavity and a sufficient quantity of the selected mount- ing medium placed on top of it. Notice that a great excess of the medium is pro- vided and permitted to rise up in a convex meniscus. After this drop has been placed in position the mount should be inspected carefully to make quite certain that the fluid is in contact with the protective ring of varnish all the way around the edge. If the slide is perfectly clean it may so happen that the meniscus does not extend to the varnish ring, leaving a small air gap which will result in a bub- ble — almost impossible to remove sub- sequently. The next step is that of placing the coverslip in position. The covershp must never be let down from one side in the manner customarily taught in making balsam mounts. It must be held between the thumb and second finger and lowered horizontally until it is in the position shown in Fig. 11. It will be seen that the object remains in the central position in which it started whereas, if the cover were lowered from the side, the object would inevitably be pulled by capillary attrac- tion to one corner whence it would be almost impossible to displace it. Fig. 12 shows that the covershp has been let down and pressed with a needle onto the surface of the tacky protective ring of gold size. The excess fluid has been pushed out and mopped up with a filter paper. Care should be taken to remove the whole of the fluid between the outer projecting edge of the covershp and the ring to which it is attached. This is one of the most critical stages in the whole procedure. The needle used to press the covershp in place should be run with a circular movement round the covershp vertically above the protective ring, and pressure should be continued until the glass is clearly in contact with the gold size at all points. The shde is then placed on the turntable, centered, and a ring of the selected second cement applied round the edge. The result of this is shown in Fig. 13, where it will be seen that the edge of the coverslip is firmly embedded in the cement which has run under as far as the pro- tective barrier. The existence of the pro- tective barrier and the overhang of the covershp insure, therefore, that there shall be a good, thick layer of this cement in position. The shde is now laid on one side until this first protective layer is thoroughly dry and then (Fig. 14) as many rings of asphalt varnish turned over the top as are required. It is an excellent thing to apply two coats of asphalt var- nish, naturally permitting the first to dry before applying the second, at the Sealing FLUID WHOLEMOUNTS — AQUEOUS TYPE 25 Cavity Gold size ring Drop of preservative Object 10 ,^ Coverslip 11 Preservative withdrawn 12 ^_ Gold size coat sealing 13 Asphalt varnish ^^^finishing coat ^^^ . 14 Figs. 9 to 14. Longitudinal section of a cavity slide showing successive stages in the preparation of an aqueous wholemount. 9. Protective ring of gold size turned. 10. Object placed in position in a large drop of preservative. 11. Coverslip held horizontally in con- tact with preservative. 12. Coverslip lowered and preservative withdrawn between protective ring and edge of coverslip. IS. Heavy sealing coat of gold size applied. I4. Finishing coat of asphalt varnish applied over gold size. 26 THE ART OF MAKING MICROSCOPE SLIDES Deep cells time of making the slide and to turn an additional coat on top every two or three years. Slides treated in this manner may be kept for as long as 20 years without any air bubbles appearing. The petroleum jelly method of Spence, which in the author's opinion is more applicable to Drop of preservative Object to the side of the cell or dissolved in the mounting medium. It is best to remove dissolved air from the medium either by boihng or by placing a small beaker of the medium under a vacuum until all the dissolved air has been removed. A pro- tective ring is turned as before (Fig. 15) Gold size ring Cell 15 Coverslip X 16 Preservative withdrawn ML 17 Gold size sealing coat 18 Asphalt varnish finishing coat 19 Figs. 15 to 19. Sections showing successive stages in the preparation of an aqueous wholemount in a deep cell. 15. The cell has been cemented to the slide, and a gold size ring turned on its inner edge, before being filled with fluid. 16. The coverslip is slid into position. 17. Coverslip pushed into place and preservative withdrawn between gold size ring and edge of cover- slip. 18. Heavy sealing coat of gold size applied. 19. Finishing coat of asphalt varnish applied over gold size. glycerol than to aqueous mounts, is given in the next chapter. For a descrip- tion of a modification of this method ai> plied to aqueous mounts, refer to Spence 1940 (il/icroscope, 4:121). The method of mounting in a relativel}' deep cell is shown in Figs. 15-19. Par- ticular care has to be taken in this case to prevent the appearance of air bubl)les which may come from air either attached but it will he seen, in this case, that this protective ring is on the inner edge of the upper surface of the cell. The cell is then filled with the preservative fluid, allow- ing an excess to rise in a concave meniscus. To make sure that no air is caught on the irregular surface of the inside of the cell one may now either place the whole under a vacuum or, more conveniently, take a clean brush and wipe the inside of Algae FLUID WHOLEMOUNTS — AQUEOUS TYPE 27 the cell with it. Particular attention should be paid to the junction of the cell with the slide, whore tnipi)ed air bubbles are often caught. The author finds it best not to lower the coverslip horizon- tally, as in the previous mount, but to shde the cover horizontally onto the cell. This is shown in Fig. 16 where the cover- slip has reached halfway across. This illustration is shghtly exaggerated since the cover may be started farther across and sUd only the last few miUimeters. Care must naturalh' be taken that the gold-size protective ring is sufficiently dry not to smear the coversUp as it is pushed. If, when the cover reaches nearly to the other side of the cell, a small air pocket is left, it may be filled with moun- tant and the coverslip pushed neatly into place. It is also possible to lower the coverslip from one side — that is, to place one edge in contact with one edge of the cell and to lower the other with a needle — provided that object is sufficiently large not to become displaced. Fig. 17 shows the covershp in place after it has been pressed in contact with the protective ring and after the mounting fluid has been wiped from the outside. This is more difficult, and must be done more carefully, than in the case of the flat mount previously discussed. A ring of the first sealing cement is then (Fig. 18) applied to fill the gap between the ovcrliip of the coverslip and the protective ring on the inner edge of the cell. It is not necessary to do this on a turntable since this cement need not come onto the top of the coverslip at all but may be applied directly from the side. After this cement has had time to dry one should then build up (Fig. 19) several layers of asphalt varnish. So many laj^ers are re- quired to fill the angle between the cell and the coversUp that it is often desirable to use some cement containing a pigment. If a pigmented cement is used it should, however, be given a coat of waterproof asphalt varnish on the top before the slide can be considered finished. The purpose of a thick layer of cement, filling the angle between the cell and the slide, is to pro- vide additional mechanical support to the cell. The most frequent cause of break- down of thick aqueous mounts is either the complete detachment of the cell from the slide, or the cracking of the ce- ment which holds the cell in place with the subsequent intrusion of small air bubbles. Specific Examples Preparation of a Wholemount of Microcystis in the Fluid of Ripart AND Petit 1884 Wholemounts of unicellular algae pre- pared in any medium except balsam are rarely seen nowadays. These balsam mounts, though they display fairly clearly the internal structure of the alga, give the student not the faintest idea of wdiat the material looks like in life. Nothing is more valuable for the laboratory instruc- tion of classes, who will subsequently study in the field, than a series of whole- mounts of phytoplankton preserved so as to resemble, as nearly as possible, the Uving material. It is the author's opinion that the solution of Ripart and Petit, used as described in this example, gives as close an approximation to the appearance of the Uving material as can be produced. Very weak solutions of formaldehyde are often used for the preservation of vials of phytoplankton concentrates for labora- tory study, but it is not, in the writer's opinion, a satisfactory medium for the preparation of a wholemount. The blue-green alga microcystis has been selected for the present example because it hajjpens to be the most common alga found in large bodies of w^ater in the district from which the author is writing. Other blue-green and green algae may just as well be prepared by the present method. It is a waste of time to endeavor to concentrate algal collections in the field. Several gallons of the greenish water containing these specimens should be collected and brought back to the laboratory for immediate processing. There are two ways of concentrating the specimens. The first is to add to the 28 THE ART OF MAKING MICROSCOPE SLIDES Algae fluid containing them considerable quanti- ties of the required preservative, to permit the algae to settle to the bottom, and then to pour off the supernatant Uquid. One of the modern plankton centrifuges will do the job twice as efficiently in half the time. These plankton centrifuges are built as miniature milk separators save for the fact that the vertical plates of the latter are missing. That is, the plankton centrifuge is merely a small cup which is rotated at high speeds while a continuous stream of the material to be concentrated is poured into the top. The plankton organisms, being the heavier, are collected round the edge while the cleared water passes out at the bottom. The material should be put through the separator twice and with its aid it is possible to con- centrate five gallons of plankton into 100 milhliters in about five minutes. These concentrates must be processed immedi- ately; within a space of ten minutes the available oxygen in the water will have been used up and the concentrate will die with a consequent degradation of its appearance. The solution of Ripart and Petit, here recommended, may be used as a fixative for animal tissues as well as a preservative for plant tissues and is accordingly given in Chapter 18 under the heading F 4000.0010. fcit is a weak solution of copper acetate and copper chloride, acid- ified with acetic acid and with a small quantity of camphor added. More modern writers (Mayer 1920, p. 232) have sug- gested the substitution of thymol for camphor, and menthol may equally well be employed. It is unwise to use a satu- rated solution of any of these compounds, for crystals are likely to form through the slight evaporation which always takes place in a mount. One therefore takes equal quantities of a saturated solution of camphor or thymol, and distilled water, and then adds to each hter of this mixture two grams each of copper acetate and copper chloride together with seven milh- liters of acetic acid. About ten times its own volume of preservative should be added to the concentrate, the bottle con- taining which is then carefully tilted backward and forward at intervals for the next twenty-four hours before the organ- isms arc allowed finally to concentrate at the bottom of the jar and the supernatant reagent poured off. This preserv^ed con- centrate may be kept indefinitely in the dark and mounts made at any time. Preparation of the actual mounts must be done on two successive days: on the first of these the cells are prepared and placed on one side to become hard; on the second the actual mount is made. Clean sHdes are absolutely necessary and may either be cleaned in the manner sug- gested in the last chapter, or may be chemically cleaned by one of the cleaning mixtures given in Chapter 28. The sKdes should be selected rather more carefully than usual for the most minute flaw in the surface of the slide will become appar- ent in an aqueous fluid mount, even though it would be in\dsible in a colloidal or resin medium of higher refractive index. Having selected and cleaned the shdes, a ring of gold size is turned on each, care being taken that the ring is smaller in diameter than is the coverslip to be em- ployed. An 18-milhmeter ring and a %-inch coverslip form an excellent com- bination. It may be pointed out that this is the reverse of what is done when mount- ing in a tin, cardboard, or plastic cell where a %-inch cell is used with an 18-milhmeter covershp. In this case the purpose of the initial ring is not only to provide support to the covershp but also to insure that the cement subsequently used for sealing shall not run in by capil- lary attraction and ruin the mount. The ring should be as narrow as can be drawn and should be about }i4 of an inch thick when in the fluid stain. With experience, and a fine sable brush, it is possible to draw these initial rings about ^i^ of an inch in width, though Ke is permissible and wiU be more hkely in the hands of the inexperienced. As many rings are prepared as mounts are to be made and placed on one side until the next day. If, through some accident, mounting cannot be con- tinued on the next day, or possibly the day after, it will be necessary to put a thin coat of fresh gold size over the dry coat and permit this to harden for 24 hours. The condition of the gold-size Rotifers FLUID WHOLEMOUNTS AQUEOUS TYPE 29 ring when mounting is critical; it must have dried to a rubbery, but not to a hard, consistency. For the final mounting one requires at hand the shdes which have been prepared, the turntable which was used to draw the original ring, some clean covershps, a pipet of the eye-dropper type, and the concentrate of algae. A slide is centered on the turntable and a twist given to the turntable to make sure that the centering is accurate. A drop of the concentrated algae is placed in the center. If the slide is clean this drop will flow outwards until it reaches the cement ring where it will be held. Under no cir- cumstances may the coverslip be placed on the preparation until the fluid touches the ring at all points. If it does not do so at once it may be brushed out with a small brush. It does not matter in the least if it flows over the ring but it will be impossible to avoid including air bubbles if it does not reach the ring. A coverslip, held between the thumb and the index finger of the right hand, is lowered horizontally until it touches the drop of algal suspen- sion. It is then dropped in such a manner that it falls onto the ring which should be centered exactly under the coverslip. If it is not centered it is still possible to ad- just it with a needle, provided it is not too far out, as long as it has been dropped and not pressed down. If it is initially pressed down, so as to make a contact with the gold size, nothing can be done and another sUde must be taken in its place. As soon as the coverslip is centered a fine needle is taken and run round immediately over the ring to press the glass into contact with the slightly tacky gold size. Any of the algal suspension which has crept onto the top surface of the coverslip is re- moved with a soft cloth, remembering to be very careful not to shift the cover- shp; any fluid which has spread out over the shde may he wiped off as far as the edge of the coverslip itself. There will still remain a small quantity of the fluid be- tween the ring, which is slightly smaller than the coverslip, and the edge of the coverslip itself. This must be removed, using the edge of a sheet of filter paper which is touched down to the fluid. The withdrawal of this superfluous fluid from between the edge of the cover- shp and the ring, is a critical part of the proceedings. The ring on which the cover- slip rests is not sufficient to prevent evaporation but is sufficient to prevent the introduction of air mechanically while this superfluous fluid is being withdrawn. If, however, the coverslip is ever so shghtly raised by the edge of the filter paper an air bubble will inevitably enter the mount which must then be thrown away. Assuming that all has gone well, and that the superfluous fluid has been with- drawn, a heavy ring of gold size is turned on and the mount placed on one side. The ring of gold size should be at least }i of an inch wide and as thick as the ma- terial can be persuaded to flow from the brush. The mount is then placed on one side and the next one taken. A single ring of gold size will hold the mount in good condition for a few months but if permanence is desired it is better to add three other rings of gold size at daily intervals, then to wait a week and to turn on top of this a coat of asphalt varnish. The degree of permanence of these mounts is variable. The writer has one in his possession which is more than 20 years old and is as good as it was the day it was made. Preparation of a Wholemount of a Rotifer by the Method of Hanley 1949 The method of Hanlev is a modification narcotic (Chapter 19, AF 50 Rousselet of the well-known method published by 1895), and the sjiibstitution of formalde- Rousselet 1895 (11479, 5:1) which has hyde for osniic^'acid in kiUing. These been quoted without alteration in the substitutions not only render the final literature for more than 50 years. Han- preparation better and more permanent ley's method involves narcotization in his l)ut also remove the difficulties both of own narcotic (Chapter 19, AF 50 Hanley working with osmic acid and of securing 1949) as a substitute for Rousselet's cocaine. This method is of great impor- 30 THE ART OF MAKING MICROSCOPE SLIDES Rotifers tance because satisfactory wholemounts of rotifers cannot be made in either resin- ous or gelatinous media, since no method of dehydration has yet been discovered which will not distort all save a very few of the toughest rotifers. The collection of rotifers is relatively simple. Planktonic forms, either marine or fresh-water, may be taken in fine plankton nets and usually occur in con- siderable quantities where they occur at all. The tube or bottle at the end of the plankton net should be emptied into a con- siderable volume of water and kept well- oxygenated unless the specimens are to be prepared immediately. The usual methods of plankton concentration are very unsatisfactory for delicate rotifers, and it is better to rely on their attraction by light, and by high concentrations of oxygen. If, on the return to the laboratory, the quart or gallon of plankton suspension be placed on a bench and one side shaded while the other is brilliantly illuminated, all the planktonic rotifers will be found to concentrate at the surface on the illumi- nated side of the bottle. They may then be picked out without difficulty with a fine pipet and transferred to a watch glass for narcotization. If there are only a few rotifers present it may be necessary to take the jar into a darkened room and to illuminate one angle of it with a small spotlight (such as the Nicholas lamp used by embryologists) which will collect all the rotifers from half a gallon of water in a few minutes. If the jar is going to be left for some time under these conditions it is desirable to use some form of heat filter between the lamp and the jar. The collection of sessile rotifers is more difficult. They will usually be found at- tached to the stems of water plants, and to the underside of water-lily leaves. It has been the author's experience that more rotifers will be found in relatively small ponds than hi large lakes, and that if one could find a body of water several feet deep but of only a few hundred square feet of surface area, and if this water is relatively choked with large water weeds but contains only a small quantity of green algae, it is likely to contain many of the rarer forms of sessile rotifers. The distribution of these forms is, however, very scattered and it is scarcely ever worth while to collect large quantities of water weeds with a drag and then to take them back to the laboratory and hunt through them. It is far more profitable to settle down and hunt the weeds as they are in the water, cutting from them short lengths of stem or small areas of leaf which bear the required forms. These are then placed in a large jar of water from the pond and brought back to the labora- tory for further treatment. The most difficult part of the prepa- ration of a mount of the rotifer is to narcotize it correctly. Hanley (Micro- scope, 7:155) has discovered that the use of alcohol in Rousselet's fixative is an- tagonistic to the cocaine in the same solu- tion and that it is, therefore, by Rous- selet's method necessary to use very large quantities of narcotic with a resultant very short interval between complete narcotization and death. With Hanley's narcotic the narcotization is relatively rapid but the interval between complete narcotization and death is relatively long. With Rousselet's fixative there is often only a period of from one to two seconds between the moment when the fixative can be applied and the moment when the rotifer dies and is then worthless. With Hanley's narcotic, this period is extended for as long as 10 to 15 seconds and only those who have mounted rotifers by Rousselet's method can appreciate how great is this advantage. For the actual process of narcotization it is necessary to have two watch glasses, one containing the rotifers swimming in their normal environment, and the other a 10% solution of formaldehyde. These two watch glasses should be sufficiently far apart that fumes from the formalde- hyde do not dissolve in the glass contain- ing the rotifers. There is also required a supply of Hanley's narcotic, a fine pipet, and a dissecting microscope having a power sufficiently high to enable the rotifers to be seen clearly. For an average watch glass containing the rotifers two drops of Hanley's narcotic are added to the water and mixed by sucking the water in and out with a rather coarse pipet. Rotifers FLUID WHOLEMOUNTS — AQUEOUS TYPE 31 It does not matter that this treatment will cause the intifei's tn contract for tlioy will have ample opportunity, at this stage, to re-expand. The watch glass is then left alone for about 20 or 30 minutes, a further drop added and vcni cautiously mixed in; after a furtiioi- five niinutes another drop is mixed in with extreme caution and the rotifers watched under a microscope. The pH of the water, as Hanley points out, very greatly affects the rai)idity of nar- cotization which may be complete in from 45 minutes to an hour and a half. No definite data are, however, available as to the adjustment of the pH in relation to the quantity of the narcotic so that one can only j)roceed by trial and error. The author differs from Hanley as to the exact momeiit at which fixation or kill- ing should take place. Hanley states that it is safe to pick out the rotifers and trans- fer them to the formaldehyde solution when they are moving sluggishly about but do not contract when they hit each other. He further saj's that it is too late to applj' the killing agent when cihary action has ceased. It has been the writer's experience that kiUing should always take place at the exact moment when the ciha cease to move. With Rousselet's narcotic this cessation of ciliary movement is fol- lowed within a second or two by death; it has been the writer's experience that with Hanley's narcotic one has at least ten seconds of leeway which permits one to flood the watch glass with a considerable quantity of 10% formaldehyde. Which- ever method is adopted, as soon as the formaldehyde has been placed in the watch glass, it is rapidly withdrawn and replaced with fresh 10% formaldehyde in which the rotifers remain until they are ready for mounting. With Rousselet's method one used to add a drop or two of 2% osmic acid to kill the rotifers and then remove them very, very rapidly from the mixture through several changes of distilled water and then in to the formalde- hyde for preservation. This method oc- casionally resulted in the destruction of the cilia, and it was also exceedingly diffi- cult to avoid retaining sufficient osmic acid to cause subsequent darkening of the mount. As Hanley's method of sealing a wet wholemount differs appreciably from the writer's, which was given in the descrip- tion of the last example, Hanley's method will 1)6 given in some detail. The following description is taken almost verbatim from the paper of Hanley cited. If cement cells are used the cell is made beforehand and allowed to dry. When mounting, rotifers are picked out with a fine pipet and placed on the floor of the cell. The slide is then placed on the microscope stage and filled to excess with 23^^% formaldehyde. The mount is examined under the micro- scope and any foreign bodies or air bubbles removed with a fine pipet — do not run a needle round inside the end of the cement ring to remove bubbles, "unless you are fond of cement scrapings in your mounts." Much of the excess fluid can be removed with the pipet, being careful not to remove the rotifers also, and a clean coversUp then placed on the mount with flat-ended forceps. The coverslip should float on the dome of fluid and then is tapped down smartly with the base of the forceps — if this is not done smartly enough the rotifers will be washed out. The surplus fluid is removed with filter paper, changing the point of application as the rotifers move, and when nearly all tlie surplus has been removed the cover can be pushed slowly into place with a bent wire. It is important to notice that no wet cement is used on the cement cell. This is quite unnecessary with cement rings. (This is Hanley's opinion not the writer's.) If the cell is properly made, the cover glass when set down adheres so firmly to the cell that it can be broken before it will move, while, when wet cement is used, the cover cannot be centered once it has been applied. The slide is then placed on a turntable and a thin ring of cement is run round and over the edge of the cover in the manner described in the last example. In the matter of sealing the writer prefers the classical method of running several rings of gold size as a seal and finishing this with a flexible black varnish. Fluid Wholemoiints in Nonaqueous Media General Principles Nature of the Process The mounting of whole objects in non- aqueous media is essentially the same process as mounting objects in aqueous media: that is, the objects are enclosed in the preservative medium in a very flat box, the floor of which is formed by the slide, the top of which is formed by the coverslip and the sides of which are formed either of cement, or by a cell. There are not, however, so many possible choices among cells and seahng media as is the case with aqueous mounts, for the choice of the medium itself dictates every subsequent step. Choice of the Medium Only three nonaqueous media are commonly used in mounting: these are glycerol, bromonaphthalene, and Uquid petrolatum. These should never be used when any aqueous substitute is available, nor should a fluid medium be used if a mountant which will harden under the coverslip (see the next three chapters) can be employed in its place. Each of these three media will be discussed in their turn. Glycerol is widely used as a mountant in those cases in which a water-miscible, high-refractive-index material is required and in which a medium of the type dis- cussed in the next chapters cannot be em- ployed. The principal reason that such media cannot be used is the difficulty of transferring delicate objects to, say, glycerol jelly without causing a collapse of their walls, while it is comparatively simple to get delicate objects into glycerol by evaporation. This technique is usually applied to nematode worms, and some- times to small arthropods or very delicate coelenterates, which should be fixed in the ordinary manner and then transferred very gradually to alcohol and from alcohol to ^ % glycerol in alcohol. The alcohol is then slowly evaporated, leaving the material in pure glycerol. It is almost impossible to seal a deep cell full of glycerol, and mounting in this material should be confined to cells built out of cement or to shdes in which a concave hollow has been ground. Sealing Glycerol Mounts with Dichro- mate Gelatin There are three ways in which a glycerol-filled cell may satisfactorily be sealed. The first is with the aid of molten gelatin, appUed from a turntable in the manner described in the last two chapters, and then varnished with any good cement; the second method involves the apphca- tion of a molten resinous medium; the third method uses petrolatum. In the case of the first method it is better to use a solution of gelatin containing potassium dichromate, which becomes insoluble on exposure to hght, than to use straight gelatin; and it is doubtful if the formula of Riiyter 1934 or 1935 (Chapter 28) can be improved. A narrow ring of material is turned on the sUde, in the manner previously de- scribed, the ring being made slightly smaller in diameter than the size of the coverslip to be used and just sufficiently thick to keep the covershp from bearing on the object. This cement shrinks on drying so that a ring must be turned 32 Sealing FLUID WHOLEMOUNTS IN NONAQUEOUS MEDIA 33 somewhat thicker than is customary with other cements. A number of slides may be prepared at the same time and left in a light place for an hour or two until the gelatin has become insolubilized. The object, together with a drop of glyc- erol, is placed in the middle of the cell and the coverslip lowered vertically as shown in Fig. 24 (Chapter 6). The covershp is then held firmly in place, either with the finger or with one of the chps shown in Fig. 25, while all traces of exuded glycerol are removed with the aid of a rag moist- ened in alcohol. The shde is then placed on the turntable and a ring of molten dichromate gelatin turned over the edges of the covershp. This ring of cement is cooled — it is not necessary to dry it — and the whole slide then thoroughly cleaned in 95% alcohol, either apphed from a rag, or by waving the slide back- ward and forward in the fingerbowl of the reagent. Great care is necessary at this stage to avoid displacing the coverslip. The purpose of the ring of gelatin, in fact, is not so much to cement the cover- slip in place as to provide a temporary seal which will hold the cover sufficiently long to permit the removal of exuded glycerol. As soon as the slide is dry, and glycerol-free, several coats of gold size are added, allowing ample time for each to dry, and then a final coat of asphalt varnish is turned on top. Slides prepared by this method have a very pleasing appearance but they require a great expenditure of time compared to the use of a thermoplastic resin cement. Sealing Glycerol Mounts with Thermo- plastic Resin Mixtures The medium most usually recom- mended for heat-sealing glycerol mounts is Noyer (Chapter 28, V 12.2 Noyer 1918), a simple mixture of rosin and lanolin. The writer prefers the formula of Fant (V 12.2 Fant 1932), containing a quantity of dried Canada balsam, which appears to make it both easier to handle and more adhesive. Whichever medium is employed, the object in glycerol is placed under the coverslip and, after crudeh' wiping away the excess fluid, a layer of molten cement is applied to the edge. For making large quantities of these preparations a most ingenious mechanism has been described by Banard (113G0, 54:29), but it is proposed here only to deal with the method of handling indi- vidual slides. This method is shown in Fig. 20 where the objects are being mounted under a square coverslip. It is the author's opinion that no satisfactory seal can be made by this method on round coverslips. The dish in the left foreground contains the objects in pure glycerol and, immedi- ately behind it in the center of the picture, there is a tin can containing the cement selected. The author always prepares the cement in such quantities as will just fill an empty boot-polish can, which is admirably adapted to the purpose. The tool being used is the same rather heavy brass tool which is shown being employed in the mounting of paraffin blocks in Fig. 65 (Chapter 12). An ordinary section lifter, sometimes recommended, is too thin and does not hold enough cement. In the illustration, it is presumed that the object has been placed under the coverslip, the coverslip lowered in place, the glycerol roughly wiped away, and the metal tool heated to about 150° to 200°C. This tool is now dipped into the can of cement, so that the edge accumu- lates molten cement along it, and then touched down on the edge of the cover- shp. It will be noticed that the edge to- ward the front of the illustrations has already been finished and that the second edge is being apphed. Before this was done, a minute drop of the cement was placed at one corner of the covershp to hold it in position. Having finished two sides in this manner, it is easy to apply cement to the third side, but the whole trick of a successful mount lies in the method in which cement is applied to the fourth side. It will be obvious that this very hot cement, when it is applied to the coverslip, will cause an instantaneous expansion of the fluid. This does not matter as long as one side remains open. The last side, however, cannot be sealed in one piece, and it is necessary to apply the cement in such a manner that about a one-milhmeter gap is left at a corner 34 THE ART OF MAKING MICROSCOPE SLIDES Sealing for the escape of the heated glycerol. The slide is then cooled, such glycerol as has been extruded from the corner is wiped away, and a small drop of very hot cement is apphed at this place. Slides sealed in this manner will last almost indefinitely and require no further finish- ing beyond a brief wash in alcohol to slip. The size of the drop is therefore critical but can onlj^ be learned by experience. The shde is now placed on a warm table, kept a few degrees above melting point of the petrolatum employed, and molten petrolatum run under the cover from a pipet. Spence prefers to take a Fig. 20. Sealing a wholemount with Fant's cement. remove excess glycerol. They are, how- ever, clumsy in appearance compared to a ringed slide made with dichromate gelatin. Sealing Glycerol Mounts with Petro- latum This method, which was developed by Spence 1940 {Microscope, 4:123) is the best yet developed, providing one is looking for chemical stability rather than mechanical strength. The object is lifted in a drop of glycerol and placed in the center of a clean shde. Three Uttle squares of petrolatum-soaked paper or card, of a thickness sufficient to prevent coverslips crushing the object, are placed round, but not in contact with, the drop. The cover- slip is now lowered vertically onto the drop which should spread out, when the coverslip is resting on the squares, until it occupies about half the area of the cover- wisp of solid petrolatum on a toothpick and to let this melt and run under the covershp. In either case, one is left with a bubble of glycerol surrounded by a thick layer of molten petrolatum. The slide is now chilled and any excess petrolatum scraped away. The mount is permanent in this form, but the petrolatum is so soft that the cover is liable to become de- tached when dust is wiped from it. A certain degree of mechanical strength can be given by turning on three or four rings of shellac, followed by three or four coats of asphalt varnish. Sealing Other Non-aqueous Liquid Mounts Mounting in liquid petrolatum is prac- tically confined to blood films, on the assumption that this inert medium pre- vents the fading of methylene blue-eosin stains. These stains are, however, best Nematodes FLUID WHOLEMOUNTS IN NONAQUEOUS MEDIA 35 kept dry, and many of the neutral niount- ing media described in Chapter 20, under the heading M 23.1, are less trouble to use, and probably just as good. Liquid petrolatum is difficult to seal, though the author has had most success with the hot- resin method described in the last para- graph. Even with this material, however, there is a slow diffusion of the brown resin through the liquid petrolatum which ultimatel}' damages the slide. Since liquid petrolatum does not evaporate, it is some- times preserved by holding a covershp in place with a drop of cement at each corner. The quantity of cement used is thus so small that diffusion through the mounting medium is negligible, while the degree of adherence is sufficiently good for all nor- mal handling. Bromonaphthalene is used only for mounting diatoms, when a medium of high refractive index is re- quired. The only satisfactory cement for sealing is a de-waxed shellac prepared by the method of Hitchcock (Chapter 28, V 11.2). Both the preparation of the cell, and the process of mounting, are special- ized procedures which are described in considerable detail in the second of the typical preparations which terminate this chapter. Specific Examples Preparation of Nematodes in Glycerol Nematodes are awkward objects from which to make v.holemounts, for their thick cuticle permits only slow diffusion of reagents, and it is almost impossible to get them into either resinous or gelati- nous media. The objection to shrinkage is not on aesthetic grounds, but on the basis that the folds and ridges of cuticle render it almost impossible to make out clearly those internal organs upon which classification depends. Nematodes are, therefore, almost invariably mounted in glycerol. No difficulty will be experienced in collecting small nematodes from the blood, or when they are free-swimming (as Anguillula). The standard method of se- curing nematodes and their eggs from feces, however, is by flotation from a strong salt solution. Fresh specimens are collected and flooded with 10 or 15 times their volume of a 20% solution of sodium chloride. This may be added directly to the cardboard containers customarily used for such samples, and the unpleasant odor may be diminished by adding small quantities of nitrobenzene both to the salt solution anfl to the feces themselves. After the sohds have settled, the top layer, on which tlie nematodes will be floating, is jerked into another dish with a quick movement of the wrist. It is almost impossible to pick the worms or eggs from the surface in a pipet so that, when a sample has been thus isolated, it should be diluted to a salt concentration of about 1 % which allows the specimens to sink to the bottom. They may then be washed with weak saline until free of fecal matter. This method of collection cannot be used with fecal specimens which have been mixed with animal charcoal as a deodor- ant, because the charcoal also floats on the surface. Worms may, however, be collected from such samples by a modified Berlese funnel (see Chapter 4, Fig. 21). In this technique a plug of glass wool is placed at the bottom of an ordinary glass funnel and the fecal material poured in. The bottom of the funnel is then lowered into a tube of 1 % salt solution until the liquid rises just to the lower edge of the fecal matter. A lamp, or some other heat source, is then placed above the feces. The worms endeavor to escape from the heat and, burrowing down through the feces, ultimately pass through the glass- wool plug and accumulate at the bottom of the tube of salt solution. The collection of small nematodes from soil samples is much more difficult than from feces. The flotation method is practically impossible because in most soil samples there are large quantities of organic matter which will also float, while the modified Berlese funnel usually permits enough clay to sift down to make it difficult to sepaiate' the worms. Pro!)- ably the best procedure is to dilute soil samples with a 1 % salt solution and then 36 THE ART OF MAKING MICROSCOPE SLIDES Nematodes survey small aliquots by strong trans- mitted light under a dissecting micro- scope. The nematodes may be recognized by their activity, picked out with a fine pipet, and transferred to fresh saline. Whatever method has been employed, one is left with a collection of nematodes in salt solution. The solution should be changed frequently, until the worms are clean; for satisfactory wholemounts cannot be made if either dirt or mucus adheres to the outside. Heat is the only fixative which will penetrate a nematode rapidly. It is conventional, therefore, to fix worms in hot 70 % alcohol, though hot water will, in point of fact, do equally well. The exact temperature is immaterial and usually 100 times as much 70% alco- hol as there is saline around the worms is warmed until bubbles appear. This is usu- ally at about 55° to 60°C. The hot alcohol is then rapidly flooded over the living worms, which are again collected by being allowed to settle to the bottom of the dish or tube. Most of the worms fixed by this method will be found to have straight- ened out, and the few which have not had better be thrown away. The worms must next, very carefully and slowly, be transferred to absolute alcohol, in which they must remain until they are completely dehydrated. This transfer is best effected through 5 % grades of alcohol; that is, from 70 to 75 to 80 to 85, etc. In the case of worms with very tough cuticles, a faster schedule may be employed. The reason the worms must be transferred to absolute alcohol before passing to glycerol is that it is almost impossible to get rid of water once it has got into the glycerol, and the high re- fractive index of the glj^cerol is lost if it is diluted. It is easy to find out how fast a schedule may be emploj'^ed by taking one of the worms from 70% and throwing it directly into, say, 95% alcohol. If, after two or three hours in this, there is no sign of tlie colla])se of the wall, the rest may follow it, but if the wall collapses one must experiment with 80% and so on until one has found the most rapid transfer which may be made. When the worms are all accumulated in absolute alcoliol, a little glycerol is added. Assuming that the worms are in 100 milliliters of absolute alcohol, it would be safe to add about 10 drops of glycerol, being very careful to shake rapidly and continuously so as to disperse the glj^cerol rapidly. The worms are left in this mixture for about 24 hours before a further 10 or 20 drops of glycerol are added and mixed. This schedule is continued until about 10 milliliters of glycerol have been added. The solution is then concentrated by evaporation, in a desiccator, at a rate which leaves the worms in concentrated glycerol at the end of about a week. It is easiest to suck air through with an aspirator, being careful that the air itself passes through a de- hydration column before entering the desiccator. This method of preparation is laborious in the extreme, but it yields a product which looks exactly like a glass model. The author knows no other method which will produce clear nema- todes without causing the collapse and wrinkUng of the cuticle. To make these cleared nematodes into permanent mounts, one now secures the necessary slides, coverslips, a metal tool of the type shown in Fig. 20, and a can either of Noyer's or Fant's cement. It is unnecessary to make a cell, or to use a concave slide, because the viscosity of the glycerol will hold the cover a reasonable distance away from the slide while the mount is being made, and the method of mounting embeds the coverslip so firmly that it does not subsequently shift. The slide is taken, cleaned by any preferred means, and the required specimen or specimens placed in the center in a drop of glycerol. Square coverslips should be employed and a little experience will soon show what amount will fill the cover- sUp to the edge. The coversHp should be lowered vertically to avoid displacing the worms, and, as soon as the glycerol has reached the edge, the heated metal tool is plunged into the cement and used to seal one edge. The success of tlie process depends on having the cement hot enough at the moment when it is applied. The opposite edge of the coverslip is then sealed, and these two seals connected by a third. The application of cement to the Diatoms FLUID WHOLEMOUNTS IN NONAQUEOUS MEDIA 37 fourth side is, however, made in such a manner that a gap of about a millimeter will be left between the cement and one of the corners of the cover. This gap is necessary to permit the heat-expanded glycerol to escape. After the slide has thus been not quite sealed it is permitted to cool and a rag moistened with 95% alcohol is used to remove excess glycerol from the httle vent which has been left. This vent is then itself sealed with a drop of very hot cement. Preparation of Diatoms in Bromonaphthalene Strewn slides of diatoms may be mounted dry in the manner described in Chapter 1. When it is necessary, however, to resolve fine structure, they should be prepared in a medium of high refractive index, and no resin has yet been found which is as satisfactory as bromonaph- thalene. No one who has ever examined diatoms mounted in bromonaphthalene will ever wish to use any other medium and, though the process is tedious, the end result justifies the trouble taken. Before mounting, diatoms must be collected and cleaned. The three great sources are fresh-water, sea-water, and fossil deposits. Diatoms occur in fresh water as part of the plankton, but are mostly found in the mud on the bottom of ponds or attached to weeds. No attempt should be made to separate diatoms from the weeds in the field; the collection should be taken back to the laboratory. The first rough separation is then carried out by cutting the plants into about ]>i- inch lengths and putting them into a flask with enough water to cover them, shaking vigorously, and then straining this water through coarse cloth into another con- tainer. More water is then added to the material, which is again shaken, and so on until after four or five washings, all the diatoms have been removed. These wash- ings may be set on one side to settle for further treatment. Diatoms may be separated from fresh mud by taking advantage of their photot- ropism. The mud, together with an ade- quate quantity of the water from which it was collected, is placed in a small saucer and a thin layer of cheesecloth is spread on the top. The mud should be sufficiently Uquid to permit diatoms to pass through readily, but sufficiently solid to prevent the cheesecloth from*sinking into it. If the dish be set in bright light for a day or two, the diatoms will migrate through tlie cheesecloth and form a d;irk greenish smear over its surface. The cloth is then removed, washed, and the diatoms ac- cumulated in a small quantity of water. The collection of diatoms from marine plants may follow the technique used for fresh-water plants, though the larger algae are better scraped with a blunt knife. These scrapings are then transferred to a jar of sea water where the diatoms and debris settle to the bottom. A rather large number of marine diatoms are, however, planktonic and can be collected from sea water with a centrifuge. A plankton-con- centrating centrifuge is described in Chap- ter- 2 and with its aid large volumes of water may be processed in a relatively short space of time. If such a planktonic centrifuge is not available, it will be neces- sary to collect the samples by towing be- hind the boat a long conical net of the finest obtainable bolting silk to the end of which is attached a small tube in which collect those specimens which have not passed through the net. Unfortunately the diatoms form a relatively small bulk, even though they may be numerous in quan- tity, of the total material collected, so that if there are many crustaceans among the plankton it is desirable to have a double net, the first layer of which will retain the crustaceans without permitting the dia- toms to pass. Whatever method of con- centration is adopted, however, one ends, as in the other processes described, by having a mixture of dirty diatoms and sludge accumulated in the bottom of the dish. Diatoms also occur in guano, and in many fossil deposits, and must be roughly separated before cleaning. If the fossil de- posit resembles guano, it is only necessary to shake it up in water and pass it through a coarse sieve to remove the sand and 38 THE ART OF MAKING MICROSCOPE SLIDES Diatoms other extraneous material. Many of the more interesting diatoms, however, are found in hard aggregates which must be broken up before the frustules can be separated. Many methods of doing this have been described, but undoubtedly one of the most useful is the technique of Swatman {Microscope, 7:132). This technique utiUzes the expansion and contraction which takes place on the sudden crystallization of a supersaturated solution of sodium acetate. The rock, or hard aggregate, containi ng the diatoms is roughly broken into j'i-inch pieces and placed at the bottom of an Erlenmeyer flask. Two or three times its own bulk of sodium acetate is then added, and thor- oughly mixed in, before adding water to the extent of about 5 % the total weight of the sodium acetate. The flask is then very carefully warmed, the flame being first ap- plied to the sides and not to the bottom, until the sodium acetate is molten. Heat- ing should then be continued until the material commences to boil and it should be maintained in a hot condition for as long as is required to cause the penetra- tion of this supersaturated solution to every part of the aggregate. The flask is then cooled slowly, care being taken to avoid jarring, and when the solution is cold a single crystal of sodium acetate is dropped into it, which causes instant crystallization. As the flask will heat up greatly during crystallization, it is then recooled in water. The mass is remelted, recooled, recrj^stallized, and so on until a sufficient disintegration of the rock has taken place. Another method of arriving at the same result is to soak the pieces of material in water, to freeze them very rapidly (either in a freezer unit or in dry ice) then to drop them into warm water, refreeze them, and so on. This process is no more effective, however, and is usually much more trouble to carry out, than the sodium acetate procedure outlined. When the mass has sufficiently disintegrated, it is strained through a coarse sieve to get rid of the lumps and the diatomacious ma- terial allowed to form a sludge at the bottom. Whatever method has been employed, one has now, from either fresh or fossil material, a sludge which should be trans- ferred to a flask. This sludge contains dia- toms together with various organic and inorganic impurities. The first thing is to get rid of any carbonates which may be present by adding hydrochloric acid cau- tiously (if there is a great deal of carbonate present effervescence may rise above the neck of the flask and cause a loss of mate- rial) until no further gas is evolved. The flask is then filled with water, the undis- solved material allowed to settle, the water poured off, and the process repeated until all the soluble chloride has been removed. If there is any appreciable amount of clay present, it will also have been removed by this process, since even the smallest dia- toms will settle relatively rapidly com- pared to the fine particles of clay. It is next necessary to remove an}^ organic matter which may be present, and many methods have been proposed for this. The conventional method, also described by Swatman (loc. cit.), is to get the diatoms into concentrated sulfuric acid which is then heated to about 120°C. This chars the organic matter which is then oxidized by dropping small crystals of potassium chlorate into the hot acid. It should per- haps be emphasized that only exceedingly small crystals should be added, and that those who do not normally wear glasses should use some form of protection against the chance of spurting acid. If there is much organic matter present, the heated acid will be from black to dark brown in color, and chlorate is added until the color is reduced to yellow. The only safe method of removing the acid is to wait until it is entirely cold and then pour it in a slow and steady stream, while constantly stirring, into a relatively large volume of water. The diatoms settle out on the bottom. Some iron may still be present, either de- rived from a fossil deposit or from the chlorophyll of the plant debris. This is best removed by suspending the diatoms in 5 % sodium hydroxide and bringing them to the boil. If there is any iron present it will ap- pear as a brownish ferric hydroxide through which the diatoms will settle readily and which may then be poured off. After repeating this process several times, the diatoms are treated with hydrochloric Diatoms FLUID WHOLEMOUNTS IN NONAQUEOUS MEDIA 39 acid to remove the last of the iron chloride and again washed by decantation. It will probably happen, howe\-er, that many of the finest markings on the dia- toms are still filled with finely divided clay which must be removed by treating the diatoms in the cold with a 10 "^t, dilution of ammonia. The frustules will be dam- aged if a hot or strong solution is used, and it is best to leave the diatoms in cold ammonia for two or three days, shaking at intervals, before washing them by de- cantation. The final stage in cleaning the diatoms is now to wash them with re- peated changes of filtered distilled water until all traces of dissolved salts have been removed. Some workers, after treating the dia- toms with ammonia, repeat the sulfuric acid-potassium chlorate treatment as a final precaution. Another variant is to precede the original treatment with sul- furic acid and potassium chlorate by treat- ment with a hot mixture of two parts of sulfuric acid with one of nitric acid. This treatment is recommended when the orig- inal collection contains very large quan- tities of vegetable matter in addition to the diatoms. Swatman (loc. cit.) points out that if diatoms are collected from mud containing coal dust this will not be satisfactorily removed by any of the pre- ceding processes and recommends that the diatoms be fused in a platinum crucible with pure potassium nitrate for removal of this contaminant. It will have been observed, either in theory or practice, that many of the proc- esses just described result in the produc- tion of noxious vapors, so that they cannot be properly carried out by anyone not ha\dng access to a chemical hood. To meet this objection Hendey 1938 (11360, 58:49) has devised a most ingenious apparatus which will permit any of the processes described to be used in a living room. All the methods so far described pre- sume that the collector has been working close to his laboratory and has, therefore, not been faced with the problem of trans- l)orting large quantities of vegetable matter. A rough method of field cleaning (Swatman 1941: 11479, 1:191) may be used to concentrate diatoms. As much water as possible is drained from the rough sludge and replaced with 10% sulfuric acid. Potassium permanganate is then added, with constant stirring, until the solution remains pink after standing for a few minutes; then enough oxalic acid is added to dissolve the brown oxide sludge. The clear solution may be poured off and the diatoms roughly washed before being transferred to a tube. By whatever method the diatoms have been cleaned, they are now presumed to be accumulated in clean distilled water. They should be roughly sorted into their kinds, since diatoms are much easier to handle under the surface of water with the aid of a fine pipet than they are when dry. The different kinds are then stored in small vials of distilled water to which a trace (about one-tenth of 1 %) of formalde- hyde is added with a view to discouraging organic growth. Larger quantities of form- aldehyde should not be used, or a fine de- posit will be found on the surface of the diatom when it is subsequently dried. To mount a strewn slide of diatoms, it is only necessary to take a drop of the dis- tilled water with the diatoms suspended in it, to let this evaporate on a coverslip, to dry the coverslip with heat, and then to mount it in the manner to be described subsequently. It may be presumed, how- ever, that the worker wishes to prepare a slide in which the diatoms are arranged in some given order on the coverslip. It must not be thought for one moment that this method of arranging diatoms on the cover- slip is of necessity confined to the produc- tion of artistic pictures. It is true that the method was developed by those who wished to build pictures, but it can also be used to line uj) in correct ranks all of the species found, for example, in one locahty. No method of arranging diatoms on, and attaching them to, a coverslip will compare with that of BelHdo 1927 (11360, 47:9). The description cited is one of very considerable complexity and goes into details not possible in the present place. It consists essentially, however, of coating a chemically clean covershp with an exceedingly thiu film of Bellido's ce- ment (Chapter 28, V 11.1 Bellido 1897) which is then dried. Bellitlo recommends 40 THE ART OF MAKING MICROSCOPE SLIDES Diatoms that the film be applied by dipping a needle into the cement and then drawing the flat of the needle sharply across the covershp. This leaves an invisible film of dry gelatin on the cover, and individual diatoms may be placed on this film to which they will not adhere until the film is slightly moistened by breathing on it. The moment this has been done the diatoms are permanently attached. Before individual diatoms may be se- lected for this technique, however, they must be dried. It is not safe to dry them on glass, to which they frequently adhere. It is better to attach a piece of mica to a slide with petrolatum and to evaporate the drop of water on this base. Individual di- atoms may then be picked up on the end of a hair under the microscope. There is usually enough grease on a normal hair to permit the diatom to adhere. Bellido de- scribes the ingenious idea of mounting a hair on the collar of a microscope objective in such a manner that the tip of the hair is in focus when the draw tube of the microscope is pulled halfway out. It fol- lows that when the draw tube is pushed home the hair will be out of focus, and also well above the plane of the object which is in focus. It is possible, therefore, to press the draw tube fuU}^ home, search one of the squares of mica for the required speci- men, pull out the draw tube until the hair is in focus, and then lower the microscope until the hair touches and picks up the object. Belhdo recommends that the hair be moistened with a little bromonaph- thalene and that the film of gelatin also be lubricated with the same reagent. He has also described, in the place quoted, a sealed chamber within which all these operations may be conducted without the risk of dust faUing on the preparation. Another device for handling individual diatoms on a mechanically operated hair has been described by Meakin 1939 {Microscope, 4:8). These mechanical de- vices are only necessary, however, for handling large quantities of rather small diatoms. A few months of practice with a hair mounted in any holder will enable the average worker to arrange diatoms di- rectly. A mechanical device is, of course, almost necessary if one is endeavoring to arrange the diatoms according to any artistic pattern. In any event, as soon as the diatoms have been arranged one breathes very gently on the coated cover- slip and pauses a moment or two. The covershp is then examined under the microscope and a few of the larger and rougher diatoms very delicately probed with a hair. If they are found to be firmly attached, it may be assumed that the smaller diatoms are also attached. If, how- ever, anything is found to be loose, one breathes again, and again probes until such a time as all the diatoms are fixed. These coverslips with the diatoms at- tached to them may now be laid on one side while the necessary cells for mounting are prepared. There will be required a turntable, shdes, a fine brush, a hot plate, and some de-waxed shellac (Chapter 28 V 11.2 Hitchcock 1884) which should be as thick as can conveniently be persuaded to flow from a brush of the size selected. A fine ring is then turned of a size shghtly smaller than the covershp. Save for the very largest diatoms a single ring of this cement will be sufficiently thick. It is ab- solutely necessary that these shellacked rings be baked if they are to become in- soluble in the bromonaphthalene used for mounting. As soon, therefore, as the alco- hol has evaporated from the shellac, the slides are placed on a hot plate, or for that matter, in an oven, and heated to just below the melting point of the shellac for at least 30 minutes. The cells, which would now be adequate for aqueous media, must be further processed for bromo- naphthalene mounting by having the top ground flat. This is done by taking some of the finest available carborundum, making it into a slurry with water, and spreading this on a sheet of fine quality plate glass. The slide, with the cell down, is then laid on this glass and, with a delicate finger placed over the center of the cell, moved gently backward and forward for a few moments. It is then picked up, washed under the tap, and examined with a strong lens to make sure that there is a flat, smooth area over the whole of the top of the cell. The slide is then washed free of Diatoms FLUID WHOLEMOUNTS IN NONAQUEOUS MEDIA 41 all traces of grit and dried in a dust-free place. As many prepared slides as are re- quired now have a small drop of bromo- iiaphthalene placed in each cell. If petro- latum was used to hold the coverslip in place while the diatoms were mounted on it, this should first be removed with ether to make quite certain that the diatom frustules are grease-free, dry, and clean. When one is satisfied as to this, the cover- slip is lowered in place on top of the bromonaphthalene and then, with some blunt instrument (Bellido recommends a toothpick) pressed on the cell until it is firmly attached. If the coversUp is flat and if the cell has been properly ground, this seal is sufficiently good to permit one to remove any exuded bromonaphthalene with a cloth before turning on an addi- tional layer of the shellac as a final seal. It is usually safer to follow this with an- other layer of some impermeable cement such as asphalt varnish. Though this process may sound labori- ous, it actually takes less time by this means to mount one each of the 200-300 species that may be found in a fossil de- posit on a single coverslip than it takes either to mount them individually on separate sHdes by any other means, or to endeavor to find them under a microscope if they are arranged at random. Wholemoiints in Gum Media General Principles Nature of the Process The preparations which have been de- scribed in the last two chapters are those in which the specimen is sealed in a pre- servative fluid. These mounts, as will be readily understood by anybody who has read the chapters, are difficult and labori- ous to prepare, so that most slides are made in a mounting as distinguished from a 'preservative medium. A mounting me- dium,, used in this sense, is one which itself hardens and holds the coverslip in place while at the same -time preserving the object contained in it. Mounting media may be divided into two large groups: first, those which are miscible with water; second, those which are not miscible with water, so that some initial treatment must be given to most objects before they are mounted. The media miscible with water are in themselves divisible into two types: first, those which are liquid at room tem- perature (dealt with in this chapter), second, those which are solid at room tem- perature and must be melted before they can be used. Water-soluble media are all colloidal dispersions of various materials, the colloids being in the sol phase for the media described in this chapter, and in the gel phase for the media described in the next. Types of Gum Media Employed Many formulas for mounting media of the sol-colloidal type are given in Chapter 27 under the heading M 11.1. They are dispersions of either natural or synthetic gums in water and must, therefore, depend for their hardening upon the evaporation of moisture from the edge of the coverslip. Were this to continue for an indefinite period, the media would naturally harden and crack; hence, most contain either glj^cerol or sorbitol to impart hygroscopic qualities. The prototype of all these media is Farrants', which is a simple dispersion of gum arable in water to which has been added a small quantity of glycerol to- gether with a preservative. All media de- rived from this follow the same pattern, differing mostly in the quantity of glycerol included and in the nature of the preserva- tive selected. The fundamental objection to gum-arabic media is that it is difficult to obtain a pure sample of the gum, and one has to go through a wearisome process of filtration to avoid having the mount filled with sand grains and pieces of stick. Another objection to this type of medium is the low index of refraction, which leaves objects mounted in it relatively opaque when they are examined by trans- mitted light. This difficulty is overcome in Berlese's medium, to which chloral hy- drate is added in considerable quantities with a view to increasing the index of re- fraction. The ordinary media of the Farrants' type have an index of refraction just over 1.3, while Berlese's medium and its modifications have indices of refraction as high as 1.47. Very few synthetic sub- stitutes for water-soluble gums are avail- able, the most promising at the present time being polyvinyl alcohol, which is used in the media of Downs, and of Gray and Wess. It is probable that the recent appearance on the market of water-soluble cellulose derivatives (for example, car- boxymethyl cellulose) may lead to the 42 Mounting GUM MOUNTS 43 ultimate suppression of gum arabic in mounting media. Types of Objects Which May Be Mounted Simple water-miscible liquid mountants are of far wider utility than is usually realized, for there has been a complete mental block on the part of most micro- scopists when faced with any mounting medium which is not a solution of a resin in a hydrocarbon. As a matter of fact, most simple objects such as the scales of fish, animal hairs, and the like, may be more readily mounted in aqueous media than in resinous ones. The actual process of mounting is so simple that it is regarded with distrust by those who have come to believe that only through complexity can good results be produced. With these media one merely takes the object which it is desired to mount, places it in the drop of mountant on the shde, and presses a coversUp onto the top. This process is not confined to relatively hard objects of the type described, but may also be apphed to many protozoa and other small inverte- brates. These do not make satisfactory permanent mounts by this method, for they ultimately reach a refractive index identical with that of the mountants and thus vanish; but a temporary mount of Paramecium, in one of these media, will show the internal structure to a class far better than will the average stained mount, and will also give a far better reaUzation of what the living object looks hke. Objects most commonly mounted however, are small arthropods of -the degree of transparency that does not re- quire that the skeleton be cleared in the manner described in Chapter 6. Gum mountants are not satisfactory in thick layers and the writer has never made a successful mount in a deep cell. There is no point in endeavoring to use shallow cells for these media, for the viscosity of the mountant is sufficiently high to pre- vent the coverslip from crushing small objects. Finishing Slides in Gum Media Exudate round the edges of the cover may be removed by washing with warm water, but it will be some time before the edges reharden. Moreover, no mounting medium containing glycerol or sorbitol can fail to absorb moisture from the air on humid days, and to lose it on dry days, so that it is usually better to finish the shde by applying a ring of varnish in the man- ner described in previous chapters. It does not matter what cement is employed, the writer's preference being for gold size, probably more from force of habit than from any other reason. Specific Example Preparation of a Wholemount op a Mite by the Method of Berlese The use of the name Berlese in the head- ing of this example is less an injunction to employ the mounting medium of that writer than a tribute to the method of col- lecting small arthropods which he intro- duced. This method is applied with the aid of the Berlese funnel which is seen in Fig. 21. This is a double-walled funnel, between the walls of which warm water may be placed and maintained at any desired temperature by applying a small flame to a projecting side arm. The temperature is not critical, so that no thermostatic mechanism is provided, but a thermom- eter may be inserted and used to read the temperature at intervals. A circle of wire gauze with a mesh of about K 6 of an inch is placed at the bottom of the inner glass funnel, and whatever material to be searched for mites is placed loosely on this gauze. The lower end of the glass funnel is then attached with modeling clay to a tube containing whatever medium is being used for the collection of the specimens. If the specimens are merely to be stored, rather than mounted at once, 95% alcohol may be placed in the tube, and it is then unnecessary to seal it to the base of the funnel. If, however, the specimens are to be mounted directly in Berlese's medium, in which better mounts can be prepared from living than from preserved material, 44 THE ART OF MAKING MICROSCOPE SLIDES Mites Fig. 21. Berlese funnel in use. the funnel must be sealed to prevent the more active forms from working their way out of the tube. After the mass has been placed in posi- tion a small lamp, certainly not of more than 15 watts, is mounted in any kind of a reflector some distance above the material. The animals in the material, therefore, find themselves surrounded by heat at the sides and plagued with hght froju above. As all of these animals are photophobic, they tend to move toward the lowest point of the mass from which they drop down the tube into the funnel. By this means it is possible in 10 or 15 minutes to collect the whole fauna from a large handful of any organic material which would, by any other means, take several hours to search. The use of the funnel is not, of course, con- fined to moss but may be used for hay, straw, shredded bark, or any other mate- rial from which small arthropods are cus- Mites GUM MOUNTS 45 tomarily collected. The only difficulty in using this equipment is in preventing the heat from getting too great. Some people use so large a lamp above, and so high a temperature around the edges, that many small arthropods are killed before they have time to fall into the trap which has been laid for them. The outer water for most uses should be at a temperature of 30° to 40°C., while the lamp above should under no circumstances raise surface tem- perature of the material above 60°C. These temperatures are for a moderately dry moss sample, and may be considerably exceeded when one is dealing with a drj^ material such as straw. Wet moss of the sphagnum type, however, requires lower temperatures. Assuming that permanent mounts are to be made, for record purposes, of all the small invertebrates which may be found in a moss sample, it is necessary to make adequate preparations to receive them while the moss is being treated. Two kinds of gum mountants are desirable: a high- refractive-index medium like Berlese, for the very heavy-walled forms such as the Oribatid mites and the Pseudoscorpion- ides; and a low-refractive-index mountant, like Gray and Wess, for the thinner-walled forms such as the Tyroglyphid and Gam- assid mites. This last medium is also suitable for Thysanura and for CoUem- boUa. Thick-walled beetles and fleas, if they are to be made into microscope slides, had better be treated as described in Chapter 6, and should be accumulated for this purpose in a tube of 95% alcohol. Sphagnum moss is also likely to yield a number of crustaceans, particularly Cla- docera and Ostracoda. These are better mounted in glycerol jelly, in the manner described in the next chapter, and should be transferred as soon as they are found to 30% alcohol where they will die with their appendages extended. They should not, however, be permitted to remain in this weak alcohol for longer than is necessary to kill them, but should then be trans- ferred to 95% alcohol. A considerable number of nematode worms are likely to turn up and should be treated as de- scribed in the last chapter, while a tube of some fixative should be provided to re- ceive any small annelids which may be found in the gathering, and which must be fixed, stained, and mounted at once. A brush will also be required, a supply of clean 3" X 1" glass slides, and a numljer of covershps. All being ready, and observation show- ing that no further forms are falling through the Berlese funnel, the collecting tube beneath it is now inspected to see roughly what one has gathered. If there are a considerable number of Gamassid mites or active insects, it is necessary gently to open a portion of the tube by pushing away the modelhng clay with the thumb and to let a minute drop of ether run down inside. When this has been done the tube is removed and the contents tipped out into a petri dish or similar con- tainer and the catch sorted. A mite, or similar form, which is to be mounted, is then picked up on the tip of a brush and transferred to a drop of the mountant. As little water as possible should be transferred with it and the mite should be pushed under the surface of the gum with the point of a needle. The mount is then inspected under the low part of the microscope and, if any large quantity of air has been carried in with the mite, the bubbles are released with the aid of a fine needle and allowed to come to the surface before the covershp is laid gently into place. The drop of gum should be of a considerable size and no endeavor should be made to press the coverslip down. If a reasonably thick layer of mountant is left almost any small arthropod will spread its legs like a textbook diagram before dying and will remain in this form indefinitely. Wholemoiints in Jelly Media General Principles Glycerol jelly is the only type of water- miscible medium known to most workers. Many objects, both plant and animal, which are usually prepared in glycerol jelly, are much better mounted by the method described in the last chapter, and the author would most warmly recom- mend to workers who have been using glycerol jelly that they try one of the methods there described. Formulas for the jelly media, the use of which is described in the present chapter, are given in section M 12.1 of Chapter 26, and it will be seen that they are all essen- tially the same. That is, they consist of a dispersion of gelatin, which has been di- luted with glycerol until the required index of refraction is obtained, and they have added to them some preservative. The older glycerol jellies, designed for use in European laboratories, do not in general contain sufficient glycerol to withstand the drying effects of an American laboratory. The author has in his possession many deep wholemouhts of fairly large crusta- ceans which remained perfect for ten years in England but which dried and cracked after only two years in the United States. There is little to choose among any of the media, apart from the consideration just given, and practice will soon permit the mounter to select the one which works best in his hands. Nature of the Process Mounts prepared in glycerol jelly may be made either on flat slides or as deep wholemounts. Glj'cerol jelly can be used for larger objects than can the gum media of the last chapter and, except for botan- ical specimens, the use of glycerol jelly is largely confined to the preparation of permanent slides of unstained crustaceans. These media are sohd at room temperature and must be melted before use. Material cannot be mounted directly from water unless the objects are soaked for a long time in molten jelly to allow some of the glycerol to penetrate. Mounts made from specimens taken directly from water are liable to have the jelly crack away from the object as it cools. Moreover, these media are not in their own right good preservatives so that material placed di- rectly from water into them is liable par- tially to decompose before the mount stabihzes. lit is conventional to transfer objects to these media from 50% alcohol, but it is better to harden the objects first in alcohol than to transfer them from alcohol to 50% glycerol and thence into the molten medium. Process of Mounting There are three separate stages in the preparation of glycerol-jelly mounts. The first is hardening the object; the second is getting the object from the hardening fluid into the 50% glycerol; and the third is the transfer from the 50% glycerol onto the slide. As these mountants cannot be used for stained specimens, there is little object in using any fixative other than alcohol. The transfer from the hardening alcohol to the glycerol, however, must be by such stages as will insure that the object does not col- lapse through osmotic pressure. This makes it impossible satisfactorily to mount the majority of nematode worms in 46 Principles JELLY MOUNTS 47 jelly, and thus necessitates the glycerol technique described in Chapter 3. The author prefers to transfer objects from 95% alcohol to 70% alcohol and then to add, by such stages as are necessary, enough glycerol to this mixture to insure that, when the alcohol has been removed by evaporation, 50% glycerol will remain. The two great difficulties in mounting material in these media are first to arrange the object on the shde before the jelly has time to solidify, and then to get the cover- in which they are left until they are thor- oughly permeated, and then placed in a stender dish on top of a water bath, seen in the left background of the picture. On this water bath, which is held about 10°C. above the melting point of the jelly, there is also placed the bottle containing the mounting medium and as many sHdes as will be required. A shde is taken, a molten drop of the medium placed on the warm shde, and the object to be mounted re- moved with a pipet and placed in this Fig. 22. Layout for jelly mounting. slip into place without disarranging the object. The writer has invented a tool for overcoming these troubles, the use of wliich will be described in some detail. The layout, when one is mounting a num- ber of objects, is shown in Fig. 22; the only object not commonly found in laboratories is the special tool shown in the left hand in the picture. This device is a short length of ^^-inch aluminum rod flattened at one end and rounded at the other. This alumi- num rod is screwed to a short length of brass tube which terminates in a wooden handle. The procedure in mounting small objects is as follows. The objects them- selves are accumulated in 50% glycerol, drop. The shde is then removed from the water bath and examined under a micro- scope while a warmed needle is used to push the object into the required position. Make sure that it is in contact with the slide and that there is a considerable amount of jelly above it. In the center of the picture will be seen three slides which have so been treated and laid on one side until the medium has hardened. As many slides as are required may be treated in this manner so that one has a series of preparations, each of which con- tains a domed drop of solid jelly with the object lying in the required position at the bottom of it. Now take a cover glass of the 48 THE ART OF MAKING MICROSCOPE SLIDES Finishing required size, moisten the underside by breathing on it, or by smearing on it a little 50% glycerol, and then lay this shde on top of the domed drop of jelly. The cylinder of aluminum is then taken, warmed in the flame until it is somewhat above the melting point of the jelly, and pressed gently on top of the center of the slide until enough jelly has been melted to permit the cover to drop down to the re- quired level. Very little practice is re- quired before one can flatten down the coverslip without in any way disturbing the arrangement of the object at the bottom, which remains throughout this whole process in a layer of solid jelly. In this way the coverslip is flattened without disturbing the object, and one avoids the constant nightmare of endeavoring to lower a coverslip onto rapidly cooling jelly without disturbing the contained object. If the specimens are so thin that pressure may be applied, a clip is attached and the slide placed for a few minutes on a hot table, to permit an equalization of the pressures between the object and the sur- rounding jelly. The method of mounting in deep cells is practically identical and is, with the de- scribed tool, just as convenient. The slide, with the cell cemented to it, is warmed on a water bath and then filled with molten glycerol jelly. Sufficient glycerol jelly should be used to leave a high domed layer protruding above the cell; air bubbles must be displaced with a hot needle. The object is then placed in the glycerol jelh', arranged in the required position within the cell, and ^then gently' laid on one side to cool. A moistened coverslip is then placed in the center of the dome and pressed down with the warmed tool until it is flattened against the top of the cell. Large cells of this nature should always be placed on a hot plate for at least two or three hours before they are finally cooled, for the commonest cause of break- down of deep jelly mounts is failure to permit the osmotic tension to equahze be- tween the object and its surrounding medium before coohng. Finishing Glycerol Jelly Mounts Most glycerol jelHes contain so much glycerol that they cannot be left unsealed. They may appear excellent for a few days but sooner or later the glycerol will spread out over the surface of the shde, even to the extent of moistening the label and causing it to fall off. The slide, however, must be cleaned before it can be sealed. Make sure that the slide is cold — a refrig- erator is very convenient — and then with a sharp razor blade or scalpel trim away all the unwanted jelly. Then remove the smears that are left with a moist cloth and use another cloth moistened with 95 % alcohol to remove any glycerol which may remain on the glass. There is always some residual gh'cerol, however, which makes it essential that the first coat of cement should be a gelatin-dichromate mixture of the type of Riiyter 1934 (Chapter 28, V 12.1). This is melted on the water bath and applied to the edges of the coverslip with a brush. The slide need not be warmed since these cements stay molten at quite low temperatures. The slide is placed on one side to dry and then given an additional coat of any waterproof ce- ment. It is a worthwhile precaution, be- fore applying the last coat of cement, to wipe off the slide with 95% alcohol to re- move any trace of glycerol. Specific Example Preparation of Small Crustaceans in Glycerol Jelly The term small crustaceans, as here used, includes all specimens up to the size of a Gammarus, as well as the numerous larvae which are found both in fresh and salt waters. This group contains a large number of fascinating forms of universal distribution. A brief word must be said on methods of collection and preservation be- fore passing to actual mounting. Free-swimming crustaceans, whether marine or fresh-water, are collected by means of a plankton net. This is a long conical net, made by the professional from bolting silk, and by the amateur from the Crustaceans JELLY MOUNTS 49 nearest woman's stocking. What distin- guishes tliese nets from all others is that the lower end of the net, instead of being tied off, is blocked by a small glass tube tied firmly in place. These nets are towed slowly through the water, a process which results in the accumulation of large quan- tities of plankton within the net. The net itself is then very slowly and with constant shaking lifted from the water with the result that all those forms which have been accumulated in the net are washed down in the small glass tube, the contents of which may then be tipped out into an- other container. Unless one is on a very long collecting trip, it is better to bring home planktonic crustaceans alive, and for this purpose the contents of the tube should be tipped into at least a gallon of well-aerated water — fresh or salt as the case may be — for transference back to the laboratory. It is almost impossible to make a satisfactory preparation from the horri- ble messes which result from endeavor- ing to preserve directly the contents of an entire tube by throwing it into formalde- hyde, a technique too often employed. When plankton samples are brought back to the laboratory they may again be con- centrated with a plankton net or other de- vice, and the still-Uving individuals are picked out with a pipet and transferred to a small bowl of clean, well-aerated water. Marine forms in particular should be washed in numerous changes of water to rid them of adherent phytoplankton which is almost impossible to remove from the fixed specimen. Though every mounter of microscope slides will go to endless lengths to narcotize many invertebrates, few ever appear to consider this necessary in the case of crustaceans. It is however much easier to identify specimens if their ap- pendages are properly spread out, and the writer always kills crustaceans either ^vith weak alcohol or with chloroform, a few drops of which are sprinkled on the sur- face of the water. As soon as they have dropped to the bottom they will be found to be flexible, and may then be picked out and placed on a sUde, the appendages ar- ranged more or less in the order required, and 95% alcohol cautiously dropped on them until they have stiffened in position. Such specimens may then he transferred directly to a tube of do% alcohol where they will retain the required shape until needed for mounting. Some small crusta- ceans are always found mixed up with weeds, both marine and fresh-water, from which they may be readily sei)arated in the laboratory. Large masses of the weeds are brought back to the laboratory, and placed in shallow dishes with just sufficient water to cover them. The lack of oxygen in the water soon forces the crustaceans to detach themselves and gather round the edges of the bowl from which they may be removed with a pipet. There are a few marine copepods {Dyspontius, for ex- ample) which are considered exceedingly rare, for the reason that nobody ever col- lects them. These forms have sucking mouth-parts which they use to extract the juices of algae. They never become de- tached from these algae in the normal course of events. They may be readily collected by taking large masses of the specific alga, placing it in weak alcohol, shaking it vigorously, and then examining the sludge which is deposited at the bot- tom of the jar. Good hauls of these forms may also often be found at marine stations by going through the sludge which collects at the bottom of the jars in which both algae and ascidians have been stored. There are also many small crustaceans which dwell in mosses, even in those which are apparently quite dry. These will not be secured if the moss is treated with a Berlese funnel in the way described in the last chapter. The only way to collect them is to soak the moss in some kind of narcotic until the crustaceans are stunned, then to rinse off the moss in a considerable volume of fluid which is allowed to settle, and to examine the sludge. The same proc- ess, apphed to marine sands between tide- marks, often discloses small forms not found by any other means. Another fruit- ful way of collecting so-called rare forms is to go netting at night, since there are many marine crustaceans (Cumacea, for example) which become planktonic only at night. Parasitic forms, particularly cope- pods, are also often overlooked, particu- larly those which inhabit invertebrates. No matter how these forms are col- 50 THE ART OF MAKING MICROSCOPE SLIDES Crustaceans lected, they should all be narcotized before killing, and should be killed in 95% alco- hol and stored in this fluid, with a trace of glycerol, until required. When it is de- cided to mount them they are removed from 90% alcohol to 70% alcohol. After they have been permeated with this, glycerol is added little by little until the total concentration of glycerol is such that, if the alcohol be evaporated, a 50% glycerol-water mixture will be left. The final evaporation is best done at a tem- perature of 30° to 40°C. and is very con- veniently conducted in a desiccator through which a current of air is drawn with any aspirator device. If the specimens are not very minute, it is desirable that the 50 % glycerol should be poured off and replaced with the molten mounting me- dium in which the specimens should re- main for an hour or two before mounting. Do not, however, transfer to the molten jelly more specimens than you are able to mount at one time, since prolonged soak- ing in the molten medium tends to soften them. The required number of slides are then warmed and a specimen, in a large drop of molten medium, placed on each. The specimen is then arranged with a needle and chilled rapidly. If there is not a large domed drop of medium over the top of the specimen, further medium is added from a pipet until there is a thick layer. When all the specimens have thus been mounted in the required position with a big dome of cooled jelly above them, a covershp thinly smeared with 50% glyc- erol is laid on each. All of the shdes may be provided with coverslips resting loosely on them before going further. The alumi- num rod shown in Fig. 22 is now warmed and pressed down on the coverslip until the latter has come to rest on the speci- men, or on the walls of the cell. A httle practice is required to be able to do this without pressing down either so hard that one crushes the specimen or so long that one melts the jelly surrounding it. If these specimens are mounted in a medium which does not contain sufficient glycerol, and which accordingly cracks and dries out when they are transferred to a drier environment, they may be re- mounted without very much difficulty by cracking the covershp, soaking the speci- men in 50% glycerol for a week or two, then removing the rest of the coversUp and remounting as though one had a fresh specimen. Wholemounts in Resinous Media General Principles Mounting whole objects by the methods described in the last five chapters involves little preparation of the specimen but a great deal of preparation of the mount. In preparing wholemounts in resinous media a great deal of attention must be paid to the preparation of material, although the actual mounting is simple. Resinous media are used for whole- mounts not only because they permit mounting stained objects but more par- ticularly because they impart to the speci- men a great degree of transparency. This transparency comes from the increase in the index of refraction when the specimen is completely impregnated with the resin. These resins are not, however, miscible with water, hence the water must first be removed {dehydration) and then the de- hydrant replaced with some material {clearing agent) with which the resin itself is miscible. Before these operations the specimen must be killed and hardened {fixed) and it is customary to stain the specimen in order to bring out those in- ternal structures which would become in- visible, were they not colored, through the increase in transparency. All of the follow- ing operations must, therefore, be con- ducted and will be discussed in turn : 1. narcotizing and fixing 2. staining 3. dehydrating 4. clearing 5. mounting Narcotizing and Fixing Specimens Hard objects such as small arthropods, hairs, and the hke may be dehydrated and mounted directly into resinous media,' but are far better prepared according to the manner described in Chapter 4. Most ob- jects which are mounted in resinous media are, however, too^soft to withstand the process of dehydration and^clearing with- out special treatment. Though hardening and fixing agents were once considered as separate, they are now usually combined into a solution known as a fixative. Before deahng with fixatives, however, it is neces- sary to point out that few small animals, on being plunged into a fixative, will re- tain their shape, so that it is necessary first to narcotize them in some solution which will render them incapable of muscular contraction. Narcotization may be caused either through the blocking of nerve impulses which cause contraction, or by some treat- ment which will inhibit the actual con- traction of the muscle. For blocking nerve impulses there are a wide range of nar- cotics available (see Chapter 19, AF 50) and making a choice between them must be a matter of experience. It is to be rec- ommended, in the absence of experience, that one of the solutions containing co- caine be first tried, since cocaine is the nearest approach to a universal narcotic known to the author. Should cocaine not be available, crystals of menthol may be sprinkled on the surface of the water con- taining the specimen. It is very important to distinguish between narcotization and kilUng, for a good wholemount cannot be made from a specimen which has been permitted to die in the narcotic. Narcotization should always proceed slowly; that is, one should add a small 51 52 THE ART OF MAKING MICROSCOPE SLIDES Narcotizing quantity of narcotic at the beginning and increase tlie quantity later, adding the fixative only after the cessation of move- ment. This is easy to judge in the case of motile forms, which may be presumed to be naiTotized shortly after they have fallen to the bottom, but in the case of sessile forms it is necessary to use a fine probe, preferably a hair, to determine the end point of narcotization. Recommended Narcotics and Fixatives for Specific Objects It must be pointed out that the primary purpose of fixing an object before making a wholemount is to retain as nearly as possible the natural shape. The fixative selected should, therefore, contain an im- mohilizing agent as well as a hardening agent. Gray 1933 (11360, 53:14), in a dis- cussion of the principles governing the selection of fixatives, came to the conclu- sion that there were only two good im- mobihzing agents. These were heat and osmic acid. It is therefore necessary, when dealing with highly contractile or imper- fectly narcotized animals either to select a fixative containing osmic acid (Chapter 18, F 1000) or to heat the fixative. Neither osmic acid nor heat are good hardening agents and should not, therefore, be used alone. The best hardening agents for ob- jects which are subsequently turned into resinous wholemounts appear to be chro- mic acid and formaldehj'de, used either singly or in combination, and these solu- tions are usuall}'' acidified with acetic acid to assist in the preservation of internal structures, particularly nuclei. Reference to the classification of fixatives at the be- ginning of Chapter IS will show a large number of solutions fulfilling these re- quirements and no specific recommenda- tion need be made here. Mercuric chloride, particularly in the solution of Gilson 1898 (Chapter 18, F 3000.0014), is another good fixative to use before wholemount- ing. The following recommendations, drawn largely from Gra}- 1935 (Microsc. Rec, 35:4), and Gray 1936 {Microsc. Rec, 37 :10), are to be taken only as suggestions representing the author's opinion and should be used as a basis for further experiment. NoNCONTRACTiLE Protozoa. These do not require nnrcotization and may be fixed directly in a weak solution of osmic acid. The writer, however, much prefers his own technique (described at the end of this chapter) for the handling of these specimens. Individual Contractile Protozoans, These are very difficult to handle. Ten per cent methanol is quite a good narcotic for Dileptus, but 1 % hydroxylamine seems better for Spirodomum. It is the writer's practice to try new forms with the follow- ing narcotics in the order given: 10% methanol, 1% hydroxylamine, 1% ure- thane, AF 51.1 Hanley 1949, AF 51.1 Rousselet 1895, and AF 51.1 Cori 1893 (Chapter 19). There are many forms, how- ever, which do not respond to these nar- cotics and of which it appears almost im- possible to make a good wholemount. Individual rhizopods, as Amoeba and Difflugia, are best fixed to a coverslip in the following manner. Take a clean cover- slip and smear on it a very slight quantity of fresh egg albumen. The solution of Mayer 1884 (Chapter 26, V 21.1), which is often recommended for the purpose, should be avoided, for the glycerol and preservative included in it inhibit the ex- pansion of the animals. Each individual protozoan is placed in the center of a covershj) and left to expand. While this is going on a flask (or kettle) is fitted with a cork. Through this cork is inserted a glass tube the outer end of which has been drawn to a fairly fine point. The water in the flask is boiled to produce a jet of steam. As soon as the protozoan is satis- factorily expanded, the coverslip is picked up very gently and the underside passed momentarily through the jet of steam. This instantly hardens the protozoans in position and at the same time cements them to the coverslip through the coagu- lation of the egg white. The coverslip should then be transferred to any standard fixative solution for a few minutes before being washed and stored in alcohol. Among the Suctoria, Acineta and Den- drocometes may be prepared by placing them in a good volume of water, sprink- ling menthol crystals on the surface, leav- ing them overnight, and then adding suffi- Fixing WHOLEMOUNTS IN RESINOUS MEDIA 53 cient 40% formaldeh3^cle to bring the total strengtli to 4%. These forms may then be transferred to alcohol for staining and preparation as resinous wholemounts or may be mounted directly in formalde- hyde as described in Chapter 2. Stalked Ciliate Protozoans. These forms are quite easy to fix provided one reahzes that double narcotization is neces- sary: once for the stalk and once for the head. The author's technique is to nar- cotize with Rousselet's solution until the snapping movements have slowed up and then very gently to add weak hydrogen peroxide. The specimens are then watched under a microscope and the selected fixative — which must contain osmic acid — is flooded onto them at the exact moment when the cilia straighten out and become stationary. This is satisfactory' with Carchesium, Zo- othamnium and Vorticella. Opercularia and Epistylis have noncontractile stalks and one need, therefore, only use hj^drogen peroxide. The writer has never made a satisfactory mount of Scalhidium or Pyxicola. CoELENTERATA. Hj'droids are usuallj' narcotized with menthol, though the writer prefers liis own mixture (Chapter 19, AF 51.1 Graj') for the purpose, and fixed in a hot mercuric-acetic mixture. A description of the narcotization of Hydra is given in Chapter 9 and a detailed ac- count of the preparation of Medusae in Chapter 20. Anthozoa, particularly the small ones likely to be prepared as whole- mounts, can be narcotized with menthol, though magnesium sulfate is better. Platyhelminthes. Some of the smaller fresh water Turbellaria (e.g. Vortex, Microstortmm) may be narcotized satis- factorily by adding small quantities of 2% chloral hydrate to the water in which they are swimming. Another good techniciue is to isolate the forms in a watch glass of water and place the watch glass under a bell jar together with a small beaker of ether. The ether vapor dissolves in the water and narcotizes these forms excel- lently. A detailed account of the method of handling the liver fluke is given in Chapter 20 and may be satisfactorily employed for other parasitic flatworms. Annelida. Small, marine, free-living Polychaetae make excellent wholemounts and do not usually need to be narcotized before killing. They should, however, be stranded on a slide and a very small quantity of the fixative dropped on them, so that they die in a flat condition which makes subsequent mounting possible. Much more reaUstic mounts are obtained by this means than if they are laboriously straightened before fixing, for they usu- ally contract into the sinuous wave which they show when swimming. There seems to be no certain method of fixing the Nereids with their jaws protruding and one has to rely on chance to obtain one in this condition. The free-swimming larvae of marine polychaetes are very difficult to fix fsatisfactorily, because the large fiota- tion chaetae usually fall out. The writer prefers for these, as for other marine invertebrate larvae, to concentrate a relatively large quantity of the plankton and then to flood over it three or four times its volume of Bouin's fixative (Chapter 18, F 5000.1010 Bouin 1897) at 70°C. The specimens are then allowed to settle, the fixative poured off, and re- placed with 70% alcohol which is replaced daily until it ceases to extract yellow from the specimens. By hunting through a large mass of plankton so fixed, one can usually obtain a considerable number of specimens in a perfectly expanded condition. Fresh water Ohgochaetes are best narcotized with chloroform, either by adding small quantities of a saturated solution of chloroform in water, or by placing them in a small quantity of water under a bell jar in which an atmosphere of chloroform vapor is maintained. Leeches are difficult to handle and the author has had most success bj' placing them in a fairly large quantity of water to which is added, from time to time, small quantities of a saturated solution of magnesium sulfate. As soon as the leeches have fallen to the bottom considerably larger quantities of magnesium sulfate can be added, which will leave the leeches, in a short time, in a perfectly relaxed, but not expanded, condition. They should then be flattened between two sUdes and 54 THE ART OF MAKING MICROSCOPE SLIDES Stains fixed in Zenker's fluid (Chapter 18, F 3700.0010 Zenker 1894). After the speci- mens have been fixed sufficiently long to hold their shape when the glass plates are removed, they are transferred for a couple of daj^s to fresh fixative and then washed in running water overnight. If the crop contains any considerable quan- tity of blood, it will be necessary to bleach this before a satisfactory stained wholemount can be made; the specimens should, therefore, be transferred to the bleach of Murdoch 1945 (Chapter 19, AF 31.1) where they should remain for a few days. Bryozoa. Marine bryozoans may be narcotized without difficulty by sprinkhng menthol on the surface of the water con- taining them. Subsequent fixation is best in some chromic-acetic mixture, for osmic acid|tends to precipitate on the test and blacken the specimen. It may be pointed out that, for taxonomic purposes, dried wholemounts of the test prepared as described in Chapter 1 are of more value than are wholemounts with the expanded animal. It is usually recommended that fresh-water bryozoans be narcotized in some cocaine solution, but the writer has found menthol just as good and much easier to use. Fresh-water bryozoans should be fixed directly in 4% formalde- hyde since they shrink badly in any other fixative. Gastrotricha. These give excellent results by the special technique for minute fresh-water animals described at the end of this chapter. Small Crustaceans. These are some- times prepared as resinous mounts, though the writer prefers to mount them in glycerol jelly in the manner described in Chapter 5. They may be narcotized in weak alcohol and fixed in almost any fixative. Other Arthropods. Wholemounts of most small arthropods are better made in gum media in the manner described in Chapter 4. A detailed description of the preparation of the skeleton of an insect for mounting in Canada balsam is among the typical preparations described at the end of this chapter. Choice of a Stain It is now to be presumed that, whatever method of narcotization and fixation has been employed, the specimens to be mounted have been washed free from fixative and accumulated either in water or 70% alcohol. The reason that so many formulas and methods for staining are given in Chapter 20, 21, and 23 is that no two people have ever agreed on the best method of staining anything. The sug- gestions which follow, therefore, are hkely to be modified by every individual reading the book; but they are included for the sake of those inexperienced in making wholemounts. Small Invertebrates and Inverte- brate Larvae, These are best stained in carmine by the indirect process : that is, by overstaining and subsequent differentia- tion in acid alcohol. For most specimens the writer prefers Grenacher's alcoholic- borax-carmine (Chapter 20, DS 11.22 Grenacher 1879). As an alternative, particularly for marine invertebrates, he has frequently used the two formulas for Mayer's paracarmine (Chapter 20, DS 11.22 Mayer 1892a and 1892b). With these stains available there are very few small invertebrates or invertebrate larvae which cannot be prepared. Larger Invertebrate Specimens, Larger specimens are better stained by the direct process: that is, exposed for a con- siderable length of time to a very weak solution of stain and subsequently not dif- ferentiated. Tills process is described in considerable detail for the fiver fluke in Chapter 20; the directions there given ap- ply equally to earthworms, leeches, or medium-sized polychaetae. Vertebrate Embryos. These seem to stain more satisfactorily in hematoxyhn than in carmine solutions, the author's preference being for the formula of Carazzi (Chapter 20, DS 11.122 Carazzi 1911). This formula is not very well known but may be used whenever the solution of Delafield is recommended. Detailed in- structions for the use of this stain on a chicken embryo are given in Chapter 20. People who wish to produce a startling, Dehydration WHOLEMOUNTS IN RESINOUS MEDIA 55 rather than a useful, mount are recom- mended to try the technique of Lynch (Chapter 20, DS 13.7 Lynch 1930). Plant Materials. Plant specimens for wholemounts often consist of only one, or at the most two, layers of cells and are easier to stain than zoological specimens. The nuclei may be stained either with saf- ranin (Chapter 20, DS 11.42) or with any of the iron hematoxyUn techniques (Chap- ter 20, DS 11.11) which in zoological procedures are rigorously confined to sec- Dehydration is carried out by soak- ing the specimen in gradually increasing strengths of alcohol, it being conventional to employ 30%, 50%, 70%, 90%, 95%, and absolute alcohol. The writer prefers to omit from this series, unless the object is very deUcate, both the 30% and the 50% steps in the process, thus starting with di- rect transfer from water to 70% alcohol. The only difficulty likely to be met in de- hydration is in the handling of small speci- mens, for if they are in specimen tubes it Fig. 23. Transferring objects between reagents with cloth-bottomed tubes. tions. A contrasting plasma stain may be used after the nuclei have been well differentiated. Dehydration It is to be presumed that the specimens, plant or animal, stained or unstained, are now accumulated either in distilled water or in 70% alcohol according to the treat- ment which they have had. It is now necessary to remove the water from them before they can be transferred .into a resinous mounting medium. Ethanol is widely used as a dehydrant and, at least in the preparation of wholemounts, only its nonavailabihty should make any sub- stitute necessary. If substitution is neces- sary, acetone or methanol, in that order of preference, may be used, but they have the disadvantage of being more volatile than ethanol and, therefore, require more care in handhng. is almost impossible to transfer them from one to the other without carrying over too much weak alcohol. The writer has long since abandoned the use of tubes in favor of the device seen in Fig. 23. This is a short length of glass tube, open at both ends, with a small piece of bolting silk or other fine cloth tied across the lower end. The specimens are placed in these httle tubes which (see illustration) are trans- ferred from one stender dish to another wdth a minimum chance of contamination. These tubes are commercially available in England but in America must either be imported or homemade. There is no means of judging when de- hydration is complete save by attempting to clear the object. It is unwise to beheve the label on an open bottle or jar if it says absolute alcohol because this reagent is hy- groscopic and rapidly absorbs water from the air. One should, therefore, keep a 56 THE ART OF MAKING MICROSCOrE SLIDES Clearing quantity of anhydrous copper sulfate at the bottom of the aljsolute alcohol bottle and cease to regard the alcohol as absolute when the salt starts turning from white to blue. More wholemounts are ruined by being imperfectly dehydrated than by any other method, and even the smallest speci- men should have at least 24 hours in abso- lute alcohol before being cleared. If the specimen is to be mounted in Canada balsam, or one of the substitutes for it, it must next be cleared, but if it is to be passed directly to Venice turpentine the reader should turn to end of the chap- ter for a detailed description of this technique. The Choice of a Clearing Agent A clearing agent must be some sub- stance which is miscible both with abso- lute alcohol and with the resinous medium which has been selected for mounting. The ideal substances for tliis purpose are essential oils for they impart just as much transparency to the specimen as does the resin used for mounting, so that one has, as it were, a preview of the finished specimen. The use of xylene or benzene, which is so widespread in the preparation of paraffin sections (see Chapter 12) has tended to spread into the preparation of wholemounts, for which purpose, in the author's opinion, they are worthless. They have a relatively low index of refraction ; hence one cannot tell whether or not the slight cloudiness of the specimen is due to imperfect dehydration until after they have been mounted in balsam. The writer's first choice is terpineol (synthetic oil of hlac) which has advan- tages possessed by no other oil. It is read- ily miscible with 90% alcohol, so that it will remove from the specimen any traces of water which may remain in it through faulty dehydration, and it has also the property of not making specimens brittle. The odor is very shght and rather pleas- ant. Oil of cloves is the most widely recommended essential oil for the prepa- ration of wholemounts and it has only two disadvantages: its violent odor and the fact that objects placed in it are rendered brittle. If a small arthropod be cleared in oil of cloves, it is almost impossible to get it into a wholemount without breaking off some appendages. Oil of cloves is, how- ever, miscible with 90% alcohol. Oil of cedar (more correctly oil of cedar wood) has been recommended in the literature and has the advantage of having a pleas- ant odor and of not rendering ol:)jects brittle. Unfortunately it is very sensitive to water so that perfect dehydration in absolute alcohol is necessary before en- deavoring to clear with it. Two clearing agents, wliich are excellent for unstained specimens, are very little known. These are turpentine and acetic acid. The acid cannot be used with stains for obvious reasons, while the turpentine is a strong oxidizing agent and cannot, therefore, be used after hematoxyhn, though it is perfectly safe with carmine. Absolute (glacial) acetic acid is miscible at all proportions both with water and with Canada balsam. If small arthropods are to be mounted in balsam, rather than in the manner described in Chapter 4, they may be dropped into acetic acid, left there until they are completely dehydrated, and then transferred directly to balsam. This little-known technique is strongly to be recommended. Mounting Specimens in Balsam Nothing is easier than to mount a speci- men in a resinous medium, provided that it has been perfectly dehydrated and cleared. A properly made wholemount should be glass-clear, but it will not be clear in balsam unless it is clear in ter- pineol or clove oil. Not more than one in a thousand wholemounts has this vitreous appearance, and the Avorker who is ac- customed to looking at rather cloudy wholemounts should take the trouble to dehydrate a specimen thoroughly, then to remove the whole of the dehydrating agent with a clearing agent, and then to mount properly in balsam. The first step, therefore, in making a mount in, say, Canada balsam is to make quite certain that the specimen in its es- sential oil is glass-clear; the second step is to make certain that one has "natural" Canada balsam and not "dried" balsam which has been dissolved in xylene. Solu- tions of dried balsam in hydrocarbons are Mounting WHOLEMOUNTS IN RESINOUS MEDIA 57 meant for mounting sections and are, for this purpose, superior to the natural bal- sam. Natural balsam is, however, just as preferable for wiiolemounts and is just as easy to obtain. If it is found to be too thick for ready use, it may be warmed gently until it reaches the desired consist- ency. A single small specimen is mounted by placing it in a drop of balsam on a slide and then lowering a covershp horizontally (Fig. 24) until the central portion touches the drop. The coverslip is then released and pressed very gently until it just touches the top of the oliject. By this means it is possible to retain the object in the center of the covershp and also, if one is using natural balsam which does not shrink much in drjing, to avoid using cells for any but the largest object. Unfortu- nately most people are accustomed to mounting sections in thin balsam by the teclmique shown in Fig. 25: that is, b}^ touching one edge of the covershp to the drop and then lowering it from one side. The objection to this is that the balsam, as is seen in the figure, immediately runs into the angle of the coverslip, taking the object with it, and it is difficult to lower the covershp in such a waj' that the object is left in the center. If one is mounting thin objects, or deep objects in a cell in which a cavity has been ground, it is desirable to hold the coverslip in place with a clip while the balsam is hardening. This proc- ess is seen in Fig. 25, the type of chp there shown being made of Phosphor bronze wire, and is far superior, in the writer's opinion, to any other type. This description presumes that one is using natural Canada balsam, which is un- questionably the best resinous medium in which to prepare wholemounts. If one is using one of the thin resinous media, many formulas for which are given in Chapter 26 under the heading M 30, a very differ- ent technique wiU have to be adopted. In the first place these media are so thin that it is almost impossible to ajiply the cover- shp as shown in Fig. 24, and one is forced to adopt the technique shown in Fig. 25. This difficulty may be avoided by placing the object on the slide, placing a drop of the medium over the object, and tlicn placing the shde in a desiccator until most of the solvent has evaporated. A second layer is then placed on toj-), and a large drop, or rather a thick coat of varnish, is thus built up over the specimen. A cover- slip is then applied and the slide warmed until the resin becomes fluid. The best use for solutions of balsam in making wholemounts is in the preparation of dehcate specimens or a large number of objects. The technique for the former is described in Chapter 20. In the latter case the objects are transferred from the clear- ing medium to a tube or dish of the solu- tion of balsam in whatever hydrocarbon has been selected, and the solvent then evaporated. When the balsam which re- mains has reached a good consistency for mounting, each specimen is taken, to- gether with a drop of balsam, and placed on a slide. A covershp is then added. By this method large numbers of shdes may be made in a short time. It is not necessary to use solutions of dried balsam, and the writer prefers, for this pur^DOse, to dilute natural balsam with benzene. Mounting large objects in a deep cell in Canada balsam is not to be recommended for the reason that the balsam becomes yellow with age and, in thick layers, tends to obscure the specimen. A wholemount of a 96-hour chicken embryo, for example, is of very doubtful value; but if it has to be made it is best first to impregnate it thoroughly with a fairly thin dilution of natural balsam. It is then placed in the cell, piling the solution up on top, and left in a desiccator. The cell is refilled as the evaporation of the solvent lowers the level. When the cell is finally completely filled with solvent-free balsam it is warmed on a hot table and the covershp applied directly. Finishing Balsam Mounts If a mount has been properly made with natural balsam, and if the size of the drop has been estimated correctly, no finishing is recjuii'cd since no balsam will overflow the edges of the coveislij). Natural balsam takes a long time to harden and, if one has a fairly thick mount the covershp of which is not supported, drying cannot be hastened by heating as this will liciuify the balsam, causing the coverslip to tip 58 THE ART OF MAKING MICROSCOPE SLIDES Finishing Fig. 24. Applying coverslip to balsam wholemount. Fig. 25 (inset). Wrong way to apply coverslip to balsam wholemount. to one side. Mounts in natural balsam are better put away for a month or two before any attempt is made to clean them. The slide should be cleaned, when it is suffi- ciently hard, first by chipping off any ex- cess balsam with a knife and secondly by wiping away the chips with a rag moist- ened in 90% alcohol. This will leave over the surface of the slide a whitish film w^hich may then be removed with a warm soap solution, and the sHde may be pol- ished before being labeled. The writer prefers to apply a ring of some cement or varnish round the edge of his balsam mounts for two reasons. In the first place such a ring diminishes the rate of oxidation so that the mounts do not start going brown around the edges. In the second place, such shdes are less Ukely to be damaged in students' hands because of the psychological effect produced by a well-finished shde. One must carefully avoid using any cement or varnish which is soluble in, or miscible with, balsam; the author prefers to use one of the numerous cellulose acetate lacquers which are avail- able on the market. The method of apply- ing such a ring has been described in Examples WHOLEMOUNTS IN RESINOUS MEDIA 59 Fig. 26. Balsam wholemount ready for drying. Chapter 1. With regard to labeUng, it may the label is to be attached should, there- be pointed out that no power on earth will fore, be cleaned more carefully than any persuade gum arable, customarily used for other. The writer prefers to moisten both attaching labels, to adhere to a greasy or sides of the label, press it firmly to the oily sKde: the portion of the sUde to which glass, and to write on it only after it is dry. Specific Examples Preparation of a Wholemount of Pectinatella Though this exposition specifically ap- pUes to preparing wholemounts of the animal named, it applies equally well to the preparation of wholemounts of any other fresh-water bryozoan or, as a matter of fact, for any small invertebrate of about the same size and consistency. Pectinatella has been picked only for the reason that it has a habit of turning up on the walls of the aquaria in the writer's laboratory. If it does not turn up in aquaria in the reader's laboratory, it will be necessary for him to collect the specimen. Fresh-water bryo- zoans are rather like gold: that is, they oc- cur where you find them. Profitable hunt- ing grounds are the underside of the leaves of large water plants and the surface of branches of trees which have fallen into the water but have not yet had time to de- cay. An old trick of European collectors was to lower a length of rope into a pond in which bryozoans were known to occur, and to leave it there for the summer. It was astonishing how frequently, when these ropes were pulled up again in the fall, they were found to be covered with colonies of bryozoans. However the bryozoans be obtained, it 60 THE ART OF MAKING MICROSCOPE SLIDES Pectinatella is necessary next that they should be nar- cotized. The material on which they are living is cut up and the pieces placed in fingerbowl or aquarium of pond water. Distilled water and tap water are lethal to these forms. There should not be so many specimens that they touch each other on the bottom of the fingerbowl, and the fingerbowl itself should be completely filled with water. Fresh-water bryozoa are a little sensitive to heat and may not re- spond well to the high temperatures found in some laboratories. In this case it is as well to put the fingerbowl containing the specimens in a refrigerator, preferably one held at about 10°C, and to leave them there overnight. They may then be brought out and narcotized before they have time to suffer from the increasing temperature. It is usually recommended that fresh- water bryozoans be narcotized with co- caine, either as a straight 2 % solution, or in one of the mixtures the formulas for which are given in Chapter 19 under the heading AF 50. The writer prefers to use menthol which is both cheaper and easier to obtain. For an ordinary fingerbowl about a gram of menthol sprinkled on the surface will be sufficient. There is no means of foreteUing how long it will take the specimens to become narcotized, therefore one must look at them at inter- vals until they are seen not to be contract- ing. This may not be due to narcotization, however, so one should take some very delicate instrument — a hair mounted in a wooden handle is excellent — and use this to push the individual polyps. If, on re- ceiving a push, they contract sharply, it is evident that little narcotization has taken place and more menthol should be sprin- kled on the surface. If, on being pushed with a hair, they contract slowly, it is evident that they are partly narcotized and one must be careful not to disturb them further for at least ten minutes, for if they contract in a narcotized condition they will not again expand. The right stage for killing has arrived when no amount of shoving with a hair will per- suade the specimens to retract, and an ex- amination under a binocular microscope shows the ciliary action on the lophophore not to have stopped. A tube is used to siphon from the fingei-bowl so much water that the remaining layer just covers the specimens. The fingerbowl is then filled with 4% formaldehyde, covered, and placed to one side. One must be careful to distinguish at this point between a killing agent such as formaldehyde, and hardening and fixing agents. In the present instance it is un- necessary, since the stain to be used con- tains in itself an adequate mordant, to use a fixative which will combine with the proteins of the specimen, but it is neces- sary that they should be hardened in order that they may withstand the treatment to which they will be subjected in staining and dehydration. Four per cent formalde- hyde hardens very slowly, and it is sug- gested that they should next be passed to alcohol for the hardening process. It is desirable to flatten the specimens before hardening into the shape that they will be required to assume after mounting. It is to be presumed that the purpose of making a microscope shde is to study the object wliich has been mounted; and the depth of focus of microscope lenses is so slight that only relatively thin objects can be studied at one time. It is extraordinary how frequently tliis simple principle is overlooked, or how frequently people en- deavor to flatten the object after it has been gotten into balsam when it is almost invariably so brittle that it will break up during the flattening process. Five min- utes' work in arranging the parts before hardening makes all the difference be- tween a first-class and a second-class mount. To arrange and flatten the objects for hardening, the 40% formaldehyde is replaced with water. The specimen is re- moved to a fingerbowl of clean distilled water where it is examined thoroughly to make sure it has no adherent dirt. The object is flattened by hardening it between two slides, but obviously, if it is just pressed between two shdes it will be squashed rather than flattened. Anything may be used to hold the two sUdes apart, though in the present instance a very thick No. 3 or two No. 2 covershps would give about the right separation. A glass shde is taken, and about an inch on each side of the center a thick No. 3 coversUp is placed and held in place by the capillary Pectinatella WHOLEMOUNTS IN RESINOUS MEDIA 61 attraction of a drop of water. The speci- men is taken from the water with a large ej^e-dropper type of pipet and placed in a considerable volume of water on the slide. It is then easy to arrange the parts with needles; but it is difficult to lower a second slide on top of the first without disarrang- ing these parts. An alternative method is to place the shde with its coverslips in the fingerbowl with the specimen, to arrange its parts under water and to place the second sUde on top. Whichever process is adopted, the slides are then tied or clipped, together and transferred to a jar of 95% alcohol, where they may remain for a week, or until next required. Each speci- men is treated in this manner; and it is better not to try to flatten two or three specimens on one slide. When it is desired to continue mounting the specimens, each slide is taken and placed in a fingerbowl of 95 % alcohol be- fore the cords which bind them together are cut, or the clips removed. Getting the two sUdes apart without damaging the specimen is not easy, particularly if the specimen tends to stick to one or the other of the sfides. The simplest method is to in- sert the blade of a scalpel into the gap between the shdes and, twisting it slightly sideways, see if the specimen is free. If the specimen shows signs of sticking to one slide, the other may be removed and the specimen washed from the shde to which it is stuck with a jet of 95 % alcohol from a pipet. If it shows signs of sticking to both shdes, it is still possible, bj' projecting a jet of 95% alcohol between them, to free it from both. Each shde is treated in due order until one has accumulated the whole of the flattened specimens in a dish of 95% alcohol. It must be understood that these specimens have been hardened flat so that no amount of subsequent treat- ment will ever swell them out again or prevent them from remaining in the re- quired position. It is recommended, if there are several specimens to handle, that a series of the Uttle cloth-ended tubes shown in Fig. 23 be used. The only alternative is to handle each specimen with the aid of a section lifter with the consequent risk of damage. Though not nearly so satisfactory, it is also possible, at least for the process of staining and dehydration, to place the specimens all together in a small vial in which the different fluids used may be suc- cessively placed. A wholemount of this type is best stained in carmine and the choice would lie between Mayer's carmalum (a detailed description of the use of which is given in Chapter 20), Grenadier's borax-carmine (also described in Chapter 20), and May- er's paracarmine, which will accordingly be selected. Preparation of this stain (the formula for which is given in Chapter 20 under the heading DS 11.22 Mayer 1892) does not present any difficulty, but it should be noted that the differentiating solution is 0.2% solution of strontium chloride in 70% alcohol. Adequate sup- plies of this should be available before one starts staining. The specimens are passed from 95% al- cohol to 70% alcohol. They will naturally float, but as soon as they have sunk to the bottom it may be presumed that they are sufficiently rehydrated and either the cloth-ended tube containing them may be transferred to the dish of stain or the 70 % alcohol may be poured out of the tube and stain substituted for it. One of the advan- tages of this stain is that it is relatively rapid in action — very few specimens will not be adequately stained in five to ten minutes — but it does not matter how long the materials remain in it. It is, therefore, often convenient to leave the specimens in overnight and to start differentiation the next morning. They are then either re- moved to the differentiating solution or, alternatively, the stain is poured off and the differentiating solution substituted for it. In the latter case three or four changes will be required, owing to the necessity of leaving some stain in the bottom of the tube to avoid pouring the specimens out with it. Unless the operator is quite ex- perienced, it is safer to shake the tube so as to distribute the specimens thoroughly in the stain, and then to tip this into a large fingerbowl of dilferentiating solution from which the specimens may be subse- quently picked out and transferred to a new tube of differentiator. It is tragically easy, in pouring off stain, to pour speci- mens with it down the sink. As soon as the stain has been washed off with the dif- 62 THE ART OF MAKING MICROSCOPE SLIDES Pectinatella ferentiating solution, a single specimen should be transferred to a watch glass and examined under a low power of the micro- scope. It is more than probable that little differentiation will be required, so that a simple rinse may be adequate. It is difficult to judge the exact degree of differentia- tion, but it must be remembered that the object will appear darker after clearing than it does in the differentiating solution. The internal organs should be sharply de- marcated when the outer surface of the specimen is relatively free from stain. This may be judged in Pectinatella by placing a covershp on the specimen and examining one of the branches of the lophophore under the high power of the microscope. Differentiation may be considered com- plete when only the nuclei in the cells of the lophophore are stained. The speci- mens are then washed in four or five changes of 70% alcohol, to remove the strontium chloride, before being placed for at least a day in 96% alcohol as the first stage of dehydration. They should then be transferred to two changes, with at least six hours in each, to a considerable volume of fresh 95% alcohol and may then be cleared. Absolute alcohol is not necessary if terpineol is the clearing agent. There is some danger, if the specimens are transferred directly from 95% alcohol to a fluid as viscous as terpineol, that the specimens will become distorted through the very violent diffusion current. This may be avoided in the following manner: one takes a fairly wide (about an inch) glass vial and fills it about half full of terpineol. Ninety-five per cent alcohol is then carefully poured down the side of the vial (or on to a spoon held in the vial in the manner of a bartender making a pousse-cafe) so as to float a layer of alco- hol on top of the terpineol. The specimens are now dropped into the alcohol and sink through it, coming to rest on the surface of the layer of terpineol into which they sink slowly without any strong diffusion currents. They will have sunk to the bottom after a little while, but there will still be alcohol diffusing upward from them. As soon as the diffusion currents have ceased, the alcohol should be drawn from the top of the tube with a pipet and the specimens transferred to clean ter- pineol. When they are in fresh terpineol, they should be examined carefully under a microscope to make sure that they are glass-clear without the least trace of milki- ness. If they appear shghtly milky they have either been insufficiently dehydrated, or the alcohol used for the dehydration has become contaminated with water. In either case they must be transferred to a tube of fresh alcohol for complete dehy- dration and then put back into terpineol. It is a waste of time to endeavor to pre- pare a balsam mount from a specimen which is not perfectly transparent in the clearing medium. When all the specimens are in terpin- eol, take some clean shdes, some clean %-inch circular coverslips, and a balsam bottle containing natural balsam. (The author's preference for the natural balsam rather than a solution of this material in some solvent has already been explained.) Place a drop of the natural balsam on each of, say, six slides, and then one at a time lift out six specimens from the terpineol and place them on top of the balsam. They will sink through the balsam slowly so that these six slides should be pushed on one side while a further six slides have drops of balsam put on them, and so on. As soon as the specimens have sunk to the bottom of the drop of balsam, a covershp is held horizontally above, touched to the top of the drop, and then pushed down with a needle until the specimen is flat- tened firmly against the slide. As these specimens have been properly hardened and flattened there is no risk of their being damaged by drying the mount under pres- sure; therefore one can then apply a clip (see Fig. 26) and place the shdes on a warm table to harden. Each slide is then cleaned, finished, and labeled as usual. Preparation of a Skeleton of an Insect in Balsam Two methods have already been de- gum media given in Chapter 4, and the scribed for preparing small insects as simple, though very little-known, method wholemounts. These are the use of the of dropping the insect directly into glacial Insect skeleton WHOLEMOUNTS IN RESINOUS MEDIA 63 acetic acid and then transferring it from this acid to balsam. Many small insects, or other arthropods, are too opaque for this method of preparation to render ap- parent any details of the endoskeleton, which is so frequently necessary for diag- nostic purposes. These forms must, there- fore, be skeletonized and rendered par- tially transparent before mounting. Insects are skeletonized witli 10% po- tassium hydroxide, which dissolves and removes the internal organs while at the same time softening the skeleton suffi- ciently for the specimen to be flattened and made into a wholemount. Some very skilled mounters of the past made a spe- cialty of preparing whole insects without pressure, but these specimens are chiefly valuable as exhibits, and not as sUdes for study. Let us suppose that we have an ant which is to be made into a transparent wholemount. It does not matter whether this specimen has been freshly collected or has been dried for some time; in either case it must be soaked for at least three or four days in 95 % alcohol. Unless this pre- caution is taken, the strong alkali will dis- solve the thin membrane which holds the joints together and the specimen will fall to pieces. Disregard of this simple precau- tion is responsible for more failures in this type of mount than anything else. After the specimen has been satisfactorily hard- ened in alcohol, it is transferred to water until it is rehydrated and then placed in the alkali. In the case of old and hardened specimens it is desirable first to drill a fine hole at the tip of the abdomen with a sharp needle. After 24 hours in the alkali, the specimen should be removed and stranded on a glass shde with the legs more or less in the position in which they will be mounted. The s{)ecimen is then gently stroked with a brush from the point of the head toward the tip of the abdomen, care being taken not to break off any of the appendages. The purpose of this stroking is to expel any of the viscera which may have been dissolved, either through the natural vent, or the small one which has been made with a needle. It has been stated that the specimen which we are examining, an ant, may be left for 24 hours before this is done, but in the case of thinner-walled and more delicate speci- mens, stroking must be done two or three times a day. The reason for this is that the hydrolysis of the internal organs causes great swelling and, unless some of this fluid is expelled at frequent intervals, the abdomen, thorax, and even the head will be swollen and stretched into an un- natural appearance. The process of gently stroking out the contained material either once or twice a day is continued until it is apparent that no further viscera remain in the animal. It must be emphasized again that it is quite impossible to make a good preparation if the specimen is just thrown into alkali and left there until it is sufficiently transparent. When all the vis- cera have been removed, the appendages are carefully arranged with needles as the specimen lies stranded on the slide, and a few drops of glacial acid dropped onto the specimen from a pipet. This instantly ren- ders the specimen transparent and at the same time partially hardens the append- ages in place. The specimen, however, has yet to be flattened and properly hardened before it can be mounted. A couple of thick covershps, or a couple of pieces of cardboard of the same thickness as that desired in the final specimen, are laid one on each side of the specimen to support a second shde which is then placed on top. It is essential that the specimen should be flattened without producing any wrinkles in the softened chitinous exoskeleton and, unless the insect is naturally flat, this can- not be done merely by dropping a shde on top of it. Instead, the brush which was previously used for stroking is turned side- ways, and rolled backward and forward along the insect, pressing out any wrinkles which may appear. A second slide is placed on top to hold the insect flat and, with the appendages in the position de- sired, two shdes are tied or chpped to- gether and placed in a jar of 95% alcohol until they are next required. They should not remain in alcohol for less than a week and may remain for an indefinite period without damage. After the specimen has been hardened and dehydrated in this manner, the two sUdes are very carefully separated, with the use of a fine pipet to 04 THE ART OF MAKING MICROSCOPE SLIDES Insect skeleton squirt in jets of alcohol to free the speci- men. It does not matter if tlie specimen should stick to one of the two shdes for it may then be mounted on this slide. Whether, however, the specimen is free in alcohol or adherent to one shde, it must now be cleared, and the use of turpentine for this purpose is strongly recommended. As soon as the specimen has cleared, it is placed in the center of the shde and a considerable quantity of natural balsam poured on top of it. Since this specimen will not be danaged by heat, it should now be warmed until the balsam is hot and as hquid as water. Tliis drives off the turpen- tine, as well as most of the natural solvent of the balsam, so that when the covershp has been placed and the slide cooled it will be finished. Excess balsam may then be scraped and washed off in the usual man- ner and the specimen labeled. The description just given is of the con- ventional method of preparing mounts of this type and may be equally well apphed to parts as well as to entire insects. The preparation of demonstration mounts with- out "pressure is now a lost art, and there appears to be no one alive to duplicate the feats of the old mounters of the last cen- tury who were able to turn an entire housefly into what appeared to be an am- ber glass model of itself. For the benefit, however, of those who may wish to resur- rect this art, the author would hke to offer a few suggestions as to a method by which he has prepared passable, but not good, shdes of this type. The insect, re- laxed exactly as if it were being prepared for a museum specimen, is then " set" on a glass slide, with the legs arranged in the required position and held in place by cementing the tip of each leg to the shde with a small drop of molten gelatin. A piece of wood is whittled down until it can be slid between the insect and the glass, thus stopping the legs from con- tracting and pulling the insect down in the next stage of hardening. The glass with its attached insect should then be placed in 95% alcohol and left for a week to harden. If, at this stage, it is placed directly in alkah, the ligaments of the legs will be softened and the specimen will no longer be set in a natural position. It is, however, possible, after the specimen has been re- hj'dratod, to drill a very small hole in the back of the ajjdomen and to work a hypo- dermic needle forward until the tip of it reaches to the head. A minute quantit}^ of 10% potassium hydroxide is then injected and the specimen left in a moist chamber for two or three hours. The needle is then reinserted and a further quantity of po- tassium hydroxide injected. By this time some of the viscera will have been softened sufficiently to come out of the hole on the edge of the needle. Care must be taken that none of the hydroxide gets onto the legs, or onto any of the fine appendages, and particularly that it does not run down and dissolve the gelatin which is holding the specimen in place. After three or four days of making injections daily, the vis- cera of the insect will have been washed out and the body itself commencing to soften. The slide is then put into alkali, where it remains until the legs of the specimen start to soften and to be trans- parent. The specimen is then taken to glacial acetic acid, and from glacial acetic acid to xylene which is used to wash the acetic acid from it. When all the acid has been removed the specimen is transferred to a weak solution of Canada balsam in xylene, in which it remains until it is thoroughly impregnated. A deep cell (see Chapter 1) is next ce- mented to a glass shde, and the specimen transferred to the cell which is filled to the brim with a solution of balsam in xylene. This is placed in the desiccator to evapo- rate, the cell being filled up as often as is necessary. The cell, with its contained in- sect, is then slowly heated to drive off the remainder of the solvent and finally closed with a covershp. The writer must again confess, reluctantly, that such specimens make magnificent show pieces that are of very doubtful scientific value. Preparation of an Alga in Venice Turpentine Venice turpentine is the natural balsam cidua). It is a thick balsam, of about the which is exuded by the larch {Larix de- consistency of natural Canada balsam. Alga WHOLEMOUNTS IN RESINOUS MEDIA 65 and takes its name, as do so many other resins, from the place from whicli it was first exported to England. The commonest commercial use of this material today is in the preparation of artist's pigments, so that it is usually better secured from an artists', than from a scientific, supply house. Man}' substitutes and impure speci- mens are on the market and, as the only value of Venice turpentine lies in its per- fect miscibility with alcohol, any specimen should be tested b}^ seeing whether an equal volume of tlie balsam and of 95% alcohol will make a perfectly clear mix- ture. If they do not, the specimen is not true Venice turpentine and is worthless for the technique which follows. Zimmerman 1896, p. 18, recommends that, in any case, the raw resin should be diluted with twice its own volume of 95% alcohol, filtered, and then heated until the alcohol is evaporated from it. Tliis method is, however, dangerous, unless the atmos- pheric humidity is practically nil. It is much simpler to get a first-class specimen of Venice turpentine in the first place. This medium was first recommended for the preparation of botanical wholemounts by Pfeiffer and Wellheim 1892 (23632, 8:29) but did not come into general use until it was reintroduced by Chamberlain 1915 (p. 97). This writer, however, com- plains that the original directions of Pfeif- fer and Wellheim were diffuse; he cites, not their original paper on the mounting of objects in Venice turpentine, but an- other paper on the preparation of fresh- water algae (Pfeiffer and Wellheim 1894; 10606, 26:674). The present description is drawn from all the sources cited. We will assume that we are dealing with Spirogyra, a form notoriously difficult to prepare as a satisfactory wholemount. It may be collected in quantity almost any- where in the world, and should be trans- ferred immediately after collection to a large volume of any chromic-acetic fixa- tive; the formula of Lavdowsky 1894 (Chapter 18, F 6000.0010) is excellent for the purpose. After the masses of algae have been in the solution for a day or two, they should be washed in running water for 24 hours and then pieces should bo selected for mounting. These pieces should be relatively straight, and about y> inch long, for it is a waste of time to take great masses of algae through the compUcated l)rocesses which follow, and then to select finally only the few pieces which one de- sires to mount. The selected pieces should be carefully passed through 15%, 30%, 50% and 70% alcohol and finally into 90% alcohol in which they are to be stained. Some specimens are so delicate that they will not stand transference from water to alcohol, no matter how gradual the transition phases may be, and for these the method of Chamberlain may be adopted. The algae are transferred from water to 10% glycerol antl the water then evapoiated until they are in pure glycerol. The glycerol is then washed out with 95% alcohol without risk of tlie specimen col- lapsing. This washing must, however, be thorough. Assuming that we now have the speci- mens in 95% alcohol, it is recommended that they be stained by the technique of Chamberlain (Chapter 20, DS 13.5 Cham- berlain 1915). For this there will be re- quired a 1% solution of phloxine in 95% alcohol, a similar strength solution of ani- lin blue in the same solvent, and a 0.1% dilution of hydrochloric acid, also in 95% alcohol. The specimens are transferred from alcohol to the 1 % phloxine solution , where they remain for about 24 hours. They are then rinsed in alcohol for a min- ute or two, or until most of the excess has been removed, and transferred to the ani- lin blue solution. They should remain in this until sufficient blue color is showing in the cytoplasm and until the cell walls themselves have just started to take this blue. They should not, however, remain in the blue for sufficiently long to obscure the bright red color of the nuclei. Cham- berlain suggests that from three to thirty minutes may be necessaiy, but the writer has never had to use a longer period than five. It is obviously desirable to experi- ment witli a few filaments until one has esta])lished the correct time and then stain all the rest of the filaments together. After the filaments have been stained in the blue, they should be transferred to the acid alcohol where they should remain 66 THE ART OF MAKING MICROSCOPE SLIDES Alga until the excess blue has been removed from them. The object of this acid is not to remove blue from tissues which have already taken it, which it will not do, but to rinse off the outside and thus leave the red nuclei bright and clear. If, through any accident, the material has been left in the blue stain too long, it should be transferred to a large volume of 95 % alco- hol and left there until the whole of the blue color has been removed. This will also remove most of the red from the nu- clei, and one must, therefore, start the whole process over again. After the specimens have been stained they must be put into Venice turpentine. The reason for the selection of this mate- rial is that they may be passed directly to it from alcohol without the intervention of any clearing agent which would cause them to collapse. They cannot, of course, be passed directly from the alcohol to full- strength balsam but must be got into it by a process of evaporation. The exact strength to which one transfers them is immaterial, but the strongest which can be safely used is a mixture of ten parts of 95% alcohol to one part of the balsam. No difficulty at all will be occasioned in evaporating off the alcohol, provided it is done in an absolutely dry place. A large desiccator is, therefore, charged with silica gel. This material was not available at the time of the descriptions already cited but it provides what the earlier workers lacked — a material to be used in a desiccator which both provides an adequately dry atmosphere and absorbs a certain amount of alcohol vapor. The weak Venice turpen- tine containing the specimens is, therefore, placed in an evaporating basin, or crystal- lizing dish, and placed in a desiccator con- taining siUca gel. As the alcohol is ab- sorbed relatively slowly by the silica gel, it is preferable to have two desiccators and to transfer the dish from one to the other daily, thus avoiding saturating the atmosphere with alcohol. The evaporation of the alcohol should l^e continued until the Venice turpentine is as thick as the original specimen; and it may be neces- sary, after about two or three days have been spent in the evaporation, to transfer it to a desiccator containing a fresh batch of siUca gel in an oven at 30° to 40°C. This also renders the Venice turpentine more fluid and permits the mount to be more easily made. When the Venice turpentine is suffi- ciently thick, a perfectly clean shde is taken and a small drop of Venice turpen- tine is placed on it. A needle is then dipped into the Venice turpentine and, with an- other needle, one of the short filaments of alga maneuvered alongside it; the first needle is then drawn slowly at an angle about 45 degrees from the Venice turpen- tine, so that the filament will remain lying along the side of it. The needle is then laid flat on the Venice turpentine on the slide and, by a roUing movement, the alga is transferred to the drop. As many fila- ments as are required should be trans- ferred and the slide containing the speci- mens in Venice turpentine should then be placed in a desiccator. One of the easiest errors to make in this technique is to leave a small quantity of alcohol in the Venice turpentine before starting to mount. If this is done, it is al- most impossible to prevent the moisture in the breath from clouding the resin and ruining the whole long complex operation that has already been undertaken. When as many mounts have been prepared as are required, they are removed one at a time from the desiccator, a little fresh Venice turpentine placed on top of each and the coverslip apphed. They may then be placed on a warm stage at a temperature of 30° to 40°C. and permitted to harden for a week or two. It is difficult to clean a Venice turpen- tine mount for if one tries to dissolve off the excess Venice turpentine with alcohol, the covershp may be dislodged. It is better to use only as much Venice turpentine as will exactly come to the edge of the cover- shp to avoid having to clean at all. Vos- seler 1889 (23632, 6:292) recommends that the mount, as soon as it is made, should be ringed (as described in Chapter 2) with a solution of Canada balsam in xylene, and that the Canada balsam should then be hardened. This gives a very attractive and clean mount and is strongly to be recommended. Gray's method WHOLEMOUNTS IN RESINOUS MEDIA 67 Preparation of Minute Fresh- water Organisms by the Method of Gray 1932 The technique here given, which is abridged from the original description of Gray 1932 (11360, 52:370), permits one to prepare permanent mounts of individ- ual microscopic organisms. It may be used equally well to mount an individual i)ro- tozoan or an individual alga. It consists essentiall}' of utiUzing a special fixative which renders a layer of albumen on the slide intensely sticky so that the selected object, immediately after fixation, adheres to it. The range of possible application of this method is very wide, the notable excep- tions to its use being for rotifers, stalked ciliates (wliicli cannot satisfactorily be narcotized), and nematode worms which cannot be mounted in balsam without great distortion. Almost anything else can be mounted, provided that its size lies be- tween a total length of about three milli- meters, and that of the smallest object which can be seen under a wide-field bi- nocular microscope. The reagents required, wloich can most conveniently be kept in drop bottles, are 70% alcohol, ether, 40% formaldehyde, glacial acetic acid, and the stock fixative solution (Chapter 18, F 3500.1010 Gray 1932) which consists of 1% each of picric acid and mercuric chloride in 90% alcohol. Before commencing to mount a series of specimens, it is also necessary to have some Mayer's albumen (Chapter 28, V 21.1 Mayer 1889), some clean shdes, sev- eral small specimen tubes or vials for the preparation of the fixative, some' strips of filter paper, a writing diamond, and, lastly, one or more coplin jars of 70% alco- hol in which the mounts may be accumu- lated as they are made. The required fixative is made up in small quantities according to the speci- mens which one desires to mount. When examining a sample of water without knowing what to expect, it is as well to accumulate three mixtures, each for spe- cific organisms. These are: A. for 'protozoans stock fixative 10 ether 3 acetic acid 2 40% formaldehyde 5 B. for heavily cidicularized forms (e.g. Gastrotricha) stock fixative 10 ether 1 acetic acid 4 40% formaldehyde 5 C. for delicate larvae {e.g. Miracidia) stock fixative 10 ether 2 acetic acid 1 40% formaldehyde 5 The parts given are by volume and the author usually uses drops in making up these mixtures since only a very small quantity is required even in making many slides. Half a dozen clean slides are now taken and smeared with a quantity of Mayer's albumen in the center of each. The layer should be considerably thicker than that which would be applied were one prepar- ing to attach paraffin ribbons. The collec- tion is now examined and any small object which it is desired to mount is taken up in a pipet with the least possible quantity of water and placed on the patch of albumen, beyond the limits of which the water should not run. The surplus fluid is then drawn off, sufficient, however, be- ing left for the animal to swim naturally. The animal is watched until it is in a fairly normal position and a large drop of fixa- tive then allowed to fall on it from above. Immediately the fixative has reached the water, diffusion currents of almost explo- sive intensity result, and considerable care nuist be taken to keep the rapidly moving object within the field of the dissecting microscope. If the object leaves the area of albumen, it must be guided back with a fine glass needle, the point of which will collect a capillary droplet of the fluid. When the animal is in the desired position over the albumen smear, all surplus fluid, which by now will have collected into drops moving slowly over the surface of the slide, is removed l)y the filter |)aper. The object is now closely watched under the dissecting microscope until the droplet of fluid, which will have collected round it, has so far evai)orated as clearly to show the outlines of the object. When evapora- 68 THE ART OF MAKING MICROSCOPE SLIDES Gray's method tion has proceeded this far, the sUde is gently flooded with 70% alcohol The correct point at which to do this is easily recognizable with practice but is difficult to describe in words other than the above. If evaporation be allowed to proceed too far, the animal, especially if it be spherical, is liable to become distorted; if it be ar- rested too soon, the animal is liable to be- come detached. A Uttle practice will, how- ever, readily allow one to determine the exact moment at which the alcohol must be added. After the alcohol has been added to the shde and left for a few moments, a small circle is cut around the object with a writ- ing diamond, as, once lost from the field of the dissecting microscope, such objects as small protozoans are almost impossible to distinguish from specks of dust acquired in the fixing process. The slide is then transferred to a coplin jar of 70% alcohol, for the specimen is firmly fixed in position and will not be detached through any sub- sequent process of staining and mounting. Staining may be carried out by any method. It is customary to stain most small fresh-water animals in alum hema- toxyhn, most small fresh-water plants in some safranin solution, while the writer prefers, for gastrotrichans, one of the alum carmines which must be allowed to act over a long period. It is possible with the aid of this method to mount 20 or 30 selected forms from a collection of fresh-water plankton in less than half an hour, and it will be found very useful to be able to examine a collec- tion of fresh-water plankton with the as- surance that any unknown form may be permanently attached to a shde for subse- quent identification. Smear PreparaUofis from Fluid Material General Principles Every chapter up to the present has been concerned with the preparation of microscope shdes from whole objects pre- served in as nearly as possible their natu- ral shape. Chapters 10 through 15 will be concerned with the preparation of thin slices or sedio^is of objects. Between these extremes of a whole object and a thin slice there are two types of preparation, which are discussed in the next three chapters. Smears, discussed in this and the next chapter, are exactly what their name indi- cate : they are prepared by smearing some substance on a clean glass slide where it may be fixed, stained, and mounted. Squashes, the name of which is also self- explanatory, are prepared by squeezing either animal or plant materials in such a way that thej^ disintegrate into their com- ponent cells, which may then be studied without reference to the relations which they previously had with each other. Smears may either be prepared from fluids or from soUd objects. A separate chapter is devoted to each. The present chapter deals with the preparation of thin layers of fluid so that the cells contained in them may be studied. Three operations are necessary in the preparation of smears of fluids: first, the smearing of the material itself into a layer of the required thickness; second, fixing this layer both to insure its adherence to the slide and to make sure that the contained cells remain in their normal shape; third, staining and mount- ing the fixed smear. Each of these opera- tions will be discussed successively. Preparation of the Smear The first thing to do in the preparation of a smear is to make sure that chemically clean slides are available. The adherence of the smeared material to the slide will be excellent if it is a fluid containing con- siderable quantities of protein (as blood), even if the shde be not clean, but there are many fluids which are used in the pro- duction of smears which will not adhere at all save to an absolutely clean glass surface. Any method may be used for cleaning slides, but for this particular purpose the author prefers to use a house- hold scouring powder, which consists of a soft abrasive together with some detergent agent. This powder is made into a thin cream with water and each slide is then dipped into this cream and stood in a rack to dry. As soon as it has dried the shdes may be returned to a box, preferably each being separated from the other with a thin paper insert. As slides are commonly sold with these paper separators, it is only necessary to take a box and to save the separators when one dips the slide, return- ing them after they are dried to the same box with the same separators. Two or three hundred slides may easily be prepared in this manner in a short time and stored against future use. For use the slide is polished with a clean linen or silk cloth. Smears often have to be made at un- expected moments, therefore it is a con- venience to have slides at hand which may be rendered fit for use in a few moments. The actual method of smearing the ma- terial varies greatly according to what is being used. Probably more smears are made of blood than of any other fluid, and the technique for the preparation of these is so well established that it will be de- scribed as a type. The material itself may either be taken from the puncture wound G9 70 THE ART OF MAKING MICROSCOPE SLIDES Smearing directly onto the slide, or (as in Fig. 27) behind the first slide and distributed more removed from the puncture wound with a or less uniformly on the under shde. A few pipet and transferred to the slide. The people still try to conduct the operation drop is placed about }'i of an inch from in the reverse manner: by placing the sec- one end of the slide, and a second shde (as ond slide on top of the first, sloping it at a Fig. 27. Making a smear preparation, a. Place the drop about an inch from the end of the slide. Fig. 28. Maicing a smear preparation — (continued), b. Apply a second slide just in front of the drop. Fig. 29. Making a smear preparation — (continued), c. Push the slide smoothly forward to spread the smear. shown in Fig. 28) placed on the drop. Capillary attraction will naturally dis- tribute the fluid along the edge of the sec- ond slide which is then (Fig. 29) pushed sharply forward until it reaches the end of the bottom slide. The material of which the smear is being made is thus spread out reverse angle to that shown, and then en- deavoring to push rather than drag the material across the lower shde. The objec- tion to this is that it results in crushing cells, though it must be admitted that it frequently gives a more uniform distribu- tion of the material. The use of a glass shde Fixation SMEAR PREPARATIONS FROM FLUID MATERIAL 71 for spreading has a great deal against it. There is a risk tliat the edge of the upper slide will scratch the lower, and though these scratches are not apparent in smears which are to be examined under an oil im- mersion objective, they are objectionable in a dry slide. It is also difficult to secure a sUde which is entirely flat and which will thus make a layer of uniform tliickness, for the tliickness of the laj^er obviously depends on the degree of contact between the upper sHde and the lower. The writer has recently been using a thin sheet of transparent plastic (methyl methacrylate) in place of glass and has obtained very much better results. To prepare such a sheet for use one cuts, or saws, a 3" X 1" rectangle from it and then polishes one of the edges by rubbing it briskly backward and forward on a sharpening stone or on a ^ piece of fine sandpaper. This turns up a feather edge on both sides of the edge which has been flattened. This is removed by taking the shp and holding it at just about the same angle at which it will be used for preparing the smear and giving it one or two quick strokes on the finest sandpaper. The use of a soft material like this not only insures that there will be no scratches on the slide, but also guarantees that the edge used for smearing will always remain in contact with the slide. After two or three hundred smears have been made the piece of plastic may be thrown away and a new one taken. The method described is the standard procedure for producing thin smears. These are necessary for those fluids, such as vertebrate blood or mammalian seminal fluid, which contain very large numbers of objects which must be separated as widely as possible if they are satisfactorily to be studied. There are a number of fluids, however, from which thick smears must be made either because, as in the case of inverte- brate blood, they contain relatively few cells or because, as in the case of malarial diagnostic smears, one is seeking for a parasite which is relatively sparsely dis- tributed through the material. These thick smears are made with the aid of a loop of wire held in a needle-holder of the type found in bacteriological laboratories. This loop is dipped into the fluid to be ex- amined, and used to spread it with a rotary motion in tlie center of the shde. This is very similar to the preparation of smears of bacterial material which is de- scribed in some detail in Chapter 21. Fixing Smears Smears may be fixed by drying, by alcohol, or in one of the conventional fixa- tives. When a smear is to be fixed by dry- ing it is, as soon as it has been made, waved in the air and then set on one side for subsequent treatment. This procedure is excellent in the case of objects such as bacteria or erythrocytes, which do not change their shape after drying, or for materials such as white blood corpuscles, which it is not desired to preserve in their normal shape. No other object can, how- ever, be considered satisfactory unless it has been fixed, and the simplest method of doing this is to pass the smear, just as it is drjdng, through a jet of steam. This technique has been described in Chapter 6 for mounting amebas and need not be re- peated here. All other smears should be fixed before they are dried and it is something of a problem to fix them without removing the material from the shde. It is obvious that if the material is freshly smeared onto a glass shde and then dropped into a fixative of some kind, it will be washed off. The logical solution to the problem is to use a fixative in a vapor phase, and nothing is better for this purpose than osmic acid. To use this material, a couple of glass rods are placed in a petri dish sufficiently far apart to permit the sUde to rest on them without the smear touching them. A drop or two of a solution of osmic acid, usually of 2 % strength is put on the bottom of the petri dish and the cover replaced. It must be emphasized that osmic acid fixes the mucous membrane of the nose and throat just as readily as it does a smear and every precaution must be taken to avoid inhal- ing the vapors. As soon as the smear is made, and before it has time to dry, it is placed face down across the two glass rods so that it is exposed to the vapor but not to the liquid. The cover is then replaced on the petri dish, and the slide left in place for 72 THE ART OF MAKING MICROSCOPE SLIDES Staining about three or four minutes in the case of a thin smear, or for five to ten minutes in the case of a thick one. It is then trans- ferred to distilled water to await staining. It occasionally happens that a slide must be fixed in one of the conventional fluid fixatives. This is done with the same petri-dish and glass-rod setup as is used for vapor fixation, but in this instance the fixative is carefully poured into the petri dish, which must be level, until it has reached such a depth that, when the slide is laid across the glass rods, the under side of the slide with the smear on it is in con- tact with the fluid while the upper part is free from fluid. If the smear is reasonably tliin and is laid carefully in place, it usu- ally will not become detached. Thick smears, particularly those made with fluids containing very little protein, will not stand this treatment; they must either be fixed in the vapor phase, or else the fluid itself must be mixed with a small quantity of an adhesive, such as Mayer's albumen (Chapter 28, V 21.1 Mayer 1884). Staining Smears Blood smears are so universally stained with one or another of the methylene blue- eosinate mixtures (Chapter 20, DS 13.1 and 13.2) that it comes as something of a surprise to most people to learn that any stain which is suitable for sections may also be employed for smears. The ad- vantage of methylene blue-eosinates for blood films is that the solvent methanol acts as a fixative so that they are stained and fixed in the same operation. When a blood smear is to be used for diagnostic purposes, these techniques are excellent, because the appearance of the various types of white corpuscle under this treat- ment is known to every technician. For research studies on the blood, however, it is strongly recommended that the worker experiment, first by fixing the blood film in osmic vapor in the manner described, and secondly by applying to it some other of the complex techniques described in Chapter 20. For materials other than blood there is no limit to the type of stain- ing which may be employed, though it must be remembered that very thin films require a stain of considerable intensity if the finer structures are to be made out. Thus, for example, a thin smear of mam- mahan spermatozoa is best stained by one of the very dense iron hematoxylin tech- niques such as that of Biitschli (Chapter 20, DS 11.111 Butschh 1892). The method for the application of these stains to smears differs very little, in most cases, from the method for the application of the same stains to slides, and no specific in- struction need be given. Bacteriological staining methods, which differ from those used in botany and zoology, are given in Chapter 23 under the heading DS 23.2. Typical Example Demonstration of Monocystis from the Seminal Vesicle of an Earthworm Few sporozoans are available for class demonstration purposes and the choice is practically limited to the inhabitants of the intestines of a cockroach or to the specimen at present under discussion, Monocystis. The advantage of Monocystis is that all the forms from the sporozoite to the trophozoite occur in the seminal vesicle of the earthworm, and may therefore be made available on a single smear. The degree of infection among earthworms varies greatly, but it has been the author's experience that the larger the earthworm the more hkely the chance of a heavy in- festation. But it is no use making a whole lot of smears for class demonstration pur- poses until one has satisfied oneself by a preliminary survey of a single smear that the material will be satisfactory. There is no need to kill or anesthetize the earthworm, which' is simply pinned down in a dissecting tray and sUt from the anterior end to about the 16th or 17th seg- ment. The edges of this slit are pulled back and pinned into place disclosing the large white seminal vesicles. There should be available, before mak- ing the smear, a petri dish in which are a couple of short lengths of glass rod, a sup- Monocystis SMEAR PREPARATIONS FROM FLUID MATERIAL 73 ply of Schauclinn's fixative (Chapter IS, F 3000.0000 Schaudiiiu 1893), an ade- quate supply of clean glass slides, an eye- dropper-type pipet, some 0.8% sodium chloride, and some cophn jars of distilled water. Enough fixative is poured into the petri dish so that when a sHde is laid on the pair of glass rods, its lower, but 7}ot its upper surface will be in contact with the fixative. This level is best established with a plain glass slide before the smears are started. The seminal vesicle of the earthworm is slit and a drop of the contained fluid re- moved with the pipet. This pipet is then used to smear a relatively thick layer of the material on the center of one of the clean shdes and, before it has time to dry, this slide is laid face down in tlie fixative for about two minutes. The sUde is then removed, rinsed under the tap, and ex- amined under a high power of the micro- scope after a covershp has been placed over the smear. It is rather difficult to see the trophozoite stages in an unstained preparation, but no difficulty will be ex- perienced in picking out the spore cases (pseudo-navicellae) owing to their rela- tively high index of refraction. It may be taken that adequate numbers of the para- sites are present if not less than three or four of these spore cases occur within the field of a four-millimeter objective in a thick smear of this nature. As soon as a satisfactorily infected worm has been found, the remainder of the material from the pipet is placed in a watch glass and diluted with 0.8% sodium chloride until it forms a dispersion about intermediate in thickness between cream and milk. As many smears as are required are made from this cream as rapidly as possible. The dilution in question will not retain the parasites in good condition for more than about five minutes, but if in- sufficient smears have been made in this time, it is easy to take a fresh supply of the seminal fluid from another vesicle and to dilute it in a fresh watch glass. The cream should be spread with two slides in the manner described above, and each shde placed face downward in Schaudinn's fixative for three or four minutes before being removed to a cophn jar of distilled water. After having been washed in water the smears should be transferred to 70% alcohol where they can remain until they are ready for staining. Any stain may be used but it is conventional to employ a hematoxylin mixture. The author prefers to use the old "triacid" stain of Biondi which is given in Chapter 20 under the heading DS 13.33 Biondi (1888). The ad- vantage of this solution is that the orange G is picked up by the cases of the sporo- cysts, while the trophozoites are red with clear green nuclei. Nuclei of the sperma- tozoa and spermatids of the earthworm occupy so much of the cells in which they are found that they give the whole stain a greenish cast. This green background shows up the red trophozoites and the brilliant orange sporozoites. The method of staining is easy. The solution, made in accordance with the directions given, is diluted to the extent of about 2% with distilled water. The slides are then placed in this diluted solu- tion and left until examination under the low power of the microscope shows them to have been adequately stained and differentiated. They are then briefly rinsed in distilled water and dried. There is no necessity to use any dehydrating agent, such as alcohol, which will interfere with the staining, because the smears should be sufficiently thin and the objects in them suflticiently well fixed that drying will not distort them. To complete the mount, a drop of the mountant selected is added and a coverslip applied. Balsam may be used, but the writer considers it to have too high a refractive index, and for that reason prefers one of the "neutral" mountants, based on gum sandarac, the formulas for which are given in Chapter 26 under the heading M 23.1. 8 Smear Preparations from Cut Surfaces General Principles The last chapter described the prepara- tion of sniears from materials which were fluid; the present chapter deals with the method by which smears may be obtained from the surface of materials which have been cut into blocks. Preparation of the Smears There are only two methods of produc- ing smears from the cut surface of sohd bodies. Either the cut surface is rubbed on a clean slide,- leaving behind a few cells which have been detached from it, or the cells are squeezed from a cut, and subse- quently pressed out in the form of a smear. The former method is used for animal tissues and the latter for plant specimens. The difficulty in preparing smears from the cut surface of animal tissue is not so much to secure material as to avoid secur- ing unwanted cells. The blood content of the majority of organs is so high that, if a freshly cut surface be smeared on a piece of glass, the few cells which become de- tached will be obscured by the red blood corpuscles. The technique is, therefore, usually applied to the central nervous system, from the cut surfaces of which cells not only detach themselves very readily but which has also the advantage of being only shghtly vascularized. The only difficulty in preparing a smear from a freshly cut surface of the central nervous system lies in finding a pair of forceps sufficiently wide and sufficiently blunt to hold the material without it disintegrat- ing. Apart from this the technique is essen- tially that described in the last chapter. A perfectly clean slide is taken and rubbed with the cut surface of the mate- rial. These smears cannot be dried satis- factorily but must always be fixed, and it is necessary to use the technique, dis- cussed in the last chapter, of placing the sUde face down across a pair of glass rods lying in the bottom of the petri dish in such a manner that the lower surface, but not the upper surface, is in contact with the fixative. The fixative to be selected naturally varies according to the material to be studied, but fixatives containing mercuric chloride are usually to be pre- ferred. Smears may also be fixed in osmic vapor, though they are not usually as satisfactory w'hen prepared by this method as are smears prepared from fluid material. The smear technique in plant micro- technique is largely confined to securing sporogenous tissue from anthers at various stages of their development. This tech- nique, which was introduced by Taylor 1924 (3430, 78:236), has the advantage that it permits the examination of chromo- some material without the trouble of de- hydrating, embedding, and sectioning. This smear technique must, however, be differentiated from the squash techniques, described in the next chapter, in which cells are dissociated. The smear technique can only be used for materials which can be squeezed out. There is some division of opinion as to whether a small quantity of adhesive (such as Mayer's albumen) should first be smeared on the sUde, or whether the material extruded from the anther has enough protein to cause ad- hesion. In either case the anther is cut with a very sharp scalpel about one third of the distance from its base and placed on the sUde with the cut end in the region where one wants the smear. The back of a scalpel is then rolled from a position about a milfimeter from the cut edge, toward the cut edge. The material which is thus ex- truded is rapidly smeared with the back of the scalpel, the crushed anther removed, and the slide inverted on glass rods in a fixative. It has been found (Kauffman 1927, 20540b, 2:88) that these smears are best stained with an iron hematoxyUn technique (Chapter 20, DS 11.111). 74 Negri bodies SMEAR PREPARATIONS FROM CUT SURFACES 75 In every other respect these smears are treated as though they had been prepared from a fluid material (see last chapter) but a single typical preparation applying one of these techniques will be given. Typical Example Demonstration of Negri Bodies by the Method of Dawson 1934 The detection of Negri bodies is, of course, used in the diagnosis of rabies. A method for the demonstration of these bodies in sections is described in Chapter 21, and the method here described is in- tended less for permanent preparations than for a rapid diagnostic procedure. The description referred to contains de- tailed directions for the dissection of the horn of Amnion, which is the portion of the brain usually selected for these tests. We will assume, therefore, that the worker has dissected, following the necessary pre- cautions as to his own safety, the brain of the diagnostic guinea pig in such a manner as to expose the horn of Ammon. For the preparation of paraffin sections, the horn of Ammon is divided into small pieces, but for the preparation of smears it must be kept whole and a sharp razor used to trim away about the lower third leaving a freshly cut surface. If there is any quantity of extravasated blood pres- ent, it must be washed off with a gentle stream of normal saline or it will tend to obscure the picture. To prepare the smear preparations there are required an adequate quantity of clean shdes, a supply of methanol, a 2 % solution of phloxine, and a quantity of Lofiler's polychrome methylene blue. The prepara- tion of the polychrome methylene blue solution is described in Chapter 20 (DS 11.44 Lofiier 1890) and need not be re- peated here. It is convenient to have the methanol and methj'lene blue solutions in drop bottles and to have the 2% phloxine and the 20% alcohol in coplin jars. If the slides are to be filed for reference, rather than used immediately for diagnosis, it is also desirable to have some neutral mountant (Chapter 26, M 23.1) The entire horn of Ammon is then taken and dabbed once or twice in the center of one of the clean shdes. If one endeavors to smear it, too much material usually (;omes off; but the vertical dabbing motion will provide a sufficiently thick film which, when moist, should appear as no more than a slight clouding of the surface of the slide. The shde is then waved gently back- ward and forward until it appears to be just about to dry. The material will be lost if methanol is added to it while it is completely wet, and the material will be distorted if it is permitted to become en- tirely dry; but only a little experience is necessary to enable one to adjudge the exact moment at which methanol should be dropped on the smear from the drop bottle. The shde may then be placed on one side to dry, if it is to be used immedi- ately; it should be dropped into a cophn jar of methanol if it is not to be stained for, say, half an hour. When the slides are to be stained they are waved in the air until the methanol has evaporated and then dropped into the 2 % phloxine where they remain from two to five minutes. Upon being withdrawn from the phloxine, a stream of water from a wash bottle should be used very gently to remove the excess phloxine, the slide drained by its corner onto filter paper, and the polychrome methylene blue dropped onto the surface from a drop bottle. The methylene blue is left in place for 15 seconds, washed off with a stream of water from the wash bottle, and the slide then dropped into 20% alcohol where it may be left until no more color comes away. It may remain in the alcohol for 10 or 15 minutes without damage. If the slide is required for immediate examination, nothing remains to be done save to remove it from the 20% alcohol, wave it about in the air until it is dry, and then examine it under the microscope with an oil immersion lens. If, however, the shde is to be filed for permanent refer- ence, a drop of neutral mountant should be placed on the smear as soon as it has been dried and a covershp added. Squash Preparations from Solid Bodies General Principles Nature of the Process Squash preparations are not, as their name might cause one to suppose, ob- tained merely by crushing an organ or animal in order that it may become thin enough to examine under the microscope. This would result in the hopeless distor- tion of the cells and their contents. A squash preparation, properly prepared, is obtained by causing the cells of animal or plant material to become separated one from the other without losing their indi- vidual shape in order that they may be spread out on a shde in a single layer for examination. This process is today much better known in botany than in zoology, though it was at one time the standard method of preparing histological speci- mens. Another fundamental difference be- tween the smear and squash technique is that the former always employs fresh un- fixed material while the latter should always employ- a material which has been fixed previously. Process of Maceration The separation of cells of fixed plant or animal material through the hydrolysis of their interstitial tissue of cement is known as maceration. It does not matter what fixative has been used, though in the author's experience fixatives containing cln-omic or osmic acid, or mixtures of these, are best. Tissues are fixed in the ordinary way and the fixative thoroughly removed ]:)y washing before maceration commences. The two most common metli- ods of maceration, either for animal or plant material, are acid hydrolysis and enzyme hydrolysis. Each will be described separately. Acid hydrolysis of plant tissues is almost always carried out in 10 % hydro- 7G chloric acid, in ^vhich the fixed tissue is soaked until a sample of it, placed under a coversUp, is found to disintegrate into its constituent cells when the coverslip is tapped hghtly with a needle. Acid hydrol- ysis of animal tissues, however, has been carried out with almost any acid used for microscop}^ and reference should be made to Chapter 19 (V 40) where many sug- gested mixtures are given. It may be pointed out that almost any acid fixative solution, if diluted with from 50 to 100 times its own volume of water, will act as a macerating agent. Enzyme hydrolysis of animal tissues may be conducted either in an alkahne or an acid environment, and reference should be made to the methods of Jonsset 1903 and Langeron 1942 for examples of each of these. Abbre\dated techniques for these methods are to be found in Chapter 19 under V 40 and need not be expanded here. Enzyme hydrolysis of plant tissues is of quite recent origin, and depends on the extraction of enzymes from sources which are customarily used in the digestion of plant material. Ensweller 1944 (20540b, 19, 109) suggests the extraction of various fungi but the method of Faberge 1945 (Chapter 19, V 41.1), of which a detailed description is given in one of the typical preparations following this chapter, is much to be preferred. The terminal point of enzyme maceration may be detected, exactly as is that of acid maceration, b}'' whether or not the organism or tissue unrler examination disintegrates into its constituent cells. Staining and Mounting Macerated Specimens Though the process of maceration is it- self quite easy, staining and mounting of Chromosomes SQUASH PREPARATIONS FROM SOLID BODIES 77 the products of maceration present many difficulties. It' the preparation is broken up under a covershp, it is difficult to remove it witliout losing the cells, or to stain them with the coverslip in place; if the macera- tion is carried out in a small tube, it is difficult to concentrate the cells readily on the slide after they have been stained. Probably the simplest method in most cases is to treat the individual cells as though they were a culture of protozoans: that is, to stain them, dehydrate them, and get them into a small quantity of balsam, and then to place a drop of this balsam under the coverslip. As an alterna- tive to this, the macerated material may 1)6 smeared over the surface of a slide which has had an adhesive applied to it, or it ma}' be diluted with an adhesive material such as Mayer's albumen and then treated as though it were a fluid smear. The ol)jection to this treatment is that the majority of fixed cells are very brittle and will be damaged when the smear is made. The selection of a stain is not difficult since each dissociated cell may be treated as a small wholemount. It is not worth while to double-stain macerated speci- mens, the true function of which is to present a clear picture of the shape, not the nature, of individual cells. Typical Examples Preparation of Microsporocytes of Crocus for Chromosome Examination In the last chapter it was pointed out that material of this nature could be squeezed from the anther and spread over a sfide with the back of a scalpel. This method inevitably leads to distortion both of the cells and of the chromosomes, and in the writer's opinion the method here de- scribed gives a better preparation. There is first the collection and fixation of the anthers, and second, the separation of the microsporocj'tes from the other cells of the anther by maceration. The advantage of the crocus for this jDreparation is that it maj^ be brought into flower in the laboratory at any season of the year. The anthers may be taken at any stage of their development, the most useful stage for demonstration prepara- tions being that which is reached when the flower is just beginning to color. The flower is removed from the corm, the petals stripped away, and the anthers placed in fixative. Many fixatives may be used, though one of the best is Navashin (Chapter 18, F 6000.1010 Navashin 1912). The anthei's are placed in several hundred times their own volume of this fluid which is, by convention, but probal^ly unneces- sarily, kept in the icebox. The anthers are removed after 24 hours and waslicd over- night in running water. The method of maceration selected for this example is that of Faberge 1945; the abbre\aated directions are in Chapter 19 under the heading V 41.1. This method uses the stomach of the edible snail {Helix pomatia) which dissolves the intei'stitial tissue of plant cells. Edible snails are ol)- tainable either by collection in the field o r from restaurant supply houses. Snails ob- tained from the latter are usually in a state of liibernation from having been kept on ice and must be revived by being kept at room temperature for a day or two, after which they may be given a meal of lettuce and used on the day following. The snails are killed by drowning in warm water overnight, which leaves them fully ex- panded, and the stomach is then dissected out. The snail is removed from the shell and pinned down through the foot. The mantle cavity is then lifted in a pair of forceps and sfit. As soon as the edges of this sfit have been pulled back the crop (often miscalled the stomach) will be seen as a carrot-shaped body filled with brown- ish fluid. The fluid within the crop must now be withdrawn, either by the insertion of a hypodermic syringe, or by figaturing the crop at each end, removing it, and then squeezing the contents into a tube. It is simplest to handle a good many snails at the same time, since the material removed may be preserved in an icebox with a drop of toluene on top. The anthers, taken either from the water in which they have been washed or the alcohol in which they are stored, are placed in a drop or two of the enzyme solu- tion. If the maximum number of sfides 78 THE ART OF MAKING MICROSCOPE SLIDES Hydra are required, it is desirable to cut the anthers into pieces before placing them in the fluid. Maceration will usually be com- plete in about eight hours so that it is con- venient to start the preparation in the evening and to examine individual pieces at intervals in the course of the next morning until one has determined that maceration is complete. The completion of maceration may be judged by the fact that the materials should be flabby, but not completely disintegrated. At this stage the microsporocytes are extracted by gently squeezing either the anther, or each individual piece of anther, into a small drop of water. The micro- sporocytes themselves will not appear to be distinct but will appear as a gelatinous mass. This mass should be accumulated in a drop of water on a single slide and then small drops taken from it and made into smears on other clean slides. These smears need not be fixed, but may be permitted to dry on the shde to which they will ad- here so well that they can be stained by any nuclear staining method. Aceto-car- mine is in general use for temporary prep- arations, and a description of the use of this stain in plant material is given in Chapter 20. Preparation of a Dissociated Hydra It is a little pathetic that the majority of elementary textbooks of biology should include an illustration showing the types of cell to be found in hydra and that in- structors should then issue to the student a series of sections in which these cells are not visible. The illustrations have mostly been taken from older textbooks dating from the period when disassociation tech- niques for animal tissues were common. It would surely be more reasonable to show students shdes wliich agree with the illustrations in the books they use. Fixation is necessary before dissocia- tion, and the only question to be settled is whether or not the finished slide should show muscle cells in an expanded condi- tion, in which case the hydra will have to be narcotized before fixation, or whether it will be sufficient to kill the hydra with- out narcotization. It seems better, how- ever, to show cells in the expanded condi- tion and the hydra should therefore be collected from the tank or pond where they are growing, and accumulated in a watch glass of water which is kept in a cool place in subdued fight so that the hydra may expand. A drop of 2% chloral hydrate is then added for each five milfi- liters of water in the watch glass. This should be mixed with the water by suck- ing in and out of a pipet. This is likely to cause partial retraction of the hydra but they will expand again. After about 10 minutes two or three more drops per five milUliters of water may be added and mixed in, as the hydra are usually by this time sufficiently narcotized not to con- tract. The hydra should then be watched until touching with a hair causes no retrac- tion. The watch glass is then very care- fully picked up and placed in a fingerbowl. The reason for this is that hydra can most satisfactorily be fixed in large cjuantities of hot fixative. The solution of Perenyi (Chapter 18, F 6000.0040 Perenyi 1888) is excellent for this purpose. Tliis fixative, which has few other uses, is made by dis- solving % of a gram of chromic acid in 135 milHliters of water and then adding to this 100 millihters of ethanol. Seventeen and a half millihters of strong nitric acid are then added and the solution placed on one side until it has turned violet. The fixative should be heated to 70°C. and then flooded suddenly over the narcotized hydra which should remain in the fixative for about two days before being removed for dissociation. An individual dissociated hydra may be prepared as a smear, but it is presumed in this case that a number of shdes are being made for class issue, so that it is better to proceed by a different technique. The hydra are taken from the fixative (it is unnecessary to wash them) and placed in a few drops of the selected dissociating agent in the bottom of a small tube. The selection among the acid, dis- sociating media given in Chapter 19 under the heading AF 41.1 is not important, but the writer has been successful in the pres- ent preparation by the method of Hopkins as quoted by Roberts [Chapter 28, VJ41.1 Hopkins (1895)]. This method requires first, 20% nitric acid, second, a saturated solution of potassium alum. Hydra SQUASH PREPARATIONS FROM SOLID BODIES 79 The tube containing the hydra is half filled with 20% nitric acid. The specimens may be left overnight for treatment the next morning or, if one is in a hurry, the tube may be very gently warmed (to a maximum of about 50°C.) for twenty minutes. In either case it will be found that the hydra, which had become hard in the fixative, are now flaccid and tender. The acid is removed, either by pouring or by withdrawing it with a pipet, and the tube filled with the saturated solution of potassium alum. The specimens will float for a brief time but, as soon as they have sunk to the bottom, the alum solution is poured off and replaced with fresh solu- tion. The tube is now shaken gently until such time as each hydra has dissociated into its constituent cells. If this does not take place after shaking for a few minutes, it is necessary to withdraw the alum solu- tion, replace it with nitric acid, and to continue macerating for a further period. It is not to be anticipated that all hydra out of a batch of 20 or 30 will disintegrate at the same time, but any large cell masses which remain may easily be picked out with the point of a needle after the tube containing the cells has been emptied into a watch glass. * Having thus secured a suspension of the cells in a solution of alum, it is necessary ,, first to wash most of the alum from them, ^' and then to get them into stain. For this purpose rinse the tube into a larger one which is filled with water, stir up, and allow the cells to settle. Any of the alum- carmine stains given in Chapter 20 (DS 11.21) may be used. Whichever one is chosen is poured over the cells after the supernatant water used in the last wash has been poured off. The time for staining is not important, three or four days being usually enough. Though the cells cannot be seen in the stain, it may be assumed that they have fallen to the bottom. The upper two-thirds of the stain is poured off before filUng the tube with a weak (1 %) solution of w^hatever alum was used in the prepara- tion of the stain selected. The cells are again allowed to settle, the supernatant liquid poured off, the tube refilled with alum solution, and so on, until the wash solution is practically colorless. The cells now have to be dehydrated; this is done by pouring off the alum solu- tion, replacing it with, say, 30% alcohol, which is itself replaced with 70% alcohol, as soon as the cells have fallen to the bottom. At this stage a few cells should be withdrawn with a pipet, placed on a sUde, covered, and examined under a high power of the microscope. Each cell should show the nucleus clearly stained dark red with a faint pink cytoplasm; if they appear too dark, a small drop of acetic acid should be added to the tube, mixed with the alcohol, and poured off after five minutes. The washing is continued until the alcohol no longer smells of acetic acid. It is easy to overdifferentiate at this point and unless the cells are grossly overstained, it is better to take them through without fur- ther differentiation occasioned by the wash in alum solution which thej^ had im- mediately after staining. The 70% alcohol is now replaced with absolute alcohol in which the cells should be thoroughly stirred and then left overnight. A second change of absolute alcohol should be given; this should not be poured off but should be withdrawn with a pipet, so as to leave the cells accumulated in the least possible quantity of absolute alcohol at the bottom of the tube. The tube is then filled with benzene and left until the cells have again fallen to the bottom, when the benzene is withdrawn and replaced with fresh benzene, which is again replaced. The cells, which are now lying at the bottom of the tube in not more than a drop or two of benzene, should be covered with three or four drops of a strong solution of Canada balsam in benzene. The specimens should now be stirred up and left for an hour or two until they are thoroughly permeated with the balsam, a drop of which may then be removed, placed on a sUde, and covered. By this method, as many slides may be made as there are drops of balsam; and, if the cell concentra- tion has been kept reasonaljly heavy, it is usually better to use a very small {% inch) covershp in order to get as many slides as possible. A preparation of this size should contain two or three hundred cells and will give the student an excellent picture of all of the cell types found in hydra. 10 Ground Sections General Principles Nature of the Process Previous chapters have dealt with the preparation of whole specimens either mounted individually, smeared, or squashed on a slide. There are many speci- mens which, in order that their micro- scopic structure may be examined, have to be cut into thin structures. The more usual methods of cutting such materials are given in this and the next four chap- ters. The present chapter, which describes the preparation of sections of materials too hard to be cut by anj^ conventional method, had better be ignored by anyone not specifically interested in this process. Sections of hard materials such as bone, the calcareous skeletons of coral, and even some of the hardest vegetable materials, must be prepared in two stages. The first of these stages is the preparation of a crude section from a half to one millimeter in thickness, while the second stage con- sists in grinding this down while leaving both sides polished. Preparation of the Crude Section It is presumed that we are dealing with material which cannot be cut with a knife and must, therefore, be cut with a saw. Most woodcutting saws are not hard enough for the purpose and the choice lies between the ordinary hacksaw, intended for cutting metal, and a jewelers' saw. The disadvantage of a hacksaAV is that it is very coarse, so that only thick sections may be cut; the disadvantage of the jewelers' saw is that, unless it is guided by an expert hand, it will not cut a parallel- sided section. Whichever saw is selected, however, it should have relatively coarse teeth, particularly if bone, or material containing very much animal matter, or material which has been embedded in resin, is to be cut. These materials choke the teeth of a fine saw; the tooth marks left by a coarse saw do not matter, for they will be ground and pohshed out. For very hard substances, such as teeth, it is often necessary to use a circular diamond saw, which is usually available in depart- ments of geology Avhere rock sections are cut. Even teeth, however, may be cut with a jewelers' saw provided that the blade be changed at intervals and that the entire operation be conducted under the surface of water. Another type of preparation is that in which it is desired to preserve both the hard and the soft portions at the same time. A standard example of this is the preparation of a coral, of which it may be desired not only to section the calcareous skeleton but also to retain in position the soft parts of the animal within. This can only be done by embedding the material in some substance nearly as hard as the skeleton itself. A number of resins have been proposed for this purpose. The au- thor always prefers, however, to use Canada balsam because, though it is gummy in the final grinding, it has the ad- vantage that it need not be removed for mounting, and thus obviates one rather laborious stage of the process. As an alternative one may employ the process of Henrichi 1916 (4349, 6:45) in wliich gum damar is substituted for balsam, though the method which is described in- volves the use of machinery not normally available in laboratories. The technique of 80 Grinding GROUND SECTIONS 81 oml)e(l(ling in balsam is described in some detail iu the .second example wiiich follows the chapter and need not be repeated here. Selection of Grinding and Polishing Agents Once the initial sections have been pre- pared it is necessary that they should have one face pohshed, that this polished face should then be attached to some mate- rial, and that tlie other face be j^round away and brought to a pohsh when the section is of the correct tliickness. One technique for doing this is described in the first example at the end of this chap- ter, but some discussion must take place at this point as to the selection of grinding and pohshing agents. The initial flattening of one side of the section is best done with the aid of carbo- rundum powder, using a grit of about 100- mesh. The 100-mesh carborundum itself is far too coarse to leave a surface wliich may be polished and an intermediate stage is required. In the writer's opinion, pumice is best used for this intermediate stage. We are now speaking of relatively soft material, such as bone or coral, and not of thin slices of rock which would require several stages of carborundum between 100-grit and pumice. One of the most diffi- cult things to determine is when the scratches have finally been removed; this can never be seen when the sections are wet from grinding. It is, therefore, neces- sary at intervals to wash the surface of the section which is being ground, to dip it into alcohol, and then to warm it until dry. The surface of the section is then ex- amined with a strong hand lens by re- flected light, and grinding is ceased when it presents a uniform, dull surface un- broken by scratches. The next stage is to bring this flattened surface to a high degree of polish. All the old directions recommend the use of rouge . The objection to rouge is its color, and the fact that it gets over everything on the bench and around the bench. The author most warmly recommends the substitu- tion of white rouge (eerie oxide), which is rapidly replacing ordinary rouge in the polisliing of glass, and is also excellent for microscopical specimens. It does not mat- ter very much against what surface the abrasive has up to this time been rubbed, though glass is conventional. It is, how- ever, impossible to get a fine surface with rouge on glass and one should, therefore, use a leather strop for the purpose. This does not mean a loose leather strop of the type used by barbers but rather a flat surface of horsehide which has been at- tached to a hardwood block. Rubbing the section up and down on this, while it is well lubricated with a slurry of white rouge, will soon bring it to a fine pohsh. There is no reason to get discouraged if, after polishing, the surface is found still to have a few fine scratches on it. The sec- tions are going to be mounted in balsam which will hide most of the scratches. The sections are then cemented, pol- ished side down, to a shde and ground on some flat surface with coarse carborundum until they are of the required thickness. It is unfortunate that there is no adequate method of judging this thickness except by eye; experience is the only guide which may be reasoriably followed. As soon as the section has been ground down to the required thickness it is then smoothed with pumice, polished with white rouge, and finally mounted. Practical appUcation of the principles here discussed will now be given in the form of two typical preparations. Typical Examples Preparation of a Section of Bone If the worker is interested only in the production of a section which will show the existence of Haversian canals, it is better to decalcify the bone (in one of the solutions given under AF 20 in Chapter 19) and to prepare sections by the paraffin technique described in Chapter 12. These sections, however, show neither the lamel- lae, lacunae, or canalicuh, which can only be demonstrated in a section prepared by grinding, in which all the calcareous parts remain intact. 82 THE ART OF MAKING MICROSCOPE SLIDES Bone The first thing is to secure a piece of dry bone. The majority of museums have old broken specimens from which they are only too glad to give away a bone or two. The example shown being sectioned in Fig. 30 is part of the femur of a horse which became accidentally broken. If no dried specimen of bone is available, and one is, therefore, forced to start with raw material, it is fii'st necessary to boil the bone for four or five hours in water in order to remove as much of the protein material from it as possible. If, moreover, handUng bone because it combines the stiffness of a hacksaw with the thinness of a jewelers' saw. The first cut is made at right angles to the direction of the cut shown and a second cut (as seen in the illustration) is then made, parallel to the free surface that has been cut, and at right angles to the present position of the saw in the figure. Several slabs are cut, as uni- formly as possible, but the saw kerf is stopped about a millimeter above the horizontal cut, and a second cut made, until (as is seen in the figure) as many Fig. 30. Sawing slabs of bone for sectioning. Note that the vertical kerfs have not been extended to meet the horizontal kerf. one is dealing with a long bone containing marrow, it is necessary that it should be cut with a hacksaw into short lengths in order that the marrow may be removed by boiUng. The bone is then taken from the boiling water, dried for a day or two, and then defatted by being soaked in any fat solvent. About the cheapest and most convenient solvent available is naphtha, though if price is a secondary considera- tion one can, of course, use xylene. Three or four changes, each lasting a week, in a considerable volume of solvent must be made, and the bone should then be baked in a low-temperature oven until the sol- vent has been removed. A series of thin slabs is then cut, as shown in Fig. 30. The saw there shown, which is a cheap imitation of a hacksaw, is the best that the author has found for slabs as are required have been outHned. Each is then successively cut off. A con- venient size slab for preparation of a microscope shde is approximately }^i inch square, which is the size shown in the illustration. Single sections may be handled without being attached to anything. Several blocks may, however, be ground down at the same time by cementing them to a slide as shown in Fig. 31. The hot table (shown in its entirety in Fig. 8) is heated to about 100°C. The slide is then hberally covered with natural balsam — not a solution in xylene — and the slabs laid in place. Each block of bone will have a jagged corner sticking from it, where it broke away just before the saw cut was complete, and these little jagged corners must be placed upper- most. One must also be careful that the Bone GROUND SECTIONS 83 thickest sections of l)oiie are placed at tlie outside of the piepaiatioii, or it will \)v im- possible to avoid the slide's \vol)blinf; while being ground. As soon as all the slabs of bone have been placed, the balsam is heated until it boils rapidly. The balsam usually catches fire during this process, but it may be extinguished by blowing on it. A pair of forceps is finally used to push each piece of bone into contact with the glass and the sUde is cooled. The scratches from the earl)orundum nuist now be polished out with pumice powder. One secures either another piece of glass or a flat hardwood board — they are etiually good -and prepares on this a paste of pumice and water exactly as the carborundum paste was prepared. The slide is washed to remove the carborun- dum grains and then rubl)ed, with exactly tiie same motion, in a slurry of pumice un- til each section of bone is uniformly Fig. 31. Mounting bone slabs on glass slide. While the section is cooling, a pool of water is poured on a slab of plate glass, and carborundum powder, of about 100 mesh, is sprinkled in until a thick cream is produced. The slide is then turned upside down in this cream, as shown in Fig. 32, and rubbed backward and forward with a circular movement, so as to grind down the bone. The grinding should be con- tinued until the pieces of bone have been reduced to a uniform thickness. It may be necessary, from time to time, to add more water, and the specimen should ))0 lifted at intervals to make sure that the abrasive fluid is underneath it, and not being pushed out by a wall of balsam. smooth on the surface. Some people prefer to use a hardwood, rather than a glass, slab for the pumice. It is best to dry the surface of the bone and to examine it un- der a lens by strong reflected light to make sure that the scratches have been removed. The sections are now polished on a horsehide strop cemented to a wood block. The strop is lubricated with a thick cream of either rouge or white rouge (eerie ox- ide). The shde should be rubbed rapidly with very little ])ressure; too much pres- sure is liable to dry the sections. If dried rouge is forced into the surface under pressure it is almost impossible to remove 84 THE ART OF MAKING MICROSCOPE SLIDES Bone unless the specimens are reground with pumice. The final polish can be judged by eye and should be such as would be acceptable on, say, a pohshed ivory ornament. The slide is then returned to the hot table seen in Fig. 31 and heated until the balsam is molten. Another slide is placed alongside the first, liberally smeared with balsam, and the sections transferred from the first shde to the second with the pol- ished side down. They must be pressed hard to make sure that the polished side necessary that the shde should not rock from side to side as it is pushed about, or the covershps will become ground at the end before the sections of bone in the mid- dle are thin enough. Experienced experts can often grind a section of bone until it is as thin as a number 1 coverslip, but this is not recommended to the beginner, for if the section is ground too thin it will sud- denly disintegrate and all the work done so far will be lost. When the sections are judged to be thin enough, the slide is very carefully washed under the tap to remove Fig. 32, Grinding down bone slabs. is in contact with the glass. The shde is then cooled and returned to the glass plate (Fig. 32) containing the carborundum paste, on which it is now slowly and stead- ily ground until the sections are thin enough. The thickness may partly be esti- mated by holding the specimen up to a Ught and seeing how transparent it is be- coming; the correct thickness has the transparency of a rather thin sheet of oiled paper. If the technician does not care to trust his judgment in the matter it is pos- sible to take two number 3 coverslips and to cement one on each end of the slide. A thick number 3 covershp is about the thickness of a good section of bone so that, if the bone is ground down until the cover- shp just starts to be affected by the grind- ing compound, the sections may be pre- sumed to be of the right thickness. To use this method satisfactorily, however, it is all traces of carborundum grit and the sec- tions then smoothed with pumice, as was done before. A certain amount of reduc- tion in thickness may also be produced by the pumice though it is usually better to use carborundum. As soon as the scratch marks of the carborundum have been re- moved, the sections are again washed care- fully under the tap and then pohshed. A coverslip is then placed on top of the prep- aration and the slide examined under vari- ous powers of the microscope, which will verj^ soon disclose whether or not it is thin enough. If it is not thin enough to show the required structures it should be taken back to the carborundum, ground some more, then resmoothed and repohshed in the manner described above. Several trials are often required before all the sections are found to be satisfactory. There are two schools of thought as to Coral GROUND SECTIONS 85 the mounting of these l)one sections. Some people prefer to mount them dry, in which case tlie shde is pkiced in a jar of benzene and left until all the Canada balsam has been removed. Each individual section is then picked up on a section lifter, trans- ferred to two or three fresh changes of benzene to remove the last of the balsam, and then dried under pressure between two glass slides; when it is dry it is then treated as any other dry wholeniount (see Chapter 1). This method undoubtedly makes it easier to see the finer structure of the bone, but it is applicable only to very thin sections, for the additional transpar- ency imparted b}' the balsam will be lost. It is usually more satisfactory to melt the balsam, to lift each section up, to place it in fresh balsam on its individual slide un- der a coverslip, and to heat it until all the air bubbles have been expelled. The coverslip is then held down with one of the clips shown in Fig. 25 and cooled. All these operations can be conducted much more conveniently on the machinery made for grinding and poUshing rock sec- tions, but this description has been given for the benefit of those who lack such machinery. It has from time to time been recommended in the literature that one should grind sections of this kind down on microtome sharpening stones, using oil as a lubricant. The writer has had the most wretched results by this method ; it is men- tioned only in order that it may be avoided. Preparation op a Transverse Section of a Coral with Polyp in Place It must be understood, first of all, that the preparation here to be described will give a very much less satisfactory trans- verse section of a coral polyp than will the paraffin method described in Chapter 12. This method is intended onl}^ as a compromise between a paraffin section of a polyp and a ground section of a hard structure of the type described in the last example. The living animal must first be narco- tized, fixed, and hardened. It does not matter what coral be selected. The north- ern coral {Astrangia danae) is convenient both because of its wide distribution and because it ma}- be obtained from biological supply houses. Supposing, however, it is to be collected fresh, a piece should be secured of about the size of an orange, or smaller, and brought back to the laboratory and left to expand in plenty of well-aerated sea water. It must, of course, be narcotized before fixation and a double process is best for this type of specimen. About 5% of its volume of a saturated solution of mag- nesium sulfate is therefore added to the water and the action of this narcotic is enhanced by sprinkhng menthol on the surface. After about half an hour the pol- yps will be extruded from the coral in a partially narcotized condition and should be fixed. Perfect narcosis will result in such a small quantity of the polj'p remaining in the coral that it is scarcely worth while sectioning it, whereas fixing an unnarco- tized coral causes such a contraction of the polyp that the sections will be hard to interpret. The fixative selected should be one which will harden the poh'p as much as possible without having any effect on the calcareous structure surrounding it. The copper sulfate-mercuric chloride of Lo Bi- anco (Chapter 18, F 3400.0000 Lo Bianco 1890) is excellenth' suited for the purpose and about a gallon will be required for the fixation of a specimen of the size described. As much of the water as possible is now siphoned off from the vessel containing the coral specimen and the fixative added. The fixative should be stirred at intervals for the next two or three days and then the specimen should be transferred to fresh fixative for a period of about another week. The coral is then washed in running water overnight and j^laced in 4 % formal- dehyde, which should be changed daily until the whole of the fixative has been washed out of the specimen. A coarse hacksaw is then used to cut the specimen into cubes of about an inch on a side, with due regard to the selection of pieces which will subsequently give good sections. These inch cubes are now washed in running water overnight, to get rid of the formaldehyde, and suspended in a con- siderable volume of 70% alcohol. It will be necessary to impregnate them with a resin ; 86 THE ART OF MAKING MICROSCOPE SLIDES Coral they must, therefore, be completely dehj'- drated and cleared as though whole- mounts were to be made of them (Chap- ter 6). This dehydration is very slow so that, after about two weeks in 70% alco- hol, they should be placed for a further two weeks in 95% alcohol before being transferred for still another week into ab- solute alcohol. As considerable volumes are required it may be found more eco- nomical to substitute anhydrous acetone for the absolute alcohol. The writer prefers to embed in Canada balsam but this must be freed of its con- tained essential oils if it is to become hard enough for grinding. It is very difficult to melt dry Canada balsam without obtain- ing a mass full of air bubbles and it is, therefore, better to take a pound or two of the natural balsam, place it in a wide evajjorating dish and heat it to about 120°C., with due precautions against fire, until such time as a small drop placed on a cold plate hardens rapidly to a material which will crack and chip, rather than bend, when a knife is applied to it. A solution of about 30% by weight of this essential-oil-free Canada resin in ben- zene is also required but may be made up from the commercial dried product. After the specimen has been completely dehydrated it must, of course, be de-alco- holized in some material which is miscible with the resin and it is suggested that benzene be used. Three changes of ben- zene, with about a week in each, will be required ; and it is desirable that some de- hydrating agent, preferably silica gel or calcium sulfate should be kept in the ves- sel to remove the last traces of water. When the specimen is completely impreg- nated with benzene it is transferred to the solution of drj' Canada balsam in benzene and left there for a week or two until it is imi:)regnated. The solution is then trans- ferred to an open vessel and warmed gently until as much as possible of the solvent has been removed. Care must be taken never to raise it to the boiling point of the solvent or bubbles may occur in the actual specimen which will wreck the sub- sequent preparation. The specimen is then immersed in molten balsam, which is maintained at about 100°C. until no fur- ther diffusion currents are seen. The beaker is then cooled until the balsam is completely hardened. The only practical method of removing this hardened block of clear balsam containing the specimen is to crack the beaker away from it with a hammer. One is now left with a solid block of bal- sam containing the coral, and a saw is used to trim the specimen to shape. This trim- ming should result in a rectangular block, if we are dealing with Astrangia danae, of about a 3'^-inch side, by one inch long, with the polyp protruding from one end. Canada balsam is not easy to saw and it is recommended that the blade be kept lu- bricated at all times with a weak soap solution. This rectangular block is now treated exactly as though it were a piece of bone and a series of slices of from 3^^ to one mil- limeter thick cut from it. These slices can- not, however, be handled all at one time in the manner described in the last example, but must be handled individually. Each slice is therefore taken and ground flat with carborundum used in the manner described in the last example. Instead, however, of having the section cemented on a slide, it is held on the ball of the first finger and rubbed backward and forward until it is flattened. When it is flat, it will not be satisfactory to wash it under the tap, because many of the carborundum grains will be embedded in the balsam and one should, therefore, take a rag moist- ened with benzene and wipe the surface carefully until the carborundum grains are seen to be removed. Great care should be taken to do this in such a manner that the flatness of the center of the section, where there are only the soft parts em- bedded in balsam, is not disturbed. The section is then rubl^ed up and down with the finger using pumice on wood, and again washed and cleaned. These speci- mens do not polish well on leather and a sheet of velvet (which may be gummed to a wooden block) should be substituted. This velvet is Uberally lubricated with white rouge in water and a polish put on the lower surface of the section. If the white rouge is sufficiently diluted with water, it is unlikely to become embedded Coral GROUND SECTIONS 87 in tlie balsam; therefore washing in water will remove it. The section must now l)e mounted on the slide which it is finally to occupy, but this is done in exactly the opposite manner to that described in the last example, in which the shde was covered in balsam and the object pressed to it. In this case the section must be placed on a flat surface, a slide warnred rather above the melting point of the balsam and pressed down on to the specimen so as to melt the minimum possible quantity of balsam to permit per- fect adherence. Only in this manner can one avoid disturbing the soft parts. The other side of the section is now ground down, smoothed, and polished exactly as described in the last examj^le. It will, how- ever, be much easier to estimate the thick- ness of a section of this type, for one can always observe the soft parts under the microscope. When the section has become sufficiently thin, it is only necessary to ])lace a drop of natural l)alsam on top, ap- ply a coverslip, and warm the whole while applying jiressure. Though this description has api^fied to the preparation of a coral, it may equally well be applied to the production of sec- tions of bone with the bone marrow and blood cells retained in position. Those who prefer to grind their sections on oilstones with an oil lubricant should consult the description of Henrichi 1916 (4349, 6:45). 11 Sections of Free Material General Principles Nature of the Process A section is a thin slice cut from biolog- ical materials with a view to studying either the cells themselves, or their ar- rangement, neither of which can well be made out from a wholemount. If the Though sections may be cut at any an- gle, they are usually taken through one of three planes (see Fig. 33), known as trans- verse, sagittal, and frontal. The purpose of this orientation of the material with rela- tion to the plane of the section is to permit Fig. 33. Standard section planes. worker's only interest is, in the shape of the cells, as distinct from their structure, he should attempt some of the prepara- tions described in Chapter 9, which will often be found better than sections for his purpose. a better visuahzation of the structure of the whole, from an examination of sec- tions. When the relation of organs is to be reproduced from sections, the process is known as reconstruction and is referred to briefly in Chapter 14. In theory a section 88 Microtomes SECTIONS OF FREE MATERIAL 89 can be made by cutting a thin slice from the object with a sharp knife. Few mate- rials, however, are suitable for this, nor does this procedure yield sections of the same thickness. It is, therefore, customary to use an instrument known as a micro- tome: a device for advancing a block of tissue a given amount, cutting a slice from it, and then re-advancing it for the same amount, and so on. A full account of all the types of mechanism by which this re- sult can be produced is to be found in Richards 1949 and need not be repeated here. Another objection to the mere cutting of slices from an object is the nature of biological specimens themselves. Few of these are stiff enough to withstand the ac- tion of the knife without bending, and many contain cavities which would be crushed out of recognition as the section was taken. This makes it necessary, for most biological work, to surround and sup- port the object to be cut with some mate- rial which will impregnate it. The medium most commonly used is wax. The tech- nique for cutting wax sections is described fully in the next chapter. Nitrocellulose is also employed and is described in Chapter 13. Another method of stiffening the ma- terial, so that sections may be cut from it without crushing, is to freeze the speci- men. This techniciue is described in Chap- ter 15. There are however, materials which may be cut without either the complicated microtomes described in these chapters or the support of impregnating substances. Sections which are so cut are known as free or freehand sections. These form the subject of the present chapter. Microtomes for Free Sections Even though the material itself is of the correct consistency to withstand the ac- tion of the knife, it is still necessary to have some mechanism which will produce sections of known thickness. The tj^pe of microtome usually employed in liard so(;- tioning is sliown in Fig. 34 and consists essentially of a disk, usually of polished plate glass, supported on a cylinder grip- ped in the hand. Within this cylinder there is some mechanism for holding specimens Avhich terminates at its lower end on a micrometer screw. When this screw is turned, the object in the holder is pushed above the surface of the glass plate. The collar of the micrometer screw is gradu- ated, sometimes in thousandths of an inch, but more usually in hundredths of a millimeter. The unit commonly used to describe the thickness of a section is a Fig. 34. Hand microtome. micron which is one-thousandth of a milli- meter; but hand sections are very rarely cut of less than 10-micron thickness and are usually better at two or three times this. Methods of Holding the Material Though the material itself may be suit- able for cutting, it is rarely of a size and shape which may be gripped in the holder of the hand microtome without additional support. It must, therefore, be held in some substance which will itself cut read- ily and which may be easily shaped to sup- port what is being cut. It is possible to embed the material either in wax or nitro- cellulose before cutting a hand section, but if one is to go to this amount of trouble, it is usually better to use a comi)lex micro- tome of the type described in Chapters 12 and 13. Vegetable tissues are usually em- ployed to support objects for hand section- ing and the two best known are elder i)ith and carrots. Klder pith has tlie advantage that it may be stored indefinitely and cuts with a clean crisp action. Unfortunately the pith of the American elderberry (Sam- bucus caiuidensis) does not ai)pear to he as suitable for the purpose as the pith of the 90 THE ART OF MAKING MICROSCOPE SLIDES Fixation European elderberry {S. nigra). This dif- ference between the two species may ac- count for the disfavor in which elder pith is held in the United States, but in the writer's experience it is far more conven- ient than the carrot. The disadvantage of the carrot is that it must be absolutely fresh, and even if it is kept in water over- night it loses much of that crispness which is necessary for the production of a good section. Almost all hand sections are cut from plant material, and most of them from leaves or stems. To support a leaf, a cylinder, of the right diameter to fit in the microtome is cut either from elder pith or carrot, split down the middle, and the leaf inserted. The holding screw is then tight- ened. Stems, however, cannot be held by this means and a hollow cjdinder must be prepared with an outer diameter con- venient to the microtome and an inner diameter which is slightlj' less than that of the stem to be gripped. This hollow cylinder is then split, the stem inserted, and the section cut. Of course, a few sul)- stances, such as cork or stiff plant stems, may be cut without any other support; these are, however, in the minority. Hardening and Fixing Materials for Cutting Many objects, which are in themselves unsuitable for sectioning by hand, may be made more suitable if they are fixed and hardened. A general discussion of the principles governing the selection of a fixa- tive is given in Chapters 6 and 12; the formulas which have been suggested for the purpose are given in Chapter 18. If, however, one is to go to the trouble of hardening and fixing material in a formula designed to preserve the structure of the cells, it is usually worth while to go to the additional trouble of embedding the ma- terial and cutting sections as described in the next two chapters. For material to be hand sectioned it is sufficient that it be preserved in 90% alcohol. This process is equally appUcable to the stems and leaves of the botanists, or to the verj' few animal materials, such as cartilage, which are suit- able for the production of hand sections. It must not be thought that objects em- bedded in nitrocellulose or wax should not, or cannot, be cut on a hand microtome. The study of the embryology of the frog is rendered much easier to elementary students if they are allowed to cut hand sections of blastulae, gastrulae and young larvae for themselves; and a word might be said at this point as to the method by which such blocks maj^ be readily pre- pared in quantity. The larvae are fixed, dehydrated, cleared, and impregnated with wax in the manner described in Chap- ter 12. Instead of casting each into an individual block, however, a large slab of wax — the cheapest paraffin is suitable — is cast in a tray. A heated iron rod of about I'^-inch diameter is then driven into the slab so as to make a little pool of molten wax. An impregnated larva, or egg, is then dropped into the hole and the process repeated. By this means a couple of hundred frog eggs may be embedded in ten minutes. The large block of wax, con- taining numerous eggs, is then cut with a saw into rectangles, the edges of which are trimmed with a knife until they will fit into a hand microtome. These blocks may even be cut without a hand microtome by placing the block on a bench and shaving off successive sections with a sharp scalpel. Staining and Mounting Sections Sections, wliich are taken individually from the knife and accumulated in a dish of 70% alcohol, should be treated as wholemounts rather than as sections. They may, that is, be directly mounted in either gum media (Chapter 4) or jelly media (Chapter 5) or they may be stained and mounted in resinous media in the manner described in Chapter 6. It might be pointed out, however, that many sections may be double- or triple- stained (a process which is impossible with wholemounts) and that in theory any method of staining described in Chapters 20, 21, or 23 for wax or nitrocellulose sec- tions may also be applied to a hand sec- tion. These are, however, much better applied to sections after they have been attached to a slide. If they are to be at- tempted, reference should be made to Leaf SECTIONS OF FREE MATERIAL 01 Chapter 28 (V 21.3), wliore are ftivon formulas and methods whicli may l)e used to attach individual sections. Sections which are not attached to shdes must he transferred from one fluid to anotlier witli the device known as a section lifter, wliicli is merely a small flattened sheet of metal held in a wooden handle. Typical Examples Preparation of a Transverse Section of the Leaf of Ligustrum The leaf of the privet is, in the opinion It is necessary to have a hand micro- of the writer, the easiest biological speci- tome of the typo already desci'ihed, an old- men from which a section maj' be pre- fashioned luuul razor, some freshly picketl Fig. 35. Inserting a leaf into a split cylinder of carrot. Fig. 36. Cutting a hand section. The razor is drawn across the plate with gentle pressure and the section then washed into a stender dish. pared and is included in this place as an introduction to the art of cutting sections. The leaves should be collected in sum- mer and stored in a jar of 90% alcohol which must be changed as often as it be- comes diluted from the extracted water or discolored by the extracted chlorophyll. If large quantities of alcohol are used in the first place it will be unnecessary to change it, but it must be agitated from time to time to avoid the accumulation of water at the bottom. carrots, and a stender dish of 70% alcohol. The razor shown in the illustration (Fig. 35) is flat on one side and hollow-ground on the other. This is the best kind for hand sectioning, but if it is not obtainable a double hollow-ground razor may be used. Next take a coi'k borer of such a size that it will bore out a cylinder which will fit reasonably well into the holder of the microtome. Then (Fig. 35) cut from a fresh carrot as many cylinders as are required. A leaf is then trimmed until it is the same 92 THE ART OF MAKING MICROSCOPE SLIDES Wood width as the cylinder. The cyUnder of carrot is spht, inserted into the holder of the microtome, and the leaf pushed down into the center of the cyUnder (Fig. 35). The holder of the cj'hnder is now tight- ened and the razor used to slice off as much of the leaf and cork as projects above the level of the plate (Fig. 36). The micrometer screw at the bottom of the cylinder is then turned as far as is neces- sary to advance the carrot and leaf the thickness of the required section above the glass plate, and a section shaved off. The razor must not be pushed straight across the material, but must be drawn side- ways, so that the whole length of the razor is used to cut the section. In Fig. 36 the razor is placed in the correct position for the beginning of the cut; but by the time it has passed through the block, the oppo- site end of the razor will be opposite the section. Notice also that the material is being sectioned with its thin edge, not its breadth, against the knife. This is neces- sary whether one is cutting sections by hand or by any other means, because the less material cut at the same time, the less is the chance that it will be torn from its support. The section on the knife is now brushed off into one of the stender dishes of alco- hol, the micrometer screw advanced the same amount, another section cut, and so on. About twice as many sections should be cut as are ultimately required, for at least half of them will either be damaged or will have one end thicker than the other. The sections are therefore examined un- der the low power of a microscope and those which are not considered satisfac- tory are thrown away. Stains used for materials of this type are given in Chapter 21 (DS 21.15), to- gether with a specific example. After the sections have been stained as there de- scribed, they should be dehydrated ex- actly as if they were wholemounts and mounted in balsam in the manner de- scribed in Chapter 6. Preparation of a Section of Wood The last process described is one of the easiest preparations which may be made in microtomy, whereas the present is one of the most difficult. The sectioning of wood belongs properly in this chapter on the preparation of freehand sections, be- cause wood does not need to be embedded. The difficulty of cutting it hes in the fact that it is too hard to be cut by regular methods while being in general too soft to be cut by the method for ground sections given in Chapter 10. Some plant materials, such as the wood of the fignum \'itae, or the ivory nut, may be cut by grinding techniques and make better sections b}' this method than by any other. A few woods, such as white pine cut parallel to the grain, are sufficiently soft to be cut in a hand microtome without any preparation. The present example deals with such woods as maple or oak, which fall between these two extremes. The first thing to be done in the prepa- ration of any section of wood is to cut a block, one end of which is of the size of the required section. A section size about ^i inch square is usually adequate to demon- strate the structure of the wood, and a number of blocks this size should be pre- pared with due regard to the plane of the section. The wood must next be softened. This would be relatively simple if mechanical means alone could be emploj'ed. Unfor- tunately, however, many woods, particu- larly oak and teak, contain silica, which can only be removed by treatment with hydrofluoric acid, and this acid naturally cannot be made to penetrate the wood until all air has been removed. The blocks of wood are therefore boiled in distilled water for about ten minutes. A disk of glass — or of wire gauze — which will just fit inside the beaker, is placed on top of the pieces of wood, and a sufficient weight added on top to cause the blocks to sink to the bottom. The beaker is now trans- ferred to some type of vacuum equipment and exhausted until bubbles are seen to cease leaving the wood. The vacuum is then released, the beaker returned to the flame, and again boiled for ten minutes. This process of alternate boiling and evac- uation is continued until the wood no Wood SECTIONS OF FREE MATERIAL 93 longer floats, a state showing that the greater part of air has been removed. The blocks are now transferred to 50 % commercial hydrofluoric acid, and a word of warning must be issued as to the danger of this material. Since it dissolves sihca, it cannot be handled in glass vessels, and the choice lies between hard rubber and lead. Not only is the vapor extremely corrosive, but a burn from hydrofluoric acid on the skin is worse than that from any other chemical known to the writer. Extreme care should be taken, therefore, in transferring blocks to hydrofluoric acid, where they may remain until the silica has been dissolved from them. A block of 1.^-inch oak will be satisfactorily desihci- fied in one day, but teak should remain for at least three or four. Observing the same precautions as before, the blocks of wood are removed from the hydrofluoric acid to running water and must be washed for at least three or four hours before thej' are safe to handle with the hand. A block ma}' now be removed from the water and mounted in any convenient mi- crotome for cutting. The harder woods can never be satisfactorily cut on a hand microtome and the best mechanism is un- doubtedly the sliding microtome described in Chapter 12. Exactly the same precau- tions in cutting a wood block on this mi- crotome should be taken as in cutting a block of anything else. That is, the knife must be sloped at an angle towards the block so that the greatest possible length of knife is used to cut a single section, and the block must be so orientated that the knife enters at one corner rather than flat on the side. The sections as they are cut ma}'- be removed to water, and any tendency which they have to curl may be counteracted by warming the water. An interesting variation of this stand- ard technique was proposed at the same time by Crowell 1928 (20540b, 5:149) and b}' Kisser (cited from Crowell). This method consists of mounting a dry block of wood in any tj^pe of microtome and di- recting onto its surface a jet of high-pres- sure steam. After the steam has acted for a moment or two, a single section is cut and the steam again apphed for another few moments before cutting the next section, and so on. It is stated that by this means sections of the hardest material may be taken without the use of hydrofluoric acid. Sections of wood are usuallj- mounted in balsam, dehydrated, and cleared as de- scribed in Chapter 6. Sections which tend to curl may be tied between sUdes. Sections of wood containing some natu- ral color, as oak and mahoganj', are best mounted unstained, but thin sections of colorless wood may become too transpar- ent under this treatment. Almost any dj'e may be used, since the purpose is not to differentiate the parts but only to render them Wsible. 12 Paraffin Sections General Principles Nature of the Process The last chapter discussed freehand sec- tions, that is, sections of material which is in itself sufficiently strong and sufficient!}' coherent to hold together when cut in thin slices. The majority of objects to be sec- tioned, however, contain cavities which would collapse under the action of the knife, or are not of a shape or consistency which would enable one of them to be cut by hand. Objects of this nature must, therefore, be supported in a matrix which ■will itself section well, and those contain- ing cavities must be impregnated through- out their whole substance with the embed- ding medium. Wax, nitrocellulose, and a variety of water-soluble materials have from time to time been suggested as im- pregnating and supporting agents, but the use of wax is so convenient and simple that only in special cases should any other material be emploj'ed. The advantage of wax is not only that it readily passes from a solid to a molten state at temperatures which do not dam- age the material, but also that it is some- what sticky, so that ribbons of sections may be prepared, each section being in the ribbon in the same order as it was cut from the object. Thus, if a rectangular block of wax is mounted in some kind of holder and then brought sharply down on a hori- zontal knife, the thin slice of wax which is cut off will adhere by its edge to the edge of the knife. If the block is then advanced by some mechanical device — such as a microtome — a small distance and again brought down on the knife, a second sec- tion will be cut off which will displace the first section, to which it will adhere on one 94 edge, while the other edge remains at- tached to the knife. By the repetition of these movements a long ribbon may be produced. A ribbon of this type is seen in all the stages of its preparation in Figs. 65-70. Preparation of paraffin sections is quite a complex operation and in\'olves the following stages: 1. Fixation of the material. 2. Dehydration, in order that the ma- terial may be impregnated with a fluid capable of dissolving wax. 3. The removal of the dehydrating agent with a material solvent of, or miscible with, molten wax. 4. The soaking of the cleared specimen in a molten wax for sufficiently long to insure that it shall become com- pletely impregnated. 5. Casting the now impregnated speci- men into a rectangular block of wax. 6. Attaching this block of wax to some holder which itself may be inserted into a suitable microtome. 7. The actual cutting of the sections of the block into ribbons. 8. The placing of these ribbons on a glass shde in such a manner that they will lie flat and that the con- tained section will be adherent after the wax has been dissolved away. 9. The removal of the wax solvent. 10. Staining and mounting. I^ach of these operations will be dealt with in due order. This chapter terminates with a series of examples which describe in de- tail the application of the principles dis- cussed to actual preparations. Fixation PARAFFIN SECTIONS 95 Selection of a Fixative Fixative formulas are given in Chapter 18; and the selection of the fixative for small invertebrates has already been dis- cussed in Chapter 6. When one intends to section a small invertebrate, with the pri- mary function of preservinj^ its parts in as natural as possible a relation to each other, the same fixative should be employed as is recommended for those invertebrates in- tended to be made into wholemounts. The purpose in each case is to preserve the ob- ject in as natural a shape as possible with- out special regard to the preservation of the fine details of the cells themselves. Something of the same consideration applies to blocks of tissue which are to be fixed in such a manner that their general structure, or histology will be displayed. In this case, however, there is no problem of contraction of parts, so that fixatives which would be quite useless for a whole animal may safely be applied to a block of tissue. Reference to Chapter 18 will show that there are between 600 and 800 solu- tions recommended for fixation, and there is no general agreement as to which is the best for any particular purpose. These notes are therefore written only for the benefit of the beginner who, presented with this bewildering display, lacks the ex- , perience on which to base his choice. The selections given below are modified from Gray 1933 (11360, 53:15). The figures ' following the name and date refer to the decimal classification of Chapter 18 in which the formulas for these fixatives will be found. Specific suggestions for the em- ployment of fixatives are to be found in many of the examples of the preparation of shdes which occur in this book. A. Recommended Fixatives for Em- bryos OR Whole Organs Exceeding 5 Mm in Thickness. 1. FOR USE when the PRESERVATION OF SHAPE IS OF PRIMARY IMPOR- TANCE. Bensley 1915 F 1700.0010 Erhtzkv 1877 F 4700.0000 Hoyer 1899 F 3700.0000 Lavdowski 1894 F 6000.0010 Maximov 1909 F 1700.1000 Miiller 1859 F 7000.1000 Orth 1S9() F 7000.1000 R^gaud 1910 F 7000.1000 2. WHEN IT IS DESIRED, AS FAR AS POS- SIBLE, TO PRESERVE BOTH SHAPE AND PROTOPLASMIC DETAIL. a. When shape is of greater impor- t(l7lC€ IlcUy 1903 F 3700.1000 Petrunkewitsch 1933 F 4900.0010 Rawitz 1895 F 5600.0040 Smith 1902 F 7000.1010 Zenker 1894 F 3700.0010 b. When protoplasmic detail is of greater importance Fol 1896 F 1560.0000 Gilson 1898 F 3000.00 14 Kohn 1907 F 3700.0010 Maver 1880 F 5000.0050 Rabl 1894 F 2300.0000 Tellysniczky 1898 F 3500.0010 B. Recommended Fixatives for Small Portions of Organs or Whole Or- gans OR Embryos Not Exceeding 5 Mm. in Thickness. 1. when a general-purpose fix.\- tive is required Carleton and Leach 1938 F 3000.1000 Gatenby 1937 F 6700.0040 Gerhardt 1901 F 3600.1010 Schaudinn 1900 F 3000.0000 2. WHEN PROTOPLASMIC DETAIL IS OF GREATER IMPORT.\NCE a. When nuclear fixation is espe- cially required Allen 1929 F 5600.1010 van Beneden (1905) F 3000.0010 Carnoy 1887 F 0000.0010 Carnoy and Lebrun 1887 F 3000.0010 Sanson (1928) F 0000.0010 b. When cytoplasmic detail is espe- cially required Champv 1911 F 1670.0000 Flemming 1884 F 1600.0010 Kultschitzkv 1887 F 4700.0010 Mann 1894 F 1300.0000 Smith 1935 F 1670.0010 There are only two general precautions to be observed in the practical application on THE ART OF MAKING MICROSCOPE SLIDES Dehydration of fixatives: first, that adequate volumes (at least 100 times the volume of the part to be fixed) be employed; second, that mixtures containing either chromic acid or potassium dichromate with formaldehyde be used in the dark. After fixation, tissues should be thoroughly washed in water if this is the solvent for the fixative, or in alcohol if the fixative is based on the lat- ter. Objects are usually stored, after fixa- tion and washing, in 70 % alcohol ; though if they are to be kept a long time before dehydration, it is recommended that 5% of glycerol be added to the alcohol. This glycerol musi,, however, be very thor- oughly washed out before dehydration commences. Choice of a Dehydrating Agent Chapter 25 discusses the numerous or- ganic solvents wliich from time to time have been proposed for the dehydration of biological specimens and the selection between them is not usually of great im- portance. The classic method of dehydra- tion is to soak the object in a graded series of alcohols, usually 10 or 15% apart. De- hydration through gradually increasing strengths of alcohol may be vital when one is dealing with delicate objects con- taining easily collapsible cavities, such as chicken and pig embryos, but a block of tissue may be taken from water to 95% alcohol without any apparent damage. Even though one uses increasing strengths of alcohol, the series normally in employ- ment at the present time is by no means satisfactory. It is customary, for example, to pass from water to 30% alcohol at one end of the series and to pass from 85% to 95% alcohol at the other. The diffusion currents between water and 30% alcohol are far greater and far more intense than those between 85 and 95%, and an inteUigently graded series for delicate ob- jects should run from water to 10% to 20% to 50% to 95% alcohol rather than through the conventionally spaced grada- tions. This is not at all in accordance with the recommendations in most textbooks but is based on the author's experience over a long time. In using this classic method of dehydration, it is not necessary to confine the technique to ethanol. Meth- anol or acetone will dehj-drate just as effectively, though they are rather more volatile. There is a considerable vogue nowadays for the substitution for a straight dehy- drating agent of some solvent which is both miscible with water and also with molten wax. The best known of these is dioxane, though n-butanol has also been recommended. The writer is not in love with these methods for, though the sol- vents involved are excellent dehydrating agents, they are relatively poor solvents of paraffin and frequently cause great shrinkage of delicate objects in the final transition between the solvent and the wax. For such purposes as the routine ex- amination of the tissue blocks in a patho- logical laboratory, or for sectioning rela- tively sturdy plant materials, they may justifiably be employed. For sections in- tended, however, to retain intact struc- tures on which research is subsequently to be conducted, it is most stronglj^ recom- mended that the standard routine of pass- ing from a dehydrating to a clearing reagent be retained. Selection of a Clearing Agent Reference should again be made to Chapter 25 for a list of the materials which have from time to time been recommended for de-alcohohzing, or clearing, biological specimens. The choice of a clearing agent in section cutting is of far more impor- tance than the choice of a dehj^drant, for there is not the shghtest doubt that pro- longed immersion in some of the volatile hydrocarbons, particularly xylene, leads to a hardening of the tissue with subse- quent difficulty in sectioning. The classic method is to pass from alcohol to xylene, but the only apparent reason for the choice of xylene over toluene or benzene lies in the work of Squire (1892, page 80) who timed the evaporation rate of these three solvents from an open watch glass and found xylene to evaporate the most slowly. There is little choice in the solvent power of any of these three hydrocarbons on wax; the writer's preference is for ben- zene, though it seems impossible to shake the faith of the conventional that the more expensive xylene is a necessity both as a Embedding PARAFFIN SECTIONS 97 solvent for emheddiiig media and as a clearing agent before them. These three hydrocarbons are so cheap, and are ob- tainable in such a pure form, that there seems no necessity to use any other clear- ing agent, unless one prefers the reagents which are supposed to combine the func- tions of both dehydration and clearing. It is still occasionally recommended that essential oils, such as cedar oil, be used for clearing objects for embedding. There is no justification for this unless it is vital that the object be rendered transparent (rather than alcohol-free) in order that some feature of its internal anatomy may be oriented in relation to tlie knife. Essen- tial oils are excellent for wholemounts, l)ut they are not readily removed from the specimen by molten wax; therefore, if they must be used, they should always be washed out with a hydrocarbon before the wax bath. Relatively small traces of any essential oil will destroy the cutting prop- erties of any wax mixture and, as they are nonvolatile, there is no chance of getting rid of them in the embedding oven. Choice of an Embedding Medium Formulas for the various wax mixtures used in the preparation of ribbons of sec- tions will be found in Chapter 27 (E 21.1). It is to be presumed at the present time that no one will endeavor to use a plain paraffin but will use one of these mixtures. If, for some strange reason, a pure paraffin is preferred, then it is necessary to buy (in the United States by importation) a care- fully fractionated and very expensive wax. Ordinar}' cheap paraffin is a mixture of a great variety of compounds of slightly dif- ferent melting points, and it is essential in the use of jnire wax that a wax of a veiy sharp melting point should be obtained. The choice of an embedding medium should be dictated less by the nature of the specimen than by the conditions under which it should be cut. If pure paraffin is to be employed, it sliould be seloct('(l with sucli a melting point, that tlie hardened wax will give a crisp section at the re- quired room temperature. In the Europe of twenty years ago, when many writers were recommending a wax with a melting point of 52°C., the average laboratory temperature in winter was between 50° and GO^F. A wax of 52°C. melting point, in an American laboratory kept between 70° and SO°F., is far too soft to cut any but the thickest sections. The use of waxes of 58°C., which are quite hard enough for cutting sections in an American labora- tory, is unfortunate, since such use re- quires an oven temperature of at least 60°C. which results in many tissues be- coming hard and brittle. As the introduc- tion of any foreign substance automati- cally lowers the melting point of the wax, it is obviously desirable to use mixtures rather than the pure material. The writer's preference is naturally for his own compo- sition (E 21.1 Gray 1944). The advantage of the mixtures there specified is that they have a relatively low melting point but soften ver}' little before reaching the melt- ing point. The degree of hardness (that is the thinness of the section which may be cut) may be controlled accurately by the proportion of resin added; and the writer has once secured, on a demonstration, a paraflftn ribbon more than 20 feet long of 1 -micron-thick sections. Media of this hardness, however, impregnate very slowly and should only be used for mi- nute objects. For ordinary routine prepa- rations the writer's jircference is for any of the paraffin-rubl)er-bayberry-wax mix- tures. The introduction of rubber un- doubtedly increases the stickiness of the wax and makes it easier to secure continu- ous ribbons, while the bayberry wax not only prevents the crystalUzation of the paraffin but also lowers its melting point. The beginner is strongly recommended to experiment with several of the rubber- bayberry-wax compositions and to select after exj)eriment that which gives him uni- formly successful results in his own la])oratory. Technique of Dehydrating, Clearing, and Embedding Before passing to tlic choice of a micro- tome and tiie method of using it, it is necessary to discuss briefly tlie actual op- erations which are involved in using the dehydrating, clearing, and embedding me- dia selected. The techniques of dehydra- tion and dc-alcoholization do not differ 98 THE ART OF MAKING MICROSCOPE SLIDES Embedding materially from those used in the prepara- tion of wholemounts which have been de- scribed in Chapter 6. The whole process could, however, be much simplified if people would only remember that water is heavier than the majority of dehydrat- ing agents, and that the majority of de- hydrating agents are lighter than most The first prerequisite is some device which will maintain wax just at its melt- ing point. Most people employ complex thermostatically controlled ovens for this purpose, but the exceedingly simple device shown in Fig. 37 has a great deal to recom- mend it. As will be seen, this consists essentially of a series of incandescent elec- i.'-,te Fig. 37. Simple radiant heat embedding oven. Height of the hood should be adjusted until the wax is vielted for about one-half its depth. clearing agents. Translating this theory into practice it must be obvious that the object to be dehydrated should be sus- pended toward the top of a tall cylinder of dehydrant in order that the water ex- tracted from it may fall toward the bot- tom of the vessel, and that an object for clearing should be held at the bottom of the vessel for the reverse reason. It is, indeed, practically impossible to dehy- drate a large object unless it is so sus- pended. The process of impregnating the tissues with wax lias not, however, provi- ousl}' been discussed and will be tlealt with fully. trie bulbs held, at a distance which may be varied, above a series of glass \'ials. Before commencing to embed one fills as many vials as one will require with wax, places them under the reflector, and turns on the current. After a little while it will be oI> served that the absorbed heat has melted the wax. The wax may be melted only at a small surface layer; it may be melted throughout the entire vial; or it may, as is required, be melted in the upper % of the vial. If this last is not achieved the height of the lamp must be varied until, after an hour or two, each of the vials contains about yi of unmolten, opaque wax at the Embedding PARAFFIN SECTIONS 99 bottom and 2j^ of the clear molten mate- rial above. Thus, when the object is placed in one of these vials it will droj) until it reaches the solidified layer, where it will remain in contact with molten wax at exactly the melting point of the wax. It is obvious that the room in which this opera- tion is to be conducted must be at a fairly constant temperature and be free of drafts, but only a very large volume of embedding work justifies the purchase of an expensive thermostatically controlled oven. If such an oven is to be purchased it is liighly de- sirable to avoid one in which the heat is distributed bv convection. Such an old- Vacuum ovens are occasionally required for the impregnation of the most dilficult material but should he avoided whenever possible. If a vacuum oven is to be em- ployed, moreover, it is necessary that all volatile solvents be removed from the material before it is placed in the vacuum so that it is always desirable to precede exposure in a vacuum oven by a consider- able period of embedding in an ordinary oven. Assuming that the material has been passed through dehydrating and clearing agents, and is now awaiting embedding, there are two main methods by which this Am INIAKC Fig. 38. Circulating air embedding oven. fashioned convection oven is seen in Fig. 52 and is to be found all too frequently in laboratories. Unfortunately these ovens, as any cook could tell any microtomist, vary enormously in temperature from top to bottom. The thermostat is usually placed at the top and, in a fairlj^ large oven, there may be as much as a ten- degree differential between the lowest shelf and the top one. The oven shown in Fig. 38 in which a circulating fan continu- ously moves the air and thus maintains a uniform temperature throughout the whole oven, is infinitely to be preferred. It is the high cost of such circulating-air ovens which leads the writer to believe that much more use should be made of the very simple rachant-heat embedding de- vice discussed previouslj'. may be done. luther be the oliject may transferred directly to a bath of molten wax, or it may be passed through a graded series of wax-solvent mixtures. The writer is strongh' in favor of the latter course. Let us suppose benzene has been selected as the clearing agent and that the object is in a vial containing a few milliliters of this solvent. Chips are then shaved from the block of embedding agent and add('(l to the vial. These usually dissolve very slowly and form a thickened layer at the bottom of the tube through which the object to be embedded sinks. The average object will be satisfactory if left overnight. The tube is then placed in the embedding oven, maintained at a temperature slightly abt)ve the melting point of the wax, and as many further shavings as possible are 100 THE ART OF MAKING MICROSCOPE SLIDES Casting block Fig. 39. Folding a cardboard box. a. The two long edges of a rectangular card are folded to meet in the center. Fig. 40. Folding a cardboard box — {continued), h. Folds are flattened out and the short edges are folded not quite to the center. Fig. 41. Folding a cardboard box — {continued), c. Corners are folded over. crammed into the tube. When these are completely molten, and most of the vola- tile solvent has evaporated, the object is removed with a pipet, or forceps, and placed in a dish of pure wax for an hour or two before being transferred to a second dish of pure wax for the time necessar}^ to secure complete impregnation. There is no method of forecasting how long an object will take to l^ecome com- pletely impregnated with wax. It is very easy to find out, when one has started to cut sections, that the impregnation is not complete; but there is no basis save ex- perience on which to base the timing in the different baths. If the object is to be transferred directly from solvent to wax, at least three baths should be employed, for nothing is more destructive to a good section than the presence of a small quan- tity of the clearing agent in the embedding medium. To an absolute beginner seeking Casting block PARAFFIN SECTIONS 101 Fig. 42. Folding a cardboard box — (continued), d. Kdije of the fold in folded back over the creased corners. Fig. 43. Folding a cardboard box — {continued), e. Box is opened and the corners pinched. Fig. 44. Folding a cardboard box — (continued), f. Finished box. a rough guess, it may be said that a block of hver tissue of three-to-five-millimeter side will be satisfactorily impregnated with wax after 30 minutes in each of three baths, while a 96-hour chicken embryo will require at least two hours in each of three baths for its successful impregnation. While the object is being impregnated with the wax it is necessary to decide what type of vessel will be used to cast the final block. This will depend more on tlie size of the object than on the preference of the worker. Very small objects may be most satisfactorily embedded in ordinary watch glasses (that is, ordinary thin-walled watch glasses not Syracuse watch glasses of the laboratory type) or in any other thin-walled glass vessel. Very large objects are often embedded with the aid of two thick L-shaped pieces of metal, which by being slid against each other may be caused to form a rectangular mold of vary- ing dimensions. The writer himself regards these as very clumsy, and always prefers to prepare a cardboard or paper box than to endeavor to maneuver metal molds which are always getting jarred out of place at the wrong moment. The prepara- tion of a paper or cardboard box is easy; the method preferred by the writer for large boxes is shown in Figs. 39-44. Take a rectangular sheet of thin card or stout paper approximately twice as long as it is wide. The area of the floor of the 102 THE ART OF MAKING MICROSCOPE SLIDES Casting block l)ox will be aliout J4 that of the sheet taken, but a little experience will soon show what size sheet to take for the box required. The sheet is laid on a fiat surface and the long sides folded inwards (Fig. 39) until they very nearly meet in the middle. These folds are well creased with the thumbnail. The sheet of paper is then flattened again and the other two edges (Fig. 40) folded in the same manner. It is necessary, however, that this fold be much larger than the first fold made. These folds are also well creased with the thumbnail. The folded sheet is then laid out (Fig. 41) Fig. 45 as there are boxes to be made. Center the sheet between the finger and the thumb (Fig. 46) and then fold up the sides (Fig. 47) creasing the paper where it is in contact with the edges of the block. Push up the end with the forefinger (Fig. 48) creasing both the paper in contact with the block and the flaps. Fold the flaps to the center (Fig. 49), being careful to get them straight and creasing them up the sides. Fold down the projecting flap (Fig. 50) and crease it firmly. Repeat these operations with the other side of the block and then slide the box off the end of the Length of box + twice height of box + twice length of flaps XI o o Xi i Fig. 45. Dimensions of sheet for folding a paper box. and the corners folded in the manner shown. Since these end folds are larger than the side folds there will be an over- hanging flap of paper at the top. After all four corners have been folded in, this overhanging flap (Fig. 42) is folded back over the triangular folded corner sections and this crease particularly firmly pressed with the thumbnail. When this has been done at each end, the box is finished and may be opened out as shown in Fig. 43. It will be found that the corners are not scjuare but may be squared by pressing with the thumb and forefinger in the manner shown. The finished box is shown in Fig. 44. There is a very convenient method of folding small boxes wliich requires a series of wooden blocks of cross-section ecjual to that of tlie boxes recjuired. Take such a block (Figs. 46-51) and as many sheets of bond paper of the dimensions shown in block (Fig. 51). It is well to have a series of these blocks made both in square and rectangular shapes. An additional advan- tage of this type of box is that one can put the data about the block on the flaps. Boxes cannot be made by this method much larger than 1" X H". Some people prefer to cast a series of rectangular boxes from plaster of Paris. This can be done by any competent crafts- man, but will not be described at this point. After the box has been prepared we come to the actual process of embedding which is shown in detail in Figs. 52-55. Before starting it is necessary to make sure that the following items are available: (1) a dish of water of sufficient size that the finished block may be immersed in it (in the illustration an ordinary laboratory fingerl)owl is in use); (2) some form of heat, an alcohol lamp being just as effec- Casting block PARAFFIN SECTIONS 103 tive as a bunsen burner; (3) a slab of plate glass; (4) a wide-moutli, oye-di-oppor ty])e pipet. It is presumed that tlie object itself is in the oven, which also contains a sup- ply of molten medium. It must l)e empha- sized that an object cannot successfully be impregnated with one kind of wax and embedded in another. Next wet the under- side of the bottom of the paper l)()x and press it into contact with the plate-glass slab. Then take from the oven (Fig. 52) a beaker of molten embedding material and fill the little paper box to the brim. The eye dropper is then heated in the flame to a temperature well above that at which the wax will melt, and is used to pick up the object from its own dish (Fig. 53) and to transfer it to the paper box. By the time this has been done, a layer of hardened wax will have been formed at the bottom of the paper box, so that the ob- ject will rest on the layer of solidified wax with a molten layer above. It will almost invariably happen that the surface has also cooled, so that a crust of cool wax will have been carried down with the object in the box. It is essential to get rid of this if the wax is to adhere through section cut- ting, and the pipet is again heated, used to melt the entire surface of the wax (Fig. 54), and to maneuver the object into the approximate position in which it is re- quired to lie in the finished block. Then blow on the surface until the wax is suffi- . ciently solidified to enable you to pick up the box carefully and (as shown in Fig. 55) to hold it on the surface of the water used for cooling. With most wax media it is desirable to cool the block as rapidly as possible; it should never be permitted to cool in air. It cannot, however, be pushed under the surface of the water, or the molten center is liable to break through the surface crust and thus destroy the block. After it has been held in the position indicated until it is fairly firm throughout, it may be pushed under the surface to complete the cooUng. The block may be left in water for an>- reasonable length of time; but if it is to be stored for days or weeks it is better kept in a 5% solution of glycerol in 70' t alco- hol. There seems to be a widespread de- lusion that because an object must be perfectly dehydrated before being impreg- nated with wax, it must subse(iuently be kept out of contact with fiuids. Nothing could l)e further from the truth. As will be discussed later, when dealing with the actual techiiiciue of sectioning, it is often desiral)le to expose a portion of the object to be sectioned and leave it under the sur- face of water for some days, in order to get rid of the brittleness which has been im- parted through the embedding process. Blocks which have been stored dry for a long jjeriod of time should always be soaked in a glycerol-alcohol mixture for at least a day before sectioning. It is, in any case, undesirable to section a block as soon as it has been made, for it is necessary for successful sectioning that the block should be the same temper- ature throughout. If a block is made in the evening, it is better to take it out of the water and to leave it lying on the bench overnight in order that the temperature may be stabilized. Assuming, however, that we have such a block at hand, the next thing to do is to mount it in what- ever holder is to be used. Choice of a Microtome Microtomes may be broadly divided into two classes. In the first of these the block remains stationary while the knife is moved past it; in the second group are those in which the block moves past a stationary knife. The first class (an exam- ple is shown in Fig. 56) is made by several manufacturers but is rarely used for the preparation of serial sections. They have the advantage that relatively large blocks may be cut, but thej' have the disadvan- tage that no ribbon can be ol)tained which is broader than the width of the knife. This microtome will not be discussed fur- ther in the i)resent place, for a detailed description of its use is given in the next chapter on nitrocellulose sections, with which this type of microtome is often to be preferred. A Minot, or rotary microtome, is shown in Fig. 57. In this type of microtome the rotation of the large wheel causes the block holder to move vertically up and down, in most instances tlirough a dis- tance of about three inches. The portion Fig. 46. Folding a paper box. a. The block is centered on the sheet. Fig. 47. Folding a paper box — {continued), b. The sides are folded up. Fig. 48. Folding a paper box — (continued), c. The end is folded up. 104 Fig. 49. Folding a paper box — (continued), d. The flaps are folded in. Fig. 50. Folding a paper box — {continued), e. The end is folded down and creased. Fig. 51. Folding a paper box — (continued), f. The cycle is repeated with the other end and the finished box removed. 105 106 THE ART OF MAKING MICROSCOPE SLIDES Casting block 1""-/ ^ W"*^' ' ' " !^ " '- " ^ ^ " " - ' ■" ' ■,' , -*i^ Fig. 52. Filling with wax an embedding box which has been attached with water to a glass slide. which slides up and down has, at the end opposite to the block, a rectangular plate of hardened steel inclined at an angle of about 45°. This plate bears, under the pressure of a powerful spring, against a hardened steel knob which is itself con- nected to a micrometer screw. As the han- dle is rotated a pawl works against a ratchet to move the micrometer screw, and thus the knob connected with it, through a given distance for each rotation. As the knob moves forward, it moves the block the required distance forward at each revolution by bearing on the diag- onal plate. This mechanism is very costly to make and is liable to a large number of minor defects which are not always appar- ent until one has started section cutting. One of the most important things that must be watched is that the knob which PARAFFIN SECTIONS 107 Fig. 53. (Top) Transferring the object from the embedding dish to the wax-filled paper box. Fig. 54. (Middle) Remelting the wax around the object with a heated pipette. Fig. 55. (Bottom) Cooling the wax block. 108 THE ART OF MAKING MICROSCOPE SLIDES Microtomes Fig. 56. Sliding microtome. Fig. 57. Rotary microtome. controls the section tliickness must be so moved that an exact number of microns is indicated. If, for example, the knob is so moved that the indicator Une hes between 9 and 10 microns, the pawl will not engage the ratchet perfectly but ^\dll chip off a small portion of brass at each revolution. It only requires a few weeks' operation under these careless conditions to destroy the ratchet wheel which will have to be replaced at the factory. No inexperienced student should ever be trusted with one of these machines until the mechanism of it has l)een explained to him and clearly demonstrated. Knives and Knife Sharpening The most important single factor in the production of good sections is the knife used in cutting. It does not matter how Knives PARAFFIN SECTIONS 109 much care has l^een taken in the prepara- tion of the block or liow complex a micro- tome is used, if the knife-edge is not per- fect there is no chance of securing a perfect section. Ordinary razors are not satisfac- tory for the production of fine sections, and it is necessary to secure a microtome knife, preferably from the manufacturer of the microtome. Another type of micro- tome knife employs the edge of a safety- razor blade in a special holder; these do not, in the writer's hands, give such good results as a sohd blade. Three types of solid blade are available : first those which are square-ground, that is, in which the main portion of the knife is a straight wedge; second, those which are hollow-ground, that is, in which both sides of the knife have been ground away to a concave surface, which results in a relatively long region of thin metal to- wards the edge; third, knives which are half-ground, that is, knives of which one side is square- or flat-ground and the other side hollow-ground. This last type of knife, which the writer prefers, is a compromise. There is no doubt that a square-ground knife is sturdier than a hollow-ground knife, a point of some importance when cutting large areas of relatively hard tis- sues; but there is equallj^ no doubt that a hollow-ground knife can be brought more readih' to a fine edge. Microtome knives must be sharpened frequenth^; but it is necessary, before discussing how to do this, to give a clear understanding of the nature of the cutting edge itself. If a wedge of hardened steel were to be ground continuously to a fine edge, as in Fig. 58, it would be utterly worthless for cutting. After only a few strokes the fine feather-edge, which would be produced by this type of grinding, would break down into a series of jagged saw teeth. A micro- tome knife, or for that matter any other cutting tool, requires to have ground on its cutting edge a facet of a relatively ob- tuse angle, whether it be a square-ground knife, as in Fig. 59, or a hollow-ground knife as Fig. 60. The process of applying this cutting facet to the tip is known as setting; it is an exceedingly difficult opera- tion to conduct, but one which must be learned by every user of a microtome knife. The actual grinding of the blade itself to the correct angle, or to the correct degree of hoUowness, cannot be done in a laboratory; the knife must be returned to the manufacturer or to some scientific sup- j)ly house adetiuatcl}^ equipped with the special machinery necessary. The cutting facet, however, must be set at least once a day if the blade is in continuous use. The nature and purpose of this cutting facet is best explained by reference to the mechanism of cutting shown in Fig. 61. Notice first that the knife blade itself must be inclined at" such an angle to the block that the cutting facet is not quite parallel to the face of the block. There must be left a clearance angle to prevent the knife from scraping the surface every time that it removes a section. This clear- ance angle should, in cutting wax, be as little as possible, and it is for this reason that the blade holder of a microtome is furnished with a device for setting the knife angle. The knife angle should not be set with reference to any theoretical con- sideration, but with regard only to secur- ing this small clearance angle. The only way to judge whether or not a satisfactory clearance angle has been obtained is to observe the sections as they come from the knife. If the clearance angle is too large, so that the section is not being cut from the block but is being scraped from it, the section will have a wrinkled appear- ance and will also usually roll up into a small cylinder. If the clearance angle is too small, so that the lower angle of the facet is scraping the block after the tip has passed, the whole ribbon of sections will be picked up on the top of the block, which will itself crack off when the knife point reaches it. It is obvious that the knife angle will be changed as the angle of the cutting facet is changed, so that it is desirable to maintain the cutting facet of as uniform an angle as possible. This angle is set onto the knife in the man- ner shown in Fig. 62. Notice that the knife has been furnished with a handle and also that a small spHt cyhnder of steel has been sUpped over the back of the blade. This spht cylinder rests flat on the stone, as does the edge of the blade, so that when the knife is pushed forward (the figure 110 THE ART OF MAKING MICROSCOPE SLIDES Knives CUTTING >i FACET BLADE Fig. 58 Fig. 59 CUTTING TIP CLEARANCE ANGLE KNIFE ANGLE BLOCK Fig. 61 Fig. 58. Knife ground as simple wedge without cutting facet. Fig. 59. Flat ground knife showing cutting facet. Fig. 60. Hollow ground knife showing cutting facet. Fig. 61. Cutting action of knife on wax block. shows it at the beginning of the stroke) the cutting facet is produced as the angle between the cutting edge lying on the stone and the enlarged temporarj' back which has been placed on the knife. Since a much blunter cutting facet is required for hard materials than for soft, it is strongly to be recommended that either two knives, or at least two sharpening backs, be secured. It does not matter what kind of stone is used for sharpening pro- vided that it is of the finest obtainable grit, that it is dead fiat, and that under no circumstancs wliatever is it used for any purpose except the sharpening of micro- tome knives. It does not matter whether Knives PARAFFIN SECTIONS 111 it be a water stone, to be lubricated witli soap and water like the yellow Belf^ian stones commonly emjjloyed in l']uro[)e, or an oilstone, to he lubricated with mineral oil like the pike stones so commonly eni- ployed in the United States. It does mat- ter, however, that it should be flooded with lubricant before staitinj;-, and that the knife should be drawn with a lij;lit pressure (notice that tlie fiii,<;er is behind and t}ot on top of the knife in the illustra- tion) the entire length of the stone at each itself. If the knife-edge is nicked to a deeper extent than about a quarter of a millimeter, the only thing to do is either to return the knife to the maiuifacturer to l)e reground, or try to avoid that por- tion of the blade containing the nick when cutting sections. It must be emphasized that the only {purpose of setting is to pro- duce a cutting facet, and that grinding, which cannot be done in the ordinary laboratory, is required for the removal of knife impeifections. Fig. 62. Setting the cutting facet. operation. If onlj- the central portion of the stone is used, it soon becomes hol- lowed out and it thus becomes impossible to maintain a uniform angle. About three strokes on each side of the knife are quite enough to produce a perfectly sharp cut- ting facet; to continue beyond three strokes will have no effect other than to diminish the length of life of the knife. This direction for the use of three strokes in setting applies, of course, only to knives which have been reasonably treated and not to those which through carelessness have acquired a nick in their edge. Where the nick is large it is almost impossible to remove it in setting, for the continual repetition of setting merely grinds away the edge of the knife and ultimately alters the thickness of the blade The next question to arise is that of stropping the blade of the knife by pulhng it backward across a leather surface in the manner shown in Fig. 63. If the knife has been set properly, stropping (the only pur- pose of which is to poUsh the facet) is quite unnecessary. The nature of the leather surface which is used for stropping makes it obviously impossible to pull the knife blade forward and thei-e is a grave risk in pulhng it backward, lest the facet, instead of becoming polished on its flat surfaces, will become rounded on its edges, and thus the work of setting be undone. Certainly no beginner should be permitted to use a strop until he has demonstrated his ability to set a knife-edge to tiie point where it will cut an excellent section without strop- ping. It is also strongly recommended to 112 THE ART OF MAKING MICROSCOPE SLIDES Mounting block the beginner that he should examine the edge of a knife under the low power of a microscope before setting, after setting, and after stropping. Mounting the Block The knife being sharpened and the microtome selected, it now remains to trim the block to the correct shape and to attach it to the object holder of the micro- tome. The rough block of wax containing the object must be first removed from the mold or, if a paper box was used, the box and it is essential that these should be exactly parallel to each other. A skilled microtomist can cut these edges parallel with a safety-razor blade without very much difficulty, but numerous devices have been described from time to time in the literature to enable one to do this mechanically. It does not matter if these two edges are exactly parallel with the plane of the object; it is only essential that they be parallel with each other. At this stage plenty of wax should be left both in front of, and behind, the object. Fig. 63. Stropping a microtome knife. cut away roughly with a knife. The block should now be held against a hght so that the outlines of the contained object can be clearly seen. The block is then trimmed until the object lies in the center of a per- fect rectangle, with the major axis of the object exactly parallel to the long sides. This is best achieved by finding first the major axis, at right angles to which the sections are to be cut, and trimming down one side of the block with a sharp safetj'- razor blade, taking off only a little wax at a time. If one tries to remove a large quantity of wax there is danger of crack- ing the block. When one side has been shaved to a flat surface, the other side is shaved parallel to it. The top and bottom surfaces of the block may now be shaved. This trimmed block has now to be at- tached to some holder which can itself be inserted into the microtome. Since the majority of sections today are cut on a Spencer rotary microtome, we will de- scribe the use of one of the holders sup- phed with this machine, though the in- genuity of man has not yet succeeded in devising a worse method of attaching a paraffin block to a microtome. The holder, which is seen in Fig. 64, consists of a disk of metal with a roughened surface at- tached to a cyhndrical shank. This disk must first of all be covered with a layer of wax and it is extraordinarily difficult to get wax to adhere to these chromium- plated surfaces. If the worker is not en- tirely bound by convention, it would be \ Mounting block PARAFFIN SECTIONS 113 much better for him to secure a series of small rectangular blocks of some hard wood like maple and to soak these for a day or two in molten wax. After they are removed, drained, and cooled it is the sim- plest thing in the world to atta(;li a paraf- fin block to them and to hold them in the jaws of the microtome. Whether the metal holder or the wooden one are used, the technique is essentially the same. A layer bring these buttresses so far up the block that thoy reach the tip of the ol)ject to be cut. The metal should now be placed on one side and allowed to reach room tem- peratui-o. Many people at this point throw the block and holder into a fingerbowl of water, which is all right provided the wa- ter is at room temperature. But there is no more fruitful source of trouble in cut- ting sections than to have the knife, the Fig. 64. Mounting the wax block on the block holder. of molten wax is built up on the surface and allowed to cool. The block (see Fig. 64) is then pressed lightl}' onto this hard- ened wax and fused with it with the aid of a piece of heated metal. Some people use old scalpels but the writer prefers the homemade brass tool shown in the figure. Care must be taken to press only very lightly with the forefinger and to perform the whole operation as speedil}^ as possible to avoid softening the wax in which the object is embedded. The metal tool should be heated to a relatively high temperature and touched hghtly to -the base of the block. If the block is very long, it is also desirable to build up small buttresses of wax against each side, being careful not to block, and the microtome at different temperatures. It is much better to mount the blocks the day before one intends to cut them and to leave them on the bench to await treatment. A final inspection is then made of the block to make certain that its upper and lower surfaces are flat, smooth, and parallel. Many people do not make the final cuts on these surfaces until after the block has been mounted in the block holder. The block and the block holder, after insertion in the jaws of the microtome, are seen in Fig. 65 and it will be noticed that setscrews on the apparatus permit universal motion to be imparted to the block so that it can be correctly orien- tated in relation to the knife. It is easy to lU THE ART OF MAKING MICROSCOPE SLIDES Cutting discover whether or not the edges are parallel by lowering the V)lock until it does not quite touch the edge of the knife, ad- justing it until the lower edge is parallel, then lowering the block again and com- paring the relation of the upper edge with the edge of the knife. Cutting Paraffin Ribbons Tlie first step in cutting sections on this type of microtome is to make sure that causes the two movable holding arms to hold the knife near its edge. The knife is now held in a pair of hemicyhnders which may be moved so as to adjust the knife angle (see Fig. 61). The knife should be set at that angle which experience has shown to be desirable — no guide other than experience can be used — and the two setscrews which lock these inclinable hemi- cyhnders in place then tightened. The two original setscrews, which hold the knife in Fig. 65. Starting the paraffin ribbon. every one of the setscrews seen in Fig. 65 is fully tight. The setscrews holding the block holder may be tightened in any or- der, provided that the result leaves the block correctly orientated, l)ut those con- nected with the knife must be done in the correct order. First the knife is inserted into the holder and fixed firmly, but not tightly, in place by the two bearings at each end. The tightening of these screws place, are now screwed up as tightly as the thumb can bear. This leaves two set- screws which come through the inclinable hemicyhnders and bear on the bottom edge of the knife. These two setscrews should then be tightened simultaneously and uniformly. The effect of this is to force the knife upward and thus wedge it with extreme firmness in the knife holder. Now that everything is tight the handle Cutting PARAFFIN SECTIONS 115 on tlie hack of tlie inierotoiue is turned until tlie block is as far back as possible, and the entire knife is moved on its car- riage until the edge of the blade is about }i inch in front of the block. A last-miiuite check is now made to make sure that the divisions of the setting device exactly co- incide with the thickness desired ; then the handle is raj^idly rotated until the block in the left hand, is slipped under the rib- bon which is then raised in the manner shown in Fig. 65. Care should be taken that a few sections always remain in contact with the blade of the knife, for if the ribbon is hfted till only the edge of the section lies on the edge of the knife, the ribbon will usualh^ break. As the handle is turned, the brush in the left Fig. 66, Laying out the ribbon. starts cutting. The front face will rarely be parallel to the blade of the knife, there- fore a considerable number of sections will have to be cut until the entire width of the block is coming against the knife. No particular attention need be paid to the quahty of this initial ribbon, which may be thrown away. We will assume that all is going well and that the ribbon is coming off in a perfect condition; if it is not, refer to Table 1. The remaining operations of preparing and mounting the ribbon are far more clearly seen in illustration than by descrip- tion. As soon as the ribbon is the width of the knife in length a dry soft brush, held hand is moved away until the ribbon is the same length as the sheet of paper on which it is to be received. Legal size (fools- cap) paper is quite commonly employed and is shown in Fig. 66. Notice that the left-hand edge of the ribbon has been laid flat some distance from the edge of the paper and that a loop, sufficiently large to avoid strain on the ribbon attached to the knife, is retained with the brush, wdiile the ribbon is cut with a rocking motion with an ordinary scalpel or cartilage knife. The larger and colder this scalpel is, the less likelihood there will be of the section adhering to it. The purpose of leaving a good margin around the edge of the paper no THE ART OF MAKING MICROSCOPE SLIDES Cutting is tliat it may he desiralile to interrujit ribbon cutting for some time and to con- tinue later. In this case the worker should furnish himself with a Httle glass-topped frame which is laid over the jiaper to pre- vent the sections from being blown about. As the inexperienced worker will soon find out, the least draft of air, particularly the explosive draft occasioned by some not he cut up until a samjile has been flattened on a slide in order to determine the degree of expansion. Though the sec- tions shown in the illustration are being mounted on an ordinary 3" X 1" slide, it would be moi'e practical (for a ribbon as wide as this) to use a 3" X IH^' or even a 3" X 2" sUde. The sections should never occupy the whole area of the slide. Fig. 67. Cutting tlie ribbon in lengths. fool opening the door, is quite sufficient to scatter the ribbons all over the room. These operations of carrying the ribbon out with the left hand, transferring the Inrush to the right hand, and cutting the ribbon off, are continued until the whole of the required portion of the block has been cut and lies on the paper. The ribbon must then be divided into suitable lengths for mounting on a slide (Fig. 67). Though in theory a section should be of the same size as the block from which it came, this practically never occurs in practice and it is usually safe to allow at least ten and sometimes twenty per cent for expansion when the sections are finally flattened. The ribbon should but at least j-i of an inch should be left at one end for subsequent labeUng. When the decision has been made as to how many sections shall be left in each seg- ment of ribbon, the first row of ribbons is then cut into the required lengths (Fig. 67). Then the worker must decide what shall be used to make them adhere to the shde. It is conventional to use the albu- men adhesive of Mayer 1880 (Chapter 28, V 21.1), and to apply a thin smear of this on a clean sUde with the tip of the httle finger. The author prefers to dilute the selected adhesive two or three hundred- fold with water, and to use this dilute ad- hesive in the next operation of flattening the sections. Mounting PARAFFIN SECTIONS 117 Fig. 68. Mounting the dry ribbon. It will have been apparent to the worker from the moment that he started cutting the sections that the}^ are not absolutely flat. They may be slightly crinkled, or slightly distorted, and must be flattened by being warmed on water heated just be- low the melting point of the wax. Some people place this water on the slide and then add the sections to it, but the writer prefers to lay the ribbons on the slide as shown in Fig. 08. This is not nearly so easy as it looks. Two brushes must be moistened with the tongue just enough to bring the hairs to a point. The two moist points are then delicately touched down (too much pressure will cause the ribbon to adhere to the paper) on each end of the selected piece of section. This l)iece is tluMi lifted as shown in the illus- tration and placed on the shde. When a sufficient number have been accumulated the slide is then picked up carefully, re- versed, and laid 'on top of the last three fingers of the left hand as shown in Fig. 69. 118 THE ART OF MAKING MICROSCOPE SLIDES Flattening It is fatal to grasp the slide by the sides; if this is done, when the water is flooded on from the pipet, the meniscus coming to the edge of the sUdes will break against the fingers, to which the sections will permanently adhere. The technique shown is quite safe and the water containing the adhesive (if none has been applied to the shde) is then flooded on froni a jiij^et in tened, the slide is gently tilted backward towards the hand so as to run off the excess water against the thumb, leaving the sections stranded in place. The sUde is now usually placed on a thermostati- cally controlled hot plate (seen at the back of Fig. 78) and dried. Most people leave their shdes overnight but frequently an hour would be sufficient. Dryness can be Fig. 69. Flooding the ribbons. the manner shown. Enough fluid should be appHed to raise a sharp meniscus at the edge of the shde. The sections must now be flattened, and this is better done rapidly with a flame than slowly on a hot plate. Fig. 70 shows the shde being held over a small alcohol lamp, but a micro-bunsen can be em- ployed equally well. The shde should be exposed to heat for a moment, withdrawn to give time for the heat to jiass from the glass to the fluid, warmed again, and so on, until the sections are observed to be flat. The utmost care must be taken at this point for, if the paraffin is i)ermitted to melt, the sections will not stick to the glass. As soon as the sections are flat- gauged without the least trouble by the fact that a moist shde shows the wax to be more or less opalescent, while on a prop- erly dried shde it is almost glass-clear. The method just described is susceptible of several variations which may be briefly noticed. Some people do not drain the water from the slide, nor do they heat the slide over the lamp; they merely place the slide, as soon as the water has been added to it, on the thermostatically con- trolled hot plate so that the sections dry and flatten at the same time. The objec- tion to this procedure is that dissolved air in the water used for flattening usually comes out in the form of bubbles which accumulate under the section, either cans- Staining PARAFFIN SECTIONS 119 ing it to fall off or at least making it very difficult to observe jwoperly when mounted. There is also the risk in this procedure that the water will not stop at the edge of the shde, but will unexpectedly flood off, (;arrying the sections with it onto the surface of the hot plate. Another procedure, frequently used by the author but not recommended for the inexperienced, is to blot the sections be- fore putting them on the hot plate. A water-saturated piece of coarse filter paper appearance, cause, and cure of the more conunon defects are shown in the pages which follow. These are by no means the only defects or the only cures which may be api^lied. Every user of the microtome should have in his hands a copy of Rich- ards 1949, which lists many suggestions beyond those here given. Staining and Mounting Sections Assuming that all difficulties have been overcome, and tliat one now has a series of i_j._^ ' .-J... i i J-i~i Fig. 70. Warming tlie flooded ribbons in order to flatten them. is placed on the drained slide and pressed hard with a rubber roller, which squeezes much of the water out of both the paper and the sections. This makes sure that the sections are perfectly flattened in contact with the slide, but requires a strong nerve to try for the first time, because most people fear that the sections will stick to the paper. This has never happened in a good many thousands of shdes which the author has made by this means. Shdes so prepared are always free of air bubbles. Before proceeding to a discussion of the next steps to be taken, it may be as well to revert to the moment when section cutting started, and to discuss the innum- erable things that may happen, other than the production of a perfect ribbon. The shdes bearing consecutive ribbons, the paraffin must next be removed in order that the sections may be stained. It is con- ventional, though probably not necessary, to warm each sUde over a flame (holding it as shown in Fig. 70) until the paraffin is molten. The shde is then dropped (as shown in Fig. 78) into a jar containing xylene, benzene, or some other suitable paraffin solvent. The jars shown in this figure are of the type known as coplin jars, which are usually employed when a rela- tively small number of 1" X 3" shdes are to be handled. Larger numbers of small slides are more conveniently handled by being placed in racks, which may be moved from one rectangular jar to an- other. Individual slides may, of course, be 120 THE ART OF MAKING MICROSCOPE .SLIDES^ Defects handled in a coi)lin jar, hut it is more con- venient for tliese to have an' ordinary round specimen tube, or vial, of just over 1-inch diameter, which maintains a single shde in an upright position without the necessity of using the relatively large quantities of fluid involved in a copUn-jar set. Coplin jars are not available for slides larger than 3" by 1"; for these one is forced to use the rectangular jars. It is necessary through the subsequent proceedings to be able to recognize in- stantly on which side of the shde the sec- tion hes. This is not nearly as easy as it sounds; a lot of good shdes have been lost by having the sections rubbed off. The simplest thing to do is to incline the shde at such an angle to the light that, if the section is on top, a reflection of the section is seen on the lower side of the shde. A diamond scratch placed in the corner is of little use because it becomes invisible when the slide is in xjdene. The greatest care should be taken to remove the whole of the wax from the slide before proceed- ing further. It is usuall}^ a wise precaution to have two successive jars of xylene, pass- ing the second jar to the position of the Table 1 DEFECTS APPEARING IN RIBBONS WHILE BEING CUT Fig. 71. Ribbon curved. Fig. 72. Sections compressed. Possible Causes 1. Edges of block not parallel 2. Knife not uniformly sharp, causing more compression on one side of block than other 3. One side of lilock warmer than other Remedies 1. Trim block 2. Try another portion of knife-edge or resharpen knife 3. Let block cool. Check possible causes of heating or cooling, such as lamps or drafts Possible Causes 1. Knife blunt 2. Wax too soft at room temperature for sections of thickness required 3. Wax warmer than room temper- ature Remedies 1. Try another portion of knife-edge or resharpen knife. Compression often occurs through a rounded cuttiing facet (see Fig. 60) produced by overstropping 2. Re-embed in suitable wax or cut thicker sections. Cooling block is rarely successful 3. Cool block to room temperature Defects PARAFFIN SECTIONS Table 1 — {Continued) 121 Fig. 73. Sections alternately thick and thin, usually with compression of thin sections. Possible Causes 1. Block, or wax holding hlock to holder, still warm from mounting 2. Block, or wax holding block to holder, cracked or loose 3. Knife loose 4. Knife cracked 5. Microtome faulty Remedies 1. Cool block and holder to room temperature 2. Check all holding screws, Remove block from holder and holder from microtome. Melt wax off holder and make sure holder is dry. Re- coat holder and remount block. Cool to room tcMiiperature 3. Release all holding screws and check for dirt, grit, or soft wax. Check knife carriage for wax chips on bearing 4. Throw knife awav Fig. 74. Sections bulge in middle. Possible Causes 1 . Wax cool in center, warm on outside 2. Only sharp portion of knife is that which cuts center of block 3. Object impregnated with hard wax and embedded in soft, or some clearing agent remains in object Remedies 1. Let block adjust to room temper- ature. This is the frequent result of cooling blocks in ice water 2. Try another portion of knife-edge or resharpen knife 3. Re-embed object 5. Return microtome to maker fur overhaul 122 THE ART OF MAKING MICROSCOPE SLIDES Defects Table 1 — (Continued) Fig. 75. Object breaks away from wax or is shattered by knife. Fig. 76. Ribbon splits. Possible Causes 1. If object appears chalky and shatters under knife blade, it is not impregnated 2. If object shatters under knife but is not chalky, it is too hard for wax sectioning 3. If object pulls away from wax but does not shatter, the wrong dehy- drant, clearing agent, or wax has been used Remedies 1. Throw block away and start again. If object irreplaceable, try dis- solving off wax, redehydrating, re- clearing and re-embedding 2. Soak block overnight in pheno- glycerol mixture, rinse thoroughly, and dry, or spray section between each cut with celloidin, or dissolve wax and re-embed in nitrocellulose 3. Re-embed in suitable medium, preferably a wax-rubber-resin mix- ture. Avoid xylene in clearing mus- cular structures Possible Causes 1. Nick in blade of knife 2. Grit in object Remedies 1. Try another portion of knife-edge 2. Examine cut edge of block. If face is grooved to top, grit has probably been pushed out. Try another por- tion of knife-edge. If grit still in place, dissect out with needles. If much grit, throw block away Defects PARAFFIN SECTIONS 123 Table 1 — {Continued) Fig. 77. Block lifts ribbon. Possible Causes 1. Ribbon electrified. (Check by test- ing whether or not ribbon sticks to everything else) 2. No clearance angle (see Fig. 60) 3. Upper edge of block has fragments of wax on it (a common result of 2) 4. Edge of knife (either front or back) has fragments of wax on it Remedies 1. Increase room humidity. Ionize air, either with high frequency dis- charge or bunsen flame a short dis- tance from knife 2. Alter knife angle to give clearance angle 3. Scrape upper surface of block with safety-razor blade 4. Clean knife with xylene No ribbon forms Defect (1) Because wax crumbles (2) Because sections, though indi- Possible Causes vidually perfect, do not adhere (3) Because sections roll into cylin- ders 1. Wax contaminated with clearing agent 2. Very hard, pure paraffin used for embedding 3a. Wax too hard at room temper- ature for sections of thickness required 3b. Knife angle wrong Remedies 1. Re-embed. {Note: Wax very readily absorbs hydrocarbon vapors) - 2. Dip block in soft wax or wax-rubber medium. Trim off sides before cutting 3a. Re-embed in suitable wax. If the section is cut very slowly, and the edge of the section held flat with a brush, ribbons may sometimes be formed 3b. Adjust knife angle first, and replacing it with fresh xylene, after about ten or a dozen slides have passed through. It must be remembered that paraffin is insoluble in the alcohol which is used to remove the xylene, so that it is no use soaking a sHde in a solution of xylene in wax and imagining that it will be sufficiently free from wax for subse- quent staining. Some people go further than this and have the first two jars con- taining xylene, and then a third containing a mixture of equal parts of absolute alco- 124 THE ART OF MAKING MICROSCOPE SLIDES Staining hoi and xylene, to make sure that the whole of the wax is removed. If even a small trace of wax remains, it will prevent the penetration of stains. Assuming that one is proceeding along tlie classic xylene- alcohol series, the shde is transferred from either the fresh xylene or the xylene-abso- lute-alcohol mixture, to a coplin jar of ab- solute alcohol. It is unfortunate that no- body seems yet to have placed on the market a coplin jar, or slide-staining dish, the lid of which is satisfactorily ground into position so that absolute alcohol, soon, however, as the slide has been in water long enough to remove the alcohol, it should be withdrawn and examined carefully to make sure that it has been sufficiently dewaxed. If the water flows freely over the whole surface, including the sections, it is safe to proceed to stain- ing by what ever manner is desired. If, however, the sections appear to repel the water, or if tliere is even a meniscus formed round the edge of the section, it is an indication that the wax has not been removed, and that the slide must again be Fig. 78. Starting a slide tlirougli the reagent series. which is very hygroscopic, remains uncon- taminated. It does not matter if xylene is carried over into the absolute alcohol, but as soon as the first trace of a white floc- culent precipitate appears in the alcohol — indicating that some wax is being carried over — the alcohol must be replaced. The writer never l)others to use a series of graded alcohols between absolute al- cohol and water. These graded series are necessary, of course, when one is dealing with the dehydration of whole objects which may be distorted, l)ut the author has never been able to find the slightest difference between thin sections which have been passed from absolute alcohol to water, and those which have laboriously been downgraded through a series. As dehydrated in absolute alcohol, passed back into a xylene-alcohol mixture, and thence again into pure xylene. In the specific examples which conclude this chapter, and in numerous places throughout Chapters 20, 21, and 23, de- scriptions are given of indi\'idual staining methods. The purpose of this chapter is to discuss only the general principles in- volved in the preparation of paraffin sec- tions, so that we may presume the section to have been already stained and returned (through such dehj'drants as are specified in the method used) to xylene, and to be ready for mounting. It is again assumed that the section will be mounted in one of the resinous media described in Chapter 26 (M 30), and Mounting PARAFFIN SECTIONS 125 Canada balsam is so conventional that it may be taken as an example. The shde is removed from the xylene and drained (Fig. 78) and then placed on any convenient flat surface. A drop of the mountant is then placed on the surface of the sections. A covershp of suitable size (Fig. 79) is then held at an incUned angle with a bent needle and slowly lowered so as to exclude all air bubbles. The edges of the slide are then roughly wiped and it is returned to the hot table shown in Fig. 78 to evapo- rate the solvent used for the resin. Though This custom of evaporating the solvents from the surface of the sUde rather than from the edge of the coverslip is nowadays considered old-fashioned; but there is no doubt that it produces a better and more durable slide than does the more usual procedure. One very common accident, which maj'' occur in the course of staining or dewaxing a slide, is that the individual sections show signs of l)ecoming detached, either through not having been perfectly in contact with the shde when dried, or through having Fig. 79. Placing the coverslip on serial section slide. this is the conventional method of opera- tion it is by no means always the best. In particular there is a tendency to ha\'e a higher concentration of solvent along the edges of the coverslip than in the center, and it also takes a surprisingly long time for the whole of the solvent to be removed. It is much bettei', if one can si)are the time, to place a relatively thin coat of mounting medium on top of the slide and then to leave the solvent to evaporate from this on the surface of a hot plate. There is no risk that the slide will dry out, for the mount- ant will act as a varnish. On the next day the slide is examined and, if it appears to be sufficiently varnished, the coverslip is placed on the surface and warmed while maintaining steady pressure. The slide will then be hardened as soon as it is cooled and may be cleaned and put away. been exposed to some reagent which has a solvent action on the adhesive. The effects of this unfortunate accident may be mini- mized by having always on hand a coplin jar of the solution of Claoue 1920 (Chap- ter 28, V 21.1). This is a most admirable lacquer into which the slide may be dipped rapidly and withdrawn. This transparent lacquer hardens readily in place and holds the section attached without seriously interfering with subsequent observation. Cleaning and Labeling Slides No slide can be considered complete un- til it has been properly labeled, cleaned, and stored. Failure to clean a shde can cause rather serious damage for, if un- wanted portions of Canada balsam are left lying about close to the edges or on the surface, and if the slide be then used 126 THE ART OF MAKING MICROSCOPE SLIDES Defects Table 2 DEFECTS APPEARING IN SECTIONS DURING COURSE OF MOUNTING Defect Cause Remedy Method of prevention Sections appear wrinkled Sections have bubbles under them 1. Blunt knife used for 1. None cutting 2. Water used for flat- 2. None tening too hot, so that folds in sec- tions fused into position 3. Sections unable to 3. None expand sufficiently: (a) because water used for flattening too cold (b) because area of water too small 1. Sections insuffi- ciently flattened, so that air is trapped 2. Air dissolved in water used for flattening has come out and is trapped under sections in drying 1. Sharpen knife and cut new sections 2. Watch temperature of water used for flattening 3(a) Watch tempera- ture of water used for flattening (b) Make sure that slide is clean, so that water flows uniformly over it Sections fall 1. Wax melted in off slide flattening 1. If sections still wet, reflood slide with water and reheat to complete flattening 2. If sections still wet, reflood slide with water, work out bubbles, and reheat to complete flatten- ing 1. None 1. Check flatness sections before draining slide of 2. Slide greasy 2. 3. Alkaline reagents 3. dissolve albumen adhesive. (Sections start to work loose in course of staining or dehydrating) 4. Sections not flat- tened into perfect contact with slide None Treat slides with Claoue's solution (Chapter 28, V 21.) 4. None 2. Use air-free (boiled) water for flattening Drain slide thor- oughly and blot off excess moisture. Squeeze sections to slide 1. Watch temperature of water used for flattening 2. Use clean slides 3. See Chapter 28. V 21 for other section adhesives not alkali-sensitive with an oil-immersion lens, the oil will dis- solve a portion of the balsam which will be found very difficult to remove either from the surface of the coverslip or from the front lens of the immersion objective. The slide cannot be cleaned until it is thoroughly dried and if it has been mounted in a solution of resin, it will re- quire several days on a hot plate or ^veeks 4. Sometimes caused by swelling of sec- tions which causes center to lift. Squeeze sections to slide and dry as rapidly as possible at room temperature before the solvent has been removed. When, however, it is' finally dried, which is when the resin on the edge can be cracked, the surface resin should be removed carefully with a blunt knife and the slide left overnight. If on examination the freshly cut edge is then found to be sticky, it is evident that more solvent has moved out and that the slide Defects PARAFFIN SECTIONS 127 Table 3 DEFECTS APPEARING IN SECTIONS AFTER STAINING AND MOUNTING Defect Cause Remedy Method of Prevention Sections distorted Sections appear opaque or have highlj' refrac- tive lines out- lining cells and tissues Sections will not take stain, or stain irregularly Sections con- tain fine opaque needles or granules 1. Blunt knife and soft wax 2. Ribbon stretched when picked up on hot day 3. Tissues not properly hardened before embedding 1. Clearing agent evaporated before mountant added 2. Sections insuffi- ciently cleared or cleared in agent not miscible with mountant 1. Wax not perfectly removed before staining 2. Section not uniform thickness 3. Tissue "old" (has been stored for a long time in alcohol or, worse still, fLxa- tive) 4. Fixative not suit- able before staining technique employed 5. Fixative not fully removed 1. Imperfect removal of mercuric fixatives None, though pro- longed flattening on warm water may help As (1) above 3. None 1. None 2. Soak off cover. Clear properly 1. Return sections through proper se- quence of reagents to xylene. Leave un- til wax removed. Restain 2. None 3. Return sections through proper se- quence of reagents to water. Wash overnight. If not effective try a "tis- sue reviver" (Chapter 22, ADS 11) 4. Try mordanting sections in recom- mended fixative 5. Consult Chapter 10, ADS 11 1. Return sections through proper se- quence of reagents to water. Treat 30 min. with Lugol's iodine, rinse, and bleach in 5 % so- dium thiosulfate. Restain 1. Use suitable knife and embedding medium Handle ribbons in short lengths or use harder wax Use more suitable fixative or fix longer. Take extra care in dehydrating, clear- ing, and embedding Obvious 2. Check quality and nature of clearing agents and mountants 1. Change first jar of xylene frequently 2. See table 1 3. Store all tissues embedded in paraf- fin blocks — never liquids 4. Obvious 5. Treat tissues as in- dicated 1. Treat tissues as in- dicated in Chapter 10, ADS 11 128 THE ART OF MAKING MICROSCOPE SLIDES T. S. intestine Table 3 — (Continued) Defect Cause Remedy Method of Prevention 2. Long storage in formaldehyde 2. None 2. Never store tissues in formaldehyde — always in paraffin blocks is not }et ready. If, however, the removal of the dried balsam leaves no sticky resi- due, it is necessary to provide two finger bowls: one of 90% alcohol and the second of a moderately strong solution of soap and water. The whole slide is then dipped in the 90% alcohol and rubbed briskh' until the excess balsam is I'emoved. It is immediately (to avoid softening the bal- sam) rinsed in the soap solution, and then polished. If the slides are unsatisfactory, tables 2 and 3 above may help to locate the trouble. When dealing with valuable series of sections it is ahvays as w'ell to write the serial numl^er of the slide, and some indi- cation of its nature, on the glass in dia- mond before attaching the label. Labels are constantly becoming detached from sUdes and it is well to have a permanent record underneath them. It has already been pointed out that no two people agree as to what label adhesive to use. The author would only reiterate the counsel he has given in previous chapters: that both sides of the label be licked thoroughly, that it then be pressed into position on the slide, allowed to dry slowdy, and the requi- site information written with waterproof India ink. The violent objections which the author has expressed in previous chapters to stor- ing wholemounts in vertical grooved filing cabinets, do not, of course, apply to sec- tions, since the section is attached to the slide and cannot drift through the mount- ant. Storing in grooved trays, which hold the shde vertically, is undoubtedly the simplest method of storing such sections, but much space can be saved if they are placed in pouches of ordinary indexing cards. If two 5 by 3 index cards be taken, and one be cut down to 5 by 2, the smaller may then be stapled to the first card in such a manner as to leave a pocket into which a slide may be inserted. The full data may then be written on the index card, and these tw^o-card pockets bearing the slides can be accumulated in ordinary card-file drawers. Typical Examples The Preparation of a Transverse Section of the Small Intestine of the Frog Stained with Hematoxylin-eosin This is the simi)lest exam]:)le of paraffin sectioning which can be imagined, and it may well serve as an introduction to this type of technique, either for a class or for an individual. The intestine of a fi'og has been selected, owing to tlie usual avail- abiUty of this form in laboratories; but any small animal may be substituted in its place. Before killing the frog it is necessary to have on hand a selected fixative and, since this is intended to be an example of the ut- most simplicity, it is suggested that the cupric-nitric-paranitrophenol mixture of Petrunkewitsch (Chapter 18 F 4900.0040 Petrunkewitsch 1933) be employed. This fixative is entirely foolproof: objects may remain in it for weeks without damage, and it also permits excellent afterstaining by almost any known technique. If only a piece of intestine is to be fixed, 100 milli- liters of fixative will be sufficient; but there is no reason why any other organ in the animal (with the exception of the cen- T. S. intestine PARAFFIN SECTIONS 129 tral nervous system) should not be pre- served in this fluid for subsequent investigation. The frog is killed by any convenient method, but it is usually best for histo- logical purposes to sever a large blood vessel and permit as much l)lood as possi- ble to drain out from the heart before opening the abdominal cavity and remov- ing the intestine. One or more lengths of about 3'3 of an inch should then be cut from the intestine and transferred directly to fixative where they may remain from a few hours to several weeks. When they are next required the speci- mens should be removed from fixative, washed in running water for a few hours, and then transferred directly to 70 'o alco- hol. The easiest method of washing objects of this size in running water is to take one of the coplin jars previoush' described, to fill it with water, insert the specimen, and then to attach a cover of coarse cheese- cloth with a rubber band. This is then placed in the sink and a narrow stream of water permitted to fall on it from the tap. It will be found that the specimen will swirl round and round in the jar in a most satisfactory manner. This simple device saves all the trouble of rigging up glass tubes and boring corks to make the cum- bersome apparatus sometimes recom- mended for the purpose. The specimen is transferred, after twentj'-four hours in 70% alcohol, to 95% alcohol. It is better to use a large volume of alcohol and to suspend the object in it than to use relatively small volumes which have to l^e frequently changed. It is recommended that a wide- mouthed stoppered jar of about 500 milli- liters capacity be fitted witli a hook in the center of its stopper, from which the ob- ject can then be suspended. The majority of stoi)pers for wide-mouthed glass jars have a hollowed undersurface which may be filled with plaster of Paris, and a glass hook (which is verj^ easily bent from thin glass rod) may be inserted in the litiuid plaster. This must naturally be done some days beforehand, and the plaster must finally thoroughly be dried out in an oven before the jar is usetl for dehydrating. If the worker does not wish to go to this much trouble, it is also easy to screw a small metal "pot hook" into the under surface of a plastic screw cover for a jar of the same size. Alcohol is, however, so hygroscopic that it is better to employ a glass-stoppered jar, the stopper being greased with stoj)C()ck grease, or petro- latum, for a permanent setup. An object as coarse as the one under discussion may be sus])ended in a loop of thread or cotton directly from the liook; or if this is not desirable, it may be enclosed in a small fold of cheese cloth for suspension. After twentj'-four hours in this volume of alco- hol, the ol)ject will be completely pene- trated, and should then be transferred to absolute alcohol using the same volume in a jar of similar construction. It is useful to place about a (luarter-inch layer of an- hydrous copper sulfate at the bottom of the absolute alcohol jar, not only to make sure that the alcohol is absolute, but also to indicate, as it changes to blue, when this jar should be removed from service. Of the mau}^ de-alcoholizing (clearing) agents which may be used, the writer would in the present case select benzene because it is less liable to harden the circular muscles of the intestine than is xylene. As benzene is fighter than an ab- solute alcohol, it is not possible to employ the hanging technique for clearing, and the object should be placed in about 25 milliliters of benzene which should be changed when diffusion currents are seen to have ceased to rise from the object. This will take about six hours for an object of the size under discussion and a secoufl bath of at least six houis should also be given. It is now necessai-y to select the medium in which em])edding is to be done and the writer would recommend the rubber par- affin of Hance (Chapter 17— E 21.1 Hance 1933) which must, of course, have been prepared some time before. The melting l)oint of this medium is about 56°C. so (hat ail oven should be availal)le whi(Oi is lliermostatically controlied at about 5S"('. This oven should contain three stender dishes as well as a 500 cc beaker contain- ing about a pound of the embedding me- dium. The ol)ject is removed from ben- zene, drained briefly on a piece of filter 130 THE ART OF MAKING MICROSCOPE SLIDES T. S. intestine paper, and placed in one of the stender dishes which has been filled to the brim with the molten embedding medium. Under no circumstances should a hd be placed on the stender dish since it is desir- able that as much as possible of the benzol should evaporate while the process of em- bedding is going on. After about an hour the specimen should be removed to fresh wax in the second stender dish, where it may remain another hour, and then to the third stender dish where it should not re- main for more than thirty minutes. Shortly before the end of this last hour a decision should be made as to what type of vessel is to be used for casting the block, and it would be difficult to improve on a paper box (Fig. 50) for this object. The box having been made (it should be of ample size) it is moistened at the bottom and placed on a slab of glass in the manner described earlier in this chapter. The box should be about half filled with embedding material from the beaker and allowed to remain until the layer of wax has con- gealed on the bottom. An object like the one under discussion is best handled with an old pair of forceps rather than with a pipet. The forceps should be warmed in a flame to well above the melting point of the wax, and moved backward and for- ward across the surface so as to melt the surface film which has formed. The object is then rapidly picked up from its stender dish, placed in the wax, and enough fresh wax from the beaker added to make sure that there will be as much solid wax above as there is underneath the specimen. Blocks of this nature shrink greatly, and it will probably be best to fill the box en- tirely full. As soon as the box has been filled, the forceps should again be warmed and passed backward and forward around the object to make sure that no film of un- molten wax (which would cause it conse- quently to cut badly) remains. The wax in its box should now be blown on until it starts to congeal on the surface, then very carefully picked up with the fingers and lowered into a dish of water at room tem- perature until the water does not quite reach the top of the box. If it be thrust under the surface at this point, all of the molten wax will come out and the block be rendered useless. As soon, however, as the block is seen to be congealed through- out, it is thrust under the surface of the water and something laid on it to keep it at the bottom. It should be left in the water for at least five or six hours and much better overnight. One now sets up the microtome, and makes sure that the knife is sharpened in the manner previously described, and then mounts the block. The block having been trimmed to size and mounted as noted earlier, there remains only the actual cut- ting. The block should be trimmed so there is at least as much wax on each side of the object as there is in object itself. This amount of wax would be excessive were we preparing serial sections, but for the preparation of individual sections of this type, in an example given for the benefit of the beginner, this quantity is desirable. The handle of the microtome should now be rapidly rotated and the be- ginnings of the sections observed. There is no need to worry if the section curls to one side or the other during this preliminary period, since the entire area of the block will not be cut until twenty or thirty sec- tions have been removed. As soon, how- ever, as the knife is seen to be approaching the object, and the block in its entirety is being cut, the ribbon must be observed most carefully to see that it is suffering from none of those defects indicated in the table of defects. Should the ribbon not be coming perfectly, various suggestions given in the table may be tried until a perfect ribbon is secured. Since we are not, in this case, preparing a series of sections, it is unnecessary to cut a longer ribbon than will contain the actual number of sections required, with a few left over for emergencies. It is, however, a great mis- take to throw i)artially cut blocks away, since they may be stored in a glycerol- alcohol mixture ciuite indefinitely, and one never knows when further sections may be required. The block, however, should be labeled before being placed in its solu- tion by writing the appropriate informa- tion on a piece of paper and fusing this with a hot needle into an unwanted por- tion of the block. Each section is now cut individually T. S. intestine PARAFFIN SECTIONS 131 from the ribbon and mounted on the shde in whatever manner has been selected. Since the use of the conventional Mayer's egg albumen (Chapter XXVIII V 21.1 Mayer 1880) has already been discussed, another medium will be used. The hydro- lyzed starch of McDowell and Vassos (Chapter 8 V 21.1 McDowell and Vassos 1940) is very little known and well worth using. The directions given in the i)lace just cjuoted should be used in the prepara- tion of this thick, viscous hquid of which about four or five drops may then be added to about 50 cc of distilled water in an Erlenmeyer flask or beaker. The slides must be cleaned before the sections are mounted, and no two people have ever agreed as to what is the most desirable method of doing this. One way is first to rub the shde briskly with 1 % acetic acid in 70% alcohol and dry it by waving in the air. Other methods of cleaning the slide, which yield equally good results can be found from the index. Several drops of the diluted adhesive are placed in the center of each slide and one of the individual sections then taken up with the tip of a moistened brush and placed on the ad- hesive. As soon as the section has been placed on the fluid, the slide is lifted up, warmed carefully over a spirit lamp until the section is flat but the paraffin not melted, and then the superfluous liquid removed carefully with the edge of a filter paper. The slide is then placed on a warm table to dry and, if the drying period is to be prolonged, it is as well to place a dust cover over it, since grains of dust falling upon the shde will adhere just as tena- ciously to the adhesive used as will the specimen itself. It is pi'ojwsed in the present example to stain the slide in the simplest possible manner with coelestin blue B followed by phloxine. Various formulas for stains of the coelestin blue B type will be found in Chapter 20 under the heachng DS 11.41; that preferred by the writer for its sim- phcity is recorded as "Anonymous 1936." There will also be required a solution of phloxine for counterstaining. Phloxine ap- pears to work best from a weak alcohol solution. In Chapter 20 under the heading of DS 12,2 will be found the suggestion that it be used in 0.2% solution in 10% alcohol. Any of the other dyes thei-e rec- ommended may, of course, be substituted. Assuming the section now to be per- fectly dry, it is turned upside down and the light is reflected from it to see whether or not the section is adherent to the glass. If there is any air gap between the section and the glass, a brilliant mirror will be formed and, in a preparation as simple as this, the shde had better be thrown away. Having selected those slides which are per- fectly adherent, they are then warmed over a flame until the wax is melted and diopped into a jar of xylene, where they remain until the paraffin appears to have been removed. They are then passed to another jar of xylene where they remain for at least five minutes, and then to a jar of equal parts xylene and absolute alcohol where they remain for a further five minutes. This treatment is followed by five minutes in absolute alcohol and then by direct transference to distilled water. After they have been in distilled water for a few minutes, each slide should be lifted and inspected to make sure that the water is flowing uniformly over both the slide and section. If it tends to be repelled by the section, or a meniscus is formed around the section, this is evidence that the wax has not been completely removed, and the slide must be transferred first to 95% alcohol to remove the excess water, then to absolute alcohol until perfectly dehy- drated, and then through absolute to xylene, where it remains until the wax has been completely removed before being brought down again as previously indi- cated. The slides may be taken down one at. a time and accumulated in distilled water until they are required. When all the slides have been accumulated in dis- tilled water, they are transferred to the coelestin B staining solution. The time in this varies, but ten to fifteen minutes will probably be sufficient to stain the nuclei. One of the most useful features of this stain is that it is almost imp()ssil)le to over- stain in it. Sections may be left overnight without staining the cytoplasm to a degree which requires differentiation. After the mu'lei are blue-black, therefore, or after a time convenient to the operator hag 132 THE ART OF MAKING MICROSCOPE SLIDES T. S. intestine elapsed, the sections are transferred to fresh distilled water where they are thor- oughly washed. Each slide is then taken individually and dipped up and down in the phloxine solution until a casual inspec- tion shows the background to be yellow- pink. The intensity of stain for the back- ground, in a case like this, is a matter of choice, some people preferring a faint stain and others a darker stain; it must be remembered, in judging the color, that the section will seem darker after it has been cleared than it does in water. As soon as it has been found from a single shde what is the time required to produce the desired degree of staining, the remainder of the slides are placed in the phloxine solution together, left the ap- propriate time, and then transferred to distilled water until no more color comes away. The shdes are then passed from dis- tilled water to 95 % alcohol where they are left for about five minutes, then to fresh 95% alcohol, where they are left for five or six minutes before being passed to ab- solute alcohol. The purpose of using the 95% alcohol is not to diminish diffusion currents but simply to save diluting the absolute alcohol by passing slides directly from water to it. After the slides have been for two or three minutes in absolute alcohol, a single slide is taken and passed into the absolute alcohol-xylene mix- ture for perhaps two minutes and then passed to xylene. This slide is then ex- amined by reflected hght against a black background and should be as nearly as possible transparent with only a faint opalescence. One of the commonest faults in mounting sections is dehydrating them imperfectly, for if there is any water which has been carried through the proc- ess into the xylene (in which water is solu- ble in the extent of about }i of 1 %) this water will be extracted by the section which is in itself an excellent dehydrating agent. There is a world of difference be- tween a i^erfectly cleared (that is glass- clear) slide and one which is only more or less dehydrated so that it appears faintly cloudy. If the shde does not appear to be sufficiently dehydrated the whole of the remaining slides should be transferred to fresh absolute alcohol and another one tried. When it has become apparent from the examination of the test slide that de- hydration is complete, the remaining slides may be run up through absolute alcohol and xylene and accumulated in the final jar of xylene. As balsam was discussed in the body of this chapter, we suggest using at the pres- ent time the medium of Kirkpatrick and Lendrun (Chapter 26 M 34.1 Kirkpatrick and Lendrun 1939). Next clean the ap- propriate number of coverslips: in the present instance a %-inch circle would be admirable. The author cleans his cover- slips in the same manner as he cleans his slides: by mping with a weakly acid, alco- hol solution. Each slide is taken individu- ally, drained by its corner, laid on flat surface, and a drop of mounting medium placed on top. The coverslip is then placed on the mounting medium and pressed down with a needle. It should not be pressed absolutely into contact with the shde or too thin a layer of mounting medium will be left; some experience is recjuired to judge when the coverslip has been pushed down far enough. If this is done skillfully the surplus mounting medium will form a neat ring around the outer surface of the cover. If it does not do so, care should at least be taken that no ])ortion of the cover is devoid of surplus mountant which will be sucked under the coverslip as the solvent evaporates. These vslides should be left to dry at room tem- perature for about one day and then placed on a warm plate for about a week. After they are dried the surplus dry mounting medium should be scraped off with a knife and the excess remaining after scraping removed carefully with a rag moistened in 90% alcohol. The shdes are again dried overnight and then should be ringed with some colored varnish using the technique described in Chapter 2. This ring does not assist the preservation of the mounting medium l)ut it has always, in the writer's experience, assured that the sUde when placed in the hands of students will be treated with more respect than a non-ringed slide. The slide, after labeling, is now complete. Frog embryos PARAFFIN SECTIOKS 133 Preparation of. Serial Sections of an Amphibian Embryo Heavily yolked embryos are among the most difficult objects from which to pre- pare satisfactory serial sections, as may be witnessed by the photographs which illustrate the work of many experimental embrj'ologists. Though the example spe- cificall}' taken is that of an amphibian embryo, the methods to be discussed may l)e used for any heavily 3'olked material such as fish, or even in the preparation of sections of the early stages (segmentation and tlie like) of bird embryos. The crux of the entire matter lies in the selection of a fixative and it is doubtful whether a worse fixative for the purpose could be found than the picro-acetic- formaldehj'de of Bouin, so generally em- ployed. Two fixatives have been devel- oped specifically for heavily yolked mate- rial: that of Gregg and Puckett (Chapter 18 F 3000.1010 Gregg and Puckett 1943) which is designed specifically for the eggs of frogs, and that of Smith (Chapter IS F 7000.1010 Smith 1912) which was originally develoiied for the eggs of Crypto- hranchus, but which the author has used successfull}^ for a large variety of heavily yolked material. The author has used Smith for so many years that he is prej- udiced in favor of this formula, and the fact that he has not been so successful with the formula of Gregg and Puckett may be due to the fact that he is less ex- perienced with it and not that it is in- herently incapable of giving equally good results. As the techniciues of fixation in- volved are altogether different they will be discussed separately. Let us assume first of all that we are using the fluid of Gregg and Puckett, which has been made up according to the formula given in Chapter 18. The mass of embryos and eggs, together with their gelatinous surrounding envelopes, are taken and placed in at least 50 times their own volume of the solution. The jar centaining them should be turned upside down at intervals to insui'e that the fluid around them does not become diluted and they are then left for 24 hours. If the eggs are to be embedded and sectioned at once, they may then be removed and waslied in running water for 24 hours or, if they are to be stored for long periods, they may be moved to 2% formaldehyde directly from the fixative and may remain in this fluid, changed possibly after the first 48 hours, until they are required for use. The mass is then removed and broken into small clus- ters which are placed in a test tul)e or small flask about one-third filled with water. The flask is then shaken vigorously, which will remove a portion of the albu- minous envelope, the dirty water poured off, fresh added, and the flask again shaken. After half a dozen treatments of this type the greater part of the albumen will have been removed. When as much of the jelly as possible has been removed by this mechanical treatment the eggs are transferred to a flask of 1 % sodium hyi)0- chlorite and shaken gently and carefully. At intervals eggs will be found to detach themselves. These should be removed either with a section lifter or a small pipet to another flask containing water; b\^ tliis means the whole of the remaining jelly will be removed. The eggs, which are now in water, should be carefully examined to see whether or not the vitelhne membrane is still adherent. If it is still adherent, those which have it should be transferred back to fresh 1% sodium hypochlorite and stirred very gently, being examined at intervals under a binocular microscope, until the membrane has been removed. The eggs are then washed thoroughly to remove all traces of hypochlorite. It is better to do this with half a dozen changes of water rather than in running water, be- cause the eggs at this stage are lirittle and portions may be flaked off the outside if they are subjected to the ))umping which seems to be an inevital)le part of washing with running water. Let us now examine Smith's method. The fixative must be made up immediately before use and large volumes are required in relation to the size of the objects. The author once fixed the entire yolk of a hen's egg, using two gallons of solution; and at least 500 milliliters should be em- 134 THE ART OF MAKING MICROSCOPE SLIDES Frog embryos ployed for a cluster of a dozen or two amphibian eggs. The eggs are transferred to the solution which is immediately placed in a dark cupboard where it re- mains for about 48 hours. The original specifies that low temperature should be employed, but the writer has been unable to find the least difference in performance between heavily yolked eggs fixed at room temperature and those which have been fixed in a refrigerator. The solution should be changed once or twice during the forty-eight hours, or at least as often as it becomes dark green. It is inevitable that it should become greenish, but by chang- ing solutions before a dark-green color ap- pears, the deposition of chromium oxides on the surface of the egg and its mem- branes may be avoided. At the end of forty-eight hours the eggs are removed to large volumes of 2% formaldehyde in the dark; the solution must be changed as often as it becomes discolored and wash- ing must continue until no further color comes away. After all possible color has been removed by the 2% formaldehyde, the jars may be taken from the cupboard and stored indefinitely at room tempera- ture in the light. Most of the eggs or embryos will be found to have become de- tached from their gelatinous membranes in the course of this treatment, but the vitelHne membrane is frequently left. It becomes brittle, however, and may be re- moved without difficulty with the aid of a couple of needles. Whichever method of fixation has been employed, we are now left with the eggs or embryos in 2% formaldehyde. The process of embedding is different according to the technique to be used. By the technique of Gregg and Puckett the eggs are dehy- drated through graded alcohols, allowing in the case of frog eggs two hours each at 35%, 50%, and 60%. It is also necessary to treat them with iodine to remove the mercuric chloride, and for this purpose they are placed in 70% alcohol, to which has been added al^out 5% of Lugol's iodine (Chap. 22 ADS 12.2 Lugol (1905)). The exact proportion of iodine is not impor- tant and technical directions usually read "add iodine until the fluid is the color of port"; but the author sees no reason why this insult to a noble wine should be per- petuated. It- requires a treatment of at least 48 hours to make sure that the mercuric residues are removed and the eggs are then transferred to 80% alcohol where they are washed until no further color comes away. They are then dehy- drated for one or two hours each in 95% and absolute alcohol. Gregg and Puckett specify clearing in xylene for 30 minutes but the writer prefers cleaning in benzene, which does not seem to render the eggs so brittle. After complete clearing, they are then transferred to a mixture of one part of the hydrocarbon employed and two parts of soft (48°C.) paraffin at room tem- perature. They may remain in this mix- ture until next required. When embedding is finally to be completed this mixture is placed in an oven at 58°C. and allowed to remain until completely liquified. The eggs are then transferred to 52° paraffin for one hour and finally to whatever medium is chosen for embedding (Gregg and Puckett prefer 55° paraffin ; the writer prefers rubber-paraffin) for another 3 or 4 hours. In the alternative technique of Smith, the procedure is rather different. After the eggs are taken from formaldehyde they are passed through 35% and 50% alcohol for two or three hours each and then placed in Grenadier's alcoholic borax carmine for about two days. The formula for this fluid is given in Chapter 20 (DS 11.22 Grenadier 1879) and it must be most strongly recommended that one use the dry stock dissolved in 70% alcohol rather than the usual solution prepared direct. The eggs are removed from the stain to one-quarter of 1 % hydrochloric acid in 70% alcohol and left there for about two hours or until the first rapid color clouds have died down. It is not in- tended to complete differentiation by this process, but only to remove the excess stain. They are then, exactly as in the pre- vious technique, dehydrated through 95% and absolute alcohol before being cleared in whatever hydrocarbon is preferred. Smith prefers embedding in 52° paraffin but this, in the writer's experience, is too soft and will only permit the tliickest sec- tions. It is again recommended that one Frog embryos PARAFFIN SECTIONS 135 of the rubber paraffins with a melting point of from 50° to 55°C. be employed. In any case we now have amphibian embryos and eggs accumulated in the embedding oven in whatever medium has been decided to use. It is usually necessary in cutting sections of this type that the orientation of the embryo in relation to the knife should be known; this is difficult to estabhsh by ordinary means when one is deahng with a more or less spherical embryo. The method preferred by the writer for indicating one of the planes is to embed at the same time and alongside the spherical embryo a little rectangular block of liver or some other soft tissue. It is easy to dehydrate clear and impregnate with wax a piece of liver and keep this permanently in the embedding oven in paraffin. When one is ready to embed the eggs a small strip (in the case of the frog embryo about 3 mm. X 1 mm. X 1 mm.) is cut from this slab of hver, and is first of all laid in the paper box to which one has added the wax in the manner already described. The egg is then transferred to the same box and is most carefuUj^ ori- entated with regard to the strip of liver, so that if the liver be cut exactly at right angles, the egg will be cut in the desired plane. A strip of hver this size does not greatly increase the total area of this block, nor does it in any way interfere with whatever staining technique is em- ployed for the sections. An identical pro- ' cedure is followed, whether one is dealing with eggs fixed by the technique of Gregg and Puckett, or fixed and prestained by the technique of Smith. After the block has been hardened under the surface of water (Smith specifies 70 % alcohol for the purpose, but it does not appear to matter) it is removed, allowed to attain room temperature, and the sides trimmed away until the strip of liver is clearly seen. The block is then attached to the holder, mounted in the block holder of a microtome, and a ribbon is prepared in the usual manner. The only difficulty that is hkely to arise is that, after the ribbons have been flattened and are dry- ing, the entire yolky center of the embryo may rise in a dome. This event usually indicates that the vitelline membrane has remained on the egg and there is nothing whatever tliat can l)e done about it. Such sections always become detached in the course of staining and are in any case worthless, for if they are varnished in place they will still be so domed as to render microscopic examination almost imi)ossi- ble. If, however, several successive batches of sections, in which one is quite certain that the vitelline membrane has been re- moved from the embryo, behave in this manner, it is sometimes possible to stop the trouble by drilling a little liole in the block until one just comes to the egg itself, and then soaking the block in glycerol- alcohol. Blocks so treated will, when cut, usually be found to lie flat on the slide. If this dexice fails it is strongly recom- mended that 70% alcohol be substituted for water used to flatten the sections (any of the customary adhesives may be mixed with alcohol of this strength just as readily as with water) and that the technique of using a wet blotter and a rubber roller be used, as described in the body of this chapter. It must be emphasized that these defects are uncommon in materials fixed in the manner described; they are men- tioned only because they occur so fre- quently in the handling of amphibian embryos fixed and prepared by other methods. After the ribbons have been flattened and dried, they are then put through the ordinary series of reagents until they are ready to be stained. The technique differs very greatly according to whether one is dealing with the technique of Gregg and Puckett or that of Smith. In the technique of the latter the slides are removed from absolute alcohol and flooded with the Lyons blue and picric acid mixture of Smith (Chapter 20 DS 12.221) for a period of about one minute. They are then returned to absolute alcohol until no color comes away, and cleared in xylene before being mounted in a resinous me- dium. The writer prefers this technique to any other because of the gross swelling which occurs in yolk when exposed to aqueous solutions of stains. The rising up of the center of the section, commented on in the last paragraph, is not confined to the time when the sections are flattening. 136 THE ART OF MAKING MICROSCOPE SLIDES S. S. mouse but may also occur during staining; the center of many an excellent section has become detached from the slide simply for the reason that it has been handled in aqueous stains. The nuclei are not so clearly shown by carmine as by many other stains, but for valuable material the author has always used Smith as an in- surance policy against losses. It is also easy with this stain to distinguish the various cells since the cell membranes themselves pick up the blue while the mass of the^^yolk retains the yellow of the picric acid. Gregg and Puckett, on the contrary, take the sections down to water in the usual manner and then stain them in Delafield's hematoxyhn [Chapter 20 DS 11.122 Delafield (1885)] differentiating in acid alcohol in the manner indicated until the nuclei alone remain clearly stained. They are then counterstained in eosin- orange (Chapter 20 DS 12.222 Gregg and Puckett 1943) before returning through the alcohols to xjdene and then to the mountant. The author has never had the least success in staining amphibian em- bryos with hematoxylin because of the very strong affinity of this stain for the albumen granules in the j^olk. After staining the sUdes are cleaned and labeled in the usual manner and will show almost incredible improvement over the usual "Bouin's fixative-hematoxylin-eo- sin" technique which most modern em- bryologists appear to employ. Preparation of a Sagittal Section of an Entire Mouse This preparation is not recommended to the stern and dedicated research worker, whose onh' interest in the preparation of microscope slides is to demonstrate a the- sis, but has been included for the benefit of those who, like the author, enjoy mak- ing a beautiful slide for its own sake. It may, of course, be argued by those who have to justify themselves that such sec- tions form an admiral^le method of demon- strating the main relationships of mam- malian anatomy to a large class. It is proposed, in fact, to prepare a sagittal (vertical-longitudinal) section of an entire mouse from the tip of its nose to the very last joint of its tail. This is a feat of great technical difficulty and requires atten- tion, at odd moments, during several months. The author does not think that the end can justify the means: the beautiful preparation must be its own justification. The mouse selected for the preparation should be of such a size that the section will fit onto a standard 3-inch by 1-inch slide, but sufficiently old to be covered in hair. A litter of freshly born white mice should, therefore, be watched until the young arc completely clothed with hair; this will be between one and two weeks after birth. The next problem is to kill and fix the mouse in such a manner as to fulfill the conditions that the tail shall \)v straight, so that it can be included in a sagittal section, and that the fixative shall shall be able to penetrate to all parts. The tail must, of course, be curled under the body if the section is to be placed on a slide, and it must also be attached to some rigid structure so as to remain straight. It is desirable to kill the mouse in a relaxed condition: and injection of sodium amytal is probably the best. For those who do not have access to h3'perdermic syringes and reagents of this tj^pe, however, it is quite satisfactory to kill with ether (not chloro- form which stiffens the animal rapidly) though one has less time to work before rigor mortis sets in. Before kilUng the mouse one should have secured the finest possible needle obtainable and some very fine silk, not Hnen or cotton, thread. There is only one way of insuring that the tail shall coincide with the nose and that is to sew the two together. Therefore, as soon as the mouse is dead, open the jaws and insert a little wedge of wood so that they are partly opened (at the tip of the jaws the gap should be about 2 mm.) and then proceed to pull the tail around until the tip of it projects just beyond the nose. Using the fine needle and the fine silk sew the skin from each side of the tail to each side of the nose. It must be remem- bered that we are concerned only with getting half a dozen perfect sagittal sec- tions and that anything outside the exact central plane will not show. Now take a S. S. mouse PARAFFIN SECTIONS 137 glass rod, or a piece of plastic, and bind the tail to it with silk, being careful to bind only loosely lest the imprint of the silk show in the final section. Great care must be tak(>n to keep the tail straight along the glass; it is then attached in front and in back to the body of the mouse exactly parallel to the spine. If this is done skilfully a median sagittal section will cut through the central portion of the central nervous system for its entire length and will show a central section of the tail to its very tip. It is just as well to kill the whole htter and to prepare them in this manner, since one or two specimens are bound to get out of alignment in the course of the subsequent operations. The writer thought at one time that the simplest method of maintaining the tail straight would be to hang the mouse from a loop of tape passed through its tail with a weight attached to the nose, but the ob- jection to this is that though the tail re- mains dead straight, it does not remain exactly parallel to the spinal cord. Before all this has been done, one should have decided on the fixative to be em- ployed and have made up a sufficient quantity of it. Fixatives containing picric acid should be avoided at all costs, since the prolonged soaking in water Avhich must inevitably accompanj^ decalcification will cause the grossest sweUing and vacuolation of picric-fixed materials. The writer's pref- erence is for the dichromate-formalde- hj'de-acetic mixtures, preference being given to those which are based on the original solution of Miiller and which thus contain sodium sulfate. Numerous foi-- mulas for these mixtures will be found in Chapter 18 under the general hea(Ung F 7000.1010, the writer has employed the mixture Bohm and Opel 1907 with success in a preparation of the present tj'pe. It must not be imagined that this, or any other fixative, will penetrate rapidly enough to fix an entire mouse before con- siderable autolysis has taken place in the internal organs; it will be necessary to make small openings in the sides if we are to have a successful preparation. As soon, therefore, as the mouse has been firmly fixed in the manner described, a sharp scalpel should be taken and a series of slits made through the skin and l)eritoneum along each side of the ab- domen. These slits should not be more than a milhmeter or so in extent, or there may be a protrusion of the internal oi'gans through them. Care must also be taken not to cut the liver, or any major blood vessel, or the entire abdominal cavity will fill up with blood, and the appearance of the finished preparation will be coni- l)letely ruined. In addition to these shts down the side, about one third of each side of the head must be cut off with a fine saw so as to expose the outer surface of the brain. The use of bone forceps or wire cutters will cause distortion; a fine jewel- ers' saw is much better for the purpose. The cutting of blood vessels in this case does not make very much difference since there are few cavities into which the blood can flow. The mouse, having thus been bound to its supports and a few small openings made, is wrapped in a fold of cheesecloth and suspended at about the center of at least one liter of the selected fixative. The jar containing the mouse and fixative should then be placed in a dark place for about two days. At the end of this time the mouse is removed and the fixative is replaced with new fixative. At this time also extend the size of the openings which have been made, since all of the blood will now be coagulated and the internal organs will be more or less firmly fixed in place. In extending these openings, re- member actually that no more than the center half-millimeter of the mouse will ultimately be required, and certainly all of the limbs and considerable areas of both flanks may be removed. Some experience is necessary in deciding how^ much to remove. This is another reason why the entire litter of young mice should have been sacrificed at the same time rather than reliance placed on a single specimen. The mouse should now be placed in fresh fixative and left in the dark for a further period of about a week, the jar being ex- amined at intervals to make sure that the fixative is not turning green. It will always turn green-brown, but should it become of a fairly dark-green color, it must immedi- ately be replaced with new fixative. It is 138 THE ART OF MAKING MICROSCOPE SLIDES S. S. mouse very difficult to overharden or overfix a specimen of this kind, and at least a month should be allowed to make sure that there is perfect fixation throughout while an exposure of six months will cause no damage. After fixation is complete, the mouse (or mice) is removed from the fixative, and without removing from the cheese- cloth bags, hung in a jar through which running water flows from a tube reaching to the bottom. It (or they) should be washed for at least three days in running water before being removed and hung in a large jar of 20% alcohol or other de- hydrant. For those who find alcohol diffi- cult to olitain, either acetone, isopropanol, or methanol are equally good for dehydra- tion, but must in each instance be used in a fairly close series. Small objects may, as has been stated elsewhere, be passed with- out danger from water into absolute al- cohol, but as large an object as this mouse will have to be dehydrated very slowly. The mouse should be left in 20% alcohol for about five days and subsequently for about five days each in 50%, 70% and 90%. The experienced reader will have noticed that we have so far said nothing of decalcification which must obviously take place before the sections can be made. On the basis of the writer's experi- ence, born out by the earlier workers but apparently nowadays ignored, it would ap- pear that hardening in alcohol after fixa- tion yields a specimen which behaves very much better under the knife than does one which has been fixed only without the sub- sequent alcohol hardening. It is for this reason that he recommends that it be taken up in the manner described to 90 % alcohol, left there for a week or two, and then brought down through the same series to water, where it is left until the whole of the alcohol has been re- moved. The specimen is now ready for decalcification. For as large an object as this, particu- larly one which has been fixed in a di- chromate mixture, the method of von Ebner would be desirable. This solution, the formula for which is given in Chapter 19 under the heading AF 21.1 von Ebner (1891), employs a strong solution of sodium chloride to diminish the swelling of the tissues caused by the nitric acid used. It is also possible to use phloroglucin for the same purpose of diminishing the swelhng; a typical formula is given in the same chapter as AF 21.1 Ferreri (1895). It has, however, been the writer's experi- ence that these phloroglucin formulas work better on smaller objects and he strongly recommends the formula of von Ebner in the present case. Following this formula, the specimen is hung in a large volume of the solution and left for three or four days. At the end of this time a further 1 % of nitric acid is added and the whole stirred up. This proc- ess of adding a milliliter of nitric acid per 100 milliliters is continued every third or fourth day until decalcification is com- plete. It is as undesirable to decalcify for too long a period as it is to leave patches of hard bone to wreck the knife, hence the worker will often find himself in a quan- dary as to how to determine when de- calcification is complete. The only way this can be done with complete success is by x-ray examination, for the least trace of undissolved calcium remaining will show clearly upon the x-ray plate or fluorescent screen. It is often possible to find some friendly dentist who will prepare an x-ray of the mouse at intervals, but if this is impossible one must judge on deU- cate probing with a needle. The two places which are usuallj^, but by no means always, the last to decalicify are the inner ear and the molar teeth, and it is a reasonably safe assumption that if a fine needle can be passed through these with- out meeting more resistance than would be occasioned by tough leather, it is safe to continue. It is not safe to probe in the direction of the vertebrae, which are often slow in decalcification, because they are too close to the central area which will subsequently be sectioned. In the ab- sence of x-ray information it is much safer to decalcify too long than too short a time, and it will be suggested later that a solution be used to mordant the sections; this will undo, to a certain extent, any excessive hydrolysis which has taken place. The decalcified mouse must now be thoroughly washed to remove all traces of acid, but water should not be used for this S. S. mouse PARAFFIN SECTIONS 139 purpose since the removal of the salt is more rapid than the acid and bad hy- drolysis may occur at this moment. The mouse should, therefore, be washed with weak (2%) formaldehyde, which should be changed at daily intervals for about a week. At the end of this time the mouse may be washed in running water overnight with safety, and may then be considered ready to be dehydrated and embedded. Before dehydration it is well to trim away as much of the material as can be removed without risk of displacing the remaining internal organs. The larger the piece to be dehydrated and embedded, the longer will the process take, and it is usually per- fectly safe to reduce the preparation at this point to a slab of about yi of an inch thick. Do not hesitate to use fine ligatures of silk to hold in place any organ showing signs of diaplacement, for these ligatures will be missed by the knife as it takes the central section desired. Dehydration and clearing can follow the ordinary procedure hanging the slab of tissue at the top of considerable vol- umes of 20%, 50%, 70%, 95%, and ab- solute alcohol before laying it at the bot- tom of a jar containing benzene, which may be changed once or twice. If the mouse has been reduced, as suggested, to a slab, possibly two or three days in each of these alcohols will be sufficient to pro- vide perfect dehydration. As the block is going to be large, plain paraffin would be a most unsuitable embedding medium, the writer warmly recommends one of the rubber-paraffin media, the formula for which is given in Chapter 27 under the heading of E 21.1. In view of the large size of the specimen, the ordinary stender dishes used for embedding will have to be abandoned in favor either of beakers or crystalhzing dishes. It is essential that the specimen should lie flat during the course of embedding, or it will inevitably become distorted, and the care thus far taken to maintain the tail in a straight line with the spine will be wasted. The specimen should first be placed in a crystallizing dish filled with benzene, and about a lialf-iiich layer of cliips of the em- bedding medium should be i)laced on to]) of the specimen. The crystallizing dish should be left at room temperature for about a day — naturally covered with a plain sheet of glass — and should then be placed in a paraffin oven or warmed to about 50°C. It is necessary to use a special oven for this purpose, because the large quantity of benzol which evaporates from the preparation will be absorbed in any other wax in the oven and render it relativel}^ useless for subsequent embed- ding. Al)out three or four hours later, after the wax has become fluid, this mixture of benzene-paraffin may be enriched by pour- ing molten paraffin into it and carefully stirring it up. One should then at intervals of a few hours — it does not matter leaving it overnight — pour off about half of the fluid contained in the dish and replace it with fresh, molten paraffin. By this means, over a space of a day or two, the specimen may be passed by reasonable gradations from benzene to paraffin. This whole proc- ess should be watched and controlled with the utmost care, for it is easy for these slabs to twist out of shape in the course of impregnation. After the changes described the specimen should finally be removed very carefully to another crystallizing dish containing clean paraffin and left for at least a day to complete the impregnation with wax. Casting of the block, which will be too large for a paper box, is one of the few cases in which L-shapecl blocks of brass can profitably be employed. Alternativelj', if L-shaped blocks are not available, take two 1-inch lengths of 1-inch-square brass to form the ends of the box which one is making and attach to them with sealing wax two thin sheets of brass along each side, thus making a metal box. This should be stood on a slab of plate glass. Now pour into this box, which should be at least three inches long by one inch wide and one-and-one-half inches deep, about a half an inch of wax and allow it to cool until it is solid. Then heat, in a small beaker, aliout a teaspoonful of wax to a temperature well above its melting j)oint — it is probabl}' safest to raise it to smok- ing heat — and then pour this suddenly onto the surface of the now hardened wax at the bottom of the box. By this means the surface is again molten and the box can be filled with wax which has been maintained in the oven at about its melt- 140 THE ART OF MAKING MICROSCOPE SLIDES S. S. mouse ing point. The object is then carefully placed in the box. At this point one may detach the tail from the rod of glass or plastic to which it has been attached. Then wait until the wax in which the specimen is lying commences to solidify and carefully fill the box to the very brim with molten wax. The whole must now be cooled as rapidly as possible. When the block has completely hardened it is slid out of its metal 1)0X and placed in a large jar of water at room temperature, where it may remain overnight or until one is prepared to deal with it. In the examples previously given use has been made of the ordinaiy rotary mi- crotome, but such an instrument is useless for the very large sections wliich we are about to cut. No microtome will serve save one of the slider type shown in Fig. 55. As these sliding microtomes have no possible justifiable use in the cutting of paraffin sections, save for very large ob- jects, it is curious that so many of them (including the one shown in the illustra- tion) have relatively small object holders provided. The jaws which are nonnally used to hold the metal object holder, how- ever, can be adapted to hold a large piece of wood. The block under discussion had better be attached by melting the wax to a piece of hard wood, previously steeped in paraffin, of a size very little smaller than the block itself. After the block has been attached — it is very dangerous to trj' it before — it must be trimmed to the shape which will be used for actual cut- ting, which differs in every particular from the shape which must be used when cut- ting serial sections of small objects. For ribbon-cutting the block is always rec- tangular and the two sides must be ex- actly parallel. In the case of a very large l)lock from which single sections are to be cut in one of these sliding microtomes, three sides of it may be left more or less rectangular, but the fourth side must come to an aiigl(> pointing to the ))lade of the knife. This angle is not important but should be between 40*^ and 60"^. It does not matter whether the sloping side extends beyond the beginning of the object or not, and it is actually of no ini|)oitance what shape the other sides are i)rovided there is a 40° to 60° angle pointing towards the knife. This angle is for the purpose of pre- senting a small area of wax to the first cut. Large sections on a microtome of this type invariably roll themselves up into a cylin- der which is \'ery difficult subsequently to unroll. If, however, there is a sloping angle pointing towards the knife, the flat portion may be held with a brush against the knife and the whole section, therefore, retained more or less flat as it comes off. Now take an old microtome knife and cut 25 or 30 micron sections from the top until one gets down t(^ that part of the ob- ject which one wishes to cut. This pre- liminary flattening of the top surface of the block, and cutting away of the un- wanted portions of the specimen, also shows how this particular block is behav- ing in I'elation to the microtome itself. If the sections curl hopelessly, in spite of the point of wax, it is evident that the knife is striking at the specimen too squarely and it should be adjusted to cut at an an- gle more like that of the knife shown in Fig. 83 which is, however, set for celloidin. A certain amount of maneuvering of the knife angle l:)ackward and forward will en- able one to secure a cut in which at least half an inch of the pointed end of the wax remains straight, thus permitting a brush held in the left hand to be pressed down while the right hand completes the move- ment of the knife. Do not imagine that sec- tions of this size will ever come off flat: it is enough if they are reasonably flat. Each section will have to be flattened independ- ently in a bowl of water heated to from 5 to 10 degrees below the melting point of the embedding medium employed. The temperature is rather critical, but it may be established by experiments on un- wanted sections, so that when the block has finallj' been trimmed down to the point where the 10 or 15 essential sections can be taken, all difficulties will have been ironed out. It is inevitable, as one cuts farther and farther into the object, that fine readjustments of the orientation will have to he made. These can only be nuule by trial and error, and one should never cut off too many sections until one has finally got the block lying in the exact plane reciuired. It may be said that this l)lane may be determined reasonably when some portion of the vertebrae are being S. S. mouse PARAFFIN SECTIONS 141 out at the same time tliat tlic skin of the tail is being cut. Remember that after fine adjustment, the knife blade will have to be used as a plane to render flat the whole surface of the block before further com- plete sections can be taken. Finally, how- ever, the moment comes which culminates all the months of work. The block is lying completely flat. One places a freshlj^ sharp- ened knife in position and prepares to take the sections required. Before this final operation it will be nec- essary to have cleaned, in any manner de- sirable, the required number of slides, and to have laid these at hand alongside the vessel of water which will be used for flat- tening. Now take the brush in the left hand, the knife in the right hand, shde the knife forward, grab the little curling tail of paraffin coming from the pointed end of the block, and with one smooth, con- tinuous movement complete the section. This section, in a more or less wrinkled condition, will now be l3'ing on the knife blade, from which it may be removed with the aid of two brushes. One l:)rush held in the left hand is moistened with the lips and applied to the upper surface of the end of the section farthest from the edge of the blade, while the other is very gently slid under the section to loosen its attach- ment from the edge of the blade. Using one brush b}^ adhesion from above and one brush to balance the other end of the sec- tion from below, now drop the section onto ' the warm water where it will completely expand. A slide is then taken in the right hand and a needle in the left, with a view to stranding the section in the right posi- tion on the shde. The slide should be placed in the water and left for a moment or two until it reaches approximately the same tempera- ture and then, while held pointing down- wards at an angle of about 45°, ap- proached to the section until that side of the section which is intended to be to- wards the upper end of the slide just touches the glass. The slide is tlien very shghtly raised so as to strand the ui)per portion, which is then held in place with a needle while the whole shde is withdrawn at an angle of from 45 to 30° from the water. It is quite impossible to pass the slide horizontally under the section and then to I'aise it so that the section remains in place. It is only by withdrawing the shde at an angle, in the manner described, that one can hope to strand the section in the correct position. If tlie section is not ill correc't position on tiie slide, no attempt can be made to rearrange it. It is only pos- sible to replace the slide in water with the hope that the section will float off so that a second attempt can be made. If the sec- tion is only slightly out of position on the slide it is much better to leave it alone, since the section usually breaks at the sec- ond attempt to strand it. As soon as the section has been stranded on the slide, the slide is removed from water, laid on a flat surface, a sheet of water-saturated coarse filter paper laid on top of it, and a rubber roller of the type used by photograjjhers pressed down with considerable force so as to squeeze the water from the paper and the section at the same time. The section is then placed on a warm table to dry. Notliing has been said about the use of an adhesive for attaching the section to the shde. Provided that the slide is per- fectly clean and that the section is pressed firmly into contact with it, there should be no necessity for any adhesive at all. For those, however, who do not care to run this risk any adhesive mentioned in Chapter 2S under tlie heading of V 21.1 may be used either by smearing it on the slide, or mixed, to the extent of about 2%, in the water used for flattening. As soon as the slides are dried they may be stained in any manner desired. For a specimen of this nature the wn-iter's first ])reference is for the stain for Patay 1934 (ab])reviated directions for wiiich will be found in Chai)ter 20 under the heading DS 12.32 Patay 1934) or for the stain of Mallory (which will be found in the same chapter under the heading DS 13.41). De- tailed descriptions of the use of both of these stains are given elsewhere. If 'the slides have to be stored for any great length of time, or if the process of decalcification has been unduly prolonged, treat each shde before staining according to the method of Mullen and McCarter, which is given in Chapter 22 under the heading ADS 12.1 Mullen and McCarter 1941. 13 Nitrocellulose Sections General Principles Nature of the Process As the name indicates, this chapter is concerned with the preparations of sec- tions of material which has been impreg- nated with a solution of nitrocellulose. This process is not to be regarded as a substitute for the paraffin method de- scribed in the last chapter; it should be used only when paraffin will not give a satisfactory result. This is usually used either for exceedingly minute objects, the orientation of which in paraffin, or the re- tention of which in paraffin sections, is almost impossible, or for very large ob- jects with numerous cavities which cannot well be supported with paraffin. Paraffin in large cavities tends to shrink away, while nitrocellulose solutions do not. There are numerous disadvantages in the use of nitrocellulose. The worst for the research worker is the difficulty of preparing serial sections with the sections in their due order. Tliis may be overcome to a certain extent by the process of double embedding (see the next chapter) but tliis itself is less satisfactory than straight em- bedding and should be used only for very small or very difficult objects. One of the advantages of nitrocellulose embedding is that the process does not in- volve the use of heat; the materials are impregnated in solutions of increasing strength at room temperature, and these solutions are subsequentl}^ hardened either by evaporation or by chemical means. The size of the nitrocellulose molecules in the dispersions (usually called solutions) em- ploj'ed is so great that the material dif- fuses slowly and the the process is a long one. Various methods have been put for- ward for using nitrocellulose at high tem- peratures, but there appears to be fittle justification for them, because, if the ma- terial to be embedded will stand boiUng, it will most certainly stand embedding in paraffin. These processes appear to have been introduced by those who are so ac- customed to celloidin embedding that they do not wish to use anything else. Materials Employed Cellulose nitrate is not, as its name might indicate, a pure chemical, but is a mixture of a great number of different compounds, the relative proportions of which depend upon the method of manufacture. Few of these mixtures are suitable for cutting sec- tions; and one should alwaj's be used which is specifically prepared for the pur- pose. The best known in the world, and for many years the only one known, was celloidin, supplied by Schering. Its place has been taken in the United States today by Parlodion, marketed by Mallinkrodt. It is unfortunate that the trade names of both of these should be so closely allied to collodion, which is a pharmaceutical solu- tion of pyroxylin unsuitable for section cutting. Cellulose nitrates, other than those marketed under brand names, are broadly classified according to the viscos- ity of the standard solution. This viscosity is expressed in terms of the number of seconds taken by a steel ball of standard size to fall a standard distance through a standard column of the solution. The low- est viscosity normally marketed is that known as 5-second nitrocellulose and is the only one which may be employed in micro- technique. Another point to be watched, 142 Solutions NITROCELLULOSE SECTIONS 143 in using other than a proprietary product, is the fact that some nitrocelhilose mix- tures are quite ^^olentl3' explosive when dry, whereas both celloidin and Parlodion, though they burn briskly if given the opportunity, do not ignite with explosive violence. Cellulose nitrates other than those indicated under brand names are always marketed in solution and should never be stored in the dry state. Many of the older books suggest that chips of nitro- cellulose material, to be used for embed- ding, be stored under water. This advice is given not to lengthen the life of the chips, but only to avoid the risk of explosion. Celloidin, which term will be used throughout the rest of this chapter when- ever a nitrocellulose-embedding medium is meant, is soluble in a great variety of modern solvents, but most techniques are based on its use in a solution of a mixture of alcohol and ether. Preparation of Solutions Celloidin is not easily soluble in the al- cohol-ether mixture usually employed, therefore a special method must be used to prepare the solutions. The chips of dried material are first removed from the bottle in which they have been kept and placed in a desiccator overnight. The only real enemy of the success of embedding in cel- loidin is water, and at no stage in the pro- ceedings may one risk contamination. It ' is usual to carry in stock a 16% solution of celloidin, therefore, 16 grams of these dried chips should be weighed. These chips are placed in a dry bottle fitted -with a glass stopper which has been tested for fit. Fifty parts of absolute alcohol are then poured over them. If this alcohol is not taken from an unopened new bottle, it is desirable that it be carefully dehydrated either with calcium sulfate or copper sul- fate before being used for this purpose. The bottle is left at room temperature overnight, in order that the celloidin may swell, and when this swelling is complete 50 parts of anhydrous ether are added. The ordinary ether of commerce and the ether used for anesthetic purposes are worthless; one must employ the variety sold as ether anhydrous by sodium. Any attempt on the part of the worker to re- move water from commercial ether with sodium in his own laboratory will produce nothing but a serious explosion. The an- hydrous ether should always be taken from a freshly opened can. The bottle is rotated slowly until the celloidin is com- jiletely dispersed through the mass. The selection of a solution of this strength is based on the fact that it is about the thick- est solution which may reasonably be poured from a bottle. Not too much of the material should be prepared at one time, since ether always evaporates through even the best fitting stopper. The only method known to the writer of keeping the material satisfactorily is to secure one of the bottles, once common in pharmacies but now difficult to obtain, in which the ground glass stopper is itself covered with a domed cap — like that on a balsam bottle — ground to the neck. If such a bottle can be obtained, the outer cap, but not the inner, may be greased with glycerol and a relatively ether-tight seal thus secured. Under no circumstances should celloidin solutions be stored in an icebox in the hope of diminisliing the rate of evaporation. If these cold solutions are then brought out into a warm room, moisture will condense all over the bottle and over the solution as it is being poured. In Chapter 27 under the heading E 22.1 will be found sugges- tions for various other solutions which have from time to time been made. Infiltration of Objects with Nitrocellu- lose Solutions In the course of embedding in paraffin, as described in the last chapter, it is just possible to get away with slightly im- perfect dehydration. In impregnation with celloidin it is absolutely impossible. The prime prerequisite to the successful infil- tration of a specimen is that it be per- fectly dehydrated. For this purpose the specimen should be brought up in the con- ventional manner through such series of alcohols as may be necessary until abso- lute alcohol is reached. It should continue dehydration for some considerable time in at least two changes of absolute alcohol, the last of which has either been drawn from a sealed bottle, or from a bottle in 144 THE ART OF MAKING MICROSCOPE SLIDES Infiltration Fig. 80. Tying paper collar round wood block. Fig. 81. Putting in first layer of celloidin. which a considerable quantity of calcium sulfate or copper sulfate has been placed as adehydrant. When the specimen is com- pletely dehydrated, it is transferred to a mixture of equal parts of absolute alcohol and ether. The worker must remember always to use anhydrous ether and not the commercial variety. Specimens should re- main in this mixture until they have been completely impregnated. The actual process of getting the mate- rial impregnated with 16% celloidin may 1)6 done in two ways. Either the ol)ject may l)e i^laced in a considerable volume of a dilute solution, and evaporated, or it may be passed through solutions of in- creasing strength. The writer prefers the latter method since it is very difficult to evaporate an alcohol-ether mixture slowly under dry conditions. Most people employ solutions of 2, 4, 8, and 16% celloidin prepared bj- dilution of the 16% stock. Every vessel used for dilution, as well as the diluent itself, must be absolutely dry. The object should be taken from the ab- solute alcohol-ether mixture, and placed in about 50 times its own volume of a 2 % dilution in a glass-stoppered bottle, which should in turn be kept in a sealed desicca- tor while the imjiregnation is going on. The time of impregnation naturall}' varies according to both the size and the nature of the object, but it is a rough and ready rule for relative time that the material should spend proportionately as long in each solution as the concentration of the Casting blocks NITROCELLULOSE SECTIONS 145 Fig. 82. Transferring object in thick celloidin. celloidin; that is, if the object were to take one hour in 2 % it should have eight hours in 16%. As a measure of absolute time it may be said that an object the size of a frog's egg will require about one day in the 2 % while a flower bud the size of a walnut should remain at least ten days. The writer prefers not to endeavor to transfer the object from one solution to another, but to pour off the weak solution and re- place it ^\'ith a stronger. There is no means of telling when impregnation is complete until one comes to cut the section; but two things must be remembered: one, that the specimen cannot be damaged no mat- ter how long it be immersed in celloidin, two, that the most frequent cause of faulty sections is imperfect impregnation. When impregnation is complete it is necessary to prepare a block for cutting. Casting Celloidin Blocks Two methods may be employed to transform the celloidin from a hquid to a solid state. Either the alcohol-ether mix- ture may be permitted to evaporate, or it may be removed with another solvent, usually chloroform, in which celloidin is not itself soluble. The removal of the sol- vent with chloroform may also be done either in the hquid or in the vapor phase. It is difficult to mount blocks of celloi- din once they have been cast on an object holder, though the solution of Apathy (Chapter 27, E 21.1 Apathy (1942)) has been specifically developed for that pur- pose. It is, moreover, very nearly im- possible to mount a celloidin block on any of the metal block holders supplied with standard microtomes. The worker is, tlierefore, advised to prepare for himself a series of small wooden blocks on which the object may be directly cast. These wooden blocks should be of hardwood and a whole series should be provided accord- ing to the size of the object which is to be cut. The smallest practical size is about J2" X H" X 1" and it should be under- stood that the block will be cast on the J-^-inch end. The largest practical size is dependent entirely upon the size of the object to be cut. These blocks should be cut, sanded as smooth as possible, baked in an oven at about 80°C. to remove as much water as possible, and then thrown directly into absolute alcohol. The abso- lute alcohol is replaced with absolute alco- hol-ether and then with 2% celloidin. The blocks are transferred for se\'eral daj's to a solution of 4% celloidin and then with- 146 THE ART OF MAKING MICROSCOPE SLIDES Cutting drawn, stood on their ends in a desiccator, and dried. Once prepared, they may be used an indefinite number of times. The process of casting the block is not difficult. First of all take a paper collar (ordinary bond paper is excellent) and tie it firmly round the edge of the block so that it projects upwards for a distance of about M inch (Fig. 80). This makes a box the floor of which is the end of the wooden block and the sides of which are of paper. Then pour into the bottom of this box about K of an inch of 16% celloidin (Fig. 81) and place it in a desiccator (at the left in Fig. 81) where the alcohol and ether are allowed to evaporate until the surface of the block is firm when touched with a blunt needle. It is not required to be hard; it is only required to be sufficiently firm that an object placed on it will not sink. Remove the block from the desiccator and fill it to the brim with the 16% celloidin containing the object (Fig. 82). The object will sink through the hquid celloidin until it comes to the firm layer underneath. Needles are used to orientate it in the desired position, and it is then either placed in a desiccator to evaporate, or, better, placed in a desiccator in the base of which the desiccant has been replaced by a quantity of chloroform (at the right. Figs. 81 and 82). There is relatively rapid vapor exchange between the chloroform and the alcohol-ether, and the block by this means may be completely hardened overnight. If speed is vital, place the whole block in liquid chloroform as soon as the object has been oriented. By this means the block will be hardened in a few hours, but with some risk that the rapid diffusion currents set up may displace the object. The block should be stored in chloroform until required. If the block is to be prepared by the method of evaporation, a box of paper is made and a layer of hardened celloidin set on the bottom. After the object has been oriented in the 16% celloidin, however, the block is placed in a desiccator to evap- orate, and is filled up from time to time with 16% celloidin as it shrinks. Blocks hardened by evaporation are denser and tougher than those hardened with chloro- form. The chloroform technique is usually more satisfactory. Cutting Sections in Celloidin There are as many methods of cutting sections from celloidin blocks as there are workers who have done it, and space does not permit all the variations to be given here. Broadly speaking they fall into two classes: those in which the celloidin is cut dry, and those in which it is cut wet. Celloidin may be cut on any kind of mi- crotome, but unless an attempt is to be made to serialize sections (which is far better done by the double embedding technique described in the next chapter) a shding microtome should be used. Celloidin cannot be cut by bringing a square edge of the block against the knife. Not only must the knife be set at an angle of about 30° to the direction of travel (Fig. 83) but the corner of the block must, as shown, be trimmed to an acute angle. The block, therefore, after being trimmed to the shape shown, is clamped by its wooden base in the holder and oriented in the desired position. If the block is being cut dry the knife is now shd forward and the sections removed to a watch glass. Do not worry if they are, as is more than prob- able, considerably curled. When enough sections have been accumulated they may be dealt with in the manner to be de- scribed later. The author much prefers to cut his blocks after they have been moistened with oil of cedar. There is a double reason for this. Not only do the sections tend to stay flatter, but if the block is thoroughly impregnated with oil it will become glass- clear so that last-minute adjustments of orientation are easy. By this technique the block, which should have been chloro- form-hardened, is transferred directly to oil of cedarwood and left until it is glass- clear. When it is removed, as much as the cedar oil as possible should be wiped off with a cloth and the block mounted in the appropriate holder. Then take a finger- bowl, or beaker, of oil of cedarwood and, after having adjusted the knife to approxi- mately the correct angle, moisten the blade of the knife with the oil. The knife is then shd forward to remove a section, Cutting NITROCELLULOSE SECTIONS 147 Fig. 83. Cutting celloidin sections. and each section is received (Fig. 83) on a brush saturated with oiL From time to time the blade of the knife should be moistened with more cedar oil, and the sections as they come onto the knife may be left to accumulate — they will be held to the blade by the cedarwood — until a considerable area of the blade is covered. These sections may then be picked up with a brush moistened with oil of cedar and transferred to a container of the same fluid; or, if there are enough of them, they may merely be washed off the knife, which has been removed, and placed in the beaker or watch glass. Both the methods just described i:)re- suppose that the object has been stained, as should usually be the case, before em- bedding, and that the sections will require no further manipulation beyond flattening and mounting. When the sections are be- ing cut with a view to staining them subse- quently, the method of cutting a block moistened with 70% alcohol is to be pre- ferred. By this method the block, which must have been hardened in chloroform, is placed directly in a considerable volume of 70% alcohol. Stronger alcohol should not be employed because it will tend to soften the block. After a day or tw'o in 70% alcohol, most of the chloroform will have been removed, the block is then mounted in the usual manner, and sections are cut from it with a knife which is moistened with 70% alcohol. The blade must be moistened with a brush dipped in 148 THE ART OF MAKING MICROSCOPE SLIDES Mounting 70 % alcohol each time a section is cut, and each section must be individually removed to a beaker of 70% alcohol in order to avoid evaporation and drying. Staining Sections It is usually desirable to stain objects before celloidin sections are cut, but when it is necessary to stain subsequently, and the sections have been prepared in 70% alcohol as described, they may be sub- mitted to the action of any staining fluids exactly as though they were freehand sec- tions. That is, they may be passed from one solution to another with the aid of a section hfter, washed in water, and in general handled with considerable rough- ness without any risk of damaging them. It must be remembered, of course, that alcohol solutions may not be employed or the celloidin will be hopelessly softened. The chief objection to this procedure is the tendency of some stains to be absorbed by the celloidin; it is cUfficult to find a plasma stain which will stain tissues with- out coloring celloidin. Nuclear staining is relatively easy, as is also metal staining, which is the process most usually applied to celloidin sections. No attempt should be made to flatten the section before it has been stained, but it should be passed through all the required techniques and then returned to 70% alcohol before mounting. A method of double staining a botanical specimen is given in the typical preparation which concludes this chapter. Mounting Celloidin Sections on Slides If the section has been cut in cedar oil from an object which has been prestained, nothing further is required than to remove the section from cedar oil, place it in the center of a clean slide, add a drop of the resinous mounting medium selected, and apply the coverslip. Anj^ sUght curl Avhich tends to lift the coverslip may be easily pressed out, either by leaving a weight on the coverslip overnight or by using a small spring clip to hold the covership down. If the section has been cut dry it will usually be found too curled to mount satis- factorily, and a different technique must be followed. In this case the section is })laced in the approximate position on the slide, and the slide, with the section, placed in a small dish containing a little ether. Within a relatively short time the celloidin will have been softened suffi- ciently to be pressed flat on the slide and mounted in balsam under the coverslip. If the section has been cut in 70% alco- hol, and subsequently subjected to various staining procedures, it is necessary that it should be dehydrated before being mounted in balsam. It may be removed from 70% alcohol to a mixture of equal parts of absolute alcohol and chloroform, the former to dehydrate the specimen, the latter to prevent the dissolution of the cel- loidin. This mixture will often appear cloudy when the section is first put in, in which case it is only necessary to replace it with fresh solution and so on until both the section and the specimen remain un- clouded. When an unclouded condition has been reached, the specimen may be dehydrated in oil of cedar, placed on the slide, and mounted as previously described. It is occasionall}^ necessary, though usu- ally undesirable, to attach a number of celloidin sections to sHdes and then to stain them in position. The reason this is unsatisfactory is that it is hard enough to remove staining dyes from the celloidin matrix when both sides of the section are free in a watchglass, and nearly impossible when one side has been pressed against a glass surface. This method, however, must be employed if it is desired to serialize celloidin sections and some cogent reason prevents the use of the double technique described in the next chapter. Of the vari- ous methods given in Chapter 28 under the heading V 21.2, the writer prefers that of Heringa and ten Berge 1923 in which clean slides are coated with a 3 % solution of gelatin and dried. When these slides are required they are soaked for a couple of hours in 5% sodium sulfate, rinsed, and again dried. The section is taken from 70% alcohol, pressed firmly to the sUde — or the sections are lined up in their order and pressed firmly to the slide — and then dipped as soon as they are partially dry once or twice in absolute alcohol and chloroform. This gives a very reasonable T.S. Lily bud NITROCELLULOSE SECTIONS 149 adhesion. Another useful method of han- dhng large numbers of celloidin sUdes is that of Linstaedt 1912, also described in Chapter 28, V 21.2. The method there given can be followed; it results, in effect, in the fusing of a large quantity of cel- loidin sections into a single sheet of celluloid, which may then be handled through stains, etc. as if it were a simple section. It will be noticed that the sheet itself is made of celluloid — not celloidin— which is a material which does not readily pick up stains. The two methods of Lon- geron involve the removal of the celloidin after the sections have been mounted, and leaves one to wonder why (celloidin should have been used, instead of paraffin, in the first place. Typical Example Preparation of a Transverse Section of a Lily Bud It has already been pointed out that one of the best uses to which celloidin maj^ be put is the preparation of sections of fine structures containing cavities which would not be held b}' paraffin. The example here selected is a case in point, for it would be almost impossible by the ordinary paraffin section technique to take a transverse section of a large flower and to maintain all the different parts in relation to each other. It would indeed be almost impos- sible to secure a section at all without gross collapse of the parts. The bud of a lily has been selected be- cause it is such admirable teaching mate- rial. Sections may be taken through a level which will show both the stamen and the pistil, and the material is sufficiently large to permit an elementary botany class to get a clear idea of the arrangement of the different parts with the use of mag- nifications no higher than those provided by a hand lens. The method of staining selected, however, is sufficienth^ good to permit the examination of the individual parts, bj- an advanced class, under the high power of the microscope. The exact species of hly, provided it is one of the trumpet varieties known to florists, is quite immaterial, and the bud should be taken about a week before it is open. The best fixative to use for this kind of thing is one of the chromic- formaldehyde-acetic mixtures known to botanists under the general term of C RAF. Several formulas for these mixtures are given in Chapter 18 under the heading F 6000.1010; the ffiiids of Navashin 1912, Belling 1930, and Randolph 1935 are the ones widely used by botanists. It makes little difference which of these formulas is employed, but they must be made up immediately before use to prevent the reduction of the chromic acid by the formaldehyde. A hly bud ly/' long X }i" in diameter will need to be fixed for about four days in one of these fluids, which should be changed daily and kept in the dark. As soon as the bud is cut from the plant it should be immersed in the fixative and the extreme tip cut off to permit the contained air to leave. After fixation in these fluids, the bud should be washed for 24 hours in running water and then transferred through 20%, 50%, 70% and 90% alcohol (about a daj- or two in each) to 95% alco- hol, which should be changed as often as it becomes discolored. It is necessary to remove the chlorophyll, or else this will subsequently diffuse into the celloidin, from which it is almost impossible to re- move it. If the process of decolorization in 95 % alcohol is too slow for the worker, he may transfer the bud to absolute alcohol until it is dehydrated, and then to chloro- form where the remaining chlorophyll will be extracted very rapidly. The risk in this procedure, however, is that the chloroform will not subsequently be sufficiently re- moved, and will thus prevent proper infil- tration by the celloidin. If chloroform is used, the bud must be removed as soon as bleached to absolute alcohol, which is changed as often as the least smell of chloroform remains. It is then put through at least 6 changes of 95 % alcohol, with one day in each, before being transferred to fresh absolute alcohol to complete the dehydration. The writer's preferred method of dehy- dration for large objects has already been 150 THE ART OF MAKING MICROSCOPE SLIDES T.S. Lily bud described (Chapter 12) and should be em- ployed in the case of the lily bud. Remem- ber that almost nothing can prevent the production of a perfect celloidin section except imperfect dehydration of the speci- men. One is only safe when the specimen has remained in a large vessel of absolute alcohol, containing copper sulfate at the bottom, for a period of 24 hours, at the end of which time not the slightest trace of color shall have been accjuired by the copper sulfate. A 16% solution of celloidin is then di- luted to a strength of 2%. If the lily bud contains very large cavities — that is, if it was taken quite late in its development — it may be necessary, in order to avoid diffusion currents and some consecjuent bending of the internal structures, to start with a solution as weak as M% celloidin rather than with the conventional 2%. It is best to fix and dehydrate several buds at one time and to take the first of these up through the conventional process. If this fails a slower method must be used. The bud is then passed to a mixture of equal parts of absolute alcohol and anhydrous ether until diffusion currents are no longer apparent. If only 20 or 30 milliliters of the fluid are used for a specimen of this size, it should be changed after about 3 hours and then left overnight in a fresh solution. There is a risk, if an object of this size is picked from alcohol-ether mixture and placed in another fluid, that the rapid evaporation will leave air bubbles; there- fore, it is best to place it in a vessel with just enough alcohol-ether mixture to cover it and then to fill this vessel with 2% celloidin. The container is then rocked gently backward and forward to mix the celloidin, and the specimen is left for about 24 hours. This weak celloidin is now poured off, leaving enough of it to cover the object, and 4% celloidin is poured in. The 4:% celloidin should be left for three or four days and then replaced in the same manner by 8% celloidin, in which the specimen should be left for at least a week. Eight per cent celloidin is sufficiently vis- cous to inhibit air bubbles when the speci- men is transferred, and it should now be lifted from this thickish celloidin and put into the 16% solution. All these operations should have been conducted in glass-stop- pered bottles kept in a desiccator. The period of time in 16% celloidin is not crit- ical but two or three weeks would be a safe period. The whole process is so long drawn out, that an extra week or two makes little difference; any endeavor to save even a few days in the final impregnation may undo all the previous work. Now take a wooden block about 1" X 1" X 2" and tie onto it a paper collar (Fig. 80) at least an inch taller than the bud. This is naturally a somewhat cum- bersome arrangement; it will probably be best to use an ordinary 5" X 3" indexing card, rather than a piece of paper, in order to get the necessary stiffness. A large box of this kind will inevitably leak so that the overlapped edges should be held together with gum arable, permitted to harden, and then dried in a desiccator. After this block, with its paper walls rising from it, has been thoroughlj^ dried in a desiccator, the paper, and about half of the wooden block, is dipped in 8% celloidin and placed back in the desiccator. This procedure not only holds the paper more firmly in position but also provides an additional assurance against leakage. When this initial coat of celloidin has hardened, about '^2 inch of 16% celloidin is poured into the bottom of the box, which is then returned to the desiccator and examined at intervals until the celloidin is found to have hardened sufficiently to bear the weight of the bud. The box is then filled with 16% celloidin and the bud is inserted. It does not matter in the least if the celloidin flows up over the side, but it will be very unfortunate if not enough of it is used. The writer finds that the best method of holding a large object like this in place in the box is to take a couple of entomological pins and drive them clear through the box and the specimen, in areas from which sections are not required. Using a long needle it is then possible to reach down and adjust both the bottom and the top of the long bud so that it lies essentially in the center of the box, held in place by the entomological pins driven through it. Fine pins of this nature do not make a sufficiently large hole to permit any leakage of celloidin. If the box is now not full of celloidin, it is T.S. Lily bud NITROCELLULOSE SECTIONS 151 topped off with the 16% solution and placed in a closed vessel (a desiccator is very convenient) in the bottom of which a small quantit}' of chloroform has l)een placed. After about a day it will be found that the block has set to a rather opaque jelly-like consistency and the whole thing — block, wood, pins, and paper — -is then thrown into a large container of anhydrous chloroform. It should remain for a few days in the chloroform and then be trans- ferred to a considerable volume of 70% alcohol, which is changed daily until no smell of chloroform is observed. The block — pins, paper, and all — may be kept in 70% alcohol until it is required. When it is decided to start sectioning, the block is removed, the pins withdrawn (a pair of phers will probably be neces- sary), and the paper shaved from the sides of the block with a sharp knife or razor. It has been presumed in the directions which have so far been given that the re- quired sections will lie about one-third of the distance from the base of the block, since it is obvious that a block of this size will not have the stability to permit cut- ting at the top. Under these circumstances the upper two-thirds of the block should be removed, and it is safer to do this with a fine saw than with a knife. If it is decided that the portions of the upper block are also required, the block may be cut with a saw into as many pieces as are wanted, , and each piece mounted on a celloicUn- impregnated wooden block with the solu- tion of Apathy given in Chapter 27 under the heading E 22.1. Blocks mounted with Apathy's cement are never as satisfactory, however, as those which have been cast directly onto a wooden block, as in the first case. The reason for the retention of the entire bud through embedding, is, of course, to avoid disturbing the exceedingly delicate relationships of the parts. This would certainly ha^Dpen if one were to en- deavor to embed one third of the bud without leaving the remainder of it at- tached for support. The lower third of the block on its wooden holder is now mounted in the object holder of a sUding microtome and oriented roughly in the position de- sired. The block will be found to be suffi- ciently clear, after one has planed off the surface with a i-azor, to see down into it and select that iM)int from which the de- sired sections will be cut. No difficult}'' will be e.xperienced in cutting these sec- tions provided the knife slopes back away from the block at an angle of about 30° and hits the corner rather than the edge of the block. Before cutting, provide a beaker containing 70% alcohol, in which the sections are to be accumulated, and another beaker and brush containing 70% alcohol, with which the knife blade and the surface of the block are to be liberally anointed while sectioning is in process. As each section comes off, it should be re- moved to the dish of 70% alcohol in which the sections may be stored until they are to be stained. It is excellent practice to accumulate a large number of these sec- tions and then to issue them to a class for staining. Celloidin embedding is such a prolonged process that it is difficult to use in class periods, but there is nothing to prevent blocks or sections from being issued to classes to whom a detailed de- scription of the manner in which they have been prepared is given. A good combination for staining these sections is Delafield's hematoxylin and saf- ranin. However, the ordinary Delafield's hematoxylin solution — the formula for which is given in Chapter 20 as DS 11.122 Delafield (1885) — cannot be used at full strength or it will be difficult to remove from the celloidin matrix. It is better to dilute the original solution \\dth about 10 times its own volume of a 1 % solution of ammonium alum. One-tenth of 1 % hydro- chloric acid in 70% alcohol and one of the solutions of safranin given in Chapter 20 under the heading DS 11.42, are also re- quired. The safranin should be either in water or in an alcohol not stronger than 50%. The solution of Johansen 1940, for example, contains enough Cellosolve to soften a celloidin section undesirably. The formula of Chamberlain 1915 is that com- monly employed. Having accumulated these reagents in three dishes, and a spare dish of 70% alco- hol, the sections are placed in safranin. It is difficult to overstain in this fluid and it is probably most convenient to leave it overnight. It should be left at least until 152 THE ART OF MAKING MICROSCOPE SLIDES T.S. Lily bud it appears to be deeplj^ stained. This will take not less than three or four hours. The next morning, if staining has taken place overnight, each section is transferred sep- arately to acid alcohol and examined un- der a low power of the microscope until the safranin is observed to be almost re- moved from the cell walls. The sections are then transferred to distilled water, preferably through two changes, to re- move the acid. As soon as the acid has been removed, they are placed in the di- luted Delafield's hematoxylin and left there until the cell walls are deeply stained. This will take from five minutes to half an hour, depending on both the thickness of the section and on the nature of the specimen. The sections are then re- moved, one at a time, to acid alcohol, and left there until the stain has been removed from the celloidin matrix but not from the cell walls. As soon as this result has been achieved the sections are transferred to 70% alcohol, in which the\" are rinsed in several changes to remove the acid. Now collect as many slides as are re- quired, a mixture of equal parts absolute alcohol and anhydrous chloroform, and a bottle of whatever resinous medium has been selected as the mountant. The sec- tions are transferred from 70 % alcohol di- rect to absolute alcohol and chloroform, in which they are allowed to remain until dehydrated. Each section is then passed to cedar oil where it remains until it is clear. The sections are now taken one at a time and drained by the corner against a piece of filter paper. A drop of the resinous medium is placed on the slide, the section placed on this, another drop placed on top, and a coverslip applied. If the sections curl to a slight extent, this may be over- come, as has already been pointed out, by placing either a weight or a clip on the cover. If, however, the sections are badly curled, it is desirable to soften the celloi- din somewhat. This may be done by using a mixture of cedar oil and clove oil (cel- loidin is readily soluble in the latter) in place of the pure cedar oil for the clearing. It is as well to try 10% clove oil in cedar oil at first and, if this does not render the sections flexible enough, to increase the quantity of clove oil until they can be flattened. 14 Sections from Double-embedded Material General Principles The onl}' purpose of embedding objects first in celloidin and then in paraffin is to secure serial sections of material which cannot be handled by the paraffin method alone. This limits its utility to small ob- jects which cannot with ease be oriented in paraffin, or alternatively, to small ob- jects of which it is quite essential to obtain series, and which like many small arthro- pods, cannot be retained sufficiently firmly in a wax matrix to permit of sections being obtained. It is possible to impregnate an object with celloidin (as described in the last chapter) and then to embed the impreg- nated object in paraffin. There are, how- ever, much better ways available which shorten to a considerable extent this long process. These methods are based on the original suggestion of Peterfi 1921 (23632, 38:342) that a solution of celloi- din in methyl benzoate could profitalily be substituted for the more conventional solutions. By this method the small objects are dehydrated in the manner described in the last chapter, just as much care being necessary as though one were running a straight celloidin impregnation, but are then passed from the absolute alcohol- ether mixture to a celloidin solution con- taining methyl benzoate or methyl saU- cylate. These solutions mostly contain 1 % of celloidin. Formulas for