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 Methods of research in microscopical ana 
 
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Methods of Research 
 
 Microscopical Anatomy 
 
 EMBRYOLOGY 
 
 BY o 
 
 CHARLES OTIS WHITMAN, MA., Ph.D. 
 
 ILLUSTRATED 
 
 S. E. CASSINO AND COMPANY 
 
 1885 
 
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PEE FACE. 
 
 The aim of this work is to supply, in a measure, a need 
 "which has been created by the rapid development of the 
 methods of microscopical anatomy and embryology within 
 the last few years. No effort has been made to gvre it 
 an encyclopsedic character, or to render it an exhaus- 
 tive treatise of the subject in any of its aspects. 
 With avalanches of methods constantly sweeping down 
 upon us, it would be as futile as it is undesirable to 
 attempt to enclose the whole field. We are already richly 
 supplied with books on the microscope, which deal with 
 its history, optical principles, forms of construction, acces- 
 sories, modes of use, etc. ; and histological descriptions 
 are furnished in numerous systematic works. The impor- 
 tant branch of methods represented by microphotography 
 has also been handled in special works. 
 
 Pre-occupation of the ground in these directions is not, 
 however, the only reason for the limitations imposed on 
 this volume. Microscopical technique is no more closely 
 connected with histological than embryological studies ; 
 and the practical considerations which would certainly 
 make it advisable not to encumber an embryological trea- 
 tise with a general survey of methods appear to me to hold 
 equally in the case of histological text-books. As the em- 
 bryologist and histologist employ, for the most part, 
 
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IV PREFACE. 
 
 identical methods and instruments, separate treatment of 
 these is obviously an advantage which may be shared by 
 both. Hitherto, most of our standard books of reference 
 on methods have been rather complex in character, deal- 
 ing with the microscope and technical methods as subor- 
 dinate and introductory to the main subject of histology. 
 Works of this description, however invaluable as histo- 
 logical manuals, are not well adapted to the every-day 
 needs of the zoological laboratory. These needs may be 
 summed up in two words, namely, material aud methods. 
 Under the head of material may be included, not only 
 the object of study, but also the times, places, and best 
 means of collecting, as well as breeding habits, food, etc. ; 
 in short, all the items of information that would aid the 
 student in making a choice of material and in controlling 
 its supply. The requirements of the histologist in this 
 direction have been bountifully provided for ; while those 
 of the embryologist have been almost wholly ignored. 
 The embryologist is not, to be sure, wholly destitute of 
 sources of information; but these sources are scattered 
 and inaccessible, and the information they offer is, for the 
 most part, too meagre to be of great assistance. Embry- 
 ologists are themselves, in a large measure, responsible for 
 the inadequate supply of this kind of information ; for it 
 is only quite recently that the matter has received any 
 proper attention at their hands, and even now it is often 
 treated with the greatest indifference. In the course of 
 any complete and thorough study of the development of 
 an animal, the investigator acquires a fund of information 
 on some or all of the points above named, which is not, as 
 a rule, to be obtained from systematic works, and which 
 may be of the highest value to the observer who next 
 takes up the same subject. Neglect to give such informa- 
 tion is, at the best, inexcusable ; and to withhold it on the 
 ground that others are not entitled to the advantages of 
 your experience is nothing less than contemptible. 
 
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PREFACE. V 
 
 The importance which I attach to this point is not 
 to be measured by the space devoted to it in the 
 following pages ; for the time which I could give to 
 it has been from necessity very limited, so that I have 
 scarcely done more than indicate what should be done 
 on a much larger scale. Should the plan commend 
 itself to the judgment of others, it may be more fully 
 developed hereafter. 
 
 This class of facts forms a part of the embryological 
 methods, embracing some notes on the occurrence of eggs 
 appended thereto. 
 
 The contents of this volume have been arranged in two 
 parts, the first embracing methods of a more general 
 nature, such as preservative fluids, dyes, macerating fluids, 
 fixatives, mounting media, the microtome with its appurte- 
 nances, methods of imbedding, etc. ; the second including 
 special applications of embryological, anatomical, and his- 
 tological methods. In an appendix will be found some 
 methods of injection, museum methods, and formulae for 
 most of the important reagents, etc. 
 
 The introduction of special methods may perhaps 
 be regarded as a not unimportant feature of the pres- 
 ent volume. This part is designed to meet the wants 
 of the beginner, as well as the more advanced student ; 
 and, under the persuasion that it will be found useful, I 
 have only to regret my present inability to make it much 
 more complete. 
 
 Sources of indebtedness have been acknowledged in the 
 text; but it should be mentioned that the matter here 
 presented has been drawn, to a large extent, from summa- 
 ries and notes that have appeared in the American Natur- j/ 
 alist during the past three years, and from a paper on the 
 methods used at the Naples Zoological Station, for the • 
 substance of which I was so largely indebted to Paul 
 Mayer. This material has, however, been entirely rear- 
 ranged, and additions and alterations have been made in 
 
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VI PREFACE. 
 
 all directions, in order to bring it up to the latest improve- 
 ments and discoveries. 
 
 In this work of revising and supplementing, I have been 
 materially aided by the excellent and comprehensive sum- 
 maries which appear in every volume of the Naples " Zo- 
 
 b/ologischer Jahresbericht," from the pen of Max Flesch of 
 Wiirzburg. In a similar manner, I have made frequent 
 use of the " Journal of the Royal Microscopical Society," 
 edited by Frank Crisp of London, and the new " Zeit- 
 ,/schrift fur wissenschaftliche Mikroskopie," published by 
 Wilh. Jul. Behrens in Gottingen. 
 
 , Fol's Lehrbuch der Vergleichenden Mikroskopischen 
 Anatomie (erste Lieferung) has also been often consulted, 
 as will appear from numerous references. 
 
 I am under obligation to Paul Mayer, Ryder, Kingsley, 
 Minot, Mark, Faxon, Osborne, and Wilson for matter 
 which appears for the first time in these pages, and to the 
 American Naturalist for most of the cuts. To Mr. Kingsley 
 I am especially indebted for correcting the proof-sheets, 
 and for many suggestions that have proved very valuable. 
 
 C. O. Whitman. 
 
 Museum of Compabative Zoology, 
 Cambridge, 1885. 
 
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CONTENTS. 
 
 PAKT I.— GENERAL METHODS. 
 
 PAGE 
 
 INTRODUCTION 1 
 
 Chapteb I. — Methods op Killing, Hardening, Pbesebving, 
 Bleaching, Macerating, Decalcifying, 
 
 and desilicifting 11 
 
 II. — Methods of Staining 33 
 
 III. — Instruments 60 
 
 IV. — Methods of Imbedding 93 
 
 V.— Fixatives foe Serial Sections 118 
 
 VI. — Mounting Media 124 
 
 VII. —The Uses of Collodion 126 
 
 PART n. — SPECIAL METHODS. 
 
 Chapter VIII. — Embbyological Methods 129 
 
 IX. — Times and Places of Ovulation . . . . 174 
 vii 
 
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Vlll CONTENTS. 
 
 Chapter X. — Karyokinetic Figures 181 
 
 XI. — Preparation of Nerve-Tissue 187 
 
 XII. — Histological Methods 200 
 
 XIII. —Reconstruction from Sections 213 
 
 APPENDIX. 
 
 1. Methods of Injection 219 
 
 2. Museum Methods 231 
 
 3. Formula foe Reagents, Dyes, etc 237 
 
 INDEX 249 
 
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Part I. 
 GENERAL METHODS. 
 
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MICROSCOPICAL METHODS. 
 
 INTRODUCTION. 
 
 In beginning the study of any branch of natural history, the 
 student of to-day finds that the first step in preparation for 
 independent investigation is to become familiar, through actual 
 laboratory work and under the guidance of experienced in- 
 structors, with the more important instruments and methods of 
 microscopical research. This is especially time in every de- 
 partment of zoology. Even in systematic zoology, it cannot be 
 neglected with impunity. Macroscopical and microscopical 
 methods of study are independent of each other only to a very 
 limited degree ; they have a hand-in-hand relation in every 
 thorough and complete morphological study, sometimes part- 
 ing company it is true, but only to supplement and strengthen 
 each other in the end. The controversies on the question of 
 using chemical reagents in the investigation of animal or vege- 
 table tissue have ceased to have more than a historical inter- 
 est. Karyokinetic figures are no longer regarded with sus- 
 picion, and the " Trugbilder " of metallic impregnations are now 
 universally approved as reliable pictures of conditions that 
 actually exist in living tissues. Methods of study are now cul- 
 tivated with as much zeal by the embryologist as the histolo- 
 gist, and the anatomist and the physiologist feels compelled to 
 supplement their methods by those of the ' plodding microto- 
 mist.' Microscopical methods are not then the monopoly of 
 any particular class of scientific investigators. 
 
 The methods of microscopical research now employed are, to 
 a very large extent, the product of the experience of the ablest 
 
 1 
 
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Z MICROSCOPICAL METHODS. 
 
 investigators in various departments of biology, during the last 
 ten or fifteen years. So rapid has been the progress in this di- 
 rection, that it has been found necessary to supplement our 
 hand-books of methods through the publication of journals 
 devoted exclusively to the work of recording and summarizing 
 new developments in zoological technique. 
 
 The rapid development of methods is at once the result and 
 one of the chief causes of the increasing activity in every field 
 of biological research. The improvement of methods leads to 
 the re-investigation of old subjects, and at the same time pre- 
 pares the way for attacking new problems. The investigator 
 who neglects to keep himself informed of the progress in meth- 
 ods of study throws away his opportunities, and has the vexa- 
 tious mortification of seeing himself outdone and his work 
 superseded by that of more skilful hands. 
 
 The microtome has come to occupy a place in the zoological 
 laboratory second in importance only to the microscope itself. 
 Many improvements in details and in accessories have followed 
 the introduction of this instrument, and a whole series of meth- 
 ods has sprung up in connection with its use. In short we 
 have a new art, which has been appropriately called microtomy. 
 
 The general favor with which the microtome has been re- 
 ceived is the best evidence of its usefulness. The use of the 
 instrument is so simple, and the methods connected with it so 
 easily acquired, that no naturalist can afford to work without it. 
 
 Within the last four or five years the improvements and dis- 
 coveries in microtomy have been both numerous and extremely 
 important. Among these may be mentioned the ribbon method 
 of cutting serial sections, discovered by Caldwell; the methods 
 of fixing sections on the object-slide, discovered by Giesbrecht, 
 Schallibaum and Mayer ; the various section-smoothers, notably 
 those invented by Mayer, Andres, Giesbrecht, Schulze and 
 Decker; the use of collodion to prevent the crumbling of 
 brittle sections, proposed by Mason; and the methods of re- 
 constructing objects from serial sections employed by His and 
 Born. A large number of new preservative and staining fluids 
 have been described ; and new methods of killing, hardening, 
 preserving, staining, and imbedding have been recommended. 
 
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INTRODUCTION. 3 
 
 Experience has shown that each different object requires a 
 special mode of treatment, and that the same object must be 
 treated differently according to the nature of the problem in 
 hand. For example, the course of preparation which has given 
 satisfactory results in the study of the development of the ova 
 of a certain species may prove quite inadequate when applied 
 to a different though closely allied form ; and it has been 
 found that different stages in the development of the same 
 ovum often require different modes of preservation. The in- 
 vestigator cannot, therefore, blindly adopt the methods em- 
 ployed by others, bat must, in by far the greater number of 
 cases, determine by experiment the method to be pursued. 
 But such experiments demand a general knowledge of methods, 
 and, above all, a knowledge of the special applications of meth- 
 ods in cognate subjects. It is in the adaptation of methods to 
 special subjects that the skill of the investigator is shown. Our 
 information of the methods employed in specific cases should 
 therefore be as extended as possible. To meet this need, entire 
 courses of methods that have led to successful results in typical 
 cases have received attention in the second part of this volume. 
 
 A few explanatory remarks and general directions may ena- 
 ble the beginner to enter upon an experimental study of meth- 
 ods with his attention at least properly oriented. 
 
 From a general standpoint, we may say that there are two 
 modes of studying an animal, an organ, a tissue, or a cell, 
 which we may distinguish as surface-observation and section- 
 observation. In surface-observation, the object may be exam- 
 ined in a living (or fresh) condition, or after treatment with 
 preservative fluids ; in section-observation — leaving the freez- 
 ing methods aside — reagents must be employed, and the vari- 
 ous steps in the preparation must of course follow some definite 
 order or sequence. After learning this sequence in a single 
 case and mastering the principles underlying it, the beginner 
 will be prepared to proceed intelligently with other cases. An 
 illustration will put this matter in a clearer light. 
 
 A small worm is a suitable and convenient object of study 
 by means of serial sections. After making the preliminary 
 
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4 MICROSCOPICAL METHODS. 
 
 Surface examination, with a view to ascertaining the external 
 features and their precise topographical relations, the prepa- 
 ration for sectioning may be undertaken. In the usual order 
 of procedure, the several steps are, (1) hilling, (2) hardening, 
 (3) staining, (4) dehydration, (5) clarification, (6) saturation 
 with paraffine, (7) imbedding, (8) sectioning, (9) arrangement 
 and fixation on the slide, and (10) inclusion in balsam. 
 
 The first two steps are often accomplished by a single 
 reagent; and sometimes the third is combined with the first 
 two, either by using a reagent (e. g. osmic acid) that will do 
 three kinds of work at the same time, or by mixing the staining 
 fluid with the killing and hardening medium (Perenyi's fluid). 
 The third step may also be suppressed, the sequence otherwise 
 remaining the same ; or it may be transposed so as to follow 
 the ninth, in which case the fourth and fifth would have to be 
 repeated before concluding with the tenth. Excepting the 
 simple modifications by combination, suppression, and trans- 
 position, here mentioned, the above sequence may be said to be 
 invariable. The real field for experimentation then lies, not in 
 the order of the steps, but in the mode of executing them : 
 and, beyond the first three steps, the method of procedure is 
 so free from complications that the beginner will need very 
 little assistance in following the course prescribed. 
 
 With so much of a general nature premised, we may take 
 up the consideration of the first three steps of the preparation 
 in their usual order, noticing, as we pass, a few of the more 
 important ways of varying the course, and calling attention to 
 such points as will tend to lessen the chances of aimless experi- 
 mentation. 
 
 1. Killing. The all-important point here is to preserve the 
 natural form in an extended condition, with as little alteration 
 of the histological elements as possible. This can usually be 
 accomplished in one of two general ways : — first, by immer- 
 sion in a fluid that kills instantly ; second, by using one that 
 operates so slowly that sensibility is lost very gradually. In 
 some cases either method may be employed with about equal 
 success, while in others the advantages may be largely or 
 wholly on one side. In order to make an intelligent choice 
 
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INTRODUCTION. 
 
 among the various agents employed for killing, it is necessary 
 to know something of their peculiarities, and then to select 
 with reference to the character and size of the animal, and the 
 special purpose in view. 
 
 If the worm be a Planarian, the method of killing instantane- 
 ously with corrosive sublimate would probably give the most 
 satisfactory results. The same method could be employed with 
 most annelids ; but here our preference might take a some- 
 what wider range, extending to one or more of the following 
 agents, — alcohol added slowly as recommended by Eisig, Mer- 
 kel's chrom-platinum solution, chromic acid, or Kleinenberg's 
 picro-sulphuric acid. These five methods are by no means all 
 that might be resorted to ; but the cases in which the desired 
 results could not be obtained through one or more of them, are 
 too exceptional to call for notice here. 
 
 Corrosive sublimate kills and hardens at the same time, and 
 accomplishes its work in a few minutes. It kills in most cases 
 so quickly that the animal has no time to contract; and it 
 leaves the tissues receptive to staining fluids. The moment the 
 solution has thoroughly penetrated all parts (10-30 m.), its 
 action should be interrupted by washing in distilled water until 
 the sublimate has been entirely removed (30-60 m.). If the 
 washing is not thorough, needle-like crystals will form and im- 
 pair or ruin the preparation. 
 
 Alcohol added by drops is a somewhat slower process, re- 
 quiring very close watching. The chief advantage of this 
 method is that it leaves no obstacles in the way of staining. 
 The dangers to be carefully guarded against are, (1) contrac- 
 tion, in consequence of a too rapid addition of alcohol, and 
 (2) maceration, resulting from an undue prolongation of the 
 process. As soon as the animal is dead, it should be placed for 
 a short time (30 m.) in 40 % alcohol, then in 50 %, 60 %, 70 %, 
 80 %, 90 %, doubling the time at each step. 
 
 Picro-sulphuric acid has the admirable quality of causing 
 little or no shrinkage, and of being easily removed in moder- 
 ately strong alcohol ; but in most cases direct immersion in this 
 fluid would kill the worm in a contracted condition. The dan- 
 ger in this direction can only be met by a preliminary treat- 
 
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6 MICROSCOPICAL METHODS. 
 
 ment with some agent that temporarily or permanently deprives 
 the worm of the power of contraction. Chloroform would 
 serve, this purpose in many, though not all cases. 
 
 Chromic acid is a convenient and much used reagent for kill- 
 ing and hardening. If a worm be placed in a weak solution, 
 it may show vigorous contractions for a considerable time, 
 but in the end it generally dies in an extended condition. 
 If it requires to be straightened, this should be done im- 
 mediately, before the hardening process has rendered it in- 
 flexible. 
 
 The great drawback in the use of chromic acid is the diffi- 
 culty of staining after it. This obstacle cannot be wholly 
 overcome, but it can be lessened considerably by interrupting 
 the action of the acid soon after the animal is completely dead. 
 This reagent hardens by virtue of chemical combinations which 
 it forms with cell substances, and it is this peculiar hardening 
 action which renders the tissues non-receptive to staining fluids. 
 The sooner, therefore, this action is interrupted by washing, the 
 easier will be the work of staining. It is advisable to use a 
 weak solution (not over £ %) and to limit its action so far 
 as possible to killing, leaving the hardening to be accomplished 
 by alcohol of 40 %, 50 %, 70 %, and 80-90 %. 
 
 Merkel's fluid is chromic acid tempered somewhat in its 
 action by platinum chloride. As a killing agent it is superior 
 to chromic acid, but as a hardening agent it has the same ob- 
 jectional feature. The suggestion made above in regard to 
 limiting the action of chromic acid, and leaving the work of 
 hardening to alcohol, holds with equal force and for similar 
 reasons in the case of Merkel's fluid. 
 
 2. Hardening. The work of hardening, after each of the fore- 
 going methods of killing, will fall mainly or wholly to alcohol. 
 The chief danger here lies in shrinkage, to avoid which it is 
 necessary to use different grades of alcohol, advancing gradu- 
 ally to 80 % or 90 % . It is generally safe to begin with 40 % or 
 50 %, and sometimes only two grades (70 % and 90 %) are re- 
 quired. The strength of the first grade, as well as the number of 
 grades to be interposed between this and 90 %, will depend 
 on the method of killing and the delicacy of the object. 
 
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INTRODUCTION. 7 
 
 The use of ascending grades of alcohol to avoid the shrink- 
 age that would follow direct transference to 90 %, is a ne- 
 cessity which finds its explanation in the attractive power of 
 alcohol for water. On exposure to alcohol the water of the 
 tissues is withdrawn more rapidly than it is replaced by alco- 
 hol ; and the stronger the alcohol the more unequal the diffu- 
 sion. The aim, then, in beginning with weak alcohol and ad- 
 vancing gradually, is to tone down the violence of the osmotic 
 action, and thus lessen the danger of collapse. 
 
 In the use of alcohols below 60 °/c, considerable care must 
 be taken to guard against maceration : this danger must be 
 avoided by transferring as rapidly as possible through these 
 grades. 
 
 It is impossible to give precise and infallible directions for 
 accomplishing this step, for no inflexible system of rules can be 
 devised to meet all the possible emergencies. The following 
 indications of time and strength are therefore to be regarded as 
 mere approximations, having very little value beyond the aid 
 they may give the experimenter in making his first trials. 
 
 Assuming that the object is not more than a few centimeters 
 long, and from two to four millimeters thick, the corrosive subli- 
 mate preparation, after being washed for an hour in water sup- 
 plied in abundance and four or five times renewed, may be 
 transferred to alcohol of 50 % one hour, 60 % two hours, 70 % 
 twelve to twenty-four hours, 80-90 % twenty-four hours. 
 
 The picro-sulphuric acid preparation, after remaining from 
 one to two hours (till completely penetrated) in the acid, may 
 be transferred directly, without washing, to 70 % alcohol for 
 twenty-four hours, and the alcohol changed four or five times 
 (until every trace of the yellow color is removed from the 
 object) ; then to 90 % for twenty-four hours or longer. 
 
 The preparations in chromic acid and Merkel's fluid, after an 
 immersion of one-half to one hour, should be thoroughly washed 
 (best in running water) for about the same time, then placed in 
 40 % alcohol fifteen minutes, 50 fo thirty minutes, 60 % one 
 hour, 70 % five hours, and 80 % ten to twelve hours. 
 
 The treatment of the alcoholic preparation has already been 
 indicated. 
 
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8 MICROSCOPICAL METHODS. 
 
 3. Staining. The work of staining will now be in order, if 
 the object admits of being stained as a whole. The postpone- 
 ment of this step till the sections have been made and fixed on 
 the slide may be considered as an alternative, to be resorted to 
 in cases of necessity, i. e., when staining in toto is found to be 
 ineffectual. If the worm is three or four centimeters in length, 
 it will facilitate the staining considerably to divide it into two 
 or three parts ; and such a division will be found to be an ad- 
 vantage in the further preparation, as well as a necessary pre- 
 liminary to imbedding. 
 
 As to the kind of dyes to be employed, it may be said that 
 alcoholic solutions, such as Grenadier's borax-carmine, Klein- 
 enberg's hsematoxylin, &c, are, as a rule, to be preferred to 
 aqueous solutions, such as Beale's carmine, picro-carmine, &c. 
 The grounds for this preference will be discussed under the 
 head of staining fluids, and need not therefore occupy us here. 
 
 Among the more important qualifications of a dye for stain- 
 ing in toto are the following : — 
 
 1. It should penetrate readily, so that its action on the 
 deeper and more superficial parts may be as equable as pos- 
 sible. 
 
 2. Its action, at full strength, should be intense — capable 
 of imparting any desired dejith of color. 
 
 3. It should give a differential rather than a diffuse stain- 
 ing ; i. e., it should act with unequal degrees of intensity on 
 the different elements of the cell, thus contributing to sharp- 
 ness of definition. 
 
 4. Its preservative qualities should be sufficiently pro- 
 nounced to enable it to act any required length of time with- 
 out causing maceration. 
 
 5. The color imparted should be permanent. 
 
 All these favorable qualities are combined in a very high de- 
 gree in Kleinenberg's hsematoxylin ; and, to a somewhat less 
 extent, in borax-carmine. In richness and transparency of tint 
 the carmine solution has the advantage ; but in point of pene- 
 trancy and freedom from danger of maceration, haamatoxylin 
 takes the higher rank. In using these dyes it must be remem- 
 bered that the hardened objects should not be transferred di- 
 
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INTRODUCTION. 9 
 
 rectly from 90 % alcohol, but should first be left for half an 
 hour or more in alcohol of the same strength as the coloring 
 solution. It should also be borne in mind that every trace of 
 acid must be removed before immersing the object in hematox- 
 ylin, for the color will inevitably fade if any free acid is left in 
 the tissues. 
 
 The preparations in sublimate, picro-sulphuric acid, and alco- 
 hol, will require only a few hours for staining, and hence the 
 choice of dyes, assuming that it is limited to these two, may be 
 made according to preference for color. The preparations in 
 chromic-acid and Merkel's fluid will stain with greater diffi- 
 culty ; but if the time required does not much exceed twenty- 
 four hours, borax-carmine can be used with perfect safety, 
 provided the hardening has been successfully accomplished, 
 The haematoxylin solution will stain quicker than the carmine, 
 and in many cases would undoubtedly give the more satisfac- 
 tory results. 
 
 No precise time-limits can be prescribed for the action of the 
 coloring fluid : the preparations must be examined from time to 
 time, and removed from the dye when they appear to be thor- 
 oughly and deeply stained. They should be washed in alcohol 
 of the same grade as the dye, and then dehydrated by treat- 
 ment with 70 fo, 90 %, and absolute alcohol. 
 
 The differential character of the stain may be strengthened 
 by washing a few minutes (before dehydrating) in slightly acid- 
 ulated alcohol. 
 
 It has already been mentioned that the difficulty of staining 
 chromic acid preparations may be lessened by cutting short the 
 action of the acid as soon as its penetration to all parts is 
 assured. There is still another precaution, the observance of 
 which will tend to reduce the evil to a minimum. The stain- 
 ing should follow at the earliest possible moment after the action 
 of the acid ; for the difficulty of staining increases with the 
 lapse of time. The second step of hardening should therefore 
 be shortened rather than prolonged, so that the first and third 
 steps may not be separated by a longer interval than necessary. 
 
 The remaining steps of the preparation present no complica- 
 tions that require to be considered here. The directions for 
 
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10 MICROSCOPICAL METHODS. 
 
 these in the following pages will, it is presumed, be found suffi- 
 ciently full to enable the beginner to proceed independently. 
 
 Microtomic methods, it should be remembered, form only a 
 part of the methods employed in the study of animal morpho- 
 logy. That they constitute a very important part is certain ; 
 but it is equally certain that surface-observation must not be 
 neglected. Cultivation of either class of methods to the exclusion 
 of the other accounts for the unsatisfactory results of many an 
 investigation. Over-confidence in section-making may prove as 
 disastrous as total ignorance of it. It would be easy to cite ex- 
 amples on both sides. Surface-observation and section-observa- 
 tion go hand in hand, and the highest advantages of either are 
 known only to those who know how to practise both. The 
 work of the microtome is supplementary in character ; its func- 
 tion is to verify and correct, to strengthen the weak points and 
 supply the deficiencies of surface-observation. 
 
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CHAPTEE I. 
 
 PRESERVATIVE FLUIDS. 
 
 Killing, hardening, and preserving are three kinds of 
 work, requiring for their accomplishment sometimes only a single 
 preservative fluid, but in most cases two, three, or even more. 
 As the same fluid often does the work of killing and hardening, 
 and sometimes of preserving too, it is impossible to divide them 
 into three classes corresponding to the kinds of work, except 
 by repeating many of them twice, and some of them three 
 times. While it is therefore more convenient to include them 
 all under " preservative fluids," as Dr. Mayer 1 has done, it is 
 none the less important to remember what kind of work each 
 fluid is expected to accomplish. 
 
 Kleinenberg's picro-sulphuric acid, for instance, is not a hard- 
 ening fluid. It serves for killing, and thus prepares for subse- 
 quent hardening. 
 
 HOT WATER. 
 
 Water heated to about 80° C, as first recommended by Bo- 
 bretzky, is an excellent mode of killing some kinds of eggs, 
 especially those of many Arthropoda. This method has also 
 been employed to advantage with the amphibian egg. 
 
 ALCOHOL. 
 In the preparation of animals or parts of animals for mu- 
 seums or histological study, it is well known that the chief 
 
 1 " Mittheilungen aus der Zoologischen Station zu Neapel," Vol. 2, p. 1, i/ 
 1880. 
 
 11 
 
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12 MICROSCOPICAL METHODS. 
 
 difficulties are met in the process of killing. Alcohol, as com- 
 monly used for this purpose by collectors, has little more than 
 its convenience to recommend it. P. Mayer has called atten- 
 tion to the following disadvantages attending its use in the case 
 of marine animals : — 
 
 (1) In thick-walled animals, particularly those provided 
 with chitinous envelopes, alcohol causes a more or less strong 
 maceration of the internal parts, which often ends in putrefac- 
 tion. 
 
 (2) In the case of smaller Crustacea, e. g., Amphipods and 
 Isopods, it gives rise to precipitates in the body fluids, and thus 
 solders the organs together in such a manner as often to defy 
 separation, even by experienced hands. 
 
 (3) It fixes most of the salts of the water adhering to the sur- 
 face of marine animals, and thus a crust is formed which pre- 
 vents the penetration of the fluid to the interior. 1 
 
 (4) This crust also prevents the action of staining fluids, 
 except aqueous solutions, by which it would be dissolved. 
 
 Notwithstanding these drawbacks, alcohol is still regarded at 
 the Naples Station as an excellent fluid for killing many ani- 
 mals designed for preservation in museums or for histological 
 work. In many cases the unsatisfactory results obtained are to 
 be attributed, not to the alcohol joer se, but to the method of 
 using it. Most of the foregoing objections do not, as Dr. Mayer 
 has expressly stated, apply to fresh-water animals; and Dr. 
 Eisig informs me that he has no better method of killing marine 
 annelids than with alcohol. 
 
 Eisig's Method of Killing with Alcohol — The process 
 is extremely simple. A few drops of alcohol are put into a vessel 
 which contains the annelid in its native element, the sea-water; 
 this is repeated at short intervals until death ensues. After the 
 animal has been thus slowly killed, it may be passed through 
 
 1 Dr. Mayer first noticed this in objects stained with Kleinenberg's 
 hematoxylin, and afterwards in the use of cochineal, where a gray-green 
 precipitate is sometimes produced which renders the preparation worth- 
 less. Such results may be avoided by first soaking the objects a few 
 hours in acid alcohol (1-10 parts hydrochloric acid to 100 parts 70 % 
 alcohol). 
 
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REAGENTS. 13 
 
 the different grades of alcohol in the ordinary way, or through 
 other preservative fluids. Objects killed in this manner show- 
 no trace of the external crust of precipitates which arises where 
 stronger grades of alcohol are first used. The action of the 
 alcohol is thus moderated, and the animal, dying slowly, 
 remains extended and in such a supple condition that it can 
 easily be placed in any desired position. The violent shock 
 given to animals when thrown alive into alcohol of 40 % to 60 
 %, giving rise to wrinkles, folds, and distortions of every kind 
 is thus avoided, together with its bad effects. 
 
 Acid Alcohol. — In order to avoid the bad effects of alcohol, 
 — such as precipitates, maceration, etc., — Mayer recommends 
 acid alcohol, — 
 
 95 volumes 70 % or 90 % alcohol. 
 3 " hydrochloric acid, 1 — 
 
 for larger objects, particularly if they are designed for preser- 
 vation in museums. The fluid should be frequently shaken up, 
 and the object only allowed to remain until thoroughly satu- 
 rated, then transferred to pure 70 % or 90 % alcohol, which 
 should be changed a few times in order to remove all traces 
 of the acid. For small and tender objects, acid alcohol, 
 although preferable to pure alcohol, gives less satisfactory 
 results than picro-sulphuric acid. 
 
 Boiling Alcohol. — In some cajses among the Arthropods, 
 Mayer has found it difficult to kill immediately by any of the 
 ordinary means, and for such cases recommends boiling absolute 
 alcohol, which kills instantly. For Tracheata this is often the 
 only means by which the dermal tissues can be well preserved, 
 as cold alcohol penetrates too slowly. 
 Glycerine and Alcohol. — 
 
 Glycerine 20 parts 
 
 Alcohol (70%) 40 " 
 
 Sea water 40 " 
 
 This mixture, poured very slowly into the containing glass, 
 often gives very good results, both for anatomical and histologi- 
 
 1 Acid alcohol, as above prepared, loses its original qualities after stand- 
 ing so'me time, as ether compounds are gradually formed at the expense 
 of the acid. 
 
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14 MICROSCOPICAL METHODS. 
 
 cal purposes. This method originated with Salvatore Lo Bianco 
 the conservator of the Naples Zoological Station. 
 
 Method of Making Absolute Alcohol. — According 
 to Sharp, absolute alcohol is pre] tared in Ranvier's laboratory 
 by adding anhydrous cupric sulphate to ninety-five % alcohol. 
 Pulverized cupric sulphate is heated to red heat, in order to drive 
 off the water of crystallization ; when cool, the white powder is 
 placed in a wide-mouthed bottle, holding about a litre, and three- 
 fourths full of alcohol. The bottle is quickly closed, and the 
 whole shaken. After standing a day or more — with occasional 
 shakings, — it is decanted, and the operation repeated, especially 
 if the cupric sulphate shows much of the blue color due to the 
 reassumption of water. As a test, a drop of the alcohol thus 
 dehydrated may be mixed with a drop of turpentine on a glass 
 slide, and examined under the microscope ; if no particles of 
 water are to be seen, the alcohol is absolute enough for all 
 practical purposes. 
 
 IODINE. 
 
 Iodine, followed by a little strong sulphuric acid, is used as a 
 test for starch and cellulose. In combination with alcohol it 
 has often been used for the central nervous system of the higher 
 vertebrates. It may be strongly recommended in the investi- 
 gation of many animals, especially transparent Arthropods, 
 where the preparations are required for immediate study. The 
 following solution is recommended by Fol : — 
 
 To a saturated aqueous solution of iodide of potassium, add 
 as much iodine as will dissolve. This gives a dark-brown fluid, 
 which, according to the end in view, may be diluted with 100- 
 500 parts of water. 
 
 Berthold 1 experimented with iodine and other reagents on 
 delicate marine alga?, and came to results that are of impor- 
 tance to zoologists as well as botanists. The aim was to find 
 solutions that would produce the least possible disturbance in 
 the structure of the cell-pi-otoplasm. It was found that sat- 
 isfactory results could not be obtained with the ordinary 
 
 1 Pringsheim's Jahrbiicher f iir Wissenscliaf tliche Botanik. Vol. XIII. 
 pp. 704^5, 1882. 
 
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REAGENTS. 15 
 
 aqueous solutions of picric acid, osmic acid, etc. The distur- 
 bance of the osmotic equilibrium, on transferring delicate 
 cells from sea-water to fresh-water solutions, resulted in 
 intra-cellular derangements. Parallel trials "were therefore 
 made of picric acid, osmic acid, and iodine, three different 
 solutions of each being made; one in distilled water, one in 
 alcohol, and another in sea-water. The solutions in distilled 
 water and alcohol proved almost worthless in each case, while 
 each of the solutions in sea-water gave good results. It was 
 found, curiously enough, that the protoplasm of the cells was 
 more easily injured than the nuclei and karyokinetic figures. 
 
 Solutions of osmic acid and corrosive sublimate in sea-water 
 gave good preparations, but the iodine solution was regarded 
 as the best reagent. A few drops of a saturated alcoholic solu- 
 tion of iodine, added to the sea-water, gives the desired results. 
 The algas remain in the solution one half to one minute, and 
 are then transferred directly to 50 % alcohol. 
 
 OSMIC ACID. 
 
 Osmic acid, one of the most important histological reagents, 
 comes in small closed glass tubes, holding a grani each. It 
 is generally used in very weak solutions (^-1 %). It is best 
 to prepare a 1 % solution by breaking the tube into a bot- 
 tle containing lOOcc. distilled water. The containing bottle 
 should be double-stoppered (stopper and cap), and should be 
 protected from the light, either by painting black or by 
 inclosure in a cylindrical pasteboard box. From this 1 % 
 solution any desired weaker solution can readily be made. 
 The action of this acid depends on its easy reduction by organic 
 substances. 
 
 Objects may be treated in two ways, — either by immersion 
 in the solution itself, or by exposure to its vapor. In the 
 latter case, the object, with little or no water about it, is 
 placed under a bell-jar near a watch-glass containing a small 
 quantity of a strong solution of the acid. The solution does 
 not penetrate more than the superficial portions of large ob- 
 jects ; while the vapor reaches even the deepest parts, and pro- 
 duces a very uniform effect. 
 
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16 MICROSCOPICAL METHODS. 
 
 The time of exposure to the action of the acid should be 
 relatively short (from one to thirty minutes). The blackening 
 of the object may be checked, and removed to a considerable 
 extent, by following the osmic acid with Merkel's fluid or 
 chromic acid. 
 
 Mayer employs osmic acid as a staining medium for the 
 hairs, bristles, etc., of the dermal skeleton of Arthropods. The 
 lustre of Sapphirina is preserved by this acid, and, according to 
 Emery, the color of the red and the yellow fatty pigments of 
 fishes. 
 
 Van Beneden found osmic acid the best preservative fluid for 
 the Dicyemidse, and my experience leads to the same conclu- 
 sion. 1 
 
 Bleaching. — It often happens that objects treated with 
 osmic acid continue to blacken, after removal from the acid, 
 until they are entirely worthless; and such results are even 
 more annoying than the difficulties in the way of staining. It 
 has been said that the blackening process can be arrested by 
 certain staining media ; but it is certain that picro-carmine will 
 not always do this, as some of my preparations of Dicyemidas 
 show. It is therefore a very important step which Dr. Mayer 
 has taken in finding a method of restoring such objects. The 
 method 2 is as follows : — 
 
 The objects are placed in 70 or 90 % alcohol, and crystals of 
 potassic chlorate (KC10 3 ) shaken into the liquid until the bot- 
 tom of the vessel is covered ; then a few drops of concentrated 
 hydrochloric acid 8 are added with a pipette, and as soon 
 as chlorine (easily recognized by its greenish-yellow color) 
 begins to be liberated, the whole gently shaken. As soon 
 as the bleaching is finished, the objects are removed to pure 
 alcohol. 
 
 1 One of the best objects for testing methods is found in Phronima 
 sedentaria. Here the cells and nuclei are so sharply defined that they 
 can be seen in the living animal, and so the effect of a preservative fluid 
 can be easily studied. 
 
 2 A slightly modified form of the method originally given in Mull. 
 Arch., 1874, p. 321. 
 
 8 Nitric acid may be used instead of hydrochloric. 
 
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REAGENTS. 17 
 
 By this method it is possible in half a day to restore large 
 Pelagia, Carinaria, Rhizostoma, etc. Small objects generally 
 require a shorter time and less acid. The process can be greatly 
 accelerated by heating on a water-bath. 
 
 Using Sapphirina as a test object, Mayer found that the 
 lustre which characterizes the living animal entirely disappeared 
 by the bleaching process. As this lustre, which has its seat in 
 the epidermis, depends on the interference of light, it is evident 
 that the cells had undergone some change, but a change so 
 slight that the tissues could hardly be said to have been injured 
 for histological purposes; besides, the removal of the osmic 
 acid leaves the animal in a good condition for staining. 
 
 Mayer's experience with Sapphirina appears to support him 
 in the following conclusions in regard to the nature of the 
 action of osmic acid ; viz., that the hardening effect of the acid 
 is due to the formation of inorganic precipitates within the 
 tissues. This is made evident by the fact that the animal be- 
 comes soft and flexible as soon as these precipitates are removed 
 by bleaching. 
 
 This method of bleaching has been used by Mayer for remov- 
 ing natural pigment. Alcoholic preparations of the eye of 
 Mysis, for instance, can be fully bleached in toto, but with better 
 success by operating with single sections. To avoid swelling, 
 which is apt to arise by the use of aqueous fluids, staining media 
 of an alcoholic nature should be used. 
 
 ACETIC ACID. 
 
 In making solutions of this acid, in all cases where it is im- 
 portant to use a definite strength, it is necessary to use the pure 
 anhydrous form (acidum aceticum glaciale), which crystallizes 
 in large, transparent glistening tables that melt at 16°-17° C. 
 Statements respecting its specific gravity cannot be taken as a 
 safe guide to the amount of water contained. It is generally 
 used in solutions oil % and 2 %. 
 
 Among reagents for animal tissues, this is one of the oldest 
 and best. It is not preservative in its action, and hence is 
 mainly used in investigations where a rapid clearing up of 
 tissues is required, and where temporary preparations alone are 
 
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18 MICROSCOPICAL METHODS. 
 
 sought. It is very useful in studying opaque ova and embryos, 
 and microscopic animals m general. Its usefulness in investi- 
 gating nuclei is well known. 
 
 It has been employed by Kolliker and others in tracing 
 nerves and nerve-terminations, for which purpose Frey recom- 
 mends — 
 
 Glacial acetic acid 1-2 drops 
 
 Water lOOcc. 
 
 The action of this acid is rapid, and often requires close 
 watching, in order to catch the most favorable of the transitory 
 conditions which it induces. Recently it has been much em- 
 ployed in combination with other reagents (Flemming, Fol). 
 
 CHROMIC ACID. 
 
 Chromic acid crystals deliquesce rapidly when exposed to 
 the air, and soon undergo a chemical change. It is therefore 
 important, on opening a bottle of the acid for making a solu- 
 tion, either to dissolve the whole at once, or to close and seal 
 as quickly as possible after taking out the desired quantity. 
 
 Solutions are seldom used above 2 % in strength ; and this 
 is a very convenient stock solution from which to prepare the 
 more common solutions, ranging from i % to 1 % . All chromic 
 solutions are best kept dark, as they undergo changes in the 
 light. 
 
 Chromic solutions have, in common with osmic acid, the 
 peculiarity of hardening by virtue of the chemical combinations 
 which they form with cell-substances, and all the consequent 
 disadvantages with respect to staining. The use of chromic 
 acid in the Zoological Station of Naples may be said to have 
 been largely superseded by picro-sulphuric acid, corrosive sub- 
 limate, and Merkel's fluid, for it is now seldom used except in 
 combination with other fluids. It is sometimes mixed with 
 Kleinenberg's fluid ; for example, when a higher degree of hard- 
 ening is required than can be obtained by the use of the latter 
 fluid alone. It is a common error to use too strong solutions of 
 chromic acid, and to allow them to act too long. Good results 
 are in some cases obtained when the objects are treated with a 
 weak solution (-J to £ %), and removed soon after they are 
 completely dead. 
 
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REAGENTS. 19 
 
 Pfitzner has made use of chromic acid, followed by osmic 
 acid, or by chloride of gold, formic acid, and safranin (or 
 hematoxylin), for the demonstration of nerve-terminations. 
 
 Flemming states that chromic acid is one of the most reliable 
 6xing reagents for the karyokinetic figures, and has proved that 
 objects hardened in this acid can be beautifully and durably 
 stained. 
 
 Chromic acid is much used in combination with other rea- 
 gents (acetic, osmic, picric, nitric acid, etc.). 
 
 The more important of these combinations are seen in 
 Perenyi's fluid ; the chrom-osmium-acetic acid mixture employed 
 by Flemming, and, in a somewhat modified form, by Fol ; the 
 chrom-formic acid used by Rabl in a recent study on karyoki- 
 nesis ; and Merkel's chrom-platinum solution, — recommended 
 by Eisig for annelids, by Brass for Infusoria, and by Whitman 
 for pelagic fish-eggs. 
 
 Dr. H. Virchow calls attention to the fact that the precipi- 
 tates which arise when preparations from any chromic solution 
 (chromic acid, Mliller's fluid, etc.) are placed in alcohol may 
 be wholly avoided by keeping in the dark until the alcohol 
 ceases to become colored. 
 
 NITRIC ACID. 
 
 This is an excellent reagent for many objects. Various 
 strengths have been employed. For chick embryos, His uses 
 10 % ; W. Engelmann found 3-4 % to work well witb the retina 
 of higher animals ; Strasburger and Flemming used 50 % in 
 studying karyokinetic figures; Rabl-Rtickhard used a 10 % 
 solution in tracing the development of the teleostean brain, 
 and Minot finds this solution well adapted for preserving the 
 external form of the brain of the human foetus. 
 
 KLEINENBERG'S PICRO-SULPHURIC ACID.i 
 
 Picric acid (saturated solution in distilled water) . . . 100 volumes 
 Sulphuric acid (concentrated) 2 " 
 
 1 Quart. Journ. Mic. Sci., Vol. XIX., p. 208-9, 1879. 
 
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20 MICROSCOPICAL METHODS. 
 
 Filter the mixture, and dilute it with three times its bulk of 
 water ; 1 finally, add as much creosote as will mix. 2 
 
 Objects are left in the fluid three, four, or more hours ; and 
 are then, in order to harden and remove the acid, transferred 
 to 70 fo alcohol, where they may remain five to six hours. They 
 are next placed in 90 % alcohol, which must be changed at 
 intervals until the yellow tint has wholly disappeared. 
 
 The advantages of this fluid are, that it kills quickly, by 
 taking the place of the water of the tissues ; that it frees the 
 object from sea-water and the salts contained in it ; and that, 
 having done its work, it may be wholly replaced by alcohol. 
 In this latter fact lies the superiority of the fluid over osmic 
 and chromic solutions, all of which produce inorganic precipi- 
 tates, and thus leave the tissues in a condition unfavorable to 
 staining. Picro-sulphuric acid does not, like chromic solutions, 
 harden the object, but simply kills the cells. 
 
 As this fluid penetrates thick chitine with difficulty, it is 
 necessary, in order to obtain good preparations of larger Iso- 
 poda, insects, etc., to cut open the body and fill the body-cavity 
 with the liquid by means of a pipette. In larger objects, care 
 should be taken to loosen the internal organs, so that the fluid 
 may find easy access to all parts. 
 
 The fluid should be applied as soon as the body is opened, so 
 that the blood may not have time to coagulate and thus bind 
 the organs together. A large quantity of the fluid should be 
 used, and it must be changed as often as it becomes turbid. 
 The same rule holds good in the use of all preservative fluids. 
 It is well also, especially with larger objects, to give the fluid 
 an occasional stirring up. 
 
 In order to avoid shrinkage in removing small and tender 
 objects from the acid to the alcohol, it is advisable to take 
 them up by means of a pipette or spatula, so that a few drops 
 
 1 Dr. Mayer uses the fluid undiluted for Arthropoda. 
 
 2 Fol gives a more precise formula, — 
 
 Picric acid 1 g. 
 
 Water 100 cc. 
 
 Sulphuric acid 6 cc. 
 
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KEAGENTS. 21 
 
 of the acid may be transferred along with them. The objects, 
 sinking quickly to the bottom, remain thus for a short time in 
 the medium with which they are saturated, and are not brought 
 so suddenly into contact with the alcohol. In a few minutes 
 the diffusion is finished ; and they may then be placed in a fresh 
 quantity of alcohol, which must be shaken u]} frequently and 
 renewed from time to time until the acid has been entirely 
 removed. 
 
 The sulphuric acid contained in this fluid causes connective 
 tissue to swell, and this fact should be borne in mind in its use 
 with vertebrates. To avoid this difficulty, Kleinenberg has 
 recommended the addition of a few drops of creosote, made 
 from beech-wood tar, to the acid. According to Mayer's expe- 
 rience, however, the addition of creosote makes no percejitible 
 difference in the action of the fluid. 1 
 
 This fluid must not be used with objects (e. g., Echinoderms) 
 possessing calcareous parts which it is desired to preserve, for 
 it dissolves carbonate of lime, and throws it down as crystals of 
 gypsum in the tissues. For such objects picro-nitric acid may 
 be used (Mayer). 
 
 PICRO-NITKIC ACID. 
 
 Water 95 parts 
 
 Kitric acid (25 % N2O5) 5 " 
 
 Picric acid as much, as will dissolve. 2 
 
 Picro-nitric acid also dissolves carbonate of lime, but it holds 
 it in solution ; and thus the formation of crystals of gypsum is 
 avoided. In the presence of much carbonate of lime, the rapid 
 production of carbonic acid is liable to result in mechanical 
 injury of the tissues; hence in many cases chromic acid is 
 preferable to picro-nitric acid. 
 
 Picro-nitric acid is, in most respects, an excellent preservative 
 medium, and, as a rule, will be found to be a good alternative 
 in those cases where picro-sulphuric acid fails to give satisfac- 
 
 1 Fol thinks the tendency to swell can be better counteracted by adding 
 to the mixture one third its volume of 1 fo chromic acid, leaving out the 
 creosote. 
 
 2 This mixture is used undiluted. 
 
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22 MICROSCOPICAL METHODS. 
 
 tory results. Mayer commends it very strongly, and states 
 that with eggs containing a large amount of yolk material, like 
 those of Palinurus, it gives better results than nitric, picric, or 
 picro-sulphuric acid. It is not so readily removed from objects 
 as picro-sulphuric acid, and for this reason the latter acid would 
 be used wherever it gives equally good preparations. 
 
 PICRO-CHROMIC ACID. 
 Picric acid (saturated aqueous solution) .... 10 parts 
 
 Chromic acid (1 %) 25 " 
 
 Water 65 " 
 
 Recommended by Fol 1 as an excellent hardening agent for 
 very small pieces of tissue. It acts slowly, having little power 
 of penetration. A little osmic acid (.005), added shortly be- 
 fore using, is said to strengthen its action much. The staining 
 capacity of objects is not impaired by this mixture. Objects 
 treated with this fluid should be washed in water. The ex- 
 traction of the acid is more complete and rapid if nearly boil- 
 ing-hot water is used. 
 
 PERENYTS FLUIDS 
 The following mixture is recommended by Perenyi for hard- 
 ening eggs and embryos. It kills instantaneously, and preserves 
 the object in its natural form and size without the least shrink- 
 age. Eggs thus preserved are not brittle, but " cut like carti- 
 lage." 
 
 Nitric acid (10 fo) 4 parts 
 
 90 fo alcohol 3 " 
 
 Chromic acid {i %) 3 " 
 
 This mixture assumes a violet hue after a short time. 
 
 Eggs may be left in this fluid four to five hours, then twenty- 
 four hours in 70 alcohol, then in 90 alcohol one or two 
 days, finally four to five clays in absolute alcohol. 
 
 The staining fluid may be best added to the fluid itself, so 
 that the eggs are hardened and colored at the same time. 
 Fuchsine and aniline red dissolve directly in the fluid, while 
 
 1 Lehrbuch der vergleich. Anat. , p. 100. 
 
 2 Josef Perenyi. " Ueber eine neue Erhartungsfliissigkeit," Zool. 
 Anzeiger, V., No. 119, p. 459, 1882. 
 
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REAGENTS. 23 
 
 eosine and purpurine are first dissolved in three parts alcohol. 
 Picro-earmine or borax-carmine mixed with the fluid give ex- 
 cellent staining. The addition of staining fluids causes a pre- 
 cipitate, so that, before using, the fluid should be filtered. 
 
 For pelagic fish ova I find that ten or fifteen minutes immer- 
 sion is quite sufficient. My use of this fluid leads me to think 
 that its action should be checked as soon as the object becomes 
 completely permeated by it. It may be followed by Kleinen- 
 berg's hematoxylin or by Bohm's carmine acetate. 
 
 MERKEL'S CHROM-PLATINUM SOLUTION. 
 
 Platinum chloride dissolved in water 1:400 
 
 Chromic acid " " 1:400 
 
 Professor Merkel, 1 who employed a mixture of these two 
 solutions in equal parts for the retina, states that he allowed 
 from three to four days for the action of the fluid. Eisig has 
 used this fluid with great success in preparing the delicate lat- 
 eral organs of the Capitellidse for sections, and recommends it 
 strongly for other annelids. Eisig allows objects to remain 
 from three to four hours in the fluid, then transfers to 70 % 
 alcohol. "With small leeches I have found one hour quite 
 sufficient, and transfer to 50 % alcohol. 
 
 For pelagic fish eggs, the best results are obtained by treating 
 first with osmic acid (i %) ten minutes, and then with a modified 
 form of MerkePs fluid for two or three days (vide methods of 
 preparing fish eggs). 
 
 BICHROMATE OF POTASSIUM. 
 Potassium bichromate acts with less energy than chromic 
 acid, causes very little or no contraction, and for many eggs 
 and embryos, 2 as well as for the central nervous system, is far 
 superior to the simple acid. As a rule, solutions are made 
 stronger and allowed to act much longer than with chromic 
 acid. The more generally used strength is 2 %. Like all 
 chromic solutions, it should be kept dark. The energy of 
 its action is increased by heating ; but, in most cases, the better 
 
 1 " Ueber die Macula ZuieadesMenschen," etc., Leipzig, 1870, p. 19. 
 
 2 C. Hoffmann has used this fluid extensively for the eggs of inverte- 
 brates and fishes. 
 
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24 MICROSCOPICAL METHODS. 
 
 results are obtained by keeping the fluid in a cool place. A 
 piece of camphor suffices to prevent the appearance of mould. 
 
 The process of hardening is so slow that it is often advisable 
 to begin by treatment with some other fluid, and then complete 
 the hardening in this. (See Betz's method of hardening brain 
 and spinal cord.) 
 
 Heidenhain {vide Special Methods) has introduced a method 
 in which the preparation is both hardened and stained before 
 treatment with bichromate of potassium. 
 
 Sectioning should not be delayed long after hardening, as 
 preparations soon become brittle. 
 
 MULLER'S FLUID. 
 
 Distilled water 100 parts 
 
 Bichromate of potassium 2 " 
 
 Sulphate of sodium 1 " 
 
 This excellent fluid was first recommended by H. Mtiller for 
 the retina, and hence often called ' Mtiller's Eye-Fluid.' At 
 least two weeks are required for hardening the retina. It is 
 now much employed for hardening the central nervous system 
 (Cf. Weigert, Max Flesch, Osborn, Birge). It hardens very 
 slowly, requiring eight weeks in the case of the vertebrate 
 brain. If kept at a temperature of 30° to 40° C, the process 
 may be completed in eight to ten days (Weigert). Henneguy 
 employed this fluid, after osmic acid, in hardening fish eggs. 1 
 Minot makes the following remarks on this fluid : — 
 In hardening in Mtiller's fluid, it is important to keep the jar 
 with specimen and fluid in a cold place — best near freezing — 
 for three or four days. The delicate tissues are then much 
 better preserved. Probably the same is true of Erlicki's fluid. 
 Using Mtiller's Fluid at a high temperature is bad for epi- 
 thelia. 
 
 ERLICKI'S FLUIDS 
 
 Distilled water 100 parts 
 
 Bichromate of Potassium 2£ " 
 
 Cupric sulphate i " 
 
 1 It has often been highly recommended for delicate embryos ; but its 
 value for such purposes has been disputed by Fol. 
 
 2 Progres me"dic, Sept. 29, 1877 ; Revue d. sc. meaic, XL, 1, p. 13 ; 
 and Warschauer med. Zeit., XXIII., Nos. 15 and 18. 
 
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REAGENTS. 25 
 
 Erlicki recommended this fluid for hardening the human 
 brain, for which fourteen days are required. During the first 
 week, the fluid was renewed every day; during the second, 
 once in three days. Thus treated, the brain is well preserved, 
 and hard enough for sectioning. 
 
 In "Weigert's method, this fluid may replace Mailer's with 
 similar results. In Freud's gold chloride method, the oblongata 
 was first treated with Erlicki's fluid. 
 
 PEKCHLORIDE OF IRON (Vulfiau, Fol). 
 
 In experimenting with a delicate class of marine infusoria 
 (Tintinnodea), Fol 1 found that the reagents in common use 
 for instantaneous killing, — such as picro-sulphuric acid, osmic 
 acid, alone or in combination with chromic and acetic acid, and 
 corrosive sublimate, — failed to give successful preparations. 
 He finally succeeded with perchloride of iron, a reagent quite 
 new in histological technique. An alcoholic solution diluted 
 to about 2 fo will answer ordinary purposes ; but a stronger 
 solution should be used in case it is desired to kill a large 
 number of animals in a large vessel. It will not do, how- 
 ever, to turn a saturated solution directly into sea-water, as 
 precipitates would be copiously formed, which would utterly 
 ruin the preparations. After the animals have sunk to the 
 bottom of the vessel, most of the water may be turned off, and 
 70 % alcohol added. In order to remove from the tissues the 
 ferric salts adhering to them, it is necessary to replace this 
 alcohol with alcohol containing a few drops of hydrochloric 
 acid. 2 
 
 For general use, the following solution is recommended : s — 
 
 Perchloride of iron (alcoholic solution) 1 part 
 
 Alcohol (70 % ) 5-10 " 
 
 1 Zeitschr. f. wiss. Zool. XXXVIII., p. 491, 1883. 
 
 2 The best method of staining such objects is to add a few drops of 
 gallic acid (one per cent solution ) to the alcohol. After twenty-four hours 
 the acidulated alcohol is turned off, and pure alcohol added. Thus 
 treated, the protoplasm will take a light brown color, the nuclei a much 
 deeper brown. Carmine stains too deeply and diffusely, and cannot be 
 successfully removed. 
 
 8 Fol, Lehrbuch, p. 102. 
 
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26 MICROSCOPICAL METHODS. 
 
 Ciliated epithelium is exposed to this solution a few minutes, 
 then washed in 50 fo alcohol, to which £ to 1 % oxalic acid has 
 been added ; and finally, after the complete disappearance of the 
 yellowish-red color, transferred for preservation to pure alcohol. 
 
 CORROSIVE SUBLIMATE. 
 
 The saturated aqueous solution, now most generally em- 
 ployed, is prepared by heating the water (with excess of sub- 
 limate) to boiling. The cold solution contains only about two 
 per cent of the salt. A three or four per cent solution can be 
 made in 50 to 60 % alcohol. 
 
 This reagent kills, in most cases, instantly, and leaves the 
 tissues in a good condition for staining and sectioning. It is 
 important, however, to cut at an early date, as objects thus 
 killed become brittle if kept long in alcohol. 
 
 Sublimate has been brought into general use through Dr. 
 Lang's recommendation. Prompted by a statement found in 
 an old paper by Blanchard,' he began experimenting with cor- 
 rosive sublimate as a medium for killing marine planarians ; 
 and his marked success led him and others to employ the same 
 with other animals. In most «ases, Dr. Lang now uses a satu- 
 rated solution of corrosive sublimate in water. A saturated 
 solution in picro-sulphuric acid, which, in some cases, gives 
 better results if a little acetic acid (five per cent or less) is 
 added, is also used. 2 Blanchard's mode of treatment was to 
 mix a quantity of the aqueous solution with the sea-water, and 
 thus poison the animals. 
 
 Lang, on the contrary, removes the sea-water so far as possi- 
 
 1 " Recherches sur 1' Organisation des Vers," by Emile Blanchard. 
 Ann.des Sci. Nat. Zool. Ser. 3, t. VIII., 1847, p. 247. 
 
 2 These solutions given in Zoolog. Anzeiger, 1879, II., p. 46. The 
 original solution (Zoolog. Anzeiger, 1878, I., p. 14-15), now little used, 
 stood thus : — 
 
 Distilled water 100 parts 
 
 Common salt 6-10 " 
 
 Acetic acid 5-8 " 
 
 Corrosive sublimate 3-12 ' 
 
 Alum (in some cases) i " 
 
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EE AGENTS. 27 
 
 ble before applying the solution. With planarians he proceeds 
 in the following manner : — 
 
 The animal is laid on its back, and the water removed with 
 a pipette ; the solution being then poured over it, it dies quickly 
 and remains extended. After half an hour, it is washed by 
 placing it in water and changing the water several times during 
 thirty minutes. It is next passed through 50 %, 70 %, 90 %, 
 and 100 % alcohol. In two days it is fully hardened, and 
 should then be stained and imbedded in paraffin as early as 
 possible, as it is liable to become brittle if left long in alcohol. 
 The time required by the corrosive sublimate varies with dif- 
 ferent objects, according to size and the character of the tissues. 
 As a general rule, it may be said that objects should be removed 
 from the fluid as soon as they have become thoroughly saturated 
 by it. In order to kill more quickly than can sometimes be 
 done at the ordinary temperature, the solution is heated, and 
 in very difficult cases may be used boiling. 
 
 Corrosive sublimate has been used with success by Dr. Lang 
 and others in the following cases : Hydroids, Corals, Nemertines, 
 Gephyrea, Balanoglossus, Echinoderms, Sagitta, Annelids, Rhab- 
 doccela, Dendrocoela, Cestodes, Trematodes, embryos and adult 
 tissues of Vertebrates, and, according to Mayer and Giesbrecht, 
 Crustacea with thin chitinous envelopes, e. g., Sapphirina, Cope- 
 pods, and larvae of Decapods. 
 
 The two great advantages of Dr. Lang's method are, (1) that 
 animals so treated are easily stained, and (2) that they are 
 killed so quickly that they are left, in most cases, in a fully 
 extended condition. Hot corrosive sublimate kills leeches so 
 instantaneously that they often remain in the attitude assumed 
 the moment before the fluid is poured over them. The color, 
 however, is not so well preserved as when killed with alcohol, 
 or even with weak chromic acid. 
 
 It should be remembered that objects lying in a solution of 
 corrosive sublimate must not be touched with iron or steel instru- 
 ments; wood, glass, or platinum may be used. 
 
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28 MICROSCOPICAL METHODS. 
 
 FLUIDS FOR MACERATION". 
 
 The separation of tissues and tissue elements may be accom- 
 plished in a great variety of ways. It may be effected through 
 mechanical processes (by the aid of dissecting needles, by shak- 
 ing, and by pressure), or by chemical means (macerating agents 
 etc.). The methods and means must be varied to suit the 
 nature of the object under study, and according to the purpose 
 in view. It may be desirable merely to dissociate certain 
 elements ; or it may be required to destroy some parts while 
 preserving others. 
 
 Most of the fluids in common use for killing and hardening 
 may be converted into macerating and dissociative agencies by 
 diluting to various degrees with water. Water alone is often 
 a valuable means of maceration. Weak alcohol (30 % or less), 
 acetic acid (1 % or less), dilute osmic acid (alone, or in com- 
 bination with other fluids), chromic acid (|— ^ %), Midler's 
 fluid, and iodized serum are among the milder agents of dis- 
 sociation ; while eau de javelle, hydrochloric acid, and caustic 
 potash belong to the more violent. The following will serve as 
 illustrations. 
 
 ANDRES' METHOD WITH ACTINL3E. 
 It is often important to see the cells of a tissue in situ before 
 freeing them with needles. In such cases Dr. Andres proceeds 
 as follows : — 
 
 1. Killed with corrosive sublimate. 
 
 2. Left in 25 % alcohol twenty-four hours. 
 
 3. Soaked for a short time in a very thin solution of gum 
 
 arabic, then in a somewhat thicker solution, and finally 
 imbedded in a very thick solution. 
 
 4. Hardened in 90 % alcohol. 
 
 5. Thick sections prepared for dissection with needles. The 
 
 sections are placed on a slide in water, which dissolves 
 the gum. 
 
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REAGENTS. 29 
 
 THE HERTWIGS' MACERATING FLUID.' 
 For the isolation of tissues in the Ccelenterates, Oscar and 
 Richard Hertwig recommend the following mixture : — 
 
 Acetic acid 1 part 
 
 Osmic acid i " 
 
 Sea water 1000 " 
 
 By means of this fluid, not only the nerve cells, muscle cells, 
 etc., can be isolated, so that the exact form of the individual 
 cells may be easily recognized, but also the tissues, in the form 
 of thin lamella, may be separated and studied as a whole. 
 Pieces of tissue or whole animals are left in the mixture five to 
 ten minutes, and then washed for several hours in £ % acetic 
 acid. The macerated parts can be further prepared and 
 afterwards colored on the slide; or they can be colored at 
 once, before preparation with needles. In the first case, picro- 
 carmine is used ; in the second, Beale's carmine, because it does 
 not harden the tissues, but assists rather the process of macera- 
 tion. Pieces of tissue may be preserved a long time in glyce- 
 rine diluted with an equal volume of water, provided a few 
 drops of carbolic acid have been added to secure against mould 
 and Bacteria. 
 
 To obtain preparations of single-cell elements of Actiniae, 
 the macerated portion must be carefully divided up into smaller 
 parts by needles, and one or more of these parts placed under 
 the cover-glass. Light blows on the cover-glass with a needle 
 will cause the cells to separate. Care should be taken to sup- 
 port one side of the cover by a hair, which is removed quite 
 gradually after the object has been reduced to very small cell 
 masses. Sliding of the cover may be avoided by placing wax 
 feet under its corners. 
 
 Dr. Mark has employed this method, and obtained excellent 
 results with it. As he remarks, the great merit of this fluid is, 
 that it separates the cell elements and hardens them at the 
 same time. The dissociative and the preservative agent are 
 combined in such proportions that the action of the former is 
 confined within desired limits by that of the latter. 
 
 i Jenaische Zeitschrift, XIII., p. 462, 1879. 
 
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30 MICROSCOPICAL METHODS. 
 
 IODIZED SEBUM. 
 
 Tincture of iodine lg. 
 
 Amniotic fluid , , lOOg. 
 
 Carbolic acid 1-2 drops 
 
 Schafer (Practical Histology, p. 45) prepares by adding to 
 the amniotic fluid " a crystal or two of iodine, and allowing it 
 to stand for a few days with frequent agitation, and then filter- 
 ing off the clear fluid from any precipitate that may have 
 formed. Almost any other fresh serous fluid can be used 
 instead, but the amniotic fluid is generally the most readily 
 obtained in sufficient quantity, and, at the same time, perfectly 
 clear and pellucid. The serous fluid serves to macerate tissues 
 which are immersed in it, whilst preserving as nearly as possible 
 the original form of their elements. The purpose of the iodine 
 is to prevent putrefaction ; it serves also, at the same time, to 
 render the tissue elements slightly firmer." 
 
 Ranvier (Traite Technique, p. 76) recommends keeping a 
 strongly iodized serum in stock, to be used for iodizing fresh 
 serum. From one to two months are required to prepare the 
 stock solution. Iodine dissolves very slowly at first ; but after 
 a time (fifteen to twenty days) more will dissolve, and at the 
 end of two months a very strong solution of a dark brown color 
 is obtained. Fresh serum may then be prepared for use by the 
 addition of a little of this strong solution, — just enough to 
 give it a light brown color. 
 
 A piece of tissue may be kept in the weak serum for several 
 weeks without danger of putrefaction, provided that a few 
 drops of the stock solution is added as often as the brown color 
 disappears. The fading of the color is due to the absorption 
 of the iodine by the object. " The addition of new quantities 
 of iodine," says Ranvier, " is the key to this method of disso- 
 ciation." 
 
 EAU DE JAVELLE (KCIO). 
 
 Perls, 1 Altmann, and Noll 2 recommend Eau de JaveUe as an 
 excellent fluid for removing the soft parts of animal and vege- 
 
 1 Arch. f. mikr. Anat., vol. XVI., 1879. 
 3 Zoolog. Anzeiger, V., No. 122, p. 528. 
 
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REAGENTS. 31 
 
 table tissues. If a piece of Spongilla, for example, is placed 
 on a slide, and a drop of this water added, all the soft parts are 
 destroyed in from twenty to thirty minutes, and the spicula are 
 left in situ. After the protoplasmic parts have been thus 
 removed, the preparation is carefully treated with acetic acid, 
 in order to remove any cloudy precipitates, then washed with 
 weak, strong, and absolute alcohol successively, and finally 
 mounted in balsam. 
 
 Very neat preparations of diatoms may be obtained with this 
 fluid. The calcareous part of shells, thus treated, is not 
 destroyed. Sections of plant buds were successfully treated, 
 and then mounted in Meyer's fluid (ten volumes glycerine, 
 twenty distilled water, and three salicyl-pyroligneous acid). 
 
 The skeletons of small animals may be easily prepared by 
 placing the bodies in Eau de Javelle, which removes the skin, 
 muscles, &c, without injuring the bones. 
 
 CAUSTIC POTASH. 
 
 Strong caustic potash (30$) is much better than weak solu- 
 tions, as it destroys the tissue elements less rapidly. Solu- 
 tions should be kept in well-stoppered bottles, and renewed after 
 some months, as they readily take up carbonic acid from the 
 air, and change into carbonate of potassium. 
 
 DECALCIFICATION. 
 
 Decalcification of bone-tissue is best accomplished by the 
 action of weak chromic acid, a reagent which preserves and 
 hardens the included soft tissues at the same time that it dis- 
 solves the earthy matter. Its decalcifying power is much 
 weaker than that of hydrochloric acid ; but the latter is de- 
 structive to soft tissues. The action of chromic acid may be 
 both improved and intensified by combination with nitric acid, 
 in the following proportions : — 
 
 Chromic acid '/i'/t.^fl-\ 70 parts 
 
 Nitric acid . '. b 3 " 
 
 Water 200 " 
 
 When chromic acid alone is used, it is well to begin with a 
 solution of about \ %, using a large quantity of the fluid 
 
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32 MICROSCOPICAL METHODS. 
 
 and renewing it often, and then follow with stronger solutions, 
 rising gradually to J % as recommended by Schafer. 
 
 In using picric acid, the solution should be a saturated one ; 
 and fresh crystals of the acid must be frequently added to 
 take the place of that which is used up in dissolving the lime 
 salts. 
 
 DESILICIFICATION. 
 
 The silicious skeletons of animals (Radiolaria, Fibrospongise) 
 may be removed, according to P. Mayer, 1 with fluorhydric acid, 
 without injury to the tissues. 
 
 The objects are first hardened in alcohol, and then the acid 
 is added in drops. The duration of the process will vary from 
 a few minutes to a day, according to the size of the object. 
 The operation should take place in the open air, in vessels made 
 of lead, silver, or gutta percha. In the employment of this 
 acid, the manipulator must take special care to avoid its poison- 
 ous fumes. The corrosive power of the acid makes it highly 
 dangerous ; contact with the skin produces wounds difficult to 
 heal. 
 
 Zool. Anzeiger, No. 97, pp. 592-593. 
 
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CHAPTER n. 
 
 METHODS OF STAINING. 
 
 Foe staining purposes, alcoholic solutions are generally to be 
 preferred to aqueous media. The disadvantages in the use of 
 the latter, in staining alcoholic preparations, — such as the tear- 
 ing asunder of fragile tissues, caused by the violent osmosis, 
 swelling, the effects of which cannot always be fully obliterated 
 by again transferring to alcohol, and maceration, which is liable 
 to result where objects are left for a considerable time in the 
 staining liquid, — may all be avoided by using alcoholic solu- 
 tions. Objects once successfully hardened may be left in such 
 solutions for any required time, and, when sufficiently stained, 
 be washed in alcohol of a corresponding strength, and then 
 passed through the higher grades without being exposed to 
 •water from first to last. As a rule, alcoholic dyes work quickly, 
 and give far more satisfactory results than can be obtained 
 with other media. They penetrate objects more readily, and 
 thus give a more uniform coloring where objects are immersed 
 in toto. Even chitinous envelopes are seldom able to prevent 
 the action of these fluids. 
 
 It is not, however, to be denied that non-alcoholic dyes may 
 often do excellent work, and, in certain cases, even better than 
 can be otherwise obtained. The swelling caused by aqueous 
 staining fluids is not always an evil, but precisely what is 
 required by some objects after particular methods of treat- 
 ment. 
 
 From experiments recently made, Mayer has found that dyes 
 containing a high percentage of alcohol stain more diffusely 
 
 33 
 
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34 MICROSCOPICAL METHODS. 
 
 than those of weaker grades, from which he infers that strong 
 alcohol robs, to a certain extent, the tissues of their selective 
 power, and renders them more or less equally receptive of 
 coloring matter. 
 
 CARMINE SOLUTIONS. 
 
 CARMINIC ACID.l 
 
 Since Gerlach first called attention to carmine as a coloring 
 matter for histological purposes, many methods of preparing 
 the carmine solutions have been published, but, so far as I am 
 aware, none which result in anything but mixtures of various 
 carmine salts with undetermined and undesirable impurities, 
 such as fatty matter, tyrosin, sugar, and salts of alkaline metals. 
 Of course, the coloration produced by these mixtures has been 
 sometimes good, sometimes bad, and the solutions have easily 
 spoiled or decomposed, while their preparation often required 
 considerable time and trouble. Several years ago I studied 
 carmine, chiefly from a chemical standpoint, and since then I 
 have often wondered why naturalists usually used carmine 
 solutions in which water, with some caustic or destructive 
 material added, was the principal solvent. Carmine of com- 
 merce, it is true, is not readily soluble, even in water, until 
 ammonia, borax, or some other aid to solution is added ; but 
 carminic acid, the basis of the coloring matter of carmine, has 
 long been stated, in the leading chemical dictionaries and hand- 
 books, to be readily soluble in water and in alcohol. Watts 
 (Diet. Chem., 1872, 1st suppl., p. 413) says of carminic acid: 
 " This acid forms a purple mass, fusible and soluble in all pro- 
 portions in water and in alcohol. Sulphuric and hydrochloric 
 acid dissolve it without alteration. It bears a heat of 136° C. 
 without decomposition." 
 
 Earlier still, Watts (Diet. Chem., 1863, vol. I., p. 804) says: 
 " The fine red pigment known in commerce as carmine is pre- 
 
 1 Geo. Dimmock. Read at the New York meeting of the Society of 
 Naturalists of the Eastern United States, 28th Dec, 1883. — Am. Nat. 
 XVIII., p. 324, 1884. 
 
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METHODS OF STAINING. 35 
 
 pared by treating a solution of cochineal with cream of tartar, 
 alum, or acid oxalate of potassium. The fatty and albuminous 
 matters then coagulate and carry down the coloring matter 
 with them." Now in preparing most carmine solutions, this 
 precipitation takes place ; and the carmine, having greater 
 cohesive (not chemical) affinity for impurities of animal origin 
 than for alcohol, its solution is not readily accomplished by that 
 medium, nor, indeed, by water. In preparing carmine solu- 
 tions for histological purposes by some of the published recipes, 
 more than one half of the coloring matter of the carmine is 
 lost in the refuse left upon the filter paper. 
 
 There are two ways commonly in use for preparing carminic 
 acid, which term I here use as distinguished from " acid car- 
 mine " (the cochineal decoction for which Grenacher, Schweig- 
 ger-Seidel, and others have given recipes), and from carmine 
 (the impure commercial powder). The first mode of prepara- 
 tion, the one which I followed in preparing the acid upon 
 which I have experimented, is that of Warren De la Rue. 
 Watts, whom I quote because he gives clear directions (Diet. 
 Chem., 1863, vol. I., p. 804) gives De la Rue's method, as 
 follows : " To separate it [carminic acid], cochineal is exhausted 
 with boiling water ; the extract is precipitated by subacetate of 
 lead slightly acidulated, care being taken not to add the lead 
 solution in excess ; the precipitate is washed with distilled 
 water till the wash-water no longer gives a precipitate with a 
 solution of mercuric chloride, then decomposed by sulphuretted 
 hydrogen ; the filtrate is evaporated to a syrupy consistence, 
 and dried over the water-bath ; and the dark purple product thus 
 obtained is treated with alcohol, which extracts the carminic 
 acid." The second mode of preparing carminic acid, that of 
 Schaller, is given by Watts (Diet. Chem., 1872, 1st suppl., p. 
 413), as follows: "C. Schaller prepares this acid by precipi- 
 tating the aqueous extract of cochineal with neutral lead 
 acetate, slightly acidulated with acetic acid ; decomposing the 
 washed precipitate with sulphuric acid, again precipitating the 
 filtrate with lead acetate, and decomposing the precipitate with 
 sulphuric acid, avoiding an excess ; then precipitating a third 
 time, and decomposing the precipitate with hydrogen sulphide. 
 
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36 MICROSCOPICAL METHODS. 
 
 The filtered solution is evaporated to dryness ; the residue dis- 
 solves in absolute alcohol; the crystalline nodules of carminic 
 acid, obtained on leaving this solution to evaporate, are freed 
 from a yellow substance by washing with cold water, which 
 dissolves only the carminic acid ; and the residue left on evap- 
 orating the aqueous solution is recrystallized from absolute 
 alcohol or from ether." 
 
 Schaller's mode of preparation gives purer carminic acid than 
 De la Rue's ; but either kind is sufficiently pure for histological 
 purposes. The precipitation by lead acetate and the dissolving 
 in alcohol free the carminic acid from animal impurities, and 
 the consequence is a purer form of pigment than can be ex- 
 tracted by any process hitherto employed for the preparation 
 of carmine for histological purposes. I will here add that car- 
 minic acid has been shown recently by Liebermann and Van 
 Dorp to be related, through nitrococcusic acid, to trinitrocresol, 
 which has been obtained from coal-tar cresol, thus showing that 
 the coloring matter of cochineal contains methylated benzine- 
 residues. 
 
 I do not know whether carminic acid, ready prepared, is 
 obtainable. Any chemical student could easily prepare it by 
 the processes given above. 
 
 It is unnecessary to explain to naturalists the advantages of 
 alcoholic solutions of carmine over aqueous ones. The alco- 
 holic solution colors preparations much quicker than the aqueous 
 solution does ; for coloring sections, I employ a solution of 0.25 
 gram carminic acid to 100 grams of eighty per cent alcohol, 
 and leave sections in the solution from two to five minutes. A 
 solution of equal carmine strength, but in absolute alcohol, can 
 be employed ; it has, however, no special advantages, since, with 
 the eighty per cent alcoholic solution, the sections can be washed 
 directly in absolute alcohol, and then put into oil of cloves or 
 turpentine. Coloring in the piece before sectioning never takes 
 as long with alcoholic carminic acid as it does with ordinary 
 carmine solutions ; and, if it did take long, the strong alcohol 
 would preserve the tissue from maceration. In coloring pieces 
 of Mollusca, or of other equally slimy animals, the slime should 
 be removed beforehand, or the coloration will be unsatisfactory, 
 
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METHODS OF STAINING. 37 
 
 because the slime, congealing in the alcohol, takes up the color- 
 ing matter, forming an almost impervious colored layer on the 
 outside, and leaving the inside of the piece nearly uncolored. 
 
 Some preparations colored in alcoholic carminic acid, and 
 then put up in glycerine, lost their color in a few months, the 
 color seeming to be entirely diffused in the glycerine, while 
 similar preparations mounted in Canada balsam retained their 
 color perfectly. I do not know if this fading would occur with 
 preparations colored with alcoholic amnionic carminate, or even 
 if this diffusion was not due to some impurity of the glycerine 
 (of the purity of which I was doubtful). Time to test this 
 matter further failed. 
 
 An alcoholic ammonic carminate, or ammonia carmine, can 
 be prepared, at a moment's notice, from alcoholic carminic acid, 
 by adding ammonia, drop by drop, and stirring until the entire 
 solution changes from its bright red to purple red. By this 
 mode, pure alcoholic ammonic carbonate can be produced, with 
 no excess of ammonia, and at any time. As the carminic acid 
 can be preserved dry without decomposition, and dissolves 
 quickly in alcohol, one can carry the ingredients of a carmine 
 solution in the vest pocket without inconvenience. 
 
 In making and using alcoholic carminic acid, pure alcohol 
 and distilled water give the best results, because a portion of 
 the carminic acid is converted to carminates by the salts of 
 impure water. In making alcoholic ammonic carminate, this 
 precaution is not as necessary, because the color of the car- 
 minates produced by the impurities of the water is so nearly 
 like that of ammonic carminate. 
 
 Alcoholic carminic acid may be used, as Grenacher's carmine 
 solution is used, to color sections from which the coloration is 
 to be afterwards partly extracted by very dilute hydrochloric 
 acid, leaving nuclei red. Another way to use carmine solutions, 
 which is especially applicable to alcoholic carminic acid, is to 
 precipitate the carmine in the tissues by some salt, the carmi- 
 nate of the base of which gives a desired coloration. I have 
 found, for example, that specimens hardened for a moment 
 under the cover-glass with an alcoholic solution of corrosive 
 sublimate (mercuric chloride) and, after washing with alcohol, 
 
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38 MICROSCOPICAL METHODS. 
 
 colored in alcoholic carminic acid, took a fine coloration of 
 mercuric carminate. So, too, specimens colored in alcoholic 
 carminic acid can be changed by a few moments' treatment 
 with a very dilute alcoholic solution of lead acetate or cobalt 
 nitrate to a beautiful purple. With lead acetate, used as above, 
 a double coloration is sometimes produced; but I have not 
 examined these colorations sufficiently to describe them ac- 
 curately. Cupric and other salts, used as above described, have 
 not given me very favorable results. Sometimes salts in the 
 tissues of the animals themselves change portions of the car- 
 minic acid to purple carminates, giving a double coloration 
 without further treatment. 
 
 Picric acid added to alcoholic carminic acid in extremely 
 small quantities (best in a dilute alcoholic solution, testing the 
 solution on specimens after each- addition) makes a double 
 alcoholic coloring fluid (a so-called picro-carmine). I have been 
 unable, thus far, to determine the proportion of picric acid 
 required for this solution, having in every case added an excess. 
 All different kinds of carmine solutions can be made from car- 
 minic acid, with the advantage of having always uniform 
 strength, of being definite mixtures, and of not spoiling as 
 readily as those made directly from cochineal. 
 
 Incompatible reagents with carminic acid are, of course, all 
 alkaline solutions, and nearly all metallic salts ; with amnionic 
 carminate, are naturally all acids ; with all carmine solutions, 
 are bromine and chlorine. 
 
 BEALE'S CARMINE. 
 
 Carmine 1 gram 
 
 Ammonia 3 cc. 
 
 Pure glycerine 96 cc. 
 
 Distilled water 96 cc. 
 
 Alcohol, 95% 24 cc. 
 
 Dissolve in the ammonia plus part of the water ; add the rest 
 of the water, and allow to stand in an open dish in a warm 
 place until the ammonia is nearly all driven off. Then add the 
 alcohol and glycerine. 
 
 For use, dilute with an equal part of glycerine, which must 
 be thoroughly mixed in by stirring. Stain in an open dish, 
 
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METHODS OF STAINING. 39 
 
 which, together with a second open dish containing acetic acid, 
 is placed under a bell glass or in a closed box. The staining 
 requires at least twenty-four hours, and much longer if the car- 
 mine is fresh. Beale's carmine improves with use, and what is 
 left, after being employed as directed, should be filtered back 
 into the original bottle. 
 
 From the carmine solution the sections are placed in 
 water and washed thoroughly, after which they are placed for 
 from one to three minutes in hydrochloric acid, diluted with 
 water until it tastes about like sharp vinegar. They are finally 
 again washed in water and are then ready to mount in the 
 usual manner. 
 
 Employed in this way, Beale's carmine is one of the most 
 valuable of histological staining fluids, both for general use and 
 also more especially for the central nervous system. If by any 
 chance the sections are over-stained, the superfluous color may 
 be extracted by a brief sojourn in very dilute ammonia — one 
 drop of strong ammonia to five cubic centimetres of water is 
 quite sufficient ; this is to be followed by a rapid washing in 
 water, and an immediate transfer to dilute hydrochloric acid. 
 (Minot). 
 
 AliCOHOL CARMINE (MAYER).' 
 
 Carmine 4 g. 
 
 Alcohol (80%) lOOcc. 
 
 Hydrochloric acid 30 drops 
 
 The pulverized carmine is added to the acidified alcohol, and 
 the mixture boiled for thirty minutes, to dissolve the coloring 
 substance. The solution is filtered warm, neutralized by adding 
 ammonia until a precipitate begins to appear. After cooling, 
 it may be necessary to filter several times. 
 
 GRENACHER'S CARMINE SOLUTIONS. 2 
 (1) Alum Carmine. — An aqueous solution of alum (1 to 
 5 fo, or any degree of concentration) boiled with \ to 1 % pow- 
 
 i Gravis. Bull, de laSoc. Belg. de Micr., 18S4, No. VII., p. 126. 
 2 Arch.f. mikr. Anat., Vol. XVI., p. 463, 1879. None of these solu- 
 tions to be used where calcareous parts are to he preserved. 
 
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40 MICROSCOPICAL METHODS. 
 
 dered carmine for 10 to 20 minutes ; allowed to cool, then fil- 
 tered. With the addition of a little carbolic acid the fluid 
 will keep for years. It colors quickly, and nuclei more strong- 
 ly than other parts. Objects washed in water after staining. 
 
 (2) Acid Borax Carmine. — a. An aqueous solution of 
 borax (1 to 2 %) and carmine (£ to § %) heated till the carmine 
 is dissolved. 
 
 b. Acetic acid added by drops to solution a, while shak- 
 ing, until the color is about the same as that of Beale's car- 
 mine. 
 
 c. Solution b left standing 24 hours, then turned off and fil- 
 tered. 
 
 This solution, which is a modification of Schweigger-Seidel's 
 acid carmine, is not recommended for coloring in toto. It col- 
 ors sections in one half to three minutes diffusely, and hence, 
 after washing in water, they are placed for a few minutes in 
 alcohol (50 % or 70 %) to which a drop of hydrochloric acid 
 has been added ; then transferred to pure alcohol. 
 
 (3) Borax Carmine. 1 — a. An aqueous solution of borax 
 (4 %) and carmine, heated till the carmine is dissolved. 
 
 b. Solution a mixed with 70 % alcohol in equal parts, left 
 standing 24 hours and filtered. 
 
 This fluid may be used for coloring objects in toto. After 
 staining, the objects are to be washed in 35 % alcohol, to which 
 a little hydrochloric acid has been added (4 to 6 drops to 100 
 cc), and allowed to remain here until the color has been suffi- 
 ciently removed. They are next passed through successively 
 higher grades of alcohol for hardening. 
 
 (4) Alcohol Carmine. — A teaspoonful of carmine dis- 
 solved, by heating about 10 minutes in 50 cc. of 60 to 80 % 
 alcohol, to which 3 to 4 drops of hydrochloric acid have been 
 added, then filtered. 
 
 Objects colored in this fluid should not be washed in water, 
 but in alcohol of a grade corresponding to that of the solution. 
 
 x Dr. Mayer prepares, for some purposes, borax carmine of 50, 60 or 
 70$. That of 70% contains little carmine, but is well adapted to stain- 
 ing delicate objects that would suffer if exposed to weaker solutions. 
 Boiling alcohol (50% or 60% ) dissolves about 1% carmine and 1 % borax. 
 
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METHODS OP STAINING. 41 
 
 For diluting alcoholic solutions of carmine, alcohol of the 
 same strength must always be used. 
 
 RANVIER'S PICEO-CAKMtNATE.l 
 
 1. Ranvier takes a saturated solution of picric acid, and adds 
 to it a saturated solution of carmine in ammonia (until a cloudy 
 precipitate begins to appear). 
 
 2. This solution is then evaporated by heat until reduced to 
 about one-fifth of its original volume, then cooled and filtered. 
 
 3. The filtered solution is then evaporated to dryness, leaving 
 a reddish powder, which, as often as occasion requires, is dis- 
 solved in distilled water (1 : 100). 
 
 In using this picro-carminate, one can have the double stain 
 of the carmine and the acid ; or the simple stain of the car- 
 mine ; the acid being washed out by soaking in distilled water. 
 
 The solution, when properly prepared, should be neutral. If 
 the solution obtained from the powder is not neutral, it can be 
 made so by adding a little carbonate of ammonia and heating 
 moderately in an open vessel, as recommended by Fol. Am- 
 monia or carbonic acid escapes, and the solution becomes at 
 once neutral. 
 
 PERGENS' PICROCARMINE.2 
 
 1. 1. Boil, for 1\ hours, 500 g. of cochineal (pulverized) in 
 30 litres of water. 
 
 2. Add 50 g. potassic nitrate, and, after a moment of boil- 
 ing, 60 g. oxalate of potash ; boil 15 minutes. 
 
 3. After cooling, the carmine settles: it is washed several 
 times with distilled water, in the course of three or four weeks. 
 
 H. 4. Pour a mixture of 1 volume of ammonia with 4 vol- 
 umes of water upon the carmine, taking care that the carmine 
 remains in excess. 
 
 5. After two days, filter, and leave the filtered solution ex- 
 posed to the air until a precipitate forms. 
 
 6. Filter again, and add a concentrated solution of picric 
 acid ; agitate, and then allow it to stand 24 hours. 
 
 7. Filter, and add 1 g. chloral for one litre of the liquid. 
 
 1 Traits' Technique, p. 100. 
 
 2 Biologie Cellulaire, by J. B. Carnoy, p. 92, 1884. 
 
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42 MICROSCOPICAL METHODS. 
 
 8. At the end of eight days separate the liquid from the 
 slight precipitate which is formed, and it is ready for use. 
 
 This fluid keeps unchanged for at least two years, and is 
 recommended by Carnoy above other picro-carmine solutions. 
 
 MINOT'S PICRIC-ACID CARMINE. 
 
 Boil one gram best powdered carmine with 200 cc. of water, 
 plus an excess of picric acid for half an hour ; allow it to stand 
 and cool ; decant the clear fluid, add fresh water, and, if neces- 
 sary, picric acid ; boil, cool, and decant ; repeat this operation 
 until all the carmine is dissolved. Place the decanted fluid in 
 an evaporating dish, add about one gram thymol, and stand in 
 a warm place until the volume is reduced to 25 cc. ; let the 
 solution cool, filter, wash out the residue, which should be on 
 the filter, with 25 cc. water ; dilute the filtrate with 50 cc. 
 water. By this means, a solution ready for use, which will keep 
 indefinitely, and contains carmine and picric acid in good pro- 
 portions, can be prepared with certainty. 
 
 It gives a stronger differential coloring than Ranvier's picro- 
 carmine ; but over-staining must be most carefully avoided. 
 For staining sections, two to five minutes are sufficient. The 
 fluid stains connective tissue (fibrous) deep red ; striped mus- 
 cle, deep, dull red ; smooth muscle, blood, and horny tissue, 
 bright yellow; glands, reddish yellow. With the kidney it 
 gives differentiation of the different portions of the tubules ; 
 for the central nervous system it seems to be of little value. 
 If rightly used, it gives a sharp, nuclear coloring. 
 
 If the aqueous solution is evaporated to dryness, the residue 
 may be redissolved in alcohol, giving an alcoholic carmine dye. 
 This I have not yet tested sufficiently. Apparently the alco- 
 holic solution will keep only a few months. The alcoholic 
 solubility of the dye offers the advantage that sections stained 
 in the watery solution can be washed in alcohol directly. 
 
 PICRO-CARMINE. 
 
 A very excellent picro-carmine is prepared by Mayer in the 
 following manner : — 
 
 To a mixture of powdered carmine (2 g.) with water (25 
 
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METHODS OP STAINING. 43 
 
 cc), while heating over a water-bath, add sufficient ammonia 
 to dissolve the carmine. The solution may then be left open 
 for a few weeks, in order that the ammonia may evaporate ; or 
 the evaporation may be accelerated by heating. So long as 
 any ammonia remains, large bubbles will form while boiling ; 
 but as soon as the free ammonia has been expelled, the bubbles 
 will be small, and the color of the fluid begin to be a little 
 lighter. It is then allowed to cool, and filtered. To the fil- 
 tered solution is added a concentrated aqueous solution of 
 picric acid (about four volumes of the acid to one of the car- 
 mine solution). 1 
 
 A small crystal of thymol, or a little chloral-hydrate (1 fo or 
 more), will serve as an antiseptic. 
 
 ACETIC ACID CARMINE.* 
 
 Pulverized carmine added to a small quantity of boiling 
 acetic acid (45 fo) until no more will dissolve; filtered, and 
 diluted to about 1 % for use. 
 
 Flemming used the concentrated solution. 
 
 BOHM'S CARMINE ACETATE. 
 
 1. Carmine (4 g.) pulverized in 200 cc. water. 
 
 2. Ammonia added by drops until the solution becomes 
 cherry-red (the carmine should now be fully dissolved). 
 
 3. Acetic acid slowly added until the cherry-red color be- 
 comes brick-red. The addition of acetic acid should be accom- 
 panied with stirring, and should cease the moment the change 
 in color is effected. 
 
 4. Filter until no trace of a precipitate remains. 
 
 If the color is not sufficiently deep, a few drops of ammonia 
 should be added before filtering, and the solution left in an 
 open vessel until the alkali has volatilized. 
 
 Objects may be left for twenty-four hours, or more, in this 
 fluid. The deep stain should be partially removed by immer- 
 
 1 The addition of the acid should cease before a precipitate begins to 
 form. 
 
 2 Schneider. Zool. Anzeiger, No. 56, p. 254, 1880. 
 
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44 MICROSCOPICAL METHODS. 
 
 sion in a mixture of water (50 volumes), glycerine (50 vol- 
 umes), and muriatic acid (£ volume), for a few minutes. The 
 karyokinetic figures are thus brought out with great distinct- 
 ness. 
 
 HEMATOXYLIN AND COCHINEAL. 
 
 GEENACHEE'S HEMATOXYLIN. 
 Saturated alcoholic solution of hematoxylin . . 4 cc. 
 Strong aqueous solution of ammonia-alum . . . 150 " 
 
 Let this mixture stand 8 days in the light ; then filter and 
 add, — 
 
 Glycerine 25 cc. 
 
 Hethylalcohol 25 " 
 
 This fluid works best after standing a few months, or, at 
 least, long enough for a sediment to form at the bottom. 
 
 KLEINENBERG'S ILEMATOXYLIN.l 
 
 1. To a saturated solution of chloride of calcium 2 in 70 <f al- 
 cohol add a little alum, and filter. 
 
 2. One volume of No. 1 mixed with six to eight volumes of 
 70 % alcohol. 
 
 3. At time of using, pour into No. 2 as many drops of a 
 concentrated solution of crystallized hsematoxylin in absolute 
 alcohol as suffice to give the required depth of color. 8 
 
 1 May be used after all hardening fluids. 
 
 2 Chloride of calcium, according to Kleinenberg, has no other use than 
 to strengthen the osmotic action between the hsematoxylin solution and 
 the alcohol contained in the tissues. As chloride of calcium and alum 
 give a precipitate of gypsum, it would probably be better to use chloride 
 of aluminum. 
 
 3 A good solution should be violet, inclining a little to blue. The red 
 tinge that arises after the fluid has stood for some time indicates that it 
 has become slightly acid, in which condition it is unfit for use. To restore 
 its proper color, it is only necessary to open a bottle of ammonia over the 
 mouth of the bottle holding the hsematoxylin, in such a manner that a 
 very small quantity of the gas will mix with the fluid. If too much 
 ammonia gas be added, a precipitate is produced which spoils the fluid. 
 
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METHODS OF STAINING. 45 
 
 If the color appears too strong, the fluid may be diluted with 
 solution No. 1. 
 
 Before immersing objects in this fluid, great care should be 
 taken to free them from the least trace of acid by frequently 
 changing the alcohol. If this is not done thoroughly, the acid 
 left in the preparation will sooner or later cause the color to 
 fade ; and such results have led to the erroneous conclusion that 
 hsematoxylin will not give durable preparations. 
 
 Small objects are best stained in a weak solution, which colors 
 more slowly, but with greater clearness, than stronger solutions. 
 After staining, Kleinenberg transfers objects to 90 % alcohol. 
 In case of over-staining, the color may be partly removed by 
 adding a little oxalic acid or hydrochloric acid (£ %, or less) to 
 the alcohol containing the objects. The acidulated alcohol is 
 allowed to work until the color is slightly reddened. On trans- 
 ferring to pure alcohol, the color passes again into a permanent 
 blue- violet. 
 
 MAYER'S COCHINEAL TINCTURE. 
 
 One gram powdered cochineal soaked in 8 to 10 cc. of 70 % 
 alcohol for several days, then filtered. 
 
 The clear, deep-red fluid thus prepared may, like hsematoxy- 
 lin, be used in all cases where it is desirable to stain with an 
 alcoholic solution, and will be found particularly useful for 
 objects that are not easily penetrated by the ordinary aqueous 
 solutions of carmine, such as the Arthropods. 
 
 It is necessary, before immersing larger objects in this fluid, 
 to leave them a short time in 70 % alcohol, otherwise there may 
 be a precipitate. The time required for staining will vary from 
 a few minutes to a day or more, according to the nature and size 
 of the object. With larger objects, requiring considerable time, 
 it is important to use a large quantity of the fluid, otherwise the 
 amount of coloring stuff in solution might not suffice to give 
 the proper depth of color. Small and delicate objects, on the 
 other hand, may be most successfully treated with a solution 
 which has been diluted with 70 % alcohol, or one which has 
 been weakened by previous use. It is always necessary to free 
 the tissues, after staining, from the surplus dye ; and this may 
 
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46 MICROSCOPICAL METHODS. 
 
 be done by washing in 70 % alcohol, which must be changed 
 until it shows no color. This process requires, for larger ob- 
 jects, considerable time and alcohol, but may be hastened by 
 using the alcohol slightly warm. 
 
 The color ultimately assumed by objects treated with cochi- 
 neal tincture varies much, and depends partly on the reaction 
 of the tissues themselves, partly on the presence or absence of 
 certain salts. It is certainly one of the best recommendations 
 of this staining agent that, varying with the nature of the ob- 
 ject and its mode of treatment both before and after staining, 
 it gives such an extraordinary diversity of results. On account 
 of the great variety of substances contained in the dried dye- 
 stuff, it is evident that the composition of the tincture must 
 vary according to the strength of the alcohol employed as a sol- 
 vent. Solutions in 90 % or 100 % alcohol have a light-red 
 color, and stain too diffusely to have any practical value. The 
 weaker the alcohol the stronger the tincture, and the stronger 
 the alcohol the more easily it penetrates objects ; the grade of 
 alcohol may therefore be selected with reference to two points, 
 depth of color and readiness of penetration ; 70 % or 60 % is 
 recommended by Mayer as combining both these qualities in 
 a very favorable degree. It is important to remember that, 
 whatever be the strength of the solution, a precipitate will al- 
 ways be produced if an alcohol of a different grade, whether 
 higher or lower, be mixed with it. It is evident, then, that a 
 tincture of any given strength contains substances that are in- 
 soluble in any other grade of alcohol, and this explains why super- 
 fluous coloring matter can only be removed from objects by the 
 aid of alcohol of precisely the same degree as that of the tincture. 
 
 Overstaining, which seldom occurs, may be easily corrected 
 by the aid of acid alcohol (-^ fo hydrochloric acid, or 1 % acetic 
 acid). Acid makes the tincture lighter, more yellowish-red, 
 while the addition of ammonia and other caustic alkalies changes 
 it to deep purple. Still more important is the fact that salts 
 soluble in alcohol give a blue-gray, green-gray or blue-black pre- 
 cipitate. For example, if a piece of cloth that has been dyed in 
 cochineal, and washed, be treated with an alcoholic solution of a 
 ferric or a calcic salt, it will assume a more or less deep blue color. 
 
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METHODS OF STAINING. 47 
 
 As the salts present in the living organism are seldom, if ever, 
 fully removed by preservative fluids, but in some cases even in- 
 creased, it will often happen that an object, though stained in 
 the red fluid, comes out blue, precisely as when stained with 
 hematoxylin. Such a result cannot, however, be obtained in 
 the presence of acids, nor in the absence of inorganic salts; 
 under these conditions the color is always red. It is not possible, 
 therefore, to know what color an object will ultimately present. 
 
 Very often the different tissues of one and the same object 
 present unlike colors. In the embryos of Lumbricus, Kleinen- 
 berg found the walls of the blood-vessels red, their contents 
 dark blue. Glandular tissues, or their contents, are frequently 
 stained gray-green. 
 
 Objects treated with chromic or picric solutions, or with 
 alcohol, usually stain without difficulty; but osmic acid pre- 
 parations should be bleached before staining. Cochineal does 
 not color so intensely as hematoxylin, and hence the latter 
 often gives more satisfactory results in the case of large objects 
 stained in toto. 
 
 As before pointed out, alcohol causes the salts contained in 
 sea water to be precipitated, thus forming a crust on the exte- 
 rior of the animal which interferes with the staining process. 
 It is therefore necessary to treat marine animals that have been 
 preserved in strong alcohol, with acid alcohol (1-10 parts hy- 
 drochloric acid to 1,000 parts 70 % alcohol), and then carefully 
 wash in pure 70 f alcohol before staining with cochineal. 
 
 ANILINE DYES. 
 
 As a rule, aniline colors cannot be employed for staining ob- 
 jects in toto. 
 
 With very small objects and sections already cut, very excel- 
 lent results can be obtained by the methods developed by Bot- 
 tcher, 1 Hermann, 2 Flemming, 8 and others ; for here diffuse stain- 
 
 1 Miiller's Archiv, 1869, p. 373. Virchow's Archiv, Bd. XL., p. 302. 
 
 2 Communicated to the Naturforscherversammlung in Graz, 1875. Tag- 
 blatt, p. 105. 
 
 8 Archiv f. mikr. Anat., Bd. XIII. p. 702, Bd. XVI. p. 302, Bd. XVIIL 
 p. 151, Bd. XIX. p. 317, and p. 745, Bd. XX. p. 1. 
 
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48 MICROSCOPICAL METHODS. 
 
 ing may generally be avoided by first overstating and then 
 withdrawing the color to any desired extent by means of alco- 
 hol. But to obtain satisfactory results, the sections must be 
 thin enough to allow uniformity of action both to the coloring 
 and the decoloring agent. It is evident that the process can- 
 not be similarly controlled in larger objects, particularly where 
 a dye is used, which, like most of those under consideration, is 
 quickly extracted by alcohol, for in this case the color would 
 be removed from the superficial layers more rapidly than from 
 the deeper ones, so that a uniform precision of color would be 
 impossible. 
 
 BISMARCK BROWN.1 
 
 Bismarck brown forms an exception to the statement made 
 in the last paragraph. 
 
 A saturated solution is made by dissolving the powder in boil- 
 ing water or weak alcohol, or, according to Mayer, in 70 % al- 
 cohol. The solution should be used undiluted, and requires to 
 be filtered from time to time. It colors very quickly objects 
 hardened in alcohol or chromic acid. 
 
 EOSIN. 
 The best strength for section-staining is 0.0005. Dissolve 
 one half gram eosin in one litre of 95 % alcohol — or dissolve one 
 gram in 100 cc. alcohol, and for use dilute one part of the 
 stock solution with 20 parts alcohol. The alcoholic solution is 
 far more convenient than the aqueous. (Minot.) 
 
 ROSE BENGALE IN COMBINATION WITH IODINE GREEN AND 
 BLEU DE LYON. 
 
 Rose bengale, according to Griesbach, 2 is the bluest of the 
 eosin dyes. An aqueous solution is very useful in staining 
 chromic acid preparations of the spinal cord, as it colors the 
 gray substance much more strongly than the white substance. 
 
 Rose bengale may be used in combination with iodine green 
 and bleu de lyon in the following manner : 
 
 i Weigert. Arch. f. mikr. Anat., XV. p. 258, 1878. 
 2 Zool. Anzeiger VI., No. 135, p. 172. 
 
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METHODS OF STAINING. 49 
 
 A section of an alooholio preparation is first placed in dis- 
 tilled water ; then quickly drawn through a deep red solution 
 ot rose bengale, and again placed in aq. dest. It is next laid in 
 iodine green for a few seconds, washed, and placed for five min- 
 utes in absolute alcohol to remove any excess of color. It may 
 be transferred directly from the alcohol to a dilute alcoholic 
 solution of bleu de lyon (two parts alcohol abs. and three parts 
 aqua dest.) and left for a few seconds, then replaced in the ab- 
 solute alcohol preparatory to mounting. 
 
 SAFBANIN. 
 
 One part safranin dissolved in 100 parts of absolute alcohol ; 
 after a few days 200 parts of distilled water is added. 
 
 Pfitzner, 1 from whom the above formula is taken, recommends 
 this solution as one of the best for staining nuclei. It is cheap, 
 easily prepared, acts quickly, and stains only the nuclei. It 
 works best with chromic acid preparations, from which the acid 
 has been removed as much as possible. 
 
 NIGROSIN COMBINED WITH PICRIC ACID.2 
 
 To a saturated aqueous solution of picric acid, add a small 
 quantity of an aqueous solution of nigrosin. This gives a deep 
 olive-green mixture which kills very quickly, and stains at the 
 same time it hardens. After a few hours immersion the object 
 may be transferred to water or alcohol for removing the acid 
 and the nigrosin which remains in solution. 
 
 This method is very good for preparing small objects that 
 have to be killed, hardened, stained, &c, under the cover-glass. 
 
 DAHLIA (MONOPHENTTL-ROSANILIN). 8 
 
 Dahlia is soluble in alcohol (one sort is soluble in water). The 
 aqueous solution stains the protoplasm blue-violet, leaving the 
 nuclei very pale. If sections thus stained are treated with very 
 dilute acetic acid, the protoplasm is decolored and the nuclei 
 
 i Pfitzner. Morph. Jahrb., VI. pp. 478-480 and VII, p. 291. 
 2 Pfitzner. Ber. Dtsch. Botan. Gessellsch., 1, No. 1, p. 44, 1883. 
 8 Ehrlieh. Arch. f. mikr. Anat., XIII., pp. 263-277. 
 
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50 MICROSCOPICAL METHODS. 
 
 become blue-violet. The staining fluid is best prepared as 
 follows : — 
 
 Alcohol (absolute) 50 cc. 
 
 Distilled water 100 cc. 
 
 Glacial acetic acid 12£ cc. 
 
 Dahlia (to near saturation). 
 
 The dye should be allowed to act at least twelve hours. 
 
 BLUE BLACK 
 
 Dissolves easily in water, with difficulty in alcohol. 
 
 Blue black -J g., dissolved in 
 
 Aq. dest 1 to 2 cc. ; then add 
 
 Alcohol 99 cc. 
 
 Recommended by Sankey, 1 especially for the central nervous 
 system. 
 
 MYETILLUS. 
 
 Dr. M. Lavdowsky 2 recommends the juice of fresh huckleber- 
 ries, Vaccinium ( G-aylussacia Gray) myrtillus, as an excellent 
 staining medium, especially for the karyokinetic figures and the 
 cellulose walls of plant cells. 
 
 Preparation. — The newly picked berries should be washed 
 in water, then the juice expressed and mixed with twice its 
 volume of distilled water containing a few grams of 90 % alco- 
 hol. The mixture may next be boiled for a short time, then 
 filtered while it is still warm. The fluid thus obtained is deep 
 red and slightly acid, and may be kept in well-corked bottles 
 for a long time if not left in too warm a place. The fluid 
 becomes somewhat thick, on cooling, and may therefore be di- 
 luted with two to three times its volume of distilled water at 
 the time of using. 
 
 Two different colors are produced by this fluid: 1. A red 
 color is produced by the action of the fluid on fresh objects, 
 and on objects hardened in chromic acid or other chromic solu- 
 tions. The best staining is said to be obtained from chromic 
 acid preparations. 2. A lilac color — more durable than the 
 
 1 Quart. Jour. Mic. Sci., 1876, p. 35. 
 
 2 Arch. f. mikr. Anat, XXIII., pp. 506-508. 
 
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METHODS OF STAINING. 51 
 
 red — may be obtained in the following manner : — Place three 
 watch-glasses on a white ground, fill one with the myrtillus 
 fluid, the second with a 1 fo solution of acetate of lead, and the 
 third with distilled water. Place the object in the dye for one 
 or two minutes, wash in the distilled water, then leave in the 
 solution of acetate of lead until the lilac color becomes pro- 
 nounced ; finally, wash and mount in glycerine, or, after treat- 
 ment with alcohol, in balsam. In case glycerine be used, a 
 little acetate of lead should be mixed with it. 
 
 DOUBLE STAINING WITH ANILINE DYES. 1 
 
 The method of experiment adopted by Vincent Harris was 
 as follows : — The blood was spread in thin layers upon cover- 
 glasses, and allowed to dry in direct sunlight. The dried blood 
 was then wet with a few drops of some dye, and, after five min- 
 utes, washed with a slow stream of water from a wash-bottle ; 
 it was then dried in the flame of a spirit-lamp, and allowed to 
 cool. A second dye was then applied in the same way ; and, 
 after washing, the preparation was mounted in balsam, without 
 having recourse to alcohol and clove oil. 
 
 The following combinations of dyes were found to give good 
 results : — 
 
 Kosein and iodine green. 
 
 Fuchsin and methylen blue. 
 
 Fuchsin and Bismarck brown. 
 
 Eosin and vesuvin. 2 
 
 Iodine green and Bismarck brown. 
 
 Hoffman's violet and Bismarck brown. 
 
 Methyl violet and methylen blue. 
 
 Rosein, followed by iodine green, stained the colored cor- 
 puscles a bright red, with bluish-green nuclei ; and the colorless 
 corpuscles were so stained that three varieties could be readily 
 distinguished. 
 
 Fuchsin and methylen formed a very successful combination. 
 The methylen blue was used as a saturated solution in absolute 
 alcohol. 
 
 i Quart. Jour. Mic. Sci., XXIII., p. 292. 
 
 a Regarded as identical with Bismarck brown. 
 
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52 MICROSCOPICAL METHODS. 
 
 Bismarck brown was prepared as a 2 fo solution in dilute 
 alcohol. In the use of this dye it was found best to immerse 
 the preparation twenty to thirty hours, as the color then re- 
 mains, even when passed through alcohol and clove oil. 
 
 An aqueous solution of Hoffman's violet was used with a 
 dilute spirit solution of Bismarck brown. 
 
 The green dyes are not permanent. The solutions should be 
 quite fresh in order to secure successful results. 
 
 THE DIFFERENTIAL ACTION OF SAFRANIN AND METHYL GREEN. 
 In studying the sexual characteristics of the oyster, Ryder 1 
 found that a mixture of these two dyes enabled him to distin- 
 guish both ova and spermatozoa in the same follicle, the nuclei 
 of the ova being stained red by the safranin, and the heads of 
 the spermatozoa bluish-green by the methyl green. The method 
 of preparation is as follows : — 
 
 1. After removing the shell, the oyster is hardened in chromic 
 acid (1 to 2 %) for several days. 
 
 2. Washed in water 2 days, and then further hardened in al- 
 cohol. 
 
 3. Soaked for 24 hours in water, to remove the alcohol; 
 then imbedded in gum arabic, and cut with free hand. 
 
 4. Sections freed from imbedding mass by washing in water ; 
 then stained in a mixture in equal parts of 
 
 Safrania (saturated alcoholic solution) 
 Methyl Green " " " 
 
 diluted with eight times its volume of water, 2 to 3 hours. 
 
 5. Decolored in 95 % alcohol until clouds of color no longer 
 appear (5 to 15 minutes). 
 
 6. Clarified in clove oil and mounted in balsam or dammar. 
 
 STAINING INTRA VITAM. 
 
 Bismarck Brown. — Karl Brandt, 2 who was the first to 
 recommend Bismarck brown for coloring living organisms, 
 
 1 Bull. U. S. Fish. Com., 1883. 
 
 2 Sitzgsber. Berl. physiol. Gesellsch., pp. 34, 35, Dec. 1878; and Biolog. 
 Centralblatt, p. 202, 1881. 
 
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METHODS OF STAINING. 53 
 
 found that, by using a weak solution (1 : 3000 or 1 : 5000) on 
 living cells (fresh-water Protozoa, etc.), the nuclei remained 
 uncolored, while fatty substances were stained brown. He suc- 
 ceeded in coloring the nuclei with a dilute aqueous solution of 
 haamatoxylin, the organisms still remaining alive. 
 
 Paul Mayer 1 mixed Bismarck brown with sea-water in which 
 Caprellidas were kept, and found that the contents of the 
 stomach and the "secretion-balls" of the liver (hepato-pancreas) 
 were soon colored brown ; and that, after a somewhat longer 
 time, the muscles were stained diffusely, and the blood-cor- 
 puscles showed one or more brown dots. When the speci- 
 mens thus treated were placed in pure sea-water, the stain 
 wholly disappeared within a few hours, leaving them quite 
 uninjured. 
 
 Cyanin (Bleu de Quinoleine, Chinolinblau). — A. Certes 2 
 recommends a weak aqueous solution of cyanin (1 : 500,000 - 
 1 : 100,000) for staining Infusoria alive. The coloring substance 
 is not completely soluble in water. The solution should be made 
 with ordinary water, filtered, as distilled water is poisonous to 
 Infusoria. Infusoria may be kept alive in the solution from 
 twenty-four to thirty-six hours. The fatty granulations of 
 the protoplasm stain deeply; the protoplasm, cuticle, and 
 cilia very feebly ; and the nuclei still less, or not at all. Solu- 
 tions of cyanin, whether in water or alcohol, are decolored 
 more or less rapidly by the light, and should therefore be kept 
 dark. 
 
 Methyl and Gentian Violet. — J. Bizzozero 3 employed 
 the two following mixtures in-the study of blood corpuscles: — 
 
 Methyl violet (concentrated aqueous solution) . . 1 part 
 Salt solution (f % ) 5000 " 
 
 Gentian violet (saturated solution) 1 part 
 
 Salt solution (f % ) 3000 " 
 
 1 Fauna u. Flora des Golfes von Neapel. VI. Monographie: Caprel- 
 Iiden, p. 153, 1882. 
 
 2 Zool. Anzeiger, IV., p. 208 and 287, 1881. 
 8 Arch. f. pathol. Anat., p. 261, 1882. 
 
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54 
 
 MICROSCOPICAL METHODS. 
 
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METHODS OF STAINING. 
 
 55 
 
 FILTERING REAGENT BOTTLE.l 
 
 A wide-mouth bottle of convenient size is fitted with a cork, 
 through which three glass tubes are passed, one of which (Fig. 
 1, a) reaches nearly to the bottom of the bottle, while the 
 other two tubes extend only just below the cork. 
 
 The long tube is curved above the cork; and at a point just 
 beyond the curve there is attached to it a short piece of tubing 
 (5) of twice the diameter of the first. The lower end of this 
 larger tube (at d ) is also fitted with a cork, through which 
 passes a short piece of small tubing, which is slightly contracted 
 at its distal end. Over the 
 cork and open end of this 
 short delivery tube, a piece 
 of fine muslin (d) is stretch- 
 ed, and the space (b) in the 
 large tube is filled with a 
 loosely packed plug of ab- 
 sorbent cotton, forming an 
 effectual filter. The sec- 
 ond tube ends above the 
 cork in a " thistle-bulb " 
 funnel (e), the opening in 
 which is formed into a 
 neck of sufficient length 
 to enable one to cork it 
 securely. Through this 
 thistle-top tube the rea- 
 gent may be readily pour- 
 ed from a dish or other vessel. The third tube (g) is bent over 
 the side of the bottle, and a piece of rubber tubing is attached, 
 which may end either in a short glass mouth-piece or atomizer- 
 bulb. In case the bulb is used, it will be found necessary to 
 loosen the cork in the funnel-tube (e) when a sufficient quantity 
 of the contents has been expelled, in order to release the pres- 
 sure and stop the flow. 
 
 When the plug of cotton becomes foul, which will not be for 
 
 Fig. 1.— Filtering bottle. 
 
 1 C. E. Hanaman. Am. Mo. Mic. Journ., March, 1883. 
 
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56 MICROSCOPICAL METHODS. 
 
 a long time, even with hsematoxylin stain, it may be replaced 
 by a fresh one by simply uncorking the lower end (d ) of the 
 large tube, pulling out the old plug with a pair of forceps, and 
 inserting a fresh one. The cotton should be but loosely packed 
 to work easily. 
 
 IMPREGNATION" WITH GOLD CHLOEIDE. 
 
 Gold chloride is one of the best, and at the same time one of 
 the most variable, agents for staining nerve fibres. Its uncer- 
 tain behavior is unquestionably due to imperfections in the 
 methods of using it ; and these imperfections are attributable 
 in part to circumstances and conditions beyond control, but 
 mainly to a failure to recognize some details in the process 
 which are essential to success. The discoverer of a new method 
 of using this agent is rarely able to claim that its application is 
 attended with uniformly successful results, even in his own 
 experience; and still less is he able to prescribe rules which 
 shall guide others to invariable success. A capital illustration 
 of the unreliableness of gold-chloride methods in general is 
 furnished in that of Prof. Flechsig (" Die Leitungsbahnen im 
 Gehirn und Riickenmark des Menschen," 1876). After stating 
 a method that has conducted him to successful results, Flechsig 
 adds, " We are not yet in a position to give directions with 
 sufficient exactness to guarantee certain success in the opera- 
 tion, since the conditions which work especially favorably or 
 unfavorably are not precisely known." Of this method, as of 
 most others, the best that can be said is, that it is very valuable 
 if it succeeds. ( Vide Chapter XIII.) 
 
 METHODS OF BLEACHING OVER-COLORED GOLD PREPARA- 
 TIONS. 
 
 Redding 1 washes the over-stained tissues in weak f erricyanide 
 of potassium. 
 
 Cybulsky z decolored with cyanide of potassium. The object 
 
 1 Proc. Amer. Micr. Soc, p. 183, 1882. 
 
 2 Zeitschr. f. wiss. Zool., XXXIX., p. 657, 658, 1883. 
 
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METHODS OF STAINING. 57 
 
 is placed in a few drops of a $ % solution on the slide, and 
 allowed to remain from one half to two minutes. As soon as 
 the color has faded sufficiently, the solution is removed by the 
 aid of filtering paper, replaced with glycerine, and then covered 
 with a cover-glass. By this process the object is not only par- 
 tially bleached, but the epithelia are loosened and easily re- 
 moved by pressure on the cover. 
 
 IMPREGNATION WITH NITRATE OP SILVER. 
 
 Nitrate of silver 1 is usually employed in solutions of -§-2$, 
 but may also be used in a solid form. In the latter form, it is 
 applicable only to dense tissues, such as the cornea and carti- 
 lage ; and the layers with which it comes in direct contact are 
 destroyed. 2 In the case of the cornea, the solid piece is passed 
 rapidly over the anterior surface of the membrane while still in 
 situ. The cornea is then detached and laid in distilled water, 
 and the epithelium removed with a brush. The nitrate of silver 
 dissolves in the water that bathes the object, passes through 
 the epithelium, and is reduced in the fibrous tissue by the 
 action of the light. 8 
 
 Solutions are employed for two purposes : first, to bring out 
 clearly cell boundaries in epithelia ; second, to demonstrate 
 small cavities and lymph-spaces. In the first case, through the 
 impregnation of the intercellular substance, so-called negative 
 images are obtained; while in the second, positive ones are 
 produced, the cavities and spaces themselves becoming black. 
 
 Pieces of fresh epithelium may be left in the solution a few 
 minutes (from 5 to 30), then transferred to a 2 f solution of 
 acetic acid, and exposed to strong daylight or direct sunlight 
 until they turn distinctly brown. 
 
 The over-blackening of the preparations may be prevented 
 by washing them in a 10 <f solution of hyposulphite of sodium 
 (Na 2 S 2 3 ), as recommended by Legros. 4 The silver compound 
 
 1 Kept in black bottles. 
 
 2 Heitzmann, Micr. BTorph. of the Animal Body, p. 9. 
 
 3 Eanvier. Traits Technique, p. 105. 
 
 * Journ. de l'Anatomie, 1868, No. 3, p. 275. 
 
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58 MICROSCOPICAL METHODS. 
 
 (insoluble in water), which has not been acted upon by the 
 light, forms with the hyposulphite a double salt, which is solu- 
 ble in water; and this double salt, together with the super- 
 fluous hyposulphite, is removed by washing the preparation 
 thoroughly in distilled water. 
 
 Bleaching. — Preparations that have become too black may 
 be bleached by the action of the hyposulphite, or by cyanide of 
 potassium (1 : 10). 
 
 In the case of marine animals, a difficulty in the way of using 
 nitrate of silver is met with, for salt precipitates the silver 
 instantly. This difficulty may be avoided in two ways : first, 
 the object may be killed in weak osmic acid, and then, after 
 washing in distilled water long enough to remove most of the 
 salt, placed in the silver solution, and treated in the ordinary 
 way ; 1 second, the object may be transferred from sea water to a 
 0.5 fo solution of potassic nitrate, in which the tissues are freed 
 from the greater part of their chlorides in a short time, and 
 from which, while still living, the object is transferred to the 
 silver solution, (v. Harmer's Method.) 
 
 Alcoholic preparations may be treated with an alcoholic 
 solution of silver nitrate ( circa 2 %)\ 
 
 In order to demonstrate the existence of small cavities in the 
 tissues (" positive images " — His), it is necessary to expose the 
 object to a rather strong solution for a longer time than usual 
 (in the dark). It is next placed in a solution of salt (2-5 f c ) for 
 a few minutes, and then washed in distilled water, and left for 
 a time in the light. The spaces appear to be filled with black 
 granules. 
 
 In the investigation of nerve-fibres, Golgi has combined 
 treatment with a mixture of bichromate of potassium and 
 osmic acid with silver impregnation, {v. Chapter XIII.) 
 
 PALLADIUM CHLORIDE (Schulze). 
 
 F. E. Schulze, 8 to whom we are indebted for the introduction 
 of this agent into histological technique, recommended it as an 
 
 1 R. Hertwig. Jen. Zeitschr. f. Nat., XIV., pp. 313, 324. 
 
 2 Fol. Lehrbuch d. vergl. mik. Anat., p. 179. 
 8 Centralbl. f. d. med. Wiss., No. 13, 1867. 
 
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METHODS OF STAINING. 59 
 
 excellent means of hardening and staining small pieces (size of 
 a bean or smaller) of organs, especially for muscular tissue. 
 Muscle-fibres, gland-cells, and epithelia stain bright yellow, 
 while fatty and connective tissue remain uncolored. 
 
 Henle and Merkel 1 employed it for sections of the central 
 nervous system, for which purpose it has since been almost 
 exclusively used in Germany. 
 
 The sections are first j)laced in a ^ — \% palladium chloride 
 for one to two minutes, then transferred directly to a strong 
 solution of ammonia carmine (a minute or more). Thus treated, 
 the axis-cylinder stains bright red, the medullary sheath yellow. 
 
 Thanhoffer recommends palladium dichloride for the nerves 
 of the cornea. Palladium has been very highly praised by 
 Bastian, and through him it has acquired general recognition in 
 England. 
 
 Schulze used a -^ % solution, and allowed it to act twenty- 
 four hours, at the end of which time the preparation is hardened 
 sufficiently for sectioning. According to Fol (op. cit., p. 180), 
 smooth muscular fibres are stained clear yellow, while striated 
 fibres take a brownish hue. 
 
 1 Henle' s Handbuch der Nervenlehre der Menschen, 1871. 
 
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CHAPTEE III. 
 INSTRUMENTS. 
 
 MICROTOMES. 
 
 Perhaps no instrument of scientific research has ever under- 
 gone more rapid alteration and variation than has the micro- 
 tome within the last ten years, and especially during the last 
 five. The rapid multiplication of different patterns, the endless 
 number of improvements and additions, all show how very 
 important, as an instrument of investigation, the microtome 
 has become in the estimation of embryo] ogists, histologists, 
 anatomists, and physiologists. The sneers that greeted its 
 introduction from some quarters have hastily subsided; and 
 now it is universally acknowledged to be an indispensable part 
 of the outfit of every respectable laboratory. 
 
 The various microtomes now in use may all be regarded as 
 modifications of two types (Oschatz and Rivet). The chief 
 difference between the two lies in the method of raising the 
 object. In one of these — the best models of which are 
 the Schanze microtome (Leipzig) and the Zeiss microtome — 
 the object is raised by a micrometer screw ; while in the sledge 
 microtome — the best form of which is the Thoma instrument 
 made by Jung of Heidelberg — it is raised by being pushed up 
 an inclined plane. In this difference lies the superiority of the 
 sledge over the screw microtome. A finer precision and a 
 more perfect uniformity in the elevation of the object are 
 attainable with the latter than with the former. It is hardly 
 possible to reach a fineness of liv™"" in the vertical elevation 
 of the object by means of the screw ; while in the sledge prin- 
 
 60 
 
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INSTRUMENTS. 
 
 61 
 
 ciple of construction the possibility of raising the object only 
 •nsW™ nas already been realized. 
 
 An interesting combination of the sliding and screw move- 
 ment is presented in the new microtome made by C. Reichert, 1 
 in Vienna. In this instrument the object is raised automati- 
 cally, which must be regarded as an important improvement. 
 
 As the Thoma, Schanze, and Caldwell microtomes are now 
 most widely known and used, they have been selected for 
 description. 
 
 THE THOMA MICROTOME. 
 
 This microtome (Fig. 2) consists of a stand of cast iron, on 
 which slide two carriers. The large knife is attached to one of 
 
 Fig. 2. — Thoma's microtome, a, carrier for the knife ; b, carrier for the object ; 
 c, micrometer-screw for fine adjustment. 
 
 these, a, which slides horizontally. The second, J, holds the 
 specimen to be cut. This second moves on an inclined surface, 
 so as to raise the specimen as required. 
 
 This, with a few modifications, is the general character of all 
 sliding microtomes ; but hitherto the carriers were constructed 
 to slide with two even surfaces between two even planes of the 
 stand, which intersect at a given angle, with the consequence 
 that all show more or less imperfect results, owing to the fact 
 that it is impossible to obtain sufficiently exact plane surfaces. 
 
 ' Described by Moeller in Zeitschr. f. wiss. Mikroskopie, I., Heft 2, 
 pp. 241-244, 1884. 
 
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62 MICROSCOPICAL METHODS. 
 
 The inconveniences appear in small, scarcely perceptible irregu- 
 larities of the movement of the carriers, and the consequent 
 impossibility of making sections as thin as with an experienced 
 hand. 
 
 This induced Professor Thoma to enter upon a consideration 
 of the geometrical and mechanical difficulties to be surmounted. 
 The question to be solved was, how many points at least of a 
 body sliding between two planes must touch the latter for this 
 body to be perfectly steady in its position. It will be found 
 that five points are sufficient, and that a carrier on five points, 
 between two plane surfaces, will slide without difficulty between 
 these planes, even if they are not absolutely geometrical planes, 
 or the angle which they include is not everywhere the same. 
 Such a carrier will always take exactly the same course ; and, 
 in consequence, a knife attached to it will cut a series of per- 
 fectly parallel sections through an object which is successively 
 raised to a higher plane after each cut. The working of the 
 instrument will therefore be far superior to any microtome with 
 large sliding surfaces which nowhere exactly fit the sliding sur- 
 faces of the stand. This indicates the desirability of construct- 
 ing the carrier for the object also on five points. 
 
 The construction resulting from these principles is simple 
 and practical, but it is necessary to take into consideration the 
 centres of gravity of the different sliding bodies. This, how- 
 ever, complicates the matter but very little. We replace the 
 two sliding surfaces of each carrier by five slightly prominent 
 points, and they will then move with exactness on any combi- 
 nation of two planes not differing too much from geometrically 
 plane surfaces. One condition only must be fulfilled, namely, 
 that the five points are so chosen as to support steadily the 
 centre of gravity of the carriers, including their accessory parts, 
 namely, the knife and object. Fig. 3 gives a more precise idea 
 of the details of construction. 
 
 In the figure, the lower surfaces of the carrier, a, which sup- 
 ports the knife, show three prominences, which give the 
 geometrical projection of the five points. Within the limits of 
 the figures these points could not be drawn exactly as they are 
 in the instrument itself. In reality, they appear only as small 
 
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INSTRUMENTS. 
 
 63 
 
 Fig. 3. — Transverse section 
 of the microtome, s s, stand ; 
 a, carrier for the knife ; &, 
 carrier for the object. 
 
 prominences upon three narrow ridges on the sliding surfaces 
 of the stand. This arrangement was desirable to facilitate the 
 action of the oil with which the sliding surfaces are to be cov- 
 ered. Two of the ridges form together parts of the oblique 
 plane, and the third corresponds to the 
 vertical sliding plane. The same ar- 
 rangement is found in the carrier, b, 
 which supports the clamp in which the 
 object is placed. 
 
 By this mode of construction the car- 
 riers will move gently and regularly, 
 even if the sliding surfaces on the stand 
 are not perfect geometrical planes. It 
 is still, however, of course, desirable that 
 as much exactness as possible should be 
 obtained in these planes, as their irregu- 
 larities cannot fail to affect the sec- 
 tions, especially as they are, in fact, multiplied in the latter. 
 
 Specimens which are well hardened will allow of sections of 
 three to four square centimeters surface and 0.015 to 0.10 mm 
 thickness. In exceptional cases pieces of so large a surface 
 may be cut of 0.005 mm thickness. If the section is smaller (for 
 instance, one centimeter square), the thickness can be reduced 
 considerably, say to 0.005 mm , or in extreme cases to half that. 
 It is not, however, all tissues and objects that will admit of 
 sections of such delicacy. Well-hardened liver may generally 
 be cut to 0.015 mm , this being about the diameter of the hard- 
 ened cell. Occasionally, however, in this tissue, sections of 
 0.010 mm can be obtained. Lymphatic glands and brain may be 
 cut to 0.010 or 0.075 mm ; embryonic tissues, well imbedded, 
 usually admit sections of 0.005 and 0.003 mm . In some cases 
 even sections of 0.002 mm thickness can be obtained. These 
 numbers refer to the largest size of the microtome, and to serial 
 sections. The two smaller sizes will give sections of the same 
 delicacy, but comparatively smaller in extent of surface. The 
 length of the sliding surfaces of the large instrument is 40"™, 
 and the edge of the knife is 23 cm . In the medium size these dimen- 
 sions are 27 and 16 cm , and in the smallest about 21 and ll cm . 
 
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64 
 
 MICROSCOPICAL METHODS. 
 
 Sliding microtomes are in general constructed for cutting 
 sections of tissues previously hardened in alcohol, picric acid, 
 chromic salts, and other agents. Fresh tissues are decidedly 
 better cut by freezing microtomes — for instance, on the simple 
 and practical instrument of Hughes and Lewis. The addition 
 of a freezing apparatus to a thoroughly exact sliding microtome 
 is neither advisable nor necessary. The differences of tempera- 
 ture produced in different parts of the instrument would be 
 apt to interfere with the perfect planeness of the sliding sur- 
 faces ; whilst, on the other hand, section-cutting with frozen 
 tissues is so simple and easy with the ordinary freezing appara- 
 tus, that any further complication in the way of a sliding sup- 
 port of the knife is superfluous. 
 
 . In cutting, the microtome is to be placed before the operator 
 as in Fig. 2, with the sliding surfaces abundantly covered with 
 oil (bone oil). In many cases it will be sufficient to simply 
 .place the hardened specimen between the arms of the clamp 
 attached to the carrier, b. The clamp should then be fixed- 
 in such a position that the specimen is as near as possible 
 to the knife-carrier. The knife will generally have to be ad- 
 justed so as to bring the whole length of its blade into action. 
 Very hard specimens are frequently cut with less difficulty by 
 
 placingthe knife 
 more obliquely 
 in regard to the 
 ?0 long diameter of 
 the instrument. 
 The inclina- 
 tion of the ob- 
 lique plane upon 
 which the car- 
 rier, b, slides is 
 1 : 20, and conse- 
 quently the sec- 
 tion will be l-20th mm thick if the carrier is moved l mm on the 
 oblique plane. A scale in millimeters with a vernier allows the 
 operations to be exactly regulated. The vernier will be found 
 sufficient for sections of 0.015 mm . Sections of greater delicacy 
 
 Fig. 4.— Micrometer-screw for delicate sections. 
 
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INSTRUMENTS. 
 
 65 
 
 should always be made by using the micrometer-screw, which 
 was designed to obtain the utmost exactitude in the manage- 
 ment of the carrier, b. Fig. 4 shows it on a larger scale. 
 
 The carrier, & slides on the same oblique plane as the carrier 
 b which holds the specimen. In all positions of the latter it is 
 therefore possible to bring the point of the micrometer screw, 
 g g, close to a small polished plate of agate, /, which is fixed to 
 the carrier, b. In this position c should be firmly screwed to 
 the stand of the microtome by d, and every revolution of the 
 micrometer-screw, g g, will then push the carrier, b, 0.3 mm . The 
 periphery of the drum, t, which is firmly attached to the screw? 
 g g, is divided into fifteen equal parts ; and consequently each 
 division marks a thickness of section equivalent to 0.001 mm . 
 The finest sections hitherto produced reach only 0.002 mm thick- 
 ness. 1 
 
 Since the first microtome was taken into use, a series of im- 
 portant improvements c 
 have been made. One 
 of them consists in a 
 clamp (Fig. 5) for 
 holding the object, 
 which can be turned 
 round three axes, and 
 admits, therefore, of a 
 very easy adjustment 
 of the object in regard 
 to the knife. It was devised to meet the desire for occasionally 
 turning the object between two successive series of sections. 
 
 The two metal plates, h h, form the jaws of the clamp. Be- 
 tween them is placed the cork which carries the specimen, and 
 
 Fig. 5.- 
 
 Clamp to be turned in three directions (as 
 seen from above). 
 
 1 Professor E. Kossman writes: "Many to whom the turning back of the 
 micrometer-screw of the microtome is an annoying delay will be thankful 
 to me for pointing out to them that in two or three seconds it can be 
 turned back its whole length by using a kind of fiddle-bow, such as is 
 used for drilling holes. The loop of the bow-string (made of strong silk 
 cord, waxed or rosined) is passed round the smooth neck of the screw, 
 and the bow is moved alternately to the left with stretched, and to the 
 right with slackened, cord. 
 
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66 
 
 MICROSCOPICAL METHODS. 
 
 the latter is fixed by turning the screws, g g. The three axes 
 are a, b b, and c c, and round these the clamp can be turned, a 
 being vertical, and b b and c c horizontal. In all positions these 
 three axes can be made immovable by the screws d, e, and f. 
 The axis, a, is formed by the vertical rod, e (Fig. 3), on the 
 carrier supporting the clamp and object. The details of the 
 construction are partly new, and are very solid and durable. 
 Their arrangement is such as to admit of a division of the cir- 
 cles in which the clamp can be turned. Another improvement 
 is the registering micrometer-screw described below. 
 
 The Registering Micrometer-Screw. — In the improved 
 form of the micrometer-screw, invented and described by 
 Mayer, Andres, and Giesbrecht, an arrangement for regulating 
 its movement has been added. It consists of a spring which, 
 after a given number of divisions of the drum, registers to the 
 ear and finger of the manipulator the number of micromilli- 
 
 meters which the object has 
 been raised. The intervals 
 between the registering clicks 
 can be varied by means of a 
 vernier-like adjustment of the 
 two halves of the drum, so as 
 to equal an entire revolution 
 of the drum, or only -fa, ^, \ 
 of a revolution. 
 
 An examination of Fig. 6, 
 which illustrates the new 
 form of the drum, will show 
 how the intervals are regu- 
 lated. The drum is composed 
 of two symmetrical halves, 
 A B and A' B', so closely 
 apposed that the dividing line 
 (dotted in the figure) is scarcely visible. The periphery of each 
 half is composed of two zones of unequal radii. The larger 
 zones, B and B', are in apposition, and together form the gradu- 
 ated portion of the drum. Each of the smaller zones are marked 
 with the figures 1, 2, 3, and 15. When the drum is in 
 
 Fig. 6. — Drum of the new micrometer- 
 screw. 
 
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INSTRUMENTS. 67 
 
 order for work, it rotates with, the screw, which is marked 
 g g in Fig. 4. 
 
 The left half of the drum, A B, is held in position by the 
 screw, s, and may be rotated independently of the right half, 
 A 1 B', or of the screw, g g, by the aid of a handle which fits the 
 holes xxx. 
 
 When the half A B is adjusted to the half A' B', in the 
 manner represented in the figure, the fifteen equal parts into 
 which the zone B is divided exactly correspond to the same 
 number of parts in the zone B 1 , so that the grooves which mark 
 these parts in one zone become continuous with those of the 
 other zone. Thus adjusted, the spring, which rides on the 
 ruffle, B B', with a sharp edge parallel to the grooves, will give 
 fifteen sharp clicks in the course of one rotation of the drum, 
 the click being heard every time the sharp edge falls into coin- 
 cident grooves. In order to adjust for fifteen clicks, it is only 
 necessary to rotate A B until groove fifteen becomes continuous 
 with groove fifteen of the opposite half (A' B'). For one 
 click in one rotation, the grooves 1 1 must be made to coincide ; 
 for two clicks, the grooves 2 2 ; and for three clicks, the 
 grooves 3 3. The intervals between successive clicks may 
 thus be made to correspond to \, J, £, or -^ of a complete 
 rotation of the drum; and the thickness of sections cor- 
 responding to these intervals should be respectively .015, .0075, 
 005, .001"" 11 . 
 
 The New Object-Holder. — The object-holder which 
 now accompanies the Thoma microtome is an invention of 
 Mayer, Andres, and Giesbrecht. The object is now movable 
 in all three directions. It is raised or lowered, and turned 
 about the perpendicular axis by free-hand ; but in the two 
 other planes it is moved by pinions, so that the plane of section 
 may be altered at pleasure during the process of cutting. As 
 seen in Fig. 7, the object, o, imbedded in a small piece of 
 paraffine, is attached to a larger mass of paraffine contained in 
 a hollow metallic cylinder. The cylinder may be slipped up 
 or down in the cubical block, A, and turned around its longer 
 axis by means of a small metallic rod applied in holes near its 
 lower end (holes not seen in figure). The position of the 
 
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G8 
 
 MICROSCOPICAL METHODS. 
 
 cylinder may be fixed by the handle, b, which works like the 
 handle of a vice. 
 
 The block, A, may be turned about the transverse axis of the 
 frame which holds it, by the pinion, D, and fixed by the screw, 
 E, the head of which is provided with holes for the metallic 
 rod. 
 
 In the same way, the frame itself may be made to turn about 
 its longer axis by means of the pinion, G, and fixed by the 
 screw, F. 
 
 The chief merit of this holder lies in the fact that the object 
 
 Fig. 7.— The Object-holder and Carrier. 
 
 may be rotated very freely about the two axes of the frame, 
 without, at the same time, being raised or lowered very much. 
 This latter advantage depends on the fact that the axes of 
 rotation are near the top of the block, A; i. e., as near the 
 object as possible. 
 
 The attachment of the object to a cylinder movable in a 
 perpendicular direction has the great advantage that pieces of 
 more than two centimeters in length may be sectioned. In 
 order to obtain room for pieces of greater length, washers of 
 
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INSTRUMENTS. 69 
 
 .5, or 1™, thickness may be placed at first under the knife, and 
 afterwards removed. 
 
 An Improvement in the Carriers. — The advantages of 
 having the carriers slide on five points (instead of two even 
 surfaces) between two even planes have been thoroughly dis- 
 cussed by Thoma. The most recent improvement consists in 
 making the so-called "points," of ivory, and the planes on 
 which the points slide of an alloy of zinc and copper (Roth- 
 guss). The result is, that these parts are no longer exposed to 
 rust, and that the plane surfaces on which the knife-carrier 
 slides are not exposed to injurious friction. 
 
 Prices of the Microtome and its Adjuncts : — 
 
 The older microtome, consisting of a stand of cast- 
 iron and the two carriers, without the micro- 
 meter-screw $24.00 ( 95 marks) 
 
 Same with ivory "points" and new object- 
 holder 27.00 (110 " ) 
 
 The registering micrometer-screw 10.00 (40 " ) 
 
 Knife for oblique cutting (16 om long), with e'tui . 4.75(19 " ) 
 
 Knife for transverse cutting, with etui, holder, and 
 
 clamp 4.75 (19 " ) 
 
 Section-smoother 2.25 ( 9 " ) 
 
 The complete set, including stand with " planes " of 
 zinc and copper alloy, and ivory " points," regis- 
 tering micrometer-screw, the two kinds of 
 knives with etuis, and section-smoother . . 53.00 (212 " ) 
 
 This microtome can be obtained from the maker, Rudolph 
 Jung, 15 Hauptstrasse, Heidelberg, or from the Educational 
 Supply Company, 6 Hamilton Place, Boston, Mass. 
 
 THE SCHANZE MICROTOME. 
 
 This instrument will be easily understood from the figure. 
 It combines both the screw and the sliding movement. The 
 knife-carrier slides, as in the sledge microtome, but the object 
 is raised by a screw, provided with a graduated drum, (Fig. 8, 
 S). The Zeiss microtome, described by Korting, 1 is constructed 
 on the same plan. 
 
 1 Jen. Zeitschr. XIV., pp. 193-195, 1880. 
 
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70 
 
 MICROSCOPICAL METHODS. 
 
 The instrument is made of brass, or, preferably, of iron, with 
 nickel-plated surfaces. The micrometer-screw moves the verti- 
 cal plate, P, which slides up and down between two planes, and 
 bears the object-holder. By means of screws, two of which are 
 placed at right angles to each other, the holder can be inclined 
 in any direction, raised or lowered, and rotated on its own axis. 
 
 bBMpo^^S BIB 
 
 *M«S 
 
 Fig. 8.— Schanze Microtome. 
 
 One rotation of the drum, S, raises the object .S nm -; division of 
 the drum into 30 degrees regulates the movement to hundredths 
 of a millimeter. 
 
 CALDWELL'S AUTOMATIC MICROTOME. 1 
 
 This machine has been devised to save labor to the histologist 
 by cutting a very great number of sections suitable for micro- 
 scopic investigation in a very short time. The machine is 
 worked by hand, and may easily be made to deliver, in one con- 
 tinuous band, accurately cut sections at the rate of 100 per 
 minute. To use it, however, to the best advantage, it is well 
 to drive it by means of some motor, the fly-wheel being already 
 
 1 Quart. Jour. Micr. 8c, XXIV., Oct., 1884, p. 64S. 
 
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INSTRUMENTS. 71 
 
 provided with a groove for the reception of the cord coming 
 from the motor. "Where there is sufficient pressure and supply 
 of water, a simple form of water motor seems the most appro- 
 priate and least expensive. 
 
 Method of using the Microtome. — Place one of the 
 cylindrical vessels supplied with the machine upon a piece of 
 paper on a glass plate, and pour into it sufficient melted paraf- 
 fine to fill it. As this cools, the paraffine will contract, and 
 will leave a hole, which must he filled up with more melted 
 paraffine. 
 
 Melt a small quantity — say an ounce — of imbedding material 
 in some suitable vessel ; a small copper pan or a porcelain cruci- 
 ble answers very well, if care is taken not to allow it to become 
 hotter than is sufficient to thoroughly melt it. Take a piece 
 of glass, and smear it with a very small quantity of glycerine, 
 to prevent the imbedding material from sticking to it. Then 
 pour the melted material on the glass in small quantities at a 
 time, so as to get a layer nearly a quarter of an inch thick. 
 This, when cut up into suitable pieces with a knife, does very 
 well for imbedding small objects. If larger objects are re- 
 quired, it is well to have two pieces of brass of the form shown 
 in Fig. 9, 5, which, when placed together, will form a cavity half 
 an inch in depth and of any desired length up to an inch or 
 more. This cavity may be filled with the melted material in 
 the manner already described, and the object to be cut must 
 then be placed in position while the material is fluid. It is well 
 to cool the material as rapidly as possible by placing it in water 
 as soon as it is sufficiently set. From the cake thus formed, or 
 from the piece cast in the mould, cut the piece of the material 
 containing the object, and with an old scalpel, heated in a 
 Bunsen flame, melt a small hole in the paraffine contained in 
 the cylindrical vessel (Fig. 9, 1, a), and insert the piece of imbed- 
 ding material containing the imbedded object ; then with the 
 heated scalpel melt a little of the paraffine round the base of 
 the projecting piece, so as to give it firm support, and allow 
 this to become thoroughly set. 
 
 Now remove the large brass plate from the top of the micro- 
 tome (Fig. 9, 1, b), and insert the vessel containing the imbedded 
 
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72 
 
 MICROSCOPICAL METHODS. 
 
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INSTRUMENTS. 73 
 
 object in the tube for its reception, having first oiled the tube 
 slightly to prevent the vessel from sticking. Next with a 
 sharp knife cut the material with the object imbedded in it so 
 that all its opposite sides are parallel. This is extremely im- 
 portant. Replace the top plate, and fix the razor in the holder 
 provided for the purpose. The clam]3 is so made, that, if a little 
 care is taken, the edge of the razor will not be injured (Fig. 9, 6). 
 The razor must be set so that its back is as high as possible, as 
 shown in Fig. 9, 6, and, above all, the razor must be extremely 
 sharp. It should be sharpened on a stone, and not on a strop. 
 The sharpness of the razor and the accurate parallelism of the 
 sides of the mass to be cut are the most important points in 
 the whole process. 1 Underneath the frame containing the 
 object is a large brass milled head (Fig. 9, 2, c). By turning this 
 the object may be raised or lowered according to the direction 
 in which it is turned. This should be done until the object is 
 just below the edge of the razor. The plate holding the razor 
 should then be moved so that the edge of the razor is close to 
 and quite parallel with the mass of material to be cut (Fig. 9, 3). 2 
 The plate should then be clamped by the screws at each side of 
 it. A few turns of the fly-wheel will now bring the razor in 
 contact with the object to be cut. The band of black ribbon 
 (Figs. 9, 1 and 3 d) is now to be placed so that the end of it 
 should be just above the razor, and clamped in that position. 
 
 1 The makers of the instrument have nearly completed an automatic 
 machine for sharpening razors, since it has occurred to them that this is 
 an operation which may be performed with much greater . ccuracy by 
 mechanical means than by hand. 
 
 1 The distance through which the sliding stage moves can he altered by 
 raising or lowering the arm (Figs. 9, 1, and 9, 4J). This distance should be 
 so arranged that the surface of the imbedding mass containing the object to 
 be cut just clears the razor when the sliding carriage is at its maximum 
 and minimum distance from either end of the machine. This is impor- 
 tant, as the speed with which the black band travels varies directly with 
 the throw of the machine. If this adjustment is made, and a little care 
 is used in adjusting the ring (Fig. 9, 1, e), — see below, — the ribbon will 
 move at each turn of the fly-wheel through a distance equal to the 
 breadth of the surface which is being cut. If, on the other hand, the 
 object swings far beyond the razor, the band will travel too quickly, and 
 probably break the string of sections. 
 
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74 MICROSCOPICAL METHODS. 
 
 When the handle is turned, the sections should come off the 
 razor in the form of a ribbon. 
 
 The ribbon of sections will not find its way to the continuous 
 black band without assistance. With a needle in a handle, or 
 with the point of a scalpel, pick up the end of the ribbon, when 
 a sufficient length of it has been cut, and place it on the black 
 continuous band, up which it will travel. When it reaches the 
 top of the band, suitable lengths may be cut off with a pair of 
 scissors. It may be found that the black band travels either 
 too slowly or too fast. Its speed may be varied by moving the 
 ring (Fig. 9, 1 e) up or down upon the vertical brass arm — up- 
 wards, if it is moving too fast ; downwards, if too slow. A 
 frequent cause of failure in the proper movement of the band 
 is that the ebonite roller at the bottom of it is allowed to press 
 against the razor ; this must be avoided. 
 
 Varying the Thickness of the Sections. — In Fig. 9, 2, 
 will be seen a milled head, f, which, when turned, controls the 
 movement of the clicks, which, acting upon the ratchet-wheel 
 attached to the micrometer-screw, regulate the thickness of the 
 sections. This may be done so as to allow the clicks to engage 
 one half, one, or several teeth of the ratchet-wheel, as may be re- 
 quired. When arranged for one half tooth, the sections will be 
 tijotto °f an mcn (.0025 mm ) in thickness ; when arranged to en- 
 gage a whole tooth, -g-fos of an inch (.005 mm ), and so on. At first 
 it is well to use a whole tooth, as when thinner sections are cut so 
 much depends on the sharpness of the razor. After cutting for 
 some timo, the machine will suddenly stop, the object ceasing 
 to rise when the handle is turned. This means that the full 
 extent of the micrometer-screw has been reached. It is neces- 
 sary then to turn the large milled head (Fig. 9, 2 c) downwards, 
 which will allow the carriage containing the object to fall to its 
 lowest limit. It will be necessary now to raise the socket (Fig. 9, 
 2 g) in which the object is held, so as to be in position to come 
 in contact with the razor. This milled head (c) is useful 
 for rapidly getting the object in proper position, and avoid- 
 ing considerable loss of time in turning the handle. The frame 
 (A) which holds the socket is arranged with two quadrants, 
 so that the socket may be set at any angle desired, and 
 
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INSTRUMENTS. 75 
 
 may be clamped with the milled head underneath it. This is 
 for use when the object has not been symmetrically imbedded. 
 The nut (Fig. 9, 1 i) is for tightening up the spring which draws 
 the carriage of the machine back after having been pulled for- 
 ward. In case this does not work properly, it is only necessary 
 to unloose the two screws, and, with some strong but blunt 
 pieces of steel placed in the two holes, to rotate the nut, so as 
 to give a proper tension to the spiral spring. When this is 
 done, the screws should be tightened up again to keep the nut 
 in place. 
 
 The lock-nuts (Fig. 9, 1 and 2 s) should be screwed up suffi- 
 ciently tight to barely prevent the carriage from falling by its 
 own weight, so that, when the milled head (Fig. 9, 2 c) is screwed 
 down, a slight pressure with the finger is necessary to make the 
 carriage fall. 
 
 To arrange the machine for cutting different-sized blocks of 
 material, it is only necessary to raise or lower the arm (Figs. 9, 1 
 and Aj). When this arm is in a vertical position, the machine 
 is arranged for its maximum traverse ; when turned to the 
 right and placed horizontally, it is at its minimum traverse. 
 The cord, however, must always be in the groove of the 
 wheel, k. 
 
 It is important to keep the strings which give motion to the 
 endless bund in proper position. The string (Fig. 9,11) should 
 go from the end of the wire, m, round the groove, n, in the 
 pulley, and thence to the elastic band, o. The elastic band, o, 
 should be stretched, and placed over the hook attached to the 
 arm,jo, care being taken that the shorter end of the arm, p, is 
 uppermost. The string, q, should be tied to the stud upon 
 which the arm, p, is supported, going thence round the 
 groove, r, of the pulley, and back again to the hook at the 
 longer and lower end of the arm, p, to which it should be tied. 
 
 Method of preparing the Slide. — Make by the aid of 
 heat a viscid solution of white shellac in light-colored creosote. 
 Spread a smooth, thin, and even layer of this solution on a 
 clean, dry slide, with a camel-hair brush or with the little finger. 
 Arrange the ribbon containing the sections on this slide while 
 moist, and place it in the dry shelf of the water bath, which 
 
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76 MICROSCOPICAL METHODS. 
 
 should be at a temperature slightly above the melting point of 
 the imbedding material used. It should be left here until the 
 creosote has evaporated, and the imbedding material melted. 
 Now allow the slide to cool, and then wash it with turpentine 
 until all the imbedding material is dissolved. Canada balsam 
 in chloroform or turpentine and the cover-glass may now be ap- 
 plied in the usual manner. For convenience of mounting, it is 
 extremely important that the ribbon of sections should be quite 
 straight ; and in order to insure this, it is necessary that the 
 sides of the imbedding material from which the sections are cut 
 should be quite parallel. The straight ribbon, when obtained, 
 should be removed to some clean surface, and there cut into 
 lengths appropriate to the size of the cover-glass used. It will 
 be found convenient to use covers at least two inches long ; 
 indeed, a useful length for slides and cover-glass is six inches 
 for the former and four inches for the latter. 
 
 CARE OF THE MICROTOME. 
 
 The planes on which the carriers slide should be cleaned and 
 oiled freely every day when in use. The registering microme- 
 ter-screw is another part that requires frequent cleaning and 
 oiling. 
 
 Neat's-f oot, black-fish, or watch oil will serve the purpose. Or- 
 dinary sewing-machine oil is not to be recommended, as it gums, 
 and thus greatly increases the wear of the instrument and the 
 labor of cleaning. Minot recommends "pure paraffine oil, 
 specific gravity 25." In drawing the knife, no pressure should 
 be exerted, for this soon injures the knife and impairs the 
 movement of the carrier. 
 
 RUTHERFORD'S FREEZING MICROTOME. 1 
 This instrument (Figs. 10, 11) consists of a plate of gun-metal, 
 B, with a circular opening at its centre. The opening leads into 
 a well, A, closed inferiorly by a brass plug (K, Fig. 11), capable 
 of being moved up or down by means of a screw, D. The tissue 
 to be frozen is placed in the well, and sections are made by 
 
 1 Outlines of Practical Histology, pp. 164-169, 1876. 
 
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INSTRUMENTS. 
 
 77 
 
 gliding a knife through the tissue that projects above the level 
 of the brass table. The thickness of the sections is regulated 
 by an indicator. The freezing mixture is placed in the box, 
 C, the water from which flows away by the tube, H. The 
 microtome is clamped to a table by the screw, F. The object 
 is seen in A, Fig. 11, and the freezing mixture in G of the same 
 figure. The brass box is covered with gutta percha. 
 
 The knife should have a back with straight edges, so that it 
 may not tilt in the process of sectioning. The surface that 
 rests on the plate should always be hollow, and it is convenient 
 
 Fig. 10.— Rutherford's Freezing Microtome. 
 
 to have the upper surface the same, unless the sections be very 
 large, when it should be flat or slightly convex. 
 
 The plug (K, Fig. 11) is unscrewed and oiled to prevent its 
 becoming fixed during the freezing. The object and an imbed- 
 ding mass are placed in the well. 
 
 A thick solution of gum answers well as an imbedding agent, 
 as it does not become crystalline when frozen, and can be cut 
 like cheese. If there be any alcohol in the tissue, it must be 
 thoroughly removed by immersion in water from six to twenty- 
 
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78 
 
 MICEOSCOPICAL METHODS. 
 
 four hours, according to the size of the tissue, before it is 
 placed in the gum. It is always advantageous to allow the tis- 
 sue to soak in the gum for twelve to twenty-four hours before 
 freezing, in order that the gum may permeate every part of the 
 tissue, and prevent the formation of a crystalline condition 
 within the frozen tissue. 
 
 Thick gum solution is poured into the well ; and, when a 
 film of ice has formed at the periphery, the tissue should be 
 introduced, and held against the advancing ice until it is fixed. 
 In this way the tissue may be secured in any position for the 
 
 Fig. 11. — Rutherford's Freezing Microtome, sectional view (for explanation see text.) 
 
 process of sectioning. A plate of cork with a weight on it is 
 placed on the microtome plate to prevent the entrance of heat, 
 and also of salt from the freezing mixture. 
 
 Small quantities of finely powdered ice and salt are placed 
 alternately, with the aid of a small spoon, in the freezing box, 
 and are stirred round the well, the tube, H, being carefully 
 kept open to allow the water to escape from the melting ice. 
 When snow can be obtained, it is, of course, used instead of 
 ice. The powdering of the ice is rather tedious, but it may be 
 readily done in that form of sausage machine where two cylin- 
 ders with spiral grooves are rolled against one another. Before 
 putting the ice into this apparatus, it should be wrapped in a 
 thick cloth and broken with a wooden mallet. The freezing 
 
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INSTRUMENTS. 
 
 79 
 
 process is really very simple, and can be fully carried out in 
 from ten to twenty minutes. Of course the period is shorter, 
 the colder the surrounding air. 
 
 It is not necessary to wet the knife, for this is thoroughly 
 done by the thawing gum. The sections often contain a num- 
 ber of air bubbles ; these, however, together with the gum, are 
 soon removed by placing them in dilute alcohol (rectified or 
 methylated spirit, 1 ; water, 2 parts). If the sections are to be 
 
 Fig. 12.— Jacobs' Freezing Microtome. 
 
 preserved for a time, until they are examined or mounted, they 
 are transferred from the dilute alcohol to rectified spirit. When 
 sections are apt to be spoiled by manipulation, they should 
 be placed on the slide before they thaw. They can then be 
 readily washed by making a pool of dilute alcohol around them, 
 which may be changed two or three times. 
 
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80 
 
 MICROSCOPICAL JIETHODS. 
 
 A NEW FREEZING MICROTOME. 
 Dr. F. O. Jacobs lias devised the freezing microtome seen in 
 Figs. 12 and 13. It consists of a copper rod, A, two inches or 
 more in diameter, and six inches high, enclosed by an inner zinc 
 (b) and an outer brass tank (c). Above is the table, D, working 
 on a fine screw (a). Through the centre of the table passes a 
 
 narrower portion of the 
 copper rod, the piston (p). 
 When the inner tank is 
 filled with a mixture of 
 salt, ice, and water, the 
 temperature of the cop- 
 per rod is so reduced as 
 to freeze any object (F) 
 placed on its upper end. 
 The size of the rod is 
 such that its temperature 
 will remain very steady 
 for from four to six hours 
 
 Fig. 13.— Sectional view of Jacobs' freezing without any further care On 
 microtome. ,. « JT 
 
 the part oi the ojDerator. 
 
 By this arrangement, — the advantages of which will be 
 readily seen, — objects can be easily frozen, and without any 
 slop or " muss." 
 
 The imbedding mass is composed of — 
 
 Gum arabic 5 parts 
 
 Gum tragacanth 1 " 
 
 Gelatine 1 " 
 
 The mixture is dissolved in enough warm water to give it 
 the consistency of thin jelly when cold. A little glycerine 
 (1 : 6) is added to the water. 
 
 AN ETHER FREEZING APPARATUS.! 
 A very simple and convenient little freezing apparatus, which 
 can be used with almost any microtome, has recently been de- 
 
 1 Zeitscb. f. Instrumeiitenkunde, Apr., 18S4, pp. 126, 127. 
 
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INSTRUMENTS. 81 
 
 scribed by W. Emil Boecker. Following the principle of the 
 Lewes and the Roy instruments,' the ether spray is thrown upon 
 the under side of the object plate, instead of on the object 
 itself. 
 
 The apparatus consists of a short cylinder (Fig. 14, A), closed 
 above by the plate, P, on which the object is placed for freez- 
 ing. A metallic tube (a), drawn to a fine point, penetrates the 
 base of the cylinder, and another (b) its side. The vertical 
 tube (a) is connected by rubber tubing 
 with a small bellows, while the horizontal 
 tube (5) is similarly connected with a glass 
 tube which passes through the stopper of 
 a bottle containing ether, reaching nearly 
 to the bottom. When the bellows is set 
 in motion, the current of air throws the 
 ether spray against the plate (P), and the 
 rapid evaporation thus produced soon fio. 14.— Boeker's freeamg 
 
 . apparatus. 
 
 lowers the temperature sufficiently to 
 
 freeze the object. The ether vapor and the air escape through 
 a series of holes in the side of the cylinder. A third tube (c) 
 connects with a second glass tube that extends barely through 
 the stopper of the bottle of ether, and thus provides for the 
 admission of air into the bottle, and for the escape of the excess 
 of condensed ether. 
 
 The cylinder is small enough to be received by the holder of 
 the microtome. 
 
 It may be obtained from W. Emil Boecker, in Wetzlar, Ger- 
 many, at sixteen and a half marks ($4.25). 
 
 The Roy microtome, referred to above, is made by Schanze 
 Pathologisches Institut, Liebig-Strasse, Leipzig. 
 
 NOTES ON THE USE OF THE FREEZING MICROTOMES 
 The tendency at the present time is to make all microscopic 
 sections by the dry method after paraffin infiltration and imbed- 
 ding ; but no doubt there is a place, and an important one, for 
 
 1 Arch. f. mikr. Anat., XIX., pp. 137-143, PI. VI., 1880. 
 
 2 From Science Record, No. 6, April, 1884. 
 
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82 MICROSCOPICAL METHODS. 
 
 the freezing microtome in practical histology, and in this note 
 I would like to call attention to what seem to me improve- 
 ments in its use. Disliking greatly the disagreeable " muss " 
 made by ice and salt, it occurred to me to take advantage of 
 the device of plumbers to thaw out water and gas pipes, — to use 
 strong alcohol with the ice or snow instead of salt. By using 
 snow, or finely powdered ice, and 95 % alcohol, a temperature 
 of twenty centigrades below zero is obtained within five min- 
 utes, and this temperature may be maintained with far less 
 trouble than with ice and salt. The microtome used is the 
 Rutherford pattern, modified by placing the drain near the top 
 instead of in the bottom. A rubber tube passing from this 
 drain to a jar preserves the overflow. It requires about 250 cc. 
 of alcohol to freeze and keep frozen one tissue for cutting ; but 
 this is not lost, as little evaporation takes place, and the dilution 
 does no harm for many purposes ; hence the method is not 
 wasteful, while it is much more pleasant and expeditious than 
 with salt. 
 
 Ordinarily tissues are infiltrated with thick gum before freez- 
 ing, and then the sections are soaked in a relatively large 
 amount of water, to remove the gum. Evidently, while soak- 
 ing, staining, and transferring the sections, especially if they 
 be of such an organ as the lungs, there is every liability of 
 their becoming folded or torn. This may be avoided by stain- 
 ing the tissue in the mass, as for dry section-cutting, and then 
 soaking in water to remove any alcohol, and finally completely 
 infiltrating the tissue in a thick solution of very clean gum 
 arabic. "When ready to make the sections, the well of the 
 microtome is filled with the thick gum, and the tissue intro- 
 duced at the proper time, as usual. Before cutting, the gum is 
 cut away from the tissue as in sharpening very bluntly a lead 
 pencil; then, as the sections are cut, they are transferred 
 directly to the slide. After several slides are filled, a drop of 
 glycerine is added to each section, and the cover-glass applied. 
 This is practically mounting in Farrant's solution. — S. H. 
 Gage, 
 
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INSTRUMENTS. 83 
 
 THE USE OF GELATINE JELLY IN THE FREEZING MICROTOME. 1 
 
 Instead of freezing in gum, as is usual, one may use gelatine 
 jelly. This is prepared and clarified in the ordinary manner. 
 It should set into a stiff mass when cold ; how stiff will best 
 be learned by experience. 
 
 The tissue to be cut is transferred from water to the melted 
 jelly, and should remain in it till well permeated. 
 
 It is then placed on the piston of a Rutherford's microtome ; 
 the " well " should not be filled ; for adherence, it is sufficient 
 to roughen the surface of the piston with a file. No more jelly 
 should be used than is sufficient to surround the specimen ; if 
 too much has been added it may be removed, when frozen, by 
 careful paring. 
 
 When well frozen, sections may be cut in the ordinary way ; 
 while frozen, they should be quickly transferred to the glass 
 slide on which they are to be mounted. On touching the glass, 
 the slice of jelly almost immediately thaws and adheres as a con- 
 sistent fibre to the surface. When enough sections have been 
 placed on the slide, they should each be covered with a drop of 
 glycerine (the sooner this is added the better) ; a cover-glass is 
 then superposed, zinc white or some similar cement is run 
 round it, and the preparation is complete. In process of time 
 the glycerine will permeate the gelatine and convert it into gly- 
 cerine jelly ; if this does not take place soon enough, it may 
 be hastened by placing in an oven kept at a temperature of 
 about 20 to 30° C. 
 
 In this way a series of entire sections may be obtained from 
 the most disconnected structures, even when they contain hard 
 silicious spicules, as in the case of sponges. 
 
 Diatoms may be cut without difficulty by this method. 
 
 MICROTOME KNIVES. 
 
 The Scientific Instrument Company of Cambridge, England, 
 are preparing an automatic machine for sharpening microtome 
 
 i J. Sollas. Quart. Jour. Mic. Sc, XXIV. pp. 163-164. Jan. 1884. 
 
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84 MICROSCOPICAL METHODS. 
 
 knives, which, it is to he hoped, may prove to be just what 
 every microtomist so much needs. 
 
 It is easy enough to find men skilled in honing ordinary 
 razors, but not so with the microtome knives. Numerous and 
 repeated trials have been made among the most hopeful ex- 
 perts in and about Boston, but in no case have the knives been 
 returned in an acceptable condition ; and sometimes they have 
 been much damaged or wholly ruined. The best test for the 
 condition of the edge is to try it on the palm of the hand. A 
 knife that will not cut a ribbon of paraffine sections .005™ 
 thick is not fit to use ; the best knives should cut as thin as 
 .001 mm . It is not often that it becomes desirable to cut so thin, 
 but it is important in making thick sections (.02-.01 mm ) to use 
 a knife that has a much finer working capacity. A thoroughly 
 sharp blade may have very nearly a horizontal position for 
 its lower (plane) surface in sectioning, while a duller one 
 requires to have its back raised a little above the level of 
 its cutting edge. It is safe to say that a knife cuts well, 
 when thin sections (.005 mm or less) agree in size with the 
 cut surface of the paraffine block. It may be possible to 
 cut a straight ribbon with a dull knife, but in this case it 
 will probably be found that the sections are shortened in a 
 direction at right angles to the edge of the knife, which shows 
 that the knife is acting the part of a plough, which crushes 
 more than it cuts. 
 
 The statement that a sharp knife may have a nearly hori- 
 zontal position must be understood to have some limitations. 
 In general it may be said that the larger and harder the object, 
 the more imperative it becomes to have the under surface of 
 the blade slant towards the object; and the necessity for this 
 is greater with a transverse than with an oblique knife. For 
 very hard objects, a relatively thick-edged knife is required, as 
 well as a slanting position. 
 
 For ordinary histological or embryological work, the upper 
 surface of the blade is ground hollow, the lower surface plane 
 (Fig. 14, Jc), the edge being left very thin, so that only an ex- 
 tremely slight bevel is made in setting. What bevel there is 
 should be mainly on the upper side. The edge, when exam- 
 
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INSTRUMENTS. 85 
 
 ined with a magnifying power of a hundred diameters, should 
 be perfectly straight and smooth. 
 
 Method of Sharpening. Microtome knives can be prop- 
 erly sharpened only by those who understand their chief pecu- 
 liarities, and who have trained themselves in this special work. 
 The difficulties in acquiring the art are not, however, insur- 
 mountable; for, with the proper 
 means and a little perseverance, 
 they can be mastered in a short 
 time. The first important step is Fle . i 5 ._ Diagram illustrating the 
 to provide one's self either with a ^X&T^^T^ 
 good razor-strop (those made by wire - 
 Zimmerman in Berlin are considered excellent), or with a 
 long and wide oil-stone of the finest quality. Strops made of 
 a leather band, unsupported by a solid base, and kept tense by 
 the aid of a screw working in a frame, should never be em- 
 ployed in sharpening these knives ; for they invariably give a 
 bi-convex edge, with which it is impossible to do fine work. 
 To secure a plane bevel of the cutting edge, the surfaces of 
 the strop must be perfectly smooth, flat, and hard. In using 
 the strop the knife is drawn back and forth, back foremost, 
 without pressure, until the edge appears sharp when tested 
 in the manner before mentioned. 
 
 In using an oil-stone, it is well to cover the surface of the 
 stone with a mixture of glycerine (2 parts) and water (1 part), 
 as recommended by Fol. 1 The blade is laid flat on the stone, 
 and pushed forward, edge foremost, in such a manner that the 
 free end of the knife finishes by resting on the more distant end 
 of the stone. Here the blade is turned on its back and returned, 
 edge in advance as before, to the place of starting. In drawing 
 the blade, the utmost care should be taken never to raise, in the 
 slightest degree, the back from the stone ; and further, the knife 
 must not be pressed on the stone, but held lightly by the finger 
 tips, and the necessary friction be left to capillary adhesion. 
 i. After drawing the knife fifteen or twenty times, it should be 
 tested as before. 
 
 1 Lehrbuch d. vergl. mikr. Anat., p. 129. 1884. 
 
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\ 
 
 86 MICROSCOPICAL METHODS. 
 
 The knives furnished with the Thoma microtome should be 
 provided with a wire support (Fig. 15, w) for the back of the 
 knife during the process of sharpening. 
 
 PREVENTING THE ROLLING OF SECTIONS. 
 The section-smoothers described below have been devised for 
 the prevention of section-rolling. 
 
 Besides the section-smoother, there are other means by which 
 the rolling of sections may be prevented. It may be effected 
 by holding a thin narrow sjjatula over 
 the edge of the knife while cutting. 
 The spatula may be made of brass, in 
 the form of Fig. 16 {A) ; or of paper 
 fastened to a flattened needle, as indi- 
 cated in C. The spatula should be bent 
 slightly (_Z?), and its convex face held 
 over the paraffine without pressure. 
 
 Fi u . .6.-See l i.. n sm ,oi.,,v S . A WUi11 b ™ Sh ' ^^ flnttened » ma J 
 
 be used for the same purpose. 
 ■ Rolling may also be prevented by making the paraffine softer 
 and less elastic, through the addition of a small quantity of 
 vaseline, and lastly — and most effectively — by placing the 
 knife at right angles to the carrier. 1 The discovery of the fact 
 that sections may be cut without rolling, by giving the knife a 
 transverse instead of an oblique position, was made by Mr. Cad- 
 well. Since the discovery of this method, it has come very 
 rapidly into general use, and now Jung's microtome is supplied 
 with ' transverse ' as well as ' oblique' knives. This method, ex- 
 cellent as it is, especially with small objects, does not suffice in 
 all cases, and does not therefore remove the necessity of a sec- 
 tion-smoother. Even with the knife placed transversely, the 
 section-smoother may often be used with advantage, and some- 
 times proves indispensable. 
 
 In this method it is important to use a moderately soft paraf- 
 fine, which may be obtained by mixing, in proper proportions, 
 soft and hard paraffine, and further, to give the piece of paraffine 
 
 l Vide Patten's method, p. 142-143. 
 
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INSTKTJMENTS. 87 
 
 to be cut a rectangular form. The piece must then be so placed 
 in the holder, that the side next to the knife is exactly parallel 
 with the cutting edge. Thus placed, every section lies flat on 
 the blade. The second section pushes on the first, adhering to 
 its adjoining side ; the third pushes on the first two, adhering 
 to the second. A whole ribbon of sections may be cut in this 
 way in a few moments, without danger of losing their serial 
 order. Thus three very important points are gained ; the sec- 
 tions remain perfectly flat, the cutting may be as rapid as the 
 hand can move, and the order of the sections is preserved with- 
 out trouble to the manipulator. Care must be taken only that 
 the opposite sides of the paraffine are parallel, otherwise the 
 ribbon will curve to the right or left, and the arrangement of 
 the sections on the slide be less easily accomplished. 
 
 The method used until quite recently at the Naples Zoologi- 
 cal Station for preventing rolling consists in holding a needle, 
 spatula, or brush lightly over the paraffine during the cutting 
 of each section. This method has now been abandoned for the 
 one described below. 
 
 The Section-Smoother 1 devised by Mayer, Andres, 
 and Giesbrecht. — The section-smoother (" Schnittstrecker ") 
 is an instrument designed to prevent the rolling of sections ; it 
 is attached to the knife itself, and thus accomplishes its work 
 unaided by the hand. 
 
 The accompanying figure represents a portion of the knife, 
 k, with the section-smoother attached, and a block of paraffine, 
 p. A section of the knife and rod,/"', is also given, in order to 
 show the position of the rod above the edge of the knife. 
 
 The most important part of the instrument is the cylindrical 
 steel rod, f, which is supported in a position exactly parallel to, 
 and close above, the cutting edge of the knife. In this position 
 the rod compels the sections to pass between itself and the 
 knife. The parallel position of the rod in the vertical plane is 
 obtained by rotating it about the axis, x, which turns in the 
 hole c or d; the parallel position in the horizontal plane is 
 
 1 "Neuerungen in der Schneidetechnik," in Mittheilungen aus der 
 Zoolog. Station zu Neapel, Vol. IV., p. 429, 1883. 
 
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88 MICROSCOPICAL METHODS. 
 
 reached through the screw, a and a'; and the vertical distance 
 of the rod from the edge of the knife is regulated by the 
 screw, b. The entire apparatus is slipped on at the end of the 
 blade, and held fast by two springs that press upon the under 
 surface of the blade. The rod and its holder, which rotates on 
 the axis, d d, can be lifted up from the edge of the knife by 
 the aid of the handle, e, and turned back far enough to admit 
 
 FIG. 17. —Section-Smoother. After Mayer, Andres, and Giesbrecht. 
 
 of cleaning the rod and the knife, 
 three interchangeable rods, differing 
 to sections of different sizes. 
 
 Schulze's Section-Smoother. 1 
 sists of a small weight supported 
 weight (w), which has the form 
 8 mm in length, and is fixed to the 
 rod, which can be turned about its 
 or down in a ferrule (f), as may 
 
 The apparatus includes 
 in thickness in adaptation 
 
 — This contrivance con- 
 by a steel spring. The 
 of a cylinder, is about 
 lower end of an upright 
 longer axis, or moved up 
 be seen from the figure. 
 
 1 Franz Eilhard Schulze. 
 No. 132, p. 100. 
 
 Eia Schnittstrecker. Zool. Anzeiger, VL, 
 
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INSTRUMENTS. 
 
 89 
 
 One end of the spring (s) supporting the weight is soldered to 
 the ferrule; the other end is held fast in the holder (&), one 
 arm of which is prolonged into an enlarged handle-like portion. 
 The holder (k) fits into a cylindrical hole, which penetrates 
 perpendicularly to a depth of 2-3 mm , the hinder portion of the 
 object-carrier. Within this hole the holder can be turned, or 
 moved up or down ; and these movements, together with those 
 which may be given to the rod (r) and the spring (s), are ample 
 for all adjustments of the weight (w). 
 
 Preparatory to cutting, the weight should be turned so as to 
 be parallel with the edge of the knife, and raised or lowered by 
 means of the rod (r) and the holder (k) until it rests lightly on 
 the upper surface of the paraffine. Slight changes in the pres- 
 
 FlG. 18. — Schulze's Section-Smoother adjusted to a Hock of paraffine (p) preparatory 
 to cutting ; o, the object ; k, parafline-holder ; s, steel spring ; w, weight. 
 
 sure of the weight can be made through the rod (»•) / greater 
 changes can be effected by bending the spring. 
 
 The weight should rest, not directly over the object, but on 
 the edge of the paraffine next to the knife. 
 
 This section-smoother, which can be fitted to any sliding 
 microtome, can be obtained of Fr. Fasching, in Graz, Buger- 
 gasse, No. 13, at a price of 3 \ marks, or 85 cents. 
 
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90 
 
 MICROSCOPICAL METHODS. 
 
 Decker's Section-Flattener. — Dr. F. Decker 1 has in- 
 vented a section-flattener which presents an important improve- 
 ment on those described above. The flattener is a cylindrical 
 glass roller (a), which rotates on a brass rod (b) 
 
 The diameter of the rod is 2 mm , slightly less than the lumen 
 of the roller, so that the latter rotates easily and evenly. 
 
 A steel plate (e), bent so that the two arms clasp the blade 
 firmly, forms a carrier for the roller and its shaft. The carrier 
 is slipped on at the free end of the blade, and the pressure of 
 its arms strengthened by means of the screw (/*), the inner 
 end of which bears against a narrow strip of steel (g). To the 
 upper arm of the carrier a brass block (h) is attached by means 
 of a hinge, which runs lengthwise along the middle of the under 
 
 Fig. 19. — Decker's Section-Flattener. 
 
 side of the block. The short arm of the brass rod (V) passes 
 through the middle of the block in a horizontal direction, and 
 is fixed in any position by means of the screw (k). The dis- 
 tance of the glass roller from the edge of the knife is regulated 
 by the screws, i and %'. If one of these screws is raised, and 
 the other lowered, one half of the block (h) will be raised, and 
 the other half lowered, and the distance between the roller 
 and the blade correspondingly altered. The length of the glass 
 
 1 Ein neuer Schnittstrecker. Arch. f. mikr. Anat., XXIII., pp. 537- 
 543, 1SS4. 
 
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INSTRUMENTS. 91 
 
 cylinder (a) is 5 cm , its diameter should vary according to the 
 size of the object to be sectioned. Three cylinders, measuring 
 4, 6, and 9 mm in diameter, will be found sufficient for all ordi- 
 nary purposes. 
 
 The apparatus above described can be adjusted to knives of 
 different microtomes. It may be obtained from Gustav Stober, 
 in Wtirzburg. 
 
 Kingsley's Section-Smoother. 1 — Led by a suggestion of 
 P. Francotte, 2 Mr. Kingsley devised a simple form of section- 
 smoother, which can readily be made by anyone, and which 
 fairly answers the purpose for which it was intended. A piece 
 of iron wire is bent in the manner shown in the cut, so that the 
 two ends will form a spring 
 clip, grasping the back of < 
 the knife. The middle por- 
 tion is so fixed that it will 
 be parallel with the edge of 
 the knife, and at a distance 
 
 » , , , , . , ~ Fig. 20.— Kingsley's Section-Smoother. 
 
 of about a hundredth of an 
 
 inch from it. For this purpose the writer has found an ordi- 
 nary hairpin, deprived of its lacquer, about the right size. 
 
 In cutting, the section passes between the wire and the blade, 
 and all tendency to curl is prevented. This form of smoother 
 is applicable to the knife when used in any form of sledge 
 microtome, or when cutting free-hand ; but for use with the 
 Sterling (well) microtome, it is evidently ill adapted, for the 
 ends which come underneath the blade would interfere with 
 the work. The form of smoother described by Drs. Gage and 
 Smith embraces the same principle, but is more complicated. 
 
 The Section-Flattener of Gage and Smith 3 — The 
 section-flattener consists of a rod (b) of spring brass, about 
 5 mm in diameter, flattened on two sides (JB and D), extending 
 parallel with the edge of the knife, and projecting about 2 mm 
 beyond it. Opposite the cutting edge, the space between the 
 
 1 J. S. Kingsley, Science Record, 11, No. 5, p. 112, March 15, 1884. 
 "■ Bull. Soc. Beige de Microsc, X., 1883-4, No. 3, p. 55. 
 8 "Section-Flattener for Dry Section-Cutting." The Microscope, Feb- 
 ruary, 1884. 
 
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92 
 
 MICROSCOPICAL METHODS. 
 
 rod and knife is about l mm , while nearer the back of the knife 
 the distance is greater (Fig. 21 D, a, b). At each end, the rod 
 is bent at right angles. Next the handle, it passes through a 
 hollow cylinder (Fig. 21, d), into which it is secured by a milled 
 nut (i). At the free end of the knife, the rod is hooked 
 over the back of the blade (Fig. 21, A), the spring of the wire 
 securing it firmly. At the two angles of the rod it rests on the 
 blade, so that, in cutting sections, any amount of pressure may 
 
 JL B CO 
 
 Fig. 21. — The section-nattener attached to a knife, (a), blade of knife ; (a/) tang; 
 (6), section-flattener ; (c), milled nut ; (d), the part of the clamp bearing the hol- 
 low cylinder ; (e), part of clamp ; (/), screw holding the two parts of the clamp 
 together. 
 
 A, section showing the end of the fiattener hooked over the blade ; B and D, sections 
 showing the form of the rod (6), and its position with relation to the edge of thf, 
 knife ; C, section of the tang, showing the manner of attaching the clamp. 
 
 be applied at these points. The rod is attached to the knife by 
 means of a clamp, which consists of two pieces clasping the 
 tang and held together by a screw (Fig. 21, C). To clean the 
 knife and rod, or to remove sections, the rod may be raised, as 
 it swings freely in the hollow cylinder attached to (d). The 
 rod may be removed by removing the milled nut (i) ; the 
 entire apparatus may be removed from the knife by loosening 
 the screw (/). 
 
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CHAPTBE IY. 
 
 METHODS OF IMBEDDING. 
 
 In every method of imbedding, the principle is the same ; 
 namely, to saturate objects with substances which not only 
 fill the larger internal cavities, but which also penetrate the 
 tissues themselves, rendering them (after cooling) sufficiently 
 hard for the process of sectioning. 
 
 PARAFFINE. 
 
 One of the most convenient and most commonly used sub- 
 stances for imbedding is paraffine. The process of imbedding 
 is, in general, very simple ; and the mass is ready for cutting a 
 few minutes after the operation is completed. Where fineness 
 and uniformity of section and rapid execution are of impor- 
 tance, the paraffine method has no equal. 
 
 In this method, the hardness of the paraffine is an important 
 consideration. There is no invariable rule to be followed in 
 this respect ; it is necessary to use different degrees of hard- 
 ness, according to the thickness of section required, the 
 nature of the object, and the temperature of the room in which 
 the object is sectioned. It is convenient to have in stock a 
 soft (fusible at 45° to 46° C.) and a hard paraffine (fusible at 
 55° to 56° O), and to mix these in proportions suited to the 
 occasion. As a general rule, for a room temperature of 68° to 
 70° F. (20° to 22° C), it suffices to mix two parts of the hard 
 with one of the soft paraffine. The harder the paraffine, the 
 easier it is to obtain very thin sections, and the greater the 
 liability to roll. The melting point of the different sorts of 
 
 93 
 
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94 MICROSCOPICAL METHODS. 
 
 paraffine ranges between 40° and 60° C. The most general rale 
 for determining the proportions of soft and hard paraffine is to 
 so mix that the point of fusion lies between 50° and 53° C. 
 
 Dr. Minot employs paraffine melting at 55° to 56° C. and a 
 soft " chewing-gum " paraffine melting at 50° C, and mixes in 
 the proportion of 20 : 9. 
 
 For cutting ribbons of sections, Adam Sedgwick uses hard 
 paraffine coated with soft. The block is coated by dipping it 
 in melted soft paraffine, and the soft paraffine is then cut away 
 from the two sides of the block, being left on the front and 
 hind faces only. 
 
 Preparation for Imbedding. — Preparatory to imbed- 
 ding, the objects are removed from absolute alcohol (96 % will 
 be sufficient) to clove oil, turpentine, creosote, or chloroform, 
 and left until thoroughly saturated. The penetration of the 
 clarifying medium may, in some cases, be advantageously has- 
 tened by warming a little. In transferring from alcohol to the 
 clarifying medium, there is often considerable danger of col- 
 lapse and distortion from shrinkage, especially with delicate 
 objects, and with such as have larger or smaller internal cavi- 
 ties, or are enveloped by impervious membranes. In the use of 
 clove oil, which is one of the most satisfactory media, this 
 danger may be guarded against by placing the object first in a 
 mixture of alcohol and the oil in equal parts, and then, after 
 some time, into pure clove oil. 1 
 
 In order to avoid shrinkage, in passing delicate objects from 
 alcohol to chloroform or an oil, Giesbrecht 2 recommends the 
 following method : — 
 
 Pour a little absolute alcohol into a small glass tube, place 
 the canular end of a pipette, containing the solvent, below the 
 surface of the alcohol, and allow a few drops to flow from it to 
 the bottom of the tube ; into this tube let fall, by the aid of 
 another pipette, or a small spatula, a few drops of absolute alco- 
 
 1 As clove oil mixes very slowly with paraffine, it is sometimes advisa- 
 ble to place the object, after clarification in clove oil, in turpentine for a 
 while, transferring from this to paraffine. 
 
 2 Giesbrecht. "Zur Schneide-Technik," in Zoolog. Anzeiger, 1881, 
 No. 92. 
 
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'methods of imbedding. 95 
 
 hoi, containing the objects to be imbedded. The objects will 
 sink through the alcohol, which, being the lighter fluid, has 
 taken a superjacent position, and rest on the upper surface of 
 the fluid expelled from the first pipette. Most of the alcohol 
 may now be removed by a pipette, and the objects left to sink 
 gradually into the heavier fluid at the bottom of the tube. In 
 this way, the replacement of the alcohol contained in the ob- 
 jects by an oil, or some solvent of paraffine, is much retarded, 
 and thus the danger from shrinkage reduced to a minimum. 
 
 Where chloroform is preferred to creosote or oil of cloves, a 
 little ether (a3ther sulfuricus, C 4 H 5 0) should be added, as many 
 objects will not sink in pure chloroform. 
 
 Dr. Giesbrecht recommends for difficult cases chloroform, 1 as 
 it is one of the best, and, at the same time, the most volatile, 
 solvent of paraffine. 
 
 After the objects have become thoroughly saturated with 
 chloroform, the containing tube is placed on a water-bath, and 
 heated to about 55° C, the melting point of paraffine ; then a 
 small piece of paraffine is added, and allowed to dissolve ; and 
 this is repeated until bubbles cease to rise from the objects. 
 To make sure that the chloroform has been fully expelled, the 
 objects may next be transferred to pure paraffine, and left for a 
 few minutes before imbedding. 
 
 1 Biitschli {Biolog. Centralblatt, B. 1, p. 591) has also recommended 
 chloroform. 
 
 For the Hydrozoa, Professor Weismann prefers turpentine to chloro- 
 form, as, where the latter has been used, the parafnne is liable to be more 
 or less spongy, in consequence of bubbles lodged in the tissues. 
 
 Turpentine renders objects brittle ; and, on this account, chloroform 
 will, in many cases, give better results. The spongy state of the parafnne 
 results from the fact that the chloroform has not been allowed to wholly 
 escape. 
 
 In the case of the Actiniae, Dr. Andres employs a mixture of turpen- 
 tine, creosote, and alcohol, using successively mixtures containing more 
 turpentine and less alcohol, thus : — 
 Mixture No. 1. 
 
 Turpentine 1 
 
 Creosote 2 
 
 Alcohol (absolute) . . . 1 
 
 No. 2. 
 
 No. 3. 
 
 No. 4. 
 
 2* 
 
 4i 
 
 1i parts 
 
 2* 
 
 21 
 
 2i " 
 
 6 
 
 4 
 
 
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96 MICROSCOPICAL METHODS. 
 
 Kossman ' says that chloroform, is the only solvent that can 
 be successfully used in the case of objects with thick chitinous 
 membranes. 
 
 In the use of chloroform, two points must be carefully 
 attended to, namely, the complete saturation of the object be- 
 fore it is placed in paraffine, and the complete evaporation of 
 the chloroform before the object is finally imbedded. If the 
 first point is not secured, of course the paraffine will not pene- 
 trate the object thoroughly; and if the chloroform does not 
 wholly escape before the process of imbedding begins, the 
 paraffine will be spongy, and consequently unfit for section-cut- 
 ting. The evaporation of the chloroform may be effected in 
 two or more ways. In all cases the object must lie in chloro- 
 form until thoroughly saturated. Then paraffine may be added 
 gradually, as recommended by Dr. Giesbrecht ; or the saturated 
 object may be placed in a solution of paraffine in chloroform, 
 as recommended by Professor Blitschli. After remaining here 
 until it is thoroughly impregnated (an hour or less), it may be 
 placed in a watch-glass with a little of the solution, and kept at 
 a temperature of about 50° C. until the chloroform has escaped. 
 In case of larger objects, they may be transferred directly from 
 the solution to pure paraffine, without undergoing the slow 
 process of evaporation. 
 
 Kossmann transfers the object directly from pure chloroform 
 to pure paraffine, and allows it to remain in the paraffine (kept 
 at a constant temperature of 50° C.) for several hours, — some- 
 times for two or three days. 
 
 Saturation with. Paraffine. — After the object has been 
 properly clarified by one of the above methods, it is to be trans- 
 ferred to melted paraffine. It should be soaked first of all in 
 very soft paraffine, or a mixture of paraffine and turpentine, 
 and then in the paraffine intended for imbedding. For this 
 purpose a convenient form of water-bath is required, such as 
 that described below, in which the two paraffines can be kept 
 in separate basins, at a temperature of about 55° C. The time 
 required for complete saturation with paraffine will vary ac- 
 
 1 Zool. Anzeiger, No. 129, p. 20. 
 
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METHODS OF IMBEDDING. 
 
 97 
 
 cording to the size and nature of the object, from 15 minutes 
 to as many hours. For objects not more than one or two milli- 
 meters in thickness, 30 or 40 minutes, divided equally between 
 the two basins, are usually sufficient. In the case of large ob- 
 jects, and such as are penetrated with difficulty, owing to mem- 
 branes or other impediments, the process may be made much 
 more expeditiously, and with far greater safety in vacuo, by the 
 aid of Hoffman's imbedding apparatus. 
 
 Imbedding Boxes. — The saturation with paraffine being 
 complete, the concluding steps in the process of imbedding 
 may be undertaken. 
 
 Boxes for imbedding may be made of rectangular pieces of 
 paper in the following manner : The paper is first broken in the 
 lines a a 1 and b b 1 , then c d and d d' (by 
 bending always towards the same side). 
 Then in every corner a break {A A 1 , B B', 
 C C, D £>'), is made by bringing A c and 
 A a together. The four sides of the box 
 are next bent up, and the corners at the 
 same time turned outward and back behind 
 the ends A B ab and G D a 1 b'. Finally 
 the upper edge of these ends is bent down 
 over the corners. 
 
 Bubbles around the object may be re- 
 moved by means of a heated wire. {Block- 
 raann.') 
 
 A more convenient box, introduced by 
 Dimmock, may be made of two pieces of 
 type metal (or better of brass). As will 
 be seen from the accompanying diagram, 
 each piece of metal has the form of a carpenter's square, with 
 the end of the shorter arm triangularly enlarged outward. A 
 convenient size will be found in pieces measuring 7 mm (long arm) 
 by 3 cm (short arm), and 7 mm high. With such pieces a box may 
 be constructed at any moment by simply placing them together 
 on a plate of glass, which has previously been wet with glycerine, 
 and gently warmed. The area of the box will evidently vary 
 according to the position given to the pieces, but the height 
 
 Via. 22. — Diagram illus- 
 trating method of mak- 
 ing paper boxes for im- 
 bedding. 
 
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98 
 
 MICROSCOPICAL METHODS. 
 
 can be varied only by using different sets of pieces. In such a 
 box the paraffine may be kept in a liquid state by warming now 
 and then over a spirit lamp, and small objects be placed in any 
 desired position under the microscope. 
 
 It is well to imbed in a thin layer of paraffine, so that the 
 object, after cooling, may be cut out in small cubical or pyra- 
 midal blocks, which may be easily fixed, 
 for cutting, to a larger block of hard par- 
 affine, or, better, to a block of wood satu- 
 rated with paraffine. [Mayer.) 
 
 Mr. Dimmock also used for the same 
 purpose ' quotations ', such as are used by 
 printers in filling blank spaces at the be- 
 ginning and end of chapters. These 
 quotations vary somewhat in size, and 
 are sold at 25 cents a pound. 
 
 Mayer, Andres, and Giesbrecht recom- 
 mend brass instead of type-metal for these 
 boxes, and a wash of thin collodion, where 
 it is desirable to keep the paraffine in a 
 melted condition for a considerable time, 
 as in imbedding small objects in definite 
 positions. The glass plate forming the 
 base of the box is first wet with glycerine, 
 and then the box is washed with collodion, and placed on a water- 
 bath, in order to evaporate the ether. In this way a box is 
 obtained in which paraffine can be kept for hours in a fluid con- 
 dition, without escaping between the glass and the metallic 
 pieces. The box is kept on a small water-bath, made for this 
 special purpose, while arranging the object under the dissecting 
 microscope. 
 
 Orientation. — With larger objects there are no difficulties 
 in the next step. It is only necessary to fill the box with 
 melted paraffine, and then to drop in the object by the aid of a 
 spatula, and place it in any desired position with a hot needle. 
 Orientation becomes difficult only with objects so small that 
 their position cannot be controlled except by the aid of a dis- 
 secting microscope. Round objects, less than one millimeter 
 
 
 \ 
 
 
 
 
 
 
 
 
 
 \ 
 
 Fio. 22. —Type-metal box 
 for imbedding. 
 
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METHODS OF IMBEDDING. 99 
 
 in diameter, e. g., many ova and embryos, — are the most diffi- 
 cult to manage. Such objects may usually be successfully ori- 
 ented in the following manner : — 
 
 1. Prepare the box; for this it will be necessary to use the 
 two triangular pieces of metal, a rectangular glass plate (2 in. 
 X 2^- in.). The plate should be cleaned and then smeared with 
 glycerine, and the pieces of metal so adjusted that the arms 
 are parallel with the edges of the plate. 
 
 2. Having warmed the box over a spirit lamp, lift the object 
 from the basin of paraffine by the aid of a small, flat, thin 
 spatula (first starting it from the bottom by shaking the jjaraf- 
 fine a little), and allow it to flow with the parafhne carried on 
 the spatula into the box. 
 
 3. Then fill the box (5-6 mm deep) with the melted paraffine, 
 and warm it a little over a spirit lamp, just enough to keep all 
 of the paraffine in a liquid condition for a few moments. Now 
 place the box on the warm table of a dissecting microscope, 
 and by the aid of a hot needle proceed to place the object in 
 the desired position. As the object is illuminated from below, 
 it can be easily seen, turned over, and moved about at pleasure. 
 If the paraffine freezes before orientation is effected, it should 
 be melted again as before, and the needle must be kept hot by 
 repeatedly holding it in the flame of the lamp. 
 
 The difficulty of finding very small objects in a basin of pa- 
 raffine will be very much lessened by keeping the paraffine free 
 from dust, and the bottom of the basin (tin) scoured bright. 
 A piece of emery cloth serves for polishing. 
 
 The necessity of rewarming the box .if paraffine, which often 
 arises in the above method, may be removed by using a hot 
 bath on the table of the microscope. This bath should be a 
 box of convenient size (not over 2 cm high), with top and bottom 
 of glass, with an opening at one end for filling with hot water, 
 and another at the opposite end provided with a rubber tube 
 and clamp, for drawing off the water as soon as the object has 
 been arranged. 1 
 
 Prevention of Bubbles. — After the imbedding process 
 
 1 A similar device has been described by Fol, Lehrb. d. vergl. mik. 
 Anat., p. 122. 
 
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100 MICROSCOPICAL METHODS. 
 
 has been carried thus far, there is still another danger to be 
 carefully guarded against. If the box is left to cool slowly in 
 the air, bubbles are very likely to appear in the paraffine, which 
 will prove a serious obstacle in cutting. Profiting by Cald- 
 well's suggestion, to cool the box in water, one may avoid all 
 such inconveniences. As soon as the paraffine cools around the 
 object, so that its position is secured, the box should be held in 
 a vessel of cold water, first at the surface (until the paraffine 
 has set), then fully submerged. In this way the paraffine is 
 quickly cooled sufficiently for removal from the box, which 
 may then be used for imbedding a second object. A dozen ob- 
 jects may be thus imbedded in a very short time. If the box 
 is plunged below the surface of the water before the paraffine 
 has become rigid, holes will arise hi the mass and fill with 
 water. 
 
 Dr. Schulgin's Mixture for Imbedding. 1 — Instead of 
 pure paraffine, Dr. Schulgin uses a mixture of paraffine with 
 ceresin, a substance somewhat similar to wax, but firmer and 
 much less brittle. Paraffine, which melts at 55° C, is recom- 
 mended ; and the amount of ceresin to be added to a given 
 amount of paraffine may be determined by experiment. The 
 finest sections of this substance are not brittle, and herein lies 
 the chief excellence of the mixture. If this mixture proves 
 too hard, it may be softened by adding a little vaseline. 
 
 Caldwell's Method of Imbedding in Paraffine. 2 — 
 After the specimen has been stained it should be left in 
 90 fo alcohol for a few minutes, and thence transferred to 
 absolute alcohol, there to remain until all the water is extracted. 
 The length of time necessary for this varies greatly with the 
 size of the specimen. A three-day chick, for instance, will re- 
 quire about an hour, larger specimens a day or moi-e, in which 
 case the absolute alcohol should be changed occasionally. 
 Some tissues may be transferred directly from the absolute al- 
 cohol to turpentine, and thence in about two hours to the 
 melted imbedding material. For delicate tissues, however, the 
 
 1 Zool. Anzeiger, VI. No. 129, p. 21, 1883. 
 
 2 Quart. Jour. Micr. Sc. XXIV., Oct. 1884. 
 
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METHODS OF IMBEDDING. 101 
 
 following process, though longer and more troublesome, is 
 preferable. With a pipette introduce some chloroform, to 
 which two or three drops of ether have been added, under 
 the alcohol in which the object is lying. The object will then 
 float for some time at the junction of the alcohol and chloro- 
 form, and will finally sink into the chloroform when saturated 
 with it. If, as often happens in the case of embryonic tissues, 
 the object is lighter than the chloroform, it is not easy to tell 
 when the saturation is complete, but generally, on shaking the 
 bottle, a saturated tissue can be temporarily covered by the 
 chloroform, while tissues containing alcohol keep steadily on 
 the surface. 
 
 When the tissue is saturated with the etherized chloroform, 
 it should be transferred to pure chloroform, and there left for a 
 few minutes. Then drop in some pellets of soft paraffine and 
 leave it for two hours or more, shaking occasionally. The 
 whole should then be poured into a small melting-pot, and a 
 quantity of imbedding material added. The melting-pot should 
 then be placed in the water-bath at a temperature of about 
 60° C, and there left until all the chloroform has evaporated, 
 which may be determined by the absence of smell of chloro- 
 form on shaking. If much imbedding material is required, 
 this process takes a day or two ; it is therefore better, when 
 the solution of imbedding material is fairly strong, to take out 
 the tissue and put it direct into pure melted imbedding mate- 
 rial. In any case no chloroform must remain in the material 
 to be cut, as it makes it brittle. Generally speaking, the more 
 gradually these processes are passed through, the better will be 
 the result. 
 
 Ryder's Method of Imbedding Fish feggs 1 — which 
 have been colored in toto with borax carmine, or borax picro- 
 carmine, is as follows : — 
 
 a. After dehydration with about forty times their own vol- 
 ume of strong commercial or 97 % alcohol, and afterwards 
 saturated with oil of cloves, the embryos are placed in a 
 watch-glass containing a melted mixture of chloroform and pa- 
 
 1 On the Preservation of Embryonic Materials, etc., p. 15. 1884. 
 
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102 MICROSCOPICAL METHODS. 
 
 raffine in equal parts, in which they may remain twenty or 
 thirty minutes, at a temperature not above 150° F. When sa- 
 turation is complete, the eggs have the same appearance in the 
 melted mixture as in alcohol. 
 
 b. From the above they are transferred to another larger 
 dish, containing pure paraffine, which melts at 158° F., but 
 which must on no account be allowed to boil. Here they re- 
 main for twenty to thirty minutes more. 
 
 c. The embryos are then transferred, one or two at a time, to 
 a common slide, such as is used for mounting objects. The 
 slide may be warmed over an alcohol lamp. A brass ring, 5 to 
 8 millimeters deep, and 24 in diameter, is then placed on the 
 slide around the object. This ring is then filled with melted 
 paraffine, and the object arranged in it in the desired position, 
 with a hot needle, when the whole is left to cool. 
 
 d. After cooling, the paraffine contracts within the ring, 
 when the latter may be removed, and the discoidal block may 
 then easily be loosened from the slide. The block may then 
 be trimmed down, with a scalpel, into a shape suitable for fas- 
 tening into the well in the carriage of a sledge microtome, or 
 the block may be marked and laid away until it is wanted for 
 
 IMBEDDING APPARATUS. 
 
 In imbedding relatively large anatomical preparations, the 
 penetration of the paraffine to all parts can be secured by the 
 aid of a good air-pump ; but this method is tedious, and robs 
 one of time. The same end may be reached with very little 
 cost of time and labor by means of a suction-pump, such as is 
 used in chemical laboratories, provided only that the water 
 pressure at command is sufficiently great to do the work. Dr. 
 F. W. Hoffman 1 has described a simple apparatus to be used 
 with the suction-pump. 
 
 The suction-pump, (Fig. 24 #), is connected with the exsicca- 
 tor, JE, by means of rubber tubing, through each portion of which 
 runs a piece of glass tubing as a protection against undue com- 
 
 l Zool. Anz., VII., No. 165, p. 230, 1884. 
 
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METHODS OP IMBEDDING. 
 
 103 
 
 pression. The exsiccator, holding the small basin of paraffine, P, 
 in which is placed the thermometer, (Th'), stands in a zinc pan 
 ( W), containing water and a thermometer (Th). The capacity 
 of the pan should be such that the temperature of the water can 
 be kept quite even during the process of imbedding. The flask, 
 .J 7 , introduced between /S and E by means of a T-shaped tube 
 serves to catch any water which may find its way into the con- 
 necting tube, in consequence of variation in the water pressure. 
 The glass tube, m, the lower end of which is in a bottle of mer- 
 
 Fig. 24.— Hoffman's Imbedding Apparatus. 
 
 cury, serves as a manometer. The pressure indicated enables 
 one to judge whether the preparation is saturated with paraf- 
 fine. 
 
 In using this apparatus, the water-bath, W, should first be 
 heated to a temperature of about 60° ; then the basin, _P, con- 
 taining melted paraffine and the object to be imbedded, may be 
 
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104 
 
 MICROSCOPICAL METHODS. 
 
 placed in the exsiccator and the pump set in operation. When 
 the mercury reaches the highest point in the tube, m, and air- 
 bubbles cease to rise from the object, the process is ended, and 
 the air admitted by loosening the screw-clamp, IT. Before ad- 
 mitting the air, the stop-cock, L, can be closed, in case it is 
 thought best to leave the preparation still longer in vacuo. 
 As soon as sufficient time has been given for complete satura- 
 tion, the stop-cock, JO, is opened 
 slowly, and the air streams in. The 
 end of the connecting tube is bent 
 upward in E, in order that the par- 
 affine may not be disturbed by the 
 inflowing air. Finally, the object is 
 taken out and placed in a box of 
 melted paraffine, and left to cool. 
 
 With a water pressure of 700- 
 720 mn >, Hg., most objects will be 
 completely saturated within 20 min- 
 utes. 
 If water pressure is wanting, a vacuum may easily be pro- 
 duced with the apparatus devised by G. P. Francotte. 1 This 
 apparatus (Fig. 25) is connected with the exsiccator (E, Fig. 24) 
 by means of the tube, t, and is provided with a barometer tube, 
 J2, which descends into a vessel of mercury. The vessel, A, 
 holding about half a litre, is partially filled with water. The 
 water is boiled by the aid of a spirit-lamp, and the air escapes 
 by the tube, c, which is provided with a pinch-cock, P. When 
 the steam passes out in a jet, P is closed, the lamp removed, 
 and A cooled slowly by a wet sponge. The vacuum thus pro- 
 duced causes the mercury to rise above 70 cm - After half an 
 hour, the air may be admitted. The flask, _R, serves to receive 
 the steam when formed in excess. 
 
 Fig. 25. — Francotte's imbed- 
 ding apparatus. 
 
 WATER-BATH. 
 
 A convenient form of water-bath, devised by Dr. Mayer, is 
 represented in Fig. 26. 
 
 1 Bull. Soc. Belg. Micr. XI., pp. 45-48, 1884. 
 
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METHODS OF IMBEDDING. 
 
 105 
 
 It is a small brass box 18™ long, 9™ wide, and 8 om high. The 
 tube, A, through which the water is received, and the rod, b, 
 serve as handles. The receiving tube is closed by a cork, pro- 
 vided with a glass tube for the escape of steam, which is bent 
 in the form of a siphon to protect against dust. One and a 
 half centimeters from the base of the box is an oven, o, 
 jcm high and 12 c,n long, which passes completely through the 
 box, and serves for warming the slides when shellac is used. 
 Above are seen two circular, basin-like pits, P, 5.5 cm in diam. 
 
 CZJb 
 
 Fig. 26. — Water-bath. 
 
 and 4 cm deep, for receiving the two tin paraffine cups. These 
 are covered by circular plates of glass. There are also six tubu- 
 lar pits, one for a thermometer t, the others for glass tubes. 
 
 This water-bath will be found useful for other purposes than 
 those of imbedding and mounting. It will, of course, be under- 
 stood that the purpose in giving its exact dimensions is simply 
 to furnish a guide where one is required. There are at least 
 two important advantages offered by this water-bath over those 
 in general use ; viz., the slides are protected from dust, and the 
 paraffine is not exposed to the water. 
 
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106 MICROSCOPICAL METHODS. 
 
 GUM AEABIC.l 
 The object, saturated with water, 2 is placed in a watch- 
 glass, and surrounded with finely powdered gum arabic. 
 The gum absorbs the water, and forms a mucilage which grad- 
 ually penetrates the object. The preparation is next placed in 
 a small cavity made in elder pith, and covered with the pulve- 
 rized gum. The whole is left to dry. A little before complete 
 dessication, it is plunged into 80 fo alcohol. After remaining 
 over night in alcohol, the solidified gum has acquired the con- 
 sistency necessary for cutting. While cutting, the knife should 
 be kept wet with 80$ alcohol. 
 
 GLYCERINE AND GELATINE. 
 
 Gelatine 1 part 
 
 Distilled water 6 " 
 
 Glycerine 7 " 
 
 For preservation, a little carbolic acid (1 gram for 100 grams 
 of the mixture) should be added. Objects are transferred 
 directly from water to the melted mixture, and, after complete 
 saturation, imbedded in paper boxes. After cooling, the objects 
 thus imbedded are hardened in alcohol, then sectioned, and 
 mounted in glycerine. 3 
 
 EGG-EMULSION (Calberla).* 
 
 The white of several eggs is separated from the yolk, freed 
 from the chalazse, cut with shears, and thoroughly mixed by 
 shaking with a 10 % solution of carbonate of sodium (fifteen 
 parts of the white to one part of the solution). The yolk is 
 next added, and the mixture shaken vigorously. After remov- 
 ing the foam and floating pieces of yolk by the aid of filtering 
 paper, the so-called " egg mass " is ready for use. It is this 
 
 ' Gravis, Bull, de la Soc. Belg. de Mic. No. VII., 1884, p. 108. 
 
 2 If the object has been preserved in alcohol, it must remain some 
 time in water before imbedding. 
 
 8 This method is recommended by Kaiser. Botan. Centralbl. I., p. 25, 
 1880. 
 
 4 Morphologisches Jahrbuch, II., p. 445, 1876. 
 
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METHODS OF IMBEDDING. 107 
 
 fluid with which the hollow in the solid block 1 is wet, the block 
 itself being only a piece of the same mixture after it has been 
 hardened in alcohol. 
 
 Calberla soaks the eggs a few minutes (5-20) in the fresh 
 white of the egg before imbedding ; Hertwig appears to omit 
 this part of the process. 
 
 After the egg has been fixed to the block, it is placed in a 
 paper box, and covered with the fresh mass (l-2 om deep). The 
 box is then placed in a vessel that contains alcohol (75-80 % ) 
 enough to bathe its lower half; the vessel, covered with a 
 funnel, is heated over a water-bath for 30-40 minutes, care 
 being taken not to boil the alcohol. The imbedding sub- 
 stance, thus hardened, is next placed in cold alcohol (90 %) 
 which should be changed once or twice during the first twenty- 
 four hours. After remaining in alcohol for about forty-eight 
 hours, the imbedded egg is ready for cutting. 
 
 Ruge's Method of making Egg-Emulsion. — According 
 to Scott, 2 Dr. Ruge prepares egg-emulsion in the following 
 manner : — 
 
 The yolks and whites of as many eggs as are needed are 
 shaken violently together, with the addition of four drops of 
 glycerine to each egg. The mass is then filtered through fine 
 flannel, which removes all the membranes and air bubbles, and 
 gives a beautifully homogeneous substance. The hardening is 
 done as in Calberla's method, and the alcohol entirely removes 
 the yellow color of the mass, leaving it as white as if the whites 
 alone had been used. This substance becomes perfectly trans- 
 parent in oil of clove or balsam, and cuts with extreme readi- 
 ness, allowing very thin sections to be made. The objection to 
 Calberla's method is that the carbonate of soda is apt to crys- 
 tallize out in long needles, and so injure the preparations. 
 
 CELLOEDIN. 
 
 Schief f erdecker's Method. 3 — Celloidin is a form of nitro- 
 cellulose (pyroxylin) prepared in ]Dlates, and may be obtained 
 
 1 See Hertwig's method of imbedding the frog's eggs, infra. 
 
 3 Science Record, II., pp. 41, 42. 
 
 8 Arch. f. Anat. u. Phys., I. Abth., p. 199, 1882. 
 
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108 MICROSCOPICAL METHODS. 
 
 from Wittich & Benkendorf, Chausee Strasse 19, Berlin, N. ; 
 from Bachrach & Brothers, Baltimore ; or from the Educational 
 Supply Company, 6 Hamilton Place, Boston. 
 
 Schiefferdecker uses two solutions, one of a syrupy consis- 
 tency, the other somewhat thinner. The celloidin plate is cut 
 into small pieces, and dissolved in absolute alcohol and ether (in 
 equal parts). Objects are transferred from absolute alcohol, 1 
 first to the thinner solution, then to the thicker. After remain- 
 ing a few hours (or days, according to the character of the 
 object) in the latter, they are imbedded in paper 
 boxes. As soon as a hardened film forms on 
 the solution in the box, the whole is placed in 
 \ - || 82 fo alcohol for 24-48 hours, and thus rendered 
 
 \ ' '_'1U=0 
 
 I ill I sufficiently hard for cutting. 
 
 Blochmann 2 recommends imbedding on a cork 
 rather than in a paper box, as less celloidin is re- 
 quired, and as the cork is held more firmly in the 
 holder. One end of the cork is made rough, 
 and surrounded by a strip of paper, which is 
 made fast by a pin, as shown in the figure. The 
 
 fBii roughened surface of the cork is wet with abso- 
 
 lute alcohol, and then the object imbedded in 
 
 Fig. 27. 
 
 the usual manner. In order that this small box 
 
 may sink in alcohol, in which it is placed for hardening the cel- 
 loidin, it may be weighted with a small lead ball fastened to 
 the cork by a needle (see figure). 
 
 In cutting, the knife is kept wet with alcohol (70 fo). The 
 sections may be placed in water or in alcohol, and afterwards 
 stained with carmine or hasmatoxylin, in which the celloidin is 
 only a little, or not at all, stained. Aniline dyes color the cel- 
 loidin, and therefore should not be used. 
 
 The sections can be mounted in glycerine or in balsam ; but 
 in the latter case they must be dehydrated with 95 % alcohol, 
 as absolute alcohol dissolves the celloidin. They should be 
 
 1 If the objects are penetrated with difficulty, they may be transferred 
 from absolute alcohol to ether, then to the celloidin solutions. 
 
 2 F. Blochmann. "XJeber Einbettungsmethoden." Zeitschr. f. wiss. 
 Mitr., I., H. 2, p. 218, 1884. 
 
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METHODS OF IMBEDDING. 
 
 109 
 
 clarified in bergamot oil or origanum oil (clove oil dissolves the 
 celloidin). 
 
 Objects imbedded in celloidin can be preserved ready for 
 cutting for a long time in 70-80 % alcohol. 
 
 Thoma on Methods of Imbedding in Egg-Emulsion 
 and Celloidin. — The method of treating tissues with gum 
 arabic, first brought into use by Rindfleisch and Ranvier, is now 
 very generally known and practised. The same may be said 
 of the method of cutting sections between two pieces of elder 
 pith or hardened liver, etc. These, in certain conditions, are 
 very useful and simple; but other methods of imbedding, of 
 more recent date, give sections of the utmost perfection and un- 
 surpassed delicacy. 
 
 The method of imbedding in emulsions containing fat and 
 albumen originated with Bunge, and was subsequently modified 
 by Calberla and Ruge. The following is very nearly the for- 
 mula of the latter : The albumen and yolk of several hens' eggs 
 is placed in a porcelain mortar, and well stirred until it forms a 
 thin yellow fluid, a result generally obtained in a few minutes. 
 This fluid is subsequently passed through thin linen, in order to 
 remove the remaining membranaceous fragments. The speci- 
 men, previously hardened in alcohol, is then fixed by pins in a 
 paper box, and covered with the fluid. 
 The preparation cannot, however, be 
 immersed directly in alcohol for the 
 purpose of hardening. It must be first 
 hardened by alcohol steam, taking 
 care never to raise the temperature of 
 the steam above 30° C. For this pur- 
 pose, Professor Thoma uses a simple 
 apparatus represented in Fig. 28. 
 
 A shallow water-bath, a, stands on 
 an iron tripod, bbb, and is heated by 
 a small flame, c. The water-bath is 
 covered by a thin plate, dd. Upon 
 this plate is a small glass vessel, e, filled with common alco- 
 hol, and covered with a perforated disk of tin, ff. On this 
 disk are placed the paper boxes, g g, containing the speci- 
 
 FlG. 28. 
 
 - Thoma's imbedding 
 apparatus. 
 
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110 MICROSCOPICAL METHODS. 
 
 mens and the imbedding fluid. The latter and the alcohol 
 vessel are again separated from the external air by a glass 
 cover, h. This apparatus, slightly heated, will harden the 
 imbedding masses within a few days, after which time they are 
 removed, and subsequently fully hardened in a bottle containing 
 ordinary alcohol. The latter process determines the degree of 
 consistence of the imbedding mass. It can be made extremely 
 hard by repeated use of strong alcohol. After a few trials it 
 will be easy to find the convenient degree of consistence for 
 each specimen. 
 
 If the temperature of the alcohol steam is more elevated, it 
 will be found that the imbedding mass, instead of shrinking, 
 will appear to increase in volume, innumerable air bubbles de- 
 veloping in the emulsion. This can be easily avoided by using 
 lower temperatures. Another danger, however, exists in the 
 holes which the pins make in the walls of the paper boxes. The 
 emulsion before hardening is so very liquid that it will pass 
 through the smallest opening ; this renders it necessary not to 
 withdraw any of the pins from the sides of the paper box, and 
 to use boxes without any openings. It will be found that this 
 mass adapts itself perfectly to all surfaces of the specimens 
 without penetrating into their interior structure, and that it 
 can be cut admirably at all thicknesses down to 0.003™ 11 . An- 
 other very agreeable quality results from the fact that the newly 
 prepared emulsion will adapt itself readily to hardened pieces. 
 This enables us to spread out fine membranes on pieces of the 
 hardened imbedding mass, and subsequently to imbed both in 
 the way just described. 
 
 After this praise of the egg-emulsion, ft will be just to men- 
 tion a property which is occasionally disagreeable. It cannot 
 be easily detached from the sections, and we have no means of 
 dissolving it in media which do not injure the objects. The 
 mass also colors in all the staining fluids generally used, and 
 therefore becomes very visible in the preparations. The latter 
 inconvenience should in all cases be avoided by coloring the 
 specimen in toto before imbedding. For this purpose the fluids 
 of Grenacher, 1 and especially alum-carmine, may be recom- 
 
 1 Arch. f. mikr. Anat., XVI. (1879), p. 465. 
 
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METHODS OF IMBEDDING. Ill 
 
 mended. The imbedding mass remains nearly absolutely color- 
 less if the specimen, after staining and before imbedding, is 
 hardened again in alcohol. 
 
 Very elegant results may also be obtained by an imbedding 
 mass originally invented by Duval, and recently much improved 
 by Merkel and Schiefferdecker. 1 This is collodion, or, prefera- K- 
 bly, a solution of so-called celloidin. If this substance cannot 
 in general be cut to such extreme delicacy as the albuminous 
 mass just described, it has a great advantage in being extremely 
 pellucid. 
 
 According to the formula of Schiefferdecker, the imbedding 
 fluid consists of a concentrated solution of celloidin in a mix- 
 ture of equal parts of absolute alcohol and ether. The speci- 
 men is soaked successively in absolute alcohol and ether, and in 
 the imbedding fluid. This requires at least several days. After 
 this time, the imbedding proper may be undertaken, and for 
 this we have the choice of two methods. 
 
 The even surface of a cork is covered with a thick solution 
 of celloidin, so as to form by evaporation a strong collodion 
 membrane on the cork. Upon this is put the specimen, covered 
 layer by layer with fresh quantities of the solution of celloidin, 
 each being allowed to dry only partially. When the object is 
 thoroughly covered, we immerse it in alcohol of 0.842 sp. gr. 
 In twenty-four hours the whole is ready for cutting. 
 
 The other method makes use of little paper boxes for the im- 
 bedding. The specimen, soaked in celloidin solution, is fixed in 
 the box by pins, and the box filled with celloidin. The prepar- 
 ation is then placed on a flat piece of glass, and covered with a 
 glass cover which does not exactly fit the glass plate. In a few 
 days the ether will have evaporated gently and slowly from the 
 imbedding mass, and the latter will shrink a little. If neces- 
 sary, further celloidin solution can be poured into the paper box 
 to fill it again. It is only necessary to moisten the surface of 
 the first mass with a drop of ether in order to allow of a per- 
 fect junction between the old and the new layers. The pre- 
 paration is again exposed to slow evaporation below the glass 
 
 1 Arch. f. Anat. u. Physiol. (Anat. Abtheil.), 1882. 
 
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112 MICROSCOPICAL METHODS. 
 
 cover and a few days later the imbedding mass will be consoli- 
 dated to an opaline body, whose consistency can well be com- 
 pared to that of the albumen of a boiled egg. The walls of 
 the paper box can now be removed, and the imbedding mass 
 placed in very dilute alcohol, which will, in a very few days, 
 produce a proper degree of consistency to admit of cutting. 
 
 This method differs, in some degree from that which Schief- 
 ferdecker gives for imbedding in paper boxes. As other ob- 
 servers have remarked, his method frequently gives rise to a 
 great number of air bubbles in the imbedding mass. Conse- 
 quent upon the altered manipulations of Professor Thoma, we 
 have to adapt the imbedded specimen to a cork for the purpose 
 of cutting. This may be done in the following way : The even 
 surface of the cork is covered by a thick layer of celloidin 
 solution. This is allowed to dry up perfectly, so as to produce 
 a hard membrane of celloidin. This is again covered with fur- 
 ther celloidin solution. In the mean time the lower surface of 
 the imbedding mass is cut even and washed with absolute alco- 
 hol, and subsequently moistened with a drop of ether. This 
 moist surface is adapted to the stratum of liquid celloidin on 
 the cork, and exposed for a few minutes to the open air. After 
 this, the whole is placed in dilute alcohol, which in a few hours 
 will unite the imbedding mass solidly with the cork. 
 
 In a great number of cases it may be regarded as a great 
 advantage of the celloidin that it penetrates the tissues thor- 
 oughly, and yet remains pellucid, so as to be more or less invisi- 
 ble in the specimen. This quality can be made use of in 
 another direction for the purpose of soaking specimens which 
 are too brittle to be cut after hardening alone. We may make 
 use of celloidin in a similar way to the gum arabic mentioned 
 above. The minute normal and pathological anatomy of the 
 lung, in particular, will derive great advantage from such a pro- 
 ceeding. Indeed, we are not able to get a perfect idea of the 
 changes produced by pneumonia, if we do not, by this method, 
 or by the following (with paraffine), prevent the loss of a great 
 part of the exuded substances which in this disease lie loose in 
 the alveolar cavities. The study, also, of micro-organisms in 
 the lung will derive great benefit from the celloidin method, 
 
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METHODS OF IMBEDDING. 113 
 
 and it will be very welcome to many to know that the tissues 
 imbedded in celloidin may be stained with the different fluids, 
 — ammonia-carmine, alum-carmine, borax-carmine, hematox- 
 ylin, analine colors, and various others. The reaction of acids 
 and alkalies — particularly acetic acid and solution of potash — 
 is, moreover, not interfered with. And further, we are able to 
 color the object before imbedding with all staining fluids which 
 are not soluble, or only little soluble, in alcohol and ether. 
 
 After staining and cutting, the sections may be mounted in 
 glycerine and various other fluids. Mounting in Canada balsam 
 requires, however, some precautions, on account of the chemical 
 character of the celloidin. Absolute alcohol and oil of clove 
 should be avoided, and replaced by alcohol of 96 fo, and by 
 oleum origani. 
 
 Mulcts Method of Imbedding in Celloidin. — The ob- 
 ject, after having been thoroughly dehydrated in alcohol, is 
 placed for twenty-four hours in a mixture of equal parts strong 
 alcohol and pure ether. If this mixture is kept long, a little 
 ether must be added from time to time. Transfer' to a thin 
 solution of celloidin, and allow it to remain for from one to 
 three days, according to the size of the object. 
 
 Imbed in a thicker solution of celloidin. The imbedding box 
 is prepared as recommended by Blochmann. Bubbles will rise 
 from the cork, and interfere with the imbedding. Two precau- 
 tions will essentially diminish this danger : 1. Pour in so much 
 celloidin that it covers the object half an inch deep, giving an 
 opportunity for the bubbles to rise above the tissue ; 2. Before 
 imbedding, cover the end of the cork with a thin layer of cel- 
 loidin (as recommended by Thoma), which is allowed to dry on 
 completely. After the object is covered, the cork is mounted 
 on a lead sinker, and allowed to stand until a film has formed 
 on the upper surface. It is then immersed in alcohol of 
 82-85 % (stronger alcohol attacks the celloidin) for one to 
 three days. I have found it best to allow plenty of time for the 
 hardening after imbedding. 
 
 The sections have to be cut under alcohol. We use Jung's 
 microtome with his largest knife placed so as to cut with as 
 much of the blade as possible. If the edge is good, then the 
 
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114 MICROSCOPICAL METHODS. 
 
 longer the draw, the thinner the section which can be made. 
 While cutting, the knife-blade should have as much alcohol 
 upon it as possible ; to secure this, we use the dripping appara- 
 tus described below. The sections should be removed from the 
 knife with a fine brush to avoid all risk to the edge. For cel- 
 loidin imbedding are needed, — 
 
 1. Mixture of ether and alcohol, equal parts. 
 
 2. A thin solution of celloidin in (1). This solution should 
 be syrupy, but still flow easily. 
 
 3. A thick solution of celloidin in (1) of about the consistency 
 of thick molasses. The usual mistake is to have the solution 
 too thick. Quantitative directions cannot be given, because 
 the celloidin varies in weight, according as it is more or less 
 dried. 
 
 4. Alcohol, 82-85%. 
 
 For mounting sections with celloidin left on them, I have 
 found none of the methods hitherto recommended satisfactory. 
 The essential oils I have tried either dissolve the celloidin, 
 like oil of clove, or cause it to shrink and distort the section, 
 like oil of bergamot. After trying various reagents, I have 
 settled upon chloroform as the most convenient medium of 
 transfer from alcohol to balsam. In using it care must be 
 taken to place the section for half a minute in perfectly fresh 
 alcohol, which is really 95-96% ; — if this is done, chloroform 
 will clear it up almost immediately. When the section is in 
 chloroform on the slide, the mounting must be expeditious, and 
 the balsam added while the chloroform is still covering the sec- 
 tion. If many sections are to be mounted, it is convenient to 
 have a dish full of chloroform, and large enough to permit 
 plunging in the slide and placing the section on it, all under 
 chloroform. The transfer, particularly of a large section, from 
 the spatula to the slide with chloroform, is often very difficult. 
 To mount a single section, put it in alcohol on the slide, wash 
 with a few drops of fresh strong alcohol ; let most of the alco- 
 hol drain off, but while the section is still covered with it, add 
 chloroform, drain off the mixture, and pour over the still moist 
 section a fresh dose of chloroform ; if the washings have been 
 really thorough, the section will clear at once. 
 
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METHODS OP IMBEDDING. 
 
 115 
 
 Dripping Apparatus for Cutting under Alcohol —We 
 
 use the form constructed by D. W. W. Gannett, as shown in 
 the sketch. A litre bottle is convenient in size ; the height of 
 the stand should be such as to bring the end of the dripping- 
 
 FiG. 29 — Dripping apparatus. 
 
 tube about one inch above the blade of the microtome knife, 
 on which the alcohol is allowed to fall continuously. To regu- 
 late the flow, an -J in. globe valve is found to be the most con- 
 venient. 
 
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CHAPTER Y. 
 
 FIXATIVES FOR SERIAL SECTIONS. 
 
 Although the first attempts at fixing sections, by Mayer and 
 Gaule, 1 were partially successful, the problem was only actually 
 solved by the discovery of liquid fixatives. It is to Dr. Gies- 
 brecht, of the Naples Zoological Station, that we are indebted 
 for the first fixative of this kind. This method, still widely 
 used, has recently been improved by a new method of dissolv- 
 ing the shellac, discovered by Mayer. The collodion method 
 of Schallibaum, is perhaps now more generally employed, as 
 it enables one to stain after sectioning, and serves equally 
 well with the shellac method for sections of objects stained in 
 toto. Mayer's albumen fixative offers some advantages. The 
 sections can be treated with turpentine, alcohol, water, staining 
 fluids, glycerine, etc., without danger of loosening. In stain- 
 ing nuclei, the film of albumen remains uncolored, which is an 
 advantage over the collodion method. 
 
 Giesbrecht's Shellac Fixative.' 2 — The shellac is prepared 
 and used in the following manner : One part of bleached shellac s 
 dissolved in ten parts absolute alcohol and filtered. The objeet 
 
 1 Franootte. "Description des differentes methodes employees pour 
 ranger les coupes en series." Bull, de la Soc. beige de microscopie, X ; 
 No. 2, p. 43. 
 
 2 Giesbrecht. "Methode zur Anf ertigung von Serien-Praparaten," in 
 Mittheilungen a. d. Zoolog. Station zu Neapel, 1881, p. 184. 
 
 8 The preparation of bleached shellac used by artists as a "fixative" 
 for charcoal pictures, as Dr. Mark first suggested, may serve equally well. 
 116 
 
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FIXATIVES FOE SERIAL SECTIONS. 117 
 
 glass is first warmed to about 50° C., 1 and then a thin film of 
 the shellac is laid on by a glass rod drawn once or twice over its 
 surface. Before using, the slide is again warmed, and the shellac 
 surface washed with oil of cloves for the purpose of softening it. 
 The wash is made with a small brush drawn back and forth until 
 the entire surface has been moderately but evenly wet with 
 the oil. Sections are now cut and arranged for the first cover; 
 this done, the slide is warmed over a spirit lamp, so that the 
 paraffine adhering to the sections melts and flows together, 
 forming an even layer, which cools almost instantly, and thus 
 secures the position of the sections, while those of the second 
 cover are prepared. The sections for the last cover having 
 been completed, the slide is warmed for ten minutes on a 
 water-bath, in order that the sections may sink into the shellac 
 and become fixed, and the clove oil evaporate. After allowing 
 the slide to cool, the process is concluded by washing away the 
 paraffine with turpentine, and inclosing in balsam dissolved in 
 chloroform. 2 
 
 Method of Fixing Collodion Sections. (Minot.) — Sec- 
 tions of objects imbedded in celloidin are " fixed " in serial 
 order, preparatory to mounting, in the following manner : — 
 
 The sections are arranged on the slide in 95% alcohol; then 
 the alcohol is poured off, and a drop of alcoholic shellac placed 
 on each section (just enough to cover the section completely). 
 The slide is next placed in the oven of a water-bath at 40°, for 
 a few minutes (5-10), until dry. The sections are then ready 
 for clarifying in clove oil, and mounting in balsam. 
 
 Mayer's Carbolic Acid Shellac. — The use of clove oil, 
 or creosote in the shellac method of Giesbrecht, leayes fine 
 granulations on the slide. In order to avoid this, Dr. Mayer 
 proposes to use a solution of shellac in carbolic acid. The fol- 
 lowing is Mayer's method of preparation, to which I have 
 added only a few details. 
 
 1 The same temperature is used throughout the operation. 
 
 2 Occasionally one experiences great difficulty in dissolving the bleached 
 lac of the stores. This is due to the fact that now the bleaching is ac- 
 complished by means of chemicals. One can, however, be assured of 
 success (we are informed) by bleaching the lac for themselves, by expo- 
 sure to the sun. (Kingsley.) 
 
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118 MICROSCOPICAL METHODS. 
 
 1. Dissolve 50 g. bleached shellac in 250 g. absolute alcohol. 
 
 It is well to powder the shellac and leave it 24 hours or more in the al- 
 cohol, shaking it from time to time. Only a small portion of the shellac 
 dissolves. Probably a much larger amount would dissolve by heating, 
 but I have not tested this. 
 
 2. Filter the solution, and evaporate the alcohol on a water- 
 bath. 
 
 The filtering paper should be wet with absolute alcohol when the so- 
 lution is poured into it. The evaporation should continue until the yel- 
 lowish residue, when cold, is quite stiff. 
 
 3. Dissolve shellac residue in pure carbolic acid, on a water- 
 bath, using from 3 to 5 volumes of acid to one of shellac. The 
 solution, after cooling, should be nearly as thick as glycerine. 
 
 It is important to use a concentrated solution of carbolic acid, and this 
 may be obtained by exposing the crystals to the air until they dissolve, or 
 by adding about 5 % water, and warming. 
 
 Following these directions, I obtained about 30 grams of the fixative. 
 
 I keep the fixative in a bottle covered with a cap, like that of a spirit- 
 lamp. A small brush is thrust through a flat piece of cork, which rests 
 on the mouth of the bottle, and allowed to dip barely below the surface 
 of the liquid. If the brush is thrust deep into the fixative, the hairs curl 
 up, and the brush is otherwise liable to be affected by the acid. 
 
 The shellac can be directly dissolved in carbolic acid, but then the 
 liquid must remain standing a long time in order to become perfectly 
 clear, for it cannot be filtered. For this reason, I prefer, with Mayer, to 
 make the solution first in alcohol. 
 
 Mayer now prepares as follows : — 
 
 The shellac is first pulverized, then heated with colorless 
 crystals of carbolic acid until it dissolves. It is next to be fil- 
 tered, the funnel being kept hot during the process, by heating 
 over a flame. 
 
 This fixative, which is much stronger than any alcoholic so- 
 lution I have been able to obtain, is applied to the cold slide 
 with a small brush, at the time of using. The brush should be 
 wiped on the mouth of the bottle in order to remove most of 
 the fixative, and then the middle portion of the slide painted 
 with a single thin coat. After the sections have been placed, 
 the slide is placed in the oven of a water-bath, kept at about 
 55° C, and left from 15 to 20 minutes. The carbolic acid eva- 
 porates, leaving a perfectly transparent stratum of shellac. 
 
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FIXATIVES FOR SERIAL SECTIONS. 119 
 
 The sections are then freed from paraffine in the usual way, 
 and mounted in balsam. 
 
 GAULE'S METHOD OF FIXING SECTIONS.l 
 
 1. Sections cut dry and placed on the slide in the order and 
 position in which they are to be mounted. 
 
 2. They are then smoothed out by the aid of a fine brush wet 
 in 50-60 fo alcohol, until all wrinkles are removed and every 
 part is in close contact with the slide. 
 
 3. Slide allowed to stand several hours (or over night) until 
 the alcohol has completely evaporated, and the sections are left 
 adhering quite firmly to the glass. The process may be has- 
 tened by gently warming to 45-50° C. 
 
 4. The paraffine may be removed by any of the solvents in 
 common use, but Dr. Gaule recommends xylol. A few drops 
 are allowed to flow over the sections, and after a few moments 
 the paraffine is fully dissolved. 
 
 5. The balsam (a mixture of balsam and xylol in equal parts) 
 is placed on the cover-glass, and this allowed to sink slowly, 
 from one side, over the sections. 
 
 Dr. Gaule finds it convenient, especially with serial sections, 
 to use large cover-glasses, often nearly as large as the slide 
 itself. Thus a single slide may often contain a large number of 
 sections closely arranged under one cover. 
 
 For large sections, this method offers one important advan- 
 tage over that of Dr. Giesbrecht ; for by the former all wrinkles 
 may be removed, while by the latter the sections must lie as 
 they fall. In the case of smaller sections, not liable to get wrin- 
 kled during the placing, I prefer the shellac method. 
 
 FRENZEL'S GUTTA-PERCHA FIXATIVE.* 
 
 The only advantage claimed for this fixative is that it allows 
 the sections to be colored after they are fixed on the slide. The 
 difficulty in dissolving the paraffine from the sections is the 
 chief objection to Frenzel's method, which is as follows: — 
 
 A solution of gutta percha in chloroform and benzine is used. 
 
 1 Arch. f. Anat. u. Phys., 1881, Phys. Abthl., p. 156. 
 
 2 Zoologisoher Anzeiger, No. 130, p. 51, 1883. 
 
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120 MICROSCOPICAL METHODS. 
 
 The solution must be filtered until it is clear and almost color- 
 less, and must not be made so thin that it will flow quickly on 
 the slide. The fluid is put on with a brush, and allowed to dry- 
 before the sections are placed. If the object is imbedded in 
 paraffine or a paraffine mixture (e. g., four parts paraffine and 
 one part vaseline), the sections are smoothed out by the aid of 
 a brush wet in alcohol, and then exposed to a temperature of 
 35° to 5C° C. for five to ten minutes, in order that the gutta 
 percha may become sticky; they are then left for five to ten 
 minutes in the air, and finally placed in a vessel containing ab- 
 solute alcohol heated to 40-50° C, in order to remove the par- 
 affine. Five to fifteen minutes are required to dissolve the 
 paraffine, and a considerable quantity of alcohol must be used, 
 as it is a poor solvent of paraffine. After the alcohol be- 
 comes saturated, it may be filtered cold, and used again. After 
 the paraffine has been completely removed, the sections are 
 transferred to 70 fo alcohol, then to lower grades, and finally to 
 water. The sections are then ready for staining. After stain- 
 ing and washing, they are soaked in absolute alcohol, and thus 
 prepared for mounting in clove oil and balsam. 
 
 If the object is imbedded in celloidin, as is now frequently 
 done, the sections may be smoothed out with benzine or chloro- 
 form, which softens the gutta percha, and thus fixes them in 
 position. After the sections have dried on, they may be 
 stained, washed, and transferred to absolute alcohol as before. 
 The application of clove oil, before the balsam, dissolves the 
 celloidin. 
 
 THRELF ALL'S CAOUTCHOUC FIXATIVE.l 
 A thin solution of caoutchouc in benzine or chloroform is 
 prepared and poured over the slide so as to form a film, in the 
 same way that collodion is poured on a photographic plate. 
 When the film is dry, the sections are arranged on it, and the 
 temperature of the slide raised to the melting-point of paraffine ; 
 the sections then fall on to the India rubber film, which has be- 
 come sufficiently sticky to adhere to them perfectly. When the 
 slide is cold, it is treated with naphtha, or any light paraffine oil, 
 
 1 Zool. Anzeigcr, No. 140, p. 300, 1883. 
 
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FIXATIVES FOE SERIAL SECTIONS. 121 
 
 the solvent action being more rapid the lower the boiling-point 
 of the oil used. 
 
 Absolute alcohol is readily miscible with the naphtha or light 
 paraffine, so that the solvent is readily removed. The slide can 
 now be placed in successive alcohols, stained, and returned to 
 absolute alcohol. It is now to be cleared with creosote or oil 
 of clove, and mounted in the ordinary way. 
 
 SCHALLIBAUM'S COLLODION FIXATIVE.* 
 The solution, which is prepared by dissolving one part collo- 
 dion in three or four parts clove oil, is applied to the slide by 
 means of a fine brush, at the time of using. The sections 
 having been arranged, the slide is warmed for a few minutes 
 (5-10) in the oven of a water-bath, in order to evaporate the 
 clove oil. The sections may next be freed from the imbedding 
 mass, and -colored according to desire. If the film of collodion 
 be too thick, cloudiness is likely to arise between the sections. 
 The cloudiness can be removed by the use of a brush wet with 
 clove oil, after the sections have been dehydrated by absolute 
 alcohol. 
 
 Gage, 2 who had begun to experiment with collodion before 
 Schallibaum's method was published, recommends that the col- 
 lodion and clove oil be applied separately : — 
 
 " A solution of collodion is prepared by adding to 2 grams of 
 gun cotton (that used by photographers is good) 54 cc of sul- 
 phuric ether and 18 cc of 95 % alcohol. After the gun cotton 
 is entirely dissolved, the solution should be filtered through 
 filter paper or absorbent cotton. The slides are coated by 
 pouring the collodion on one end, allowing it to flow quickly 
 over the slide and off the other end into the bottle. The pre- 
 pared slides should be kept free from dust. As the collodion 
 will not deteriorate after drying on the slide; any number of 
 slides may be prepared at the same time. Before using a slide, 
 it should be dusted with a camel's-hair brush ; and with another 
 brush the collodionized surface of the slide should be thinly 
 painted with clove oil. 
 
 1 Arch. f. mik. Anat., XXII., p. 689. 1883. 
 
 2 H. Gage and T. Smith. The Medical Student, p. 14, Nov., 1883. 
 
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122 MICROSCOPICAL METHODS. 
 
 "... The sections are arranged as in the shellac method. 
 The slide is warmed over an alcohol lamp and then heated in a 
 warm chamber so as to drive off the clove oil. After cooling, 
 it may be placed in a wide-mouthed vial of turpentine, chloro- 
 form, xylol, or refined naphtha, to remove the paraffine. Naph- 
 tha is very cheap, and is the best agent we have yet tried for 
 dissolving the imbedding mass. The sections are usually freed 
 from the imbedding mass within half an hour, though the slide 
 may remain in any of the solvents mentioned for two or three 
 days, or perhaps indefinitely, without loosening the sections. 
 When the slide is removed from the naphtha, the sections are 
 washed with 95 % alcohol by means of a medicine-dropper, or 
 by immersing the slide in alcohol. If the sections are to be 
 stained in Kleinenberg's hematoxylin, or in any other stain 
 containing 50 % or more acohol, the slide is transferred directly 
 from the alcohol used for rinsing to the staining agent, other- 
 wise it should be first transferred to 50 % alcohol, and from that 
 to the staining agent. Whenever the sections are sufficiently 
 stained, they may be mounted in any desired mounting medium. 
 In case Canada balsam is to be used, the slide must be immersed 
 in alcohol to wash away the stain, and finally in 95 % alcohol 
 to completely dehydrate the sections. They are cleared with a 
 mixture of carbolic acid, 1 part ; turpentine, 4 parts. The bal- 
 sam to be used is prepared by mixing 25 grams of pure Canada 
 balsam with 2 CC of chloroform and 2 CC of clove oil. The latter 
 very soon removes any cloudiness that may have appeared in 
 the collodion film." 
 
 MAYER'S ALBUMEN FIXATIVE.i 
 
 The new fixative proposed by Dr. Mayer is prepared by mix- 
 ing the filtered white of eggs with an equal volume of glycerine. 2 
 A little carbolic acid may be added as an antiseptic. A very 
 thin and even layer of the fixative is painted upon the object- 
 slide, which is then ready for receiving the sections. After the 
 
 1 Mitth. Zoolog. Station zu Neapal, IV., p. 521, 1883. 
 z Glycerine serves to keep the fixative moist while the sections are being 
 placed. 
 
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FIXATIVES FOB SERIAL SECTIONS. 123 
 
 sections are placed, the slide is warmed 1 a few minutes in the 
 oven of a water-bath, just long enough to melt the parafline. 
 The parafline is next dissolved away from the sections by tur- 
 pentine, the turpentine removed by alcohol, and the sections 
 then colored in situ on the slide. 
 
 As a coloring fluid, a strong alcoholic carmine is recom- 
 mended. It is prepared as follows : — 
 
 Dissolve four grams carmine in 100 ccm alcohol (80 fo) byboil- 
 ing*about thirty minutes, adding thirty drops of strong hydro- 
 chloric acid during the boiling. The solution should be filtered 
 while hot, and the acid carefully neutralized by adding ammo- 
 nia until carmine begins to be precipitated. 
 
 This fluid, which may require to be filtered a second time 
 after cooling, stains uncommonly quick, deep, and diffuse. To 
 obtain a differential staining, it is well to stain deeply, and then 
 partially decolor by washing with acidulated alcohol. 
 
 The decoloration should be checked at the moment when the 
 film of the fixative becomes nearly or quite colorless. 
 
 This fixative may be employed to great advantage in the 
 process of staining with aniline dyes (vide Flemming's Meth- 
 ods of Treating Nuclei) . After the sections have been fixed on 
 the slide and colored with some aniline dye, the slide may be 
 washed quickly in 70 % alcohol, then placed in 95-96 % alcohol 
 for a few moments until the fixative has lost nearly all its color. 
 The decoloration of the fixative can be followed so readily that 
 it seems to mark the precise moment when the process of de- 
 coloring; should cease. 
 
 1 Care should be taken not to cause coagulation of the film by over- 
 heating. 
 
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CHAPTEE VI. 
 
 MOUNTING MEDIA. 
 
 Although the preservative and optical qualities of glycerine 
 make it indispensable in many ways as a mounting medium, 
 still the inconveniences attending its use for permanent histo- 
 logical preparations and its evident non-adaptability to the needs 
 of the mierotomist have tended to give balsam and damar the 
 preference as mounting media. 
 
 There has been some difference of opinion on the question 
 whether Canada balsam or damar varnish is the better mount- 
 ing medium. It is generally known that uncolored structure 
 is best seen in damar, and that balsam clarifies more strongly 
 than damar. It is not, therefore, advisable to limit one's self 
 to either medium exclusively, but rather to make a selective 
 use of both. 
 
 Balsam is usually dissolved in turpentine, xylol, benzole, or 
 chloroform. Benzole, when used as a solvent of balsam, should 
 be chemically pure and free from water, otherwise very fine, 
 drop-like formations arise, which give the preparation a cloudy 
 appearance to the naked eye. Such results are most likely to 
 appear in preparations mounted at the seashore. Such prepara- 
 tions can be restored by placing the slides in a bottle with ben- 
 zole enough to cover them. The bottle should be laid on its 
 side, and the slide inverted so that the preparation may fall 
 freely, and thus escape crushing by the cover-glass. In the 
 course of an hour or two the preparations are set free, and may 
 then be transferred to clove oil and remounted in balsam. 
 124 
 
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MOUNTING MEDIA. 125 
 
 Canada balsam is prepared by heating for several hours over 
 a sand-bath, until it is quite hard when cold. It is then dis- 
 solved in an equal volume of one of the above solvents. The 
 balsam should be so hard before dissolving it that it will break 
 like glass when cold. 
 
 Damar may be prepared by dissolving the gum in pure 
 turpentine or in a mixture of turpentine and benzole in equal 
 parts. This method is preferable to that given by Schaffer, 
 according to which — 
 
 One part of gum damar is dissolved in two parts of oil of 
 turpentine, and one part of gum mastic in two parts of chloro- 
 form. The two solutions are mixed and filtered. Schaffer 
 states that the opalescence which often arises after a time dis- 
 appears permanently on boiling the fluid. 
 
 GLYCERINE JELLY. 
 
 The two following solutions, similar in composition to Bach- 
 mann's, are employed by Fol. 
 
 1st. Gelatine 30 parts. 
 
 Water 70 " 
 
 Glycerine X00 " 
 
 Camphor (alcoholic solution) 5 " 
 
 2d. Gelatine 20 parts. 
 
 Water . 150 " 
 
 Glycerine 100 " 
 
 Camphor 15 " 
 
 The solutions are prepared by first softening the gelatine a 
 few hours in the water, then melting on a water-bath. The 
 glycerine is next added, and finally the camphor solution by 
 drops while stirring briskly. 
 
 For use, the jelly is to be warmed en masse, or a piece may 
 be cut out and melted on the slide. Over-heating will produce 
 bubbles, which must be carefully avoided. The cover-o-lass 
 must be encircled by some cement, otherwise evaporation will 
 give rise to air bubbles. 
 
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CHAPTEK VII. 
 
 THE USES OF COLLODION. 
 
 In modern histological technique, collodion has come to serve 
 a variety of important purposes. Duval 1 was the first to call 
 attention to its advantages as an imbedding mass. He found 
 that it penetrated preparations easily and thoroughly ; that it 
 could be quickly brought to the proper degree of hardness in 
 alcohol of 36° (80 %); that objects thus imbedded could be 
 preserved in this alcohol for an indefinite length of time ; that 
 the imbedding mass preserved its transparency, so that the 
 preparation could be easily examined ; that the sections did not 
 require to be freed from the mass, since they could be colored 
 and mounted in glycerine, and the mass remain unaffected by 
 the process. 
 
 As soon as Duval's discovery became known, Merkel and 
 Schiefferdecker 2 began to experiment with collodion, and 
 greatly improved and extended its use. It was found desirable, 
 first of all, to be able to vary the concentration of the collo- 
 dion, an end veiy conveniently reached by Merkel through the 
 use of a solid preparation called celloidin, which he dissolved in 
 absolute alcohol and ether in equal parts. 
 
 Duval mounted sections of objects imbedded in collodion in 
 glycerine, and was at first unsuccessful in his experiments with 
 balsam. Schiefferdecker has shown that by dehydrating the sec- 
 
 1 Journ. de l'anat. et de la physiol., XV., p. 185, 1879. 
 
 2 Arch. f. Anat. u. Physiol. Anat. Abth., p. 199, 1882. 
 
 126 
 
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THE USES OF COLLODION. 127 
 
 tions with 95 % alcohol, and clarifying in oil of origanum or oil 
 of bergamot, the sections could be mounted in balsam. 1 
 
 Some improvements of minor importance in the process of 
 imbedding have been made by Thoma, Blochmann, and others. 
 
 The importance of collodion in microtomy was much in- 
 creased by the important discovery that in combination with 
 clove oil it could be used as a fixative for serial sections, and 
 that the latter could be colored after they had been arranged 
 and fixed on the slide. This invaluable method, discovered by 
 Schallibaum, 2 presents all the advantages of the shellac method 
 of Giesbrecht, and offers, at the same time, the best means of 
 meeting the difficulties of staining objects in toto. The only 
 other fixative, thus far known, which claims to accomplish simi- 
 lar results, is that introduced by Mayer (v. Fixatives, Chap. 
 VII.). 
 
 Prof. Gage, 3 who began to experiment with collodion as a 
 fixative prior to the publication of Schallibaum's method, has 
 given some valuable directions respecting its preparation and 
 application. Gage applies the collodion and clove oil sepa- 
 rately, first coating a number of slides with collodion, which 
 is poured on to one end of the slide, and allowed to flow 
 quickly over it and off into the bottle, and then at the time of 
 using adding a wash of clove oil. In order to remove any 
 cloudiness that may arise in the collodion film, a little clove oil 
 is added to the balsam. 
 
 The use of collodion to prevent the crumbling of brittle sec- 
 tions was discovered independently by Duval 4 and Mason. 6 
 The same method was employed in Semper's Laboratory by 
 Timm, 6 Will, 7 Sharp, and others ; and Mark has found it indis- 
 pensable in sectioning the ova of Lepidosteus. Mason applied 
 the collodion by means of a fine brush, taking up a small drop 
 
 1 Celloidin is a good imbedding mass for injected objects that will not 
 bear heating, and especially for such large objects as the vertebrate brain. 
 s Arch. f. mik. Anat., XXII., p. 689, 1883. 
 
 3 The Medical Student, p. 14, November, 1883. 
 
 4 Society de Biologie, 1880. — , 
 
 6 American Naturalist, 1880, p. 825. — 
 Sempers Arbeiten, VI., p. 110, 1883. 
 
 7 Sempers Arbeiten, VI., p. 7, 1883. 
 
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128 MICROSCOPICAL METHODS. 
 
 and placing it " in the centre of the object, so as to allow it to 
 flow out on all sides, to prevent the formation of air bubbles. 
 After being allowed to harden a. minute, the section may be cut 
 and placed on the slide with the film of collodion underneath" 
 Mark and others who have used collodion for the same purpose 
 simply paint the cut surface of the object with a thin film a 
 few seconds before making each section. 
 
 Celloidin has also been recommended by Schiefferdecker for 
 forming injection masses. It penetrates easily, and its viscosity 
 protects the vessels from injury in the process of making corro- 
 sion preparations (v. Methods of Injection). 
 
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Part II. 
 SPECIAL METHODS. 
 
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CHAPTEE YIH. 
 
 EMBRYOLOGICAL METHODS. 
 
 DRAWING APPARATUS. 
 
 In Part I. of his "Anatomie menschlicher Embryonen," 
 pp. 8, 9, Professor His has described a drawing apparatus simi- 
 lar to the one here represented. 
 
 For anatomical and embryological work, an apparatus of this 
 kind is simply indispensable. As every working naturalist 
 knows, an apparatus that admits the use of the camera lucida 
 with a low magnifying power, varying from five to forty diame- 
 ters, offers many advantages that are not to be obtained from 
 any system of microscopical objectives. In the absence of 
 such an instrument, one is compelled to draw by measurement 
 and " by the eye," a process which at best is slow and tedious, 
 and liable to many inaccuracies. The foundation of every 
 thorough embryological work consists, as Professor His remarks, 
 of exact drawings of the entire embryos as well as the sections 
 obtained from them. Any one acquainted with the embryo- 
 metrical investigations of Professor His on the chick, will hardly 
 require to be told that such surface views as he employed for 
 orientation in microtomic sections could not be obtained with- 
 out the aid of photography, or the camera lucida, or by both. 
 The instrument here described offers the same facilities for 
 obtaining the exact topographical relations of a complicated 
 object with low magnifying powers that the microscope affords 
 with higher powers. Further, only a single plano-convex lens 
 2.5 0m in diameter, is required for an enlargement varying at 
 pleasure from five to fifty diameters. Professor His employs 
 
 131 
 
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132 
 
 MICROSCOPICAL METHODS. 
 
 as an objective a stereoscope-head (of Dallmeyer), or a small 
 Steinheil aplanat (No. 1). 
 
 The instrument consists of a heavy circular iron base, 1 from 
 the centre of which rises a brass rod, marked to centimeters, 
 half centimeters, and millimeters. On this rod are seen the 
 mirror (M), the object-table (T), the objective (0), and the 
 
 camera lucida (P), all sup- 
 ported by horizontal bars 
 that move on sliding fer- 
 rules. The mirror is placed 
 as near the base as conve- 
 nience will allow, and its 
 supporting bar is 7.5 cm long. 
 The bars bearing the other 
 pieces are all of correspond- 
 ing length, and the sliding 
 ferrules can be fixed at any 
 point by the aid of set- 
 screws. The ferrules of the 
 mirror and the object-table 
 are made of such length that 
 when in contact with each 
 other and resting on the 
 highest part of the base, 
 they are in the position re- 
 quired for the lowest mag- 
 nifying power. In this 
 position the object-table has 
 an elevation of ll cm , the ob- 
 jective 18.5 cm , and the cam- 
 era 22 em above the lower face 
 of the base, or what may be 
 called the drawing plane. 
 Thus placed, the focal distance is 7.5 cm , and the camera is 
 considerably lower than it is possible to have it in ordinary 
 microscopes. The magnifying power is 10 diam., but it may 
 
 1 This form has been found more convenient than the rectangular form 
 seen in the figure. 
 
 Fig. 30. — Drawing apparatus. 
 
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EMBRYOLOGICAL METHODS. 133 
 
 be reduced to 5 diam. by elevating tbe drawing plane. It is of 
 course possible to obtain a lens that will magnify only 4 or 5 
 diam. in the normal position of the drawing plane, and en- 
 deavors will be made to provide the instrument with such a 
 lens. 
 
 The magnifying power may be increased in several ways, but 
 most conveniently by shortening the focal distance and raising 
 the camera until it is properly adjusted. Starting with the 
 parts placed as above given, in order to raise the magnifying 
 power from 10 to 15 diam., the objective must be lowered 7 mm 
 and the camera elevated 2 cm ; and for 20 diam., the objective 
 must be lowered 10 mm and the camera elevated 3.5 cm . Keep- 
 ing the object-table in its first or normal position, the mag- 
 nifying power may be increased to 50 diam. by lowering the 
 objective 2 0m and raising the camera 15 cm . The camera thus 
 has an elevation of 37 cm above the normal drawing plane. 
 The magnifying power may also be increased by lowering the 
 drawing plane, or what is the same thing, by raising the object- 
 table. 
 
 The positions which the objective and camera must have for 
 a given magnifying power will probably vary slightly in two 
 instruments made as nearly alike as possible, but their determi- 
 nation is a very simple matter ; and having been once accurately 
 ascertained, they may be tabulated and kept for subsequent use. 
 To ascertain these positions for any given magnification, a 
 millimeter scale may be placed on the object-table, and the 
 camera and objective moved until the picture projected on the 
 drawing plane has the desired enlargement. When the scale 
 is replaced by the object, care must be taken to have the sur- 
 face which is to be outlined in the plane previously occupied 
 by the scale. To this end it may be necessary to move the 
 object-table a very little, in order to give a sharply defined 
 picture, the j>ositions of the camera and objective being left 
 unaltered. 
 
 The object-table measures 8 X 10 cm , and has a central perfo- 
 ration 2.5 0m in diam. The whole apparatus is completed by a 
 movable shade, designed to cut off the light falling on the lens 
 and on the drawing plane. 
 
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134 MICROSCOPICAL METHODS. 
 
 It is hardly necessary to remark that opaque objects require 
 direct sunlight or light from a lamp supplied with a hull's eye 
 condenser. 
 
 This instrument, including lens, and Oberhauser's camera 
 lucida, may be obtained from The Educational Supply Co., 
 7 Hamilton Place, Boston, for thirty dollars. For everything 
 except the camera, the price is fifteen dollars. 
 
 MR. IIJIMA'S METHODS OF PREPARING PLANARIANS AND 
 THEIR EGGS.i 
 
 In the preparation of Planarians for histological study, Mr. 
 Iijima recommends corrosive sublimate as the only good pre- 
 servative agent. The worms are placed in a shallow plate, 
 without water, and a saturated solution of corrosive sublimate, 
 heated almost to boiling, is poured over them. In this way 
 they are killed so quickly that they do not have time to con- 
 tract. They are left thirty minutes or less in the sublimate; 
 then placed in water for an hour or more. The water should 
 be changed several times, in order to remove all of the subli- 
 mate ; otherwise it forms needle-like crystals, which impair or 
 ruin the preparation. Three grades of alcohol ("weak, strong, 
 and absolute ") are used in hardening, in each of which the 
 object should be left at least forty-eight hours before staining. 
 Borax-carmine (probably the alcoholic solution) is recom- 
 mended as a staining agent ; a dilute solution is used in prefer- 
 ence to the full strength, and allowed to act from three to 
 four days. 
 
 For preservation as museum specimens, they are killed with 
 strong nitric acid (about 50 %), in which they die fully extended. 
 
 Preparation of the Ova. — The egg-capsules of fresh-water 
 Planarians are generally attached to water-plants by means of 
 a white secretion. The ova are very small and few in number, 
 and are scattered among an immense number of yolk-cells. 
 The ova are completely naked, and a little -smaller than the 
 yolk-cells, and are not easily isolated. When cleavage begins, 
 
 1 " Entwicklungsgeschichte der Siisswasser-Dendroccelen." Zeitschr. f. 
 wiss. Zoologie, XL., p. 359, 1884. 
 
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EMBKTOLOGICAL METHODS. 135 
 
 a large number of yolk-cells surround the ovum, and form 
 with it a mass large enough to be seen with the naked eye. 
 Mr. Iijima adopts the following mode of isolation and prepara- 
 tion : By the aid of two sharp dissecting needles, the egg- 
 capsule is opened on an object-slide in dilute acetic acid 
 (2 fo). The contents flow out, and the empty capsule is 
 then removed. The slide is next shaken in order to isolate the 
 ova so far as possible from the yolk-cells. This process de- 
 taches many of the yolk-cells, but not all; each ovum will still 
 have yolk-cells adhering to it, and will now appear to the 
 naked eye as a minute white mass. A cover-glass, supported 
 by wax feet or by slips of paper, is now placed over them. 
 After about thirty minutes, the acetic acid is carefully removed 
 by the aid of small pieces of blotting paper placed at one side 
 of the cover, and replaced by alcohol (70 fo). The with- 
 drawal of the acetic acid must be as slow as possible, 
 otherwise the ova will be lost. After an hour the alcohol is 
 replaced by a stronger grade (90 fo), in which the ova 
 should remain two hours, Finally, the alcohol is replaced 
 by a mixture of glycerine and water in equal parts, and this in 
 turn by pure glycerine. The preparation is now complete, 
 and the cover-glass may be fixed in the usual way by means 
 of lac. 
 
 In order to obtain sections of embryos which are too small 
 to be treated individually, the contents of the capsule may be 
 hardened in toto in chromic acid (1 fo), which renders them 
 less brittle than corrosive sublimate. 
 
 The changes which take place in the ovum initiatory to 
 cleavage are very difficult to trace, as they are generally com- 
 pleted before the cocoon is laid. In some cases ova were found 
 in fresh-laid capsules, which showed the germinal vesicle still 
 unchanged ; others were found to have two nuclei, supposed 
 to be derivatives from the first cleavage-nucleus. This stage 
 of two nuclei was also found in some cocoons taken directly 
 from the penial sheath, in which the cocoon formation takes 
 place. It is therefore not quite certain when fecundation 
 takes place, whether in the cocoon or before its formation. 
 
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136 MICROSCOPICAL METHODS. 
 
 THE EGGS OF THE BRACHIOPODA. 
 
 The eggs of Terebratulina septentrionalis are found, accord- 
 ing to Morse, 1 throughout the summer months. 
 
 Specimens may be collected at low-tide mark, and by dredg- 
 ing at various depths. Morse found them abundant at East- 
 port, Me. 
 
 For the following on methods of obtaining the eggs I am 
 indebted to Dr. "Wilson : — 
 
 Sexual Distinction. — The sexes are distinguished by the 
 color of the pallial sinuses as seen through the valves of the 
 shell, the males being white, the females yellowish or cream- 
 colored. The eggs are usually discharged at night, probably 
 in the morning hours, and may be obtained in abundance by 
 keeping males and females in about equal numbers (20-30 
 each) in a vessel of water over night. The jars were kept 
 in a dark, cool cellar in plates of water, and wrapped in 
 cloth. The cloth soaks up the water, and the evaporation 
 keeps up a uniformly cool temperature. Thus kept, the 
 water requires to be changed only once a day. 
 
 The eggs will be found at the bottom of the jar, and may be 
 gathered with a dipping tube. 
 
 By tearing open the valves of females, embryos may also be 
 often found in two whitish masses at the side of the body 
 behind the mouth. 
 
 In the active, free-swimming stage the water cannot be 
 changed, but the larvae appear to flourish. 
 
 THE OVA OF CLEPSINE.2 
 
 The eggs of Clepsine are in many respects very favorable 
 objects of study. They are easily obtained in abundance, 
 can be kept alive through all stages of development without 
 difficulty, and are large enough to permit of following the de- 
 velopmental changes with a magnifying power of 20-30 diame- 
 ters. In order to observe the earliest changes, it is not necessary 
 
 i Mem. Bost. Soo. Nat. Hist., II., p. 30, 1869. 
 
 a Whitman. Quart. Journ. Micr. Sci., XVIII., 1878, pp. 2, 3, 9-11. 
 
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EMBRYOLOGICAL METHODS. 137 
 
 to watch for the deposit of the eggs ; for I have found that 
 eggs taken from the ovary at the time they are about to be laid 
 develop in the normal manner, so that material may be had at 
 any moment by simply cutting a leech with ripe eggs in two in 
 the middle. These are rare advantages in the study of the 
 phenomena of maturation and cleavage. In addition to all 
 this, certain extremely interesting phenomena display them- 
 selves at the two poles of these eggs, — phenomena which have 
 no parallel, so far as at present known, in the rest of the ani- 
 mal kingdom ; and all the principal types of cleavage are exhi- 
 bited in rapid sequence, beginning with the regular, and then 
 shifting at successive stages into the unequal, partial, and super- 
 ficial (or plasmodial). 
 
 Habitat. — In ponds, and in brooks and rivers where the 
 current is very slow, on the under side of stones, pieces of 
 wood, dead leaves, fallen branches, and other objects lodged in 
 muddy places. 
 
 Food. — Most of these animals require only one or two 
 meals a season, and, beyond this, demand only clean water. 
 Some suck the blood of fishes, others live on the blood of tor- 
 toises. Many of the smaller species derive their sustenance from 
 fresh-water snails, and one (Japan) lives habitually on Anodonta. 
 
 Time of Deposit. — C. sexoculata lays in April ; the allied 
 species of the Eastern States the first week of May. O. mar- 
 ginata lays from the 1st of May to the middle of August, while a 
 closely allied species of Massachusetts deposits early in May. 
 C. bioculata, a less favorable species for the study of the devel- 
 opment, produces eggs from April to September. 
 
 Egg-Sacs. — In most species, the eggs are deposited in thin, 
 transparent sacs (1-8) ; but in C. marginata they are covered 
 with a transparent secretion, which hardens in the course of a 
 few minutes, and thus binds the eggs together and to the ob- 
 ject on which they are placed. 
 
 Preparation for Sectioning. — The best results were ob- 
 tained from eggs treated with osmic acid (tV%) 15-30 minutes, 
 then, after passing through the different grades of alcohol, 
 stained in toto with Beale's carmine. 
 
 Hoffmann recommends bichromate of potassium. 
 
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138 MICROSCOPICAL METHODS. 
 
 Surface Views of the Germ-Bands. — The linear ar- 
 rangement of the cells composing the germ-bands is well seen in 
 eggs hardened in chromic acid (J- fo) 5-10 hours. 
 
 For views of the inner surface, it is necessary to free the 
 germ-bands from the yolk. The fresh embryo is placed in a 
 drop of water on an object-slide ; a little acetic acid (as much 
 as will adhere to a needle) is added, and all placed under' the 
 dissecting microscope. With a pair of sharp needles a rupture 
 is made along the dorsal side. By careful manipulation of the 
 needles, the embryo, in most cases, can be led away from the 
 yolk, and stretched out on the slide. 
 
 After partially removing the water by the aid of a bit of 
 filtering paper, a few drops of osmic acid are added, with care 
 not to disturb the object. At the end of an hour, the embryo 
 is washed and stained with Beale's carmine. It is again washed, 
 and, after dehydration, mounted in balsam or glycerine. Dur- 
 ing all this, the object is not once removed from the slide. 
 
 The Vascular System. — The course of the blood-vessels 
 cannot be fully made out in adult specimens. For this purpose 
 it is best to take very young individuals (10-15 days old), in 
 which the pigment is little developed. 
 
 The live-box enables one to apply pressure sufficient to render 
 distinct the main channels and branches with a low magnifying 
 power. 
 
 SPIEOEBIS BOREALIS. 
 
 The adult Spirorbis lives in a small, coiled, calcareous tube, 
 which has the form of a round, flat disk. These little white 
 coils are found in great numbers attached to seaweed and other 
 foreign bodies. They are easily kept in a small aquarium, and 
 the young may be reared in multitudes. The cleavage stages 
 and earlier larval stages are passed before the tube of the 
 parent is abandoned, and this renders it possible to make a 
 complete study of the development. The necessity of break- 
 ing the shell of the adult to obtain the eggs is a drawback. 
 
 Fewkes * placed the shells in a watch-glass, and crushed them 
 
 1 J. W. Fewkes, Am. Nat., XIX, March, 1885, p. 247. 
 
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EMBKYOLOGICAL METHODS. 139 
 
 with a spatula. On removing the fragments of the case, the 
 reddish-brown eggs are found in short strings containing from 
 one to four rows of from ten to fifteen eggs each. 
 
 Mr. Agassiz 1 states that the development begins in the body 
 of the parent before the egg-sacs are deposited in the cavity of 
 the tube, where most of the stages are completed. Fewkes did 
 not find the early stages of cleavage, but always found the later 
 stages outside the body of the parent. 
 
 MYZOSTOMA. 
 
 The reproduction of this parasitic chastopod, two species of 
 which are found on Comatula, is not limited to any particular 
 season of the year, — at least in Naples, according to Mr. Beard. 
 Its food is limited to some portion of that of its host, and con- 
 sists of infusoria, algse, and other minute organisms. 
 
 In studying the development, Mr. Beard 2 obtained his results 
 mainly by examining the different stages in a living condition. 
 
 To obtain naturally fertilized ova, a number of Comatulse 
 infested with full-grown M. glabrum are taken, and the arms 
 cut away not far from the disc. The Comatulse, with their 
 parasites, are then placed in small, deep glasses filled with sea- 
 water, through the upper part of which a gentle current is 
 maintained. The ova, which are laid nearly every day, sink to 
 the bottom, and may be readily found by removing the Comar 
 tulaa, and pouring off most of the water. The ova may be kept 
 alive four or five days after removing the Comatulse. 
 
 Artificially fecundated ova may be easily obtained in abun- 
 dance in the following manner : A number of mature Myzos- 
 toma are carefully removed from their hosts, and placed in a 
 watch-glass holding two or three teaspoonfuls of clean sea- 
 water ; they are then teased with clean needles, which results 
 in setting free a large number of ova and spermatozoa. The 
 contents of the glass are well stirred up, and allowed to stand 
 for two or three hours. The fragments of the Myzostoma are 
 removed by the aid of needles, and what remains thrown into a 
 
 1 A. Agassiz, Ann. Lye. Nat. Hist., VIII., p. 318. 
 
 2 Mitth. d. zool. Station z. Neapel, V., p. 545-547, Nov., 1884. 
 
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140 MICROSCOPICAL METHODS. 
 
 glass full of clean sea-water. This glass should be covered, and 
 the water kept well aerated by means of an air-apparatus such 
 as has been devised by Dr. Andres. As the ova and larvss 
 always remain at the bottom, the greater portion of the water 
 can be poured off, and so renewed every second or third day. 
 
 It is well in each experiment to take at least four or five 
 Myzostoma; for they are hermaphrodite, and it is therefore 
 necessary to guard against self-fertilization as far as possible. 
 The number of instances in which it can occur will, of course, 
 be diminished by increasing the number of individuals in each 
 experiment. 
 
 The post-larval stages may be obtained by slowly killing the 
 Comatulfe in a mixture of sea-water with 10 % alcohol. When 
 dead, each individual is shaken in the mixture by the aid of 
 forceps, in order that the Myzostoma may fall off. The most 
 of the water is next poured off, and alcohol gradually added 
 until a strength of 90 % is reached. A large number of para- 
 sites may then be stained together, and mounted for micro- 
 scopic examination. 
 
 THE EGGS OF SAGITTA." 
 
 To obtain the eggs of Sagitta, it is only necessary to place 
 freshly-captured animals with gravid reproductive organs in a 
 small vessel for a few hours. The eggs will be found floating 
 just under the surface of the water. Although the eggs are 
 comparatively large, it requires favorable light to see them, as 
 they are perfectly transparent. They can be fished up by 
 means of a glass tube or a large pipette. 
 
 The earliest stages may be best obtained by cutting open 
 gravid specimens, allowing the sexual products to flow out 
 through the opening thus made. In this way, the process of 
 fecundation and the production of polar globules may be wit- 
 nessed. 
 
 They can be easily studied in a living condition, and the 
 views thus obtained may be supplemented by treatment with 
 1-2 % acetic acid. 
 
 1 O. Hertwig. Jenaische Zeitschrift, XIV., 1880, p. 271. 
 
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EMBRYOLOGICAL METHODS. 141 
 
 PREPARATION OF THE OVA OF THE CRAYFISH." 
 
 A. Surface Preparations of the Early Stages. — 1. Heat 
 the ova in a vessel partially filled with water, raising the tem- 
 perature gradually to 80° C. 
 
 2. After cooling, make an incision in one side of the chorion 
 with a sharp scalpel, and then remove the chorion with needles. 
 The thin membrane inside the chorion may then be easily 
 removed. 
 
 3. With a very sharp razor, wet with strong alcohol, while 
 holding the ovum between the fingers, cut off the embryonic 
 side of the ovum in the form of a thin, plano-convex disc. 
 
 4. Stain the disc in Ranvier's picro-carmine (about thirty 
 minutes). 
 
 5. Wash, treat with alcohol, and mount in balsam. 
 
 B. Preparation for the Microtome. — 1. After heating 
 in water to 80°, place the ovum (still in its chorion) for 24 
 hours in bichromate of potassium (1-2%) or in chromic acid 
 
 2. Wash for 24-48 hours in water, which must be often 
 changed. 
 
 3. Harden in weak alcohol (60-70%) 24 hours, and for the 
 same time in absolute alcohol. 
 
 4. Remove the chorion, and stain in toto in dilute Beale's 
 carmine, or in carmine acetate. 
 
 5. After treating with alcohol, cut off the embryonic surface 
 and imbed it in paraffine. 
 
 THE EGGS OF THE PHRYGANIDS.2 
 
 Time of Ovipositing. — The eggs of Neophalax are prob- 
 ably laid after dark. They were found in abundance from the 
 middle of August till late in October. It was not ascertained 
 how much earlier than the middle of August they may occur. 
 
 Place of Deposit. — According to Rathke, Zaddach, and 
 
 *H. Reichenbach. Zeitschr. f. wiss. Zool., XXIX., pp. 124-126, 1877. 
 2 Wm. Patten. Quart. Jour. Mic. Sc, XXIV., Oct. 1884, pp. 549-558. 
 
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142 MICROSCOPICAL, METHODS. 
 
 McLachlah, the Phryganids lay their eggs, arranged in a single 
 layer, and enveloped in a transparent, gelatinous substance, on 
 the under side of various water plants. The eggs of Neophalax, 
 however, are deposited on the muddy bottom of slow running 
 streams. They are found in greatest abundance in water four 
 or five inches deep, and where there are a great man) - twigs 
 lying on the bottom and projecting above the surface. 
 
 The Eggs and their Gelatinous Envelopes. — About 
 150 eggs are found in a single spherical, vesiculated mass of 
 gelatinous substance. The eggs are greenish yellow, and their 
 gelatinous envelopes have a strong odor of musk, which is 
 perceptible even after they have been hardened by heat and 
 preserved in alcohol for several months. 
 
 Preparation of the Eggs. 1. The best method of killing 
 is to j>lace the eggs in cold water and raise the temperature 
 very gradually to about 80° C. ; the proper time to check the 
 rise in temperature is indicated by the eggs becoming hard and 
 white. 
 
 2. After cooling, they are removed to 20% alcohol, which is 
 increased by 10% once or twice a clay until it has reached the 
 required strength (about 96$;). 
 
 3. Only alcoholic stains will penetrate the chorion, and of 
 these Kleinenberg's haematoxylin and a 70% solution of cochi- 
 neal gave by far the best results. Partial and varied success 
 was obtained with safranin dissolved in 90% alcohol. The 
 eggs were allowed to remain in hasmatoxylin 5-6 days, and 
 were then decolorized very gradually in acidulated alcohol 
 (1 drop of strong hydrochloric acid to 20 grams of alcohol), in 
 which they remained several days. They were then transferred 
 to pure alcohol, which was changed once or twice until the 
 object regained its violet color. 
 
 Imbedding. — The eggs are clarified in benzole instead of 
 clove oil, as the former penetrates almost instantly, and is less 
 likely to cause shrinkage or to render the yolk hard and brittle. 
 
 After remaining 30 minutes in benzole, they are placed in a 
 saturated solution of jDaraffine in benzole, and then in pure 
 melted paraffine (melting at 58° C). 
 
 Cutting. — The blocks of paraffine in which the objects are 
 
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EMBRYOLOGICAL METHODS. 143 
 
 imbedded should have the form of a triangle, the base, which 
 is the shortest side, being just wide enough to hold the object, 
 and the altitude about five or six times the length of the base. 
 The apex of the triangle is directed towards the edge of the 
 knife, which, if placed at an angle of 10° or 12° to the direction 
 in which it slides, will reach the apex of the triangle first, and 
 cut a closely rolled shaving of paraffine. The section of the 
 object, since it lies in the last portion of the section cut, will 
 occupy the outside roll ; and, if the triangle is long enough, 
 will not cover more than one half the circumference of the 
 roll. The whole roll is placed on the slide with the section of 
 the object down. When the slide is warmed, the melting 
 paraffine flattens the section, and thus the trouble of unrolling 
 is avoided. 
 
 By this method, very thin sections (about one four-hundredth 
 of a millimeter) may be obtained. 
 
 TREATMENT OF THE OVA AND EMBRYOS OF THE APHIDES. 
 
 Witlaczil : gives a lengthy paper on the development of the 
 Aphides, and in it the following information on methods : — 
 
 The embryos of the viviparous A]3hides were examined in a 
 weak salt solution (1£ per cent), in which they live for about 
 an hour. The ovaries contain embryos in different stages of 
 development, and have to be isolated for study. 
 
 The early stages in the development of the ova may be 
 studied to advantage after treatment with hydrochloric acid 
 (3 %), or acetic acid, as these reagents partially dissolve the 
 yolk elements and thus render the preparation more trans- 
 parent. The later stages, on the contrary, are rendered more 
 opaque by the same treatment. 
 
 The ovaries of viviparous Aphides were prepared for section- 
 ing, by Ludwig Will, 2 in the following manner. 
 
 The Aphis is killed in water heated to about 70° C, then 
 hardened in successively higher grades of alcohol. In order to 
 color in toto, the cuticula must be punctured with a sharp 
 
 i Zeitschr. f. wiss. Zool., XL, Heft 4, p. 563-564. 
 2 Semper's Arbeiten, VI. 1883. 
 
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144 MICROSCOPICAL METHODS. 
 
 needle, and then the dye will penetrate easily to all parts. As 
 very thin sections are required for the study of such small 
 elements, it is best to use dyes that stain very deeply, such as 
 borax-carmine and ha3matoxylin. It is further necessary to 
 employ either the shellac or the collodion method of fixing the 
 sections, otherwise important parts are liable to drop out of place. 
 
 THE EGGS OF LIMULUS. 
 
 The eggs of Limulus are laid between and above tide-marks 
 about the first of June, on the south side of Cape Cod. It 
 appears probable that the period of ovulation is rather pro- 
 longed, as I have found at Annisquam eggs well matured in 
 the oviduct the last week in July. In hunting for the eggs no 
 indications can be followed. The eggs are deposited in the 
 sand and must be searched for at random. In general appear- 
 ance, as they lie loosely in the sand, they resemble seed pearls ; 
 the general color is a dirty green or ashy gray, but pink eggs 
 are common, and these develop as well as any. My attempts 
 at artificial impregnation were apparently unsuccessful, though 
 I learn from Dr. E. B. Wilson, that Mr. H. L. Osborn, of 
 Johns Hopkins University, tried it and found that the early 
 stages developed slowly, and possibly I did not keep the 
 eggs long enough. 
 
 The eggs obtained from the sand possess great vitality and 
 will live for months in confinement. Embryos from eggs ob- 
 tained in June were kept in a small aquarium until November, 
 the loss of water from evaporation being made good by daily 
 additions of fresh water. In Dr. Lockwood's experience, eggs 
 passed through the winter, hatching in the spring. I obtained 
 my best results in the following ways : — 
 
 For surface views, a light staining with osmic acid (-^ %) 
 proved best, rendering visible details not otherwise readily 
 seen. For sections, successive grades of alcohol (50, 75, 90 fc, 
 and absolute) were at least equal to chromic acid, Merkel's 
 fluid, Perenyi's fluid or Midler's fluid. With chromic acid they 
 show a tendency to shrink. Corrosive sublimate renders the 
 yolk very hard and brittle, while the coagulation of the albu- 
 men by heat, so successfully employed in studying many arthro- 
 
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EMBRYOLOGICAL METHODS, 145 
 
 pods, proved in my hands worthless. The greatest difficulty 
 with the earlier stages was in freeing the egg from the chorion. 
 In most cases I had to cut chorion and egg together, and the 
 results were far from satisfactory. The eggs stain very slowly. 
 My best preparations were obtained by over-staining the sections 
 on the slide with eosin or haBmatoxylin, and then withdrawing 
 the excess of color by acid alcohol. — J. S. Kingslet. 
 
 MOLLUSCAN EGGS. 
 
 For embryological study, Fol 1 recommends Planorbis as the 
 most favorable object among fresh-water molluscs. 
 
 Planorbis contortus deposits in May and June ; P. margina- 
 tus, from July to September. 
 
 Helix deposits its eggs in the earth and then covers them. 
 If one marks the spots where they are seen preparing their 
 nests and does not disturb them, the eggs may be found on the 
 following day. The first stages of cleavage can only be ob- 
 tained by killing specimens ready to deposit. The eggs, if 
 carefully removed from the cocoon, and placed in a compres- 
 sorium where they can be kept moist, and supplied with air, 
 will develop normally for a week or more. 
 
 THE EGGS OF THE SLUG (Umax campestris)? 
 
 Place of Deposit. — The eggs are found in moist places, 
 protected from the drying influences of direct sunlight and cur- 
 rents of air. They are frequently met with in the vicinity of 
 a small stream, some stagnant pool, or in low meadow-land. In 
 such localities, loose piles of decaying wood are often chosen 
 for the deposit of eggs. At other times, when the ground is 
 less protected, they may be found under loose stones, or even 
 in the bed of some spring-time water channel, where crevices 
 in clumps of earth afford protection. They are laid at all hours 
 of the day, but, as a rule, early in the morning. 
 
 Mode of Culture. — The most convenient way of obtain- 
 
 1 Arch, de Zool. exp6rimentale, VIII., p. 103. 
 
 2 Mark. Bull. Mus. Comp. Zool., VI., Part 2, No. 12, 1881. 
 
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146 MICfiOSCOPIOAL METHODS. 
 
 ing material is to keep the slugs in confinement. If properly 
 cared for, they will thrive, and produce eggs, even during the 
 winter. They may be kept in glass or earthen dishes covered 
 with gauze or with perforated tin covers, and supplied with 
 moss, which must be kept wet. 
 
 Food. — Lettuce, plantain leaves, cabbage, &c. During the 
 winter they may be kept on wheat bran. To avoid the rapid 
 growth of mould which follows when the bran is sjirinkled over 
 the wet moss, the bran may be kept in a watch-glass. If they 
 are not kept supplied with food, they will devour one another. 
 
 Appearance of the Eggs. — The eggs are usually found 
 in clusters of about a dozen, though the number may vary from 
 one to thirty or forty. Sometimes they are loosely collected 
 together, or even scattered over an area of a few inches ; at 
 other times, they are closely packed in more or less rounded 
 masses. They are often arranged in rows in the narrow cracks 
 of moist, decaying wood, or in the chinks in caked earth. 
 They are frequently united in the form of a rosary. 
 
 Egg-Envelopes. — The ovum proper is just visible to the 
 unaided eye as a whitish dot, having not more than one twenti- 
 eth the diameter of the whole egg. The ovum is imbedded in 
 a yellowish, viscid substance, called the albuminous envelope. 
 This central mass, occupying from § to § the diameter of the 
 egg, is invested by a very firm, structureless membrane (mem- 
 brana alhuminis). External to this are two thick, trans2)arent 
 envelopes, the inner of which is semi-fluid and homogeneous, 
 while the outer is laminated and elastic. 
 
 Preparation of the Ova. — 1. Treat with acetic acid 
 (1-2 %) four hours or more. 
 
 2. Remove the outer envelopes in the following manner : — 
 
 Hold the egg between the thumb and finger of the left hand, 
 thrust the point of fine scissors through the two external enve- 
 lopes, and then make a cut sufficient to allow the resistant 
 membrane (membrana albuminis) with its contents to escape. 
 Place the egg in a drop of water on a slide, and remove the 
 external envelope. Portions of the semi-fluid envelope will 
 remain attached to the surface of the resistant membrane. 
 The next step is more difficult. Thrust a sharp needle, under 
 
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EJIBRYOLOGICAL METHODS. 147 
 
 the dissecting microscope, quite through the resistant membrane, 
 and thus hold the egg, while, with another needle, a second 
 puncture, followed by a gash (by gently pulling in opposite 
 directions with the needles), is made, which will allow the ovum 
 to escape with the albumen. Float the ovum, by the aid of one 
 needle, into a fresh drop of water near one end of the slide, 
 strand it, and quickly wipe away (with a cloth wound over the 
 tip of the fore finger) all remaining albumen and water. Add 
 a drop of fresh water, and float the ovum back to the centre of 
 the slide. Arrange a cover-glass with wax feet, and adjust it 
 under the microscope. 
 
 3. Allow a drop of Beale's carmine to flow under the cover, 
 and examine at once. The ovum may remain from 1 to 2 hours 
 in the staining fluid. 
 
 4. Draw away the carmine with the aid of a brush, and at 
 the same time allow dilute glycerine to replace it. 
 
 5. Replace the dilute with pure glycerine. 
 
 This method will serve for studying the earlier changes in 
 the ovum. In later stages osmic acid works better than acetic 
 acid, as it hardens the albuminous envelope as well as the ovum. 
 
 EGGS OF THE STARFISH. 
 
 The following extract is from A. Agassiz's Embryology of 
 the Starfish 1 : — 
 
 Sexual Distinction. — The males and females of our com- 
 mon species of Starfishes, Asterias vulgaris and A. arenicola, 
 can readily be distinguished by their difference in coloring, all 
 those having a bluish tint being invariably females, a reddish or 
 reddish-brown color indicating a male. When cut open, so as 
 to expose the genital organs, the difference between the males 
 and females is still more striking. The long, grape-like clusters 
 of reproductive organs, extending from the angle of the arms, 
 on both sides of the ambulacral system, to the extremity of the 
 rays, present very marked differences in the two sexes. The 
 ovaries are bright orange, while the spermaries are of a dull 
 cream-color. At the time of spawning, which is very different 
 in the two species mentioned above, the genital organs are dis- 
 1 Mem. Mus. Comp. Zool., Fart First, pp. 3-5. 
 
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148 MICROSCOPICAL METHODS. 
 
 tended to the utmost, filling completely the whole of the cavity 
 of the ray, the abactinal system itself being greatly expanded 
 by the extraordinary development of these organs. 
 
 Artificial Fecundation. — If we take a male and female 
 starfish in this state, and cut a portion of the genital organs 
 into small pieces, we shall find that the eggs and spermatozoa 
 escape in such quantities as to render turbid the water in which 
 they are placed. Throwing these small pieces of the genital 
 organs into shallow dishes containing fresh sea-water, and stir- 
 ring the mixture thoroughly to ensure the contact between the 
 spermatozoa and the eggs, will be sufficient to fecundate the 
 latter. In order to make the operation perfectly successful, some 
 precautions are necessary : all the pieces of the genital organs 
 which are left after repeated stirring must be carefully removed ; 
 there must not be too many eggs in one dish, so that the water 
 can have free access to them in every direction. The removal 
 of the remnants of the ovaries and spermaries is very necessary, 
 as the pieces which remain clotted together decompose very 
 rapidly, and endanger the safety of the eggs, even when the 
 water can be changed with the greatest facility. As soon as the 
 fecundation is fulfilled, the water in the dishes must be repeat- 
 edly changed, until it becomes perfectly clear ; for the presence 
 of too many spermatozoa, rendering the water milky, prevents a 
 favorable result. It is best only to use one male and one female 
 for the mixture in each vessel, as eggs taken from many indi- 
 viduals lessen the chances of success. The eggs sink to the 
 bottom, so that the water can be poured off and changed with- 
 out much danger of throwing them away. Immediately after 
 the mixture is made, the water should be changed three or four 
 times in succession ; after that, every half hour until the fourth 
 hour, when an interval of two to four hours may elapse before 
 renewing the water. As it is extremely difficult to change the 
 water after the embryos have hatched and are swimming freely 
 about in the jar, without losing many of them, it is advisable 
 before they hatch, which is about ten hours after the fecunda- 
 tion, to reduce the water to a minimum volume, and then simply 
 to add a little fresh sea-water and remove the contents of the 
 vessel to larger and larger jars. In this way the water can 
 
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EMBEYOLOGICAL METHODS. 149 
 
 be maintained sufficiently pure until the young embryos have 
 taken the habit of swimming near the surface, when it may all 
 be drawn off by means of a siphon. A great deal of time and 
 trouble will be saved by this mode of procedure, and fewer 
 specimens lost. The jars containing the eggs should be kept 
 in a cool place ; the most convenient method of securing a low 
 and even temperature is to place the small jars in large tubs 
 filled with cold water. 
 
 BALANOGLOSSUS. 
 
 The specimens first discovered were found at Charleston, 
 S. C, by Stimpson, over thirty years ago, and described by 
 Girard, under the name of Stimpsonia aurantiaca. 1 Mr. Agas- 
 siz has found it at Beverly, Mass., and at Newport, R. I. 
 
 Packard collected it at Beaufort, N. C. ; and Verrill reports it 
 from Fort Macon and Naushon Island. 
 
 It is found at low-water mark, buried in the sand to a depth 
 of eight to fourteen inches. Its burrowings are recognized by 
 peculiar elliptical coils of sand which are thrown out at the 
 surface. 2 The burrow is lined by a "thick mucous layer," 
 which forms a sort of case in which the worm is able to move 
 up and down. 
 
 The earliest free-swimming larva? are found, according to 
 Mr. Agassiz, about the first of August, at Newport, and are 
 captured by surface skimming. 
 
 According to Bateson, 3 who has recently studied the species 
 occurring at Hampton, Va., the sexual products escape by a 
 rupture of the body-wall in both sexes. The eggs are small, 
 ovoid, opaque, grayish yellow in color, and about f mm in length ; 
 they are enclosed each in an elastic, transparent membrane. 
 The spermatozoa dehisce in lobate spermatophoric masses. 
 
 Bateson * made use of the following method for obtaining 
 the larva? and embryos : — 
 
 1. The mud inhabited by Balanoglossus is placed in a glass 
 
 i Verrill. Eeport 1871-1872, p. 351-2. 
 
 2 A. Agassiz. Mem. Am. Acad. Arts and Sciences, IX., p. 431, 1S73. 
 
 8 Quart. Jour. Mic. Sci., XXIV., Ho. 94, p. 209, 1884. 
 
 4 Quart. Journ. Micr. Sc, XXV. (Supplement, 1885), pp. 115, 116. 
 
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150 MICROSCOPICAL METHODS. 
 
 vessel of water, and stirred up, avoiding rotatory movement, 
 until the whole is in suspension. 
 
 2. A number of adult individuals cut into very fine pieces 
 are next thrown in, and the whole left to settle for a few min- 
 utes. These pieces sink to the same depth as the larvae, and 
 thus serve to mark the important layer. Practice enables one 
 to dispense with this step, the right layer being recognizable by 
 the size and character of the particles composing it. 
 
 3. The water and lighter particles in suspension are then 
 siphoned off until the layer of chopped Balanoglossus is 
 reached. This layer is drawn off, and examined separately. 
 In this manner all the animals living in several hundredweight 
 of mud may, in an hour or two, be collected into about a pint 
 of water, and sorted with a simple microscope. This was gen- 
 erally done by rotating a little of the water in a shallow saucer 
 with a slight peripheral groove. The larva? then all lie in the 
 groove, which may be passed under the lens by rotation. 
 
 Preservation. — The larvae are placed for less than a min- 
 ute in — 
 
 Corrosive sublimate (sat. sol.) 2 parts 
 
 Glacial acetic acid 1 " 
 
 After washing with water, they are passed through 30 %, 50 %, 
 70 %, and 90 % alcohol. While this method gave the best 
 results, on the whole, the softer parts were found to be best 
 preserved in specimens that were treated with Perenyi's fluid 
 one hour, and then transferred to 90 % alcohol. 
 
 THE EGGS OF DOLIOLTJM. 1 
 The ripe egg of Doliolum passes through the genital orifice 
 into the atrium or cloacal chamber, and is soon carried out of 
 this chamber with the water. The egg does not swim on the 
 surface of the water, but sinks to the bottom and there remains 
 until the larval tail is formed. There is no way, therefore, of 
 obtaining material for the study of the development except 
 
 1 B. Uljanin. Fauna u. Flora des Golfes von Neapel. X. Monogra- 
 phic: Doliolum, pp. 46-49. 1884. 
 
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EMBRYOLOGICAL METHODS. 151 
 
 that of keeping the two sexes in confinement. TJljanin kept 
 them in glass vessels filled with filtered sea-water and covered 
 with a glass plate. The vessels were placed in tanks, and a 
 small stream of water allowed to flow steadily on to the glass 
 cover. The evaporation of the water from the sides of the 
 vessel kept it at a low and even temperature, which was found 
 to be favorable for keeping the animals and the eggs alive. 
 
 The fresh-laid egg is inclosed in a follicle, consisting of a 
 single layer of cells. The fecundation and the metamorphosis 
 of the germinal vesicle take place after the egg has escaped 
 from the cloacal chamber. The development is remarkably 
 rapid, the tailed larva appearing in about 6 hours after fecunda- 
 tion of the egg. 
 
 PELAGIC FISH EGGS. 
 
 The transparent eggs of various Teleostei found floating on 
 the surface of the sea during the summer months are ex- 
 tremely favorable objects for the study of vertebrate develop- 
 ment, notwithstanding the difficulties in the way of hardening. 
 
 These eggs are found at Newport all through June, July, 
 August, and September, and probably in April and May. 
 They occur in greatest abundance in June, falling off considera- 
 bly by the middle of July, and becoming less and less abundant 
 from this time onward. 
 
 The eggs are readily collected by surface skimming with a 
 fine hand net or a tow net. The calm streaks formed by tidal 
 eddies are the best collecting ground. 
 
 To find the eggs, after they have been collected, the water 
 containing them must be poured into glass vessels and exam- 
 ined on a black ground. Mr. Agassiz employs glass pans, and 
 has one end of the table covered with black tiles (the rest with 
 white tiles), which makes a very convenient ground for pur- 
 poses of this kind. 
 
 Various species of eggs will of course be found together, 
 and the distinguishing marks are often more or less difficult to 
 recognize. But the difficulties in the way of identifying 
 species are incomparably greater than those of "sorting" the 
 eggs. 
 
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152 MICROSCOPICAL METHODS. 
 
 The most important guides to identification Lave been thus 
 stated by Mr. Agassiz : — 
 
 "The pigment spots of the surface of the yolk, and those 
 characteristic of different species of fish embryos, begin to 
 make their appearance at very different times in the many 
 species we have examined. Hence, until the characteristic 
 pigment pattern of an embryo is pretty well known, it is easy 
 to confound the eggs of very different species. The position 
 and shape of the otoliths and the degree of development of the 
 pectorals become also excellent guides to the identification of 
 eggs well advanced in their development. The differences in 
 the young embryos on hatching are very considerable, and in 
 these earlier stages the degree of development of the head, the 
 proportional size of the yolk-bag, the shape of the embryonic 
 fin, the position of the vent, and the pattern of the pigment 
 spots, are all of great use in the identification of the species." 
 
 " The presence or absence of an oil globule is an excellent 
 guide in the identification of the egg ; the size of this globule is, 
 however, quite variable. In one of the species of Cottus there 
 are many globules present, and the number of these varies from 
 sixteen to thirty-two for this species. In another species, 
 (Hemitripterus) in which there is generally only one globule, it 
 is not an uncommon occurrence to find two globules." 
 
 The size of the egg varies, as a rule, within narrow limits 
 (.01-.05 mm ), and is therefore an important aid in identification. 
 
 In studying the living egg, profile views and optical sections 
 are obtained by simply tilting the microscope, the tube being 
 inclined at different angles between the vertical and horizontal 
 positions, according to the view desired. The egg should be 
 confined in a live-box, in a drop of water, without pressure. 
 Thus confined, the egg will live till the time of hatching with- 
 out change of water, and its position is under perfect control. 
 
 I have experimented with all the hardening reagents in com- 
 mon use, and have failed to find any completely satisfactory 
 method of preserving the vitellus. Even the germinal disc 
 cannot be well preserved by any of the ordinary hardening 
 fluids. Kleinenberg's picro-sulphuric acid, for instance, causes 
 the cleavage products to swell, and in many cases to become 
 
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EMBKYOLOGICAL METHODS. 153 
 
 completely disorganized. The embryonic stages can be hard- 
 ened in chromic acid (1%), but the yolk contracts considera- 
 bly without becoming well hardened. The best preparations 
 of the cleavage stages have been obtained with osmic acid, 
 followed by a modified form of Merkel's fluid. This fluid con- 
 sists of chromic acid {\%) and platinum chloride (J$) mixed 
 in equal parts. Thus prepared, it causes maceration of the 
 embryonic portion of the egg. By using a stronger chromic 
 acid (1%) and combining it as before with the same volume of 
 platinum chloride (i%), everything may be well preserved and 
 hardened except the yolk. But this fluid cannot be used with 
 success unless the egg has been first killed by another agent ; 
 for eggs placed in this fluid continue to live for a considerable 
 time, and may even pass through one or two stages of cleavage. 
 It is therefore necessary to use some reagent that kills in- 
 stantly. For this purpose a weak solution of osmic acid may 
 be used. 
 
 The eggs are placed in a watch-glass with a few drops of 
 sea-water, and then a quantity of osmic acid Q-%) equal to that 
 of the sea-water is added. After five to ten minutes the eggs 
 are transferred to the mixture of chromic acid and platinum 
 chloride, and left for twenty-four hours or more. This fluid 
 not only arrests the process of blackening, but actually bleaches 
 the egg to a considerable extent. After this treatment it is an 
 easy matter to separate the blastoderm from the yolk by nee- 
 dles ; and the preparations thus obtained may be stained at 
 once, and then treated with alcohol and mounted in balsam. 
 
 The value of such preparations must be measured by the 
 accuracy and clearness with which they present the conditions 
 in the living state. A careful study of the preparations shows 
 that the method can be relied on in every particular. The 
 osmic acid fixes the living conditions more perfectly than any 
 other reagent at present known, and the chrom-platinum mix- 
 ture completes the work of hardening without shrinkage or 
 swelling. The finest details of the cleavage lines, the cleavage 
 cavity, and the nuclear figures are well preserved. The rela- 
 tion of the blastoderm to the protoplasmic mantle enveloping 
 the vitellus, and all the particulars in regard to the origin of 
 
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154 MICROSCOPICAL METHODS. 
 
 the so-called " free nuclei," are satisfactorily shown. No vio- 
 lence is required in order to free the blastoderm from the yolk, 
 as a clean separation is usually effected by the action of the 
 acids. In this separation the protoplasmic mantle invariably 
 goes with the blastoderm, in the older as well as in the younger 
 stages of development. 
 
 For sectioning, the embryonic portions of the egg need not 
 be separated from the yolk. But before transferring the eggs 
 from the chrom-platinum solution to the different grades of 
 alcohol (50 to 100%), the egg-membrane should be broken or 
 perforated by the aid of needles on the side opposite the blasto- 
 derm, in order that the alcohol may reach the egg readily, as 
 otherwise the membrane wrinkles badly, and often injures the 
 embryonic portion. For the embryonic stages, the above 
 method of hardening has not been altogether satisfactory. 
 The embryos are brittle, and the boundary lines between the 
 different parts are not always sufficiently clear. A few of 
 these stages were hardened in Perenyi's fluid, and the sections 
 have proved much more instructive than any obtained from 
 eggs hardened in other fluids. 
 
 Perenyi recommends leaving eggs from four to five hours in 
 this fluid. Two hours' immersion is certainly sufficient for 
 pelagic fish eggs, and probably even a shorter time would do. 
 Among the methods of staining recommended by Perenyi may 
 be mentioned that of mixing borax-carmine or picro-carmine 
 with the hardening fluid. The addition of the staining fluid 
 produces a precipitate which should be removed by filtering. 
 The filtered mixture both hardens and stains at the same time, 
 which is certainly an advantage. After an immersion of a few 
 hours in this fluid, the eggs should be transferred first to 50% 
 alcohol (5 hours), then to successively higher grades. This 
 method of hardening does not render the eggs brittle. 
 
 EGGS OF THE SALMONIDJS. 
 
 The following method is recommended by Henneguy 1 for 
 the eggs of Salmonoids : — 
 
 1 Bull, de la Soc. PMlomathique, No. 1, 1879, pp. 75-77. 
 
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EMBRYOLOGICAL METHODS. 155 
 
 1. Osmio acid (1%) for a few minutes till the eggs show a 
 light brown color. 
 
 2. Transferred to Muller's fluid and left for a few days. 
 
 The egg-membrane is cut with a fine pair of scissors while in this 
 fluid. The yolk dissolves, leaving the hardened embryo and the cortical 
 layer (periblast) free, so that they can be easily removed and prepared 
 for sectioning or for mounting in toto. 
 
 4. Stained in picro-carminate of ammonia. 
 
 5. Dehydrated with alcohol of 80% followed by absolute 
 alcohol. 
 
 6. Immersed in collodion 24 hours. 
 
 7. Placed on a piece of elder-pith soaked with alcohol, and 
 covered with a layer of collodion. 
 
 8. Sectioned as soon as the collodion has hardened suffi- 
 ciently, then mounted in glycerine. 
 
 Trutta fario. 1 — 1. Chromic acid (%%) 24 hours. 
 
 2. Distilled water, 2 hours. The egg membrane expands, 
 and may now be easily removed. 
 
 3. Washed in distilled water 12 hours. 
 
 4. Absolute alcohol, glycerine, and aq. dest. in equal parts, 
 4 hours. 
 
 5. Absolute alcohol. 
 
 6. Bohm's carmine acetate, 1-2 days. 
 
 7. Mixture of water (50 vols.), glycerine (50 vols.), and muri- 
 atic acid Q- vol.), for a few minutes. 
 
 8. Washed in water 4-5 hours. 
 
 9. Absolute alcohol 12 hours, preparatory to imbedding in 
 paraffine. 
 
 THE EGGS OF THE AMPHIBIA. 
 Our common frogs and toads begin to spawn soon after 
 the first warm days of spring. The eggs of Rana are gen- 
 erally found first, and the spawning season lasts for about a 
 month, beginning (for this climate) the first or second week 
 in April. The spawning season begins about the same time 
 with Amblystoma, but not until two or three weeks later with 
 the bull-frog (Rana catesbeiana). 
 
 1 Kupffer. His and Braune's Arcliiv, Anat. Abth., 1882. 
 
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156 MICEOSCOPICAL METHODS. 
 
 The eggs of the frog cohere in a mass, and only a few min- 
 utes are required for their deposit ; while those of the toad are 
 excluded in a long string of jelly, the process lasting for hours. 
 The eggs of Amblystoma are laid in gelatinous masses a little 
 smaller than that of the frog. The eggs are light brown, and 
 thus easily distinguished from the dark-brown eggs of the frog 
 or the almost black eggs of the toad. 
 
 Our common newt, Diemictylus miniatus, deposits its eggs 
 singly on aquatic plants. The whitish ectosac is elliptical, and 
 much harder and tougher than the envelope of the frog's egg. 
 The egg is pale brown or flesh-colored, and, as it is closely 
 wrapped up in leaves, which are glued together by its adhesive 
 coating, it is not easily found. If specimens are captured at 
 the beginning of the spawning season, and the pairs found copu- 
 lating are isolated in vessels containing only a few water-plants, 
 the eggs are readily obtained. 
 
 The process of copulation, in which the male clasps the 
 female around the neck with his hind legs, generally lasts from 
 one to two hours. I have never seen eggs extruded at the time 
 of copulation, and am inclined to think, from what I have seen, 
 that fecundation takes place before deposit. The spermatozoa 
 would easily find their way to the cloaca of the female through 
 the fanning movement kept up by the tail of the male ; but it 
 is more difficult to account for their actual entrance, as there is 
 no contact of the labia of the cloaca in the two sexes. 
 
 Preparation for Sectioning. — I have obtained good 
 results with the frog's eggs by the following method : — 
 
 1. Small pieces of the gelatinous mass — each containing 
 from 5 to 15 eggs — are cut free with scissors, and placed in 
 i % osmic acid for 20-25 minutes. 
 
 2. Transferred directly to the chrom-platinum solution re- 
 commended for fish eggs (24 hours). 
 
 3. The eggs are next placed in water, and freed from their 
 gelatinous envelopes by the aid of sharp needles and a dissect- 
 ing microscope. The removal of the envelopes is accomplished 
 without much difficulty after a little experience. 
 
 4. Wash in flowing water, or in water often renewed, for at 
 least two hours. 
 
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EMBRYOLOGICAL METHODS. 157 
 
 5. Treat with 50 % alcohol (1-2 hours), 70 % (5-10 hours), 
 then preserve in 80 % or 90 %. 
 
 If sectioned soon after hardening and dehydration with alco- 
 hol, the eggs will cut without crumbling when imbedded in 
 paraffine. They may be stained, after passing through 90 % 
 alcohol, with borax-carmine ; but the staining will be of little 
 or no help in the earlier stages. 
 
 HEETWTG'S METHOD OF PREPARING AND CUTTING AMPHIBIAN 
 
 EGGS. 1 
 
 Although the amphibian egg has long been a favorite object 
 of study among embryologists, — and quite as much so since 
 section-cutting came into vogue as before, — comparatively little 
 progress has been made in overcoming the difficulties that attend 
 its preparation for the microtome. The chief difficulties are 
 found in freeing the egg from its gelatinous envelope, and in 
 preparing it so as to avoid brittleness. 
 
 The best method that has thus far been proposed for these 
 eggs is unquestionably that of O. Hertwig, and I shall there- 
 fore give it in detail. 
 
 1. In order to facilitate the removal of the gelatinous enve- 
 lope, the eggs are placed in water heated almost to boiling 
 (90-96° C.) for 5-10 minutes. The eggs are thus coagulated 
 and somewhat hardened, while the envelope separates a little 
 from the surface of the egg, and becomes more brittle. The 
 envelope is then cut under water with sharp scissors, and the 
 egg shaken out through the rupture. With a little experience 
 a single cut suffices to free the egg. 
 
 2. By the aid of a glass tube, the egg is taken up and trans- 
 ferred to chromic acid (£ %), or to alcohol of 70, 80, and 90 %. 
 Chromic acid renders the egg brittle, and the more so, the 
 longer it acts ; therefore the eggs should not be allowed to 
 remain in it more than twelve hours. While eggs hardened in 
 chromic acid never change their form or become soft when 
 transferred to water, those hardened in alcohol, when placed in 
 water or very dilute alcohol, lose their hardness, swell up, and 
 often suffer changes in form. 
 
 1 Jenaische Zeitschrift fur Naturwissensckaft, XVI., p. 249, 1882. 
 
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158 MICROSCOPICAL METHODS. 
 
 3. Alcoholic preparations are easily stained ; but chromic acid 
 preparations are stained with such difficulty and so imperfectly 
 that Hertwig omitted it altogether. 
 
 There is an important difference between alcohol and chro- 
 mic acid in their effect on the pigment of the egg. Chromic 
 acid destroys the pigment to some extent, and thus obliterates, 
 or at least diminishes, the contrast between pigmented and non- 
 pigmented cell-layers. As the distribution of the pigment is of 
 considerable importance in the study of the germ-lamellse, it is 
 well to supplement preparations in chromic acid with those in 
 alcohol, in which the pigment remains undisturbed. 
 
 4. Eggs hardened in chromic acid were imbedded almost ex- 
 clusively in the egg-mass recommended by Calberla. The great 
 advantage offered by this mass is, that it supplies a sort of anti- 
 dote to the brittleness of the egg. It glues the cell-layers 
 together, so that the thinnest sections can be obtained without 
 danger of breaking. 
 
 5. As the dorsal and ventral surfaces and the fore and hind 
 ends can be recognized in very early stages, it is important to 
 know precisely how the egg lies in the egg-mass, in order to 
 determine the plane of section. In order to fix the egg in any 
 given position in the imbedding mass, Hertwig proceeds as 
 follows : — 
 
 a. A small block of the hardened mass is washed in water to 
 remove the alcohol; and in the upper surface of the block, 
 which has been freed from water by the aid of filtering paper, 
 a small hollow is made. This hollow is then wet with the 
 freshly prepared fluid mass. 
 
 b. The egg is washed in water, to remove the alcohol, placed 
 on a piece of filtering paper to get rid of the water, turned on 
 the paper by a fine hair brush, until it has the position desired ; 
 the point of the brush is next moistened and pressed gently 
 on the upper surface of the egg; the egg adheres to the brush, 
 and may thus be transported to the hollow prepared for it in 
 the block. 
 
 c. After the egg has thus been placed in position, a drop of 
 absolute alcohol, carefully applied, will coagulate the "fluid 
 mass " with which the hollow was wet, and thus fix the egg to 
 
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EMBRYOLOGICAL METHODS. 159 
 
 the block. The block is again washed, and finally imbedded 
 in the egg-mass (cf pp. 106-107). 
 
 THE INFLUENCE OF GRAVITATION ON THE DIRECTION OF 
 CLEAVAGE-PLANES. 
 
 The slimy envelope of the frog's egg undergoes peculiar 
 changes the moment it comes in contact with water. It swells 
 up very quickly, assumes a gelatinous consistency, and loses its 
 adhesive property. The cavity of the envelope thus becomes 
 somewhat larger than the egg and fills with water, in which 
 the egg can rotate easily. Under these conditions, the egg 
 takes its normal position, with its pigmented pole uppermost, 
 very soon after deposit. In this position the axis of the egg is 
 vertical, and the first cleavage-plane is meridian, coinciding 
 with the axis. It has generally been supposed that the direc- 
 tion of the first plane of cleavage is determined by some neces- 
 sary relation to the axis of the egg ; but the means of testing 
 the validity of this view is a recent discovery, for which we are 
 indebted to E. Pfltiger. 1 A series of brilliant experiments was 
 undertaken by this author in the hope of settling the question, 
 whether the direction of the first cleavage-plane is determined 
 by the direction of the axis of the egg, or by the direct influ- 
 ence of gravitation. These experiments were made possible 
 by the discovery of a method by which the egg could be pre- 
 vented from rotating and confined in any desired position. 
 This was accomplished in the following manner : — 
 
 Pfliiger. — Mature eggs were taken from the uteri and 
 placed in dry watch-glasses (a single egg in each glass) ; then 
 a drop of water containing spermatozoa was added, and, after 
 a few seconds, drained off. Thus treated, the mucous envelope 
 does not swell sufficiently to allow the egg to rotate ; but it 
 adheres to the glass, and, at the same time, presses upon the 
 egg just enough to hold it in position. If the eggs are pro- 
 tected against drying by keeping in a moist chamber, develop- 
 ment aj>pears to go on as well as under normal conditions. 
 
 1 Arcliiv. f. d. ges. Physiologie, XXXI., p. 312, 1883. 
 
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1G0 MICROSCOPICAL METHODS. 
 
 The experimenter finds thus a very ready means of giving the 
 axis of the egg any desired inclination, and is in a favorable 
 position for studying the effect on the direction of the cleavage 
 plans. 
 
 Born.— Born, 1 who has followed up the line of investigation 
 started by Pflilger, gives some valuable details of the method 
 of procedure. A number of shallow, round, flat-bottomed 
 glasses of different sizes served for receiving the eggs. These 
 were provided with glass covers, which were so adjusted as to 
 admit air without allowing much evaporation. 
 
 The eggs were fixed on round glass plates, somewhat smaller 
 than the flat bottoms of the dishes on which they rested. In 
 one diameter of each plate was etched an arrow, and on each 
 side of the arrow four or five eggs were placed, and their 
 position noted in a diagram. In addition to this, two diagram- 
 matic sketches of each egg were made on two rectangular 
 pieces of paper, one to show the upper, the other the lower 
 aspect of the egg, at the moment it was fixed on the plate. 
 Each egg was further designated with its number and time. 
 In order to view the eggs from below, the jjlate was simply 
 inverted over a smaller glass dish containing a little water. A 
 damp chamber was thus formed, so that the eggs were not 
 exposed to evaporation during the preparation of the diagrams. 
 The sketches were made for the most part without the aid of 
 the camera lucida and microscope. 
 
 Fecundation was accomplished by adding, with a brush, to 
 each egg a small drop of water containing spermatozoa. If too 
 large a quantity of spermatic fluid was added, the excess was 
 removed by the aid of a bit of blotting-paper. 
 
 Born remarks that most of the eggs in his first experiments 
 were lost by undue exposure to evaporation ; and adds that 
 danger in this direction may be guarded against by covering 
 the plate which carries the eggs with a fine spray of water, as 
 often as it is exposed for the purpose of making drawings. 
 
 Eggs were prepared for section-cutting in the following 
 manner : — 
 
 i Arckiv f. mik. Anat., XXIV., H. 4, pp. 476-4S0, 1S85. 
 
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EMBRYOLOGICAL METHODS. 161 
 
 1. For orientation, the gelatinous envelopes were marked 
 with red and green size-colors, which were liquidized by heat- 
 ing. The melted coloring substance was applied in drops with 
 a very fine brush in such a manner as to indicate the position 
 of the two poles of the egg. The diagrams were marked with 
 the same colors at corresponding points, in order to render 
 identification easy. 
 
 2. After marking, the eggs were killed by placing them 
 (together with the glass plate) in oil heated to about 90° C, 
 then transferred to 75% alcohol, and, after a few hours, to 80% 
 alcohol. They were next separated from the glass plate by 
 means of a thin knife pressed against the plate, and then left 
 in 80% or 90% alcohol for preservation. 
 
 3. The eggs were imbedded in paraffine in the usual way, 
 the color-marks, still visible on the envelopes, serving for orien- 
 tation. 
 
 4. Eggs thus treated were brittle, and complete sections 
 could only be obtained in one of two ways — either by passing 
 a hot spatula quickly over the cut surface of the paraffine block, 
 or by covering this surface with a drop of melted paraffine, be- 
 fore drawing the knife each time. Painting with thin collodion 
 would undoubtedly have served equally well, had the necessary 
 precautions been observed. 
 
 The startling conclusion reached by Pfltiger, that gravitation 
 determines the direction of cleavage-planes, opened at once a 
 new field of investigation, and called out other important meth- 
 ods of testing this question. 
 
 Rous. — Roux 1 placed frog's eggs, immediately after fecun- 
 dation, in moist cotton in a centrifugal apparatus, the turning 
 of which was sufficiently rapid to overcome the directive influ- 
 ence of gravitation, so that the axes of the eggs, instead of 
 taking a vertical direction, remained in whatever position they 
 chanced to be at the start. The result was that the first cleav- 
 age-plane invariably coincided with the axis of the egg, and 
 that the development was in all respects the same as under nor- 
 mal conditions. 
 
 1 Breslauer iirztlichen Zeitschrif t, No. 6, 1884. 
 
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162 MICROSCOPICAL METHODS. 
 
 Rauber. — Rauber J endeavored to confine the trout's egg in 
 abnormal positions by means of peculiarly constructed silver- 
 plated clamps, but was unsuccessful. In the centrifugal appara- 
 tus the axis of the trout's egg took a horizontal position, with 
 the germinal disc directed centripetally, and the yolk centrifu- 
 gally. In this position, the eggs developed normally, from 
 which Rauber inferred that gravitation could be replaced by 
 centrifugal force, but that a directive force of some kind must, 
 in all cases, be present, under the influence of which develop- 
 ment takes place. 
 
 0. Hertwig. — The small, transparent eggs of the sea-urchin 
 are perfectly spherical and free from food-yolk, and in them the 
 centre of gravity may be assumed to coincide with the geomet- 
 rical centre. In these respects they were regarded by O. Hert- 
 wig 2 as favorable objects for studying the main question raised 
 by Pfltiger's investigations. For examination, the fecundated 
 eggs were placed in a hanging drop of water on the under side 
 of a cover-glass. The first cleavage-plane exhibited inclinations 
 in every direction, varying between the vertical and horizontal 
 position, from which it was inferred that its direction was not 
 controlled by gravitation. 
 
 EEPTILIAN OVA.8 
 
 1. The ova taken from the oviduct are opened in a dilute 
 solution of osmic acid (1 %), and then the white removed as far 
 as possible. 
 
 2. The osmic acid is then turned off, and a weak solution of 
 chromic acid (£ %) added. 24 hours. 
 
 3. With a sharp, fine pair of scissors, cut around the germinal 
 area, just outside its margin ; and, after it has been completely 
 encircled with the incision, float it carefully off from the body 
 of the yolk. 
 
 4. The yolk and acid are next removed, and a copious supply 
 of clean water added, which must be several times renewed. 
 
 1 Berichte der naturforsebenden Gesellschaft zu Leipzig, Feb. 12, 1884. 
 
 2 Jenaische Zeitschrift, XVIIL, pp. 17S-182, 1885. 
 
 8 Kupffer. His and Braune's Archiv, Anat. Abth., 1882, p. 4. 
 
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EMBKYOLOGICAL METHODS. 163 
 
 5. Calberla's fluid (glycerine, -water, and absolute alcohol in 
 equal parts) 3 hours. 
 
 6. Hardened in 90 % alcohol. 
 
 7. Stained in Bohm's carmine acetate 24 hours. 
 
 THE HEN'S EGG. 
 
 The very complete instructions given by Balfour 1 for study- 
 ing the development of the chick may be assumed to be in the 
 hands of every student of avian embryology, and need not, 
 therefore, be summarized here. A very important addition to 
 this branch of technique has recently been made by Duval, 2 
 whose methods I propose to give in detail. First in order and 
 importance are the means and method of orientation. After 
 the appearance of the primitive streak, at about the twelfth 
 hour of incubation, it becomes easy to distinguish anterior, pos- 
 terior, and lateral regions in the blastoderm. Hitherto it has 
 been a matter of conjecture whether anterior and posterior 
 regions became morphologically defined at any considerable 
 time before the formation of this streak ; and no one, before 
 Duval, attempted to clear up the question, simply because it 
 appeared impossible to find any means of orienting sections at 
 an earlier date. Duval addressed himself to the task of finding 
 out the transformations of the blastoderm, which lead up to the 
 establishment of the primitive streak, and to this end he was 
 compelled to seek, first of all, for some reliable means of exact 
 orientation. 
 
 Method of Orientation. — It was noticed by Balfour, and 
 confirmed by Kolliker, that the axis of the chick embryo lies 
 constantly at right angles to the longer axis of the egg. If an 
 egg, after one or two days' incubation be opened, while held in 
 such a position that its large end is turned to the left and its 
 small end to the right of the operator, it will be found that the 
 caudal end of the embryo is directed towards the operator, 
 while the cephalic end is turned in the opposite direction, as in- 
 dicated in Fig. 31. Out of 166 cases Duval found only three that 
 
 1 Appendix to the "Elements of Embryology." 
 
 2 Ann. des sc. nat., XVIII., Nos. 1, 2, 3, 1884. 
 
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164 
 
 MICROSCOPICAL METHODS. 
 
 could be regarded as exceptions to the rule. Assuming that 
 the orientation is thj same before the appearance of the primi- 
 tive streak, we have then a very reliable means of recognizing, 
 even in the fresh-laid egg, when the blastoderm has a homoge- 
 neous aspect, the future 
 anterior and the future 
 posterior region. But 
 this fact alone is not all 
 that is required for com- 
 plete orientation : the 
 blastoderm must be 
 hardened, and the 
 means of orientation 
 must be preserved. 
 That portion of the 
 
 Fig. 31. — Diagram of hen's egg. y, yolk ; b, blasto- vitelline sphere which 
 derm; a, anterior ^.posterior end of embryo. bears ^ bla8toderm 
 
 must be so marked that the anterior and posterior regions of the 
 blastoderm may be recognized after the process of hardening, 
 and after the blastoderm, together with some of the circum- 
 jacent yolk, has been cut free from the rest of the egg. This 
 may be done in different ways, according to the method em- 
 ployed in hardening. 
 
 I. Osmic-Acid Method. — 1. Make a triangular box with- 
 out bottom, like that seen in Fig. 32, by folding a 
 strip of paper 5 mm wide and 50 mm long. 
 
 2. After opening the egg carefully from the 
 upper side, remove with a pipette the thin layer 
 of albumen which lies above the cicatricula, so far 
 as this can be done with safety. 
 
 3. Place the triangular box over the blastoderm 
 in such a manner that the base corresponds to the 
 future anterior region, and the apex to the future 
 
 posterior region. While pressing slightly on the box in order 
 to bring it into close contact with the surface of the yolk, fill it 
 by means of a pipette with osmic acid (£-]%), and allow the 
 acid to act for some minutes. 
 4. As soon as the area inclosed by the box begins to blacken, 
 
 Fig. 32. 
 
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EMBRYOLOGICAL METHODS. 165 
 
 the whole should be immersed in a vessel of chromic acid, in 
 which the paper box may be detached, and the vitelline sphere 
 freed from the albumen and the shell. 
 
 5. The vitellus may now be transferred, by the aid of a very 
 deep watch-glass, to another vessel of chromic acid, where it is 
 allowed to remain one or two days, until the peripheral layers 
 harden and form a sort of shell around the central portion 
 which is still soft. 
 
 6. A triangular piece of this shell, inclosing the triangular 
 area browned by the osmic acid, is next to be cut out with 
 a pair of sharp scissors. The excised piece is then left a 
 day or more in the chromic solution before treatment with 
 alcohol. 
 
 II. Alcohol Method. — 1. Open the egg as before, and, 
 without attempting to remove the albumen, place the triangular 
 paper box over the blastoderm ; slight pressure causes the box 
 to sink into the albumen till it is brought into contact with 
 the yolk. By the aid of a pipette, fill the box with absolute 
 alcohol ; this coagulates rapidly the inclosed albumen, while 
 the albumen outside the box remains fluid. 
 
 2. After cutting the chalazeae close to the vitellus, the fluid 
 portion of the albumen is carefully drained off, leaving only 
 the vitellus and the box with its coagulated contents in the 
 shell. 
 
 3. The shell may now be filled with absolute alcohol until 
 the yolk is completely covered, and then left for some hours, 
 during which the more superficial layers of the yolk harden 
 sufficiently to form a shell-like envelope of the softer central 
 portion. 
 
 4. The triangular mass formed by the box and the hardened 
 albumen is now ready to be cut out, in the same manner as in 
 the osmic acid method. During this process, the paper box 
 may become detached, either spontaneously or with some assis- 
 tance ; or it may adhere so firmly that it cannot be safely 
 removed. There is no inconvenience in leaving it in place, as 
 it will cut easily when the piece is ultimately sectioned. 
 
 5. The piece is further hardened 24 hours in absolute alcohol, 
 then preserved in alcohol of 36° (80%). 
 
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166 MICROSCOPICAL METHODS. 
 
 lit Hot Chromic Acid. — 1. Treat with osmic acid as 
 in No. I. 
 
 2. Place the whole in a solution of chromic acid, and heat to 
 the point of boiling over a water-bath. 
 
 3. After cooling, cut out the triangular piece as in No. I. (6), 
 leave it for a few days in chromic acid, then transfer to 
 alcohol. 
 
 Imbedding and Cutting. — Duval imbeds, after each of the 
 foregoing methods, in collodion (see p. 170). The surface of 
 each section is collodionized some seconds before drawing the 
 knife, by allowing a drop or two of thin collodion to flow 
 over it. 1 
 
 Staining. — The sections are placed in serial order on a 
 slide, and then covered with picro-carmine strongly diluted 
 with glycerine. The sections may be left in the staining fluid 
 24-48 hours, the admixture of glycerine preventing drying. 
 After they are sufficiently colored, the staining fluid is allowed 
 to drain off, and the slide is carefully washed with a pipette. 
 The sections, still in place, are treated with successive grades 
 of alcohol, and then mounted in balsam after being clarified in 
 benzine ("benzine Collas"). 
 
 Mounting the Blastoderm in toto. — During the first 
 three or four days of incubation I have obtained good surface 
 preparations of the blastoderm in the following manner : — 
 
 1. Break the shell by a sharp rap of the scissors at the broad 
 end; then carefully cut away the shell, beginning at the place 
 of fracture and working over the upper third or half. 
 
 2. After removing as much of the white as possible without 
 injury to the blastoderm, place the rest of the egg, while still 
 in the shell, in a dish of nitric acid (10 %), deep enough to 
 cover it. 
 
 3. The coagulated white should next be removed from the 
 blastoderm by the aid of a brush or a feather, and the egg then 
 allowed to remain in the acid 30 minutes. 
 
 1 This method was recommended by Duval early in 1880. ' ' De quelques 
 perfectionnements a l'emploi du collodion en technique histologique." 
 Society de Biologie, 1880. Later in same year came a similar account 
 from Mason (Nov. 1880). 
 
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EMBRYOLOGICAL METHODS. 167 
 
 4. Cut around the blastoderm with a sharp-pointed pair of 
 scissors, taking care to cut quickly and steadily. After carry- 
 ing the incision completely round, float the blastoderm into a 
 watch-glass, keeping it right side up and flat. 
 
 5. Remove the vitelline membrane by the aid of dissecting 
 forceps, and the yolk by gently shaking the watch-glass and by 
 occasional use of a needle. The yolk can sometimes best be 
 washed off by means of a pipette. 
 
 6. Wash in water (several times changed). 
 
 7. Color deeply with carmine or hematoxylin. 
 
 8. Remove excess of color by soaking a few minutes in a 
 mixture of water and glycerine in equal parts, to which a few 
 drops (about 1%) of hydrochloric acid have been added. 
 
 9. "Wash and treat 30 minutes with a mixture of 
 
 Alcohol (70%) 2 parts 
 
 Water 1 " 
 
 Glycerine 1 " 
 
 10. Transfer to pure 70% alcohol, then to absolute alcohol. 
 Clarify with creosote or clove oil and mount in balsam. 
 
 The above method of treatment will also serve for blasto- 
 derms which are to be sectioned. 
 
 THE EGG OF THE EABBIT.i 
 
 The period of gestation for the rabbit is four weeks, and im- 
 pregnation takes place immediately after littering. At the end 
 of pregnancy, the ovary presents mature follicles containing 
 eggs that are ready to be fecundated. The marks of maturity 
 in the egg may be learned by comparing such eggs with eggs 
 taken from the ovary at different times during gestation, and 
 noting the differences. If a rabbit is killed two days before 
 littering, eggs will be found which would have attained their 
 complete development two days later. It is not, then, a diffi- 
 cult task to analyze the series of successive modifications which 
 the egg undergoes during the four weeks of gestation. 
 
 In a similar manner, the age of eggs taken from the uterus 
 may be ascertained by reckoning forward from the time of 
 
 1 E. Van Beneden. Kecherches sur l'Embryologie des Mammiferes. 
 Archives de Biologie; I., pp. 138-139, 146-153, 1880. 
 
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I" •,- 1 
 
 168 MICROSCOPICAL METHODS. 
 
 copulation. The fecundation of the egg takes place generally 
 9 hours after coition, and the first cleavage appears from 10 to 
 12 hours later. 
 
 Method of Taking the Eggs. — After opening the abdo- 
 men along the white line, a ligature is fastened around the 
 vessels running to one of the uterine tubes, and then the 
 uterus is cut out. The abdominal cavity is then quickly closed 
 by means of a few stitches. If the operation is performed 
 with care, the rabbit endures it very well, and development 
 progresses normally in the uterus which is left in place. The 
 second uterus may be cut out several hours later, and from it 
 more advanced stages of development may be obtained. 
 
 As soon as the first uterus is removed, it is opened by an 
 incision passing from the vaginal extremity along the middle 
 of the side opposite the insertion of the peritoneal fold. 
 
 It requires some experience to be able to find the eggs readily 
 during the first few days of their sojourn in the uterus ; while 
 from the fifth day onward they become sufficiently large to be 
 easily seen with the naked eye. For a short time after enter- 
 ing the uterus, the eggs are all found near the termination of 
 the oviduct, and are best discovered by searching with a lens 
 between the. folds of the mucous membrane. The egg is at 
 this time enveloped by a thick layer of albuminoid substance, 
 which makes it easier to find. From the moment of entering 
 the uterus, the eggs, which are already in the metagastrula 
 stage, begin to increase in size, and at the same time to take up 
 positions at longer and longer intervals, until at length they 
 are ranged along the entire length of the tube. 
 
 As the eggs are found, they are raised on the point of a scal- 
 pel, and placed on a slide in aqueous humor or some other 
 fluid. By the end of seven or eight days, the eggs become so 
 firmly attached to the epithelium of the mucous membrane that 
 they can no longer be detached in an unmutilated condition. 
 
 Method of Hardening. — According to Van Beneden, the 
 best method of treatment for the stages of cleavage, the 
 metagastrula, and young blastocysts, is as follows : — 
 
 1. Place the egg on a slide in a drop of 1 % osmic acid, and 
 leave it a few moments only. 
 
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EMBKYOLOGICAL METHODS. 169 
 
 2. Remove the egg on the point of a scalpel to another slide 
 provided with a few drops of Mtiller's fluid (or bichromate of 
 ammonia). 
 
 3. After an hour renew the fluid, and then leave the prepara- 
 tion 2-3 days in a damp chamber. 
 
 4. Transfer to a drop of very dilute glycerine, then to a 
 stronger glycerine, and finally mount in pure glycerine. 
 
 For surface views, treatment with nitrate of silver (-J fo) J-2 
 minutes gives good preparations. 
 Method of Preparing Somewhat Later Stages. 1 — 
 
 1. After removing the uterus, as before, cut it into as many 
 segments as there are inclosed embryos, taking care to cut 
 transversely midway between two swellings. 
 
 2. Place one of the pieces in a dissecting trough, and pin it 
 in such a position that the mesometrical side rests on the bot- 
 tom of the trough. The convex surface is then turned towards 
 the operator. 
 
 3. Whatever be the fluid in which the operation is performed, 
 the egg is opened by seizing the wall of the uterus near the 
 middle of the convex side of the swelling with forceps, and 
 then making a small incision at this point. From the opening 
 a quantity of fluid escapes, which produces a precipitate the 
 moment it comes in contact with picro-sulphuric acid. 
 
 4. After removing the coagulated mass from the preparation, 
 one lip of the opening is raised by the aid of forceps, and then 
 a crucial incision of the uterine wall is made with a sharp, fine 
 pair of scissors. By pinning back the four strips, the embryo 
 is brought into view. The placenta is always on the mesomet- 
 rical side, so that one is certain, in operating as above directed, 
 not to injure the embryo. In order to avoid cutting the termi- 
 nal sinus of the vascular area, the crucial incision must not be 
 extended too far. 
 
 5. By using Kronecker's artificial serum, at a temperature of 
 the body, the embryo may be kept alive for hours. 
 
 1 E. Van Beneden and Charles Julin. Kecherches sur la formation 
 des annexes fa 1 tales cliez les Mammiferes. Archives de Biologie, V., p. 
 378, 1884. 
 
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170 MICROSCOPICAL METHODS. 
 
 The liquid that escapes when the incision of the wall is made 
 comes from the blastodermic cavity. 
 
 THE USE OF COLLODION IN SECTIONING BRITTLE EGGS. 
 
 The crumbling of sections of brittle eggs imbedded in paraf- 
 fine may often be prevented by adding a thin film of paraffine 
 with a hot spatula before making each cut, as recommended by 
 Born (see p. 161); but this can often be acromplished with great- 
 er ease and certainty by the use of collodion. The following is 
 a detailed account of the collodion method, as employed by 
 Mark : — 
 
 A small bottle, covered with a closely fitting glass cap, is 
 kept about half full of thin collodion, into which dips the tip 
 of a camel's-hair brush. The quill of the brush is thrust 
 through a hole in a thin, flat cork, which rests on the mouth of 
 the bottle, inside the glass cap, and which serves at once as a 
 temporary cover and as a support to the brush, the latter being 
 adjusted to any height of the collodion by simply pushing it up 
 or down through the cork. 
 
 The collodion should be quite thin, and used so sparingly 
 that it will dry almost instantly (within 2-3 seconds after being 
 applied) without leaving a trace of glossiness on the surface of 
 the paraffine. As soon as it begins to leave a shining surface, 
 it should be thinned with ether, which is kept in a small bottle 
 near at hand. 
 
 Method of Using. — 1. Place the object, imbedded in 
 paraffine, in the receiver of the microtome, and cut away the 
 paraffine to within l-2 mm of the object, leaving a rectangular 
 surface, as in the ribbon method. 
 
 2. Lift the brush, wiping it on the mouth of the bottle, in 
 order to remove the most of the collodion, and then paint the 
 paraffine and object by quickly drawing the brush across the 
 surface, taking care that the brush be evenly applied, and that 
 the collodion is not carried on to the vertical faces of the block. 
 
 3. A moment after the temporary moistening vanishes 
 (within 5 seconds after painting), the knife may be drawn and 
 returned, leaving the section on the edge of the blade. 
 
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EMBRYOLOGICAL METHODS. 171 
 
 4. Paint again, and leave the surface to dry while removing 
 the section already cut to a slide prepared with Schallibaum's 
 mixture of collodion and clove oil. Place the section upside 
 down, so that its painted face comes in contact with the fixa- 
 tive. 
 
 5. Make the second section, and repeat the steps in No. 4. 
 If the precautions before named are observed, it will not be 
 
 necessary to wait for the drying of the collodion film, and the 
 section may therefore be cut at once, — i. e., within 5 seconds 
 after painting. It is thus possible to cut as fast as one can 
 paint the surface ; and with some practice it becomes possible 
 to cut continuous ribbons of sections which may be transferred 
 at intervals. It will be found most convenient to cut enough 
 for one row, or half a row, of sections at a time, and then 
 transfer to the slide, rather than to cut the whole object with- 
 out interruption. 
 
 Precautions. — 1. Especial care should be exercised to pre- 
 vent the painting of the vertical face nearest the operator, since 
 the section is then liable to cling along its whole edge to this 
 vertical film, and be carried under the knife-blade. If by 
 chance this should occur, the section should be removed from 
 the block beforeUhe knife is shoved bach, as it is liable to be 
 caught and lacerated between the face of the block and the 
 under surface of the returning blade. The possibility of the 
 section being thrown under the knife-blade may, however, be 
 obviated, either by carefully trimming (to allow for which the 
 nearer margin of the paraffine may be left broader than the 
 other three) the vertical face, in case it is accidentally painted ; 
 or by drawing the knife slowly, so that the first indication of a 
 failure to cut through the vertical film may be recognized, and 
 the section held in place on the blade by a slight pressure with 
 a soft brush, whereupon the knife will cut through the film and 
 leave the section free. 
 
 2. If, by chance, the paraffine block has been painted with 
 too much collodion, or with collodion that is too concentrated, 
 thus leaving a shining surface, the film should be at once broken 
 by pressing it gently two or three times in quick succession 
 with the end of a rather stiff, blunt, dry brush. This enables 
 
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172 MICROSCOPICAL METHODS. 
 
 the collodion to dry quickly, and thus prevents the softening of 
 the paraffine. 
 
 3. If the sections have a tendency to curl, they may be flat- 
 tened out on the slide by means of a brush ; for a section thus 
 impregnated with collodion may be handled during the first 
 few seconds after contact with Schallibaum's fixative with much 
 greater freedom than one not so treated. If the sections are 
 cut sufficiently thin (e. g., 5 fi), there is no curling. 
 
 This method is useful in preventing the folding of thin sec- 
 tions, as well as the crumbling of brittle ones. 
 
 METHODS OF TRACING THE DEVELOPMENT OF THE TELEOS- 
 TEAN BRAIN. 
 
 I. Rabl-Rvickhard's Method. 1 — The eggs are killed in a 
 10 % solution of nitric acid, and the membrane removed as 
 soon as possible (about 15 minutes after immersion), before its 
 contraction has brought it into contact with the embryo. If 
 this precaution is neglected, the embryo is liable to become 
 flattened and otherwise deformed. 
 
 After remaining in the acid an hour or more, the preparation 
 is transferred to a 1-2 % solution of alum for the same time, and 
 then hardened in the usual grades of alcohol. 
 
 The embryo is in the most favorable condition for drawing 
 on the day of preparation. Later, it becomes cloudy white, 
 and the details of configuration are not so easily made out. 
 For drawing, the embryo is placed on a black ground, and 
 illuminated by artificial light concentrated by a condenser. 
 
 II. Gi-oronowitsch's Method. 2 — For surface views, the egg 
 is immersed in a 5 % solution of nitric acid until the outlines 
 of the embryo begin to be visible through the membrane (about 
 3 minutes), and then removed, before coagulation of the yolk, 
 to a 5 % solution of alum. After the lapse of an hour, the 
 yolk appears quite transparent, the embryo alone appearing 
 white. 
 
 While in the alum solution, the egg membrane is cut, and 
 carefully removed from the embryo. 
 
 i Arch. f. Anat. u. Entw'gesch., 1882, pp. 67, 68. 
 2 Morph. Jahrb., X., H. 3, p. 381, 1884. 
 
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EMBRTOLOGIOAL METHODS. 173 
 
 The object thus prepared is ready for examination, which was 
 usually made by the aid of artificial light. 
 
 In case it becomes desirable to mount the object for later 
 study, this can best be done in 10 % glycerine, to which a little 
 sublimate has been added. 
 
 For sectioning, the egg is treated with Kleinenberg's picro- 
 sulphuric acid three hours, then passed through 40, 70, and 
 90 % alcohol. 
 
 It is very important to remove the membrane as early as 
 this can be done without injury to the embryo. The operation 
 may be undertaken usually at the end of ten minutes, at which 
 time the embryo begins to be seen through the membrane. 
 
 The preparations were imbedded in paraffine, and cut with 
 Thoma's microtome. 
 
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CHAPTEE IX. 
 
 TIMES AND PLACES OP OVULATION. 
 
 The Rock-Barnacle {Balanus balanoides). — Extremely 
 abundant along our rocky shores, sometimes completely whiten- 
 ing the rocks for long distances. Also very common on the 
 piles of wharves and bridges. Faxon found Balanus larvae 
 July 10. 
 
 Hyperia. — This very interesting little amphipod, which 
 lives in jelly-fishes (Cyanea), was found with eggs, at Newport, 
 from May 24th to September. The eggs are transparent 
 (E. N.Whitman). 
 
 Phronima. — Only an occasional visitor at Newport ; usually 
 found in company with Salpa. It is quite transparent, and 
 lives in a barrel-shaped case, open at both ends. The case 
 agrees in size and general appearance with Salpa. The trans- 
 parent eggs are placed in the middle of the case, around the 
 inner surface, forming a more or less perfect ring (E. N. Whit- 
 man). 
 
 The Prawn (Palcemonetes vulgaris). — Abundant in the 
 eel-grass, and especially in the estuaries, occurring far up in 
 the mouths of rivers. Carries eggs in July and August (Faxon). 
 
 Hippa emerita. — Lives in shallow water on sandy bot- 
 toms. It burrows head first, instead of backward. Eggs pro- 
 duced in July and August (Faxon). 
 
 The Lobster (JBbmarus americanus). — " There is a great 
 
 difference in the breeding season on different parts of the coast. 
 
 The lobsters from New London and Stonington often lay their 
 
 eggs as early as the last of April or first of May; while at 
 
 174 
 
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TIMES AND PLACES OF OVULATION. 175 
 
 Halifax Mr. Smith found females with, recently laid eggs in 
 September. At Eastport, Me., the females carry their eggs in 
 mid-summer. In the male, the genital orifices are in the bases 
 of the last pair of legs ; in the female, they are at the bases of 
 the middle pair. This will always serve to distinguish the 
 sexes ; but they also differ in the structure of their abdominal 
 appendages" (Verrill). 
 
 The Green Crab ( Carcinus mcenas). — Very common along 
 our coast, from Cape Cod to New Jersey (Verrill). The 
 ground color exhibits all shades between a bright green and a 
 dark brown, and is varied by spots and blotches of yellow. 
 The bright orange-colored eggs, carried under the tail, were 
 found at Newport from May 28 to July 20 (E. N. Whitman). 
 
 The Rock Crab ( Cancer irroratus). — Usually larger than 
 the preceding, and easily distinguished by its dull reddish color, 
 sprinkled over with darker, brownish dots, and by having nine 
 blunt teeth along each side of the front edge of its shell (Ver- 
 rill). One or two specimens were found with eggs in June, at 
 Newport. 
 
 The Mud Crab (Panopeus Sayi). — I found this crab with 
 eggs throughout the month of June ; and Faxon found eggs 
 nearly ready to hatch as late as August 28. 
 
 The Blue Crab (Neptunus hastatus). — Common on muddy 
 shores, in brackish streams, and estuaries. Easily distinguished 
 from other species by the sharp spine on either side of the 
 back. Eggs found by Faxon August 16. 
 
 Occurrence of Eggs at Annisquam. — The following 
 notes on the occurrence of eggs at Annisquam have been 
 kindly furnished by J. S. Kingsley : — 
 
 Xiineus lays its eggs in masses, surrounded by a glairy 
 matrix, June 1 to June 10. The eggs undergo a regular devel- 
 opment without metamorphosis. From three to ten eggs in a 
 mass, eggs slightly whitish, matrix colorless and transparent. 
 
 Meckelia ingens contained ripe eggs July 12. 
 
 Tergipes despectus lays its eggs in rounded masses of from 
 ten to thirty eggs, attached to hydroids, June 26. The mass 
 is composed of a transparent, colorless jelly, in which the white 
 eggs are closely packed. 
 
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176 MICROSCOPICAL METHODS. 
 
 Molgula manhattensis. The eggs, in the earliest stages and 
 up to the tadpole stage, were found in the cloaca June 1st ; the 
 same stages and later ones about the middle of July of the 
 same year, indicating a breeding season of some extent. The 
 eggs, embryos, and adults live readily in confinement, and seem 
 to require no change of water. Have kept them for six weeks. 
 
 Crangon vulgaris oviposits from July 5 to the middle of 
 August. 
 
 Gammarus omatus oviposits the first two weeks in June. 
 The eggs are best obtained by taking couples attached to each 
 other. They live well in confinement, and the young can read- 
 ily be hatched. 
 
 Mossia lays its eggs in water of several fathoms. The eggs 
 are laid singly, usually in the cavities of a large yellow sponge, 
 but sometimes freely on the bottom. The eggs are about the 
 size of a French pea, are spherical, and dark-colored. "We 
 found them at Annisquam the first week in August. For other 
 details, see Verrill in report on Cephalopoda of the Northeast- 
 ern Atlantic in Rep. U. S. F. C. for 1878. 
 
 Loligo pealii oviposits from at least July 5th to the 15th. 
 (For description of capsule, vide Brooks & Verrill.) The cap- 
 sules are attached below low-water mark, but are frequently 
 thrown up by the tide. They withstand exposure to the air 
 over one tide jierfectly, but do not live in confinement long 
 after hatching. During the limits given above they are abun- 
 dant on Long Beach (Coffin's Beach) opposite Annisquam. I 
 have not had good results with sections. They may be readily 
 freed from the matrix and the egg envelopes with needles. 
 The great difficulty lies in the fact that the thin germinal por- 
 tion readily separates from the yolk during the process of hard- 
 ening. The yolk cuts tolerably well, and does not crumble 
 badly. I have had as good results with alcohol of successive 
 grades as by any mode of treatment. 
 
 FISH EGGS. 
 I am indebted to John A. Ryder for the following notes and 
 suggestions: — 
 
 The Cod. — The eggs of the cod may be obtained from the 
 
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TIMES AND PLACES OF OVULATION. 177 
 
 living fishes at Fulton Market wharves during the winter, and 
 artificially fertilized. The ova of the cod are very transparent, 
 without oil drops ; the germinal disk is formed very slowly, 
 and occupies an inferior position in relation to the lighter 
 vitellus. They measure about ^ g to T V of an inch in diameter. 
 The microscope must be so arranged as to enable the student 
 to study the living germ disk from below, from the side, 
 and from above. The instrument, when using the camera, 
 should be utilized in the upright and horizontal position, so as 
 to get views of optical sections. In order to get surface views 
 from below, a Nachet inverted microscope or an inverting 
 prism must be used, as the vitellus and germ cannot be kept in 
 the position desired, or be turned, on account of the tendency 
 of these two parts to arrange themselves, the former above and 
 the latter below the other, without such violence as will injure 
 the egg. This is an excellent type in which to study the early 
 phenomena of development, or the changes which take place 
 during fertilization. 
 
 The Shad. — Alosa sapidissima. Anadromous shad come 
 from the sea to the weedy flats of rivers to spawn. When 
 captured in the seines, the eggs may be taken from the female 
 fishes and artificially fertilized with the milt pressed from the 
 male. Vitellus, ^ inch in diameter ; egg membrane, when it 
 has absorbed water and the egg is fertilized, measures about 
 \ inch in diameter. Vitellus not very transparent. Hatches 
 in three to five days. This is one of the most accessible 
 forms of fish ova for the embryologist. Germ disk lateral in 
 position, egg heavier than water. May be hatched artificially 
 in a jar with a central inlet tube reaching to near the bottom, 
 and an outlet tube reaching a little way through the cork, a 
 gentle current being allowed to pass through the vessel, as 
 shown in the diagram below, the current being regulated by a 
 single stop-cock in the inlet or feed-pipe. 
 
 Other heavy ova, such as those of the trout, salmon, and 
 white-fish, are easily hatched during the colder part of the year 
 in this apparatus. 
 
 The eggs of Belone longirostris, £ inch in diameter, and 
 those of Chirostoma half as large, are provided with filamen- 
 
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173 
 
 MICROSCOPICAL METHODS. 
 
 (M 
 
 
 K 
 
 
 tary appendages, by which they are fixed to stationary objects 
 in the water. The eggs of the former are found adhering in 
 clusters to the meshes of the pound nets in the southern part 
 of the Chesapeake Bay in July and 
 August. The ova of Chirostoma or 
 Menidia may be taken artificially 
 about the middle of July. Both are 
 rather troublesome to hatch by means 
 of artificial methods. 
 
 The adhesive eggs of the File or 
 Fool-fish, Monacanthus broccus, may 
 be taken at the pound nets during the 
 middle of July. These ova are very 
 small and greenish, and contain scat- 
 tered oil-globules. 
 
 Stickleback. — The four-spined 
 stickleback, Apeltes, may be obtained, 
 in the month of April, from weedy ditches, and transferred 
 in single pairs, male and female, to aquaria, where, if they 
 are provided with live crustacean food, Copepoda, and 
 water plants, the male will build his nest and place the eggs 
 therein and hatch them out. The eggs are amber colored, a 
 line in diameter, and adhesive, and are easily hatched out 
 artificially in dishes, if the water is simply changed two or 
 three times a day. The eggs will stand a great deal of hand- 
 ling during development, and are to be commended to the 
 student mainly for the purpose of sectioning, as the vitellus is 
 too opaque for the purposes for which the cod's egg is suitable, 
 though much may be observed in relation to the development 
 of the vitelline circulation which develops in a remarkable if 
 not in a unique manner. 
 
 G-ambusia. — The viviparous minnow, Gambusia patruelis, 
 met with in the little streams connecting with the Chesapeake, 
 in the vicinity of Cherrystone, Va., is an interesting form. The 
 female is found with gravid ovaries about the middle of 
 August. It is easily captured with a dip net; is about 1| 
 inches long, while the male is only a little over an inch. The 
 whole fish may be hardened and the embryos studied in sec- 
 
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TIMES AND PLACES OF OVULATION. 179 
 
 tions as they lie in the ovarian follicles, which latter have a 
 very large opening at one side, which answers to the micropyle 
 of ordinary fish ova. 
 
 The Yellow Perch. — Perca flavescens lays its eggs in 
 ribbon-shaped masses in April. The egg is remarkable for its 
 complex membrane, which is composed of a thin, outer adhe- 
 sive layer, a very thick middle canaliculated layer, and an inner 
 zona radiata. 
 
 The White Perch has small adhesive ova, which may be 
 pressed in the mature state from the parent fishes in April 
 and May, and hatched on threads which have been drawn 
 through the freshly extruded spawns, the threads being sus- 
 pended in a hatching jar, through which water is kept flowing. 
 
 Gold-Fish. — The eggs of the common gold-fish ( Carassius) 
 may be had during the months of May, June, and July, in an 
 aquarium provided with aquatic plants, upon which the fishes, 
 if in good condition, will attach their adhesive ova singly. 
 This cyprinoid seems to breed for a considerable period during 
 the warm months. 
 
 Cat-Fish. — The cat-fish (Amiurus) under favorable condi- 
 tions will spawn in an aquarium about the middle of July. 
 The ova are £ inch in diameter, adhesive, in a mass ; each egg 
 has an outer adhesive envelope and an inner zona radiata, 
 separated by a thin space from the outer envelope, but con- 
 nected with the latter by irregularly disposed columns of 
 adhesive matter. The male hovers over the eggs and forces 
 the water through them. They hatch in six days, but the 
 vitellus is not wholly absorbed until about two weeks have 
 elapsed. This form is remarkable for the suddenness with 
 which the ordinal characters of the type appear in the embryo, 
 the barbels, in fact, grow out on the second day of develop- 
 ment. It is also remarkable in another respect, viz., the pres- 
 ence of fine, loose granules between the vitellus and the egg- 
 membrane, these granules being stirred about in the breathing 
 chamber of the egg as soon as the embryo is far enough devel- 
 oped to move its tail about. The egg-membranes are exceed- 
 ingly elastic. 
 
 Myriapods. — Geophilus? Strigamia? I have found the 
 
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180 MICROSCOPICAL METHODS. 
 
 nests of these creatures as little burrows or cavities under logs 
 and clumps of loose, damp, friable turf in low, swampy flats, 
 along the mouth of the Susquehanna River, near Havre de 
 Grace, Md. The female was apparently guarding a cluster of 
 about 20 beautiful amethyst-colored ova, each about -fa inch 
 in diameter. 
 
 Polyxenes fasciculatus. I have found this neat little myria- 
 pod in the chinks of the bark of the sycamore and red cedar in 
 Fairmount Park, Philadelphia. Its eggs are found in the same 
 situation in April and May amongst the detached caudal bris- 
 tles and ecdyses of the adults as very small oval bodies. It is 
 also found in abundance along the beach of the Chesapeake 
 Bay in St. Mary's County, Md., under fragments of cedar bark ; 
 this form is much paler than the inland form, and may be 
 another species. 
 
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CHAPTEE X. 
 NUCLEI (KARYOKINETIC FIGURES, &c). 
 
 FLEMMING'S METHOD. 
 
 The method employed by Bottcher and Hermann of over- 
 staining objects with aniline dyes, and then removing the color 
 to any desired extent by the aid of alcohol, formed the starting- 
 point of some of the methods recently published by Flemming. 
 The following is a summary of the more important of these 
 methods : — 
 
 A. For Nuclei in general 1 — 1. Objects hardened in 
 chromic acid (-fa-^ %)• 
 
 The concentration of the acid and the time of immersion vary 
 according to the character of the tissue. The process of staining is not 
 influenced perceptibly by the time of action of the acid; but objects be- 
 come very brittle if left a long time in the acid. 
 
 2. Carefully washed in distilled water. 
 
 3. Transferred directly, in small, thin pieces or sections, to a 
 small quantity (l ccm ) of the dye, and left (covered) 12-24 hours. 
 
 Safranin, Magdala red (rose de naphthaline), and dahlia 
 (monophenylrosanilin) give the best staining. Safranin pre- 
 pared as given on p. 49 ; magdala in the same way ; dahlia 
 best dissolved in water or acetic acid. 
 
 Only very small objects or thin sections can be successfully 
 stained. 
 
 4. Dye with the object placed in a shallow watch-glass ; the 
 watch-glass, with contents, placed in a dish of 70 <f alcohol, and 
 agitated a little, in order to remove most of the free dye ; then 
 
 i Flemming. Archivf. mik. Anat., Vol. XIX., p. 321, 1881. 
 
 181 
 
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182 MICROSCOPICAL METHODS. 
 
 transferred at once to absolute alcohol for half a minute or 
 longer, till no visible clouds of color are seen about the object. 
 
 The process of decoloring is now completed, and the objects must be at 
 once removed from the alcohol; otherwise, the color will be too much 
 weakened. If it be required to examine the objects before mounting, they 
 may be removed to distilled water, in which the color of the nuclei will 
 remain unchanged for a considerable time. They must then pass through 
 alcohol again before mounting. 
 
 As the aim in decoloring is to bring out a strong, differential staining 
 of the nuclei, the process should be checked the moment it reaches the 
 nuclei. No precise time-limits can be given, as the length of the process 
 varies with the thickness and kind of tissue. 
 
 5. Clarified in clove oil, and mounted in damar-lac. 1 
 
 Clove oil withdraws the color a little, and hence it must not 
 
 be allowed to work too long. Creosote extracts the color still 
 
 more rapidly. 
 
 B. Eggs of Echinoderms." — In somewhat later researches 
 on karyokinesis, Flemming states (p. 5) that he obtained ser- 
 viceable staining of nuclei in the two following ways: — 
 
 1st. Living eggs colored on the slide, either with safranin or 
 aniline dyes, followed by acetic acid (1 fc), which is allowed to 
 flow under the cover and thus replace the staining medium, 
 or with Acetic acid carmine (after Schneider) used undiluted. 
 
 The last, mentioned staining agent causes swelling, but still 
 gives the typical features of the karyokinetic figures. 
 
 2d. Eggs first hardened in strong nitric acid (40-50 to aq. 
 dest. 60-50), then washed in distilled water until the yellowish 
 color, due to the presence of the acid, disappears. Colored 
 with acetic acid carmine. 
 
 C. Chrom-Osmium- Acetic Acid and Safranin. 8 — 
 Among the various methods of studying karyokinetic figures 
 (" mitosis ") which have been given by Flemming, the latest, 
 most detailed, and most highly recommended is the follow- 
 
 1 Flemming prepares this by dissolving (warm) one part of gumdamar 
 in one part of turpentine and one of benzole, and then evaporating to a 
 syrupy consistency. 
 
 2 Arch. f. niik. Anat., XX., p. 1, 1881. 
 
 8 Zeitschr. f. wiss. Mikroskopie, I., Heft 3, pp. 349-361, 1884. 
 
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NUCLEI (KARTOKINETIC FIGURES, ETC.). 183 
 
 1. Place fresh-cut pieces of tissue (best not more than 
 £ an thick) in a mixture of 
 
 Chromic acid (1 %) 15 parts 
 
 Osmicacid (2%) 4 " 
 
 Glacial acetic acid 1 part (or less) 1 
 
 They should remain in this fluid at least one day ; for complete 
 hardening, better 2-3 days, or even weeks or months. They 
 are not injured by exposure to light. 
 
 2. Wash in water for an hour or more. This is best done by 
 placing the object, enclosed in a small box of wire netting, in 
 a strong current of water. 
 
 3. Harden in absolute alcohol a few hours. 
 
 4. Imbed, while still saturated with alcohol, in very soft 
 paraffine, elder pith, or in celloidin. 
 
 This method of imbedding is considered best and safest for obtaining 
 clear and well-preserved nuclear figures; but, as Flemming remarks, it is 
 not to be adopted in cases where it is important to have a uniform series 
 of thin sections. 
 
 5. Cut with the knife flooded with alcohol. 
 
 6. Wash the sections in water, and then transfer them to a 
 strong solution of safranin (or gentian violet) for 24 hours or 
 more. 
 
 7. After washing in water, place in absolute alcohol to which 
 a little hydrochloric acid ( \% or less) has been added, leaving 
 them for only a few moments, until clouds of color are no 
 longer seen. 
 
 8. Transfer to pure absolute alcohol, and finally clarify with 
 clove oil, and mount in damar or balsam. 
 
 Special stress is laid on this method as a convenient means of 
 finding karyokinetic figures, and of determining, by their fre- 
 quency, p/aces of most rapid growth. 
 
 STRASBURGER'S METHODS.* 
 
 In the study of nuclear metamorphosis, Strasburger made 
 use of 1 % acetic acid, with which a little methyl green was 
 
 1 A much stronger mixture than was recommended in his " Zellsub- 
 stance, Kern, and Zelltheilung, 1882, p. 381. 
 
 * Arch. f. mik. Anat., XXI., p. 476, and "Zellbildung und Zelltheil- 
 ung," 3d ed., p. 141. 
 
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184 MICROSCOPICAL METHODS. 
 
 mixed. The figures are " fixed " and colored at the same time, 
 but the preparations are not permanent. 
 
 Preparations were also made by treating with alcohol, or 
 50 % nitric acid, and then staining with the coloring substance 
 mixed with dilute glycerine. Rapidity of preparation and 
 clearly defined spindles compensate, in these methods, for the 
 lack of permanency. 
 
 EABL'S METHODS.i 
 
 Material — The skin and kidney of Proteus and the epithe- 
 lium of the mouth of Salamander larva?. The epithelium is 
 the more favorable object, as the very large nuclei can be ex- 
 amined in surface preparations. The achromatic spindles are 
 seen to best advantage in the renal tissue. 
 
 Preparation. — a. Place small, fresh pieces of the object in 
 chrom-formic acid (200 g. of a £ % solution of chromic + 4 to 
 5 drops of strong formic acid) 12-24 hours. 
 
 b. Wash thoroughly, and harden slowly, first 24-36 hours in 
 60-70 % alcohol, then in absolute alcohol. 
 
 Instead of the chrom-formic acid, a J % solution of platinum chloride 
 may be used; the preparation otherwise remaining the same. Chrom- 
 formic acid causes the chromatin filaments to swell somewhat, so that 
 their longitudinal division generally becomes obliterated ; while plati- 
 num chloride causes a slight shrinkage which brings out very dis- 
 tinctly the division of the filaments, as well as the chromatin spherules 
 of Pfitzner. 
 
 c. Stain in either of the three following ways : — 
 
 1. Grenadier's hcematoxylin (strongly diluted with distilled 
 water) 24 hours, followed, after washing, with acidulated alco- 
 hol (few drops of HCL). 
 
 2. Pfitzner 's safranin 2-4 hours, followed ' by absolute alco- 
 hol, in which the object is left until no visible cloud of color 
 remains upon turning it over (generally about two minutes), 
 clove oil a few minutes, and damar. 
 
 3. Double stain with hematoxylin and safranin ; stain very 
 feebly with haematoxylin ; wash and treat with acidulated alco- 
 hol ; and then stain with safranin as in No. 2. 
 
 i Morph. Jahrb., X., H. 2, pp. 215-219, 1884. 
 
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NUCLEI (KARYOKIXETIC FIGURES, ETC.). 185 
 
 Examination. — High powers are required in the study of 
 the mounted preparation, either the homogeneous immersion 
 T \ of Zeiss, with Abbe's condenser, or that of Hartnack, No. 
 III., %\. Nachet's camera was employed in drawing. 
 
 It is well to work with green light, which can be ob- 
 tained by inserting a green-colored glass plate beneath the 
 table of the microscope, as was first recommended by Engel- 
 mann. 
 
 The slide devised by Rabl enables one to examine a prepara- 
 tion from both sides. It consists of four pieces of glass, of the 
 shape and size seen in the figure (a, b, c, d), and a cover-glass, 
 g, which serves as the object-bearer. The two glasses, a and b, 
 
 -J- 
 
 are painted on one side with chloroform balsam, and then con- 
 nected by means of c and d. The frame thus formed is com- 
 pleted by adding the pieces, e and/, between c and d. The frame 
 is next turned over, and the middle portions of the glass bars, 
 c and d, painted with balsam; and a thin glass cover, g, 
 placed so as to rest on the painted sides of c and d. This 
 glass (g) bears the object in damar, which is covered by 
 another very thin glass. The object, lying between two 
 thin cover-glasses, can be viewed from both sides with the 
 highest powers. 
 
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186 MICROSCOPICAL METHODS. 
 
 HEIDENHAIN'S METHOD OF USING HEMATOXYLIN.! 
 
 1. Pieces of organs are first well hardened in alcohol. 
 
 2. Transferred to a ^-\% aqueous solution of hematoxylin 
 8-10 hours. 
 
 3. Treated with bichromate of potassium (£-1%) 8-10 
 hours. 
 
 4. After becoming thoroughly black, they are washed in 
 water, dehydrated with alcohol, imbedded in paraffine, and 
 mounted in balsam. 
 
 Nuclei are thus stained black, while other parts of the cells 
 appear dark gray, or likewise black. Sections require to be 
 very thin. 
 
 This method of staining may be followed equally well with 
 sections (Minot). 
 
 DEMONSTRATION OF NUCLEI IN THE PSEUDO-CHLOROPHYLL 
 BODIES (ZOOCHLORELLA) OF HYDRA.2 
 
 1. Color the green corpuscles alive, after setting them free 
 by pressure, in an aqueous solution of hematoxylin. Remove 
 the excess of coloring substance by washing in water, and then 
 kill in absolute alcohol. The chlorophyll is generally extracted 
 in about two hours, at the end of which time the nuclei, one 
 or more in each cell, appear as violet-colored dots. 
 
 2. First kill with osmic or chromic acid (%%), then stain 
 with hematoxylin. 
 
 3. (After Flemming). Leave pieces of Hydra in \% chromic 
 acid 1 hour; wasb, and stain with a solution of magdala in 
 50% alcohol (24 hours). Place the pieces in absolute alcohol, 
 several times renewed, in order to remove a part of the color. 
 Isolate the green cells (in water or balsam) by pressure. 
 Nuclei are colored as before. 
 
 1 Arch. f. mik. Anat. XXIV., H. 3, pp. 468-470, 1884. 
 
 2 K. Brandt. Arch. f. Anat. u. Phys. phys. Abthg., 1882, pp. 133-134. 
 
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CHAPTER XI. 
 
 PREPARATION OF NERVOUS TISSUE. 
 
 GOLD CHLORIDE METHODS. 
 
 Flechsig's Method. — 1. Place the organ (brain, oblongata, 
 spinal cord, etc.,) in a 1 % solution of ammonium bichromi- 
 cum, and let it remain until it becomes hard enough to section 
 (about 6 days). 
 
 2. Wash the sections for a short time in distilled water, and 
 then place in a.^% solution of gold chloride 15-30 minutes. 
 
 3. Transfer, after washing again in distilled water, to a solu- 
 tion of caustic soda (10^ or less), in which the white matter 
 immediately assumes a dark violet color, while the gray matter 
 remains apparently uncolored. A few hours to one or more 
 days. 
 
 4. After washing, treat with alcohol, and mount in the usual 
 way. 
 
 One of the most reliable methods of using gold chloride is 
 that employed by Dr. Sigm. Freud, 1 in investigating the course 
 of the nerve-fibres in the oblongata of the human fcetus. 
 
 Freud's Method. — 1. Harden in Erlicki's fluid (2£ parts 
 of bichromate of potash, ^ sulphate of copper, and 100 aq. 
 dest.) until the preparation has become sufficiently firm for 
 cutting ; then transfer to alcohol, and section at convenience, 
 taking care only that this be done before it becomes brittle. 
 
 2. Wash the sections well in distilled water, in order to free 
 
 1 Arch. f. Anat. u. Physiol., Anat. Abth., 1884, pp. 453-460. 
 
 187 
 
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188 MICROSCOPICAL METHODS. 
 
 tliem from the alcohol with which the knife of the microtome 
 is wet, and place them in a \% solution of gold chloride 3-5 
 hours. If the sections are thoroughly washed, no film or preci- 
 pitate will form in the gold chloride, and the same solution will 
 bear repeated use. 
 
 3. After washing, place in strong caustic soda (1 part natrurn 
 causticum fusum, and 5-6 parts water) 3 minutes. The sec- 
 tions, for the most part, become at once transparent. 
 
 4. Remove the sections, one at a time, to a glass slide, and 
 free them from the lye, so far as this can be done by a careful 
 use of blotting-paper. 
 
 5. Place them in a 10-12$ solution of iodide of potassium 
 5-15 minutes, in which they flatten out, and gradually assume 
 a red color in consequence of a reduction of the gold. 
 
 It is well to have the sections as free as possible from the lye 
 before placing them in the iodide solution, and to renew the 
 latter after staining from 10 to 25 sections. 
 
 6. Place the sections again on a glass slide, and dry them by 
 the aid of filtering paper ; loosen them from the slide by dij»- 
 ping it in a glass of distilled water ; change the water ; treat 
 With the ordinary grades of alcohol ; then clarify and mount in 
 the usual way. 
 
 This method can be varied to advantage, in many cases, by 
 adding to the 1% gold-chloride solution an equal volume of 94 fi 
 alcohol. 
 
 G-aule's Method for Nerves. — 1. Object treated with 
 chromic acid Q%) 12 hours. 
 
 2. Washed 12 hours. 
 
 3. Inlfo gold chloride (dark) 1 hour. 
 
 4. Washed in distilled water, and placed in formic acid 24 
 hours (or until a violet color is obtained). 
 
 5. Washed and treated with alcohol. 
 
 6. Imbedded in paraffine. 
 
 Rammer's Methods for Nerve-Terminations. — A. 1. 
 Muscles of salamander placed direct in a mixture consist- 
 ing of : — 
 
 Gold chloride (1%) 4 parts 
 
 Formic acid 1 " 
 
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PREPARATION OF NERVOUS TISSUE. 189 
 
 The two fluids are mixed, heated to boiling point, and used immedi- 
 ately after cooling. This process renders the gold chloride more easily 
 reducible. 
 
 2. Washed at the end of 20 minutes in 
 
 Formic acid 1 part 
 
 "Water 4 " 
 
 -B. 1. Muscle of frog placed in fresh filtered lemon juice 5-10 
 minutes. 
 
 2. Washed in water, and then left 20 minutes in 1% gold 
 chloride. 
 
 3. After washing again, placed in acidified water (water 
 50 cc. + acetic acid 2 drops) in daylight for 24-48 hours. 
 
 Such preparations grow darker with time. 
 
 In the method of Loewit, 1 the object was first treated with 
 formic acid before being exposed to the action of gold chloride ; 
 the result was that the terminal ramifications of the nerves 
 were deleteriously affected • by the first reagent before they 
 could be fixed by the second. In combining the two, as in A, 
 Ranvier hoped that the gold chloride would accomplish its 
 work before the nerves were injured by the acid. The result, 
 although not quite satisfactory, showed that the method was 
 far better than that of Loewit. As the injurious effect of 
 formic acid could not be wholly avoided, Ranvier attempted to 
 replace it by a more favorable acid, which he found in lemon 
 juice. This led to method B. 
 
 Motor Nerve Endings. — Ciaccio 2 has investigated the 
 motor nerve-plates in the depressor muscle of the jaws of 
 Torpedo marmorata by treatment with double chloride of gold 
 and cadmium. From the anterior third of the muscles, strips 
 one millimeter thick were cut with scissors, the strips were 
 then left for five minutes in fresh filtered lemon juice, washed 
 in distilled water, and placed for half an hour in 1 % solution of 
 gold and cadmium, being kept dark; washed again in 1% 
 aqueous solution of formic acid, in which they were left 12 
 hours in the dark, then 12 in the light ; finally, kept in the 
 
 i Cf. Quart. Jour. Micr. 8c, XX, No. 80, pp. 456-458, 1880. 
 2 Arch. Ital. Biol., III., 75. 
 
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190 MICROSCOPICAL METHODS. 
 
 dark in stronger formic acid for one day, and preserved in 
 glycerine. The fibres of such strips may easily be dissociated. 
 Pfitziier's Method of Studying Nerve-Terminations. 1 
 The material for investigation consists of a fresh piece of the 
 epidermis (circa 6 mm in diam.) taken from the ventral (unpig- 
 mented) side of a well-fed salamander larva. The methods of 
 treatment are as follows : — 
 
 1. Hardened in chromic acid (|-£ fo) for a number of days. 
 
 2. Imbedded in elder pith, and cut with the microtome into 
 sections 10-15 ft. thick. 
 
 3. Sections washed in distilled water for at least 30 minutes, 
 (not injured by remaining in water 24 hoars). 
 
 The further treatment of these sections may take either of 
 the following courses : — 
 
 A. 1st. Colored with safranin and mounted in damar. 
 
 2d. Colored with hematoxylin and mounted in damar. 
 3d. Mounted unstained in glycerine. 
 
 B. 4th. (a) Transferred to a 1 % solution of gold chloride 
 (15-30 minutes) to which has been added a trace of hydrochlo- 
 ric acid. Kept dark. 
 
 (b) Carefully washed, half an hour or more, and then placed 
 in 5 % formic acid 12-24 hours (exposed to light). 
 
 (c) Again thoroughly washed in distilled water, and then 
 mounted directly in glycerine, or, after staining with safranin, 
 in damar. 
 
 C. 5th. (a) Transferred to \ f osmic acid for 30 minutes or 
 longer. 
 
 (b) Carefully washed and mounted in glycerine. 
 
 GOLGI'S SILVER METHOD." 
 
 For the investigation of nerve-fibres, Golgi has employed 
 with success the following method : — 
 
 1. A fresh nerve (rabbit) is placed for one hour in a mix- 
 ture of 
 
 Bichromate of potassium (2 % ) 10 parts 
 
 Osmic acid (1 fo ) 2 " 
 
 i Morph. Jahrb., VII., pp. 291, 292, and 731, 1881. 
 2 Arch, per le sc. med., IV., p. 221, 1880. 
 
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PREPARATION OP NERVOUS TISSUE. 191 
 
 2. The nerve is then cut into pieces £-1° in length, and again 
 returned to the mixture for some hours. 
 
 3. Transferred to nitrate of silver (-J- fo) for 8 hours. 
 
 4. Sectioned and mounted in damar. 
 
 Golgi made preparations in still another way : — 
 
 1. Peripheral nerves treated with bichromate of potassium 
 4-8 hours (Central organ 10-15 days). 
 
 2. Transferred to silver nitrate 12-24 hours (in dark). 
 
 3. Sectioned and mounted as before, and then exposed to 
 light for reduction. 
 
 WEIGERT'S METHOD OF STAINING THE CENTRAL NERVOUS 
 SYSTEM WITH HEMATOXYLIN.* 
 
 Weigert's hsematoxylin method is much more expeditious, 
 simple, and easy than his fuchsin method; and, as it gives 
 equally good or even better results, and is far less capricious, it 
 has superseded the latter method. 
 
 1. Object hardened in Milller's or Erlicki's fluid. 
 
 The hardening process in Miiller's fluid may be completed in 8-10 
 days, instead of 8 weeks, by heating in an incubator kept at 30°-40° C. 
 
 In Erlicki's fluid, the hardening requires ordinarily 8-10 days; by heat- 
 ing it may be accomplished in about 4 days (Centralbl. f. d. med. Wiss., 
 XX., No. 46, p. 819, 1882). 
 
 2. Transferred directly, without ever coming in contact with 
 water, to alcohol. 
 
 If the object is allowed to remain in alcohol until it becomes green, it 
 must be returned to the original hardening fluid, and left until the brown 
 color is restored. 
 
 3. Best imbedded in celloidin. 
 
 4. The brown (not green) sections are stained in 
 
 Hsematoxylin 1 part 
 
 Alcohol 10 " 
 
 Water 90 " 
 
 This staining mixture is boiled and allowed to stand some days before 
 using. The sections remain 24 hours (1-2 hours, if kept at a tempera- 
 ture of 35°-40° C.) in the dye, but may remain without injury any length 
 of time before being transferred to the decoloring fluid. The sections 
 must not be exposed to water before staining. 
 
 l Fortschritte der Medicin, II., No. 6, p. 190, 1884. Cf. Edinger's Re- 
 view in Zeitschr. f. wiss. Mikroskopie, I., H. 2, pp. 290-292, 18S4. 
 
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192 MICROSCOPICAL METHODS. 
 
 5. The black-colored sections, after being rinsed in water, 
 £.re decolored in 
 
 Borax 2 parts 
 
 Ferricyanide of Potassium (red prussiate of potash) 1\ " 
 
 Water 100 " 
 
 The first traces of a distinction between white and gray substance will 
 appear in about 30 minutes; but the decoloration must be allowed to go 
 on until the gray substance becomes plainly yellowish, the white sub- 
 stance remaining black. 
 
 6. The sections are next washed thoroughly in water, then 
 treated with alcohol, clarified in xylol, and mounted in balsam. 
 
 Max Flesch 1 has made some important modifications of 
 Weigert's method. In the first jslace, he discovered that prepa- 
 rations which had been washed in water in the usual way, after 
 coming from Mtlller's fluid, could be stained, provided the 
 sections (made in celloidin) were treated a few minutes in 
 % % chromic acid, and then, after being washed in water, placed 
 in the coloring fluid. The sections stain very much quicker 
 than in Weigert's method. The decoloring process of Weigert 
 is followed. Creosote is said to be superior to xylol as a clari- 
 fying medium. 
 
 According to Minot, Weigert's hsematoxylin method may be 
 used after any method of hardening and cutting, provided the 
 sections are treated 5-15 minutes in 1 % bichromate of potas- 
 sium, then washed in water, and transferred to the staining 
 mixture. Instead of bichromate of potassium, the following 
 mixture may be used with equal success, but with somewhat 
 different results : — 
 
 Water 100 cc. 
 
 Alum 1 g. 
 
 Bichromate of potassium 1 g. 
 
 THE NUMERICAL RELATION OF NERVE FIBRES TO GANGLION 
 CELLS IN THE SPINAL CORD OF THE FROG. 
 
 In the very difficult task of determining the precise numeri- 
 cal relations of nerve fibres to ganglionic cells, Prof. E. A. 
 Birge, 2 working under the direction of Dr. Gaule of Leipzig, 
 employed the following methods with success : — 
 
 1 Zeitschr. f. wiss. Mikroskopie, I., Heft 4, pp. 564-566, 1S84. 
 
 2 Arch. f. Anat. u. Phys., Phys. Abthg., 1SS2, pp. 437-440 and 440-448. 
 
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PREPARATION OF NERVOUS TISSUE. 193 
 
 A. Method of Counting Nerve Fibres. — a. Prepara- 
 tion. 1. Lay bare the spinal cord in its whole extent by the aid 
 of bone-nippers ; then, after cutting off the roots close up with 
 sharp shears, remove the cord, in order to give the hardening 
 agent free access to the vertebral canal. 
 
 2. Cut off the nerves at some distance outside the interver- 
 tebral canals, through which they pass* and remove the entire 
 vertebral column, together with the roots and nerve portions, 
 to a 1 % solution of osmic acid, in which, protected from the 
 light, it is allowed to remain six hours. Before treating with 
 the acid, it is well to remove the muscular tissue as far as pos- 
 sible. The pleuroperitoneal membrane covering the nerves 
 should not be disturbed. 
 
 3. After washing 2-3 hours in distilled water, place the pre- 
 paration in 70 % alcohol for 24 hours, then in 96 % alcohol. 
 
 4. After dehydration, the nerves with their roots may easily 
 be removed from the vertebral column. In order to avoid con- 
 fusion, each should be isolated in a small open box, made of 
 tin-foil and provided with a number. 
 
 5. Preparatory to imbedding, transfer the preparations from 
 alcohol (each in its tin-foil box) to clove oil, then to turpentine, 
 then to a mixture of turpentine and paraffine, heated to 45° C, 
 for one hour, and finally to pure paraffine, at 55°-G0° C, for 20 
 minutes. 
 
 6. Sections of the imbedded nerves should be made as thin 
 as sitr™" 1 - They are mounted as they are taken from the knife 
 (without removing the paraffine) in zylol balsam, after Gaule's 
 method. 1 
 
 b. Counting. An examination of the thin transverse sections 
 of the nerves with the microscope shows how important a part 
 osmic acid plays in the above method of preparation. The 
 medullary sheaths are stained black, and appear in sections, as 
 small black rings, at the centre of each of which is seen a 
 minute gray dot (the axis cylinder). Each individual fibre, 
 however small, is thus rendered perfectly distinct. The exact 
 
 1 The methods since introduced of fixing sections on the slide would 
 doubtless be more convenient and satisfactory in every way. 
 
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194 MICROSCOPICAL METHODS. 
 
 number of fibres in the nerve may be determined by the use of 
 a net-micrometer, which divides the section into conveniently 
 small areas for counting. 
 
 B. Method of Counting Ganglion Cells. — a. Prepara- 
 tion. 1. Having laid bare the central organs of the nervous 
 system, cut off the roots as far as possible from the spinal cord, 
 and then remove the brain and cord to Muller's fluid (7 days). 
 
 2. Wash carefully and thoroughly, and then transfer to 
 70 fo alcohol for 24 hours. 
 
 3. Stain with Grenacher's alum-carmine. 
 
 4. Wash, treat with alcohol, clarify, and imbed in paraffine. 
 Sections need not be thinner than 7 y llm , and should not be 
 thicker than ^ mm . They must be mounted in serial order, and 
 without any loss. 
 
 b. Examination. The nerve fibres are colored very little, the 
 gray substance deeply so. The counting of the ganglion cells 
 is comparatively easy, as the number of cells on either side of 
 a section is only from two to ten. The number of cells is tabu- 
 lated separately for each side and for each section, and the 
 difference between the two sides noted. As the total number 
 of cells varies with the weight of the frog, a record of the 
 weight must also be kept. 
 
 BETZ'S METHOD OF HARDENING THE BRAIN.l 
 
 1. Harden in 70 % alcohol containing enough iodine to give 
 it a yellowish-brown color. After a few days the color disap- 
 pears, and a little iodine tincture is again added (6-10 days). 
 
 2. Transfer to a 3 % solution of 'bichromate of potassium, 
 and leave until completely hard. The time for removal from 
 the fluid will be marked by the formation of a reddish-brown 
 precipitate on the surface of the preparation. 
 
 During the entire process of hardening, the preparation 
 should be kept in a cool place. 
 
 THE BRAINS OF URODELA. 
 The following method of preparation is extracted from Dr. 
 
 1 From Fol's Lehrbuch, p. 105. 
 
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PREPARATION OF NERVOUS TISSUE. 
 
 195 
 
 H. F. Osborn's papers 1 on the brains of American TJrodela, 
 and from a letter in which the details were more fully given : — 
 "Before hardening, the brains were inflated with Miiller's 
 fluid, so as to preserve the natural proportion of the cavities. 
 After treatment with alcohol, they were placed for a week in 
 dilute carmine. Calberla's egg-mass was employed as before, 
 except that the ventricles were injected with the mass before 
 hardening. The delicate parts of the brain-roof were thus 
 retained. It appears now that celloidin may be used for this 
 purpose to equal, if not to greater, advantage in results, and with 
 considerable economy of time. The sections were cut in absolute 
 alcohol, then floated upon a slide in consecutive order, from 
 
 Glass dish an& 
 
 Paper ^Sotte_^ 
 and Arum 
 
 65%Mca7iolf 
 
 MVJra netting 
 support 
 
 -Large \wcterj"»ft 
 
 Fie. 35. — Bath for hardening egg-emulsion. 
 
 20 to 50 at a time, and covered with a delicate slip of blotting- 
 paper during treatment with oil of cloves." 
 
 Imbedding. — 1. The "egg-mass was prepared by shaking 
 the white and yolk of egg together, with three drops of glyce- 
 rine to each egg, and then well filtered through coarse cloth. 
 
 2. The bath is then prepared as follows : — 
 
 a. Outside is a large water-pan, for boiling with a Bunsen 
 burner, etc. 
 
 b. Inside this, supported on rests to prevent jarring, is a 
 covered glass dish, filled to about 1 inch in depth with 85^ 
 alcohol. 
 
 i Proc. Acad. Nat. Sc. of Philadelphia, 1883, p. 178, and 1884, p. 262. 
 
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196 MICROSCOPICAL METHODS. 
 
 c. Within the glass dish is placed a piece of coarse wire 
 netting which supports the imbedding box, raising it above the 
 alcohol. 
 
 3. The box, made of paper in the usual way, and one-fourth 
 filled with the imbedding mass, is kept in the bath until the 
 mass is hardened enough to support the brain. The brain is 
 next placed on the hardened stratum, and covered with the 
 fresh mass. The second stratum is hardened just enough to 
 hold the brain in place, and then a third is added, filling the box. 
 
 4. The whole mass must now be allowed to harden through 
 and through, requiring about fifteen minutes. 
 
 5. The hardening is completed by passing the box through 
 three grades of alcohol, — 80, 90, and 100$;, — allowing it to 
 remain 24 hours in each. 
 
 When the mass becomes nearly white, and ceases to discolor 
 the alcohol, it is ready for cutting. 
 
 MOUNTING AND PHOTOGRAPHING SECTIONS OF CENTRAL 
 NERVOUS SYSTEM OF REPTILES AND BATRACHIANS.l 
 
 Both the brain and spinal cord were entirely separated from 
 the body, and, with their membranes, placed in iodine-tinted 
 alcohol until they had acquired a slight degree of consistency, 
 — from 6 to 12 hours. They were then transferred to a 3% 
 solution of bichromate of potash, with a small piece of camphor, 
 in a tightly-corked, wide-mouthed bottle, and allowed to remain 
 until ready for cutting, renewing the solution every two 
 weeks. 
 
 The time required for the hardening process varies consid- 
 erably in different animals, and this variation is more dependent 
 upon the class of animal, than upon the relative dimensions of 
 the specimens. 
 
 For example, on the same day I placed the brain of a large 
 rattlesnake with that of a small salamander in the same bottle, 
 and at the end of six weeks the former was ready for sectioning, 
 whilst the latter was not sufficiently hard until a month after- 
 
 1 J. J. Mason. Minute Structure of the Central Nervous System of 
 Certain Reptiles and Batrachians of America, 1879, 1882. 
 
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PREPARATION OF NERVOUS TISSUE. 197 
 
 wards. By thus employing the same reagent in all cases, I 
 have been able to note constant differences in the action of 
 both the hardening and the coloring agent, carmine. 
 
 Perhaps the most striking illustration of this is furnished by 
 the nervous centres of tailed batrachians, which, while they 
 stain very readily, invariably require about a third more time 
 to harden than specimens from the other orders. Specimens 
 from ophidians stain less satisfactorily than those from any 
 other of the classes which I have studied, while with the spinal 
 cords of alligators, turtles, and frogs, failure to obtain good 
 results in this particular is very rare. 
 
 In all cases the sections have been stained after cutting, 
 injury from excessive handling being wholly avoided by the 
 use of siphon tubes to remove the alcohol and washings. For 
 producing transjaarency, oil of cloves has been used, and the 
 mounting has been done under thin, clear covers, in a solution 
 of Canada balsam in chloroform. 
 
 All the negatives have been made on glass thoroughly 
 cleaned and lightly coated with a solution of wax and benzole, 
 so that the collodion film, previously made adherent to thin 
 sheets of gelatine, could be safely removed from the plate. 
 The flexible negatives thus obtained are well adapted to the 
 artotype process, and, as they can be indefinitely preserved 
 between the leaves of an ordinary scrap-book, are very desira- 
 ble for a series of illustrations. In making the original nega- 
 tives on glass, the " wet collodion process," with the sulphate 
 of iron developer, has been exclusively employed. 
 
 The prints correspond exactly with the negatives, both in out- 
 line and detail. No distinction occurs, as in silver printing, in 
 which process the paper is subjected to prolonged washing. 
 
 In many of the photographs the gray substance appears 
 lighter in shade than the white substance. This appearance is 
 due to a greater degree of transparency of the gray substance 
 in these sections, resulting from the action of the oil of cloves, 
 followed by an increased action of the transmitted light on the 
 sensitive collodion film of the negative, and hence by a thinner 
 deposit of ink over corresponding parts of the positive plates 
 from which the artotypes are printed. 
 
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198 MICROSCOPICAL METHODS. 
 
 With regard to the process employed, Dr. Mason says that 
 after experimenting with various methods he found that satis- 
 factory prints could be made in ink directly upon plate-paper, 
 and that these impressions were as perfect in fine detail as any 
 of those obtained by the silver process of printing. The plates 
 (all printed by the autotype process), are as durable as steel 
 engravings. While a photograph often cannot show all that 
 can be discovered by more direct microscopic observation with 
 a judicious working of the fine adjustment, high authority has 
 stated, and perhaps correctly, that a good photograph with a 
 low power, say from three to one-half inch, is a better means of 
 illustrating the anatomical structure of the nervous tissues 
 than hand-drawing. Some of the plates with high powers 
 leave much to be desired both in distinctness and tone, and in 
 general it may be affirmed that the same defect as regards 
 distinctness always exists, and for obvious reasons, in photo- 
 graphs of sections with powers much above one-half inch. 
 
 METHOD'S OF STUDYING THE NERVOUS SYSTEM OF ANNELIDS.' 
 
 Maceration is the best means of demonstrating the existence 
 of a peripheral nervous system (Polygordius, Protodrilus, and 
 Saccocirrus) and of showing its relation with the central ner- 
 vous system. As macerating agents, Fraipont employed weak 
 alcohol (36-48 hours), chromic acid ( T J ¥ %, 24 hours), and a 
 weak solution of bichromate of potash (48 hours). 
 
 After treatment with one of these agents, a definite portion 
 of the annelid may be placed on a slide, teased apart under the 
 dissecting microscope with fine needles, and then examined in a 
 drop of the macerating fluid ; or it may be freed from its cuti- 
 cula, and subjected to gradual pressure under a cover-glass. 
 This treatment causes the preparation to flatten out, but does 
 not dissociate the tissues so far as to obscure the relations 
 existing between the different layers and their constituent ele- 
 ments. 
 
 In some cases, good results may be reached by giving light 
 taps on the cover-glass with the point of a needle for ten min- 
 
 i Julien Fraipont. Arch, de Biol., V., F. 2, pp. 251-254, 1884. 
 
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PREPARATION OF NERVOUS TISSUE. 199 
 
 utes or more. In either case, the progress of dissociation can 
 be followed with the microscope. 
 
 Specimens to be sectioned with the microtome should be so 
 killed that they remain straight and extended. They may be 
 killed by adding very slowly alcohol to the water. As soon as 
 they cease to move, they should be taken out, and extended on 
 a slide, and then hardened with alcohol, osmic acid, picro-sul- 
 phuric acid, chromic acid, or corrosive sublimate. 
 
 Another method of killing is to pour hot corrosive sublimate 
 over the worm after it has been stretched out on a dry slide. 
 A mixture of osmic acid (1 fo) and chromic acid (i %) in equal 
 parts was also employed with some success. 
 
 For coloring, borax carmine was employed after sublimate 
 and chromic acid ; picrocarminate of ammonia after alcohol, 
 osmic, and picric acid ; haematoxylin and aniline dyes after 
 chromic acid. 
 
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CHAPTEK XII. 
 
 HISTOLOGICAL METHODS. 
 
 METHODS OF INVESTIGATING ANIMAL CELLS.l 
 
 As most Protozoa move very rapidly when hungry, it is well 
 to feed them before attempting to study them with the micro- 
 scope. If well fed with powdered pieces of plants, etc., they 
 usually remain quiet after a short time, and begin to assimilate 
 the food material which they have appropriated. In this con- 
 dition of comparative quiet they can be easily examined with 
 high powers. For this purpose they may be placed under a 
 cover-glass with considerable water and a number of small 
 green algse to keep the water supplied with oxygen. 
 
 For higher powers, Abbe's illuminating apparatus is ex- 
 tremely useful. In some cases, it is desirable to have a com- 
 pletely one-sided illumination, and this is best effected by 
 inserting beneath the illuminating apparatus a circular dia- 
 phragm-plate perforated with a slit 3 mm wide that runs parallel 
 to the edge of the plate. It is best to have about 2 mm between 
 the slit and the edge of the plate. Several diaphragm plates 
 should be prepared in which the slit varies in extent from a 
 half to a whole of a quadrant or more. 
 
 The following mixture, which is Merkel's fluid with the addi- 
 tion of a little acetic acid, is recommended above all other 
 reagents as a preservative medium : — 
 
 Chromic acid 1 part 
 
 Platinum-chloride 1 " 
 
 Acetic acid 1 " 
 
 Water 400-1000 " 
 
 1 A. Brass. Zeitschr. fiir wiss. Mikroskopie, No. I., pp. 39-51, 1884. 
 
 200 
 
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HISTOLOGICAL METHODS. 201 
 
 Unicellular animals die very slowly in this mixture, and 
 suffer very much less alteration in structure than when killed 
 in osmic acid or picro-sulphuric acid. 
 
 A special method is required for Protozoa filled with opaque 
 food material. In many cases the nucleus and the structure of 
 the cell body are completely obscured by foreign bodies. The 
 method adopted in such cases is as follows : — 
 
 (1) Placed in picro-sulphuric acid 3-4 minutes. 
 
 (2) Transferred to boiling hot water for a short time. 
 
 (3) Placed in water, and a little ammonia added ; this causes 
 the contracted object to swell up to its original size and form. 
 
 (4) Neutralize the ammonia with a little acetic acid, and then 
 
 (5) Color with borax-carmine or ammonia-carmine. 
 
 (6) Wash and examine in dilute glycerine. 
 
 The picro-sulphuric acid destroys the nutritive material; the 
 ammonia dissolves any particles of fat that may be present ; 
 and thus the object becomes transparent so far as possible. 
 
 A concentrated solution of corrosive sublimate may also be 
 used with success for killing Protozoa ; but care must be taken 
 to wash thoroughly. 
 
 Dr. Brass has obtained his best results without reagents or 
 dyes. 
 
 BLANC'S METHOD OF PRESERVING AND STAINING PROTOZOA. 
 
 For preserving these small organisms, various reagents have 
 been recommended. Certes 1 and Landsberg 2 employ osmic 
 acid ; Korschelt," chromic acid or osmic acid ; and Entz, 4 Klein- 
 enberg's picro-sulphuric acid. 
 
 Finally, Blanc 6 recommends the following very dilute picro- 
 sulphuric solution : — 
 
 Picric acid (saturated solution in dist. water) . . 100 vol. 
 
 Sulphuric acid (concentrated) 2 " 
 
 Distilled water 600 " 
 
 To this solution, which may be employed as it is for the 
 larva? of Echinoderms, Medusae, and Sponges, a little acetic acid 
 
 1 Compt. Rend. Acad. Sc, Paris, t. 88. 2 Zool. Anzeiger, No. 114. 
 
 8 Zool. Anzeiger, No. 109. * Zool. Anzeiger, No. 96. 
 
 6 Zool. Anzeiger, No. 129. 
 
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202 MICROSCOPICAL METHODS. 
 
 (1 %) is added for Rhizopods and Infusoria — two or three 
 drops for 15 grams of the solution. The acetic acid is 
 added in order to sharpen the outlines of the nuclei and 
 nucleoli. 
 
 This liquid is preferable to osmic acid, because it does not 
 render the objects non-receptive to staining fluids. 
 
 The entire process of hardening, washing, staining, and 
 mounting can be more expeditiously performed under the cover- 
 glass than otherwise. The acid is allowed to work until the 
 objects have become thoroughly yellow. The acid is then 
 replaced by 80 % alcohol, frequently renewed until the yellow 
 color entirely disappears ; 96 % alcohol is next used, and then 
 absolute alcohol. 
 
 The hardened objects may be stained with picro-carmine, or, 
 better, with an alcoholic solution of safranin. Five grams of 
 safranin are dissolved in 15 grams of absolute alcohol; the solu- 
 tion left standing a few days, then filtered, and diluted with half 
 its volume of distilled water. 
 
 This solution of safranin is preferable to picro-carmine, be- 
 cause it colors more quickly, and because one can so regulate 
 its action as to give a sharp definition to the protoplasm or the 
 nucleus. 
 
 After the object has been more or less deeply stained, accord- 
 ing to the end in view, it is washed in 80 % alcohol, which is 
 renewed until a moment arrives when no visible clouds of color 
 appear ; at this moment the 80 fo alcohol is replaced with abso- 
 lute alcohol, and this by clove oil. 
 
 As safranin is soluble in alcohol, the process of washing will 
 of course remove or weaken the color; but decoloration is 
 gradual, so that one needs only to watch and apply the clove 
 oil when the color has been reduced to the desired intensity. 
 This process, then, consists in overstating and then 
 removing the color to any desired degree. The process of 
 decoloration is not entirely arrested by the application 
 of clove oil, contrary to Blanc's assertion, hence it should 
 be replaced by Canada balsam as early as possible. The 
 same method is adapted to other microscopic animals. 
 
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HISTOLOGICAL METHODS. 
 
 203 
 
 METHODS OF PREPARING THE ALCYONARIA 
 Wilson 1 has employed with success the following methods 
 in preparing the Alcyonaria for histological purposes : — 
 
 " After testing many preservative and staining fluids, the fol- 
 lowing methods were adopted as giving, upon the whole, the 
 best results : The animals were suddenly killed by momentary 
 immersion in a mixture of one part strong acetic acid and two 
 parts of a concentrated solution of corrosive sublimate in fresh 
 water. After being quickly washed, they were transferred to 
 a concentrated solution of sublimate in fresh water, and left 
 two or three hours, the internal cavities being injected with the 
 solution where this was possible. They were then thoroughly 
 washed in running sea-water, then in distilled water, and finally 
 preserved in successive grades of alcohol. 
 
 "A weak solution of iodine in alcohol and sea- water also 
 gives beautiful results, but is less certain in its action. For 
 staining, I have used Grenacher's alum-carmine, borax-carmine, 
 picro-carmine, and Kleinenberg's haematoxylin. Much the best 
 results are obtained by the use of alum-carmine ; but it must be 
 used as quickly as possible, since the gelatinous tissue of the 
 mesoderm is apt to shrink if the object be left too long in 
 aqueous fluids. The tissues were decalcified with very weak 
 nitric or hydrochloric acid in 90 % alcohol. For maceration, 
 the Hertwig's well-known mixture of osmic and acetic acid 
 gives good results." 
 
 DR. ANDRES' METHODS OF TREATING ACTINLE. 
 
 Among the various methods employed by Andres in killing 
 the Actinias, the three following, given in the order of their ex- 
 cellence, are said to have worked most satisfactorily : — 
 
 A. Corrosive sublimate. — With small animals, a hot solu- 
 tion, used in the manner recommended by Lang, gives good 
 results ; with larger animals, where this mode of treatment 
 fails, the fluid must be injected. The cannula of a glass syringe, 
 filled with the hot fluid, is inserted into the mouth at the mo 
 
 1 Mitth. a. d. ZoSL Station zu Neapel, V., p. 3, 1884. 
 
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204 MICROSCOPICAL METHODS. 
 
 ment it opens, which act habitually follows on gently touching 
 the lip. After injecting, the hot solution is poured into the 
 glass containing the animal and a small quantity of sea-water. 1 
 
 If the operation is cleverly performed, the animal remains 
 fully expanded, as the mechanical pressure of the injected fluid 
 prevents contraction. 
 
 After from five to fifteen minutes, the animal is washed in 
 distilled water, and allowed to remain twelve hours in 50 % alco- 
 hol, 1 then passed through the higher grades of alcohol. Borax- 
 carmine and haBmatoxylin used for staining. 
 
 B. Glycerine and Alcohol. 8 — 
 
 Glycerine 20 parts 
 
 Alcohol (10 fe) 40 " 
 
 Sea water 40 " 
 
 This mixture, poured very slowly into the containing glass, 
 often gives very good results, both for anatomical and histo- 
 logical purposes. 
 
 C. Nicotine and Tobacco Smoke. — a. A solution of 
 nicotine (1 g.) in sea water (1 1.), conducted into the vessel 
 containing the animal fully expanded in a half litre of sea-water, 
 by means of a thread sufficiently large to empty the flask hold- 
 ing the nicotine solution in the course of twelve hours. 
 
 b. The vessel containing the animal in an extended condition, 
 covered by a bell-jar in which tobacco smoke is confined, until 
 the animal becomes completely benumbed. 
 
 After being deprived of sensibility by either of these meth- 
 ods, the creature may be killed in corrosive sublimate or in 
 pier o-sulph uric acid. 
 
 D. Andres finds that in the use of chloroform, dropped 
 slowly into the water, or administered in form of vapor, macera- 
 tion usually sets in before the power of contracting is lost. 
 Good preparations of the internal parts may be obtained by 
 injecting a weak solution of osmic acid. The method of freez- 
 
 1 Andres. "Intorno all'Edwardsia Claparedii," in the Proceedings of 
 the "Keale Accademia dei Lincei," Vol. V., Ser. 3, Mar. 7, 1880, p. 9. 
 
 3 A little camphor (1:100) added to the alcohol will facilitate the re- 
 moval of the sublimate. 
 
 8 This method originated with Salvatore Lobianco. 
 
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HISTOLOGICAL METHODS. 205 
 
 ing has also been employed with some success. For this pur- 
 pose, three vessels are placed one within the other, the central 
 one containing the Actinia, the middle one ice and salt, and the 
 outer one cotton. 
 
 The ice containing the congealed animal is dissolved in alco- 
 hol or an acid. 
 
 E. Maceration. — It is often important to see the cells of a 
 tissue in situ before freeing them with needles. In such cases 
 Dr. Andres proceeds as follows : — 
 
 1. Killed with corrosive sublimate. 
 
 2. Left in 25 % alcohol twenty-four hours. 
 
 3. Soaked for a short time in a very thin solution of gum 
 arabic, then in a somewhat thicker solution, and finally im- 
 bedded in a very thick solution. 
 
 4. Hardened in 90 f„ alcohol. 
 
 5. Thick sections prepared for dissection with needles. The 
 sections are placed on a slide in water, which dissolves the gum. 
 
 PREPARATION OF THE ECHINORHYNCHI.! 
 
 It is a very difficult matter to kill Echinorhynchi instantly. 
 This cannot be done either with corrosive sublimate or strong 
 osmic acid, even after preliminary treatment with tobacco 
 6moke or chloroform. Thus treated, they contract strongly, 
 and remain so after death. 
 
 Much the best results are obtained by killing gradually with 
 ■jijy fc osmic acid, in which they contract during the first hours, 
 but stretch out again and die fully extended. This method 
 causes slight swelling, but does not seriously injure the object 
 for histological investigation. In specimens left for twenty- 
 four hours in the osmic acid, it is easy to isolate under the 
 dissecting microscope the subcuticula, and the two layers of 
 muscle fibres (circular and longitudinal). For the study 
 of the internal organs, the Echinorhynchi should be cut 
 open immediately after death and transferred to a T J 5 % solu- 
 tion of osmic acid. The preservation of specimens thus treated 
 may be accomplished in the following manner : after carefully 
 
 1 A. Ssefftigen, Morph. Jahrb., x., Heft 1, pp. 120-163, 1884. 
 
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206 MICROSCOPICAL METHODS. 
 
 washing away the osmio acid, place the objects in a very dilute 
 solution of potassic acetate in an open vessel, and leave them 
 for two or three days, during which much of the solution evap- 
 orates. Finally transfer to a saturated solution in order to 
 clarify so far as possible. Very beautiful preparations are said 
 to be thus obtained. 
 
 The course of the nerves may be easily traced in specimens 
 that have lain several days in 1^ formic acid. The tissues 
 swell up strongly and become quite transparent so that the 
 nerves can be seen. If the muscular layers be separated from 
 the subcuticula in specimens thus treated, and then stained in 
 gold chloride, the lateral nerve trunks may be clearly shown. 
 For the histological study of the nerves, the Echinorhynchi 
 should be treated with chromic acid and then stained deeply 
 with borax-carmine. 
 
 Chromic acid preparations are also best for the study of the 
 subcuticula. Echinorhynchi live for clays in a ^% solution of 
 chromic acid, but eventually die in a fully extended condition. 
 Such preparations, after treatment with alcohol, maybe colored 
 at once ; or, after washing a day or more in running water, ex- 
 posed to the action of osmic acid, and then colored in borax- 
 carmine. 
 
 For the study of the sexual organs, a very dilute picro- 
 sulphuric acid (1 part of the acid to 8-10 parts of water) is rec- 
 ommended. 
 
 The tissues of the Echinorhynchi are not easily stained. 
 Borax-carmine, which, according to Ssefftigen, is the best stain- 
 ing fluid, must be allowed to act a long time (often one or 
 more days) ; after a deep stain has been taken, the preparation 
 should be partially decolored by the use of hydrochloric acid 
 in the ordinary way. 
 
 METHODS OF STUDYING THE SO-CALLED LIVER OF THE 
 CRUSTACEAN 
 
 For the study of fresh tissues, Dr. Frenzel places a small 
 piece of the organ on the slide, in the blood of the individual 
 from which it was taken ; or, in sea-water diluted until the 
 
 2 Johannes Frenzel. Mittheil. a. d. Zool. Station, v., p. 51, 1884. 
 
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HISTOLOGICAL METHODS. 207 
 
 salt contained amounts to about l£- 2% (1 part distilled water 
 and 1 part sea-water from the Bay of Naples). The so-called 
 "physiological salt solution" (f %) worked unfavorably, causing 
 maceration. 
 
 Various fluids were employed for killing and hardening, 
 partly for determining the effect of different reagents on the 
 nuclei and the protoplasm, and partly for finding the best 
 means of preparing the object for sectioning. 
 
 Very good preparations were obtained with warm alcohol 
 from 1Q% to 90$ ; while direct immersion in absolute alcohol 
 did not pro've advantageous. This treatment gave good results 
 for the cell protoplasm, but destroyed the structure of the 
 nuclei. Still better results were obtained for the cells (not for 
 the nuclei) by adding a few drops of iodine to 70% alcohol; 
 
 The most satisfactory results were reached by immersing 
 the object in a saturated aqueous solution of corrosive subli- 
 mate from 10-30 minutes, then washing with water, and finally 
 replacing the water gradually with alcohol. 
 
 Perenyi's fluid gave best results when combined with corro- 
 sive sublimate. The object was left from 5-10 minutes in the 
 first-named fluid, then transferred to the second and left for the 
 same time. 
 
 While these methods were good for the Decapods, Amphi- 
 pods, and Phronimidee, the Isopods required a different treat- 
 ment. "With these, Kleinenberg's picro-sulphuric acid, diluted 
 with an equal volume of water, and allowed to act 15-20 min- 
 utes, gave much better preparations than the sublimate solu- 
 tion. 
 
 A METHOD OF SILVER STAINING FOR MARINE OBJECTS.! 
 
 "The principle of this method was suggested to me by Dr. 
 W. H. Ransom, and consists in the replacement of distilled 
 water in the ordinary process of silver staining by a solution of 
 a neutral salt not precipitable by silver nitrate, and of the same 
 specific gravity as sea-water. Loxosoma and JPedicellina were 
 the first objects investigated, and these animals are not killed 
 
 1 S. F. Hanner. Mitth. d. zool. Station z. Neapel, v., p. 445, Nov., 1884. 
 
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208 MICROSCOPICAL METHODS. 
 
 by an exposure of as much as half an hour to a 5 % solution of 
 potassic nitrate in distilled water. It is thus quite easy to free 
 the tissues from the greater part of their chlorides by washing 
 with the above-mentioned solution of potassic nitrate; from 
 this, the objects are transferred (naturally without the forma- 
 tion of any precipitate) for 4 or 5 minutes to a solution of 
 silver nitrate (£-1%, according to circumstances). After re- 
 duction of the silver during exposure to light in the nitrate 
 solution, the tissues may be mounted permanently either in 
 glycerine or in Canada balsam. Very beautiful preparations of 
 Xioxosoma were easily obtained by the use of osmic acid and 
 picro-carmine after treatment with silver nitrate. The animal 
 may be transferred directly from the silver solution to osmic 
 acid (i %) and thence to picro-carmine, reduction taking place 
 during the process, or the osmic acid may be added after the 
 silver has been already reduced in the potassic nitrate. In 
 successful preparations made in the above manner, the limits of 
 all the cells of the epidermis and of the alimentary canal are 
 exceedingly sharply marked out, the nuclei of these cells, as 
 well as of the muscle cells, connective tissue corpuscles and other 
 tissue elements being very distinctly stained. Besides the 
 above-mentioned forms, I have obtained good results with the 
 epidermis of Meditsce, Hydroids, Saxitta and Appendicularia 
 (tail). 
 
 " Amongst sponges, Dr. G. C. J. Vosmaer has, by means of 
 the above method, easily demonstrated an outer epithelium in 
 Chondrosia, where F. E. Shulze first has been unable to detect 
 a cell layer of this kind even by the use of silver nitrate ; * in 
 Thenea where Sollas 2 has observed no cell outlines, and in 
 many other sponges. Dr. Ed. Meyer has successfully applied 
 the same method to the epidermis and peritoneal epithelium of 
 Annelids (Tomopteris, Amphictenidae), to teleostean ova, etc. 
 He has obtained good nuclear coloration by transferring the 
 tissue after reduction in potassic nitrate to alcohol, and subse- 
 quently staining with Paul Mayer's alcoholic carmine. 3 In 
 
 i Zeitschr. f. wiss. Zool. XXIX., p. 107, and XXXI., p. 290. 
 
 2 Ann. and Mag. of Nat. Hist., 6th Series, IX., p. 445. 
 
 3 Mitth. d. Zoolog. Stat, zu Neapel. IV., p. 521. 
 
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HISTOLOGICAL METHODS. 209 
 
 Brachiopoda, Dr. J. F. Van Bemmelin has had no difficulty 
 in using the method for the investigation of the epidermis and 
 peritoneal epithelium. 
 
 " Few animals seem to resist the action of potassic nitrate to 
 so great an extent as Loxosoma and Pedicettina, most forms 
 being either immediately, or after a few minutes, killed by an 
 immersion in a 5f solution of this substance. Even in many 
 of these cases, the tissues suffer very little histological change, 
 and can be easily stained by silver nitrate. It is possible that 
 many other salts may be used more advantageously than po- 
 tassic nitrate in washing the chlorides from the tissues without 
 killing the animal. A 4£% solution of sodic sulphate may be 
 used instead of the potassic nitrate, over which, however, in 
 most cases it has no obvious advantages. 
 
 " R. Hertwig 1 has described a method for the silver staining 
 of marine animals, consisting in treating with dilute osmic 
 acid, washing with distilled water, until the chlorides are 
 removed, subsequently placing in a silver nitrate solution, and 
 reducing in distilled water. By this method, Hertwig has been 
 able to obtain sharply defined cell limits in the ectoderm of 
 Ctenophora, but not over the whole surface of the epithelium. 
 I have myself employed Hertwig's method in Loxosoma and 
 Pedicellina, but with results not nearly so satisfactory as in 
 the method I have described, the limits of the epidermic cells 
 being not visible over the whole surface, whilst after the em- 
 ployment of Dr. Ransom's method in these two genera, I have 
 invariably found that every epidermic cell was distinctly 
 marked out by a sharp contour." 
 
 THE DEMONSTRATION OF CELLS IN THE CORPUS VITREUM 
 OF CYPRINOID FISHES. 
 The vitreous body of the eye of the fish is, in many 
 species, according to H. Virchow, 2 entirely destitute of cells ; 
 while in others its whole surface is covered with them. The 
 form of these cells varies for different species, the peculiari- 
 
 1 Jenaische Zeitschr. XIV., p. 324. 
 
 2 Arch. f. mik. Anat., XXIV., H. 2, pp. 106-108, Oct., 1884. 
 
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210 MICROSCOPICAL METHODS. 
 
 ties being sufficiently well marked to serve as a means of dis- 
 tinguishing groups, families, and even genera and species. 
 
 In order to study the vessels and cells of the vitreous humor, 
 it is necessary to make a preparation of a portion of the periph- 
 eral zone ("Grenzhaut "), free from surrounding tissues. 
 Place the eye, freed from the sclerotic and choroid investments, 
 (1) in a 1 fo solution of corrosive sublimate, heated to about 
 30° C, and leave it here 7 hours, the solution meanwhile being 
 allowed to cool ; or (2) in Mtlller's fluid for 24 hours. Either 
 method of treatment will secure, in most cases, the separation 
 of the retina from the vitreous body, which is the first impor- 
 tant step in the preparation. The advantages of the first 
 method are that it hardens just enough to render further prepa- 
 ration easy, and preserves well the color of the tissues ; but it 
 produces shrinkage in the nuclei. Miiller's fluid is a better pre- 
 servative for the nuclei, but hardens less, and injures the clear- 
 ness of the color. 
 
 The process of staining will be most successful if undertaken 
 immediately after washing in distilled water — before hardening 
 in alcohol. For this purpose the preparation should be left in 
 a weak solution of hematoxylin 24 hours, washed for an hour 
 or more in a \ % solution of alum, and then placed in Ogatha's 
 solution of eosin 12 hours (eosin + 60 alcohol + 140 water.) 1 
 
 After the preparation is stained, it may be washed in abso- 
 lute alcohol, and then examined in glycerine (?). 
 
 Good preparations for showing the distribution of the cells 
 and blood vessels may be obtained in the following manner : — 
 
 After treatment with corrosive sublimate, cut off the hinder 
 portion of the preparation, and remove the retina ; then, by the 
 aid of two p>airs of forceps, separate a piece of the superficial 
 layer of the vitreous body from the remaining portion, and 
 place it (external side clown) on a slide ; after placing the cover- 
 glass, press it lightly, and then put slide and all into a dish of 
 alcohol. After some hours, the cover-glass, with the prepara- 
 tion adhering to it, may be loosened from the slide, and the 
 remaining steps of staining, washing, anhydrating, clarifying, 
 and mounting in balsam accomplished without difficulty. 
 
 i Arch. f. Anat. u. Phys. Abth., 1883, p. 409. 
 
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HISTOLOGICAL METHODS. 211 
 
 THE MUCOUS MEMBRANE OF THE STOMACH 
 
 In dealing with this subject, Nikolai Trinkler 1 has improved 
 considerably the methods of preparation hitherto employed. 
 
 1. As soon as the animal is dead, the stomach, with a portion 
 of the oesophagus and duodenum, is cut out very quickly, and 
 opened along the large curvature. The surface of the mucous 
 membrane is first tested in different places with litmus paper, 
 and then washed with a stream of distilled water. 
 
 2. In animals with a thick-walled stomach, the external mus- 
 cular layer is separated, and then the mucous membrane cut 
 into small pieces, some of which are placed in hardening fluids, 
 while others are placed in some indifferent fluid (humor aquaeus, 
 iodized serum, £ % NaCl) for examination in fresh condition. 
 In order to prevent putrefaction, in the latter case, they may be 
 kept in an atmosphere of water vapor and carbolic acid. 
 
 For Hardening. — Milller's fluid (1 or 2 days) is the best 
 agent. Preparations hardened in chromic (£-1 % ) or picric acid 
 should not be transferred directly to alcohol, but first left for a 
 day or moi-e in Miiller's fluid, then treated with alcohol of suc- 
 cessively higher grades. 
 
 Preparations with osmic acid (tV-J %) should be washed a 
 long time in distilled water, then placed in a saturated solution 
 of potassic acetate, and finally examined in glycerine. 
 
 For Maceration of the epithelium and glandular elements, 
 Miiller's fluid was used, either alone or in combination with a 
 weak solution of NaCl, as employed by Kutschin. Alcohol 
 (33^ %), chloral hydrate (5 % ), chromate of ammonia, salt solu- 
 tion ("35 %), and a mixture of 1 vol. chromic acid (^ %), 1 vol. 
 chloral hydrate (5 %), and a few drops of acetic acid, were also 
 employed for the same purpose. This mixture and that of 
 Kutschin give excellent results. 
 
 In Staining, Heidenhain's method 2 of using weak dyes and 
 allowing them to act a long time may be followed, as a gen- 
 eral rule. Litmus and chlorophyll were also used as dyes. A 
 
 1 Arch. f. mikr. Anat., XXIV., Oct., 1884, pp. 174-176. 
 
 2 Arch. f. mikr. Anat., VI., p. 402. 
 
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212 MICROSCOPICAL METHODS. 
 
 strong solution of litmus, of absolutely neutral reaction, must 
 be employed, and this serves both as a staining and as a micro- 
 chemical agent. Chlorophyll was obtained by soaking the 
 leaves of Syringa vulgaris in strong alcohol 24 hours ; the dark 
 green fluid thus obtained was filtered, and then evaporated. 
 The dry residue was dissolved in a little distilled water, and 
 again filtered. 
 
 Following in the line of Heidenhain and Rollett, the different 
 functional conditions of the glands, induced by feeding, starv- 
 ing, etc., were investigated; and more extreme conditions were 
 reached by feeding with phosphor and alcohol. 
 
 THE CONTINUITY OF PROTOPLASM IN VEGETABLE STRUCTURES. 
 
 W. Gardiner 1 gives the following methods of detecting pro- 
 toplasmic threads, which pass from cell to cell: — 
 
 Chlor-Zinc-Iod Method. — 1. Sections of endosperm are 
 first stained with iodine and mounted in chlor-zinc-iod, in which 
 they remain about 12 hours. 
 
 2. After washing well, stain with picric Hoffmann's blue 
 (prepared by dissolving the dye in 50 fo alcohol, which has been 
 saturated with picric acid). 
 
 3. Wash again, and mount in glycerine ; or, after treating 
 with the usual grades of alcohol, and with clove oil, mount in 
 balsam. 
 
 In cases where the tissue swells quickly under the action of chlor-zinc- 
 iod, the exposure need not be so prolonged ; and excessive swelling must 
 be prevented by the use of alcoholic iodine at the outset, and by washing 
 with alcohol instead of water. 
 
 As to the time of action, the manipulation of the reagents must be 
 varied to suit the requirements of different kinds of tissue. 
 
 Molybdic Acid Test. — If a section of some living endo- 
 sperm is treated with a solution of molybdic acid in strong 
 sulphuric acid, the cell-wall will swell up, and the threads 
 which traverse it will soon assume a blue color, while the main 
 mass of protoplasm becomes intensely blue. The cell-wall 
 itself remains uncolored. This very delicate reaction demon- 
 strates the protoplasmic nature of the threads. 
 
 1 Arbeit. Bot. Inst. Wurzburg, III., pp. 53-60, 1884. Cf. Journ. Eoy. 
 Micr. Soc, IV., Part. 5, pp. 637-641, 1884. 
 
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CHAPTER XIII. 
 
 RECONSTRUCTION FROM SECTION'S. 
 
 ORIENTATION IN MICEOTOMIC SECTIONS. 
 
 If an object has been cut into serial sections and mounted, 
 the value of the series for microscopical investigation will 
 depend not only on the success with which each step in 
 the preparation has been attended, but also on our ability to 
 grasp all the topographical relations of each section. It is not 
 enough to know the region through which a section passes ; we 
 must have the means of ascertaining to within a very small 
 fraction of a millimeter the exact path of the knife. Such pre- 
 cise orientation can only be arrived at in an indirect way ; but 
 the improved instruments and methods of section- cutting make 
 its attainment a no very difficult task. To determine the locus 
 of sections with accuracy, several conditions must be fulfilled. 
 The sections must be made of uniform thickness, arranged in 
 serial order, and all similarly disposed. With these conditions 
 satisfied, the plane of section determined, and an accurate sur- 
 face view of the object obtained prior to imbedding, it becomes 
 an extremely simple matter to know what portion of the surface 
 view is represented by any given section. The following data 
 will furnish an illustration : — 
 
 Blastoderm of the chick, 5 mm long. 
 
 Surface view magnified 20 diameters. 
 
 Thickness of each section ,05 mm . 
 
 Plane of section at right angles to the long axis of the blastoderm. 
 
 From these data we know that there should be just 100 sec- 
 tions, and that each section must correspond to l ram of the sur- 
 face view. 
 
 213 
 
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214 MICROSCOPICAL METHODS. 
 
 Now if we draw a line at one side of the surface view, paral- 
 lel to, and of equal length with, its long axis, and divide this 
 line into 100 equal parts, the number of the section will corre- 
 spond to the same number on the scale, and the exact position 
 of the section be recognized at a glance. 
 
 Of the conditions above named as essential to an exact 
 knowledge of the locus of any given section, the only one likely 
 to present any serious difficulties is that of obtaining sections 
 of uniform thickness. Where the object-carrier and vernier 
 are combined, and moved directly by the hand, it is extremely 
 difficult, if not impossible, to obtain that degree of uniformity 
 required for exact topographical study. In the best microtomes 
 now in use, the carrier is moved only indirectly by the hand, 
 through a micrometer screw, and its movements are thus brought 
 under perfect control. 
 
 THE RECONSTRUCTION OF OBJECTS FROM SECTIONS. 
 
 The importance of attending to all available means of orien- 
 tation will be best understood by those who know how to make 
 use of sections in the reconstruction of objects or parts of 
 objects. Suppose the only material at the disposal of an inves- 
 tigator to be a single small object, and that the rarity of the 
 object renders its replacement extremely improbable. How 
 shall the object be treated in order that the most exhaustive 
 knowledge of all the details of its inner structure may be 
 obtained? One might be tempted to lock it up as a cabinet 
 rarity, if he did not know how to make a single series of sec- 
 tions tell the whole story. If the preliminary steps have been 
 correctly taken, it is possible to construct from serial transverse 
 sections a median sagittal (longitudinal and vertical), or frontal 
 section, or a section in any desired plane. From the same 
 series may be constructed also surface views of internal organs, 
 which are inaccessible to, or unmanageable by, any of the ordi- 
 nary methods of dissection. 
 
 It frequently happens that sections can be obtained by con- 
 struction that could not be obtained by any direct means. For 
 example, we may desire a frontal section of a vertebrate em- 
 bryo that will show all the parts that lie in the same level with 
 
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RECONSTRUCTION FROM SECTIONS. 215 
 
 the chorda, or a sagittal section that will represent a median 
 plane. It is evident that no such sections can be directly 
 obtained, owing to the axial curvature of the embryo ; but they 
 can easily be constructed from transverse sections. It is here 
 that we see some of the great advantages to be derived from 
 the use of the microtome. It not only overcomes the opacity 
 of objects, but it also enables us to represent curved and twisted 
 surfaces in plane surfaces. The ability to construct sections at 
 right angles to the actual planes of section is the key to the 
 next and final step, — " the plastic synthesis " of the sectioned 
 object. 
 
 METHOD OF RECONSTRUCTION. 
 
 Professor His was the first to make known a method of recon- 
 struction. 1 Others have since made use of the same method 
 for different purposes. A. Seessel, a former pupil of Professor 
 His, employed it in a work on the development of the fore-gut. 2 
 Rosenberg made use of it in the construction of frontal views 
 of the sacrum ; 8 and Krieger, in the investigation of the central 
 nerve-system of the crayfish. 4 The method is well illustrated 
 by two figures (11 and 12, PI. XXXI.), given by Krieger; and 
 these figures are well worth examination, as they show how to 
 proceed when the plane of section is not quite at right angles 
 to the axis of the object. Professor His has also constructed 
 frontal and profile (sagittal) views of the human embryo by 
 the same method, and has explained the process in Part I., p. 10, 
 of his " Anatomie menschlichen Embryonen." 
 
 For an illustration, we will take the data given under the 
 head of orientation, and indicate how a surface view could be 
 constructed from a series of transverse sections of the germinal 
 disc of the chick. We should first draw 100 parallel zones on 
 
 1 His. " Untersuchungen ii. d. erste Anlage des Wirbelthierleibes," 
 p. 182, 1868. " Neue Untersuchungen ii. d. Bildung des Hiihnerembryo, 
 in Arch. f. Anat. u. Physiol., anat. Abth.," p. 122, 1877. 
 
 2 Seessel. " Arch. f. Anat. u. Physiol., anat., Abth.," p. 449, 1877. 
 8 Rosenberg. Morph. Jahrb. "Vol. I., p. 108, 1875. 
 
 * Krieger. Zeitschrift f. wiss. Zool. Vol. XXXIIL, p. 531, 1880, and 
 Zool. Anzeiger, p. 369, 1878. 
 
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216 MICROSCOPICAL METHODS. 
 
 a sheet of paper, each zone corresponding in thickness to a 
 single section (l mm ). 
 
 A median line would then be drawn at right angles to these 
 zones ; this line would represent the length of the disc magni- 
 fied 20 diameters (100 mm ). We should next make an outline 
 drawing of the first section enlarged the same number of diame- 
 ters as before. The width of this drawing and its parts (primi- 
 tive streak, embryonic rim, etc.), could then be indicated in 
 the first zone by dots placed at the proper distance on the 
 right and left side of the median line. The dots for each suc- 
 ceeding section having been placed in their corresponding 
 zones, nothing further would remain to be done, except to 
 connect the dots of corresponding parts in the several zones, 
 and shade according to the requirements of the case. 
 
 If the plane of section is not quite perpendicular to the axis 
 of the object, one has only to determine the angle which the 
 axis makes with the plane of section, and draw the median line 
 so that it forms the same angle with the parallel zones. Such 
 a case has been clearly illustrated by Krieger. 
 
 In the construction of sagittal sections, a profile line (dorsal 
 line, etc.), will serve as the ground line. 
 
 BOEN'S METHOD OF RECONSTRUCTING OBJECTS FROM 
 MICROSCOPIC SECTIONS.! 
 
 Dr. G. Born describes in detail a very ingenious method of 
 constructing models of objects from serial sections. By the 
 aid of the camera, the outlines of the sections are transferred 
 to wax plates, which are then cut out so as to correspond in out- 
 lines as well as dimensions to the sections equally magnified in 
 all three directions. "With plates thus prepared, it is only 
 necessary to put them together in the proper order to obtain a 
 complete model. The method is simple and extremely useful, 
 especially in investigating objects with complex internal cavi- 
 ties. 
 
 Born has made use of the method in studying different parts 
 
 1 G. Born. "Die Plattenmodellirmethode." Archiv f. mik. Anat. 
 XXII., p. 584, 1883. First described in Morphol. Jahrbuch, II., p. 578, 
 1876. 
 
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EECONSTEUCTION FROM SECTIONS. 217 
 
 of the vertebrate head ; Swirski, in elucidating the develop- 
 ment of the shoulder-girdle of the pike ; Stohr, in tracing the 
 development of the skull of Amphibia and Teleostei ; and TTskow 
 in studying the development of the body cavity, the dia- 
 phragm, etc. 
 
 An Illustration of the Method. — Born makes use of 
 three rectangular tin boxes of equal size, each measuring 
 270 mm X230 mm X2£ mm . Sections should be made about 2 y" m 
 thick (never thinner than ^jy mm ). If we desire to construct a 
 model of an object from serial sections ^u mm thick, which shall 
 be magnified 60 diameters, then the wax plates must be made 
 60 times as thick as the sections, i.e., 2 mm thick. 
 
 The surface of a plate that could be made in a box of the 
 above named dimensions contains 62.100D mm ; and the vol- 
 ume of such a plate 2 ram thick would therefore be 124.2 ccm . The 
 specific gravity of common raw beeswax amounts to .96-97. 
 For use, it requires only to be melted and a little turpentine 
 added to make it more flexible. Thus prepared, its specific 
 gravity is about .95 ; and this number has been found suffi- 
 ciently accurate in all cases. The weight of the wax required 
 to make one plate of the above size, will, accordingly, be 
 117.99 gr., or, in round numbers, 118 gr. The wax having 
 been weighed and melted, the tin box is first filled l£ em deep 
 with boiling water, and then the melted wax poured upon the 
 water. If the water and the wax are quite hot, the wax will gen- 
 erally spread evenly over the surface ; if gaps remain, they can 
 be filled out by the aid of a glass slide drawn over the wax. As 
 soon as the plate has stiffened, and while it is still soft, it is 
 well to cut it free from the walls of the tin box, as further 
 cooling of the water and the box might cause it to split. By 
 the time the water becomes tepid, the plate can be removed 
 from the water to some flat support, and left till completely 
 stiffened. Half a hundred plates may thus be prepared in the 
 course of a few hours. 
 
 The outlines of the section are transferred to the plate in the 
 following manner : A piece of blue paper is placed on the plate 
 with the blue side turned toward the wax, and above this is 
 placed a sheet of ordinary drawing paper. The outlines are 
 
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218 MICROSCOPICAL METHODS. 
 
 drawn on the latter by the aid of a camera, and, at the same 
 time, blue outlines are traced on the wax plate. The plate can 
 then be laid on soft wood and cut out by the aid of a small knife. 
 Thus a drawing and a model of each section are prepared. 
 The plates thus prepared can be put together in the proper 
 order, and fastened by the aid of a hot spatula applied to the 
 edges. 
 
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APPENDIX. 
 
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APPENDIX. 
 
 METHODS OF INJECTION. 
 
 THE PREPARATION OF DRY INJECTION MASSES. 1 
 
 The variously colored gelatine emulsions in common use as 
 injections keep for only a short time, and have therefore to be 
 prepared as occasion arises for their use. The dry emulsions 
 recommended by Fol are very easily prepared and convenient 
 in use. As they will keep for any length of time, they can be 
 prepared in quantities, and thus be ready for use at any moment. 
 
 Carmine Emulsion. — One kilogram gelatine (softer kind 
 used in photography) soaked in water for a few hours until 
 thoroughly softened ; after turning off the water, heat the gela- 
 tine over a water-bath until liquified ; and then add to it, little 
 by little, one litre of a strong solution of carmine in ammonia. 2 
 The mixture, stiffened by cooling, is cut up, and the pieces 
 packed in a fine piece of netting. Vigorous pressure with the 
 hand under water forces the emulsion through the net in the 
 form of fine strings or vermicelli. These strings are placed in 
 a sieve, and washed until they are free from acid or excess of 
 ammonia, then collected, and re-dissolved by heating. The 
 liquid is poured upon large sheets of parchment which have 
 been saturated with paraffine, and these sheets are then hung up 
 to dry in an airy place. The dried layers of the emulsion, 
 which are easily separated from the parchment, may be cut into 
 
 1 Fol. Beitrage zur histologischen Technik. Zeitschr. f. wiss. Zool., 
 XXXVIII., p. 491, 1883. 
 
 2 Cf. Lehrbuch d. vergl. mik. Anat., p. 13, when a somewhat modified 
 process is given. 
 
 221 
 
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222 
 
 MICROSCOPICAL METHODS. 
 
 strips, and placed where they are protected from dust and 
 dampness. 
 
 The carmine solution used in this emulsion is prepared as 
 follows : — 
 
 A strong solution of ammonia is diluted with 3-4 volumes of 
 water, and carmine added in excess. After filtering, the solu- 
 tion is mixed with the gelatine, and then enough acetic acid 
 added to change the dark purple-red into blood-red. It is not 
 necessary to completely neutralize the ammonia. 
 
 The dry emulsion requires only to be placed in water for a few 
 minutes, and melted over the water-bath, to be ready for use. 
 
 Blue Emulsion. — A slightly modified form of Thiersch's 
 formula . — 
 
 1. To 300 ccm of melted gelatine add 120 ccm of a cold saturated 
 solution of green vitriol (ferro-sulphate). 
 
 2. To 600 ccm of melted gelatine add first 240 ccm of a saturated 
 solution of oxalic acid, then 240 ccm of a cold saturated solution 
 of red prussiate of potash (potassic ferricyanide). 
 
 3. No. 1 poured slowly into No. 2 while stirring vigorously ; 
 the mixture heated for fifteen minutes. 
 
 4. After cooling, the emulsion is pressed through netting, the 
 vermicelli washed and spread on waxed paper for drying. In 
 this case the vermicelli must be dried directly, as they do not 
 melt well without the addition of oxalic acid. 
 
 The dry vermicelli are prepared for use by first soaking in 
 cold water, and then heating with the addition of oxalic acid 
 enough to reduce them to a liquid. 
 
 Black Emulsion. — 1. Soak 500 g. gelatine in two litres of 
 water in which 140 g. of common salt have previously been 
 dissolved, and melt the mass on the water-bath. 
 
 2. Dissolve 300 g. nitrate of silver in 1 litre distilled water. 
 
 3. No. 2 poured very slowly into No. 1 while stirring. An 
 extremely fine-grained emulsion may be obtained by using 3-4 
 times as much water in Nos. 1 and 2. 
 
 4. No. 3 pressed into vermicelli, as above, and then mixed 
 with No. 5 by clear daylight. 
 
 5. Mix lj litres cold saturated potassic oxalate with 500 ccm of 
 a cold saturated solution of ferro-sulphate. 
 
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APPENDIX. 223 
 
 6. No. 4 mixed with! No. 5 gives a thoroughly black emul- 
 sion, which should be washed several hours, again melted, and 
 finally poured in a thin layer on waxed paper. 
 
 A gray-black emulsion may be obtained by using 240 g. 
 potassic bromide in the place of common salt in No. 1, the 
 remaining operations being the same. 
 
 A STARCH INJECTION MASS. 1 
 
 A coarse injection mass, which is cold-flowing, may be forced 
 nearly to the capillaries, rapidly hardens after injection, leaves 
 the vessels flexible, does not dull dissecting instruments, is suit- 
 able for permanent dry or alcoholic preparations, is simple in 
 its manipulation, cleanly and economical, seems to be fully 
 realized in the starch mass introduced by Ad. Pansch, of Kiel, 
 and since recommended, with various modifications, by Wiks- 
 zemski, Dalla Rossa, Meyer, and Browning. 2 
 
 As starch is insoluble in alcohol and cold water, it becomes 
 hard when injected into the blood vessels simply by the exuda- 
 tion of the liquid with which it is mixed. (That the starch 
 grains forming the mass remain entirely unchanged may be 
 easily demonstrated by making a microscopic examination of 
 the contents of an injected vessel.) 
 
 The mass originally recommended by Pansch consisted of 
 wheat flour and cold water, to which was added a sufficient 
 quantity of the desired coloring matter. Later experiments 
 have shown that pure starch is better than flour. 
 
 Mass for Ordinary Injections. — 
 
 Dry starch ("laundry" is good) 1vol. 
 
 2k % aqueous solution of chloral hydrate .... 1 " 
 
 95 % alcohol 3 i " 
 
 Color i " 
 
 1 S. H. Gage. New York Medical Journal, June, 1884. 
 
 2 See Ad. Pansch, "Archiv fur Anatomie und Entwickl.," 1877, pp. 
 480-482, and 1881, pp. 76-78; Wikszemski, same Journal, 1880, pp. 232- 
 234; Dalla Rossa, same, pp. 371-377; Herm. von Meyer, same, 1882, pp. 
 60, 61, and 1883, pp. 265, 266; Browning, " Annals of Anatomy and Sur- 
 gery," 1884, pp. 24, 25. 
 
 8 The chloral and alcohol prevent fermentation in the mass when it is 
 kept in stock; the alcohol also increases the fluidity, and likewise the 
 more rapid hardening in the vessels; both, of course, act as a preservative 
 upon the animal injected. 
 
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224 MICROSCOPICAL METHODS. 
 
 Since almost any animal injected may afford some organ 
 worth preserving, it seems better to employ permanent colors 
 for tinging the mass. Among those which are available, proba- 
 bly vermilion, red lead, ultramarine, chrome orange, yellow, or 
 green are preferable. 
 
 Preparation of the Color. — 
 
 Dry color 1 vol. 
 
 Glycerine 1 " 
 
 95 % alcohol 1 " 
 
 To avoid lumps, which would clog the cannulas, or small ves- 
 sels, the color is thoroughly ground with the liquid in a mortar. 
 It is stored in a well-stoppered bottle, and is prepared for use 
 simply by shaking. 
 
 Special Mass. — For the injection of brains, and, perhaps, 
 for other rapidly perishing specimens, it seems best, as sug- 
 gested by Professor "Wilder, to use strong preservatives in pre- 
 paring the mass : — 
 
 Corn starch (that used for food) lvol. 
 
 5 % aqueous solution of chloral hydrate .... -J- " 
 
 95 % alcohol f " 
 
 Color i " 
 
 For convenience and economy, a considerable quantity of 
 either of the masses described above may be prepared at once, 
 and kept in a wide-mouthed specimen or fruit jar. A smooth 
 stick in each jar is convenient for stirring the mass, which 
 should always be done just before using. The syringe may be 
 filled directly from the jar, and any mass remaining in the 
 syringe after the injection is finished may be returned to the 
 jar. 
 
 If it is desired to have the mass enter very fine vessels, some 
 of the stock mass, as given above, diluted with an equal volume 
 of water or chloral solution, may be injected first, and imme- 
 diately followed by the undiluted mass, or, for large animals, a 
 mass containing twice the usual amount of starch. In whatever 
 form the starch is used, it is necessary to work somewhat expe- 
 ditiously, because the exudation of the liquid in the smaller 
 vessels takes place so rapidly that Ihe mass hardens very quickly 
 in them. The larger the vessel, the more slowly, of course, do 
 
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APPENDIX. 225 
 
 the exudation and, consequently, the hardening, take place. It 
 sometimes happens that large vessels, like the aorta, are not 
 fully distended after the exudation of the liquid. In this case 
 some mass containing double the ordinary amount of starch can 
 be advantageously injected in two hours, or longer, after the 
 first injection. 
 
 Dry Preparations. — Finally, if vessels injected with the 
 starch mass are dissected free, soaked a day or two in Wicker- 
 sheimer's preservative, and then dried, they retain their form, 
 and, to a great degree, their flexibility. 
 
 CELLOIDIN INJECTIONS. 
 
 In the formation of injection masses, collodion plays still 
 another important role, for the discovery of which we are in- 
 debted to Schiefferdecker. 1 It can be made to penetrate easily 
 very fine blood-vessels ; and its viscosity protects them against 
 injury. Its application is simple and easy, and in all these 
 respects it is said to be superior to the masses hitherto employed 
 for "corrosion preparations." It has a slight shrinkage, but 
 not enough to form a serious drawback. It is prepared in dif- 
 ferent ways, according to the color to be given to the injection. 
 
 A. Asphalt-Celioi&in Injection. — 1. Pulverized asphalt 
 placed in a well-closed bottle of ether, and allowed to remain 24 
 hours, during which the mixture must be several times shaken. 
 
 2. The brown-colored ether is turned off, and small pieces of 
 celloidin dissolved in it until the solution flows like a thick oil. 2 
 
 B. Vesuvian-Celloidin Injection. — 1. Make a saturated 
 solution of Vesuvian in absolute alcohol. 
 
 2. Dissolve in this pieces of celloidin until the desired con- 
 sistency is reached. 
 
 The brown injection thus obtained is less satisfactory than 
 that formed from asphalt, as its color fades somewhat. 
 
 C. Opaque Celloidin Injections. — 1. Dissolve celloidin 
 in absolute alcohol and ether in equal parts. 
 
 2. Add vermilion or Prussian blue to color. 
 
 1 1. c, p. 201. 
 
 2 The pulverized asphalt can be used many times over for coloring the 
 ether, as very little of it will dissolve in 24 hours. 
 
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226 MICROSCOPICAL METHODS. 
 
 The coloring substance should be mixed with a small quan- 
 tity of absolute alcohol, and then reduced to great fineness by 
 continued trituration in a mortar. To the thick, paste-like mass 
 thus obtained, the solution of celloidin is next added. The 
 amount of coloring substance should be as little as possible, as 
 the mass will otherwise be too brittle. If a fine injection is 
 required, the mass should be filtered through flannel moistened 
 with ether. 
 
 The syringe employed must be entirely free from fatty sub- 
 stances, as these render the injection-mass brittle. If the piston 
 does not fit the syringe-tube sufficiently closely, it may be 
 wound with a little gauze. The cannula should be filled with 
 ether before it is inserted, and tied in place, and again filled 
 just before it is joined to the syringe. 
 
 In using a mass dissolved in alcohol and ether, it is well to 
 add a little ether, which will spread over the surface, and thus 
 prevent the formation of a film. The injection should be made 
 moderately quickly, as the mass stiffens soon after contact with 
 the tissues. After injection, the syringe and cannula should be 
 cleaned with ether. 
 
 The injected organ is placed in hydrochloric acid, diluted 
 more or less, according to the danger of shrinkage. It is left 
 in the acid, which is occasionally renewed, until the tissues are 
 sufficiently corroded to be easily washed away by a slow and 
 steady stream of water, conducted through rubber tubing con- 
 nected with a water pipe. The preparation may then be left 
 in water for some days or weeks, in order to free it .from 
 remaining fragments of tissue by gradual maceration. The 
 preparation, when finished, may be preserved either in glyce- 
 rine or a mixture of glycerine, alcohol, and water in equal 
 parts. 
 
 The asphalt-celloidin mass is the one most highly recom- 
 mended by Schiefferdecker. 
 
 OSBORNE'S METHOD OF INJECTING THE ARTERIES AND VEINS. 
 
 In this method two injecting fluids are employed, the first 
 having a density that will allow it to pass the capillaries easily, 
 while the second is of such a density that it will be arrested at 
 
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APPENDIX. 
 
 227 
 
 the capillaries. The whole vascular system may thus be in- 
 jected from the arterial bulb. 
 
 The method is employed in the following manner : — 
 
 1. The animal to be injected is immersed in tepid water, and 
 the heart laid bare. 
 
 2. The apex of the single ventricle, in the case of an am- 
 phibian, or of the left ventricle, in the case of higher animals, 
 is then widely opened, and the blood allowed to flow from the 
 auriculo-ventricular aperture. 
 
 3. The cannula is next inserted so that it reaches into the 
 arterial bulb, and two ligatures made at the points indicated by 
 1st L and 2d L. 
 
 4. When the body is thoroughly warmed, an ordinary red or 
 purple gelatine mass is slowly 
 injected. The second ligature 
 having been left loose, a quan- 
 tity of blood, gradually follow- 
 ed by the injecting mass, flows 
 from the auriculo-ventricular 
 opening. 
 
 5. As soon as the gelatine 
 mass runs quite clear, the sec- 
 ond ligature is fastened, and 
 the syringe replaced by another 
 containing a red plaster-of -Paris 
 injecting mass. The latter 
 drives the gelatine mass con- 
 tained in the arteries before it, Fla 
 as far as the capillaries. "When 
 the gelatine is well cooled, the 
 animal is ready for dissection. 
 
 The preparation may be well preserved for an indefinite time 
 in Wickersheimer's fluid. Alcohol shrinks the gelatine, and 
 thus produces breaks in the veins. 
 
 ■Illustrating method of prepar- 
 ing the frog's heart. V, ventricle ; LA, 
 left auricle ; p, auriculo-ventricular open- 
 ing ; 1st L and 2rf L, first and second 
 ligatures ; C, cannula. 
 
 HAY'S METHOD OF MAKING DOUBLE INJECTIONS. 
 
 A. For Dissecting Purposes. — A cannula is fitted into 
 the aorta of a cat, and a gelatine mass colored with carmino is 
 
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228 MICROSCOPICAL METHODS. 
 
 injected until it is seen to flow from the right side of the heart; 
 then, the tube conveying the red mass being detached, a tube 
 conveying a blue gelatine mass is slipped over the same can- 
 nula, and the pressure again applied. Into this blue mass is 
 mixed thoroughly a quantity of starch — preferably from 
 wheat. This starch-bearing mass pushes the carmine mass be- 
 fore it until the starch grains enter the capillaries, and effectu- 
 ally plug them up. The arteries are thus left blue and the 
 veins red. The first mass injected need not be unusually thin. 
 
 B. For Histological Purposes. — The capacity of the 
 capillaries is so great, as compared with that of the arteries, 
 that any commingling of the two colors is concealed in them. 
 Carmine is used for the veins because of the ease with which it 
 may be prepared, its permanence, and the facility with which 
 it passes through the capillaries. On the other hand, the gela- 
 tine for the arteries may be colored with the coarser pigments, 
 such as Prussian blue or ultramarine. The latter furnishes a 
 beautiful blue. Vermilion is not suitable for the first injected 
 mass, since, on account of its high specific gravity, it readily 
 sinks to the lowest side of the vessels, drags behind, and causes 
 a commingling of the colors. An additional reason for filling 
 the veins with red rather than with blue is found in the agreea- 
 ble and natural color given to the preparation. 
 
 Of course, a mass of plaster of Paris, injected after a gelatine 
 mass, will drive it on until the plaster reaches the smallest 
 vessels, thus producing a double injection. The starch mass 
 recently proposed as a filling for blood-vessels will readily lend 
 itself to the production of a double injection according to the 
 method detailed above. 
 
 So far as I am aware, the usual method of producing a 
 double injection of the blood-vessels, preparatory to making 
 sections for the microscope, is to inject first a gelatine mass of 
 one color into the artery until the increasing pressure gives 
 notice that the mass is entering the capillaries, and immediately 
 after to inject a differently colored mass into the vein. The 
 injection being thus accomplished, one of two things, it seems 
 to me, is likely to happen : either the vessels will not be well 
 filled, or the mass intended for one set of vessels will be dxiven 
 
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APPENDIX. 
 
 229 
 
 through into the other. To avoid these accidents, I have prac- 
 tised the method of filling both sets of vessels at the same mo- 
 ment and under exactly the same pressure. 
 
 The pressure is kept low at the beginning, so that all the 
 arteries and veins shall be thoroughly filled before either mass 
 begins to enter the capillaries. Then, as the pressure is in- 
 creased, the differently colored masses meet each other in the 
 capillaries, and, if the pressure on each is equal, the vessels 
 may be filled as full as compatible with safety without danger 
 of either color being driven from one set of vessels into the 
 other. The way in which this result is accomplished will be 
 understood better by 
 reference to the ac- 
 companying draw- 
 ing (Fig. 37). The 
 desired pressure is 
 secured by allowing 
 a stream of water 
 from a hydrant or 
 from an elevated cis- 
 tern to flow into a 
 tight vessel. A two- 
 gallon petroleum can 
 does quite well. As 
 the water flows in, 
 the air is forced out 
 through a rubber 
 tube, A, into the wide-mouthed bottle, F, whose tightly fitting 
 cork gives passage to two other glass tubes. These extend below, 
 just through the cork, and above, connect respectively with 
 the rubber tubes C and D. Into the side of F, near the bottom, 
 is fitted another tube, E, reaching to a height of ten inches or 
 more, open above and graduated into inches. If preferred, 
 this tube may also pass through the cork and extend down well 
 into the mercury with which F is partly filled. B is a bottle 
 of suitable size, in which is contained a blue injection mass for 
 filling the veins, and R, a similar bottle containing a red mass 
 for the arteries. The interiors of these bottles are connected 
 
 Fig. 37. — Apparatus for making double injections. 
 
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230 MICROSCbPICAL METHODS. 
 
 with the bottle F by the tubes D and C. Each of the bottles 
 B and R has a tube, which, starting from near the bottom, 
 passes through the cork, and is, a little above this, bent at 
 right angles. With these are connected the rubber tubes, 
 H and I. Now when water is allowed to flow into the reser- 
 voir mentioned above, the air is forced out through A into 
 F, and thence along the tubes D and C into B and R. As 
 soon as the pressure in these bottles becomes sufficiently great, 
 the liquid which they contain will be driven out through the 
 tubes H and I. If there should be any obstacle to the escape 
 of these fluid masses, the pressure in all the vessels will rise 
 and be registered by the height of the mercury in E. 
 
 If now it is desired to inject, for instance, the kidney of a 
 pig, a cannula made of a glass tube must be fitted securely into 
 the .renal artery, and a similar one into the renal vein. The 
 cannulas must be of such .a size that the rubber tubes H and I 
 will fit them well. Heat the gelatine masses in the bottles 
 B and R to the proper temperature, and keep them so heated 
 until the injection has been finished. Special care must be 
 taken with the tubes H and I to prevent the gelatine passing 
 through them from becoming frozen. Now, having clamped 
 the tube H, have an assistant turn on a small stream of water 
 until the gelatine begins to flow slowly from I. If the diame- 
 ter of the cannula is not too small, it may be held with the free 
 end directed upward and filled with gelatine allowed to drop 
 from the mouth of I. Then slip I over the cannula, unclamp 
 the tube H, and when the gelatine from B has begun to flow, 
 slip it over the cannula inserted in the vein. Then increase the 
 pressure gradually until it has reached as high a point as 
 experience has taught to be safe for the organ operated on. 
 
 By means of this apparatus, which will require the expendi- 
 ture of only a few cents and a little ingenuity, double injections 
 may easily be made of any organs whose veins are not pro- 
 vided with valves. I have made injections of the kidney, 
 whose arteries and glomeruli became uniformly filled with the 
 red mass, and whose veins and the system of capillaries sur- 
 rounding the renal tubules became filled with the blue. The 
 lungs and the liver are easily and successfully injected. I 
 
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APPENDIX. 231 
 
 have been less successful in injecting the organs that send 
 away their blood current through the portal vein; but I have 
 no doubt that they too may be injected. 
 
 Triple injections of the liver may be made by first injecting 
 the hepatic artery with a green mass until the whole liver 
 assumes a green tint, and afterwards injecting the portal vein 
 and the hepatic vein with red and blue as above directed. 
 
 The same apparatus may be employed to make either single 
 injections or the double injection described under the second 
 head of this paper, by simply clamping one of the tubes C or D. 
 As a matter of course, care must be taken that all the corks 
 fit tightly in the bottles, otherwise the internal pressure may 
 force them out at the very moment when an accident will do 
 the most damage. 
 
 MUSEUM METHODS. 
 
 ANESTHETIC AGENTS. 
 
 Carbonic Acid. — Fol * has made the important discovery 
 that Coelenterates and Echinoderms may be rendered insensible, 
 and kept so for hours and even days, without injury, by satu- 
 rating the water with carbonic acid. The containing vessel 
 must, of course, be hermetically closed. The animal at once 
 becomes insensible and motionless, but preserves its natural 
 appearance, and recovers at once when again placed in pure 
 sea-water. This method may be used, not only for obtaining 
 life-like photographs, but also, as Dr. Fol suggests, for trans- 
 porting animals alive. Fishes and molluscs do not survive this 
 treatment, and crustaceans for only a short time. 
 
 Fol tried various narcotics, but found that small doses would 
 not bring the animals to rest, while large doses acted as poisons. 
 The same proved true of tobacco smoke and aqueous solutions 
 of ether, chloroform, and ethyl bromide. Sulphydric acid and 
 carbonic oxide gave satisfactory results in only a few cases. 
 
 Chlorhdyrate of Cocaine. — In order to kill in an ex- 
 
 1 Zoologischer Anzeiger, No. 128, p. 698, 1882. 
 
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232 MICROSCOPICAL METHODS. 
 
 panded condition such animals as the fresh-water Hydra, Bryo- 
 zoa, etc., it is only necessary to treat them in the manner 
 recommended by G. J. Richard. 1 For example, place a colony of 
 Bryozoa in a watch-glass with about 5 C0 of water. When they 
 are well expanded, add slowly £ cc of a 1 % solution of chlorhy- 
 drate of cocaine. After five minutes, another like quantity of 
 the solution is added. At the end of ten minutes more, the 
 animals are perfectly insensible, and may be treated with the 
 ordinary hardening reagents without danger of contraction. 
 
 WICKERSHEIMER'S FLUID. 2 
 
 (1) 3000 g. boiling water ; in this are dissolved : — 
 
 (2) 100 g. alum. 
 
 (3) 25 g. salt. 
 
 (4) 12 g. potassic nitrate (KN"O s ). 
 
 (5) 60 g. potash (KHO). 
 
 (6) 10 g. arsenic trioxid (As 2 2 ). To 10 litres of this mix- 
 ture, after it is filtered and cooled, are added 
 
 (1) 4 litres glycerine. 
 
 (2) 1 litre methyl alcohol. 
 
 Dried Specimens. — If specimens are to be later preserved 
 in a dried condition, they are allowed to soak in the fluid 6 to 
 12 days, according to their size, then taken out, and dried in 
 the air. Muscles, tendons, etc., remain thus soft and pliable. 
 
 Hollow organs (lungs, intestines, etc.) are first filled with the 
 fluid, before being placed in it. 
 
 To Preserve Color. — Specimens must not be dried, but 
 kept in the fluid. 
 
 Preservation of Bodies for Dissection. — Injection of 
 the fluid suffices to keep corpses for years. They will lose 
 their fresh appearance, and the epidermis will brown. This can 
 be prevented by rubbing the fluid over the surface and then 
 preserving in air-tight vessels. 
 
 1 Zool. Anzeiger, VIII., No. 196, p. 332, 1885. 
 * Zool. Anzeiger, Vol. II., No. 45, p. 669, 1879. 
 
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APPENDIX. 233 
 
 KOCH'S METHOD OF MAKING SECTIONS OF CORALS.l 
 
 (1) A small piece of the object is stained in any coloring 
 fluid. 
 
 (2) Washed and treated with alcohol. 
 
 (3) Placed in a thin solution of gum copal in chloroform. 
 (The copal is ground in a mortar with fine sand ; the chloroform 
 is poured over it, and the solution is then filtered.) 
 
 (4) The solution with the object is heated gently, in order 
 to evaporate the chloroform. 
 
 (5) When the evaporation has proceeded so far that the 
 solution can be drawn out in threads which become brittle on 
 cooling, the object is taken out, and dried slowly by artificial 
 heat until it is stony hard. 
 
 (6) Sawed with a fine saw into thin sections. 
 
 (7) Sections ground smooth and flat on one side with an 
 ordinary whetstone. 
 
 (8) The section is then fastened to an object-slide by means 
 of Canada balsam or the copal solution (must be warmed 
 gently). When firm, it is 
 
 (9) Ground down, first with grindstone, then with whetstone, 
 until the required thinness is attained. 
 
 (10) Washed and mounted in balsam. 
 
 The object can be decalcified by first freeing it from the 
 copal or balsam with chloroform, then fixing it, by means of 
 thick Canada balsam, on a slide, and then carefully decalcify, 
 wash, stain, and mount as before. 
 
 This method might be employed in other cases where it is 
 desired to preserve the relations between hard and soft parts ; 
 e. g., sections of star-fishes, bones, crinoids, etc. 
 
 SEMPER'S METHOD OF MAKING DRIED PREPARATIONS. 
 Semper's method, published in the Sitzungsber. d. phys.-med. 
 Ges., Wtirzburg, 1880, and in the Zoolog. Jahresbericht for 
 1880, has been redescribed in detail by Dr. Sharp. 2 
 
 i Zool. Anzeiger, No. II., 1878. Cf ; Q. J. M. S. (N. S.), XX., p. 245, 
 1880. 
 
 2 Proc. Acad. Nat. Sci. Philad., 1884, pp. 24-27. 
 
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234 MICROSCOPICAL METHODS. 
 
 1. Place the object in a weak solution of chromic acid 
 (£-2 fo) 6-24 hours, according to its size and nature. For 
 small animals, such as annelids, gastropods, frogs, mice, etc., 
 6-8 hours are sufficient. 
 
 2. Transfer to a large quantity of clean water, which must 
 be often renewed until the acid has been so far withdrawn that 
 the water remains uncolored by it. This part of the process 
 may be much shortened by allowing a current of water 
 to flow through the vessel. The usual time is from 10 to 20 
 hours. 
 
 3. Treat with 30-40 % alcohol 10-24 hours; with 60-70 % 
 alcohol 2 or 3 days (with larger objects a week) ; with 
 90 % alcohol 2 or 3 days, or more ; and finally with absolute 
 alcohol. 
 
 The treatment with absolute alcohol is the most critical part of the 
 whole process. Absolutely every particle of the -water must be removed; 
 for any tissue in which it remains will become spotted, and eventually 
 spoil. Dr. Sharp always takes the precaution of changing the absolute 
 alcohol once or twice, and leaves the object in it 3-7 days. 
 
 4. Transfer to turpentine, and leave it until it becomes thor- 
 oughly saturated (2-3 days). With large objects it is best to 
 change the turpentine once. 
 
 5. Place the preparation in the air, in order to evaporate the 
 turpentine, protecting it carefully from dust. 
 
 The preparation soon becomes white, resembling the whitest 
 kid. It is light, stiff, and, on account of the resin contained, 
 perfectly insect-proof. 
 
 If hollow organs (stomach, bladders, lungs, etc.) are pre- 
 pared, they may be inflated with air after they have remained 
 a short time in turpentine ; by so doing, much space and con- 
 sequently much alcohol are saved. 
 
 Prof. Semper keeps his preparations in dust-proof glass 
 boxes, in which Jhey can be seen from both sides. 
 
 To the five steps of the process, a sixth, discovered by Sem- 
 per a few years ago, is given by Dr. Sharp. It consists in plac- 
 ing the prepared object in a solution of glycerine and sugar, 
 which brings back almost entirely the original color in many 
 
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APPENDIX. 235 
 
 O. P. HAT'S METHOD OF MAKING DRY PREPAEATIONS. 
 
 While it may be true that in many cases the preparations 
 made according to Semper's method have an appearance simi- 
 lar to a gypsum model, they quite often present a dingy, 
 weather-beaten aspect that is by no means agreeable. The 
 thin membranes and the connective tissues of dissections are 
 left in a loose, woolly condition that grows worse by hand- 
 ling. 
 
 The microscopist completes his work by mounting his prepa- 
 rations in a solution of balsam. In like manner, Semper's 
 method may be completed by saturating the preparation with 
 some solid that will fill up the pores, bind the parts together, 
 and restore the natural appearance. The solid which I have 
 employed for this purpose is a mixture of Canada balsam, paraf- 
 fine, and vaseline ; but it is probable that a soft paraffine will, 
 in most cases, do quite well. It is necessary that the mixture 
 shall melt at about 46° C. (115° F.) "While yet saturated with 
 the turpentine, it is to be immersed in the mixture, heated a 
 little above the melting point, and kept there until all the tur- 
 pentine has been replaced. In many, if not in most, cases, 
 however, the turpentine may be allowed to evaporate before 
 the preparation is put into the melted paraffine mass. The 
 latter then quickly penetrates the tissues, and the work is sim- 
 plified. The preparation is then to be kept in an oven warm 
 enough for the excess of paraffine to melt and drain off. It 
 may then be wrapped in cloths, or in bibulous paper, until the 
 whole of the paraffine mixture adhering to the outside has been 
 dried off. 
 
 The advantages to be derived from pushing the process to 
 this stage are the attainment of a greater degree of firmness 
 and strength in the specimen, the obviation of the bleached 
 appearance assumed on the escape of the turpentine, and the 
 restoration of the natural colors. Probably any colors will 
 reappear that will endure immersion in alcohol. In the case of 
 anatomical preparations made in the way described, injected 
 vessels show to advantage. I have also prepared specimens of 
 lizards, small turtles, fishes, mussels, and earthworms; and 
 
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236 MICROSCOPICAL METHODS. 
 
 whenever the tissues have been thoroughly saturated with the 
 wax mass, the results have been satisfactory. 
 
 ON THE USE OF VASELINE TO PREVENT THE LOSS OF ALCO- 
 HOL FROM SPECIMEN JARS.i 
 
 The petroleum preparation known as vaseline is known 
 to be practically unaffected by ordinary temperatures and 
 by most substances. In the Journal of the Chemical So- 
 ciety, July, 1882, p. 786, it is said to be "sparingly soluble 
 in cold, strong alcohol, and completely in hot, but separates 
 out on cooling." After trying various substances — wax, 
 paraffine, oil, and glycerine — with but partial success, the 
 use of vaseline was suggested by the two authors indepen- 
 dently and nearly at the same time. The experiments tried 
 this spring indicate that, during three months, at ordinary 
 spring and summer temperatures, there is no appreciable loss 
 of 95 % alcohol from glass vials or jars, whether upright, or in- 
 verted, or on the side, provided corks are anointed on the 
 bottom as well as on the side ; provided ground-glass stoppers 
 are anointed and firmly inserted ; and provided the rubber 
 rings of fruit jars and the specimen jars made by Whitall, 
 Tatum & Co. are anointed on both sides, and the covers well 
 screwed down. 
 
 "We have also used the vaseline for preventing the loss of 
 other liquids, excepting chloroform and spirits of turpentine ; 
 as a lubricator of drawers, and to prevent the sticking of the 
 covers or stoppers of cement vials ; and for the prevention of 
 rust upon steel instruments. 
 
 1 Wilder and Gage. Proc. A. A. A. S., XXXII. , p. 318. 
 
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APPENDIX. 
 
 237 
 
 FOEMUL^E FOE EEAGENTS. 
 
 PRESERVATIVE FLUIDS, etc. 
 
 ACID ALCOHOL (Mayer). 
 
 Alcohol (70 % or 90 % ) 95 parts 
 
 Hydrochloric acid 3 
 
 For large objects. 
 
 BOILING ABSOLUTE ALCOHOL (Mayer). 
 For killing arthropods instantly, where other agents fail. — 
 
 GLYCERINE AND ALCOHOL (Salvatore Lobianco). 
 
 Glycerine 20 parts 
 
 Alcohol (70%) 40 " 
 
 Sea water 40 " 
 
 KLEINENBERG'S PICRO-SULPHURIC ACID. 
 Picric acid (saturated solution in distilled water) . 100 parts 
 
 Sulphuric acid (concentrated) 2 " 
 
 Filter the mixture, and dilute it with three times its bulk of 
 water ; finally, add as much creosote as will mix. Use undi- 
 luted for Arthropoda (Mayer). Blanc dilutes with six times 
 its bulk of water, and then adds a little 1 % acetic acid (2-3 
 drops for 15 cc of the solution) for Infusoria. 
 
 MAYER'S PICRO-NITRIC ACID. 
 
 Nitric acid (25 % W O 2 ) 5 parts 
 
 Water 95 " 
 
 Picric acid, as much as will dissolve. 
 
 To be used instead of picro-sulphuric acid for objects pos- 
 sessing calcareous parts. 
 
 PICEO-CHROMIC ACID (Fol). 
 
 Picric acid (saturated aqueous solution) .... 10 parts 
 
 Chromic acid (1 % ) 25 " 
 
 Water 65 " 
 
 Penetrates well only the smallest pieces of tissue.. 
 
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238 MICROSCOPICAL METHODS. 
 
 PERENYTS FLUID. 
 
 Nitric acid (10 fo) 4 parts 
 
 Alcohol (90%) 3 " 
 
 Chromic acid ( \ % ) 3 " 
 
 An excellent fluid for eggs that are brittle when hardened by 
 other reagents. 
 
 MERKEL'S CHROM-PLATINTJM SOLUTION. 
 
 Platinum chloride dissolved in water 1:400 
 
 Chromic acid 1:400 
 
 For the retina (Merkel) ; annelids (Eisig). 
 
 MODIFIED SOLUTION (FOB FISH OVA). 
 
 Platinum chloride 1:400 
 
 Chromic acid 1 : 100 
 
 Mixed in equal parts. 
 
 WITH ACETIC ACID (FOB PBOTOZOA Brass). 
 
 Platinum chloride 1 part 
 
 Chromic acid 1 " 
 
 Acetic acid 1 " 
 
 Water 400-1000 " 
 
 PERCHLORIDE OF IRON (Fol). 
 
 Perchloride of iron (alcoholic solution) 1 part 
 
 Alcohol (70%) 5-10 " 
 
 Recommended for cilia, pseudopodia, small pelagic animals, 
 etc. 
 
 CORROSIVE SUBLIMATE (Mercuric chloride). 
 
 /Saturated aqueous solution (prepared by heating the water 
 to boiling). 
 
 Employed where it is desirable to kill instantly in an ex- 
 panded life-like condition; also much used for embryological 
 purposes. 
 
 G-oadby's Conserving Liquor: — 
 
 Aq. dest 1,500 grms. 
 
 Salt 120 " 
 
 Alum 60 
 
 Sublimate .25 " 
 
 Preserves marine animals well for a considerable time. Un- 
 satisfactory for histological preservation. 
 
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APPENDIX. 239 
 
 Pacini's Fluids: — 
 
 A. Aq. dest 200 parts 
 
 Salt 2 " 
 
 Sublimate 1 " 
 
 Serves for instantaneous killing. 
 
 B. Aq. dest 300 parts 
 
 ' Salt 1 " 
 
 or 
 
 Acetic ac 2 " 
 
 ' Sublimate 1 " 
 
 Glycerine (25° Beaum^) 15 " 
 
 For nuclei, and dissolving red blood corpuscles (leaving the 
 white). 
 
 IODINE SOLUTION (Fol). 
 Iodide of potassium (saturated aqueous solution). 
 Iodine (as much as will dissolve). 
 
 Water 100-500 parts 
 
 For temporary preparations. 
 
 Rabl's Fluids (for nuclei) * : — 
 
 A. Chromic ac. (\fo) 200 g. 
 
 Formic ac 4-5 drops 
 
 Causes chromatine filaments to swell somewhat. 
 
 B. Platinum chloride (\% ). 
 Shrinks the filaments slightly. 
 
 Both fluids may be followed, after treatment with alcohol, 
 by Grenacher's hematoxylin (dilute), safranin (after Pfitzner), 
 or by hematoxylin and safranin. 
 
 FOL'S MODIFICATION^ OF FLEMMING'S FLUID A. 
 
 Osmic ac. 1% 2 parts 
 
 Chromic ac. 1% 25 " 
 
 Acetic ac. 2% 5 " 
 
 "Water 68 " 
 
 Erlicki's Fluid: — 
 
 Potassium bichromate 2J g. 
 
 Cupric sulphate i g. 
 
 Aq. dest 100 g. 
 
 i Morph. Jahrb., X, 2, p. 215, 1884. 
 2 Lehrbuch d. vergl. Mik. Anat., p. 100, 1884. 
 
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240 MICROSCOPICAL METHODS. 
 
 Mullens "Eye-Fluid": — 
 
 Aq. dest 100 parts 
 
 Bichromate of potassium 2 " 
 
 Sulphate of sodium 1 " 
 
 Flemming's Fluids (for nuclei) : — 
 
 A. 1 Aq. dest 100 parts 
 
 Chrom ac i " 
 
 Osmie ac A " 
 
 Glacial acetic ac -fa " 
 
 For nuclear filaments omit the osmic acid. 
 
 B. s Chrom. ac. (1%) 15 parts 
 
 Osmic ac. (2%) 4 " 
 
 Glacial acetic ac 1 " 
 
 Stain with safranin or gentian violet. 
 
 C 8 Nitric acid (40-50 to aq. dest. 60-50). 
 Stain with Schneider's acetic acid carmine. 
 
 Strasburger's Fluid (for nuclei) 4 r — 
 
 A. Acetic acid ( 1 % ) and a little methyl green. 
 Preparations are not permanent. 
 
 B. Nitric acid (50% ) followed by dye mixed in dilute glycerine. 
 
 Fr. Meyer's Preservative Fluid ( u SalicylrIfolzessig ,, ): b 
 Pyroligneous acid (acetum lignorum) .... 100 parts 
 Salicylic acid 1 " 
 
 This mixture is diluted with glycerine and water in three 
 ways : — 
 
 1. For Ziarvce, Nematodes, Hydra, etc.: 
 
 Glycerine 10 parts 
 
 Water 20 " 
 
 Salicyl-pyrolig. ac 3 " 
 
 2. For Infusoria : 
 
 Glycerine 10 parts 
 
 Water 40 " 
 
 Salicyl-pyrolig. ac 5 " 
 
 3. For Alga: 
 
 Glycerine 1 part 
 
 Water 20 " 
 
 Salicyl-pyrolig. ac 1 " 
 
 1 Zelsubst. Kern u. Zelltheilung, p. 382, 1882. 
 
 2 Zeitschr. f. wiss. Mikroskopie, I., 3, p. 349, 1884. 
 8 Arch. f. mik. Anat., XX., p. 1, 1881. 
 
 4 Arch. f. mik. Anat., XXI., p. 476. 
 
 6 Arch. f. mik. Anat. XIII., 1876, p. 868-869. 
 
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APPENDIX. 241 
 
 MACERATING AGENTS. 
 
 1. Alcohol 30$ (or less) 
 
 2. Acetic acid 1 % " 
 
 3. Osmicacid ^% " 
 
 4. Chromic acid 05%-%. 1 
 
 ( Acetic acid 1 part 
 
 5. J Osmic acid £ " 
 
 (Sea water 1000 " 
 
 ( Tincture of iodine 1 g. 
 
 6. -) Amniotic fluid 100 g. 
 
 I Carbolic acid 1-2 drops 
 
 Fresh serum may be iodized by the addition of a few drops 
 of a strongly iodized serum kept in stock. « 
 
 (Eaude Javelle (KCIO) 8 drops 
 
 7 " | Water 100 cc. 
 
 ( Caustic potash 30-32 parts 
 
 8 ' \ Water 70-68 " 
 
 DECALCIFYING MIXTURE. 
 
 Chromic acid 70 parts 
 
 Nitric acid 3 " 
 
 Water 200 " 
 
 DESILICOTCATION {Mayer). 
 Fluorhydric acid added in drops to the alcohol containing the ohject. 
 
 STAINING MEDIA. 
 
 CARMINE. 
 
 Beale's Carmine. 1 
 
 Carmine 6 grms. 
 
 Strong liquor ammonise 2 cc. 
 
 Glycerine 60 cc. 
 
 Aq. dest 60 cc. 
 
 Alcohol 15 cc. 
 
 Having placed the carmine and ammonia together in a test- 
 tube heat over a spirit lamp, and the carmine will soon dis- 
 solve. The solution should be boiled for a few seconds, and 
 
 i How to Work with the Microscope, p. 109. 
 
 Digitized by Microsoft® 
 
242 MICROSCOPICAL METHODS. 
 
 then allowed to cool. After the lapse of an hour, during 
 which much of the excess of ammonia has escaped, the glyce- 
 rine, water, and alcohol may be added and the whole filtered, 
 or allowed to stand for some time, and the perfectly clear 
 supernatant fluid poured off and kept for use. 
 
 ALCOHOLIC CARMINIC ACID (Dimmoch). 
 
 Carminic acid 1 g. 
 
 Alcohol (80% ) 400 g. 
 
 ALCOHOLIC AMMONIA CARMINE {Dimmoch). 
 Alcoholic carminic acid. 
 Ammonia (added by drops until color changes to purple red). 
 
 ALCOHOLIC PICRO-CARMINE {Dimmoch). 
 Alcoholic carminic acid. 
 Picric acid (dilute alcoholic solution) in very small quantity. 
 
 ALCOHOL CARMINE (Mayer). 
 
 Carmine (pulverized) 4 g. 
 
 Alcohol (80% ) 100 cc. 
 
 Hydrochloric acid 30 drops 
 
 Dissolved by boiling 30 minutes ; filtered warm and neutral- 
 ized with ammonia. 
 
 PICRO-CARMINE (Mayer). 
 
 Carmine (powdered) 2 g. 
 
 Water 25 cc. 
 
 Ammonia (enough to dissolve carmine). 
 
 Picric acid (saturated aqueous solution — 4 times volume of the car- 
 mine solution). 
 Hoyer's Pier o -Carmine: — 
 
 1. Dissolve 1 g. carmine in 6 ccm. ammonia, diluted with an equal 
 volume of water. 
 
 2. Dissolve i g. picric acid in 50 ccm. of warm water. 
 
 3. Mix the two solutions and dilute so as to make 100 ccm. 
 
 4. Then add 1 g. chloral hydrate for preservation. 
 
 CARMINE ACETATE (Bbhm). 
 
 Carmine (pulverized) 4 g. 
 
 Water 200 cc. 
 
 Ammonia (added by drops till the solution is deep red, and the 
 
 carmine is dissolved). 
 Acetic acid (added until the solution turns to brick red). 
 Filter until no trace of a precipitate remains. 
 
 Digitized by Microsoft® 
 
APPENDIX. 243 
 
 ACETIC ACID CARMINE {Schneider). 
 
 Acetic acid (45$) 1 part 
 
 Carmine (as much as will dissolve). 
 
 "Water 99 " 
 
 GRENACHER'S CARMINE SOLUTIONS. 
 
 Alum Carmine: — 
 
 Alum (1-5% aqueous solution) 100 cc. 
 
 Carmine 1 g. 
 
 Boil 10-20 minutes, and filter when cool. 
 
 Acid Borax Carmine: — 
 
 Borax (1-2% aqueous sol.) 100 cc. 
 
 Carmine |-§ g. 
 
 Heat till carmine dissolves. 
 
 Acetic acid (added by drops while shaking, until the color is about the 
 same as Beale's carmine). 
 Filter after standing 24 hours. 
 
 Borax Carmine: — 
 
 Borax (4% aqueous sol.) 100 cc. 
 
 Carmine 1 g. 
 
 Heat till carmine dissolves. 
 
 Alcohol (10% — equal to the vol. of carmine sol.) 
 Filtered after 24 hours. 
 
 Alcohol Carmine: — 
 
 Carmine 4 g. 
 
 Alcohol (60-80%, with 6-8 drops HC1) 100 cc. 
 
 Dissolve by boiling, then filter. 
 
 HEMATOXYLIN. 
 
 GRENACHER'S HEMATOXYLIN. 
 
 Hematoxylin (saturated alcoholic solution) ... 4 cc. 
 
 Ammonia-alum (concentrated aqueous solution) . 150 cc. 
 Let stand 8 days in the light, then filter and add 
 
 Glycerine 25 cc. 
 
 Methyl alcohol 25 cc. 
 
 This dye works best after standing until a sediment forms. 
 
 Digitized by Microsoft® 
 
244 MICROSCOPICAL METHODS. 
 
 WEIGERT'S HEMATOXYLIN (for nerves).* 
 
 Hematoxylin 1 part 
 
 Alcohol 10 " 
 
 Water 90 " 
 
 Boiled and allowed to stand a few days. 
 
 WEIGEET'S DECOLORING FLUID.* 
 
 Borax 2 parts 
 
 Ferricyanide of potassium (red prussiate of potash) 2£ " 
 Water 100 " 
 
 Differentiates white and gray substance. 
 
 HEIDENHAIN'S HEMATOXYLIN.' 
 
 Hsematoxylin £-1 g. 
 
 Water 100 cc. 
 
 Preceded by alcohol and followed by bichromate of potassium {\-\% ). 
 Stains nuclei black. 
 
 KLEINENBERG'S HEMATOXYLIN. 
 Chloride of calcium (saturated sol. in 70% alcohol) 1 part 
 Alum ( a little added, then filter) ...... 1 " 
 
 Alcohol (70% ) 6-8 " 
 
 Hsematoxylin (saturated sol. in absol. alcoh., — enough to give the 
 desired depth of color). 
 
 MAYER'S COCHINEAL TINCTURE. 
 
 Cochineal (pulverized) 1 g. 
 
 Alcohol (70%) 8-10 cc. 
 
 Filter after standing several days. 
 
 CZOKOR'S ALUM-COCHINEAL.* 
 
 Cochineal 7 g. ; pulverized with 
 
 Alum 7 g. ; then add 
 
 Water 700 cc. ; and boil down to 400 cc. 
 
 Add a trace of carbolic acid and filter. This stain may be used after 
 any method of hardening; it differentiates well, giving the nuclei a 
 ha5matoxylin shade, while staining the other parts of the cell red. 
 
 1 Fortschritte d. Med., II., No. 6, p. 190, 1884. 
 
 2 I.e. 
 
 8 Arch. f. mik. Anat., XXIV., H. 3, p. 468, 1884. 
 1 Arch. f. mikr. Anat., XVHI., p. 413. 
 
 Digitized by Microsoft® 
 
APPENDIX. 245 
 
 ANILIN DYES. 
 
 Bismark Brown: — 
 
 Dissolved in boiling water, weak alcohol, "70% alcohol (Mayer), or 
 dilute acetic acid (Flemming). 
 
 Stains well chromic acid or alcoholic preparations. 
 
 Eosin: — 
 
 Eosin 1 g. 
 
 Alcohol (95%) 100 cc. 
 
 For use, dilute with 20 parts alcohol (Minot). 
 
 Ffitzner's Safranin: — 
 
 Safranin 1 grm. dissolved in 
 
 Absolute alcohol 100 ccm. 
 
 After standing one or two days, filter, and dilute with 
 Distilled water 200 ccm. 
 
 PICB.O-NIGROSIN (PJitzner). 
 Picric acid (saturated aqueous sol. ). 
 Kigrosin (small quantity). 
 
 Hardens and stains at same time. 
 
 PICRIC-HOFFMANN'S-BLUE.i 
 Dissolve Hoffmann' s-blue in a saturated solution of picric acid in 50% 
 alcohol, until the fluid assumes a dark greenish-blue tint. 
 
 DAHLIA (Ehrlich). 
 
 Alcohol abs 50 cc. 
 
 Aq. dest 100 cc. 
 
 Acid. acet. glac 12^ cc. 
 
 Dahlia (to near saturation). 
 
 BLUE-BLACK (SanJcey). 
 
 Blue black £g. 
 
 Aq. dest 1-2 cc. 
 
 Alcohol 99 cc. 
 
 SAFRANIN AND METHYL GREEN (Ryder). 
 
 Safranin (saturated alcoholic sol.) 1 part 
 
 Methyl green (saturated alcoholic sol.) 1 " 
 
 Water 16 " 
 
 i Arbeit. Bot. Inst. Wiirzburg, III., pp. 53-60, 1884. 
 
 Digitized by Microsoft® 
 
246 MICROSCOPICAL METHODS. 
 
 FIXATIVES. 
 
 ALCOHOL, SHELLAC (Giesbrecht). 
 
 Bleached shellac 10 g. 
 
 Absolute alcohol 100 cc. 
 
 Leave 24 hours, then filter. 
 
 CARBOLIC ACID SHELLAC (Mayer). 
 
 Bleached shellac 50 g. 
 
 Absolute alcohol 250 cc. 
 
 Filter, and then evaporate the alcohol. Dissolve the shellac resi- 
 due in 
 
 Carbolic acid 3-5 part3 
 
 COLLODIOK (Schallibaum). 
 
 Collodion 1 part 
 
 Clove oil 3-4 " 
 
 ALBUMEN (Mayer). 
 
 White of egg (filtered) 1 part 
 
 Glycerine 1 " 
 
 MOUNTING MEDIA. 
 
 GLYCEBLNE JELLY (after Fol). 
 
 Strong solution : — 
 
 Gelatine 30 parts 
 
 Water 70 " 
 
 Glycerine 100 " 
 
 Camphor ( alcoholic sol. ) 5 " 
 
 Weak solution : — 
 
 Gelatine 20 parts 
 
 Water 150 " 
 
 Glycerine 100 " 
 
 Camphor 15 " 
 
 CANADA BALSAM. 
 
 Prepared by heating on a bath until it becomes so hard on 
 cooling that it cannot be indented by the finger-nail, and breaks 
 like glass ; then dissolved in an equal volume of pure benzole 
 (beware of benzole that contains water). 
 
 DAMAE LAC. 
 
 Prepared by dissolving the gum in pure turpentine, or in a 
 mixture of turpentine and benzole in equal parts. 
 
 Digitized by Microsoft® 
 
APPENDIX. 
 
 247 
 
 TABLE I. 
 Comparison of the Units of Measure. 
 
 
 1 
 
 MM. 
 
 1 
 PABIS 
 
 LINE = 
 
 1 
 
 ENGLISH 
 LINE = 
 
 1 
 
 RHEIN 
 LINE = 
 
 1 
 
 VIENNA 
 LINE = 
 
 Millimeter .... 
 
 1.0000 
 
 2.2558 
 
 2.1166 
 
 2.1802 
 
 2.1952 
 
 Paris Line .... 
 
 0.4433 
 
 1.0000 
 
 0.9384 
 
 0.9964 
 
 0.9732 
 
 English Line . . . 
 
 0.4724 
 
 1.0659 
 
 1.0000 
 
 1.0299 
 
 1.0371 
 
 Rhein Line .... 
 
 0.4587 
 
 1.0347 
 
 0.9710 
 
 1.0000 
 
 1.0070 
 
 Vienna Line .... 
 
 0.4555 
 
 1.0275 
 
 0.9642 
 
 0.9930 
 
 1.0000 
 
 TABLE II. 
 
 Reduction of the Units of Measure to Mioromillimeters. 
 
 MICROMILLIMETER. 
 
 PARIS LINE. 
 
 ENGLISH 
 LINE. 
 
 RHEIN LINE. 
 
 VIENNA 
 LINE. 
 
 1 11 (0.001 mm.) = . 
 
 
 
 O.000443 
 
 0.000472 
 
 0.000459 
 
 0.000455 
 
 2 n = 0.002 mm. = 
 
 
 
 0.000887 
 
 0.000945 
 
 0.000917 
 
 0.000911 
 
 3 it = 0.003 mm. = 
 
 
 
 0.001330 
 
 0.001417 
 
 0.001376 
 
 O.001366 
 
 4 ii = 0.004 mm. = 
 
 
 
 0.001773 
 
 0.001890 
 
 0.001835 
 
 0.001822 
 
 5 n = 0.005 mm. = 
 
 
 
 0.002216 
 
 0.002362 
 
 0.002293 
 
 0.002277 
 
 6 ft = 0.006 mm. = 
 
 
 
 0.002660 
 
 0.002834 
 
 0.002752 
 
 0.002733 
 
 7 \i = 0.007 mm. = 
 
 
 
 0.003103 
 
 0.003307 
 
 0.003211 
 
 0.003188 
 
 8 ji = 0.008 mm. = 
 
 
 
 0.003546 
 
 0.003779 
 
 0.003670 
 
 0.003644 
 
 9 ii = 0.009 mm. = 
 
 
 
 0.003990 
 
 0.004252 
 
 O.004128 
 
 0.004099 
 
 10 (i = 0.010 mm. = 
 
 
 
 0.004433 
 
 0.004724 
 
 0.004587 
 
 0.004555 
 
 20 ii = 0.020 mm. = 
 
 
 
 0.008866 
 
 0.009448 
 
 0.009174 
 
 0.00911O 
 
 50 n = 0.050 mm. = 
 
 
 
 0.022165 
 
 0.023620 
 
 0.022935 
 
 0.022775 
 
 100 in = 0.100 mm. = 
 
 
 
 0.044330 
 
 0.047240 
 
 0.045870 
 
 0.045550 
 
 Digitized by Microsoft® 
 
Digitized by Microsoft® 
 
INDEX. 
 
 Acetic acid, 17. 
 
 for tracing nerves, 18. 
 
 after staining, 182. 
 Acid, acetic, use of, 17. 
 
 for decoloring, 46. 
 
 alcoholic carminic, 37. 
 
 carminic, 34-38. 
 
 fluorhydriCj 32. 
 
 hydrochloric with alcohol, 
 for marine animals, 12. 
 for bleaching, 16. 
 for decoloring, 39, 46. 
 
 nitric, 19. 
 
 osmic, 15, 164. 
 
 oxalic, 45. 
 
 and alcohol, 25. 
 
 picric, 15. 
 
 picro-chromic, 22. 
 
 picro-nitric, 21, 22. 
 Actinia, 28, 29, 203, 204. 
 Albumen, 122, 123. 
 
 advantage of, in staining with aniline 
 dyes, 123. 
 Alcohol, for killing, 5. 
 
 for hardening, 6. 
 
 the action of, 7. 
 
 danger of maceration, 7. 
 
 disadvantages attending its use, 12. 
 
 Eisig's method of killing with, 12. 
 
 acid alcohol for hardening, 13. 
 
 boiling alcohol for killing, 13. 
 
 with glycerine for killing, 13. 
 
 method of making absolute, 14. 
 
 iodine dissolved in, 15, 207. 
 
 absolute, for hen's egg, 165. 
 Alcyonaria, 203. 
 Alosa, 177. 
 
 Amblystoma, 155, 156. 
 Amiurus, 179. 
 Amphibia, 155-162. 
 Amphietenidas, 208. 
 Anaesthetic agents, 231. 
 Aniline dyes, 47-54. 
 
 double staining with, 51. 
 
 solubilities of, 54. 
 
 not used with celloidin sections, 108. 
 
 differential staining with, 123. 
 Annelids, 19, 198, 208. 
 Antiseptics, 
 
 carbolic acid, 122, 211. 
 
 chloral, 41, 43, 223. 
 
 thymol, 43. 
 
 Apeltea, 178. 
 Aphides, 143, 144. 
 Appendicularia, 208. 
 Arthropods, 
 
 eggs of, killed in hot water, 11. 
 
 killed in boiling absolute alcohol, 13. 
 
 hairs, etc. , stained with osmic acid, 16. 
 
 treatment with picro-sulphuric acid, 
 20. 
 Artotype process, 196-199. 
 Asphalt-cell oidin; 225. 
 Asterias, 147-149. 
 
 B. 
 
 Balanoglossus, 149, 150. 
 Barnacle, 174. 
 Batrachians, 196. 
 Beale's carmine, 38-39. 
 
 macerating action, 29. 
 Belone, 177. 
 
 Bergamot oil, 109, 114, 127. 
 Bismarck Brown, 48, 51, 52. 
 Bleaching, 
 
 after osmic acid, 16. 
 
 natural pigment, 17. 
 
 after gold chloride, 56-57. 
 
 after silver nitrate, 58. 
 Bleu de Lyon, 48-49. 
 Blue black, 50. 
 Brachiopoda, 126, 209. 
 Bryozoa, 232. 
 
 c. 
 
 Calberla's fluid, 163. 
 
 Caoutchouc, 120. 
 
 Capitellidae, 23. 
 
 Caprellidae, 53. 
 
 Carbolic acid, 122, 211. 
 
 Carbonic acid as an anaesthetic, 231. 
 
 Carminate, alcoholic amnionic, 37. 
 
 Carmine, alcohol (Mayer), 39, 123. 
 
 alum (Grenacher), 39. 
 
 acid-borax (Grenacher), 40. 
 
 borax (Grenacher), 40. 
 
 acetic acid (Schneider), 43. 
 
 emulsion, 221. 
 
 solutions, 34-44. 
 Carminic acid, 34-38. 
 Cat-fish, 179. 
 Celloidin, 107-114, 117, 120. 
 
 advantages of, 112-113, 127. 
 
 injections, 225. 
 
 249 
 
 Digitized by Microsoft® 
 
250 
 
 INDEX. 
 
 Central nervous system, 
 
 Weigert's method, 191-192. 
 
 method of counting fibres and gang- 
 lia, 192-194 t 
 
 Betz's method, 194. 
 
 Osborn's method, 194-196. 
 
 photographing sections, 196-198. 
 
 of annelids, 198, 199. 
 Chirostoma, 177. 
 Chloral, 41, 43, 223. 
 Chloride of calcium, 44. 
 Chloroform, 6, 95, 96 t 114. 
 Chlorophyll, 212. 
 Chlor-zinc-iod, 212. 
 Chondrosia, 208. 
 Chromic acid, for killing, 6. 
 
 principal drawback and best means of 
 meeting it, 6, 9. 
 
 use of, 18. 
 
 for nuclei, 19. 
 
 combined with other acids, 19. 
 
 precipitates avoided by keeping dark, 
 19. 
 
 for decalcification, 31. 
 
 for frog's egg, 157, 158. 
 
 hot, for hen's egg, 166. 
 Clarifying media, 94. 
 
 penetration hastened by -warming, 94. 
 
 mode of avoiding shrinkage, 94. 
 
 chloroform preferred to creosote or 
 oil of cloves, 95. 
 
 Andres' mixture, 95. 
 
 for celloidin sections, 109. 
 
 clove oil followed by turpentine, 94. 
 
 benzole, 142. 
 
 creosote, xylol, 192. 
 Clepsine, 136-138. 
 Clove oil, 94, 109, 113, 182. 
 
 followed by turpentine, 94. 
 Cocaine, chlorliydrate of, 231. 
 Cochineal, 12, 35, 41. 
 
 (Mayer), 45-47. 
 Cod, 176, 177. 
 Coelenterates, 29, 231. 
 Collodion, 
 
 method of fixing sections, 117. 
 
 as a fixative, 121, 122. 
 
 uses of, 126-128, 170-172. 
 Comatuh©, 139. 
 Corals, sections of, 233. 
 Corpus vitreum, 209. 
 Corrosive sublimate, 5, 7, 15, 18, 28, 37, 134. 
 
 use of, 26. 
 
 removal of, facilitated by camphor, 
 204. 
 
 combined with Perenyi's fluid, 207. 
 Crab, 175. 
 Crangon, 176. 
 Crayfish, 141. 
 
 Creosote in picro-sulphurie acid, 20, 21. 
 Ctenophorse, 209. 
 Cyanide of potassium, 56, 58. 
 Cyanin, 53. 
 
 D. 
 
 Dahlia, 49-50. 
 Decalcification, 31. 
 Decoloring, 
 
 with hydrochloric acid, 39, 45, 46. 
 
 with ammonia, 39. 
 
 with oxalic acid, 45. 
 
 with 70 per cent alcohol, 46, 181, 182. 
 
 with acetic acid, 46, 49. 
 
 Decoloring — continued. 
 
 with alcohol, 48. 
 
 with HC1 and alcohol, 142. 
 Desilicification, 32. 
 Dicyemida^, 16. 
 Diemictvlus, 156. 
 Doliolum, 150, 151. 
 Drawing apparatus, 129-134, 
 Dried preparations, 
 
 Semper's method, 233. 
 
 Hay's method, 235, 236. 
 Dripping apparatus, 115. 
 
 E, 
 
 Echinoderms, 21, 201, 231. 
 Echinorhyncni, 205. 
 Egg-emulsion, 106, 107, 109. 110. 
 
 bath for hardening, 195. 
 Elder pith, 106, 109. 
 Embryological methods, 129-173. 
 Emulsion, carmine, 221. 
 
 blue, 222. 
 
 black, 222. 
 Eosin, 48, 51. 
 
 Ogatha's, 210. 
 Erlicki's fluid, 24, 25, 187, 191. 
 
 F. 
 
 Ferricyanide of potassium, 56. 
 Fibrospongise, 32. 
 Filtering bottle, 55. 
 Fishes, fatty pigments, 16. 
 
 eggs of, hardened in Merkel's fluid, 19. 
 
 nitric acid for hardening the brain, 
 19, 172. 
 
 Perenyi's fluid, 23. 
 
 osmic acid and MUller's fluid for the 
 eggs, 24. 
 
 pelagic fish eggs, 151-154. 
 
 trout's egg, 162. 
 
 eggs, 176-179, 208. 
 
 corpus vitreum, 209. 
 Fixatives, for serial sections, 116-123. 
 
 alcoholic shellac, 116, 117. 
 
 carbolic acid shellac, 117, 118. 
 
 Gaule's method, 119. 
 
 gutta-percha, 119, 120. 
 
 caoutchouc, 120, 121. 
 
 collodion, 121, 122. 
 
 albnmen, 122, 123. 
 Fluorhydric acid, 32. 
 Formulae for reagents, 237-246. 
 Fuchsin, 51. 
 
 G. 
 
 Gambusia, 178. 
 
 Gammarus, 176. 
 
 Ganglion cells, method of counting, 194. 
 
 Glycerine and alcohol for killing, 13, 204. 
 
 and gelatine for imbedding, 106. 
 Glycerine jelly for mounting, 125. 
 Gold chloride, 56, 187-190. 
 
 Flechsig's method, 187. 
 
 Freud's method, 187-188. 
 
 Gaule's method, 188. 
 
 Eanvier's methods, 188-189. 
 
 Ciaccio's method, 189. 
 
 Pfitzner's method 190. 
 
 after chromic acid, 188. 
 Gold-fish, 179. 
 
 Gravitation, influence of, on cleavage, 
 159-162. 
 
 Digitized by Microsoft® 
 
INDEX. 
 
 251 
 
 Gum arabic, for imbedding, 28, 106, 109. 
 Gutta-percha, 119, 120. 
 
 H. 
 
 Hematoxylin, 12. 
 
 (Grenacher), 44, 184. 
 
 (Kleinenberg), 44, 45. 
 
 Heidenhain's method, 186. 
 
 Weigert's method, 191. 
 Hardening, 6, 7, 11. 
 
 with acid alcohol, 13. 
 
 with boiling alcohol, 13. 
 Hatching jar, 177, 178. 
 Helix, 145. 
 
 Hen's egg, 19, 163-167. 
 Hippa, 174. 
 
 Histological methods, 200-212. 
 Hoffman's blue, picric, 212. 
 Hoffman's violet, 51. 
 Hydra, 186, 232. 
 Hydrochloric acid, 
 
 for removing ferric salts, 25. 
 
 for decalcifying, 31. 
 
 for decoloring, 39. 
 Hydroids, 208. 
 Hyperia, 174. 
 Hyposulphite of sodium, 68. 
 
 Imbedding, methods of, 93-115. 
 
 ribbon method, 94. 
 
 melting point of paraffine, 93, 94. 
 
 preparation for, 94, 142. 
 
 apparatus, 102-104, 109. 
 
 boxes, 97, 108, 111, 112. 
 
 orientation, 98, 99. 
 
 prevention of bubbles, 99, 100, 113. 
 
 Schulgin's mixture for, 100. 
 
 Caldwell's method of, 100, 101. 
 
 Ryder's method of, 101. 
 
 in gum arabic, 106, 109. 
 
 in glycerine and gelatine, 106. 
 
 in egg emulsion, 106, 107, 109, 110. 
 
 in elder pith, 106, 109. 
 
 in liver, 109. 
 
 in celloidin, 107-109, 111-115. 
 Infusoria, 19, 25, 53, 202. 
 Injection, 
 
 methods of, 221-231. 
 
 dry masses, 221-223. 
 
 starch 223. 
 
 special mass, 224. 
 
 dried preparations, 225. 
 
 celloidin, 225. 
 
 double injections, 225-231. 
 < apparatus for double injections, 229. 
 Iodine, use of, 14, 15, 30, 207. 
 Iodized serum, 30. 
 
 K. 
 
 Karyokinetic figures, 15, 19, 44, 60, 181-186. 
 
 as a guide to intensity of growth, 183. 
 
 chrom-formic acid contrasted with 
 platinum cbloride, 184. 
 Killing, general statements regarding, 4, 
 11. 
 
 planarians, 5. 
 
 annelids, 5. 
 
 with hot water, 11, 157. 
 
 with alcohol, 12. 
 
 with boiling alcohol, 13. 
 
 Killing — continued, 
 
 with glycerine and alcohol, 13. 
 
 with perchloride of iron, 25. 
 
 with corrosive sublimate, 26-27. 
 
 with nigrosin and picric acid, 49. 
 Kleinenberg'spicro-sulphuric acid, 5,7, 18. 
 
 not a hardening fluid, 11. 
 
 composition of, 19-21. 
 
 L. 
 
 LeecheSj 27. 
 Lemon juice, 189. 
 Lepidosteus, 127. 
 Limax, 145-147. 
 Limulus, 144, 145. 
 Lineus, 175. 
 Lobster, 174. 
 Loligo, 176. 
 Loxosoma, 207, 208. 
 Lumbricus, 47. 
 
 M. 
 
 Maceration, 28-31. 
 
 Andres' method with Actimse, 28, 205. 
 
 the Hertwigs' mixture for ccelenter- 
 ates, 29. 
 
 with iodized serum, 30 
 
 with eau de javelle, 30. 
 
 with caustic potash, NaCl., 211. 
 
 with alcohol, 33| %, 211. 
 
 with chloral hydrate, 211. 
 
 with chromate of ammonia, 211. 
 
 with chromic acid and chloral hy- 
 drate, 211. 
 Meckelia, 175. 
 Medusse, 201. 
 Menidia, 178. 
 
 Merkel's cbrom-platinum solution, 5, 7, 
 18, 19. 
 
 arrests blackening by osmic acid, 16. 
 
 composition and use, 13. 
 
 modified form of, 153, 200. 
 
 for nuclei, 184. 
 Methods, macroscopical and microscopi- 
 cal, 1. 
 Methyl green, 52. 
 
 violet, 51. 
 
 and gentian violet, 53. 
 Metbylen blue, 51. 
 Meyer's fluid, 31. 
 Micrometer-screw, 64. 
 
 registering, 66. 
 Microtome, the, in zoology, 2, 10. 
 
 price of, 69. 
 
 two types of, 60. 
 
 Thoma's, 61-69. 
 
 Schanze, 69, 70. 
 
 Caldwell's, 70-74. 
 
 care of, 76. 
 
 the freezing, 
 
 Rutherford's, 76-79. 
 
 Jacob's, 79-80. 
 
 ether-freezing apparatus, 80, 81. 
 
 notes on, by Gage, 81, 82. 
 
 gelatine jelly instead of gum, S3 
 Microtome knives, 83-86. 
 
 method of sharpening, 85, 86. 
 Microtomist, 1. 
 Microtomy, 2. 
 Molgula, i76. 
 Mollusca, 36. 145-147. 
 Molybdic acid test, 212. 
 
 Digitized by Microsoft® 
 
252 
 
 INDEX. 
 
 Monacanthus, 178. 
 Mounting media, 124, 125* 
 balsam in xylol, 119. 
 balsam in chloroform and clove oil, 
 
 122. 
 balsam, solvents of, 119, 124. 
 
 preparation of, 125. 
 damar, 124, 125. 
 
 preparation of, 125. 
 glycerine jelly, 125. 
 Mucous membrane of stomach, 211. 
 Muller's fluid, 24, 191, 211. 
 Muscular fibres stained with palladium 
 
 chloride, 59. 
 Museum methods, 231-236. 
 Myriapods, 179, 180. 
 Myrtillus, 50, 51. 
 Mysis, 17. 
 Myzostoma, 139, 140. 
 
 N. 
 
 Naphtba, 122. 
 Neopbalax, 141-143. 
 Nerve-endings, 18, 19, 188, 189, 190. 
 Nerve-fibres, 58, 193. 
 Nerves of cornea, 59. 
 Nicotine, 204. 
 
 Nigrosin and picric acid, 49. 
 Nitrate of silver, 57, 58. 
 
 with marine objects, 58, 207. 
 
 Golgi's method for nerve-fibres, 190, 
 191. 
 
 Hertwig's method for marine ani- 
 mals, 209. 
 Nitric acid, 19, 172. 
 
 combined with chromic acid for de- 
 calcifying, 31. 
 
 for nuclei, 182. 
 Nuclei, 181-186. 
 
 stained with safranin, 49. 
 " " dahlia, 49. 
 
 differentially stained, 123. 
 
 in general, 181, 182. 
 
 ova of Echinoderms, 182. 
 
 O. 
 
 Object-holder and carrier, 67-69. 
 Oil for microtome, 64, 76. 
 Opaque-celloidin, 225. 
 Origanum oil, 113, 127. 
 Orientation, 
 
 in imbedding, 98, 99. 
 
 of frog's eggs, 161. 
 
 of hen's egg, 163-165. 
 
 in sections, 213. 
 Osmic acid, 15. 
 
 as a staining medium for hairs, etc., 
 16. 
 
 blackening process arrested, 16. 
 
 bleaching after, 16, 17. 
 
 for strengthening picro-chromicacid, 
 22. 
 
 for hen's egg, 164. 
 
 after silver nitrate, 208. 
 
 followed by silver nitrate, 209. 
 Oxalic acid, 26. 
 t Oyster, 52. 
 
 P. 
 
 Palinurus, 22. 
 Palladium chloride, 58, 59. 
 
 Parafnne, 
 
 cooled in water to prevent air-bub- 
 bles, 71. 
 
 for imbedding, 93-104. 
 
 saturation with, 96. 
 
 solvents of, 94-96, 122, 123, 142. 
 
 and vaseline, 120. 
 
 and ceresin, 100. 
 Pedicellina, 207. 
 Perca, 179. 
 
 Percbloride of iron, 25. 
 Perenyi's fluid, 19, 22, 154. 
 Phronima, 16, 174. 
 Phryganids, 141-143. 
 Picric acid, 15. 
 
 for decalcification, 32. 
 
 for making picro-carmine, 38. 
 
 combined with nigrosin, 49. 
 Picro-carniine (Ranvier), 41, 42. 
 
 (Pergens), 41. 
 
 (Minot), 42. 
 
 (Mayer), 42, 43. 
 Picro-chromic acid, 22. 
 Picro-nitric acid, 21, 22. 
 Planarians, 134, 135. 
 Planorbis, 145. 
 Polygordius, 198. 
 Polyxenes, 180. 
 Potash, caustic, 31. 
 Potassium bichromate, 23. 
 Prawn, 174. 
 Preservative fluids, 11. 
 Protodrilus, 198. 
 Protoplasm, continuity of, 212. 
 Protozoa. 200, 201. 
 Pseudo-chlorophyll bodies, 186. 
 
 Babbit, 167-170. 
 Radiolaria, 32. 
 Rana, 155. 
 Reagents, 
 
 formula 237-246. 
 Reconstruction, from sections — 
 
 His and Born 2, 215-218. 
 
 general remarks on, 213-215. 
 
 illustration, 213, 215-218. 
 Reptiles, 198. 
 Reptilian ova, 162. 
 Rhizopods, 202. 
 
 Rose Bengale with Iodine Green, 48. 
 Rosein, 51. 
 Rossia, 176. 
 
 S. 
 
 Saccocirrus, 198. 
 Safranin (Pfitzner), 49. 
 
 with methyl green, 52. 
 Sagitta, 140, 208. 
 Salmonidaa, 154-155. 
 Salt solution, 143, 207, 211. 
 Sapphirina, 16, 17. 
 
 Sectioning, order of steps preparatory 
 to, 4. 
 
 combination, suppression, and trans- 
 position of steps, 4. 
 Section-observation, 2, 10. 
 Sections, ribbon method, 2, 
 
 cut under alcohol, 106. 113-116, 
 
 dripping apparatus, 115. 
 
 fixatives, 116-123. 
 
 collodionized, 127, 166. 
 
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INDEX. 
 
 253 
 
 Sections — continued. 
 
 prevention of rolling, 142, 143, 170-172. 
 
 orientation, 213. 
 
 of corals, 233. 
 Section-smoothers, 8&-92. 
 
 of Mayer, Andres, and Giesbrecht, 87, 
 88. 
 
 Schulze's, 88, 89. 
 
 Decker's, 90. 
 
 Kingsley's, 91. 
 
 of Gage and Smith, 91, 92. 
 Shad, 177. 
 Shellac, alcoholic, 116, 117. 
 
 carbolic acid, 117, 118. 
 Slide devised by Eabl, 185. 
 Skeletons prepared in eau de javelle, 31. 
 Spinal cord, 
 
 staining with rose Bengale, 48. 
 
 " " palladium chloride, 59. 
 
 Spirorbis, 138, 139. 
 Sponges, 201, 208. 
 Spongilla, 31. 
 Staining, 8. 
 
 alcoholic and aqueous solutions^ 8, 33. 
 
 qualifications of a dye for staining in 
 toto, 8. 
 
 with gallic acid, 25. 
 
 methods of, 33-54. 
 
 intra vitam, 52. 
 
 Haidenhain's method, 211. 
 Stickleback, 178. 
 
 Stomach, mucous membrane of, 211. 
 Surface-observation, 2, 10. 
 
 T. 
 
 Teleostei, 151-155. 
 
 Temperature, methods of keeping uni- 
 formly cool, 136, 151. 
 Tergipes, 175. 
 Thymol, 43. 
 Tobacco smoke, 204. 
 Tomopteris, 208. 
 
 U. 
 
 Urodela, 194-196. 
 
 Vaseline, 
 
 to prevent loss of a'lcohol from speci* 
 men jars, 236. 
 Yesuvian-celloidin, 225. 
 Yesuviu, 51. 
 
 w. 
 
 "Water, hot, for killing, 11. 
 "Water-bath, 104, 105. 
 "White perch, 179. 
 "Wickersheimer's fluid, 225, 227, 232. 
 
 Xylol, 119, 122, 192. 
 
 T. 
 
 Yellow perch, 174. 
 
 Zoochlorella, 186. 
 
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AUTHORS. 
 
 USmV 147-149,151, 152 
 inSeM,^, 66, 87, 95, 98, 140, 203, 204. 
 
 Bachmahn, 125. 
 
 Bachrach & Brothers, 108. 
 
 Balfour, 163. 
 
 Bastian, 59. 
 
 Bateson, 149, 150. 
 
 Beard, 139. 
 
 Bemmelin, 209. 
 
 Beneden, Ed. Tan, 16, 167-170. 
 
 Berthold, 14. 
 
 Betz, 194. 
 
 Bianco, 204. 
 
 Birge, 24, 192-194. 
 
 Bizzozero, 53. 
 
 Blanc, 201. 
 
 Blanchard, 26. 
 
 Blochmann, 97, 168, 113, 127. 
 
 Boecker, 81. 
 
 Boebm, 43, 155, 16-3. 
 
 Bottcher, 47, 181. 
 
 Born, 2, 160, 161, 170, 216. 
 
 Brandt, 52, 53, 186. 
 
 Brass, 19, 200, 201. 
 
 Brooks, 176. 
 
 Browning, 223. 
 
 Butschli, 95, 96. 
 
 Bunge, 109. 
 
 Calbtsbla, 106, 107, 109, 158, 163, 195. 
 Caldwell, 2, 61, 70, 86, 100. 
 Carnoy, 42. 
 Certes, 53, 201. 
 Ciaccio, 189. 
 Cybulsky, 56, 57. 
 
 Decktsb. 2, 90. 
 Dimmock, 34-38, 97, 98. 
 Dorp, Tan, 26. 
 Duval, 111, 126, 127, 163-166. 
 
 Eisig, 5, 12, 19, 23. 
 Emery, 16. 
 Engelmann, 19. 
 Entz, 201. 
 Erlicki, 24, 25. 
 
 Easchthg, 89. 
 Faxon, 174, 175. 
 Fewkes, 138, 139. 
 Flechsig, 56, 187. 
 
 Elemming, 18, 19, 47, 123, 181-183 186. 
 Fol 14, 18, 19, 20, 21, 22, 24, 25, 41, 59, 85, 
 
 99,125,145,219,231. 
 Fraipont, 198, 199. 
 Erancotte, 91, 116.. 
 Frenzel, 119, 120. 
 Freud, 25, 187. 
 
 Gagts, 81, 82, 91, 121, 127, 223, 224, 236. 
 
 Gannett, 115. 
 
 Gardiner, 212. 
 
 Gaule, 116, 119, 188, 192. 
 
 Gerlaeh, 34. , „„ ftrt „ D . 1c 
 
 Giesbrecht, 2, 27, 66, 87, 94, 95, 96, 98, 116, 
 
 119, 127. 
 Girard, 147. 
 Golgi, 58, 190. 
 Goronowitsch, 172. 
 Gravis, 106. 
 Grenadier, 35, 39, 110. 
 Griesbach, 48. 
 
 Hanamajn', 55. 
 
 Harmer, 58, 207. 
 
 Harris, 51, 54. 
 
 Hay, 227-231, 235, 236. 
 
 Heidenhain, 24, 186, 211, 212. 
 
 Heitzmann, 57. 
 
 Henle, 59. 
 
 Henneguy, 24, 154, 155. 
 
 Hermann, 47, 181. .„ 
 
 Hertwig, bsear, 29, 107, 140, 157-159, 162, 
 
 209. 
 Hertwig, Richard, 29, 58. 
 His, 2, 19, 58, 129 215. 
 Hoffman, 97, 102, 103, 137. 
 Hughes, 64. 
 
 IUIMA, 134, 135. 
 
 Jacob, 79, 80. 
 
 Julin, 169. 
 
 Jung, 60, 69, 86, 113. 
 
 Ifn!sley W 91, 117, 144, 145, 175, 176. 
 
 Kbrting, 69. 
 
 Koch, 233. 
 
 KBlliker, 18, 163. 
 
 Korschelt, 201. 
 
 Kossman, 65, 96. 
 
 Kleinenberg, 19-21, 44, 45, 47. 
 
 Krieger, 215. 
 
 Kronecker, 169. 
 
 254 
 
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AUTHORS. 
 
 255 
 
 Kupffer, 155, 162, 163. 
 KutscMn, 211. 
 
 Landsberg, 201. 
 Lang, 26, 27. 
 Lardowsky, 50. 
 Legros, 57. 
 Lewes, 81. 
 Lewis, 64. 
 Liebermann, 36. 
 Lockwood, 144. 
 Loewlt, 189. 
 
 Mark, 29, 116, 127, 128, 145-147, 170-172. 
 
 Mason, 2, 127. 
 
 Mason, J. J., 196. 
 
 Max Flesch, 24, 192. 
 
 Mayer, Paul, 2, 11, 12, 16, 17, 20-22, 27, 32, 
 
 33, 39, 40, 45, 46, 48, 53, 66, 87, 98, 104, 
 
 116-118, 122, 123, 127, 208. 
 McLachlan, 142. 
 Merkel, 23, 59, 111, 126. 
 Meyer, 208, 223. 
 Mlnot, 19, 24, 39, 42, 48, 76, 94, 113-115, 186, 
 
 192. 
 Moeller, 61. 
 Morse, 136. 
 Miiller, 24. 
 
 Noll, 30. 
 
 Ogatha, 210. 
 
 Osborn, 24, 194-196, 225-227. 
 
 Oschatz, 60. 
 
 Packard, 149. 
 
 Pansch, 223. 
 Patten, 86, 141-143. 
 Perenyi, 22, 23, 154. 
 Perls, 30. 
 Pfitzner, 19, 190. 
 Pfliiger, 159, 160. 
 
 Kabl, 19, 184, 185. 
 
 Rabl-Riickhard, 19, 172. 
 
 Ransom, 207. 
 
 Ranvier, 14, 30, 40, 57, 109, 188, 189. 
 
 Rathke, 141. 
 
 Rauber, 162. 
 
 Redding, 56. 
 
 Reichenbacb., 141. 
 
 Reicbert, 61. 
 
 Riebard, 232. 
 
 Rindfleisch, 109. 
 
 Rivet, 60. 
 
 Rollett, 212. 
 
 Rosenberg, 215. 
 
 Rossa, 223. 
 
 Roux, 161. 
 
 Roy, 81. 
 
 Rue, De la, 35, 36. 
 Ruge, 107, 109. 
 Rutberford, 76, 82. 
 Ryder, 52, 101, 102, 176-180. 
 
 S^fftiqen, 205, 206. 
 
 Sankey, 50. 
 
 Schafer, 30, 32, 125. 
 
 Schallibaum, 2, 116, 121, 122, 127. 
 
 Scballer, 35, 36. 
 
 Schanze, 60, 61, 69. 
 
 Schiefferdecker, 107, 108, 111, 112, 126, 128, 
 
 225. 
 Schulgin, 100. 
 Schulze, 2, 58, 59, 88. 
 Schweigger-Seidel, 35, 40. 
 Scott, 107. 
 Sedgwick, 94. 
 Seessel, 215. 
 Semper, 233. 
 Sbarp, 14, 127, 233, 234. 
 Smith, S. I., 175. 
 Smith, 91, 121. 
 Sollas, 83, 208. 
 Stimpson, 149. 
 Stohr, 217. 
 
 Strasburger, 19, 183, 184. 
 Swirski, 217. 
 
 Thanhoffer, 59. 
 Thoma, 61-65, 109-113, 127. 
 Threlfall, 120, 121. 
 Timm. 127. 
 Trinkler, 211, 212. 
 
 Uljakin, 150, 151. 
 Uskow, 217. 
 
 Verrtll, 149, 175, 176. 
 Virchow, H., 19, 209. 
 Vosmaer, 208. 
 
 Watts, 34, 35. 
 
 Weigert, 24, 25, 191, 192. 
 
 Weismann, 95. 
 
 Whitman, C. O., 19, 136-138. 
 
 Whitman, E. N., 174, 175. 
 
 Wikszemski, 223. 
 
 Wilder, 224, 236. 
 
 Will, 127, 143. 
 
 Wilson, 136, 144, 203. 
 
 Witlaezil, 143. 
 
 Wittich and Benkendorf, 108. 
 
 Zaddach, 141. 
 Zeiss, 60, 69. 
 Zimmerman, 85. 
 
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