551 
 K91 + 
 1913 
 v.l 
 
 HISTOLOGY-I MICROSCOPY 
 
 
 KRAUSE-SCHMAHL 
 
 ■ 
 
 RMMMMM 
 
551 
 
 QJattteU Untuwaitg Siihrarg 
 
 Jltliaai, &T CU1 fl orl; 
 
 BOUGHT WITH THE INCOME OF THE 
 
 SAGE ENDOWMENT FUND 
 
 THE GIFT OF 
 
 HENRY W. SAGE 
 
 1891 
 
Cornell University Library 
 QM 551.K91 1913 
 v.1 
 
 Course in normal histolog 
 
 3 1924 024 791 935 
 
M 
 
 %S 
 
 Cornell University 
 Library 
 
 The original of this book is in 
 the Cornell University Library. 
 
 There are no known copyright restrictions in 
 the United States on the use of the text. 
 
 http://archive.org/details/cu31924024791935 
 
A COURSE 
 
 IN 
 
 NORMAL HISTOLOGY 
 
PUBLISHERS' ANNOUNCEMENT 
 
 RUDOLPH KRAUSE— HISTOLOGY 
 
 We offer this book to teachers and students alike as a proper guide 
 in Histology. 
 
 Professor Krause strikes a keynote in his preface to the book. The 
 study of Histology should go hand in hand with that of Anatomy. The 
 one cannot be separated from the other nowadays without serious injury 
 to the student. 
 
 The microscope is the hand-maid of anatomy. For this reason it has 
 been deemed expedient to publish the book in two separate sections. 
 
 The first part is simply a guide to the technique of microscopy, and 
 may be used by students of medicine as well as by those who pursue sub- 
 jects of science foreign to medicine. 
 
 The second part deals exclusively with histology appertaining to medi- 
 cine. Either part may be obtained independent of the other. 
 
 In bringing this work before the public it is the author's aim to give 
 to the student a book of reference which is both practical and theoretical, 
 as well as to furnish the teacher with a text-book which provides him with 
 a detailed, yet clear, concise and methodical guide through the course of 
 Microscopy and Histology. 
 
 So far as the student of medicine is concerned, he will find therein much 
 valuable information appertaining to the microtechnique which must neces- 
 sarily be taught to the exclusive student of Pathology. The subject of 
 microtechnique is in most all English-written books on this topic treated 
 but very briefly. This work not only teaches the student, in a most thor- 
 ough fashion, the theory and manipulation of the microscope, but enables 
 him to acquaint himself with all the methods employed in preparing a spec- 
 imen for microscopical examination from start to finish. 
 
 REBMAN COMPANY. 
 
 New Yokk, 1123 Broadway. 
 
A COURSE 
 
 IN 
 
 NORMAL HISTOLOGY 
 
 A GUIDE FOR 
 
 PRACTICAL INSTRUCTION IN HISTOLOGY 
 AND MICROSCOPIC ANATOMY 
 
 BY 
 
 RUDOLF KRAUSE 
 
 A. O. PROFESSOR OF ANATOMY AT THE UNIVERSITY OF BERLIN 
 
 TRANSLATION FROM THE GERMAN BY 
 
 PHILIPP J. R. SCHMAHL, M.D. 
 
 NEW YORK 
 
 WITH 30 ILLUSTRATIONS IN TEXT AND 208 COLORED PICTURES, 
 
 ARRANGED ON 98 PLATES AFTER THE ORIGINAL 
 
 DRAWINGS BY THE AUTHOR 
 
 PART I. 
 
 NEW YORK 
 REBMAN COMPANY 
 
 1123 BROADWAY 
 
Copyright, 1913, by 
 
 REBMAN COMPANY 
 
 New York 
 
 All Rights reserved 
 
 PRINTED IN AMERICA 
 
DEDICATED 
 
 TO 
 
 PROFESSOR DR. WILHELM WALDEYER 
 
 SEC. MED. COUNCILLOR 
 
 IN GRATEFUL ESTEEM 
 BY THE AUTHOR 
 
Preface 
 
 The brilliant progress made in microscopy during the last three to four 
 decades has gradually brought with it a total change in the course of histologic 
 instruction. In former days the student himself prepared his own microscopic 
 specimens with razor and needle and with the aid of a few solutions for macera- 
 tion, hardening and staining. To-day, after recognizing the inefficiency of such 
 methods, this work has almost entirely been taken from the student's hands. 
 Instead he receives from the assistant or preparator more or less immaculate 
 specimens, preserved, cut and stained; some only for a single observation, 
 others as his property. The practical course has been replaced by one of 
 demonstration ; at any rate in all its essential phases. 
 
 Special courses have been designed for instruction in the technique of 
 microscopy, with the result that such courses have but a minimum percentage 
 of attendance. By far the majority of our students enter the clinical semesters 
 without being able to prepare a useful microscopic specimen. Not until later,, 
 during a course in pathologic histology or in the laboratories of clinics, will 
 they learn those micro-technical manipulations so essential to them as prac- 
 titioners. 
 
 This, in my estimation, is not a normal state of affairs. Every medical 
 student, upon entering the clinical semesters, should be able to master micro- 
 technical methods to such a degree that he is able to prepare a good specimen 
 for microscopic examination from any organ given to him. This will be possi- 
 ble only when the course in histology occupies that rank of which it is deserv- 
 ing; that is to say, when the student ceases to be granted the meagre allowance 
 of only four, at the utmost six hours per week; but, on the contrary, as has 
 already been attempted in several places, when he is permitted to make use of 
 one entire summer semester to such advantage as was proffered heretofore dur- 
 ing two preparatory winter semesters. Then, such courses as "Technical In- 
 struction," "Advanced Course," or whatever they may be called, will be dis- 
 pensed with. The average medical man must receive an adequate technical 
 education in histology. The scientist should seek the laboratory. 
 
 During the time that the student does practical histologic work, he should 
 be bound to improve his theoretical knowledge also, which at the very best, 
 those who know will admit, leaves much room for improvement under present 
 conditions Without doubt the student takes a greater interest in a specimen 
 
 vi 
 
Vll 
 
 made by himself than in one already prepared, whether presented to him or 
 only loaned. 
 
 The aim of this book is to direct the student to a practical education in 
 histology and to microscopic anatomy, by the simplest methods. Most of the 
 specimens described can be prepared from beginning to end by the student him- 
 self, if he employs the technique of freezing, which has long since acquired the 
 title of "standard" in pathologic institutions, but was wofully neglected by 
 anatomists in the past. 
 
 By dividing a larger class of students into separate divisions, each one of 
 which being supplied with a freezing microtome, every participant will find 
 ample opportunity during the summer period to attain experience in microt- 
 omy. Furthermore, it will be possible for every student of each single group 
 to go through all the methods of embedding in paraffin and celloidin at least 
 once, from beginning to end, during the semester. Moreover, it must be under- 
 stood that unstained paraffin sections are given out on isinglass. The staining 
 must be done in all cases by the student himself, whether frozen, paraffin or 
 celloidin sections are used. 
 
 It goes without saying that staining of living tissues with methylene blue, 
 injection of blood-vessels and lymph-ducts, and similar complicated technical 
 manipulations cannot at once be performed by the student himself. He will 
 learn by witnessing these procedures. And this, too, can be easily accomplished 
 when the classes are grouped into subdivisions. 
 
 I assume, against custom, that the student is supplied with a modern micro- 
 scope having a condenser and homogeneous immersion. The latter, of course, 
 may be replaced by high power dry system to some extent. I hold that what 
 has been an established factor in the bacteriological course hitherto, should also 
 be made possible in histology. When we look at the time-honored Schicks, 
 Ploessls, Hartnacks, etc., employed in some of the histologic courses, we cannot 
 help but notice that we have not kept in step with other lines of our science. 
 Granted that the experienced eye can see most things well enough even with the 
 aid of such instruments, we should not add to the difficulties which confront the 
 beginner, the additional tax of inadequate tools. On the contrary we should 
 seek to assist him in his studies by placing at his disposal the most perfect in- 
 struments. It is of the greatest importance to use the best drawings that can 
 be made of the specimens, and every student should be obliged to make faithful 
 sketches of his slides. Microscopic drawing is not an art in the strict sense 
 of the word. In order to stimulate drawing by students, I have undertaken 
 the task of sketching every preparation myself. These drawings, no more than 
 those of students, are not intended nor expected to be works of art, but solely 
 true representations of the microscopic findings. Each sketch has been pre- 
 pared with the aid of Abbe's drawing apparatus, and the enlargement is marked 
 
vm 
 
 on every one. The fraction, which is often annexed, indicates how much the 
 original has been reduced in the reproduction. 
 
 To obtain the truest image possible of the original drawing, I have em- 
 ployed the autotype, which has attained such a standard of perfection to-day 
 as to bring out the finest details of a microscopic image. The cuts have been 
 made in a most excellent manner by the Angerer and Goeschl Art Printing Co., 
 of Vienna. The printing was done by Christoph Reisser's Sons, also of Vienna. 
 To both firms I am indebted for their readiness in giving my wishes the closest 
 attention, and also for the superior execution of the plates. 
 
 A text is furnished with every drawing. It precedes each plate, explains 
 the technique, and gives a brief description of the specimen. If the latter is 
 not confined entirely in all the details to the drawing, but deals also with other 
 matters, not depicted, it does not follow that this guide-book pretends to 
 represent a text-book of histology, which it cannot be any more than the his- 
 tologic course can be an adequate substitute for a course of lectures in his- 
 tology and microscopic anatomy; they supplement one another, but the one 
 cannot replace the other. For that reason it is desirable that text-books on 
 histology should cast off that technical ballast which they carry in contra- 
 distinction to other departments. 
 
 In fine I deem it a pleasurable duty to heartily thank my publishers. They 
 have met my wishes in every detail and have spared neither pains nor money in 
 order to equip this volume in the most lavish manner. 
 
 Berlin. Rudolf Kbattse. 
 
Translator's Preface 
 
 At the request of Mr. F. J. Rebman, I have undertaken the translation of 
 Rudolf Krause's Histology into the English language. I have adhered to the 
 German text as closely as possible, without, however, making sacrifices to 
 idiomatic expressions. My aim has been to make the book as useful to the 
 student of medicine in English-speaking countries, as is compatible with the 
 spirit in which the author has written it. 
 
 Professor Krause seeks to raise the level of instruction in Histology to that 
 obtained during recent years in other departments of medical education. He 
 deserves encouragement in this praiseworthy endeavor, and the authorities 
 entrusted w,ith the education of aspirants in medicine at the various colleges 
 are supporting him in his claim. 
 
 It affords me pleasure, therefore, to be enabled to place at the disposal of 
 teachers and students of Histology an English version of Professor Krause's 
 teachings, and I sincerely trust that the book will find a proper place in the 
 curriculum of the medical schools of all countries in which the English language 
 is spoken. 
 
 I take pride in expressing my obligation to Mr. Rebman for the aid he has 
 given me in preparing my manuscript, especially in the translation of the 
 preface by the author. I likewise cheerfully acknowledge the assistance derived 
 from the perusal of- Edward Bausch's "Manipulation of the Microscope." 
 
 P. J. R. SCHMAHL, M.D. 
 
 New York City. 
 
 IX 
 
Table of Contents 
 
 PAGE 
 
 Preface vii 
 
 Translator's Preface xi 
 
 Introduction 1, 
 
 General Part ■ • • 3 
 
 The Microscope 5 
 
 The Objective 12 
 
 The Eyepiece 17 
 
 Illumination 18 
 
 The Micrometer-Screw , 21 
 
 Rules for the Use of the Microscope 21 
 
 General Microtechnique 2-1 
 
 Methods of Preservation 25 
 
 Preparation of Sections 34 
 
 Dissociation Methods 34 
 
 Chopping Method 35 
 
 Cutting Methods 36 
 
 (a) Frozen Section 37 
 
 (b) Paraffin Section 41 
 
 (c) Celloidin Section 46 
 
 Selection of Method 47 
 
 Appendix 48 
 
 Staining Methods 48 
 
 Stains of Animal Origin 54 
 
 Stains of Vegetable Origin 55 
 
 Artificial Stains 58 
 
 Basic Dyes 58 
 
 Acid Dyes 63 
 
 Indifferent Stains 65 
 
 Staining Mixtures 65 
 
 Appendix 68 
 
 Methods of Inj ection 73 
 
 Mounting and Finishing 76 
 
 Mensuration and Drawing 79 
 
 x 
 
Introduction 
 
 THE AIM OF A COURSE IN HISTOLOGY 
 
 The object of the histologic course is a dual one: on one hand it should 
 serve as a completion of the theoretical lectures on general and systematized 
 anatomy, i.e., it should demonstrate ad oculos what the student has been told 
 of the microscopic structure of the human body and enable him to understand, 
 recognize and differentiate for himself the organs and the tissues composing 
 them. The daily practical work with the microscopic preparation should give 
 him a fixed and definite idea of the normal structures of the various organs, 
 in order to enable him in later semesters to recognize and diagnose the patho- 
 logic changes found therein. 
 
 If this were the only object, it would suffice to assign a number of good 
 specimens to the student, to discuss them thoroughly with him, and to let him 
 reproduce them with the pencil on paper. But such is not the case. Our 
 course of instruction has a secondary aim, equally important and a good deal 
 more practical, viz., to initiate the student in micro-technique, which latter 
 is of the greatest importance for advanced study as well as general practical 
 education. At the present day the microscope is probably the most important 
 and necessary requisite of a medical instrumentarium. A thorough training 
 is needed for handling it in the proper manner, and the basis of this training 
 rests with the histologic course, at the end of which the student should be 
 thoroughly familiar with the microscope and its use. 
 
 But this is by no means sufficient. The use of the microscope presupposes 
 the knowledge how to prepare a specimen for examination, and for this reason 
 it becomes necessary for the student to acquire skill in what is commonly 
 termed microscopic technique or micro-technique. This, however, is 
 an extremely large field, which requires years of study to master, and no sane 
 person will ask the student to familiarize himself with all the innumerable 
 methods and details of procedure. Nevertheless he should attain that amount 
 of technique to enable him to prepare, from any given organ, a specimen which 
 clearly shows its structural peculiarities. 
 
 To be a guide in accomplishing this end is the object of this volume. 
 Primarily it will familiarize the reader with the microscope and its use. Fur- 
 thermore, it will bring to his attention the most important reagents, stains', and 
 microtechnical methods; and lastly, in the Special Part it will give detailed 
 instructions for the production of good specimens, suitable to the study of each 
 tissue and organ, and to elucidate in words and pictures what may be learned 
 
 from these specimens. 
 
 1 
 
Untiring pains have been taken to reproduce the most characteristic 
 parts of a preparation. The cuts should stimulate the reader to draw speci- 
 mens for himself. If the first endeavors should prove unsatisfactory, there is 
 hardly anything else that will be so easily and quickly learned with a little 
 perseverance and a bit of adroitness, than is microscopic drawing, which does 
 not, as the beginner generally fears, necessarily require any special talent. 
 Not until drawing it, do we fully appreciate a specimen and learn all about 
 it, and our drawing, when completed, will forever remain vivid in our memory, 
 an indelible picture. 
 
GENERAL PART 
 
The Microscope 
 
 The elements constituting the human and animal organism are of such 
 minute size that in most instances they cannot be recognized by the unaided 
 eye ; the necessity arises therefore, to employ certain instruments which will 
 present to our eye a magnified image. The knowledge of such agents dates far 
 back in history. The fact that lenses, i.e., transparent spherical bodies, will 
 under certain conditions magnify the articles viewed through them, has been 
 known for ages. 
 
 Lenses as a Means 
 of Magnifying. 
 
 In ancient times lenses were made from more or less valuable natural crys- 
 tals. This is on rare occasions done even to this day ; but as a rule lenses are 
 now made of artificial glass of a fixed chemical combination, for it has been 
 found that the composition has a great influence on the optic properties, i.e., 
 on refraction and dispersion power. Lenses with moderate dispersion and 
 greater refraction power are designated as crown glasses, those with high 
 dispersion as flint glasses. In the preparation of the latter a large per- 
 centage of lead oxide is added to the liquid glass. They also contain arsenic, 
 manganese, and, of course, silicon dioxide in small amounts. Crown glasses, 
 on the other hand, contain also a good percentage of silicon dioxide, the alkali 
 metals, alkaline earth metals and boric acid anhydride. It follows that flint 
 glasses are, as a rule, heavier than crown glasses. The dispersion power of the 
 flint is in direct proportion to its weight, i.e., amount of lead contained in it. 
 
 Classification of Lenses. 
 
 According to their form lenses may be divided into two main divisions: 
 those which are thicker in their centre, the optic axis, than on the periphery, 
 and those in which the thickness decreases from periphery to optic axis. The 
 
 Fig. 1. 
 Positive Lenses. 
 
 Fig. 2. 
 Negative Lenses. 
 
 former are called positive or convex lenses, while the latter are known as 
 negative or concave lenses. Under the positive lenses we again distinguish 
 between biconvex, plano-convex and positive meniscus (Fig. 1). All parallel 
 
 5 
 
rays falling on a positive lens are known to unite in a point on the other side 
 of the lens, situated on the optic axis and called the focus. The distance of 
 the focus from the lens is termed focal distance. Parallel rays falling on a 
 negative lens are dispersed in such a fashion as if emanating from a point this 
 side of the glass. Here also we speak of focus and focal distance, but prefix 
 them with negative or virtual. Among the negative lenses we also speak of bi- 
 concave, plano-concave and negative meniscus (Fig. 2). 
 
 Image-Formation Through 
 a Biconvex Lens. 
 
 According to known optic laws a biconvex lens furnishes an inverted real 
 picture of an object, situated beyond its focal distance, i.e., in our case (Fig. 3) 
 
 Fig. 3. 
 Reproduction through a Biconvex Lens. 
 
 Fig. 4. 
 Eeproduction through a Biconcave Lens. 
 
 an image situated to the right of the lens (a x b 1 —A 1 B t ). If the object 
 approaches the focus, the picture recedes from the lens, becoming larger 
 (« 2 b 2 —A 2 B 2 ), and finally, when the object occupies the focus, is lost ad 
 infinitum. If the object encroaches on the focal distance itself, we get an erect, 
 enlarged, virtual image, situated to the left of the lens (a 3 b 3 —A 3 B 3 ). The 
 closer the object approaches the lens the smaller the picture will be, though 
 always remaining larger than the object itself (a 4 b 4 — A 4 S 4 ). 
 
The focal dis- 
 
 ji\: 
 
 
 A 
 
 
 / 4 
 i ? \ 
 
 if" 
 
 
 
 \ / H 
 
 
 \k j 1 */ 
 
 
 
 / 
 
 
 TTie Biconcave Lens. 
 
 Much simpler is the action of a biconcave lens. It gives us under all con- 
 ditions a reduced erect image, which becomes larger as the object is approxi- 
 mated to the lens (Fig. 4). 
 
 Magnifying. 
 
 If we want to distinctly perceive with our eyes a picture, reflected by a 
 lens, it becomes necessary to establish a distance between image and eye of 
 approximately 250 mm, i.e., to place it in distinct visual distance. The mag- 
 nifying power furnished by a certain lens is dependent upon its focal dis- 
 tance and is found by dividing the distinct visual distance of the particular 
 observer by the focal distance, both expressed in millimetres, 
 tance in turn is dependent upon the curvature of the 
 planes of the lens and the refraction index of the par- 
 ticular glass. For example, we find a focal distance of the 
 lenses, represented in Fig. 1, of 50 mm for the biconvex, 
 100 mm for the plano-convex and 150 mm for the menis-> 
 cus, presuming the refraction index to be 1.5. These 
 lenses, therefore, would possess a magnifying power of 
 5, 2.5 and 1.6 respectively. With the negative lenses of 
 Fig. 2, on the other hand, we get a focal distance of — 50 
 mm for the biconcave, — 100 mm for the plano-concave 
 and — 150 mm for the meniscus. These lenses, therefore, 
 will reduce correspondingly. 
 
 Very often two or more lenses are combined and ce- 
 mented together by a transparent resin in a fashion that they are intimately 
 approximated. They may be considered as one lens with a reciprocal focal 
 distance equal to the sum of the reciprocal focal distances of the component 
 lenses ; e.g., the biconvex lens will gain in focal distance by adding a positive 
 meniscus (Fig. 5, a), while it will lose when combined with a plano-concave 
 lens (Fig. 5, b). Again, the components may be made of the same sort of 
 glass or, what is done much more frequently, two different kinds of glass with 
 different refraction and dispersion power are selected; e.g., a combination is 
 made of a plano-concave lens of flint glass and a biconvex crown glass. 
 
 After what has been said it would seem that the strongest enlargement 
 could be obtained by the use of a simple or a combined positive lens, since it 
 would be only necessary to use lenses with a corresponding short focal dis- 
 tance. A biconcave lens of 1 mm focal distance, i.e., 1 mm curvature radius, 
 should give us an enlargement of 250 magnifying power. Practically this can- 
 not be accomplished, since such small lenses, aside from the difficulty of their 
 manufacture, are too weak, the distance between object and eye would become 
 too small, and finally such lenses would possess the two great shortcomings of 
 every spherical, refractive plane, namely, spherical aberration and chromatic 
 aberration, to such a degree as to make them useless in practice. 
 
 Fig. 5. 
 Compound Lenses. 
 
 Spherical Aberration. 
 
 By spherical aberration we understand that peculiarity of every 
 spherical, refractive plane by which only those of the parallel rays of light that 
 
are nearest the optic axis meet in the focus, while those nearer the periphery 
 are united nearer the lens, approximating the latter more and more the nearer 
 they are to the periphery. 
 
 Chromatic Aberration. 
 
 Since red rays are refracted less than blue, the former will have a greater 
 focal distance than the latter. This phenomenon has been called chromatic 
 aberration. 
 
 To avoid spherical aberration the peripheral rays are deflected, but also 
 can be obliterated by correct construction and position of the lenses. For 
 instance, a biconvex lens with equally curved planes will show a greater aberra- 
 tion than one in which the curvature radii varies, bearing a fixed relation to 
 one another. A plano-convex lens will give less aberration than a biconvex, if 
 the piano-surface of the former is directed toward the incoming rays. To 
 avoid chromatic aberration the optician combines lenses of different dispersion 
 power. He will, for instance, correct the dispersion power of the biconvex 
 
 Fig. 6. 
 Reproduction by Two Piano-Convex Lenses. 
 
 crown-glass lens (Fig. 5 fo,) by a plano-concave lens of flint glass, thereby sacri- 
 ficing, however, the refractive power. 
 
 The Combined System. 
 
 In practical work one never goes below a focal distance of 10 mm in a 
 single lens, hence, with such a lens, no enlargement greater than 25 X is ever 
 obtained. If greater power is desired, two or more lenses, separated a certain 
 distance from one another, are used. Such a combination we find illustrated in 
 Fig. 6. Here we have a plano-convex lens (L t ) of 20 mm focal distance. 
 The object a o, being situated right in front of the anterior focus, the lens pro- 
 jects a reversed enlarged real image o 1 b 1 . This image lying again within the 
 focal distance of a second plano-convex lens (L 2 ) of 40 mm focal distance, a 
 second, still more enlarged, virtual and also reversed picture (referring to the 
 object) a 2 b 2 is produced. Hence, by projecting the image of a primary posi- 
 tive lens within the focal distance of a second positive lens, we are enabled to 
 produce a second enlargement of the image, and from what has been said it is 
 evident that this enlargement will become greater as the distance between the 
 two lenses is increased, i.e., dependent on how near we bring the first image to 
 the anterior focus of the second lens. 
 
 Course of Rays in the 
 Compound Microscope. 
 
 In Fig. 7 things are still more complicated. Here we have taken three 
 lenses into Our optic system: the plano-convex L x (20 mm), the convex-piano 
 
9 
 
 L 2 (40 mm) and the convex-piano L 3 (30 mm). The distance between the 
 second and third lenses has been so chosen as to amount to half the sum of 
 their focal distances, viz., 35 mm. The size of the object and its distance from 
 the anterior focus of the first lens being the same as in the previously men- 
 tioned case, the image obtained would also be the same, if lens L 2 had not been 
 interposed in the course of rays, converging the rays and producing the much 
 smaller image a. 2 b 2 , instead of a 1 b x . This second picture is projected within 
 the focal distance of the much more curved lens L 3 , which finally furnishes the 
 greatly enlarged image a z b s . 
 
 A similar arrangement we find in our compound microscope. Here, 
 too, an enlarged reversed real image of the object is projected through a lens 
 or rather a system of lenses, the focal distance of which ranges between 1.5 
 and 40 mm, since the object is situated closely anterior to the focus of this 
 system. The latter, being so proximal to the object, is termed the objective 
 or object-glass. The lenses L 2 and L 3 are united into a system, which, being 
 
 Fig. 7. 
 Course of Rays in the Compound Microscope. 
 
 proximal to the eye of the observer, has been termed the ocular or eye- 
 piece. The lens L 2 again is called collective lens, while L 3 receives the 
 name eye-lens. Here, too, as in the previous case, the distance between the 
 eye-lens and the objective lens equals about half of the sum of their focal dis- 
 tances. On the other hand, in the microscope the eyepiece is much more dis- 
 tant from the object-glass, as in our given example, the distance of the upper 
 focus of the object-glass from the lower one of the eyepiece being 160 mm. 
 This distance has been designated as the optic or reduced tube-length 
 (A), and is chosen so as to project the image produced by the eye-lens of the 
 evepiece at a distance of 250 mm, i.e., in the distinct visual distance of the 
 observer. 
 
 Magnifying Power of the 
 Compound Microscope. 
 
 The enlargement produced by such a combined microscope is always 
 easily figured out if the focal distances of the object-glass (Fj) and the eye- 
 piece (F 2 ) are known, viz.: 
 
 350 , A 
 
 After these initiating remarks let us proceed to the description of a micro- 
 scope, which seems especially adapted for our purpose. 
 
10 
 
 Description of the Microscope. 
 
 Our instrument (Fig. 8) rests on a horseshoe-shaped foot or base, sur- 
 mounted by the barrel or body. Generally we find in the latter, right over 
 
 eyepiece " ' " ' 
 
 
 
 
 obturator ~~ "~" 
 
 
 rack and pinion 
 
 
 
 
 
 _ milled-heads (coarse adjustment) 
 
 
 - 
 
 
 ^H 
 
 
 
 i 
 
 
 
 
 draw-tube _____^___ 
 
 
 
 ] ^0m 
 
 3k^ 
 
 
 
 
 
 
 nosepiece _ * Ss ^ w 
 
 an. yiL ^ W 
 
 
 
 
 /%M§T' ■ 
 
 
 
 
 
 
 | : J XV 
 
 
 
 
 Opening - in object-table 
 
 ^^gJaU-g^J 
 
 
 
 
 
 QH object-table or stage 
 
 
 condenser ; 
 
 
 
 ■ jr»Iti+- 
 
 
 
 
 ecrew for condenser — 
 
 
 
 nillar 
 
 
 
 
 
 
 \w 
 
 
 
 
 
 
 
 
 
 
 ^^w*88 
 
 
 
 
 
 foot 
 
 
 
 
 Fig. 8. 
 
 
 
 
 The Microscope. 
 
 
 
 the foot, a joint, which allows an oblique position of the instrument, thus per- 
 mitting of a more convenient examination in the sitting posture. Closely above 
 this joint is the square stage, intimately connected with the barrel and having 
 a round opening in its middle. Here the specimen, fixed by clips, is deposited 
 
11 
 
 for examination. The table divides the microscope into a lower and upper 
 part. 
 
 On the inferior surface of the stage a projection is found, the condenser, 
 a system of lenses set in metal, for illuminating purposes. By means of a 
 screw the condenser can be adjusted. By turning it to the right, you ap- 
 proximate the apparatus to the stage, gradually introducing it through the 
 opening in the stage, until the upper surface of its superior lens is on a level 
 with the upper surface of the table. Turning to the left, the condenser will 
 recede from the table, and finally swings out to the left. 
 
 On the lower portion of the condenser two knobs are seen, one serving to 
 open and close the diaphragm, the second being attached to a swinging ring, 
 into which colored (e.g., blue) glass plates may be inserted, such as are neces- 
 sary, when working in artificial light. 
 
 Below the condenser the mirror will be found, a silvered double mirror, 
 having one plane surface on one side and a concave on the other. It is con- 
 nected with the pillar by- a movable lever and suspended in a metal crescent so 
 as to allow of any position. 
 
 Above the object-table the pillar continues as a trigonal metal prism, 
 visible only for a few millimetres, as it is enveloped by a hollow metal cylinder, 
 the barrel carrier. The hollow of the carrier corresponds exactly to the size 
 of the prism, so that the two are in intimate relation. On top the carrier is 
 surmounted by a hoodlike screw head, the head of the micrometer-screw, 
 in front it is elongated into an armlike projection, which supports the barrel. 
 
 The barrel or tube is a hollow brass cylinder. Above it is mounted 
 by a ring, through which glides a second graduated tube, the draw-tube. 
 The scale is graduated in millimetres, ranging from 140 to 200 mm or 
 14 to 20 cm, starting on top with the smallest figure and ending below with the 
 largest. These figures represent the tube length, figured from the inferior 
 outlet of the barrel, to which the objective is attached, up to the superior bor- 
 der of the draw-tube, on which the eyepiece rests. If we draw out the draw- 
 tube we increase the distance between objective and eyepiece, and hence be- 
 tween their respective foci. Thereby the magnifying power of the microscope 
 is increased. 
 
 This fact may be made use of when employing a weak objective, but is 
 objectionable with the stronger object-glasses, as the latter have been con- 
 strued for a certain tube length, viz., 160 mm. We must also take into con- 
 sideration that in our instrument a nosepiece, 8 mm thick, is inserted between 
 barrel and objective, which must be allowed for, so that for all the stronger 
 enlargements the barrel must be drawn to 152 mm, i.e., the draw-tube must 
 register 15.2. 
 
 Body and arm are connected by rack and pinion, the rack forming part of 
 the body posteriorly, the pinion part of the arm, the teeth engaging in recesses 
 on a transverse rod, which has screw-heads on either end, the coarse adjust- 
 ment. By turning these knobs the barrel is lowered or raised, thus approxi- 
 mating or receding from the stage. 
 
 On the lower end of the barrel the so-called nosepiece is found, for the 
 purpose of quickly changing objectives. It consists of a metal plate having 
 three arms, each arm being provided with a threaded ring, the whole revolving 
 
12 
 
 on a common axis. The plate itself has a threaded hole, by virtue of which it 
 is attached to the barrel. When screwed in place the three-armed piece will 
 revolve around the plate in such fashion as to fit the rings exactly to the 
 lower barrel opening. A spring ( X ) in the back of the nosepiece indicates 
 whether the position is accurate, so that one may look right through the barrel 
 and one ring, the other two being covered by the plate. 
 
 The eyepiece is attached to the upper aperture of the draw-tube, while 
 below, the objectives are screwed to the rings of the nosepiece. The latter 
 should be placed in consecutivcorder, viz., assuming objective 3 to be in place, 
 first objective 6 and then objective 1 / 12 should follow, when the nosepiece is 
 turned to the right. 
 
 Several of the parts of the microscope spoken of require a more thorough 
 discussion, principally the objectives, the eyepieces, the condenser and 
 finally the micrometer -screw. 
 
 THE OBJECTIVE 
 
 The Significance of the 
 Objective. 
 
 As already explained in our general discussion of refraction of rays in the 
 compound microscope, the purpose of the objective is to furnish a real, re- 
 versed, enlarged image of the object to be examined. The latter simply being 
 enlarged again by the eyepiece, it must needs be free of all refractive errors, 
 free of all spherical as well as chromatic aberration. This* leaves no question 
 that the objective is the most important part of the entire microscope, being 
 of much greater consequence than the eyepiece, since we can merely enlarge 
 the objective image considerably by a strong eyepiece, without bringing any 
 new elements into the microscopic picture. A structure not previously re- 
 vealed by the objective will never be disclosed by an eyepiece, no matter how 
 strong the latter may be. 
 
 Structure of the Objective. 
 
 In order to comply with all the requirements, the objective in the compound 
 microscope cannot, as we have supposed in our earlier discussion, be repre- 
 sented by a single lens, but is composed of a series of parts, arranged in a 
 common metal setting in such a fashion that all are exactly concentric, i.e., all 
 lie in the same optic axis. 
 
 As a rule, the different members are connected to one another at a fixed 
 distance, although in rare instances the distance may be altered within certain 
 limits. 
 
 Each member is attached to the lower end of a brass tubule, and these 
 tubules, sometimes separated by an interposed metal tube, are screwed one 
 into the other. According to the number of lenses we have double, triple and 
 quadruple systems. Commonly each successive member is larger than the pre- 
 ceding, making the first member, the so-called anterior or front lens, the 
 smallest of all. 
 
 This front lens is the principal or sole agent in magnifying, while the re- 
 maining lenses or correction lenses have as their object the clearing out of 
 errors from the image furnished by the front lens. Hence the size of the front 
 
13 
 
 lens will give us an estimate of the power of the entire objective, so that we 
 are able to say that, the larger the front lens is, the lower will be the power of 
 the objective. Since low-power objectives have a greater focal distance that 
 high power, the object, however, necessarily being without the. focal distance 
 of the objective, it will be seen that during a sharp focus the distance between 
 object and front lens of objective, the so-called free object distance, is 
 greater in low-power objectives than in high. For example, with an objective 
 of 42 mm focal distance we will get an object distance of 40 mm, while one of 
 1.8 mm will have only 0.17. In the latter, therefore, the front lens must be 
 brought very near to the object. 
 
 In low-power objectives this front lens is mostly composed of several single 
 lenses; in high-power object-glasses, however, there is generally one single 
 plano-convex (hemispherical) lens, the plane surface being turned toward the 
 object. Most of the weaker objectives are doublets, one combination lens fol- 
 lowing the combination front lens. Here both members take part in the mag- 
 nifying N as well as correcting process. Medium objectives have two combined 
 correction lenses above the front lens, the latter furnishing the power, while 
 in the most powerful object-glasses we find between front lens and the correc- 
 tion systems a fourth simple lens, either plano-convex or a positive meniscus. 
 
 Achromatic and Apochromatic 
 Objectives. 
 
 The individual systems, and the lenses composing them, are made of dif- 
 ferent artificial glass compounds, each possessing its own refractive and dis- 
 persive qualities. Recently objectives have been constructed with lenses made 
 partly of a single mineral, namely Ruor spar. As regards refractive index, 
 this mineral only equals crown glass, but it excels all artificial glasses in its 
 dispersive power. By the use of fluor spar the greatest color purity of the 
 microscopic image has been obtained. Objectives made of glass solely are 
 known as achromatic, those in which fluor spar is used as apochromatic. 
 For our purpose the latter may be disregarded, being much more expensive 
 than the former on account of the high-priced raw material, without yielding 
 much better results for our work. 
 
 Oil Immersion Objectives. 
 
 Finally, there is one more very important point to deal with. Heretofore 
 we have always assumed that the rays emanating from the object enter the 
 front lens directly through the air. In reality this is not so, as in most cases 
 our object is covered with a thin plate of glass, the cover-glass. The rays of 
 light therefore are first refracted by this cover-glass before reaching the front 
 lens or, to be more exact, they emanate from the object, pass through the 
 cover-glass, thence into the air and finally enter the front lens. The sealing 
 medium possessing about the same refractive index as the cover-glass, the 
 rays of light emerging from the object will pass the cover-glass unrefracted, 
 but become refracted at the surface of the cover-glass, when the following 
 takes place : since they pass from glass to air, they are everted from the optic 
 axis, so that the angle of exit is greater than the entrance angle, hence a much 
 smaller part of the light- rays enters the objective than we have heretofore 
 supposed. This fact is not of great importance when dealing with objectives 
 
14 
 
 having a large front lens, i.e., low-power objective, but is very noticeable when 
 a small front lens is used. This condition is illustrated in the right half of 
 Fig. 9, while the left half shows the remedy for it. All we have to do is to 
 interpose between front lens and cover-glass a medium of the same refractive 
 index as the latter, and the ray a b, otherwise refracted, will now reach the 
 objective unrefracted. 
 
 The interposing of such a medium, which necessarily must be liquid, is 
 done without difficulty, since the high-power objectives have but a small object 
 distance. A drop of condensed cedarwood oil is placed on the cover-glass, the 
 barrel is lowered so far as to allow the front lens to dip into the drop and thus 
 a homogeneous connection between object and objective is effected. Such ob- 
 jectives are called homogeneous immersion objectives or oil-immer- 
 sion lenses, and are to be differentiated from immersion objectives, where 
 
 front lens -^^ 
 
 
 \ 
 
 
 
 immersion 
 
 liquid or air 
 
 respectively — ^^ 
 
 
 5 \ 
 
 
 
 cover-glass _____^ 
 mounting- 
 
 
 \ 
 
 
 
 
 
 I ■ ' \ ■ 
 
 
 J 
 
 medium — — 
 
 
 
 
 slide ^ . 
 
 
 
 i 
 
 
 ^ ■ 
 
 
 
 
 
 
 
 Fig 
 
 9. 
 
 
 
 
 The Effect of Immersion. 
 
 
 
 no oil but some other liquid is used as a medium. We thus hear of immersion 
 systems, where water is used, viz., a liquid of lower refractive index than oil, 
 or those in which monobrotne naphthalin is the medium, the latter of a 
 higher refractive power, which is used when a front lens of strongly refractive 
 flint glass is engaged. 
 
 Angular Aperture 
 
 From previous remarks it will be seen that an immersion lens is superior 
 to a dry lens of the same focal distance and aperture, since in the latter the 
 available aperture of the objective is not fully utilized. The angular aper- 
 ture, or, in short, the angle of an objective, are terms used to designate the 
 angle, having its apex at the central point of the object, its arms extending to 
 the periphery of the front lens of the objective, when the object is in sharp 
 focus. Its size is dependent upon the free object distance of the objective and 
 the diameter of the front lens. The medium separating objective and cover- 
 glass must also be taken into consideration when calculating the angle. The 
 figure obtained when considering both, is the numerical aperture found by 
 
15 
 
 multiplying the refractive index of the interposed medium with the sinus of 
 half of the angular aperture: 
 
 «=« . sin u. 
 
 Since in the dry system n equals 1, the aperture in such is equal to half 
 the opening angle. With water immersion this value must be multiplied with 
 1.33, with oil immersion by 1.51, and with monobrome naphthalin by 1.62. As 
 the angle u can never attain 90°, its sinus, and hence the aperture of every dry 
 system, is always less than 1. On the other hand a may be larger than 1 when 
 immersion systems are used. 
 
 The Requisites of 
 an Objective. 
 
 A good objective should possess the following qualities : 1. It must be 
 achromatic, i.e., red and blue rays should unite absolutely. 2. A planatic 
 
 image should be obtained. 3. All rays emanating from one point of the 
 object should, as far as possible, be united in one point of the image, i.e., 
 sharp pictures should be furnished; no blurring should take place. 4. It 
 should furnish detail, i.e., the finest structures of our specimen must be re- 
 produced. 
 
 The achromatic correction of the objective, as we have seen, is brought 
 about by employing lenses of varying dispersion properties. To procure 
 aplanasia and the greatest sharpness, spherical aberration must be, as far as 
 possible, obliterated. As has been previously mentioned, the different mem- 
 bers of the objective are made up of lenses of various refractive powers and 
 thus the preceding member is corrected by the succeeding one. The optician 
 at times even goes as far as to overcorrect such a member, subsequently bal- 
 ancing this overcorrection by the following lens. The detailing power finally 
 is wholly dependent upon the aperture and is in direct proportion with the 
 increase of the angle. 
 
 Objectives are marked by their manufacturers either with figures (Arabic 
 or Roman) or with letters, giving a higher number or a later letter of the 
 alphabet to the stronger objective, and vice versa. Immersion objectives, on 
 the other hand, and also the briefly mentioned apochromatics, are known by 
 their focal distance, the former expressed in inches, the latter in millimetres. 
 Objective 1/12 therefore will possess a focal distance of 1/12 inch, i.e., 
 2.1 mm. 
 
 Objective 8. 
 
 After these general preliminaries let us consider more closely those ob- 
 jectives which are of special interest to us (Figs. 10-12). Turning to figure 
 10 we find a section through objective 3. Here we have the type of a weak 
 objective, consisting of only two lenses, these two members, however, being 
 double lenses. The first member has a diameter of 6 mm and consists of a 
 plano-concave flint-glass lens and a biconvex crown-glass lens, the focal dis- 
 tance being 15 mm. The second member, which is of the same combination, 
 has a diameter and curvature radius 3 mm greater than the former, the focal 
 distance being 29 mm with a distance from the first member of 14 mm. Thus 
 the entire objective has a focal' distance of 16.2 mm, a free object distance of 
 
16 
 
 5.5 mm and a numerical aperture of 0.3. The objective alone furnishes an 
 enlargement of 10.3, with the weakest eyepiece, however, one of 41 ; in the 
 latter case it still covers a field of 2.1 mm diameter. 
 
 Objective 6. 
 
 Fig. 11 (objective 6) represents a system of medium strength. The three 
 members composing it are placed in separate metal shells at distances 0.15, 
 respectively 0.65 mm. The second shell is screwed over the third, while the first 
 envelops both the former, being screwed to the base of the third, the upper 
 extremity of which is in direct contact with the body proper of the objective. 
 The first division consists of a hemispherical crown-glass lens of 3.5 mm focal 
 distance and about the same diameter; the two succeeding members are made 
 up each of a plano-concave flint-glass and a biconvex crown-glass lens. The 
 focal distance of 12.5 mm in the second is raised to 17.6 mm in the third 
 member, while the diameters are 6 and 7 mm, respectively, bringing the total 
 
 Fig. 10. 
 
 Objective 3 
 (Leitz). 
 
 Fig. 11. 
 
 Objective 6 
 (Leitz). 
 
 Fig. 12. 
 
 Objective 1/12, 
 
 Homogeneous Immersion. 
 
 (Leitz). 
 
 focal distance down to 4.0 mm. The magnifying power of the objective itself 
 is 48, with a free object distance of only 0.42 mm, while its angular aperture 
 has increased to 0.82. With the weakest eyepiece one may obtain an enlarge- 
 ment of 192 with this object-glass, i.e., it is five times more powerful than 
 objective 3, but the diameter of the visual field here only amounts to 0.48 mm, 
 i.e., one-fourth of the objective 3. 
 
 Objective 1/12. 
 
 Fig. 12 acquaints us with a powerful objective, an immersion system. It 
 is composed of four members, the first two being single, the last two double 
 lenses. The distances are very minute: 0.02, 0.1, 0.15 mm respectively. Each 
 division has its own shell; the setting of the first is made of aluminum. The 
 various shells are attached to one another in such fashion that 1 is screwed 
 to 2, 2 to 4, 3 to 4, and 4 to the body proper of the objective. The first mem- 
 ber consists of a single hemisphere, made of crown glass with a diameter of 
 1.96 mm and a focal distance of 1.55 mm. The second is a positive meniscus 
 with a focal distance of 4.92 mm, while the third and fourth are again piano- 
 
17 
 
 convex flint-crown-glass double lenses of 11.14 and 29.28 mm focal distance 
 respectively. The entire object-glass possesses a focal distance of 1.85 mm, 
 a free object distance of only 0.17 mm and an aperture of 1.30. It has per 
 se a magnifying power of 105, which increases to 420 with the lowest eyepiece, 
 and a visual field of 0.24 mm is covered with this objective. 
 
 Objectives for Very Low 
 Variable Enlargement. 
 
 Often a want is felt for a lower system with a correspondingly larger visual 
 field. Objective la is most adapted for that purpose. Since this type is new 
 to us, we will give it brief mention here. It consists of two achromatic double 
 lenses in a movable setting, so that they may be approximated or withdrawn 
 from one another by means of a screw to the extent of about 5 mm. The front 
 member consists of a biconcave flint-glass lens in back of a plano-convex 
 crown-glass lens, and has a negative common focal distance of 14.8 mm. The 
 upper member is made up of a plano-convex flint-crown-glass double lens of 
 24.0 mm focal distance. When both systems are at a maximum distance (30 
 mm), the common focal distance is 24 mm. When approximated as far as 
 possible, this increases to 33 mm. Here the magnifying power proper falls 
 from 3.1 to 2.0 and the object distance is thus increased from 2.0 to 14.0 mm, 
 while the numerical aperture decreases from 0.07 to 0.05. With the weakest 
 eyepiece a power of 8 — 12 is obtained with a visual field of about 10 mm diame- 
 ter. The distance of the two lenses from each other can be read on a scale 
 marked externally. 
 
 THE EYEPIECE— (Ocular) 
 
 The eyepiece consists of a metal cylinder fitting snugly into the draw-tube, 
 the component lenses being mounted on either side. On its upper extremity 
 we find a metal ring protruding, preventing the ocular 
 from sliding through the tube. 
 
 The Huyghen Eyepiece. 
 
 There are various sorts of oculars, principally the 
 Huyghen, the Ramsden, and the so-called compen- 
 sation eyepiece. We will only consider the first. The 
 Huyghen ocular (Fig. 13) is composed of two plano-con- 
 vex lenses, one of which, the eye-lens, is placed at the 
 upper, the other the collective lens, on the lower end, 
 the convexity of both being toward the objective. Be- 
 tween the two lenses we find a diaphragm on the in- 
 terior of the ocular. 
 
 The eye-lens is always the stronger, having only half 
 the focal distance of the collective lens. As previously 
 mentioned, the distance between the two lenses is half 
 
 of the sum of their focal distances. Hence these focal distances, and 
 thus the total focal distance of the ocular, may easily be determined by 
 measuring the distance, i.e., the length of the eyepiece. For example, the dis- 
 
 Fig. 13. 
 
 Huyghen's Eyepiece 
 (Leitz). 
 
18 
 
 tance (e) of the two lenses in our eyepiece /// measures 36 mm in round 
 
 f 4- f 
 figures ; using the equation : e= J ■ and granting that f 2 =®fn /i» l - e -> 
 
 At 
 
 the focal distance of the eye-lens, must be 24 mm ; f 2 , i.e., that of the collective 
 lens, will be 48 mm, and the total focal distance of the ocular, f, according 
 
 to the formula j=-h-Jj- will be found to be 32 mm. The lower focus of the 
 e 
 
 ocular lies between its lenses, the upper externally above the eye-lens. It is 
 of great importance, as far as the Huyghen ocular is concerned, that it is con- 
 structed in accordance with formula e= x ■ as nearly as possible, for it is 
 
 At 
 
 due to that fact only, that, in spite of the eye-lens as well as the collective lens 
 being simple non-achromatic, the image furnished by the objective is enlarged 
 without aberration. 
 
 Significance of Eyepiece. 
 
 As we have previously observed, the image produced by the objective is 
 first reduced by the collective lens, the latter collecting the rays coming from 
 the former. The diaphragm is found in the plane, where this reduced real 
 image is produced, which latter may be demonstrated as follows : Focus some 
 object, and after removing the eye-lens place a properly cut piece of thin 
 tissue-paper on the diaphragmatic opening; if the collective lens is now also 
 removed, a much enlarged image will be projected on the diaphragm, which 
 can be focused sharply by adjusting the draw- tube. 
 
 Thus our real image is simply enlarged by the eye-lens and brought into 
 exact visual distance of the observer. At the same time, of course, the rim of 
 the diaphragm is projected, so that our field is now bordered by a sharply 
 defined periphery. 
 
 Since this enlargement distributes the same amount of light over a much 
 greater space, the picture thus produced will be short of light, and this loss 
 naturally will increase with the strength of the eyepiece. Hence a certain limit 
 is set to ocular magnifying, and a power greater than twelve should not 
 be used, at least not with a Huyghen eyepiece. 
 
 Eyepieces are generally numbered in Roman or Arabic figures, and naturally 
 a corresponding decrease in size of the lenses and in length of the eyepiece is 
 noted in proportion with the decrease in strength. 
 
 ILLUMINATION 
 
 Illumination by Mirror. 
 
 So far we have assumed that our object itself sends out rays of light spon- 
 taneously, which in reality is not the case. We therefore illuminate our speci- 
 men artificially in order to enable it to radiate light. This illumination may 
 be brought about in two ways : either from above when the section is imper- 
 meable to light, or from below through the opening in the stage. The former 
 method, using reflected light, 1 is of little interest to us, as most all examina- 
 
 a In this method the Bull's eye condenser, so-called, is used. — (The Translator.) 
 
19 
 
 tions are made with the aid of the latter, the transmitted light, method.- 
 Illumination here takes place from below by means of the movable mirror, 
 which, as stated before, is plane on one side, concave on the other. 
 
 Assuming that we are using the plane mirror, a luminous cloud furnishing 
 the source of light, the parallel rays emanating from the latter are caught by 
 our mirror and reflected into the specimen. Each point of the latter thus 
 sends its slightly diverging bundle of rays into the objective. As each bundle 
 must needs completely fill out the angular aperture of the objective, in order 
 to obtain good illumination, we can use this plane mirror only with objectives 
 of a small aperture, to wit, weak objectives. When using stronger objectives, 
 we must have bundles of light, which have a greater divergence, and these will 
 only be furnished by a concave mirror, which causes the parallel rays to con- 
 verge and which is so adjusted as to focus in the objective. Rays of about 40° 
 divergence can be obtained from this mirror, which is absolutely sufficient for 
 medium strength objectives. 
 
 Illuminating Apparatus. 
 Condensers. 
 
 For high power enlargements still more divergent bundles will be required, 
 in order to exhaust the high apertures of the ob- 
 jective, and it is in these cases that an extra sys- 
 tem of lenses must be interposed between mirror 
 and specimen, commonly known as the Abbe 
 condenser. 
 
 This illuminating apparatus is composed of 
 two lenses (Fig. 14), which are snugly inserted 
 in a metal case. The entire system can be ad- 
 justed higher or lower and even everted entirely 
 by a screw, seen in Fig. 8. The upper of the two 
 lenses is more than a hemisphere, while the lower 
 one is a biconvex lens. The two lenses combine to form a strong collecting sys- 
 tem, rendering the rays, coming from the plane mirror, strongly convergent, 
 so that they emerge strongly divergent. Our condenser has an aperture of 
 1.20, furnishing bundles of rays of 104° divergence. The minute loss of lumi- 
 nosity, caused by the air between condenser and slide, may be obviated by a 
 drop of immersion oil so applied as to fill out this air-space. 
 
 Since, in most cases, the entire amount of light furnished by mirror or con- 
 denser is not required nor even desirable, the cone of light is regulated by an 
 adjustable device, named the diaphragm, which is interposed between mirror 
 and condenser and is capable of narrowing the cone of light in its course from 
 the mirror to the condenser to a minimum. 
 
 The Iris Diaphragm. 
 
 We utilize for this purpose the so-called Iris diaphragm, which is found 
 closely below the lenses of the condenser, being attached to the metal case con- 
 taining the latter. Such an Iris diaphragm consists of a number (12 — 18) of 
 thin small metal plates, each resembling a sickle or crescent with a short handle, 
 the point being obliquely cut off. These are arranged in a thin metal drum in 
 such a manner, that they cover one another almost entirely when the diaphragm 
 
20 
 
 is open (Fig. 15 a), leaving only their ends visible, thus forming a broad ring. 
 Each crescent forms a lever, its fixed point being located in the handle. A 
 small rod is found on the distal end of each crescent, which plays in a cor- 
 responding minute moat. These depressions are arranged in a radiating man- 
 ner on a metal ring, which is provided with a lever, the diaphragm lever 
 (Figs. 8 and 16 a). If the lever be turned to the right, all the crescents ad- 
 vance evenly toward the middle, thus bounding a polygon, having as many 
 
 Fig. 15. 
 
 Iris Diaphragm. The Cap and Lever have been 
 Removed, a, Completely Open; i, Closed. 
 
 sides as there are crescents and being nearly circular (Fig. 15 6). The more 
 the lever is turned to the right, the smaller will be the diaphragmatic opening, 
 
 Fig. 16. 
 Iris Diaphragm, a, Lever-Ring; b, Cap. 
 
 the minimum being one millimetre diameter. This well-devised apparatus thus 
 enables us to regulate the light ad libitum. 
 
 Under certain circumstances, especially when using low-power lenses, it is 
 of advantage to be able to switch out the condenser entirely. The place of 
 the diaphragm will then be taken by the so-called cylinder diaphragm, 
 which can be fitted into the opening in the stage and which is manufactured 
 with variously sized openings. The light can thus be regulated by the various 
 sizes of cylinder diaphragm used, but aside from this the amount of illumina- 
 tion can also be reduced by lowering the same cylinder to a desirable level. 
 
 Source of Light. 
 
 The best source of light for our microscope is a light cloud, a blue sky 
 being unfavorable, while direct sunlight must be absolutely avoided. Of the 
 
21 
 
 artificial lights the most serviceable and convenient is the gas-mantle burner. 
 It is a good plan to place a reflector behind the lamp and to interpose a sheet 
 of tissue-paper on a wooden frame between lamp and mirror, at least when low 
 power is used. 
 
 THE MICROMETER-SCREW 
 
 Micrometer-Screw. 
 
 Lastly, it behooves us to speak of the micrometer-screw, which constitutes 
 
 by far the most important mechanical part of the microscope and requires 
 
 the most careful manipulation. Looking at Fig. 17 we will easily understand 
 
 its construction. Here we find the upper end 
 
 of the pillar, the hoodlike covering of the 
 
 micrometer-screw having been removed. As 
 
 previously mentioned, the pillar above the 
 
 stage takes on a prismatic shape and is en- 
 closed by, the carrier. This prism ends in a 
 
 cylindro-conical knob, projecting from the 
 
 carrier, as shown in our figure. This plug 
 
 as well as the prism are hollow, the former 
 
 being slit in addition on its top. Through 
 
 this slit a bridge is inserted, which is screwed 
 
 to the top of the carrier. A strong spiral 
 
 spring, lodged in the hollow of prism and 
 
 plug, exerts pressure against this bridge, 
 
 thus pressing the bridge and the intimately 
 
 connected carrier upward. This pressure 
 
 is counteracted by the micrometer-screw 
 
 proper, a short, strong steel screw of per- 
 fect execution, its freely movable tip pressing 
 
 against a depression in the bridge. Turning 
 
 the screw to the right we will compress the spring and thus iower the tube- 
 carrier. In this manner the carrier may be lowered until its lower end ap- 
 proximates the enlargement of the pillar found just above the stage (Fig. 8), 
 in other words, until the entire prism is covered. Turning to the left, the car- 
 rier, pushed upward by the spring, is raised ; this can be done until the bridge 
 touches the ceiling of the slit, after which the carrier will cease to rise, and if 
 the turning is continued, the micrometer-screw will simply be taken out. 
 
 We may add that the different makes of micrometer-screws vary essentially 
 in their construction. 
 
 Fig. 17. 
 
 Micrometer-Screw. 
 
 SOME RULES FOR THE USE OF THE MICROSCOPE 
 
 The Procuring of 
 a Microscope.. 
 
 In the manufacture of microscopes Germany has taken first place for years 
 past, and such renowned firms as Zeiss, in Jena, Leitz and Seibert, in Wetzlar, 
 Winkel, in Goettingen, Voigtlander, in Brunswick, and Reichert, in Vienna, 
 
22 
 
 furnish excellent instruments, which can be said to be equally efficient in their 
 essentials. It is therefore partly a matter of personal preference or, perhaps, 
 partly a pecuniary question, which make should be selected by the prospective 
 buyer. 
 
 An instrument answering all the requirements of a beginner may be bought 
 nowadays at a price of 300 to 400 marks (75 to 100 dollars), and such an in- 
 strument will, moreover, prove satisfactory for the work of the general prac- 
 titioner. 
 
 The objectives should be thoroughly tested as to their efficiency, and their 
 selection should best be left to an expert. One should also convince himself of 
 the faultless construction of the micrometer-screw by sharply focusing at 
 different heights. 
 
 The Care of the Microscope. 
 
 It scarcely needs mention that an instrument of such minute precision as 
 the microscope requires the most scrupulous care and delicate manipulation. 
 It should be protected from dust by replacing it in the case after use, or, still 
 better, by keeping it under a bell-glass jar. In transporting the microscope 
 one should make it a rule to always grasp the foot and not the tube-carrier, as 
 is generally practised. 
 
 The Cleaning of the Objective. 
 
 The objectives require the most rigid care. If the front lens is soiled, the 
 entire objective should be unscrewed from its nosepiece and the lens cleaned 
 with a very soft cloth, preferably a well-laundered handkerchief. We hesitate 
 to recommend the chamois leather, which, although very useful, is soon soiled. 
 If the lens cannot be cleaned satisfactorily with the dry cloth, the latter should 
 be moistened with alcohol. If the front lens is clean and still the objective does 
 not furnish a clear picture, it will be best to have the optician look it over. 
 Under no circumstances should the beginner attempt to disarticulate the com- 
 ponent lenses with a view of cleaning the same separately. It goes without 
 saying, that the objective, or more particularly the front lens, must be pro- 
 tected from all acids, alkalies or such solutions as are solvents of fats and 
 resins. 
 
 The microscope should never remain without an eyepiece or without proper 
 apposition of the nosepiece, as otherwise dust will accumulate on the interior, 
 making it difficult to keep the lenses clean. 
 
 The Focussing of a Field. 
 
 It seems advisable to say a few words regarding the focussing of a slide, a 
 process which often proves quite difficult for the beginner. We found that each 
 objective has its own fixed object distance, and that the latter decreases with 
 the increasing strength of the lens. When handling a high-power lens it may 
 thus easily happen that the front lens comes in too intimate relation with the 
 cover-glass. One of two things, or both, may happen. Either the cover-glass 
 will be crushed and the specimen ruined or, what is more serious, the front lens 
 may be loosened from its setting. This might especially happen when using 
 an immersion lens, where, as we have found, the metal setting of the minute 
 hemisphere lies attached only to a very small ring. 
 
23 
 
 It is an excellent plan for the beginner to establish the rule to examine 
 first with a low-power lens. Any particular part of the field should then be 
 moved to the middle. It will then only be necessary to shift the nosepiece and 
 to use the fine adjustment, since the length of the objectives has been graduated 
 in a manner so as to make the coarse adjustment for all objectives the same. 
 
 Low-power objectives, e.g., as No. 3, have such a great distance as to 
 render the focussing safe. If trouble should be encountered here also, it is 
 best to proceed by raising the barrel rather than lowering it. The front lens 
 is first brought down close to the cover-glass, then, while looking in the micro- 
 scope, the barrel is raised by means of the coarse adjustment screw, until the 
 picture appears. 
 
 When the oil-immersion lens is to be used, that portion of the specimen is 
 brought to the middle of the field, focussing first with a low-power and then 
 with a medium lens, the barrel is then raised and the nosepiece turned. A 
 small drop of immersion oil is placed in the middle of the cover-glass and the 
 barrel lowered until the front lens comes in contact with the drop of oil. If 
 a small drop of oil has been used, the image will now be visible, though indis- 
 tinct, and all that remains necessary is the use of the micrometer-screw. 
 When the examination has been completed, the front lens and cover-glass are 
 cleansed with a cloth moistened with concentrated alcohol. 
 
 During the entire examination the hand should be kept on the micrometer- 
 screw, the latter following each glance of the observer and making it possible 
 for the eye to penetrate the various depths of the specimen ; hence its great im- 
 portance to our instrument. The micrometer-screw should never be used for 
 coarse adjustment, and one should always observe that the prism, mentioned 
 before, is visible at all times. If any resistance is felt when focussing, the 
 cause of same should be found before any further adjusting is attempted. The 
 micrometer-screw will be found lowered to its limit or the front lens has touched 
 the cover-glass. If the turning of the micrometer-screw has no effect on the 
 focus, it will be found to have passed its upper limit. Too great a part of the 
 prism will be visible in this case, and the fine adjustment must be lowered until 
 the correct distance is attained. 
 
General Microtechnique 
 
 Definition and Purport 
 of Microtechnique. 
 
 By microtechnique we understand the totality of all those methods which 
 serve to produce a microscopic slide. This technique depends on the one hand 
 on the peculiarities of our microscope, on the other it is governed by the prop- 
 erties of the specimen about to be examined. We will best make ourselves clear 
 hy citing a simple practical example. 
 
 Let us suppose that we are to prepare a specimen of the human kidney. 
 First, we are confronted with the proposition of making a section. This 
 section must not be made in a coarse, superficial manner, but must be so 
 perfected that a transparent preparation is obtained, since we want to ex- 
 amine our slide with transmitted illumination. We can either make thin sec- 
 tions with the cutting instruments designed for this purpose, or we may sepa- 
 rate minute particles by means of scissors, and then still reduce their size by 
 teasing with teasing needles. We now put our specimen on a thin glass plate 
 (the slide), and cover it with a still thinner small glass plate (the COVer- 
 glass). When using high-power objectives, the latter must only be a fraction 
 of a millimetre in thickness, on account of the small object distance. 
 
 As all these manipulations require time and sometimes a good deal of time, 
 our specimen will in the meantime suffer changes. In all animal organism great 
 changes take place soon after death, which alter its original structure to a 
 high degree, and which occur all the sooner if the organism has been finely 
 divided and separated from its component structures. The question thus 
 arises, how may we delay these changes or, if possible, prevent them, i.e., fix 
 our specimen. But supposing that we have made fine transparent sections of 
 a kidney and fixed them in their original structure, we will meet with still 
 another difficulty. The various tissues comprising the specimen, e.g., epithe- 
 lium, connective tissue, nerve tissue, etc., differ so little in their optic proper- 
 ties, that their differentiation will be extremely difficult to the untrained eye, 
 if not impossible. The examination will therefore be materially facilitated by 
 subjecting the preparation to a dyeing process, i.e., by staining them. 
 
 We may thus divide microtechnical methods into three great groups, 
 namely : 
 
 1. The Method of Preservation. 
 
 2. The Method of Making Sections. 
 
 3. The Method of Staining. 
 
 For all these methods scores of more or less complicated- instruments have 
 been devised during the last fifty years, and a host of chemicals, reagents and 
 
 24 
 
25 
 
 dyes have been recommended. We shall now acquaint ourselves with these, at 
 least as far as their practical value for us is concerned. 
 
 METHODS OF PRESERVATION 
 
 Observation of the 
 Living Object. 
 
 The preparation of a specimen is not always as unfavorable and difficult 
 as in the case of the human kidney. Going down the scale of organisms, we 
 find thousands of forms, which are so minute and transparent that we can 
 put them on our slide without further preparation and keep them there alive 
 under observation, for hours and even days. But also higher organisms, even 
 human, can be cut and their sections kept alive and studied for some time. For 
 instance, it is not very difficult to observe living human blood or spermatic 
 fluid under the microscope. 
 
 Indifferent Liquid Media. 
 
 The great importance in these observations is to examine the object under 
 conditions as nearly normal and natural as is available. Just as we would 
 examine infusoria, obtained from a sweet-water aquarium, in this same me- 
 dium, we likewise proceed with human blood corpuscles, i.e., we do not put them 
 in water, where they would instantly undergo haemolysis, but select as a medium 
 either blood serum or at least a liquid of similar properties, i.e., an isotonic 
 fluid. Of course, care must be taken that this fluid does not evaporate and 
 thus become concentrated. If, on the other hand, a hypotonic fluid should 
 be selected, for example, distilled water, osmosis would take place, the liquid 
 entering the corpusles and distending them. If a hypertonic solution was 
 used the result would be a loss of fluid from the corpuscles with consequent 
 shrinkage. 
 
 The most satisfactory results for mammalian tissues are obtained with 
 blood serum, which may be secured by centrifuging cooled blood, or with 
 amniotic fluid, which may easily be saved from the gravid uterus of the cow 
 or pig. The aqueous humor from the anterior chamber of the eyes of 
 larger animals may also be utilized for this purpose, and can be procured by 
 puncture with a Pravaz hypodermic needle. 
 
 Of the artificial indifferent media the normal or physiologic saline 
 solution is the best known. It should contain 0.9% of table salt for exami- 
 nation of mammalian or human tissues. A still better solution is Ringer's 
 fluid, containing NaCl, 8.0 gms., KC1, 0.2 gm., NaHC0 3 , 0.2 gm., and 
 CaCl 2 , 0.2 gm. in 1,000 cm 3 of distilled water. 
 
 Granted that the study of living organism is of great importance in the 
 physiological and biological research into the animal body, the fact remains 
 that morphology deals to a greater extent with specimens which have pre- 
 viously been killed and preserved in the proper manner. 
 
 Significance of Fixation. 
 
 Naturally the first prerequisite is, that the tissues are preserved as nearly 
 as possible in their original structure and relation. The object is, therefore, 
 
26 
 
 to fix the structure, and the methods and reagents employed for this purpose 
 have been termed fixation, and fixing solutions or fixatives. If the ob- 
 ject is thus killed in a state truly resembling that of life, it can easily be pre- 
 served for an unlimited time. 
 
 In order to familiarize ourselves with the fixation of animal tissue, it will 
 be a good plan to branch off for a short excursion into physiologic chemistry. 
 The cells and the intercellular substance composing the body tissue, consist 
 chiefly of water, minerals, carbohydrates, fats and albumins. The latter are 
 for us the most important of the constituents of tissues, high-molecular, very 
 complicated compounds, which are partly neutral, partly acid and partly basic 
 in reaction, being soluble in water, weak alkalies or acids. As far as micro- 
 technique is concerned, their most important property is that they are made 
 insoluble (coagulate), when treated with certain physical or chemical agents. 
 One of the best known and most common physical coagulating agents for 
 albumin is heat. If an albumin solution is heated to nearly 100°, all the 
 albumin becomes coagulated, provided the solution be slightly acidified. The 
 albumin now has become insoluble in its former solvent, it is denatured. If a 
 mineral acid or certain organic acids are added to an albumin solution, 
 coagulation will result with the production of an acid albumin, which, how- 
 ever, is soluble in the diluted acid. Another important group of substances 
 coagulating albumin consists of the salts of the heavy metals. These 
 change the albumin into metal albuminates, which are insoluble in water, i.e., 
 loose compounds of metal oxides with albumin. Alcohol also deserves men- 
 tion as a coagulating agent. If an albuminous solution is treated with alcohol, 
 the albumin coagulates, again becoming soluble when water is added. In this 
 case, therefore, it is not denatured. If, on the other hand, the alcohol is 
 allowed to act for a longer time, it also will render the albumin insoluble. 
 Formalin, a solution of formaldehyde in water, of late more widely known in 
 science and practice, forms methylene compounds with most albumins, which 
 compounds are insoluble in water. 
 
 This property of albuminous substances is of great interest in microscopy, 
 enabling us to fix the inconstant tissue picture by coagulating the otherwise 
 soluble albumins. 
 
 Thus, fixing means for us nothing more than the coagulation of albuminous 
 bodies, wherefore every good fixing agent must be a good coagulant of albu- 
 mins. But again, an efficient albumin coagulator need not be a good fixing 
 reagent ; e.g., tannic acid, while causing albumin to coagulate readily, is totally 
 useless as a fixative. 
 
 Practical experience during the last fifty years has demonstrated to us 
 many methods and reagents, which might be used for fixation, but we have also 
 learned that it is often of advantage not to depend upon a single reagent for 
 fixation, but to combine the good properties of a number of them ; thus alcohol 
 or metal salt solutions are used in conjunction with an acid, commonly acetic 
 acid, each of which would yield poor results if used alone. 
 
 General Rules for Fixation. 
 
 Before proceeding to the special discussion of the various fixatives, it 
 might be well to dwell on some general and practically important points. If a 
 
27 
 
 true reproduction of our preparation is desired, it follows that we must fix 
 our specimen in the living state, which proves to be quite a difficult task. 
 When dealing with animals, the living object is more often available and the 
 specimen can be taken directly after the animal has been killed and may imme- 
 diately be put in the fixing solution. With human material the conditions 
 are rarely so favorable. 
 
 AncBsthetizing and 
 Killing of the Animals. 
 
 Most commonly chloroform is used in the killing of animals. Smaller 
 animals, up to the size of a small dog, should best be put under an adequately 
 large glass globe or bell glass. A tuft of cotton, saturated in chloroform, is 
 introduced, the animal carefully watched and the glass held fast, since many 
 animals, foremost among them the cat, exhibit a state of tremendous excite- 
 ment. When the animal has become motionless and reactions have ceased, it 
 is attached to an operating board ; the thorax is quickly opened and death is 
 brought on by removing or opening the heart. Larger dogs better receive an 
 initial dose of morphine (6 cm 3 of a 1% solution of morphine muriate) sub- 
 cutaneously and, after half an hour has elapsed, will easily succumb to chloro- 
 form administered on ■ a cloth. A very convenient and humane method of 
 killing smaller animals is the administration of carbonic acid by means of 
 the so-called carbonic acid bomb, whence the gas is led through a tube into the 
 bell glass. In small animals like frogs, mice, rats, etc., death is generally 
 induced by cutting off their heads with strong scissors. When using a 
 frog, it is best to destroy the spinal cord in addition, employing a medium 
 sized probe for this purpose. Another very useful but dangerous agent is a 
 2% alcoholic solution of hydrocyanic acid, which is kept in stock in 
 most drug stores. A few drops, injected subcutaneously, kill even larger ani- 
 mals almost instantaneously. 
 
 Specimens from the 
 Human Body. 
 
 In the examination of human material much time is often lost before it 
 reaches us, unless we are dealing with an operative case or, perhaps, an 
 autopsy. Material taken two to three days post mortem is utterly useless for 
 our purpose. The maximum time which may elapse between death and the 
 taking of a section is twelve hours. A very efficient and easily obtainable mate- 
 rial, which serves well for most purposes, is the body of a child during the first 
 few weeks after birth. 
 
 .Size and Treatment 
 of the Specimen. 
 
 Since the penetrating power of most of our fixing agents is moderate, it 
 is best to use -small or at least thin pieces, unless other reasons make this im- 
 practicable. A section of 1 cm thickness might be considered as very thick. 
 
 The selected section should not be handled unnecessarily with instruments, 
 and, in order to avoid any possible crushing, the section should only be made 
 with sharp instruments, scissors or razor. 
 
 The amount of fixing solution should be plentiful, the volume being at 
 least twenty times that of the object to be fixed. 
 
28 
 
 To allow free access of the fixative from all sides, the specimen is best sus- 
 pended in the solution, or else the floor of the jar should be covered with glass 
 wool, cotton or several layers of tissue-paper. 
 
 Duration of Fixation. 
 
 The duration of the fixing process naturally depends on the size of the 
 object and the nature of the fixing agent. A general rule may be adopted that 
 the process should not last longer than is necessary for complete saturation of 
 the specimen. The speediest result is obtained from acids ; alcohol and formalin 
 taking slightly longer, while the metal salts work slowest of all; e.g., after 
 acting four hours, a 5% nitric acid solution 1 will have penetrated 6 to 7 mm, 
 10% formalin, 5 to 6 mm; 96% alcohol 3 to I mm, 0.3% platinum chloride 
 only 0.5 to 1 mm. Only a few fixing solutions can act on a specimen for an 
 indefinite period without injuring it, i.e., can also serve as a preservative. Of 
 
 ■ these we may mention primarily 5 to 10% forma- 
 lin, in which a specimen can remain almost un- 
 changed for an indefinite time. To a certain ex- 
 tent this can be said of alcohol, which should be 
 used 70 to 80% strong. All other agents change 
 the specimens under prolonged use ; they partly 
 render them hard and brittle, or partly deposit 
 themselves in crystal form, thus rendering the 
 staining difficult, if not impossible. 
 
 Jar for Washing of Fixed 
 Specimens. 
 
 After-Treatment. 
 
 We find, therefore, that after most fixation 
 methods we must seek to extract the fixing solu- 
 tion after it has done its work. This is generally 
 done by means of a wash-glass (Fig- 18), which is suspended on the knee- 
 shaped limb in a water reservoir, fed from the faucet. Our specimen thus lies in 
 a continuous current and within a few to twenty-four hours the fixative will be 
 entirely washed out. Some of our solutions cannot be treated that way, and 
 foremost among them are the acids. Here we can use 70% alcohol, which must 
 be renewed frequently, or 5% formalin. 
 
 We will now proceed to discuss a number of the most important fixing 
 solutions which are of interest to us. 
 
 1. Nitric acid, HN0 3 , when pure and concentrated, is a colorless liquid 
 of 1.54 specific gravity; when exposed to air, it soon assumes a yellow color, 
 due to the formation of nitrous acid. The commercial concentrated nitric acid, 
 which is generally used, contains 68% HN0 3 and has a specific gravity of 
 1.41. The C. P. nitric acid, obtained at druggists, contains only 25% of 
 HN0 3 , having a specific gravity of 1.15. When nitric acid is mixed with 
 alcohol, the main compounds formed are: Nitric acid-ethyl ether (C, 
 H 5 O.N0 2 ), nitrous acid-ethyl ether (C 2 H 5 .0N0) and acetaldehyde 
 (CHg.CHO). It must be borne in mind that the ethers of nitric acid are 
 quite explosive compounds under certain conditions. 
 
 1 When speaking of any per cent, solution, we naturally always mean that per 
 cent, solution in distilled water. 
 
29 
 
 Nitric acid itself, or its compounds, is one of our most valuable fixing 
 agents, and especially for certain organs, e.g., the retina, it cannot be excelled. 
 It is mostly used in a 7.5% solution, i.e., 7.5 cm 3 nitric acid of 1.41 specific 
 gravity and 92.5 cm 3 of water. 1 This agent penetrates the tissues very well, 
 indeed better than any other fixative. Nitric acid must not be allowed to act 
 too long on tissues, as it will otherwise become a solvent of certain elements 
 of the cellular tissue — five to six hours may be considered the maximum, 
 although ordinarily three to four hours will suffice. If the object should be 
 removed from the acid and directly placed in water, a swelling of the connective 
 tissue will take place, wherefore the acid must first be taken out with other 
 solvents, e.g., 70% alcohol, 5% solution of Glauber salt, or 5 to 10% formalin. 
 
 2. Acetic acid, CH 3 .COOH, a colorless liquid of intensely pungent 
 odor. Specific gravity, 1.05. It crystallizes at about 70° F., whence it is 
 often called ice- vinegar. The boiling-point is approximately 298°, and the 
 acid burns with a blue flame. It must be kept in a well-corked bottle, since it 
 is extremely hygroscopic when exposed to air. 
 
 By itself this acid makes a poor fixative, but as an ingredient of other 
 reagents it plays an important role by virtue of the rapidity with which it 
 diffuses through the tissues, rendering them acid and thus preparing them for 
 the action of the actual fixing agent. The vapors of acetic acid are also used 
 for fixation in certain instances. 
 
 3. Trichlor-acetic acid, CC1 3 — COOH, a compound in crystal form, 
 dissolving when exposed to air and having caustic properties. 
 
 A 5% solution is quite a useful fixative, acting much slower than nitric acid, 
 hence a specimen must be allowed to remain up to ten or even twelve hours. 
 The acid is easily washed out with 95% alcohol. 
 
 4. Picric acid, C 6 H 2 (N0 2 ) 3 .OH, a yellow crystalline powder, slightly 
 soluble in water (1.1%), more readily in alcohol, forming a bright yellow 
 solution. 
 
 Picric acid solutions, which are strongly poisonous, do not readily pene- 
 trate the tissues and per se are of little significance for our purpose. On the 
 other hand, picric acid becomes a potent factor when forming a constituent of 
 a solution, and is especially important as a stain. 
 
 5. Chromic acid, Cr 3 , a reddish brown crystalline compound, soluble 
 in damp air. It is strongly caustic and is reduced by alcohol into brown 
 chrome hyperoxide, respectively green chrome oxide. Chromic acid solutions 
 should not be filtered through paper. 
 
 The acid is used in a 0.25 to 1% solution, but rarely. Its penetrating 
 power is low, although greater than that of picric acid. The object is usually 
 left in the solution for twenty-four hours, after which it is washed in running 
 water for an equal length of time. Any precipitate which may have formed in 
 the tissues can be removed by treating the section with a 5% solution of 
 potassium cyanide. An objection to the use of chromic acid lies in the fact 
 that specimens so treated become brittle and are stained with difficulty. 
 
 6. Osmium tetroxid, Os 4 — incorrectly named osmic acid, is manu- 
 
 1 When speaking of water we invariably mean distilled water; if well or tap 
 water should be used, it will be specified as such. 
 
30 
 
 factured in the form of slightly yellow crystals, which are put up in small 
 glass tubes of 0.5 or 1.0 gr, on account of its great diffusibility. 1 Its vapors 
 are very irritating to the mucous membranes. After thoroughly cleansing the 
 tubes, they are filed on one side and opened by touching them with a glass 
 rod heated to white heat. In order to waste nothing, the entire tube with 
 contents is put in an absolutely clean bottle and corked with a tightly 
 fitting glass stopper. The necessary amount of water can now be added — 3 
 to 4% will make a saturated solution. The solution must be protected from 
 dust. 
 
 Osmic acid has attained great importance in microtechnique owing to its 
 properties as a fixing agent. Although only moderately able to coagulate 
 albumin, it makes an excellent fixative for the cell, particularly the cell body. 
 It is used in 0.5 to 2% solution, or in the form of vapor. Thin objects are 
 exposed to the vapors of a 20% solution for a few minutes. Osmic acid solu- 
 tions penetrate the tissues only slowly, although not as slowly as is commonly 
 stated. Two to three millimetres are saturated in a few hours. The staining 
 of osmic acid preparations is not so unsatisfactory, as is often supposed, pro- 
 vided that the acid has been thoroughly washed out in water for twenty-four 
 hours, and that it has not been treated with alcohol afterward. To preserve 
 such specimens 5% formalin is to be recommended. 
 
 Another very important advantage of osmic acid lies in the fact that it is 
 reduced to different degrees by animal tissues, thereby furnishing a finely 
 shaded picture. It is most strongly reduced by fat and some deutoplasmatic 
 bodies, which in consequence appear totally black, while the remainder of the 
 picture shows gray and grayish brown hues. A secondary staining process 
 can therefore often be dispensed with. 
 
 To decolorize osmic acid preparations, the sections are best treated with 
 an alcoholic solution of hydrogen peroxide (one part of the commercial per- 
 oxide to 5 — 6 parts of 70% alcohol). 
 
 7. Chrome-Osmio' Acetic Acid. — A mixture of 15 cm 3 of chromic 
 acid (1%), 4 cm 3 of osmic acid (2%), and 1 cm 3 of acetic acid, which has 
 received the name of Flemming's solution, has a wide range of usefulness in 
 microtechnique, and constitutes one of our best fixing solutions. In rapidity of 
 penetration it excels chromic acid as well as osmic acid. The staining proper- 
 ties of specimens so treated are excellent, inasmuch as no alcohol is used for 
 after-treatment. The section is usually fixed for twenty-four hours, but might 
 be left in the solution for several days without any harm resulting therefrom. 
 Careful washing in running water for twenty-four hours after completion of 
 fixation is indispensable. 
 
 8. Potassium Bichromate, K 2 Cr 2 7 . — Red trigonal crystals, 10% 
 soluble in water, giving a faintly acid reaction. Per se potassium bichromate 
 is an inadequate fixing agent, since it is not able to fix those albuminous parts 
 of tissues, which are most valuable to us. It penetrates slowly but steadily. If 
 one desires to use this reagent, it must be acidified first, and this can be accom- 
 plished with acetic acid. To a 3% solution add 5% of acetic acid and fix for 
 
 1 Osmic acid is at the present time a very expensive reagent, selling at about 
 $2.00-$2.25 per gram. 
 
31 
 
 twenty-four to forty-eight hours, after which the section must be washed in 
 running water for the same length of time. 
 
 9. Mueller's fluid, a reagent formerly used extensively, is to this date 
 a very efficient fixing solution. To prepare it, 2.5 gms of potassium bichro- 
 mate and 1 gm of sodium sulphate are dissolved in 100 cm 3 of water. It is 
 especially adapted for the preparation of the central nervous system for sub- 
 sequent staining of its nerve fibres. But even as a fixing agent solely Mueller's 
 solution gives better results than the bichromate solution alone, which is prob- 
 ably due to a liberation of small amounts of chromic acid by the addition of 
 the Glauber salt. The solution is allowed to act for days, weeks and even 
 months, but it must be changed as soon as it becomes turbid. Entire human 
 brains used to be treated in this fashion for months, a procedure which has to- 
 day been abandoned as coarse and primitive. After fixation the running water 
 bath is essential. Staining properties of Mueller preparations are good for 
 the usual carmine and hematoxylin solutions. In some cases the preparation 
 with Mueller's fluid is even a prerequisite. The chrome salt present acts as a 
 corrosive. 
 
 10. Potassium-Bichromate-Gsmic Acid. — Of the other potassium 
 bichromate mixtures the only one of interest to us is the potassium-bichromate- 
 osmic acid, usually designated as Altmann's mixture. It consists of equal 
 parts of 2.5% potassium bichromate solution and 2% osmic acid solution. 
 Small pieces are fixed in it for twenty-four hours and are washed equally long 
 in running water. 
 
 11. Corrosive Sublimate, HgCl 2 , occurs in white crystals, soluble in 
 water to the extent of 7%, 25% in alcohol, giving an acid reaction. The solu- 
 tion is not stable and easily breaks up on exposure to light with the production 
 of calomel. More stable compounds can be produced by using normal saline 
 solution instead of distilled water, the former giving us a neutral bichloride 
 solution by the formation of a double salt compound, chloride of mercury- 
 chloride of sodium. If the tincture of iodine - is added to a sublimate solution, 
 a decolorization of the latter with the production of mercury iodine takes place. 
 
 Corrosive sublimate energetically coagulates albumin. Its aqueous solu- 
 tions, which are acid in reaction, reduce the genuine albumins to insoluble albu- 
 minates. Albuminates furnished by the mercury chloride-sodium chloride solu- 
 tion become insoluble only on addition of an acid. The penetrating power of 
 corrosive sublimate is medium, pieces of 5 mm thickness being completely fixed 
 within four to five hours; 2.5 to 5% watery solutions are used, and it is to be 
 recommended to make your solution fresh just shortly before using it. Such 
 recent solutions give decidedly more constant results as do older table salt- 
 sublimate solutions. The solution can be used with or without additional 
 acidification. 
 
 12. Corrosive Sublimate with Acids. — For acidifying, various acids 
 can be utilized. One to two per cent, acetic, 7.5% nitric or 10% trichloracetic 
 acid all work well. Specimens should not remain longer than necessary in 
 neither the pure sublimate nor the acidified solutions. For small sections 
 one hour should suffice; larger specimens may remain six to eight hours, but 
 twelve hours should never be exceeded, as the shrinking caused by this solu- 
 tion increases with the time consumed in fixing. Washing of the section in 
 
32 
 
 running water for twelve to twenty-four hours should always be practised 
 with sublimate as well as sublimate-acetic acid solutions, while the remaining 
 acid mixture methods are best followed by strong alcohol, to avoid a swelling 
 of the connective tissues. Material for frozen sections should be washed and 
 placed in 5% formalin. Corrosive sublimate always causes coagula in the con- 
 nective tissues, which cannot be removed even by the most scrupulous washing. 
 They can be removed either in the section or in the bulk. The former is done 
 in frozen sections, where the coagula are dissolved by placing the section 
 overnight in a 5% solution of sodium sulphate and afterward washing it, chang- 
 ing the water frequently. When dealing with specimens which are to be em- 
 bedded in paraffin, the coagula are best removed by the use of 70 or 80% 
 alcohol, to which has been added enough of the tincture of iodine to give it a 
 good brown color. The iodide of mercury is thus formed, which is soluble in 
 alcohol, giving us a colorless solution. If, therefore, a zone of colorless alcohol 
 is found around the specimen on the following morning, the process must be 
 repeated, until no more decolorization is observed. 
 
 Corrosive sublimate, despite the fact that of late it has lost some of its 
 reputation, still remains one of our very best fixing agents; objects prepared 
 by its use, aside from excellent preservation of structure, show great staining 
 properties. 
 
 13. Corrosive Sublimate - Mueller's Fluid - Acetic Acid Combi- 
 nation, known as Zenker's fluid. — To 100 cm 3 of hot Mueller's fluid 5 gms of 
 bichloride of mercury are added, and just before use this solution is acidified 
 with 3 cm 3 of acetic acid. Duration of action and after-treatment are the 
 same as with corrosive sublimate, but the staining properties of the latter are 
 far superior. 
 
 14s. Corrosive Sublimate - Chrome - Osmium - Acetic Acid. — To 
 100 cm 3 of a 1% bichloride solution we add 10 cm 3 of a 10% chromic acid 
 solution, 5 cm 3 of a 2% osmic acid solution and 2 cm 3 of acetic acid. Duration 
 of fixation is twenty-four hours, to be followed by washing in running water 
 for an equal length of time. 
 
 15. Platinum Chloride. — The product sold under this name is really 
 platinum chloride — hydrochloric acid, Pt Cl 4 -j-2 H Cl+6 H 2 O ; it forms red- 
 dish brown crystalline masses, which are highly soluble in water, making a 
 strongly acid solution. Owing to the fact that this compound is acted on by 
 light, it is best to keep it in well-corked brown bottles. 
 
 16. Platinum Chloride-Osmium- Acetic Acid. — Platinum chloride 
 acts well on albumin and therefore is an excellent fixing agent ; however, it 
 possesses little penetrating power. It is generally made use of in the form of 
 platinum chloride-osmium-acetic acid or Hermann's fluid, which consists of 
 15 cm 3 of 1% platinum chloride, 4 cm 3 of 2% osmic acid, and 1 cm 3 of acetic 
 acid. In its action it is very similar to Fleming's solution. Specimens are 
 fixed from twenty-four hours to several days, and then washed for twenty-four 
 hours. The staining properties are not as good as they are obtained from 
 Fleming's solution. 
 
 17. Alcohol. — Ordinary ethyl alcohol, C 2 H 5 OH, when free from water, 
 is an excellent fixative of- albumin and, if allowed to act long enough, furnishes 
 durable denaturation products. At the same time it dehydrates the specimen, 
 
33 
 
 so that the section may be rendered free from water by the repeated use of 
 alcohol. Alcohol, when judiciously used, is a very good and, as we will see 
 later, a very convenient fixative. As regards its penetration alcohol takes a 
 medium rank, ranging somewhere after potassium dichromate in speed. The 
 staining properties are good, although they are generally overestimated. A. 
 good fixation can only be obtained from absolute or at least very strong alco- 
 hol, and here again we find a disadvantage in that the water is extracted too 
 quickly from the section, so that delicate moist tissues are apt to shrink 
 enormously. 
 
 The so-called absolute commercial alcohol contains 99.4 — 99.6% of pure 
 alcohol, which is absolutely sufficient for our purpose. It must be kept in 
 well-corked bottles, since it takes up water assiduously from the atmospheric 
 moisture. 
 
 Large quantities should be used and the sections suspended freely ; in that 
 fashion it will penetrate 8 — 10 mm within twelve hours. If a specimen is left 
 for several days, it becomes very hard ; it is well, therefore, to transfer it to 
 *70 — 80% alcohol for the purpose of preserving a section. After a time the 
 staining property of such preparations is materially decreased. 
 
 18. Alcohol Acetic Acid. — Combinations with alcohol are innumerable, 
 all designed for counteracting its shrinking property. The most important 
 adjuvant is acetic acid. One part of the latter is added to 4 parts of alcohol 
 (absolute), the mixture is allowed to act for twenty minutes, after which the 
 section is transferred to a mixture of 1 part of the acid to 8 parts of alcohol, 
 and hence to pure alcohol, which must be renewed after a few hours. Of the 
 remaining alcohol-acetic acid mixtures Carnoy's is the best known. It con- 
 sists of 60 parts of absolute alcohol, 30 parts of chloroform and 10 parts of 
 acetic acid. It penetrates well and furnishes a good nuclear and plasma 
 fixation. Fifteen to twenty minutes for ordinary, an hour for larger pieces, 
 suffices for fixation, after which the object is transferred to absolute alcohol, 
 which must be changed several times within the next few hours. 
 
 19. Formalin. — Formalin is a 40% solution of formaldehyde, H.COH, 
 in water ; it is a colorless fluid with a pungent odor, not only irritating mucous 
 membranes, but even producing an itching erythema on susceptible skin. When 
 fresh, formalin is neutral in reaction, but it gradually becomes acid, due to the 
 liberation of formic acid. 
 
 As previously mentioned, formalin can coagulate a large number of albu- 
 minous bodies. It possesses, a good penetrating power and in no way diminishes 
 the staining properties. 
 
 Formalin is used in 10% solution, and specimens can be kept in this fluid 
 for an indefinite period, so that this solution or, perhaps better, a 5% strength, 
 makes a good preserving agent as well. After fixation specimens can be 
 directly transferred to diluted alcohol, since it is not necessary to wash out 
 the formalin. 
 
 While formalin cannot be considered among the very best, it certainly is a 
 good fixing agent, and for certain tissues, e.g., muscle, it excels all others. 
 When properly used it does well in almost all instances. 
 
 20. Formalin-Mueller's Fluid. — Of the various compounds of for- 
 malin Mueller's fluid with formalin, in the proportion of 9 : 1, is the best known. 
 
34 
 
 By the addition of formalin the fixing property of Mueller's fluid is materially 
 increased. When the mixture becomes milky it must be changed. Specimens 
 should be fixed for twenty-four to forty-eight hours, washed in water and pre- 
 served in 5% formalin. 
 
 21. Formalin- Alcohol. — Combinations of alcohol with formalin are 
 also used extensively. Absolute alcohol, containing 10% of formalin, will fur- 
 nish a mixture which shrinks the tissues less than does alcohol alone. Twenty- 
 four hours are allowed for fixation, and according to what after-treatment is 
 desirable, the specimen is transferred to absolute or diluted (70%) alcohol. 
 
 PREPARATION OF SECTIONS 
 
 The methods of making microscopic sections, which we will now deal with, 
 all have in common the object of reducing the voluminous organs of the body 
 into small, thin, transparent sections, which allow the rays of light to pass 
 through them, thus giving us a specimen which is adapted to microscopic ex- 
 amination. This end may be attained in many different ways. Specimens can 
 be teased, crushed, chopped, shaken, cut, etc. All these methods have two 
 objects in view: either it is desired to preserve the relationship of constituent 
 parts of a tissue, or to dissolve such relation by dissociating or isolating the 
 component factors. The former purpose is best accomplished by the cutting 
 method, which enables us to make sections of any desired thickness, either 
 of the entire organ or of smaller parts of same. The various cutting methods 
 are by far the most important and therefore the most thoroughly understood 
 means of preparing a specimen. Aside from these, the dissociation methods 
 enjoy some of our interest by making it possible for us to study the indi- 
 vidual forms of the tissue elements. The chop-method takes a position 
 midway between cutting and dissociation methods, and may be used with ad- 
 vantage in some cases. 
 
 THE DISSOCIATION METHODS 
 
 Most tissues resist the mechanical dissociation of their component parts ; 
 e.g., it is impossible to separate a muscle fibre from the tendon fibre to which 
 it is attached, both being united by a cement substance. The same is true 
 of many other tissues, so that our first aim will be to dissolve this cement 
 chemically, after which the mechanical isolation can take place without diffi- 
 culty. 
 
 To accomplish the former, so-called isolation- or maceration-agents are 
 used, of which we have quite a large selection. We will only mention those 
 which are most important for our purpose. 
 
 Natural (Spontaneous) Maceration After Death. — After tissue 
 death the tissue juices furnish a medium, which, for instance, dissolves the 
 cement substance, gluing together the epithelial tissue. For this reason we 
 find that in objects which are fixed only after the elapse of some time after 
 death the epithelial lining has been stripped from the underlying tissue. 
 
35 
 
 Alcohol. — A fine isolation agent for epithelium is found in dilute alcohol, 
 1 part of 90% alcohol to 2. parts of water. Epithelial tissues may easily be 
 macerated after twenty- four hours. 
 
 Acetic Acid Vapors. — Better still are acetic acid vapors. The mucous 
 membrane in question is spread on a wax or glass plate, with which we cover 
 a suitable bowl containing a small amount of acetic acid. In a few minutes the 
 epithelium can be removed in large pieces from the basement membrane. 
 
 Potassium Hydroxide enjoys quite a reputation, and is prepared by 
 adding to 32.5 gms of caustic potash 67.5 cm 3 of water. The mixture is 
 agitated well in a beaker until the potash is entirely dissolved. Since much 
 heat is generated in this process the glass beaker must be kept in cold water. 
 This strong solution will isolate non-striated and striated muscle fibres in 
 fifteen to thirty minutes, but shrinks such tissues considerably. 
 
 Hydrochloric and Nitric Acids. — For the isolation of the tubules, 
 ducts and alveoli of different glands, concentrated mineral acids are by far the 
 best, such as nitric acid and hydrochloric acid, which are allowed to act on 
 thin sections for from twelve to twenty-four hours. 
 
 Mechanical Maceration. — After tissues have thus been prepared by 
 chemical agents, they can be isolated mechanically, e.g., after epithelia have 
 been treated by the above-mentioned alcohol, they are thoroughly shaken in 
 their container and the coarse pieces of tissue are removed. After a few hours 
 the cells will settle on the bottom of the glass and can be taken out with the 
 pipette. Sedimentation, however, is better and quicker accomplished by 
 centrifuging. 
 
 When a mucous membrane has been prepared with acetic acid vapors, 
 small pieces of the epithelial lining are picked up with a scalpel or spatula, and 
 the cells thereafter isolated on the slide with teasing needles or by stirring. 
 
 Muscle treated with potassium hydroxide solution is transferred to pure 
 glycerine and tincture of iodine is added until, on shaking, the fluid ceases to be 
 decolorized. The next day the specimen can be transferred to water and the 
 fibres separated by vigorous shaking. 
 
 When dealing with glands subjected to mineral acids, small pieces are taken 
 and spread carefully on the slide in thin glycerine (1 part of glycerine to 2 
 parts of water). 
 
 THE CHOPPING METHOD 
 
 The Chopping Method. 
 
 Although somewhat primitive, this method yields excellent results and has 
 the advantage of being less destructive to the tissues than other methods. It 
 is especially indicated in the examination of the retina and striated muscle, no 
 matter whether a fresh or a fixed specimen is used. It can, however, be em- 
 ployed for other tissues and organs. 
 
 A small piece of the organ is placed on the slide, and either in the dry 
 state or after moistening the same it is carefully chopped with a sharp razor. 
 Small particles will be found to adhere to the knife, and must be removed from 
 time to time. The operator must take care not to spread the material all over 
 the slide, but aim to keep it as closely together as possible. In this fashion 
 
36 
 
 excellent transverse sections of the retina as well as of muscle fibres are ob- 
 tained with the additional convenience of enabling us to study innumerable 
 longitudinal sections. 
 
 THE CUTTING METHODS 
 
 Cutting of the Fresh Specimen. 
 
 As observed in the dissociation method of fresh specimens, it is also diffi- 
 cult to make cut sections, unless the material has previously been subjected 
 to a hardening process. An object must possess a considerable degree of hard- 
 ness to make a thin section possible, and in the natural state tissues are en- 
 dowed with very little stability, excepting very few. Either they are much too 
 soft and do not furnish enough resistance to the knife, as is generally the case, 
 or the deposits of lime salts make them so hard that the knife is unable to 
 penetrate (e.g., bone, tooth, cartilage of older individuals). A really suitable 
 consistency in man and the mammalia is found only in the cartilage of the 
 young. 
 
 Cutting of the Fixed 
 and Hardened Specimen. 
 
 When dealing with a fixed specimen the prospects are much more favorable, 
 most fixing agents acting to a certain degree in a hardening capacity. It is 
 for that reason, that fixation and hardening are often used promiscuously. This 
 is not entirely justified, since we have fixing solutions which not only fail to 
 harden the specimen, but, under protracted use, even decrease their consist- 
 ency, at the same time fulfilling all their requirements as fixatives. The best 
 hardening is obtained from the prolonged use of alcohol, which is for this 
 reason often continued after the fixation has been completed ; e.g., alcohol 
 formerly was extensively and to-day is to some extent used for preparation of 
 the central nervous system. 
 
 Frozen Sections. 
 
 Cuts obtained in the said fashion, by hardening with alcohol, do not, how- 
 ever, fulfil all the requirements, and the necessity arises to find other more 
 efficient means for the hardening of our objects. The oldest method, known 
 for nearly a hundred years, is freezing. The specimen is frozen and sections 
 are then made. This, the oldest hardening process, is used to this day with 
 good results. 
 
 Embedding. 
 
 In this method our specimen is saturated with a substance, originally solid, 
 which has been liquefied in a certain manner and will afterward again enter into 
 the solid state. Of these substances paraffin and celloidin play the most im- 
 portant role to-day. To embed a specimen in paraffin, the latter must be 
 heated to above its melting-point. The specimen immersed in this molten 
 paraffin will become saturated and, after cooling, will attain a consistency 
 which is mainly dependent upon the melting-point of the kind of paraffin se- 
 lected. Celloidin, on the other hand, is liquefied by dissolving it in a mixture of 
 alcohol and ether. Here, too, the specimen is immersed in the solution, and 
 
37 
 
 hardening is secured by the evaporation of the solvent and suitable after-treat- 
 ment. 
 
 Cutting Instruments. ' 
 
 Formerly the razor was used exclusively ; it requires, however, great skill 
 and an excellent knife to follow this practice. Hence the razor is not to be 
 recommended to the beginner. Modern times have evolved instruments which 
 make it possible for him to make useful sections without special skill. Such 
 instruments are designated as microtomes. 
 
 Following we will discuss in detail the three most important cutting 
 methods. 
 
 Frozen Section Method 
 
 It is a well-known fact that freezing does not in any way injure the greater 
 number of tissues, not even the highly developed organisms. We need only 
 call attention to the experiments of Raoul Pictet, who froze frogs to a tem- 
 perature of 28° C. below the freezing-point,- so that they were as hard as 
 glass, and yet after thawing them up, the animals recuperated and lived. 
 Muscle of mammalia can be exposed to a temperature of — 50° C. for a certain 
 length of time ; it will contract vigorously after the temperature has been 
 brought up to normal again. Freezing fixed specimens does not deteriorate 
 the latter in the least. It is evident therefore that the freezing process fur- 
 nishes an excellent and unharmfu] method of hardening. 
 
 In former years it was a difficult or expensive task to secure the low tem- 
 peratures necessary for the freezing, but all obstacles have been surmounted 
 by the simple and inexpensive method found in the use of solid carbonic 
 acid. 
 
 Preparation of 
 Solid Carbonic Acid. 
 
 The solid acid may be prepared from the commercial liquid carbonic acid 
 in the following manner: Use a bag made of strong silk velvet or chamois, 
 which is open on one side, where it can be drawn together after the fashion of 
 a tobacco-pouch. After taking off the cap, which closes the carbonic acid 
 drum, this pouch is drawn over the mouth of the drum and secured tightly. 
 The tank is placed on a chair, the head being slightly lowered, and the valve is 
 opened for about ten to fifteen seconds. After removing the bag we will find 
 it filled with a light white snow, the solid carbonic acid. The snow is col- 
 lected on a cloth and thence transferred into a wooden container, provided 
 with a funnel, and here it is ground together and compressed with hammer and 
 rod (Fig. 19«). After a few seconds a cartridge of carbonic acid can be 
 pressed from the wooden container, which can be kept for hours in an isolated 
 glass cylinder, the so-called Dewar — or thermos bottle (Fig. 19 b). 
 
 The Freezing Cylinder. 
 
 To freeze a specimen, the aim must be to guide the cold furnished by the 
 cartridge exclusively to and into the specimen. To accomplish this purpose a 
 freezing cylinder (Fig. 20) is used. It consists of a metal cylinder (a), 
 which contains a Dewar bottle (c), the latter being protected by a felt 
 
38 
 
 coat (d). The Dewar bottle is a double-walled glass cylinder, the walls of 
 which are silvered, the space between the walls having been reduced to a 
 vacuum. This serves to reduce the loss of heat by conduction and radiation to 
 a minimum. The metal cylinder is closed on top by a threaded cover, on the 
 under surface of which we find attached a second metal cylinder (A;), which 
 projects into the cavity of the Dewar bottle. In this smaller cylinder the car- 
 bonic acid cartridge is placed from below and the cover I is tightly adjusted. 
 To the inner side of this cover 
 a spiral spring is attached (m) 
 which serves to press the car- 
 bonic acid tightly against the 
 undersurface of the upper large 
 cover. Into the latter the freez- 
 
 Fig. 19. 
 
 Press, a, and Retainer, h, for Solid 
 Carbonic Acid 
 
 Construction of Freezing Cylinder. 
 Explanation in text. 
 
 ing-table proper is screwed (o), being separated from the outer metal cylinder 
 by a hard rubber ring. 
 
 The Freezing Microtome. 
 
 The described apparatus can be adjusted to most microtomes. We pre- 
 fer an instrument furnished by M. Schanze (Leipzig), which combines stability 
 with greatest precision and is not very expensive. In Fig. 21 we see the in- 
 strument with the freezer attached. It rests on a heavy cast-iron plate (gpl), 
 from which arises a vertical plate (vpl), which in turn gives off a third plate 
 at an angle of 45°. This latter, together with the upper part of the vertical 
 
39 
 
 plate, thus forms an horizontal track in which glides the knife-carriage, a 
 heavy triangular metal block (mschl). Its upper surface serves for the attach- 
 ment with bolt and nut of the knife (m), which is provided with a forklike 
 handle. On the other side of the vertical plate (the one turned toward us) 
 we find the specimen-carrier. It glides on two vertical rails, the left only- 
 being visible in our picture (opl). A lever (h) serves to move the specimen- 
 carriage (oschl) up and down. In close approximation with the specimen- 
 carriage is the specimen-holder. It consists of two shafts, place.d at right 
 angles to each other, which can both be secured by means of a screw (fschr). 
 The specimen-holder proper (oh) is connected with the upper shaft; it mainly 
 consists of a ring, which receives the freezing apparatus (gfr). The latter 
 can be raised or lowered in the ring by means of a screw. The finer adjust- 
 ment of the specimen-carriage can be procured by the use of the micrometer- 
 screw. It is only partly visible in our picture, the lower end being hidden in 
 
 Fig. 21. 
 The Freezing Microtome. Paraffin Clamp at the Eight of the Microtome. 
 
 the clamp (zw), while the specimen-carriage rests on its upper extremity. The 
 section scale (tsch) serves for the adjustment of the thickness desired. It is 
 divided into 50 whole or 100 half degrees, respectively. If the disc is turned 
 one whole degree toward the left, the specimen will be raised 10 ^, so that thick- 
 nesses of 5 n and its multiples can easily be obtained. 
 
 The Practical Use of 
 the Freezing Microtome. 
 
 Unscrew the cover of the freezing apparatus, which, being intimately con- 
 nected with the freezing cylinder, will lift the latter out, then open the lock- 
 cover on the lower end of this cylinder, insert a cartridge and close it again. 
 The cover is now screwed on again and the specimen, moistened with a few 
 drops of water or normal saline, is placed on the freezing-table. After five to 
 ten minutes it will be entirely frozen. The knife should be attached to the 
 knife-carriage so that it stands vertical to the long axis of the microtome. The 
 screw designed to hold the knife (mschr) must necessarily be drawn tight. By 
 rotating the freezing cylinder up and down, the upper surface of the specimen 
 is closely approximated to the edge of the knife, the apparatus fixed in this 
 
40 
 
 position, and we can now proceed to cut. The knife-carriage is moved for- 
 ward with the right hand, evenly and without exerting any pressure. Preced- 
 ing each cut the left hand turns the section scale a corresponding number of 
 degrees to the left, according to what thickness may be required. The sections 
 obtained either cling to the knife, becoming soft, or they drop to the freezing- 
 table, where they will lie in the frozen state. All these manipulations are much 
 easier executed than they are described. 
 
 How to Obtain a 
 Favorable Consistency. 
 
 The consistency of our specimen is naturally of prime importance for the 
 securing of useful sections, and the former is mainly dependent upon tempera- 
 ture, though also influenced by the structure of the specimen. Our apparatus 
 produces a temperature of — 12 to — 15° on the freezing plate. It is generally 
 used for fresh specimens, those retaining their own natural tissue juices ; for 
 objects that have been taken out of water and are therefore also saturated with 
 it, the temperature is too low, since such objects would split on cutting. This 
 objection can be obviated by placing the specimen in a fluid having a lower 
 freezing-point than water, e.g., very dilute alcohol or, better yet, dilute for- 
 malin, where it should be kept for several hours. The author recommends 5% 
 formalin, which should not be removed before placing the object on the freez- 
 ing-table. This procedure will almost invariably give good results, and only 
 very seldom will one have to resort to 10% formalin. 
 
 Time and care should be taken in cutting, as our apparatus will keep the 
 specimen frozen for at least one-half hour, and if signs of thawing should be 
 noticed, it will only be necessary to insert a fresh carbonic acid cartridge, and 
 the work can be continued in leisure. 
 
 Thickness of Sections. 
 
 As regards the thickness of sections, we can easily obtain cuts of 5 — 10 |A, 
 even with a large cutting surface, by the use of this freezing microtome ; but 
 we do not recommend such thin sections, unless especially required, since the 
 further treatment of sections becomes more difficult the thinner the cut. 
 Sections of 15 — 25 y. thickness should answer our purpose and will always show 
 even the finest structural details, if they are stained in the proper manner. 
 
 Treatment of the Sections. 
 
 If the section curls up and drops to the freezing-table, the transport is 
 very simple ; it is grasped with a pair of fine tissue forceps and thrown into a 
 bowl of water, where it will generally unfold itself without any aid. Should the 
 section adhere to the knife and thaw, it will have to be brushed off with the 
 tip of the finger, which is then dipped into the water. Each section may be 
 taken off separately, or the operator may wait until a number have collected 
 and then take them off en masse. The finger should be thoroughly dried each 
 time to avoid bringing water to the cutting surface. 
 
 When handling a very small and thin specimen it is best to first freeze 
 enough water to make a layer of ice 2 — 3 mm in thickness, on top of which the 
 object is placed. If we want to remove the latter again, the freezing-table must 
 be unscrewed from the cover and water poured on its reverse surface for a few 
 
41 
 
 seconds, or the whole of it dipped in water, after which the specimen, still 
 frozen, can be detached. 
 
 The Paraffin-Section Method 
 
 Much more complicated than the freezing is the paraffin-section 
 method. The first consideration will be to embed the specimen in paraffin, 
 to wit, not only to give the object a coating of paraffin, but to saturate every 
 part of the specimen. This is practicable only if the specimen is first thor- 
 oughly penetrated by a liquid, which is a good solvent of paraffin, and for this 
 reason quite a complicated preparatory treatment is necessitated. 
 
 Paraffin. 
 
 As paraffin are classed mixtures of hydrocarbons of the general formula 
 of C n H 2n + 2 in which the value of n fluctuates between 20 and 27. It is a 
 coal-tar product, but has also other sources, e.g., the hardest variety has its 
 origin in ozocerite. 1 It is a white, fatty substance of a specific gravity of 0.9. 
 The melting-point may be anywhere between 40° and 85° C, according to the 
 origin of the paraffin. Paraffin is absolutely insoluble in water and almost 
 insoluble in cold alcohol. It is soluble in carbon disulphide, chloroform, ben- 
 zine, benzol, xylol, toluol, petroleum ether, etc. The boiling-point is 300°. 
 
 Preparatory Media. 
 
 The reader will notice that the above-mentioned solvents of paraffin do not 
 mix with water in any proportion, but can easily enter into a mixture with 
 alcohol in any proportion ; if, therefore, we want to saturate our watery speci- 
 men with such a solvent of paraffin, which we will call a preparatory me- 
 dium, it will first be necessary to dehydrate the tissues by the use of alcohol. 
 Let us select chloroform as a standard preparatory medium, since it pos- 
 sesses many advantages over the others. The preparatory treatment will be 
 as follows : 
 
 Dehydration. 
 
 The fixed and washed specimen, which we suppose to be in water, is 
 gradually transferred to alcohol of increasing strength. Starting with 10% 
 alcohol we increase at the rate of 10%. We make the desired strength by 
 diluting absolute alcohol with the corresponding amount of water. The length 
 of time required for each grade of alcohol depends wholly on the size of our 
 specimen. Pieces of 1 cm 3 will require approximately twelve hours, while two 
 to three hours suffice for smaller pieces. The specimen should always stay in 
 its container, for which a wide-mouthed bottle will serve best ; the latter should 
 be corked and labelled as to origin of specimen, manner of fixation, and the 
 respective alcohol in use. In this fashion specimens, lest they be very large, 
 can be dehydrated in five days, if at the end we rechange the absolute alcohol 
 once. 
 
 The introduction of the specimen into the preparatory medium should also 
 take place gradually. As a rule, three steps will suffice. First, 3 parts of 
 
 1 of<»= smell, — Kyp6s= wax; a natural paraffin, found in Galizia, having a green, 
 brown or red color and an odor of petroleum. — (The Translator.) 
 
42 
 
 for our purpose. 
 
 alcohol are mixed with 1 part of chloroform, then equal parts are used and, 
 finally, 3 parts of chloroform to 1 part of alcohol. In each of these mixtures 
 the specimen stays from six to twelve hours, although a longer period will do 
 no harm, after which it is transferred to pure chloroform overnight. 
 
 The saturation with paraffin can only be effected by placing the specimen 
 in paraffin, previously heated until liquid, and by keeping the same liquid 
 during the entire process. To do this we must use a thermostat, similar in 
 construction to those used for culturing bacteria and for the artificial breeding 
 of chicken eggs. Fig. 22 represents such an apparatus, which is suitable 
 It is made of strong copperplate, has double walls and a 
 door, the space between the walls being 
 filled with water, or, better still, with 
 I acid-free glycerine. A water-gauge will 
 I indicate the depth of the water. A 
 second box surmounts the former, also 
 equipped with a door, but having only 
 simple metal walls. The temperature 
 obtained in either compartment is indi- 
 cated by separate thermometers. The 
 heating is done by a burner placed be- 
 neath the lower box ; this burner has a 
 safety device which shuts off the gas 
 supply in case the flame should suddenly 
 be extinguished. Before entering the 
 burner the gas passes through a regu- 
 lator situated within the double wall. 
 The temperature can thus be regulated 
 at will, so that only so much gas will 
 be admitted to the burner as is neces- 
 I sary to obtain the desired temperature. 
 There are also similar thermostats on 
 the market which can be heated with 
 petroleum or by means of electricity. 
 The temperature of the thermostat 
 should exceed the melting-point of the paraffin in use by about 1 — 2°. As will 
 be shown later, we select, as a rule, a paraffin having a melting-point of 54 — 
 56°, and should therefore regulate the temperature of our thermostat to 
 56 — 58°. This temperature will be maintained in the lower compartment, 
 while in the upper, which is indirectly heated from the lower, the temperature 
 obtained will range between 40° and 45°. 
 
 The Melting-Point 
 of Paraffin. 
 
 An important factor in the paraffin method is the correct selection of the 
 paraffin as regards its melting-point. The following considerations should be 
 borne in mind : The thinner we desire the sections to be made, the harder the 
 paraffin must be, or, in other words, the greater must be the difference between 
 the room temperature and the melting-point of the paraffin ; e.g., with a paraffin 
 
 Fig. 22. 
 
 Thermostat for Embedding in 
 Paraffin. 
 
43 
 
 of 52° melting-point sections of 5 [>■ or even thinner can be made in a cool 
 room, which would be impossible in a warm room or on a hot summer day. 
 Again, in the former case it would be hard to make sections as thick as 
 25 — 50 |a, which would be an easy task in the latter instance. We might say 
 that the preparation of thin sections requires a difference between room tem- 
 perature and paraffin melting-point of approximately 40°. If this difference 
 decreases, thin sections will be compressed during the cutting process, and will 
 consequently be deformed. For thick cuts the difference must not exceed 
 30 — 35°. If it is raised in this instance the section will break. 
 
 The best plan is to select two sorts of paraffin, one, the soft paraffin, with 
 a melting-point of 42°, and the other, the hard paraffin, with a melting-point 
 of 56°. The latter is used for thin sections, while in the preparation of thicker 
 sections we will have to add more or less of the former quality to the latter. 
 As a receptacle for the paraffin, round dishes with a flat bottom, lined with 
 porcelain or enamelled tin, will serve best. 
 
 Saturating the 
 Specimen with Paraffin. 
 
 Great care must be used and the temperature changes must be gradual 
 when the specimen is to be transferred from the preparatory medium into the 
 liquid paraffin. After the specimen has been saturated with chloroform, the 
 excess of the latter is decanted until only sufficient is left to cover the object; 
 then a few pieces of soft paraffin are added and the uncovered glass is placed 
 in the upper compartment of the thermostat. After a few minutes (15 to 30) 
 the paraffin will dissolve and a few more pieces can be added, and so on until a 
 sufficient quantity of paraffin is in the glass to cover the specimen, while in the 
 meantime the chloroform is slowly evaporating. The latter process will be 
 complete the next morning, and, instead of chloroform, our specimen will now 
 be saturated with the liquid soft paraffin. 
 
 During this time we can prepare our liquid hard paraffin in the following 
 manner : A suitable basin is filled to the brim with small pieces of hard paraffin, 
 and placed in a lower compartment of the thermostat. During the night the 
 paraffin will be liquefied. The specimen is now brought from the upper division 
 into the lower and after reaching the temperature -of 58° in about half an 
 hour, it is transferred to the hard paraffin with a heated spatula. It should 
 not be kept there unnecessarily long, but not less than two hours, when reason- 
 ably large. Sudden temperature changes are deleterious, more so than too pro- 
 longed, but gradually completed exposure to hard paraffin. 
 
 Paraffin Embedding. 
 
 This brings us finally to the embedding pro-l 
 cess proper. This can be accomplished in little 
 paper boxes, watch glasses, or, better than either, 
 in small metal boxes. They consist of two brass -p 1G 23 
 
 strips bent at right angles, one limb being shorter Frame for Embedding in 
 than the other (Fig. 23). When the two strips are Paraffin. 
 
 brought together they form, with the underlying 
 
 glass plate, a little box, which is open on top and the length of which can be 
 adjusted as desired. The glass plate and inner surfaces of the brass are thinly 
 
44 
 
 coated with vaseline. The box is now filled with paraffin and the specimen im- 
 mersed in the same, and then deposited in a position most suitable for cutting. 
 Several specimens can be embedded in this manner in one box by placing them 
 at a proper distance. 
 
 Cooling of the Block. 
 
 The paraffin must needs be cooled now, and this can easily be accomplished 
 by placing the box including the glass plate in a large basin containing cold 
 water. Care must be taken here, since the lighter paraffin tends to rise to the 
 water-surface. This can be overcome by carefully inclining the bed, and let- 
 
 FiG. 24. 
 
 Position of Knife in the Cutting of Paraffin Sections. 
 
 I, correct; II, too acute; III, too obtuse. 
 
 ting the water flow over it gradually. After a few hours our block is completely 
 solidified and can easily be removed from the bed. 
 
 The Microtome for 
 Paraffin Sections. 
 
 The microtome, mentioned previously, can also be used here; we simply 
 remove the freezing-table and substitute the paraffin clamp, which is depicted 
 in Fig 21, lying next to the microtome. The paraffin block itself may be 
 properly shaped and adjusted in the clamp, or it can preferably be mounted 
 first on a small wooden block and the latter fastened in the clamp. Of course 
 the clamp can be left out all together, and the paraffin block molten directly 
 to the freezing-table. 
 
 The Cutting of 
 Paraffin Sections. 
 
 This proves more difficult to the beginner than the preparation of frozen 
 
 sections, for the reason that in this method the position of the knife is of 
 
45 
 
 great importance. While it does not matter whether our knife is in an oblique 
 or sloping position, the angle formed by the cutting edge and the horizontal 
 plane is the vital issue. The microtome knife is wedge-shaped and acts like 
 a wedge, the surfaces becoming abruptly converging near the cutting edge, thus 
 practically forming a composition of two wedges, the two converging surfaces 
 near the edge receiving the name of cutting facettes. If we picture an object 
 being placed before this double wedge (Fig. 24), it will be possible to make 
 a section only if the lower facette c d either coincides with the cutting surface 
 a b (I) or forms with it an angle above 180° (II). If the angle should be less 
 than 180° (III) no cut will result, since the facette will simply glide over the 
 surface of the object, polishing it smooth. Only, if the specimen is elevated 
 enough to let its surface be on a higher level than the knife-edge, an irregular 
 section may be accomplished. To make a correct cut, the knife must be rotated 
 upon its long axis and adjusted more steeply. Larger instruments possess for 
 this purpose a separate knife-holder, but we can accomplish the same purpose 
 by using two small wooden plates, placing one under the anterior, the other 
 over the posterior handle of the knife. A metal ring is placed on top and the 
 whole fastened tightly in the clamp. Several sizes of wooden plates should be 
 kept on hand, so that an appropriate thickness may be selected, since the angle 
 of the cutting facettes changes with almost every honing of the knife. Again, 
 the position may be too steep ; this will be indicated by the presence of numer- 
 ous breaks in the section, running parallel with the edge of the knife. 
 
 The Curling of Sections. 
 
 All paraffin sections have more or less tendency to curl upon themselves. 
 This must be obviated by the use of a camel's-hair brush. While the right 
 hand moves the knife-carriage, the left holds the brush, and as soon as the knife 
 enters the substance of the block, but not before, the section is gently pressed 
 against the knife. 
 
 Mounting of Sections. 
 
 The next step is the mounting of the section on the slide, although the 
 specimen may also be mounted on the cover-glass or on isinglass. The cover- 
 glass or slide or isinglass, as the case may be, must first be thoroughly 
 cleansed. If new, they should first be moistened with dilute (20 — 30%) alco- 
 hol, and dried with a clean cloth. Then a minute drop of egg albumin glyce- 
 rine (cut up the white of an egg, filter and mix with an equal part of glycerine 
 and a large crystal of thymol) is placed on the centre of the slide and spread 
 thoroughly with the tip of one finger. A large drop of water is quickly placed 
 on this layer of egg albumin and allowed to spread and the section placed 
 therein. The slide is now held over a flame, the heat being estimated by the 
 hand, until the sections flatten out completely, without being burned, the super- 
 fluous water is then decanted, the sections arranged, and the slide placed in the 
 upper part of the thermostat to dry. A few hours will suffice, after which the 
 sections will be found to adhere completely. 
 
 For sections which are not to be stained another method is more suited. 
 The slide is coated with a thin layer of a mixture of oil of cloves and collodium 
 (collodium 1 part, oil of cloves 3 — 4 parts), the sections are pressed down 
 
46 
 
 with a brush or dry finger-tip and can then be transferred to xylol to dissolve 
 the paraffin. 
 
 The Celloidin Section Method 
 Cettoidin. 
 
 Celloidin, a highly concentrated solution of guncotton, dinitrocellulose, 
 in a mixture of alcohol and ether, is sold in the form of rectangular plates of 
 a finger's thickness, having a consistency of cartilage. They are saturated 
 with alcohol and packed in hermetically sealed tin boxes to avoid drying. 
 
 The Preparation of 
 the Celloidin Solution. 
 
 To prepare a solution suitable for embedding, the celloidin must first be 
 carefully dried. The plate is cut into strips, which are in turn divided into 
 smaller pieces and put in a glass beaker. After protecting the contents from 
 dust by covering with a sheet of paper, the beaker is put in the thermostat. 
 After two to three days the celloidin particles will have dried and shrunk to 
 opaque hornlike chips. Care must be taken in the following steps, as the 
 celloidin in this dried state is explosive. The chips are put in a glass-stoppered 
 bottle (of 800 — 1,000 cm 3 volume), which must be absolutely dry, and are 
 covered with 250 cm 3 of absolute alcohol. The mixture is frequently shaken 
 or stirred with a glass rod. After twenty-four hours the chips will be softened 
 and swelled to a glassy mass. 250 cm 3 of water-free ether are added and, if 
 promptly stirred and shaken, the solution will be complete in from two to three 
 days. We now have a mass of honeylike consistency, the celloidin stock 
 solution. From the latter we prepare two dilutions : Celloidin / ( 1 part 
 of stock to 3 parts of alcohol-ether), and Celloidin II (1 part of stock to 1 
 part of alcohol-ether). Both solutions should be kept in well-corked bottles. 
 
 Dehydration and Saturation. 
 
 As regards celloidin embedding we find that here, too, thorough dehydration 
 is of prime importance; we therefore have to gradually transfer our specimen 
 into stronger and finally absolute alcohol, the latter being changed at least 
 once. It is then brought into a mixture of equal parts of absolute alcohol and 
 water-free ether, and from there into Celloidin I, i.e., the thin celloidin solu- 
 tion. The length of time spent in the latter (of course in a well-closed bottle) 
 depends entirely on the size of the specimen. For very small objects two days 
 are sufficient, larger ones must remain a correspondingly longer period up to 
 two or three weeks. From this thin solution the specimen is transferred to the 
 thicker Celloidin II, and thence to the stock solution, remaining in each an 
 equal length of time. 
 
 Preparation and Preservation 
 of the Celloidin Block. 
 
 After saturation with the stock solution the specimen with the retained 
 celloidin is placed on a correspondingly large dry wooden block, exposed to 
 the air for about ten minutes and preserved in a large quantity of 70 — 80% 
 alcohol. After being acted on by the alcohol for twenty-four hours the speci- 
 men will have attained a consistency adapted for cutting. Before using, the 
 wooden blocks must be treated for several days with 95% alcohol, preferably 
 
47 
 
 warm, to free them from tannic acid and resin. Stabilit, a material often 
 used in electro-technique, may be used instead of wood ; it can easily be divided 
 with a saw. 
 
 Preparation of 
 
 the Celloidin Section. 
 
 The microtome, previously mentioned, with attached clamp, can readily 
 be used for the preparation of celloidin sections. The knife is adjusted as 
 obliquely as possible, forming an acute angle with the long axis of the micro- 
 tome, so that in cutting the entire edge is made use of. This presupposes, of 
 course, that the cutting edge is immaculate. During the cutting the knife is 
 constantly moistened with 70 — 80% alcohol by means of a large camel's-hair 
 brush, so that the sections float on the edge. Tissue forceps will serve to 
 transfer the sections from the knife to the alcohol dish. As soiling of the 
 microtome with alcohol cannot be avoided during all these procedures, a 
 thorough cleaning of the instrument afterward is essential. 
 
 SELECTION OF THE CUTTING METHOD IN SPECIAL CASES 
 
 Each of the methods described has its own merits as well as disadvantages. 
 The freezing method excels the other two without doubt, on account of its 
 ready execution and rapidity. Here our specimen does not come in contact 
 with any extraneous reagents, excepting the formalin solution, and therefore 
 this represents the most conservative method, altering the staining properties 
 to the least extent. On the other hand, this method presents a great disad- 
 vantage, met in objects in which the component parts are only loosely con- 
 nected or are separated by interspaces, where this connection is easily, often 
 unavoidably, destroyed, and this fault will not be remedied as long as we fail 
 to discover a suitable embedding method for fresh objects or those kept in 
 water. 
 
 The paraffin method is doubtless the most aggressive of the three methods, 
 necessitating not only a thorough treatment with alcohol, but also the heat- 
 ing of the specimen to a considerable degree. The advantage in this procedure 
 lies in the fact that specimens so treated allow of the most delicate cuts, and 
 that in these sections the original position of the parts is absolutely preserved, 
 and furthermore that the after-treatment is the easiest imaginable. Its present 
 supremacy in our histologic and zoologic laboratories is due to these ad- 
 vantages. 
 
 Midway between freezing and paraffin methods stands the celloidin section 
 method, which avoids heating the specimen, but requires a longer saturation 
 and the most careful manipulation. Thin sections, i.e., 5 — 10 n, may be ob- 
 tained in either of the three methods. The thinnest are undoubtedly fur- 
 nished by the paraffin method, at least when the specimen is small. When deal- 
 ing with larger objects the freezing method will answer as well or often better. 
 
 The reader can deduct from these suggestions which method be best suited 
 for a particular case. We always give the freezing method the first rank, as 
 being the simplest and least aggressive, and choose the others only when sec- 
 
48 
 
 tions of the finest precision are wanted, or when the structure of the tissue 
 calls for embedding. 
 
 APPENDIX TO THE PREPARATION OF SECTIONS 
 
 DECALCIFICATION 
 
 The hard structures of the human body, viz., bones and teeth, due to de- 
 posits of lime salts, possess such firmness that they resist cutting. To render 
 them soft, we must aim to remove these salts by treating them with their 
 solvents, viz., acids. This process has received the name of decalcification. 
 
 In many cases the fixing solution can be used as such by protracting its 
 application; e.g., chrome-osmium-acetic acid and sublimate-nitric acid are 
 excellent decalcifiers and yield good results if small specimens are suspended 
 in large amounts of the liquid and the latter is changed frequently. 
 
 As a general rule, however, we recommend to fix the object first thoroughly 
 and then expose it to the action of a decalcifier. As such only those acids can 
 be considered which can change the carbonates or phosphates of the hard 
 structure in question into their respective soluble salt. 
 
 Among these acids nitric again ranks foremost. As a decalcifier we use it 
 in a 5% watery solution, immersing the fixed and washed specimens in large 
 quantities of same. To expedite decalcification, the specimen is frequently 
 shaken, or it may be suspended in the upper layers of the fluid and the latter 
 changed repeatedly. The duration of this process of course depends on the 
 size of the object and the amount of lime contained in it. In order to test the 
 progress of decalcification the specimen may be pierced with a needle or an 
 attempt at cutting with the razor may be made, unless such procedures are 
 contraindicated for other reasons. With fair sized pieces of only moderately 
 old bone two to four days will usually complete the process, 1 for whole teeth 
 eight to ten days will suffice. After decalcification the acid must not. be 
 directly washed out with water, but the specimen first immersed in 10% forma- 
 lin (which is changed frequently) or in 5% Glauber salt solution (also to be 
 changed repeatedly) for twenty-four hours, after which the washing can be 
 done without any harm resulting. 
 
 Of the other decalcifiers we will only mention the trichloracetic acid, the 
 method of procedure being identical with that of nitric acid. 
 
 STAINING METHODS 
 
 Staining. 
 
 By staining we mean that process by which an unstained body, treated 
 with the solution of a stained body, appears stained itself. The color result- 
 ing will usually be the same as that of the solution used ; however, under certain 
 circumstances this may not be the case. Dyeing is a primeval art, owing its 
 development to the beauty sense of mankind. The methods employed for the 
 staining of our microscopic specimens are largely drawn from the practical 
 dyeing process, but not with a view as to beauty in colors, but solely because 
 
49 
 
 the staining is an important, if not the most important, aid in the diagnosis 
 of tissues and their component parts. 
 
 Stains. 
 
 All stains used in microtechnique are organic compounds, viz., they contain 
 carbon and hydrogen. They either owe their origin directly to the animal or 
 vegetable kingdom, or, as in the majority of cases, they are artificially pre- 
 pared (synthetic), so that we must differentiate between natural and arti- 
 ficial stains. 
 
 The Essentiality 
 of Staining. 
 
 It has not been decided as yet with accuracy what the process is that 
 causes an organic body to appear colored, i.e., to let certain rays of the spec- 
 trum pass through, and reflect others, and absorb still others, but so much 
 we may safely assume, that this property is founded in the chemical constitu- 
 tion of the body, and a number of atom groups are already known which pos- 
 sess the capability, when entering an unstained body, to change the same into 
 a dye. In the same manner the essentials of staining have remained rather 
 obscure. Three theories have so far been expounded. The mechanical 
 theory assumes that physical powers alone are concerned in driving the 
 staining solution into the tissues, namely, osmosis and capillary attraction. 
 Once in the tissues the dye is condensed by absorption in a similar manner, as 
 animal charcoal may detract all the dye out of a stain solution and render 
 the latter colorless. The chemical theory, on the contrary, claims that 
 chemical, saltlike compounds are formed during the process of staining be- 
 tween the stain, respectively, its components and the albuminous substances of 
 our specimen. As we have discussed previously, these albuminous bodies partly 
 take on the character of acids, partly that of bases, and it therefore seems 
 quite reasonable that by their combination with the stain acids, respectively, 
 stain bases of our dyes, saltlike compounds result. The third theory finally con- 
 siders the stained preparation as a solidified solution of the stain in the tissues. 
 This is not the place to enter into a discussion of the merits of each of these 
 theories. Suffice it to say that without doubt chemical processes are con- 
 cerned in the phenomenon of staining, which, however, do not exclude the pos- 
 sibility of processes of a physical nature occurring at the same time. 
 
 Preparation of the 
 Color Bath. 
 
 A stain to be of use in our work must be soluble. Solvents are primarily 
 water, but also alcohol, rarely glycerine ; furthermore, salt solutions, weak 
 acids and alkalies are all used. To increase the staining power of some of the 
 watery solutions of dye, aniline is added, or instead of dissolving the coloring 
 matter in water, we use aniline water, which can be prepared by mixing a 
 small amount of aniline with distilled water, thoroughly shaking it and filtering 
 the opaque mixture through a previously moistened filter. 
 
 Concentration of the Stain. 
 
 As a rule, we prepare staining solutions of a fixed strength, i.e., a certain 
 amount of dye is dissolved in a given amount of solvent, so that all of the dye 
 
50 
 
 will go into solution. The dissolving takes place either at room temperature 
 or the solvent is previously heated. Often, however, we use the so-called con- 
 centrated solution, which is made by introducing an excess of the dye into the 
 solvent and shaking repeatedly; after a while we find above the sediment of 
 the excess a solution of maximum concentration. Owing to the fact that in 
 most cases the dyes are more highly soluble at a higher temperature than at 
 some lower, the strength of such solutions must needs fluctuate with the tem- 
 perature in which they are kept. When using concentrated solutions the de- 
 canting must be done with great care, to avoid picking up some of the small 
 solid particles of dye from belcw. 
 
 It is difficult to say in general which concentration would be apt to yield 
 the best results. We often hear it mentioned that thin solutions work better 
 than the concentrated. That may be the case with some certain dyes, but can- 
 not be adopted as a general principle. In short, the concentration must de- 
 pend upon the nature of the dye and the particular case in which it is to be 
 used. 
 
 Temperature of the 
 Staining Bath. 
 
 As a rule, we stain at room temperature, but special circumstances may 
 demand a higher temperature, when the staining solution must be warmed ; this 
 is identical with the custom of heating the solutions in practical dyeing. Beyond 
 doubt the heated dye will penetrate tissues in a shorter period of time and 
 the staining will be more intensive than if lower temperatures prevail. 
 
 Staining of the 
 Living Specimen. 
 
 Living Specimens, as well as preserved, may be stained. The former is 
 called vital staining or staining in the living state. A vital staining, 
 in the strict sense of the word, can only be executed in the living animal body 
 itself, by incorporating into the latter the desired dye in some appropriate 
 fashion. Vital staining is of great importance in research, and, although still 
 in its infancy of elaboration, the results obtained have been brilliant. In the 
 higher animal the incorporation of dye may be accomplished by means of 
 feeding, the dye being mixed with the food. The dye having been dissolved 
 in the chyme, absorption takes place in the intestines. A quicker method is to 
 inject the watery solution hypodermatically , and finally the intravenous 
 method may be resorted to, by introducing the stain into a vein of the 
 anaesthetized animal. Of course the dye must not be poisonous enough to cause 
 a cessation of heart action. If such should be the case, the animal is allowed 
 to bleed to death, after which the circulatory system is flooded under pressure, 
 the place of introduction being the heart or an artery. The latter method can 
 be used to good advantage in the human body, e.g., an amputated extremity. 
 
 Staining of the 
 Surviving Specimen. 
 
 We often speak of vital staining, when small sections are taken from the 
 living or recently killed animal and subjected to the action of a staining 
 fluid. It is a question whether we have here really vital staining, since there 
 are no criteria which would show us that such a section of tissue or its com- 
 
51 
 
 ponents at the moment of staining are still living or have died, a question 
 which might also enter into the vital staining proper. Hence it might be wiser 
 to designate such procedures as staining of fresh specimens, i.e., such as are 
 not preserved. Weak solutions should be used for this purpose and, if possible, 
 the dye should be dissolved in some indifferent fluid, e.g., normal saline or 
 Ringer's fluid. We can either take small particles .of the organ with scissors or 
 razor, or we can first make frozen sections of these particles and stain the 
 latter. The chopping method is also useful, the specimens being quickly 
 chopped on the slide, after which the stain is added, the specimen put in a 
 moist chamber and the whole eventually placed in the thermostat at a tem- 
 perature of 38—40°. 
 
 Staining of the 
 Preserved Specimen. 
 
 The staining of preserved objects is far more common. Here we must 
 always bear in mind that our fixation and preserving process causes invariably 
 radical changes in the chemical composition of the albumins, which make up 
 cells and tissues. One should therefore be guarded in promising results, which 
 are to be obtained with these methods. 
 
 Lump and Section Staining. 
 
 Two separate methods of procedure are to be distinguished in the staining 
 of preserved objects; on the one hand we may stain larger or smaller lumps 
 of tissue in toto, on the other we subject the sections made of the piece of 
 tissue to the dye. The lump staining requires a dye of great power of diffu- 
 sion, unless we deal with membranous structures. Most of the watery solutions 
 lack this quality, whence it is necessary, with few exceptions, to resort to alco- 
 holic solutions when staining in lump is desirable. Another disadvantage of 
 lump staining lies in the fact that we are not able to control the staining pro- 
 cess sufficiently. For these reasons this method is used only seldom in his- 
 tology, while embryology employs it frequently to this date. In section stain- 
 ing there is no difficulty in controlling the progress of the staining process and 
 to interrupt it at the proper time, and therefore it has been adopted as the 
 superior of the two methods. 
 
 Staining of Frozen Sections. 
 
 For the staining of frozen sections watch-glasses of different sizes with 
 ground edges, convex on the surface and straight on the bottom, on which .they 
 rest, are most serviceable. As a cover a glass plate or a similar watch-glass 
 of the same size may be used. The sections are placed into the staining fluid 
 by means of a curved, well-polished needle or, better still, with slightly curved 
 glass needles, which can be easily made from a glass rod or a thick-walled 
 glass tube (barometer tube) in any desired size. It is important that these 
 needles be smoothly polished; if they lack in even smoothness or present rough- 
 ness in the least degree, sections may easily adhere and tear. The specimens 
 are removed in the same manner. When the stain is opaque, difficulty in re- 
 moving the sections may be encountered. When staining but a few sections, 
 only a few drops of the solution are necessary ; when staining a larger amount 
 
52 
 
 in a correspondingly larger quantity of dye, the curved needle is guided along 
 the bottom of the dish and the sections are fished out gradually. 
 
 Staining of Paraffin 
 Sections on the Slide. 
 
 The staining of paraffin sections previously pasted on the slide is best 
 performed in cylindrical glasses, which must be slightly higher and a trifle 
 broader than our slides. The first aim will be to remove the paraffin from the 
 section, since its presence renders staining difficult, although not impossible. 
 A good solvent is found in xylol, C 6 H 4 (CH 3 ) 2 , a waterlike transparent fluid, 
 lighter than water and unable to mix with water in any proportion. The 
 paraffin will dissolve in a few seconds, if the slide is lightly agitated to and fro, 
 and if the sections are not too thick. In order to be able to transfer the speci- 
 men into our watery staining solution, we must now remove all the xylol from 
 it, which is accomplished by the use of absolute alcohol; from the latter 
 the sections are first transferred to a more dilute grade of alcohol, say 90%, 
 before the staining proper can begin. First the watery stain will not penetrate 
 the section, but after moving the latter to and fro a few times, it will enter the 
 tissue. Practically it is best to have several of these glasses side by side, the 
 first containing xylol, the second absolute alcohol, the third 90% alcohol, and 
 the fourth the staining fluid. It is of advantage to insert a fifth glass between 
 the third and fourth, containing water. Specimens are transferred from one 
 fluid to the other, several slides being placed in one glass if necessary, by plac- 
 ing them in pairs with their backs approximated. The film sides of course 
 must not be touched in any way in order to avoid laceration of the section. 
 
 Staining cf Cclloidin Sections. 
 
 Celloidin Sections are treated in a similar manner as are frozen sec- 
 tions, with this exception, that less care need be taken in transferring. The 
 specimen is simply put from alcohol to water and thence into the stain. If 
 the latter is an alcoholic solution, the water, of course, is omitted. 
 
 Simple and 
 Multiple Staining. 
 
 Depending upon whether we stain with one or with several dyes we 
 differentiate between simple and multiple staining. In multiple staining 
 we naturally select dyestuffs which differ widely, in general, contrast-stains. 
 When several stains are used in succession we speak of successive mul- 
 tiple Staining; again, when a mixture of several dyes is used for staining 
 
 we call the process simultaneous multiple staining or compound 
 staining. 
 
 Progressive and 
 Regressive Staining. 
 
 If the staining process is interrupted at its height, i.e., the optimum of 
 the process, we speak of progressive staining; in that case we need only 
 wash out the excess staining solution from the specimen. When using the 
 regressive Staining method, wc overstain the section intentionally, bringing 
 it to the maximum of the process, the optimum being gained by treating the 
 
53 
 
 specimen with a solvent of the dye used, or by partly destroying the stain. 
 Such agents are known as differentiation agents. 
 
 Substantive and 
 Adjective Staining. 
 
 According to practical dyeing we can also differentiate between substan- 
 tive and adjective method. The process is substantive when a simple solu- 
 tion of a dye is used without any adjunct agent. In the adjective method 
 we have besides dye and specimen a third factor, without which the dye will 
 fail to stain the tissue. Such agents are designated as mordants or bases, 
 and we speak of a mordant dye, when it alone has little or no staining power, 
 the latter only being developed when the base is added. Here, too, chemistry 
 has given us the key to the relation existing between the composition of a dye 
 and its staining power, inasmuch as we know that all dyestuffs containing two 
 hydroxyl groups in ortho position are mordant dyes. 
 
 Mordants. 
 
 Agents of all sorts of chemical structures are used as bases in commercial 
 dyeing, e.g., acids, oils and the product of the action of the former on the 
 latter, tannins, metal oxides and others. In microtechnique we use almost ex- 
 clusively metal oxides for this purpose. The mordant 1 dyes possess the prop- 
 erty of forming with metal oxides saltlike compounds of high staining 
 power, being soluble in water only to a small degree. Such compounds are 
 designated as color lakes. We can prepare the latter in vitro, put them into 
 solution in the proper manner, and then stain with this solution of the lake ; 
 i.e., simultaneous mordant dyeing, or we first let a solution of our base 
 act on the specimen and stain it thereafter, succedaneous mordant dye- 
 ing. Then the lake is created in the specimen. Mordant staining plays an 
 important role in microtechnique, the simultaneous process as well as the suc- 
 cedaneous. 
 
 Diffuse and Elective Staining. 
 
 The result of a staining process may be diffuse or elective. It will be 
 diffuse if all the component parts of the specimen appear equally stained. 
 Staining is called elective when either certain parts of a tissue are affected, 
 while others remain unstained, or when all the parts are stained but differ in 
 shade of the same color or under the most favorable circumstances appear ac- 
 tually in different colors. It is evident at once that diffuse staining is of small 
 value, and that our aim must be to accomplish elective staining. This end may 
 be accomplished in various manners, since experience has taught us that the 
 different dyes have a different affinity to certain kinds of tissue, so that, when 
 treated progressively, they are stained more quickly, while under the regressive 
 method they retain the particular dye correspondingly longer. 
 
 A specimen treated with the simultaneous or succedaneous method will show 
 that, under proper technique, certain dyes are taken up by certain parts of 
 tissues ; e.g., certain dyes only stain the nuclear chromatin, the basic sub- 
 
 1 The terms, "mordant" and "base," are used interchangeably to represent the Ger- 
 man "Beize," the word mordant being of French origin: mordre = to etch. — (The 
 Translator.) 
 
54 
 
 stance of cartilage, mucus, certain kinds of cellular granulations; others, 
 again, will affect the nucleoli, the protoplasm of the cell body, the haemoglobin 
 of the red blood corpuscles, the connective tissue shreds, the basic substance of 
 bone, etc. We have thus found that the group mentioned first is stained by 
 basic dyes, i.e., combinations of color bases with any desired acid, while the 
 latter is affected by acid dyes, i.e., color acids or their alkaline salts; we 
 may therefore call the first group of tissues or parts thereof basophilic, the 
 
 latter acidophilic. 
 
 Metachromatic Staining. 
 
 It is not always necessary to treat a specimen with two different colors, 
 say red and blue, in order to obtain red in certain tissue elements and blue in 
 others ; we have certain chemically uniform dyes, which possess the property 
 of staining in two different colors, or even will bring out different shades of 
 these colors in various tissue components. Such dyes are known as meta- 
 chromatic. Metachromasia is a virtue possessed by many, but principally 
 by basic dyes, where the free base has a different color than the salt repre- 
 sented by the dye ; e.g., not infrequently we find that a color base is staining 
 red, while its hydrochloric acid salt will give a blue color. The latter we use 
 as a dye, since the color bases are, as a rule, insoluble in water. If we now pro- 
 ceed to stain in the watery solution of such a blue dye, certain elements will be 
 stained orthochromatic, i.e., in the color of the solution, viz., blue, others meta- 
 chromatic, in the color of the base, viz., red. Metachromatic staining will 
 produce the greatest contrasts on the fresh, unprepared tissue and is greatly 
 influenced by our various fixing methods. A dye, in order to develop meta- 
 chromatic properties, must generally be in watery solution, since alcoholic 
 solutions show little or no metachromasia, the latter also being usually de- 
 stroyed if the specimen is afterward treated with alcohol. A strong meta- 
 chromasia and a decided basophilia may in many tissue elements go hand in 
 hand, e.g., mucus, cartilaginous substance, certain cell granulations ; on the 
 contrary the nuclear chromatin is decidedly basophilic, but shows less meta- 
 chromasia as does the greatly acidophilic collagenous connective tissue. The 
 cell protoplasm is always orthochromatic. Definite knowledge of the cause 
 of metachromasia is up to the present not claimed ; perhaps the phenome- 
 non is due to a tautomeric combination of the color base in question with the 
 acid. 
 
 After this preliminary discussion of dyes and staining we will proceed to 
 study singly the most important stains. 
 
 STAINS OF ANIMAL ORIGIN 
 
 Cochenille (Cocheneal) 
 Carmine. 
 
 The mother substance of carmine is the cochenille. The latter is a name 
 given to the dried female of the Coccus Cacti, which habitates several species 
 of the Opuntia Cacti in Mexico and Central America. The female insects are 
 gathered from the plants shortly before shedding the eggs, they are then killed 
 
55 
 
 in hot water and dried in the oven. Approximately 140,000 animals will yield 
 1 kg. 1 of cochenille. 
 
 Carmine. 
 
 Carmine is manufactured from the cochenille by extraction with alum and 
 tartar and occurs as a deeply red, earthy, friable mass, being put into com- 
 merce in smaller or larger lumps. It is totally insoluble in water and in alcohol, 
 soluble in alkalies, ammonia, acids, borax, and alum solutions. 
 
 Carminic Acid. 
 
 Chemically we must consider carmine as a combination of clay-lime- 
 albumin with carminic acid. The latter is the coloring principle of carmine 
 and in its pure state forms a beautiful red crystalline powder, readily soluble 
 in water and alcohol. With alkalies it forms soluble, with earthy alkalies and 
 the heavy metals insoluble salts. 
 
 In microtechnique the staining solutions are prepared from either cochin 
 nille or carmine, or carminic acid, as well. The best and most constant results 
 are without doubt obtained from solutions made of chemically pure carminic 
 acid, and for this reason we shall use such solutions almost exclusively. 
 
 Carmalum. 
 
 Make a hot solution of 5% potash alum, cool and filter; heat the filtrate 
 and dissolve in it carminic acid to the extent of 0.5%. A deep bluish red solu- 
 tion results, which after cooling is filtered and preserved in 0.5 — 1% formalin, 
 this solution will stain sections very rapidly and intensively, imparting a bluish 
 red tone. After staining for usually ten to fifteen minutes the sections are 
 placed in a 5% alum solution for a few seconds and then washed in water. In 
 this way an almost pure staining of the nucleus is obtained. This stain is also 
 adaptable to block staining. The blocks must not be too large and should re- 
 main in the staining fluid from twenty-four to forty-eight hours, after which 
 they are washed in water. 
 
 Paracarmine. 
 
 This preparation is of still greater intensity and can be made by dissolv- 
 ing 1 gm of carminic acid, 0.5 gm of chloraluminum 1 and 4 gms of chlorcal- 
 cium 2 in 100 cm 3 of 70% alcohol, heating carefully. When the precipitate has 
 settled, filter and keep in a well-corked bottle. For staining, this solution is 
 diluted with five to ten times its volume of 70% alcohol, and acetic acid is 
 added to the small extent of 1 — 2 drops to each. Paracarmine stains more 
 rapidly and intensively than carmalum and can be used to the same advantage 
 for sections or blocks. Alcohol, 70%, is used for washing; 2% acetic acid can 
 be added to the alcohol, if an absolute pure staining of the nucleus is desired. 
 
 STAINS OF VEGETABLE ORIGIN 
 
 Flora furnishes us with dyes of great variety and number, which formerly 
 were of decided importance to the practical dyer and are even to this day used 
 
 1 Two pounds. * Chlorides. 
 
56 
 
 frequently by him. For our purpose only two deserve mention: haematoxylm 
 and indigo. 
 
 Hoematoxylin. 
 
 It is an extract made from the wood of the hematoxylon campechianum, a 
 caesalpiniacea found in Central America, which is extracted in the form of 
 small rhomboid, colorless crystals. It is not very soluble in cold water, but 
 readily so in hot water, alcohol and ether. If a watery or alcoholic solution of 
 hematoxylin is exposed to light and air, it will first assume a yellow tinge; 
 later it will become brown. 
 
 Hasmateln. 
 
 The cause of this change in color is found in the fact that haematoxylin is 
 wholly or partly oxidized to haematein. The latter is on the market in the 
 form of a brown powder, which is soluble in water, alcohol, ether, glycerine, 
 alkalies and ammonia in particular. 
 
 Hematoxylin per se, substantively, cannot be used as a stain ; it is a typical 
 representative of a mordant dye, forming, with most metal oxides, strongly 
 staining lakes, their hue varying between blue and black. During the lake for- 
 mation the hematoxylin is either at once or gradually oxidized to hematein, 
 and the more advanced the oxidation is, the better the lake will stain. There 
 is, of course, an optimum of staining power, which may and often is trans- 
 gressed, since the oxidizing process does not stop with the formation of haema- 
 tein, but will finally lead to the formation of oxalic acid. Haematoxylin lakes 
 can be applied in either the simultaneous or the succedaneous method, i.e., we 
 can stain with the ready solution of the lake or we may produce the lake in 
 our specimen by successive applications of the mordant and the hematoxylin 
 solution. To make the solution we may use either hematoxylin or hematein. 
 
 Mcemalum. 
 
 This is a clay hematein lake. Fifty grams of alum are dissolved in 1 litre 
 of hot water, and the solution is cooled and filtered. One gram of hematein is 
 dissolved in 50 cm 3 of 95% alcohol under gentle heating, and both these solu- 
 tions are mixed. A dark bluish violet color reaction takes place at once. A 
 sediment is common and renders it advisable to filter before using the stain. 
 Hemalum will stain sections in a very few seconds ; the specimen is washed in 
 water, which will impart to it a dark blue color. If overstained, a specimen 
 can be reduced by being placed in slightly acidified water (2 drops of muriatic 
 acid to 50 cm 3 of water). The latter solution extracts any superfluous dye, 
 causing at the same time a change in color from the blue to a red tint. After 
 being thus reduced the specimen is washed until it again assumes a blue color. 
 
 Iron Hematoxylin 
 
 probably furnishes a still stronger nuclear color result than the preceding 
 stain; it is prepared as follows: 10 gms ammonium ferrosulphate, iron-alum, 
 so-called, are dissolved in 150 cm 3 of hot water; in the same manner 1.6 gm 
 hematoxylin are added to 75 cm 3 of hot water. Both solutions are cooled, 
 one is poured into the other and the mixture is carefully heated over a small 
 flame, stirring gently until the seething point is well reached. When cooled, 
 
57 
 
 the solution, now dark brown, is ready for use. It stains very rapidly and 
 thoroughly. If overstaining has taken place reduction is induced in the same 
 manner as in hasmalum. 
 
 Hcematoxylin-Iron-A lum. 
 
 Two solutions are used for this succedaneous mordant staining, which is 
 generally spoken of as Heidenhain staining, after its discoverer: a 2.5% solu- 
 tion of ammonium ferro-sulphate (iron-alum) and a 1% solution of hematoxy- 
 lin, which latter should be of some time standing and brown. The sections are 
 first placed in the mordant for at least two hours or more, if necessary, after 
 which they are washed for a short time and transferred into the color solution, 
 where they remain overnight. At first they take on a gray color, later a gray- 
 ish blue, then deep blue and finally they become entirely black. If reduction 
 is necessary they should, after a brief wash, be replaced into the mordant, 
 which gradually renders them lighter, with the appearance of black color clouds 
 in the solution. This reduction or differentiation must be closely watched and 
 should be controlled under the microscope. The nucleoli and the chromatin 
 of the nuclei resist the extraction the longest, hence this procedure is a means 
 of presenting these elements absolutely distinct. After the desired effect is 
 obtained we transfer to a large vessel of hydrant water for at least ten 
 minutes, changing the water repeatedly. 
 
 Hcematoxylin-Potassium 
 
 Dichromate, 
 
 a lake of hematoxylin, is used to differentiate the medullated fibres within the 
 
 central nervous system, pieces of which are first saturated with the chrome 
 
 salt, sections made thereof, and in these sections the lake is produced by 
 
 introducing them into a haematoxylin solution. For further details of the 
 
 method we refer the reader to the special part under Nervous System. 
 
 Hosmatoxylin-Phospho- 
 Molyhd c Acid. 
 
 Dissolve 1.75 gm of haematoxylin in 200 cm 3 of hot water, and add 
 10 cm 3 of a 10% solution of phospho-molybdic acid and 5 cm 3 of phenol 
 (carbol. acid cryst.), previously liquefied by heating. Sections, previously 
 treated with 10% phospho-molybdic acid for ten minutes and briefly washed in 
 water, are kept in this dark blue solution for from fifteen to twenty minutes, 
 washed in water and transferred to alcohol. 
 
 The entire specimen appears soft grayish blue, not very distinctly stained, 
 but from this background the collagenous fibres contrast widely in their deep 
 blue stain. By this method the connective tissue fibres are distinctly stained 
 even in such places which are hard to bring out with ordinary stains. 
 
 Indigo. 
 
 A dyestuff known in the most ancient times, is found widely distributed in 
 the floral kingdom, but its chief source is the Indigofera, a papilionacea culti- 
 vated in Bengal. We have here a very light, deep blue, friable mass, insoluble 
 in water, alcohol and ether, but soluble in aniline, phenol and acetic acid. Con- 
 centrated sulphuric acid will dissolve it, with the formation of indigo-sulpho- 
 acids. When treated with any reducing agent, indigo will be changed to indig- 
 
58 
 
 white, which is soluble in water. The formation of indig-white and indig- 
 sulpho-acid is responsible for the extensive use of indigo in commercial dyeing. 
 
 Indig carmine. 
 
 In microtechnique we use a preparation produced by sulphuric acid acting 
 as indigo, indig-carmine, the sodium salt of indig-disulpho-acid, occurring as a 
 deep blue powder, quite readily soluble in water, slightly or not soluble in 
 alcohol. This preparation in a 0.25% solution makes an excellent plasma 
 stain. 
 
 ARTIFICIAL STAINS 
 
 Artificial Stains. 
 
 Besides the coloring material furnished us by fauna and flora there is an 
 abundance of others, artificially prepared, made synthetically by the chemist. 
 They are therefore called artificial, although we must always bear in mind that 
 all dyes, natural or artificial, can be traced back to a motherbody, benzol, so 
 that we can take the latter point of view and deny any difference between them. 
 Accordingly we have already succeeded in preparing synthetically some of the 
 natural dyes, e.g., indigo. 
 
 Tar and Aniline Dyes. 
 
 All artificial dyes are made from substances contained in tar, and are there- 
 fore also known as tar dyes. The term "aniline dyes" is not comprising 
 enough, since aniline is by no means the mother substance of all artificial dyes. 
 
 Varieties of Artificial Dyes. 
 
 A rational classification of artificial dyes according to their chemical com- 
 position gives rise to considerable difficulty. The substances in which we are 
 interested are best divided into basic, acid and indifferent dyes. 
 
 Basic Dyes 
 Basic Dyes. 
 
 As basic dyes are known the salts of color bases, principally those of muri- 
 atic acid, but also the salts of sulphuric, nitric, acetic and oxalic acids. Most 
 of them are more soluble in alcohol than in water. If an alkali is added to a 
 watery solution of a basic dye, the color base will be thrown down, being, as a 
 rule, insoluble in water. Tannic acid added to a basic dye will form insoluble 
 tannate. If a basic dye in solution is treated with a strong reducing agent, 
 decolorization takes place, a leuko-compound being formed, which may by 
 oxidation be again transformed into the original dye. 
 
 Basic dyes possess an extraordinary affinity for the nuclear chromatin, for 
 which reason they may be called nuclear dyes. Other structures affected 
 by them preeminently are cartilaginous substances, granules of mast cells and 
 mucus. 
 
 Fuchsin. 
 
 This is a nitric acid salt of rosaniline, and in commerce is also known as 
 rubin, or magenta red, occurring in the form of small red crystals with a 
 metallic lustre. It is prepared with an aniline water solution, an excess of the 
 dye being suspended in aniline water (see p. 49) for several days under 
 
59 
 
 repeated vigorous shaking. This solution will stain in from ten to fifteen 
 minutes, acting more rapidly when warmed. After staining, the excess is 
 extracted with 95% alcohol, to which 10% aniline may be added. 
 
 Methyl Violet. 
 
 The nitric acid salt of hexamethyl pararosaniline is a metallic shining crys- 
 talline powder, dissolving in water and alcohol with the production of a deep 
 blue color. In microtechnique we generally use in its stead the impure gentian 
 violet. This stain is also used in an aniline-water solution. It stains in from 
 ten to fifteen minutes intensively. For washing we use first 95% alcohol, but 
 when that seems insufficient, Gram's reduction method must be resorted 
 to. One gram of iodine and 2 gms of potassium iodide are mixed with a few 
 cubic centimetres of water and shaken until dissolved, after which enough water 
 is added to make 300 cm 3 . A few drops of this mixture are placed on the slide 
 after decanting previous washing liquids, and are allowed to act until a brown 
 color results, a few seconds being required ; then the slide is quickly transferred 
 to 95% alcohol. Here the slide will again assume a blue color, discoloration 
 setting in at the same time. If this does not readily take place another treat- 
 ment with the iodine solution should be given. By this method of decoloriza- 
 tion, which is an important one in bacteriology, a wholly pure chromatin stain- 
 ing is made possible, giving excellent results, for instance in the recognition 
 and study of nuclear changes. 
 
 Methyl Green, 
 
 a derivative of the previous dye, forms a green micro-crystalline powder, is 
 easily soluble in water, not so readily in alcohol, giving a deep green color. 
 One of our best nuclear stains, it is seldom used alone, but generally in con- 
 nection with acid dyes. 
 
 Thionine. 
 
 Thionine, known also as Lauth's violet, is one of the oldest coal-tar dyes 
 belonging to the thiazines. It occurs as a brilliant crystalline powder, having 
 a metallic lustre ; in water it will slowly dissolve with production of a blue- 
 violet color. It is used in an 0.1% solution for nuclear staining. After stain- 
 ing (ten to fifteen minutes) we wash in 95% alcohol. Its principal importance 
 lies in the fact that this dye stains metachromatically, e.g., principally mucus, 
 cartilaginous substances and the mast cell granules ; they are stained red, 
 while the nuclei appear blue. If this metachromatic stain is to be maintained, 
 no after-treatment with alcohol is permissible (see p. 78), but simple water is 
 used for washing, and the specimen mounted in levulose. 
 
 Methylene Blue. 
 
 This most important of all coal-tar dyes is formed by the entrance of four 
 methyl groups into the molecule of the preceding one. It occurs in various 
 forms on the market. For our purpose the chemically pure crystallized methyl- 
 ene blue of the Hoechster Farbwerke is the most serviceable. It consists of 
 small, glittering, brilliant crystallic needles, which are quite soluble in water 
 and alcohol, to a small degree only is it soluble in normal saline, Ringer's solu- 
 tion, sea-water. If an alkali be added to a solution of methylene blue, the latter 
 
60 
 
 is broken up into methylene azure and methylene violet. If a watery solution 
 of methylene blue is treated with a reduction agent, it will be decolorized, the 
 methylene blue changing to leuko-methylene blue. Reoxidation will take place, 
 when the solution is shaken, by the mere action of the oxygen in the air. 
 Living animal tissues, blood and urine will exert this same reducing action on 
 methylene blue. 
 
 Methylene Blue for 
 Nuclear Staining. 
 
 Methylene blue is an excellent nuclear stain. For such purpose it is used 
 in a 0.5 — 1% solution; staining takes from ten to fifteen minutes, preferably 
 in the warmth of the paraffin oven, after which the specimen is treated in 95% 
 alcohol, to which 10% aniline can be added. 
 
 Methylene Blue in the 
 Staining of Nerve Tissue. 
 
 The greatest value of methylene blue lies in its property of vital staining 
 of nerves. Of the divers methods previously discussed, the following will give 
 the best and. most constant results for the central nervous system. As con- 
 cerns the material, not all animals are equally well adapted to vital staining 
 with methylene blue; rabbits, guinea-pigs and cats are most suitable. A 1% 
 watery solution of the above-mentioned chemically pure methylene blue is 
 filtered just before using, and heated to body temperature. The animal is 
 anesthetized with chloroform, the thorax quickly opened in the median line 
 with bone shears, the pericardium slit and the left ventricle opened by cutting 
 off the apex of the heart. The protruding blood is carefully aspirated in moist 
 cotton and the thoracic cavity cleansed from blood coagula. We now search 
 for the aorta, which emanates from the left ventricle closely behind the pulmo- 
 nary artery and by means of curved forceps or, better still, an aneurism needle, 
 we place a ligature loosely around this blood-vessel. As a cannula we use a 
 glass tube, slightly bent in the middle, about 10 cm in length and 2 — 3 mm in 
 bore. The end to be introduced is in the form of a bulb which terminates in a 
 point, so that the ligature may not slip. Such a cannula can easily be made in 
 any desired size (see p. 76). The point must be well polished or molten in a 
 way so as not to injure the vessel wall. The other end of the cannula is at- 
 tached to a rubber tube, which is closed by a glass cock or a pinch cock. The 
 cannula as well as the rubber tube are first filled with Ringer's solution by 
 aspiration, so that no air-bubbles may be contained within; the cock is then 
 closed and the cannula introduced with the right hand into the root of the 
 aorta by piercing through the left ventricle, while the left hand fixes the heart 
 by means of mouse-toothed forceps. When the bulb of the cannula is seen 
 lying in the aorta, an assistant will tighten the ligature placed previously, 
 fixing the cannula in place. If no assistant is at hand, the cannula is intro- 
 duced into the aorta as far as possible, then releasing his hold, the operator 
 tightens the ligature himself. For an injection apparatus we may use a sim- 
 ple funnel suspended in a filter-stative. The funnel is connected with the can- 
 nula by a rubber tube. Fig. 25 shows an apparatus which is more efficacious. 
 It consists of a graduated burette, provided with a stopcock, which is enclosed in 
 a wide glass cylinder, the latter being closed at both ends with rubber stoppers. 
 
61 
 
 A funnel is seen on top, through which the outer cylinder can he filled with 
 warm water. Below an opening leads into a drain pipe, which is provided with 
 a pinch-cock. The upper cork has a third opening for the exit of air, when the 
 cylinder is being filled. The outer cylinder is filled with warm water, the burette 
 with Ringer's solution, finally the lower end of the burette is opened to allow the 
 fluid to escape and therewith expelling all air, after which it is attached to the 
 rubber tube leading to the cannula, and all is ready for the injection. This 
 has to be done slowly and without undue pressure. 
 First the cannula-cock is opened, after which the 
 one guarding the contents of the burette is re- 
 leased. All spurting vessels must be caught with 
 forceps and tied off. Owing to the pressure 
 caused by the return flow through the veins the 
 right heart will become distended and must now 
 be opened, which is best accomplished by cutting 
 through the right auricular appendix. In this 
 manner the entire vascular system is flushed with 
 Ringer's fluid, until the latter, which is constantly 
 taken up with cotton sponges from the right 
 auricle, returns absolutely clear. In the case of a 
 cat this will consume at least 300 cm 3 of the fluid. 
 The burette is now filled with the staining solution 
 and the injection proper begins. Here, too, we 
 should work with the least pressure possible and 
 slowly. The burette should be opened only so much 
 as to admit a flow of 1 — 2 cm 3 per minute. During 
 the entire duration of the injection the staining 
 solution should be kept at a temperature of about 
 42°, which can be easily accomplished by draining 
 off the water in the outer cylinder and replacing 1 
 fresh warm water. As soon as the return flow 
 from the right heart takes on a deep blue color, 
 we tie or clamp the auricular appendix off, there- 
 with closing the vascular system once more. It is 
 hard to say how much of the stain should be in- 
 troduced, at any rate we cannot very well overdo it — 120 — 150 — 200 cm 3 , 
 when injected within two to three hours, should suffice for a large rabbit or a 
 cat. After the injection is completed, the animal is left undisturbed for an- 
 other half hour, and now the central organs are excised. 
 
 Protection of the Hands. 
 
 Since soiling of the hands with the dye is unavoidable during the injection, 
 and more so during the ensuing preparation, it is a good plan to protect them 
 by rubber gloves, such as are now in wide use in pathologic laboratories. 
 
 Reduction and 
 Resulting Blue. 
 
 If the injection has been properly executed, the organs will appear per- 
 fectly colorless on opening the skull or the vertebral column respectively, but 
 
 Fig. 25. 
 
 Apparatus for the Injection 
 of Methylene Blue Solutions. 
 
62 
 
 as soon as the air comes in contact with them, they will assume a blue color, so 
 that gray and white matter can be distinctly differentiated, the former appear- 
 ing deep, the latter light blue. The organs should be sectioned into not exces- 
 sively small pieces, say 3 — 5 mm in thickness and transferred immediately to 
 the fixing bath. 
 
 Fixing of Methylene Blue 
 Preparations. 
 
 We fix with a 10% solution of ammonium molybdate. The salt is pulver- 
 ized in a mortar, dissolved in hot water, and the clear solution cooled well 
 (3 — 5°). During the process of fixing, which should be carried on in the cool, 
 the specimens will assume a still bluer hue. The molybdic acid forms a salt 
 with the methylene blue base, which is readily soluble in water, but poorly in 
 alcohol. Hence the specimens should on the following morning be thoroughly 
 washed in running water for several hours, while the dehydration must be 
 shortened and should be instituted under as low a temperature as possible. 
 Four grades of alcohol suffice: 50%, 75%, 95% and 100%. In each of the 
 first three mentioned the sections remain from three to four hours, being re- 
 peatedly shaken, after which they are placed in absolute alcohol overnight, 
 which is changed once on the next morning. During the day they are trans- 
 ferred to xylol, in which they may remain for days. 
 
 To demonstrate the peripheral nerves it is necessary in most cases, and 
 insures better success, to expose these tissues to the air for some time before 
 fixing them, as such a precaution will bring out the optimum of nerve-staining. 
 Coarse razor sections are made of the thicker structures, moistened with 
 Ringer's solution and placed in a moist chamber. Now and then we make a 
 control examination under the microscope. If after two hours no progress has 
 been made in the nerve-staining, further waiting will be useless. 
 
 Organs which have been removed from the body or severed limbs can of 
 course be injected in a similar manner. 
 
 M ethylene- Azure. 
 
 This is the name of a dye which is produced by an alkali acting on methyl- 
 ene blue, being readily soluble in water with a blue color. It is seldom, per- 
 haps never, used alone, but in conjunction with eosin (see p. 67). 
 
 Cresyl Violet B. B., 
 
 an oxazin dye, coming into commerce as a bluish violet powder, dissolving 
 with the same color production not so readily in water, but very easily in 
 alcohol. 
 
 The importance of the dye lies in its metachromasia, which in this case is 
 accentuated and finely shaded to such a degree that it is second to no other dye. 
 A concentrated watery solution is made and for staining is diluted about ten 
 times with distilled water. Staining will take place in from fifteen to thirty 
 minutes, overstaining being impossible, even should thin sections be left for 
 twenty-four hours. Metachromasia is evident most beautifully in the fresh 
 specimen ; of fixed material, frozen sections should be used exclusively. Forma- 
 lin fixation is suited excellently, but sublimate and nitric acid give also good 
 
63 
 
 results. After staining, the section is washed in water and mounted in levulose 
 (see p. 79). A blue color will be imparted to the cell protoplasm, the inter- 
 stitial granules of the muscle fibres, reddish violet to the nuclear chromatin, 
 very light red to the collagenous tissue, deep red to mucus, cartilaginous sub- 
 stance, mast-cell granules, a yellowish red to the nerve-sheaths, a pure yellow 
 to the haemoglobin of the red blood cells. 
 
 Safranine. 
 
 Safranine O. Here we have a red powder, not very soluble in water, more 
 easily in alcohol. A concentrated solution in aniline water (see p. 49) is pre- 
 pared, in which the sections are stained for at least half an hour. To differen- 
 tiate we transfer to 95% alcohol, to which a minute amount of hydrochloric 
 acid may be added, a few drops of a 1% solution to 100 cm 3 of alcohol. 
 
 The metachromatic properties of safranine are not very pronounced. The 
 best results are obtained on mucus, cartilage and sheaths of hair roots. 
 
 Resorcin Fuchsin. 
 
 This dyestuff deserves mention here, its chemical composition being little 
 understood. Four grams of resorcin are added to 200 cm 3 of a 1% solution 
 of fuchsin and the mixture warmed. When the boiling-point is reached, we add 
 25 cm 3 of liquor ferri sesquichlorati, which is immediately followed by a volumi- 
 nous precipitate. The mixture is allowed to boil for a few minutes, is then 
 cooled and filtered. The precipitate, a bluish black, muddy mass, together with 
 the filter paper is brought to the vessel first used, 200 cm 3 of 95% alcohol are 
 poured over it, the whole is heated to boiling over a water-bath, subsequently 
 cooled and filtered; i cm 3 of hydrochloric acid are added and enough 95% 
 alcohol to make 200 cm 3 . 
 
 The bluish black solution so obtained will stain within fifteen to thirty 
 minutes, elastic fibres will appear deep blue black, connective tissue a very 
 weak blue, the basic substance of hyaline cartilage reddish violet. Excess of 
 dye is washed out thoroughly with 95% alcohol. 
 
 Acid Dyes 
 Acid Dyes. 
 
 The dyestuffs considered under this heading are mainly the alkaline salts 
 of color acids, with the possible exception of one staining acid, which must 
 needs be mentioned here, namely, picric acid. All are invested with strongly 
 pronounced acid properties, which are due to the presence of certain atom- 
 complexes (N0 2 , SO R H, COOH, OH). Most of them are readily soluble in 
 water and stain wool directly. Acid dyes may be decolorized by reduction 
 agents, but a reoxidation thereafter is jn most cases impossible. 
 
 While basic dyes show a pronounced predilection for the chromatin of 
 nuclei, the acid dyes possess an affinity for protoplasm, for the achromatic 
 portions of the nuclei and to connective tissue, to varying degrees ; e.g., picric 
 acid has a greater affinity for the cell protoplasm, while acid fuchsin selects the 
 connective tissue for its action. For this reason acid stains are seldom used 
 alone, but almost always in conjunction with basic dyes, either in mixtures or 
 by using the succedaneous process. The mixtures will be spoken of later ; as 
 
64 
 
 regards succedaneous staining, the rule should be followed to stain first with 
 the nuclear stain and secondarily with the acid dye, an excess of which latter 
 can be extracted with alcohol, 70—95%. Often the acid dye will act as an 
 agent of differentiation on the basic, so that the after-treatment with alcohol 
 will extract more of the latter dye. 
 
 Picric Acid. 
 
 We have already been acquainted with trinitrophenol as a fixing agent ; it 
 plays a much more important part as a stain. Picric acid is an excellent 
 plasma stain ; its main use is for double staining after carmine and haema- 
 toxylin. We must, however, always bear in mind, that being a monobasic acid, 
 it will strongly attract the nuclear stains of our specimen, similar to hydro- 
 chloric and acetic acid. It is necessary, therefore, to overstain in the nuclear 
 dye, if a good double staining is desired. It is used in a 1% watery solution, 
 staining cell bodies, connective tissue, muscle, red blood cells intensively yellow 
 in a few minutes. 
 
 Acid Fuchsin 
 
 is the alkaline salt of rosaniline-trisulpho-acid, the latter being prepared by 
 treating fuchsin with fuming sulphuric acid. It is a dark red powder, easily 
 soluble in water, not quite so readily in alcohol. The watery solution is in- 
 stantly decolorized by an alkali. 
 
 Acid fuchsin has attained great importance in histology, since it forms a 
 component of many important staining mixtures. It is also very serviceable 
 for staining after nuclear staining has been accomplished, a 0.05 — 0.1% solu- 
 tion in water being very efficient for this purpose. A possible overstaining can 
 be corrected by washing in hydrant water. It plays an especially important 
 part in the demonstration of Altmann's bioblasts. 
 
 Bleu de Lyon 
 
 (Lyon blue) is made also by sulphurizing a basic dye and occurs as a dark 
 
 blue powder, readily soluble in water, more difficultly in alcohol. 
 
 It is well adapted for afterstaining of carmine preparations, for which pur- 
 pose it is used in a 0.1% solution. Staining is continued until the sections 
 begin to assume a blue color, when we transfer them into 70% alcohol. In 
 well fixed specimens we can demonstrate nerves and differentiate them from 
 the surrounding connective tissue by this method. 
 
 Light Green. 
 
 Light green is a dark green powder, readily soluble in both alcohol and 
 water. It is used in the same manner as the preceding. 
 
 Orange 0. 
 
 Orange G. is a representative of that large group of dyestuffs known as 
 azo colors, which are of great importance in commercial dyeing. It forms 
 small yellowish red crystal plates, which are soluble in water and to some extent 
 in alcohol. Orange G. is one of our best plasma stains. It is especially used 
 after hasmalum staining, in a 1% watery solution; it stains in a few minutes. 
 The washing is done in 70% alcohol. 
 
65 
 
 Bordeaux B., 
 
 also an azo dye, is a reddish brown powder, readily soluble in water. 
 
 It is made use of in a 0.2% watery solution, which must be applied before 
 the nuclear staining takes place; it stains in about five minutes, after which 
 specimens are washed briefly in water and, as previously stated (see p. 57), 
 are subjected to hasmatoxylin-iron alum. The more the differentiation ad- 
 vances the more will the red color due to the Bordeaux reappear. 
 
 Eosin. 
 
 Eosin is the sodium salt of a color acid, tetrabromfluorescin. In commerce 
 it is found as a red powder, easily soluble in water. When a mineral acid is 
 added to the watery solution, the insoluble color acid will be precipitated. 
 
 Eosin is extensively used as a plasma stain, especially following haematoxy- 
 lin. The simplest manner of using it is to make a concentrated solution of the 
 preparation called "eosin yellowish," and diluting the same with ten to twenty 
 times its volume of water. The thinner the solution, the longer the staining 
 will last. After staining we wash in 70% alcohol. Eosin is of great value in 
 the staining of blood and blood parasites. 
 
 Indifferent Stains 
 
 Indifferent Stains. 
 
 Such is the name applied to a small group of dyes, the most important 
 properties of which are their relative solubilities. They are totally insoluble in 
 water, dissolve with difficulty in alcohol, but are very readily soluble in fats. 
 Chemically these dyes are indifferent, i.e., they possess no salt-forming groups. 
 Under this group belong, for instance, sudan III and scarlet R. (Scharlach R.), 
 both of which are azo dyes. They are reddish brown or red powders, difficultly 
 soluble in alcohol. 
 
 Sudan III and Scarlet R. 
 for Fat Staining. 
 
 A concentrated solution is made in 70% alcohol, the frozen sections are 
 taken from water and placed in 50% alcohol for a few minutes, then into the 
 staining fluid for fifteen to thirty minutes, after which they are washed in 
 water. Eat will appear intensively red, likewise the sheaths of peripheral and 
 central nerves. This process may be combined with a nuclear stain, e.g., 
 haemalum, as well as with a plasma stain, e.g., picric acid. In such a case we 
 first stain with hasmalum, wash in water, transfer into 50% alcohol and thence 
 into sudan ; the specimen is then washed again in water, stained in a watery 
 picric acid solution and finally rinsed with water. In the after-treatment we 
 must of course avoid strong alcohol and all solvents of fat. 
 
 STAINING MIXTURES 
 
 Staining Mixtures, Homogeneous 
 and H-eterogeneous. 
 
 As we have previously observed, multiple staining (excepting metachro- 
 matic staining) can be accomplished by either using the various dyes in succes- 
 
66 
 
 sion or by staining with a mixture of the same. Three possibilities must be 
 considered in the making of such a mixture: The component dyes may be 
 basic, they may be acid, or they may partly be basic and partly acid. The first 
 two varieties are easily prepared; such homogeneous mixtures are com- 
 pounded of numerous basic and likewise a number of acid dyes. In the third 
 variety, the heterogeneous mixtures, we are confronted with certain diffi- 
 culties. If we mix the watery solutions of a basic and an acid dye, a reaction 
 will take place, the color acid of the acid and the color base of the basic dye 
 uniting to form a new dye, which we name neutral. These neutral dyes are 
 generally insoluble in water, therefore a precipitate will be thrown down. We 
 thus must expect a precipitate when mixing solutions of both acid and basic 
 dyes. In order to bring such precipitate into solution we must employ other 
 solvents, such as ethyl and methyl alcohol, acetone or methylal. Another way 
 of procedure is to use an excess of the acid dye or to add an extra acid dye. 
 What the nature of the solution is in the latter case cannot be stated with cer- 
 tainty, the probability, however, is that aside from the neutral dye it will also 
 contain both the acid and the basic dyes in their original form. 
 
 Of the homogeneous mixtures only those of acid dyes are of interest to us ; 
 they are extensively used for afterstaining following nuclear dyes, in which 
 case they will furnish an excellent differentiation between connective tissue and 
 muscle. Sections stained with a heterogeneous mixture will show the chromatin 
 of the nuclei, the basic substance of cartilage, mucus and the granules of mast 
 cells in the basic colors, while all other elements will take up the acid dye or 
 dyes, as the case may be. 
 
 Picrofuchsin. 
 
 Here we deal with a mixture of picric acid and acid fuchsin ; it is prepared 
 by mixing 45 cm 3 of concentrated watery picric acid solution with 5 cm 3 of a 
 2% solution of acid fuchsin. It is used after nuclear staining with hfemalum 
 or iron-hsematoxylin ; it acts in five to ten minutes, the excess dye being ex- 
 tracted with 70% alcohol. Connective tissue will take on a bright red stain, 
 while cell protoplasm and muscle will appear yellow. 
 
 Picro-Indigcarmine, 
 
 a solution of indigcarmine in picric acid, is prepared by dissolving 1 gm of the 
 former in 300 cm 3 of a concentrated watery solution of the latter. This mix- 
 ture is suitable for the after-staining of carmalum, paracarmine and safranine 
 specimens. In five to ten minutes it will impart a brilliant blue color to con- 
 nective tissue, cell protoplasm, and muscle taking on a grass-green hue. 
 After staining, the specimens are washed in 70% alcohol. 
 
 Picrocarmine. 
 
 Its preparation necessitates magnesia water, which we can make by adding 
 well water to magnesia usta, leaving this mixture stand for eight days, vigor- 
 ously shaking it from time to time, so that we can always be sure to have an 
 excess of magnesia present. Pulverized carmine (0.2 gm) is boiled in 100 
 cm 3 of magnesia water for half an hour in a flask loosely plugged with cotton. 
 After cooling we filter and add a few drops of formalin. 100 cm 3 of a 0.5% 
 watery solution of picric acid with 0.15 gm of magnesia carbonate are also 
 
67 
 
 heated to boiling, cooled and filtered. The two solutions combined will give us 
 the stain ready for use. Five to ten minutes will suffice to have nuclei stained 
 red, all other structures taking a yellow stain. 
 
 Azureosin Solution. 
 
 This stain, also known as "Giemsa solution," contains 3 gms of eosin and 
 0.8 gm of methylene-azure dissolved in 250 gms of glycerine and 250 gms of 
 methyl alcohol. One drop of the solution in 1 cm 3 of water will give a ready 
 stain ; its main use is for blood slides. 
 
 Biondi Solution. 
 
 This is the name given to a mixture containing one basic color, methyl 
 green, and two acid dyes, acid fuchsin and Orange G. Its efficiency depends on 
 the care taken in its preparation, which is as follows : The colors to be used 
 should be ordered from the "Aktiengesellschaft fur Anilinfabrikation" at Ber- 
 lin, the following names being asked for: Methyl green N.M.P., acid fuchsin 
 S.M.P., and Orange G.M.P. Of the former we take 3.4 gms, of the second 4.2 
 gms and of the third 3.0 gms ; all three are placed in a small porcelain mortar 
 and carefully triturated, so that a uniform powder results, which is dissolved 
 in 100 cm 3 of distilled water. If a permanent solution is desired, the container 
 must be made of the very best Jena glass. The best plan is to use an Erlen- 
 mayer flask, place in it the dry powder mixture, add the prescribed amount of 
 water, cork well and, shaking it repeatedly, let the mixture stand on the 
 paraffin oven for two to three days. After this period complete solution will 
 have taken place, resulting in a dark brown fluid with a slight red tinge. This 
 stock solution, when properly taken care of, will keep for a long time, and 
 before using must be acidified and diluted. The following procedure, though 
 approximate only, is to be recommended as practically very efficient for acid- 
 ulation. Place 1 cm 3 of acetic acid in a 100 cm 3 graduated tube and add dis- 
 tilled water up to the 100 cm 3 mark, mix well and empty the glass ; the traces 
 of acid adhering to the wall will suffice for the acidulation. In a second smaller 
 graduate 5 cm 3 of the stock solution are placed, and rinsing thoroughly with 
 distilled water the contents are transferred to the acidulated tube, adding 
 enough rinsing water to bring the solution up to 50, and now our stain is ready 
 for use ; it will keep well for a few days. As regards the dilution, one need not 
 literally cling to the stated proportion. As far as the material is concerned, 
 we must differentiate between frozen and paraffin sections. In the former case 
 nearly all fixatives will yield good results, e.g., formalin, the various corrosive 
 sublimate mixtures, nitric acid, also chrom-osmium-acetic acid, if the specimens 
 have not been subjected to its action for too long a period. Of the paraffin 
 sections only those fixed in sublimate or its mixtures are to be recommended as 
 suitable. Celloidin preparations are only then admissable, if the celloidin has 
 been extracted prior to the staining. The duration of the staining has little 
 influence on the results ; ten minutes will suffice ; again we may stain for twenty- 
 four hours with impunity. The staining is followed by a short wash with 0.5% 
 acetic and the specimens are transferred into 70% alcohol, where thick color 
 clouds will be seen to emerge. The use of the alcohol must not be protracted 
 for very long, but as soon as the section takes on a red color, it should be put 
 
68 
 
 through absolute alcohol and thence transferred to xylol. The results obtained 
 are most excellent. A bluish green is imparted to nuclear chromatin, basic 
 substance of cartilage and mucus ; a bluish violet to the mast-cell granules ; a 
 red color appears in the nucleoli, the homogeneous juice of the nuclei, the 
 centrioles, spheres and achromatic spindles, the cell protoplasm, collagenous 
 and elastic tissues, contractile substances', oxyphilic cell granules, basic sub- 
 stance of bone, uncalcified dentine. The hemoglobin of the erythrocytes will 
 appear orange-colored. The shading of the red is so fine as to differentiate all 
 various elements among themselves. In facility and simplicity of execution 
 and variety of applicability this method is not excelled by any other. One dis- 
 advantage of Biondi solution stands out against its many valuable properties, 
 the fact that specimens prepared in such manner will not keep indefinitely. 
 This hampering objection, however, may be remedied by strictly observing the 
 rules for finishing and mounting microscopic specimens, as laid down on pages 
 76-79. 
 
 APPENDIX TO METHODS OF STAINING 
 
 THE IMPREGNATION METHODS 
 
 Essentials of Impregnation. 
 
 Metal impregnation is the term applied to a process in which we aim to 
 saturate a block or a section of our specimen with a solution of a metal salt, 
 the latter undergoing such reduction in certain parts of the tissue, which 
 enables it to form colored compounds or to be changed to the metal itself, re- 
 spectively. Thus the metal might be precipitated in these places as fine 
 granules, or these certain tissue elements may appear distinctly colored, with- 
 out any precipitate being formed. In the latter case a differentiation between 
 impregnation and staining is impossible. 
 
 Metals Concerned. 
 
 Of the metals used for this purpose only the noble metals and of these only 
 silver and gold will interest us. Another impregnation, that with osmic acid, 
 has been discussed among the fixation methods and impregnation in that case, 
 when applied to fresh tissues, naturally serves as a fixation as well, since the 
 salts of the noble metals will all coagulate albumin. 
 
 The metal salt solution may be allowed to act on either the fresh speci- 
 men or the fixed specimen or finally on sections made from the latter. Each of 
 these three methods will answer the purpose. 
 
 The Reduction 
 of the Metal Salt. 
 
 Reduction, the most important phase in the process of impregnation, may 
 be induced without our aid by the tissue itself; ordinarily, however, we aug- 
 ment the process by the use of some aiding factors. One such factor is light — 
 diffuse daylight or the direct rays of the sun. Under its influence the changes 
 will take place much more rapidly and more intensely. Another such factor is 
 found in acidulation ; the reduction does not take place in distilled water, but 
 
69 
 
 to the latter is added a greater or lesser amount of acid. Warming may in 
 certain instances also hasten reduction. Finally we resort in many cases to 
 measures such as used by the photographer in the developing of plates, namely, 
 to the use of strong reducing agents, e.g., sulphurous acid, arsenious acid, for- 
 maldehyde, resorcin, aniline, pyrogallic acid, hydrochjnon, etc. 
 
 Frequently we imitate the photographer so far as to follow our reduction 
 with a hyposulphite of soda bath, to remove any metal salt which has not been 
 reduced. 
 
 Impregnation with 
 
 Silver Salts. Silver Nitrate. 
 
 Of the silver salts the only one of importance is silver nitrate. In com- 
 merce we find it either in the form of crystals or in sticks ; it may be dissolved 
 in water to the extent of 200%, forming a strongly corrosive fluid with neutral 
 reaction. A stock solution of 20% strength should be made and kept in a 
 clean, glass-stoppered bottle. If impurities are present, silver salt will soon 
 be thrown down. , 
 
 Silvering. 
 
 A solution of 0.5 — 1% (0.5 — 1 cm 3 of our stock solution to 20 cm 3 of water) 
 will suffice to silver thin, membranous sections. The membranous tissue is taken 
 from the animal, spread quickly on a fiat plate, which is lined with paraffin and 
 then fastened with hedgehog bristles ; it is carefully rinsed just once with dis- 
 tilled water. According to the size of the specimen 10 — 20 cm 3 of the silver 
 solution are poured over the sections and spread evenly by slight and even bal- 
 ancing over all the parts, which latter gradually become opaque. After ten 
 minutes the solution is decanted, and we rinse in water several times, fill the 
 plate with acidulated (1% acetic acid) water and expose it to light, preferably 
 sun-rays, for reduction. The latter will take place very rapidly, the specimen 
 becoming gradually brown. This silvering process preeminently brings out 
 the cement-substance, being the classic method for that purpose. 
 
 Another use to which silver nitrate is put is the injection of the solution 
 into hollow structures, such as the vascular system, lungs, etc. The technique 
 ■will be described in the special part. 
 
 Chrome Silver Method. 
 
 The Golgi method, so called after its discoverer, consists in the successive 
 treatment of the specimen with a chrome salt and silver nitrate solution. It is 
 fair to assume that through the action of the bichromates on the tissues, chrom- 
 albumins are formed, the composition of which depends a great deal on the 
 duration of the process. If the specimen is now brought into a silver nitrate 
 solution, a compound of silver albumin-bichromate is formed in certain tissue 
 parts, appearing black when viewed before a light. We will thus find these 
 parts prominently black in a yellow field. Not all the tissue elements of the same 
 kind are equally impregnated, since this process favors a certain amount of 
 them, while it utterly disregards others. Besides this regular impregnation the 
 entire surface of the specimen and some of the deeper parts will be found 
 studded with granular or crystalline deposits, which act very disturbingly. 
 
70 
 
 Rapid Chrome-Silver 
 Method. 
 
 Of the many variations of the method the rapid method gives the best 
 results, and we will therefore devote special attention to it. The fresh speci- 
 mens are cut into small pieces and placed in a mixture of 8 parts of 2.5% bi- 
 chromate of potassium and 2 parts of 1% osmic acid for two to five days; they 
 are then dried with tissue paper and transferred to a 0.75% solution of silver 
 nitrate, which must be changed after a few minutes. They will be ready for 
 cutting the next day. The following details may well be observed : 
 
 Material for the 
 Chrome-Silver Method. 
 
 Mostly every organ is suited for the chrome silver method, and in every one 
 some part or other will be stained black: Connective tissue shreds, elastic 
 fibres, muscle fibres, capillaries, neuroglia -cells, nerve-endings, etc., but fore- 
 most among all others nerve-cells and their branches, peripheral and central 
 nerve-fibres. The importance of this method rests with this property, and for 
 this reason its main use is found in the study of the central and peripheral 
 nervous system. The material should be as fresh as possible ; however, we fre- 
 quently find this requisite greatly exaggerated. Six to twelve hours after 
 death we will still have tissues which give excellent results. 
 
 A more important fact is, especially when dealing with the central organs, 
 that only very young specimens can be used. When human material is desired, 
 infants one week old are to be recommended ; of animals : mice, dogs, cats, two 
 to ten days old, guinea-pigs at full term. The central organs of the calf are 
 excellent, if they can be procured early enough. 
 
 Moderately sized pieces, not too small, are selected, perhaps 2 — 3 mm in 
 thickness and 5 — 10 mm long. Several pieces should be taken from each speci- 
 men and all be placed in a brown vessel containing 50 cm 3 of fixing solution. 
 
 Duration of Impregnation. 
 
 It is of vital importance to know how long the pieces should remain in the 
 solution. There are no set rules for each organ, the process being empirical. 
 On the morning of the third day the pieces are dried superficially with filter- 
 paper and thereafter placed in 10 cm 3 of silver solution, where a red precipi- 
 tate of silver bichromate will instantly be formed. After a few minutes fresh 
 solution is added liberally until the fluid remains clear. The following morn- 
 ing some razor sections are made and examined. If the impregnation is well 
 established, the entire material can be silvered in like manner. If the result 
 was not satisfactory, a few more pieces are selected and experimented on, etc. 
 After seven or eight days a success is improbable. 
 
 Further Treatment of 
 Impregnated Specimens. 
 
 Embedding can usually be omitted. The following is the best after-treat- 
 ment. After remaining in the silver solution for from twenty-four to twenty- 
 eight hours, the pieces are briefly rinsed in distilled water and in groups are 
 placed at a proper height upon stabilit-blocks (p. 47), with a thick solution 
 of gum arabic. After a few minutes the blocks are immersed in 70% alcohol, 
 
Fig. 26. 
 
 71 
 
 after a few hours in a 95% solution of alcohol, where within a few hours they 
 will attain the proper cutting consistency. The next day sectioning with the 
 microtome may begin. The knife is placed at an obtuse angle and diligently 
 moistened with 95% alcohol; the sections, which should not be too thin, are 
 transferred to 95% alcohol. They are then transferred to carbolxylol (see p. 
 79), and finally to pure xylol, where they may remain a long time with im- 
 punity. 
 
 Mounting. 
 
 The mounting cannot take place in the usual manner, as will be described 
 later; they will soon spoil if so treated. They are subjected to the following 
 technique. Cover-glasses of corresponding numbers and sizes are cleansed and 
 placed on a specimen board ; the sections are taken from the xylol and placed 
 on the cover-glasses, leaving a good margin of the glass free. As soon as the 
 xylol begins to evaporate, a large drop of Canada balsam is placed on each 
 section and the latter put in a dust-free place to dry. The following day the 
 specimens are looked over and if any have risen in the balsam, another drop is 
 applied. After a few days the specimens, 
 now completely dry, are placed upon 
 slides, which may be made of cardboard, 
 wood, metal or glass, an opening being cut 
 out in the place where the specimen is to 
 rest (Fig. 26). Such slides can be bought, 
 but on the other hand are very easily made Wooden Slide for Chrome-Silver 
 from cigar-box wood with a little inge- I Specimens. 
 
 nuity. The cover-glass is placed in the ' 
 
 opening, specimen side down, and secured with a little cement (see p. 77). If 
 mounted in this fashion, specimens will keep for a long time and can even be 
 examined with the oil-immersion lens. 
 
 The Pyrogallol Method. 
 
 We will give this name to the method, since, similar to the photographer, 
 we use pyrogallic acid to create the silver image in the tissues. Specimens are 
 placed in 90% alcohol overnight, 8 drops of spirits of ammonia (specific 
 gravity, 0.91) having been added to each 100 cm 3 of alcohol; rinse with water 
 and transfer to a 2% solution of silver nitrate for five to eight days, preferably 
 in a thermostat to keep the temperature even. The pieces are now washed in 
 water again and brought into the following reduction mixture : pyrogallic acid, 
 1 gm; formalin, 10 cm 3 ; water, 100 cm 3 - After twenty-four hours they are 
 dehydrated in the usual manner and embedded in paraffin. This method gives 
 good results for the study of the neuro-fibrils in nerve-cells and fibres. It is 
 best suited for the central nervous system, less efficient for the peripheral. The 
 paraffin sections, mounted in the usual manner, can be gilded by placing them 
 in a 0.1% gold chloride solution for several minutes, succeeded by ten minutes' 
 treatment in 5% -fixing soda, after which they are washed in water. 
 
 The Silver-Ammonia Method 
 
 can be used for frozen sections as well as for entire tissue blocks previously 
 
 fixed in formalin. We will only discuss the latter. The blocks are washed in 
 
distilled water for twenty- four hours, impregnated in a 2% silver-nitrate solu- 
 tion for six to eight days, quickly rinsed in water and placed in the following 
 solution of silver-ammonia, which must be freshly prepared; 20 cm 3 of a 
 2% silver nitrate solution are placed in a large beaker with 3 drops of 40% 
 sodium hydroxide, shaken and the resulting precipitate of silver oxide is dis- 
 solved by adding ammonia drop by drop until the solution is absolutely clear. 
 The brown staining of tissues will be materially intensified by this solution. 
 The following morning we wash once more and transfer to 20% formalin for 
 reduction. 
 
 This method gives similar results as the one preceding. The collective 
 staining of the connective tissue is a disadvantage. 
 
 Gold Chloride. 
 
 Impregnation with Salts of Gold. 
 
 Gold chloride is the salt used almost exclusively in microtechnique. In 
 commerce we find it in the form of reddish-brown amorphous lumps or as yellow 
 crystalline needles. The chemical composition of the former is AuCl 3 +2H 2 0, 
 while the latter forms a compound with hydrochloric acid and is represented 
 by AuCl 3 +HCl+3H,0. Both salts are highly hygroscopic, hence they are 
 sold in sealed glass tubes. Of either one of the salts a 2% stock solution is 
 prepared and kept in a well-stoppered white bottle. If cleanliness is observed, 
 it will keep indefinitely. In the handling of all gold solutions metal instruments 
 must be avoided, wooden sticks or glass needles being used in their stead; the 
 latter can easily be made over the gas-flame from barometer tubes and drawn 
 out to any desired shape or thickness. 
 
 Of all the numerous gilding methods, which have been advocated in the 
 ■course of time, we will only discuss three. 
 
 Gold Chloride- 
 Lemon-Juice Method. 
 
 This method is especially adapted for thin organs, rich in connective tissue. 
 The pieces, according to their thickness, are placed for from five to fifteen 
 minutes in freshly expressed lemon-juice, which contains, besides its citrates, a 
 goodly amount of free citric acid. The specimens will be found to swell con- 
 siderably ; they are now rapidly washed in water and placed in 1 % gold 
 chloride for a half to one hour. Again they are rinsed and then reduction is 
 induced by placing them into 1 % acetic acid, preferably on a white background 
 and exposed to the direct rays of the sun. When taken from the gold solution 
 the pieces present a light or dark yellow color ; exposed to the light they will, 
 after a shorter or longer period, assume first a dirty brown, later a red, and 
 finally a bluish violet hue, depending on the intensity of the light. The last 
 color mentioned might become so deep that the specimen appears black. 
 Axiscylinders are most prone to be affected by the reduction, appearing, in a 
 good specimen, distinctly black on a brilliant red background. 
 
 In order to make sections, the pieces are subjected to 5% formalin, 
 and later are cut with the freezing microtome. Such preparations will last 
 well. 
 
73 
 
 Gold Chloride- 
 Formic Acid Method. 
 
 A process most suited to the demonstration of muscle nerves. Thin layers 
 of the muscle are spread and fastened on wax plates and placed in 30% formic 
 acid for ten to twenty minutes. We follow with 1% gold chloride for one-half 
 to one hour, and finally reduce with the previously named acid in the dark. 
 Reduction will be largely completed the next day. We now transfer to glycer- 
 ine which must contain the same acid to the extent of 50% by volume, and 
 substitute pure glycerine on the following day, which will act as a ureservative. 
 
 Gold Ghloride-Iodine- 
 Iodine-Potassium Method. 
 
 In conclusion we will deal with this process, which is used, not on fresh, but 
 on preserved material, viz., sections. The gilding of such sections, whether 
 frozen or paraffin, will always offer difficulties inasmuch as they must primarily 
 be rendered susceptible to the gold salt. Frozen sections, fixed in any of the 
 methods, are first placed in an iodine-iodine-potassium solution, made by 
 diluting our laboratory solution (see p. 59) with an equal part of water, where 
 they remain for thirty minutes. They will take on a prominent reddish brown. 
 We now rinse them briefly in water and transfer them to a 0.2% solution of 
 gold chloride for thirty minutes, where they are at first decolorized and later 
 assume a yellow color. After washing again reduction takes place in a 2% 
 solution of resorcine. Reduction will be complete in from one to two hours, 
 but may be interrupted, if desired. Rinse in water and place in a 5% solution 
 of sodium hyposulphite, which will, in the course of fifteen to thirty minutes, 
 dissolve out all the unreduced gold. The sections are finally thoroughly 
 washed. This method gives excellent results for many purposes, but chiefly 
 serves to demonstrate connective tissue fibres, neuroglia giving the next best 
 results. 
 
 METHODS OF INJECTION 
 
 As an appendix to general microtechnique the technique of injection de- 
 serves special reference at this juncture, a process which the microscopist has 
 largely borrowed from the art of macroscopic procedure. By "injection" we 
 understand the filling of any hollow structures of the animal body with foreign 
 material by the aid of proper instruments. 
 
 The spaces may be of the most varied kind, e.g., the communicating 
 channels of bony structure, joint cavities, and the excretory ducts of glands. 
 As a rule, however, the term injection is applied exclusively to the filling of the 
 blood and lymphatic systems. 
 
 Material Used for Injection. 
 
 As injection-material we can use all sorts of matter, which must needs 
 be liquid (or gaseous) at the time of injection. For obvious reasons we select 
 generally a colored injection material. They can be classified in different ways 
 and may be spoken of as unstained and stained; the latter can be again 
 divided into opaque and transparent. We can furthermore differentiate be- 
 
74 
 
 tween liquid (i.e., remaining liquid) and coagulating. Liquid material is 
 chosen for the injection of very fine blind ducts, such as the excretory ducts of 
 glands, while coagulating fluids are better suited for blood-vessels. 
 
 Properties of an Efficient 
 Injection Material. 
 
 When a colored mass is to be used, it must contain the particular color in 
 an indiffusible form, so that it will not diffuse through the vessel-walls, staining 
 the proximal tissues and thereby rendering the preparation useless. Hence 
 dyes, which are insoluble in water, can always be used to advantage, provided 
 we use a very fine suspension thereof. Soluble dyes give much nicer results, but 
 of these only very few are not diffusible ; here we can mention the soluble Berlin 
 blue and the carmine, the latter, however, only under certain conditions. 
 
 Soluble Berlin Blue. 
 
 A concentrated watery solution of the soluble Berlin blue, previously 
 filtered, makes the best watery injection material for glandular ducts. 
 
 For the injection of blood-vessels we always use a coagulating mass, i.e., a 
 mass which is injected warm and liquid, coagulating when it becomes cooled. 
 For this purpose a sufficient amount of glue is added to the color solution. 
 Such a glue-mass coagulates easily and completely, and will not interfere with 
 subsequent microtechnical procedures. 
 
 The Blue Glue-Mass. 
 
 An efficient blue glue-mass is prepared as follows : 50 gms of the best 
 gelatine are soaked in distilled water for several hours ; the water is thoroughly 
 decanted at the expiration of this time and the gelatine placed in a beaker of 
 1,000 cm 3 capacity, and warmed over a water-bath until completely liquid. 
 500 cm 3 of a concentrated solution of Berlin blue of the same temperature are 
 carefully poured into the gelatine, constantly stirring and raising the tempera- 
 ture until all of the dye has been dissolved. The hot mass is then filtered 
 through flannel with the aid of a hot- water funnel. 
 
 The Red Olue-Mass. 
 
 A good red glue-mass is made by using 50 gms of gelatine, leaving it to 
 swell in distilled water for several hours, after which time we decant the water 
 carefully. The gelatine is now stained for two to three days in a solution 
 of 15 gms of carmine in 2 / of 10% borax solution. The gelatine bars, after 
 being soaked, should be transferred singly to the solution. After the staining 
 the excess dye solution is washed off with hydrant water, and again the plates, 
 now deep blue red, are singly transferred to a vessel containing 2% hydro- 
 chloric acid. The bars are agitated in this solution, until the bluish red 
 changes to a cherry red. Subsequently they are freed from the acid by washing 
 in running water ; all water is removed or evaporated over the water bath. For 
 purposes of preservation a piece of camphor, the size of a hazel nut, is added 
 to the mass, while hot. 
 
 The Injection Syringe. 
 
 As an instrument for injection we recommend for most cases a strong metal 
 syringe, holding 100 — 200 cm 3 , the piston of which should have a washer of 
 
75 
 
 leather or asbestos (Fig. 27). The anterior conical extremity serves for the 
 attachment of an intermediate piece with stopcock, preferably a glass cannula, 
 instead of the impractical metal cannula, as depicted I 
 in our illustration. 
 
 Preparation of Glass Cannula. 
 
 These can be made in any desired form or size, to | 
 suit each particular case, in the following manner I 
 (Fig. 28). A glass tube of the proper calibre is 
 selected and heated on one spot over a small gas- 
 burner (microburner so-called), the tube being con- 
 stantly rotated. When the glass has softened, it is] 
 drawn out according to how thin a cannula is de- 
 sired. The flame is now turned very small and ro- j 
 tating, we again heat on the spot designated in Fig. 
 28 a, draw out slightly, thus molding the neck of the ] 
 cannula ; now we heat on the place shown in Fig. 28 b 
 and draw out completely, so that the head terminates 
 in a point. The point is now severed closely behind 1 
 the head with the glass file and the opening polished 
 smooth on a hone. The cannula shown in Fig. 28 c 
 would answer better for many purposes if it would J 
 come to a finer point. 
 
 The cannula is connected with the intermediate 
 piece by means of a strong rubber tube, which is j 
 secured on both ends with strong silk or thin wire. 
 
 To inject cold liquids the apparatus described on 
 p. 61 can be utilized. By raising and lowering the en- 
 tire apparatus, the pressure can be regulated at will. 
 
 Technique of Injecting. 
 
 It can be stated, as a rule governing all injec-' 
 tions, that the smaller the area to be injected, the 
 better will be the results obtained. Total injection I 
 of an entire animal body requires much care and ex- I 
 perience. If a glue mass is to be injected, the speci- 
 men must have a temperature above the melting-point 
 
 of the mass. Cold parts of a body must therefore be warmed for hours in 
 water of 40 — 45°. When using recently killed animals, this is, of course, un- 
 necessary. If the object is warm, we first introduce the cannula. 
 
 The Introduction 
 of the Cannula. 
 
 A double, strong silken thread is placed around the vessel and left untied. 
 After severing the latter, one-half is left in the hand of the operator to serve 
 as a guide, while the assistant makes a loose loop of the other half. The 
 operator now opens the vessel along its longitudinal axis to the extent of about 
 5 mm and introduces the cannula, which has previously been connected with 
 rubber-tube and intermediate piece and filled with warm salt solution or, better 
 
 <f 
 
 » 
 
 Fig. 27. 
 Injection Syringe. 
 
76 
 
 still, with an 8% solution of Glauber salt, all air-bubbles being expelled and the 
 stopcock being closed at the time of introduction ; as soon as the head of the 
 cannula is within the vessel, the assistant secures it by tying the previously 
 placed ligature. We can now inject our solution, which must be warmed to 
 40°, or we may first remove blood and clots by injecting a warm 8% solution 
 of Glauber salt and then follow with the injection mass. By the latter pro- 
 cedure we warm our specimen and obtain a more complete injection; of course 
 more of the mass is needed. 
 
 As soon as the injection material is seen to come from the efferent vessels, 
 
 Fig. 28. 
 Preparation of a Glass Cannula. 
 
 the latter are closed with artery clamps (Fig. 29), and we continue carefully 
 with our injection until the entire vascular system appears distended. It is 
 
 Fig. 29. 
 Artery Clamps. 
 
 a matter of practice and experience to know just when to discontinue . the 
 injection and how much pressure to use during it. 
 
 After-Treatment. 
 
 This is much the same as the routine with which we are familiar. The 
 injection being complete, we cool the specimen in order to coagulate the solu- 
 tion, which may require hours in the case of voluminous organs. Smaller pieces 
 are subsequently excised and fixed with any method desired, formalin par 
 excellence. 
 
 MOUNTING AND FINISHING OF THE MICROSCOPICAL 
 
 SPECIMEN 
 
 Durability of 
 Stained Specimens. 
 
 After having stained our specimen according to one of the methods de- 
 scribed, the question arises how to preserve it in a manner which is least apt to 
 allow any changes to take place and will make the specimen a durable one. In 
 
77 ' 
 
 many cases this remains a hope, since among all our dyes there are few only 
 of which it can he said that specimens stained therewith are absolutely lasting 
 in color. Perhaps we can rightfully claim this for carmine and hematoxylin, 
 but most all will be bleached in the course of years, and this is especially true 
 of the artificial dyestuffs. 
 
 A great deal depends on the mounting and care of the slide thereafter. Any 
 agent, to serve for mounting, must be chemically pure and above all must not 
 contain a trace of any acid, which would destroy any stain in the course of 
 time. Many artificial dyes being only slightly fast to light, specimens should 
 be protected from light. 
 
 Mounting Media. 
 
 The agents used to enclose, i.e., mount our specimens, mounting media so- 
 called, are partly liquid, partly solid. In the latter case they must naturally 
 be liquefied by a proper solvent before using ; by spontaneous or induced evapo- 
 ration of this solvent the specimen, covered by the cover-glass, is thus enveloped 
 in a layer of the solid medium, which serves also as a cement between slide and 
 cover-glass. 
 
 Mounting in Liquid Media. 
 
 When using a liquid medium, the latter must be protected against evapo- 
 ration, i.e., the specimen must be prevented from drying up, by the use of a 
 strong cement between slide and cover-glass. 
 
 Cover-Glass Cement. 
 
 Such a cement can be prepared by melting 10 gms of wax in a porcelain 
 dish and, while stirring, adding 35 gms of rosin. To make the mass more 
 pliable a small amount of turpentine may be added. The mixture should be 
 applied with a broad and not too thin spatula. The spatula should be warmed 
 first and then dipped in the solid mass ; the necessary amount of cement will 
 thus adhere to the spatula and the latter can be heated to such a degree as to 
 render the cement thinly liquid. The groove formed by cover-glass and slide is 
 touched with the edge of the spatula, and thus a band of cement can be drawn, 
 which solidifies at once. The surface to which the cement is applied must be 
 absolutely clean and dry, lest the cement will not adhere. It is important there- 
 fore to use a small drop of mounting fluid, so that none will be protruding from 
 under the edges of the cover-glass. 
 
 Mounting in Solid Media. 
 
 The process is simpler, when using solid media. A small drop of the 
 liquefied medium should be used, for the smaller the drop, the thinner will be the 
 layer enveloping the section. The cover-glass is grasped with forceps, the free 
 edge is approximated until it touches the slide and the glass then slowly 
 lowered, being careful not to enclose any air-bubbles. The specimen is then 
 left to dry in a dustproof place. In certain instances it is of advantage to 
 hasten the drying by the use of the thermostat. 
 
 Thickness, Size and 
 Cleansing of the Cover-Class. 
 
 The cover-glasses with which we cover our specimens must be sufficiently 
 large to cover the tissue entirely. The latter must never come close to 
 
78 
 
 the edge of the glass, lest it shall be bleached more readily. This is especial- 
 ly true of hematoxylin preparations. The size of the cover-glass is naturally 
 dependent upon the size of the slide, the latter generally measuring 26 X 76. 
 mm, the so-called English type. In order to leave a narrow margin, the cover- 
 glass must therefore not exceed 20 X 22 mm. Generally those measuring 20, 
 mm square are well suited for histologic specimens, 20 X 40 mm for larger 
 sections. The thickness should be of an average of 0.12 — 0.15 mm; thinner 
 glasses are unnecessarily difficult to clean, while thicker glasses will not admit 
 the use of the oil-immersion lens. They are best kept in a covered vessel, con- 
 taining 30% alcohol. The cover- glass is taken out with forceps and dried with 
 a clean cloth before using. 
 
 Influence of the Mounting 
 Medium on the Microscopic Picture. 
 
 Of the innumerable media recommended for mounting we are interested in 
 just three, glycerine, Canada balsam and levulose. Before going into details, 
 we must make a few general remarks about their refractive properties. Our 
 microscopic preparations possess a certain refraction index ; if they should be 
 placed in a medium with the same refraction index, all structural details will be 
 lost, will be made invisible. Before being stained, the entire specimen appears 
 equally illuminated. Staining will bring out the different hues in elegant 
 fashion. If we desire to demonstrate the structural details in the unstained 
 specimen, we must select a medium with low refractive index, hence unstained 
 specimens are best to examine in a low refractive medium, e.g., water, which 
 has a refractive index of 1.3. It is different with stained preparations, where 
 the colors will not appear prominently enough in a low refractive medium ; 
 here it is better to select a medium with a higher index, say about 1.5. 
 
 Glycerine. 
 
 C a H 6 (OH) 3 , a thick colorless fluid, mixing with water and alcohol in, any 
 proportion, is a good solvent for inorganic and organic substances. Its re- 
 fractive index is 1.45. It is used a great deal as a mounting medium, but does 
 not prevent the bleaching of the stain for any length of time. Pure glycerine, 
 acting hygroscopic, will shrink the specimen ; it is therefore advisable to use 
 a mixture of glycerine and water first, and then follow with pure glycerine. 
 
 Canada Balsam. 
 
 This is made of the resin of several North American fir-trees, has a weak 
 acid reaction and is soluble in ether, benzol, xylol and chloroform. The refrac- 
 tive index is 1.53. After the acid reaction has been neutralized by potassium- 
 carbonate, Canada balsam represents about the best mounting material for 
 stained specimens known to this day. It should be dissolved in chemically pure 
 xylol sufficiently to allow the solution to drop easily and be kept in glasses with 
 a surmounting, closely fitting glass top. 
 
 Specimens to be mounted in balsam must first be thoroughly dehydrated 
 by absolute alcohol and transferred to xylol. The slide is dried on its under 
 surface and around the specimen, and a small drop of balsam is dropped on the 
 section, which latter must never be allowed to become entirely dry. The cover- 
 glass is now placed in the manner described above. 
 
79 
 
 Celloidin sections must not be dehydrated in absolute alcohol, lest the cel- 
 loidin should be softened too much. We therefore treat it with up to 95% 
 alcohol, after which the small amount of water is extracted with carbol xylol, 
 i.e., a solution of 1 part of phenol, liquefied by heating, in 3 parts of xylol. 
 After the sections have become entirely transparent, they can be transferred 
 to xylol and are then ready for balsam. 
 
 After a few days the xylol on the edge of the cover-glass will have evapo- 
 rated sufficiently to make a strong connection between cover-glass and slide, 
 while the balsam in the proximity of the specimen will remain liquid for months. 
 It is to be recommended, especially in dealing with delicate stains such as 
 Biondi solution, to place the slide in the thermostat for a few days immediately 
 after it has been finished. 
 
 Levulose, Fruit Sugar. 
 
 The purest form is found on the market in form of light yellow crystals, 
 readily soluble in water and alcohol to form a yellowish sirup ; it has a refrac- 
 tive index of 1.5. 
 
 The only useful preparation is the crystallized levulose, 1 of which 20 gms 
 are placed in a balsam glass containing 15 cm 3 of water, the glass to be kept in 
 the paraffin oven overnight. Again we dry the cover-glass in the manner de- 
 scribed above, trying, however, to withdraw any excess of water from the 
 specimen itself by means of filter paper. A small drop of levulose is now placed 
 on the specimen, which is left uncovered for one to two minutes. The cover- 
 glass is now adjusted and the slide kept in the thermostat for several hours to 
 allow the cover-glass to settle evenly ; lastly we enclose with cover-glass cement. 
 Levulose is an excellent mounting medium, possessing the advantage over bal- 
 sam of dispensing with alcohol following the staining. It preserves better than 
 glycerine, being inferior to balsam in this respect. 
 
 MENSURATION AND DRAWING OF MICROSCOPIC 
 
 PREPARATIONS 
 
 In order to ascertain the actual size of the specimen seen in our micro- 
 scopical image, we can proceed in different ways. We may either measure the 
 specimen itself by the aid of proper devices or we can primarily project a true 
 picture of it on paper by means of the drawing apparatus, measure this sec- 
 ondarily and, with due consideration of the magnifying power used, calculate 
 the true size. 
 
 Ocular Micrometer. 
 
 Apparatus and devices for the measuring of microscopic specimens are 
 called micrometers. They are constructed after various principles and in dif- 
 ferent forms. Simplest and most efficient for our purpose is the ocular microm- 
 eter. It consists of a small round glass plate, provided with scale; the scale 
 is 5 mm long and divided into 50 equal parts. Special eyepieces are necessary 
 for the use of the ocular micrometer, in which the eye-lens can be moved toward 
 
 1 C. A. F. Kahlbaum, Berlin. Not inexpensive. 
 
80 
 
 and away from the collective lens by means of a draw-tube. The latter is 
 removed and, scale down, the micrometer is placed on the ocular diaphragm. 
 When the eye-lens is replaced, we will thus obtain a magnified picture of the 
 scale. The eyepiece being adjusted in the barrel, the images of scale and 
 specimen will cover each other, since the former is projected through the eye- 
 lens in the same plane as the latter. By moving the specimen we can place any 
 desired field under the scale and measure how many of the subdivisions on the 
 scale it will cover. Each segment of the scale has a certain value, which de- 
 pends on the size of the image produced at the level of the ocular diaphragm, 
 viz., it depends on the focal distance of the objective. The more powerful the 
 latter, the smaller will be the value of each division of the scale. The optician 
 furnishes tables with the micrometer, stating the values for the different ob- 
 jectives in a sufficiently accurate manner, so that we simply have to multiply 
 the number of subdivisions with the value stated in the table. It goes without 
 saying that during such calculations the prescribed barrel length must be 
 strictly observed. 
 
 Drawing of 
 Microscopic Specimens. 
 
 Of the significance and importance of drawing we have already spoken in 
 the preface ; suffice it to say that it is of the utmost importance that the be- 
 ginner should draw his specimens repeatedly and in an exact manner. Most 
 every beginner is possessed with a disliking for drawing, which we can often 
 trace to an underestimation of the student's own faculties. The first attempt, 
 if made at all, is generally such an utter failure, that the student becomes dis- 
 couraged and fails to try again. For this reason it is well to aid the first 
 attempts by the use of a drawing apparatus. Noticing how the picture develops 
 under his hands by the simple tracing with pencil, he will acquire the ambition 
 to complete the sketch, and after several such attempts he will have developed 
 an interest and pleasure in this sort of work. 
 
 Drawing Apparatus. 
 
 Of the various makes of such apparatus we prefer the simple and cheap 
 Abbe drawing apparatus (Fig. 30). It consists of two metal rings, the lower 
 of which (rj) is fastened centrically with three screws (schr) upon the upper 
 extremity of the barrel; the other, upper ring (r 2 ), supports the apparatus 
 proper and is connected with the first ring by a joint (gel), allowing of a for- 
 ward movement of the apparatus. Upon the upper ring we find a small glass 
 cube, enclosed in a little metal drum (tr), which is open on the side; the cube 
 consists of two prisms, their diagonal planes being in approximation. The 
 diagonal plane of the upper prism has been silvered mirrorlike, with the excep- 
 tion of a small central pupil of about 2 mm diameter. In front a horizontal 
 arm (a) is seen emerging from the upper ring; at a distance of 7 — 8 cm a 
 rectangular mirror is attached to this arm, which can be rotated on a hori- 
 zontal axis. After the apparatus is properly attached, the mirror is adjusted 
 at an angle of 45° to the arm, and now the observer, looking through the 
 opening (o) in the drum and thence through the pupil of the prism, can see 
 the microscopic picture and at the same time the point of a pencil, which is 
 
81 
 
 held under the mirror, both pictures uniting in the eye, thus making it possible 
 to trace the contours of the microscopic picture. 
 
 The use of the apparatus is not quite as simple as it may at first appear 
 to be, since differences in light between the two images may result in one out- 
 balancing the other. The apparatus being thrown back, we first focus our 
 specimen sharply, and close the iris diaphragm; the point of the pencil must 
 contrast on the white drawing paper. The diaphragm is now opened slowly, 
 until specimen and pencil point appear equally distinct. Should the drawing 
 surface be too light, which seldom is the case, smoked glasses should be inter- 
 posed between prism and mirror. If we desire correct size, we must be sure to 
 
 
 
 a 
 
 
 
 
 O "»»»,_ 
 
 
 
 
 
 
 tr , 
 
 gel • 
 
 
 
 
 
 
 . — • SB 
 
 
 ;•;; S^JtK ^ 
 
 r i 
 
 
 =#j^r 
 
 
 
 
 schr | 
 
 
 
 
 
 
 r l 
 
 
 Wt* A 
 
 
 
 
 eehr —~~~~ 
 
 
 
 
 
 
 
 
 
 mm 
 
 
 
 
 
 
 fr 
 
 ■l^o 
 
 
 
 
 
 Fig. 
 
 30. 
 
 
 
 
 Drawing 
 
 Apparatus after 
 
 Abbe. 
 
 
 
 have the drawing surface on a level with the stage, and to avoid distortions our 
 mirror must be kept at the angle of 45°. Only those places vertically under 
 the mirror should be drawn ; if larger drawings are desired, the drawing surface 
 must be moved accordingly. 
 
 This apparatus serves only for the drawing of contours, the details must 
 be worked in by free hand, reading from the microscopical picture. For this 
 purpose we may use pencils of different strengths or, better still, a brush and 
 india ink or colors. The drawing pen should be avoided, as it will impart a 
 certain hardness to the picture which is foreign to the specimen. Of the 
 brushes the Japanese are the best, results being obtained with them which are 
 even more delicate than could be produced by the pen. Of the stains we would 
 recommend the Gouache colors, manufactured by Schoenfeld & Co., in Diissel- 
 dorf . When minute details are desirable, it will be a good plan to draw through 
 a simple lens, magnifying two to three times. 
 
Index 
 
 Abbi's condenser, 19 
 
 Abbe's drawing apparatus, 80, 81 
 
 Aberration, chromatic, 7, 8, 12 
 
 spherical, 7, 12, IS 
 Acetic acid, 29, 30, 35 
 Achromatic objective, 13, 15 
 Acid dyes, 54, 63 
 Acid fuchsin, 64, 67 
 
 S. M. P., 67 
 Acidophilic, 54 
 Adjective staining, 53 
 Adjustment, coarse, 11, 23 
 After-treatment, 28 
 
 Aktiengesellschaft fur Anilinfabrikation 3 67 
 Alcohol, 26, 28, 32, 35, 52 
 
 shrinking due to, 33 
 Alcohol-acetic acid, 33 
 Altman's bioblasts, 64 
 Altman's mixture, 31 
 Amniotic fluid, 25 
 Anaesthetizing of animal, 27 
 Angle, 14 
 
 of section, 44, 45 
 Aniline dyes, 58 
 Aperture, 14, 15 
 Aplanasia, 15 
 . Apochromatic objective, 13 
 Aqueous humor, 25 
 Artery clamps, 76 
 Artificial stains, 58 
 Azureosine, 67 
 
 Barrel of microscope, 10, 11 
 
 Base of microscope, 10 
 
 Basic dyes, 53, 54, 58 
 
 Basophilic, 54 
 
 Berlin blue, 74 
 
 Bichloride of mercury, 31, 48 
 
 Biondi solution, 67 
 
 Bleu de Lyon, 64 
 
 Blocking, 44 
 
 Blood serum, 25 
 
 Blood, staining of, 65 
 
 Blue glue mass, 74 
 
 Body of microscope, 10 
 
 Bordeaux R., 65 
 
 Bull's eye condenser, 18 
 
 Canada balsam, 78 
 Cannula, 75, 76 
 
 introduction of, 75 
 Carbonic acid, 27, 37 
 Carmalum, 55 
 Carmine, 54, 55 
 Carminic acid, 55 
 Carnoy's mixture, 33 
 Carrier, 11 
 Cedarwood oil, 14 
 Cell, chemical ingredients of, 26 
 
 Celloidin, 36, 46 
 
 blocking, 46 
 
 section method, 46, 47 
 
 staining, 52 
 Cement, 77 
 
 Changes in tissue after death, 24 
 Chloroform, 27, 41 
 Chop method, 34, 35 
 Chromatic aberration, 7, 8, 12 
 Chrome hyperoxide, 29 
 Chrome-osmio-acetic acid, 30, 48 
 Chrome silver method, 69, 70 
 
 rapid, 70 
 Chromic acid, 29 
 Clips, 10 
 
 Coagulation of cellular contents, 21 
 Coarse adjustment, 11, 23 
 Coccus cacti, 54 
 Cochenille, 54 
 Collective lens, 9, 17 
 Collodium, 45 
 Color bath, 49 
 
 temperature of, 50 
 Color lakes, 53 
 Combined system, 8 
 Compensation eyepiece, 17 
 Compound lens, 7 
 Compound microscope, 8, 9 
 Concave mirror, 19 
 Condenser, 11, 12, 18, 19 
 
 Abbi's, 19 
 
 bull's eye, 18 
 Correction lens, 12, 13 
 Corrosive sublimate, 31, 48 
 
 chrome-osmium-acetic acid, 32 
 
 Mueller's fluid-acetic acid, 32 
 Cover-glass, 13, 24, 77 
 
 cement, 77 
 
 size of, 77 
 Cresyl violet R. B., 62 
 Crown glass, 5, 7 
 Curvature radius, 7 
 Cutting instruments, 37 
 Cutting method, 34, 35 
 Cutting methods, special, 47 
 Cutting of paraffin sections, 44 
 Cylinder, diaphragm, 20 
 
 Decalcification, 48 
 Dehydration, 41, 46 
 Detailing power, 15 
 Dewar bottle, 37, 38 
 Diaphragm, 11, 17, 19, 20 
 
 cylinder, 20 
 
 iris, 19, 20 
 
 lever, 20 
 Differentiation agents, 53 
 Diffuse staining, 53 
 Dispersion, 5, 8 
 
 83 
 
84 
 
 Dissociation method, 34 
 Distance, visual, 7 
 Drawing apparatus, 80 
 Drawing of specimen, 80 
 Draw-tulle, 11 
 Durability of specimen, 76 
 
 Elective staining, 53 
 Embedding, 36, 43 
 
 frame, 43 
 Eosine, 65 
 
 Erlenmayer flask, 67 
 Ethyl alcohol, 32 
 Eye-lens, 9, 17 
 Eyepiece, 9, 12, 17, 18 
 
 marking of, 18 
 
 significance of, 18 
 
 Fat, staining of, 65 
 Fixation, after-treatment, 28 
 
 amount of solution, 27 
 
 duration of, 28 
 
 general rules for, 26 
 
 reagents for, 26, 62 
 
 significance of, 25 
 Fixed specimen, cutting of, 36 
 Fleming's solution, 30, 32 
 Flint glass, 5, 7, 8 
 Fluor spar, 13 
 Focal distance, 6, 7, 8 
 Focus, 6 
 Focussing, 22 
 Foot of microscope, 10 
 Formalin, 26, 28, 29, 33, 48 
 
 alcohol, 34 
 
 Mueller's fluid, 33 
 Freezing cylinder, 37, 38 
 Freezing microtome, 38, 39 
 Fresh specimen, cutting of, 36 
 Front lens, 12 
 Frozen section, 36, 37 
 
 method, 37, 47 
 
 staining of, 51 
 Fuchsin, 58 
 
 Gentian violet, 59 
 Giemsa solution, 67 
 Glauber salt, 29, 31, 48, 76 
 Glue mass, 74 
 Glycerine, 35, 78 
 
 for mounting, 78 
 Gold chloride, 72 
 
 formic acid method, 73 
 
 Gram method, 73 
 
 lemon juice, 72 
 Golgi method, 69 
 Gouache colors, 81 
 Gram's method, 59 
 Green chrome oxide, 29 
 
 Haemalum, 56, 65 
 Haematin, 56 
 Hematoxylin, 56 
 
 iron-alum, 57 
 
 phospho-molybdic acid, 57 
 
 potassium-dichromate, 57 
 Heat, a fixing agent, 26 
 Heidenhain staining, 57 
 Hermann's fluid, 32 
 
 Heterogeneous staining mixtures, 65, 66 
 High power, 13 
 
 Hr-echster Farbwerke, 59 
 Homogeneous immersion, 14 
 Homogeneous staining mixtures, 65, 66 
 Huyghen eyepiece, 17, 18 
 Hydrochloric acid, 32, 35, 63 
 Hydrocyanic acid, 27 
 Hydrogen peroxide, 30 
 Hypertonic fluids, 25 
 Hypodermatic method of staining, 50 
 Hypotonic fluids, 25 
 
 Illumination, 18, 19, 24 
 Image, 6 
 
 planatic, 15 
 Immersion objective, 13 
 Impregnation, 68, 69 
 
 after-treatment, 70 
 
 duration of, 70 
 
 with gold salts, 72 
 
 with silver salts, 69 
 Indifferent stains, 65 
 Indigcarmin, 58 
 Indigo, 57 
 
 Injection apparatus, 60, 61, 74 
 Injection material, 73, 74 
 Injection method, 73, 74 
 Injection syringe, 74, 75 
 Injection technique, 75 
 Intercellular substance, 26 
 Intravenous method of staining, 50 
 Iodin, 31, 35 
 Iris diaphragm, 19, 20 
 Iron-hoematoxylin, 56 
 Isolation-agents, 34 
 Isotonic fluids, 25 
 
 Jena glass, 67 
 
 Joint of microscope, 10 
 
 Lakes, 53 
 Lauth's violet, 59 
 Leitz microscopes, 21 
 Lens, 5 
 
 classification, 5 
 
 collective, 9, 17 
 
 composition of, 5 
 
 compound, 7 
 
 correction, 12 
 
 eye, 9, 17 
 
 negative, 5 
 
 positive, 5 
 Levulose, 79 
 Light green, 64 
 Light, source of, 20, 21 
 Living object, observation of, 25 
 Low power, 13 
 Lump staining, 51 
 Lyon blue, 64 
 
 Maceration, 34, 35 
 Magnifying, 7, 9, 12 
 Maximum of staining, 52 
 Media for preservation, 25 
 Meniscus, 5, 6 
 Mensuration, 79 
 Metachromasia, 54, 62 
 Metachromatic staining, 54 
 Metal oxides, 53 
 Metals for impregnation, 68 
 Metals, heavy, 26, 28 
 Methylene azur, 62 
 
85 
 
 Methylene blue, 59, 60 
 Methyl green, 59 
 
 N. M. P., 67 
 Methyl violet, 59 
 Micrometer, 79 
 
 screw, 11, 12, 21, 23 
 Microscope, 5, 23 
 
 care of, 22 
 
 compound, 8, 9 
 
 description of, 9, 10 
 
 illustration (Fig. 8), 10 
 
 prices of, 22 
 
 procuring of, 21 
 
 rules for use of, 21 
 Microtechnique, definition of, 21 
 
 general, 24 
 Microtome, 37, -14 
 Mineral acid, 26 
 Mirror, 11, 19 
 
 concave, 19 
 
 plane, 19 
 Mixtures of stains, 65 
 
 heterogeneous, 65, 66 
 
 homogeneous, 65, 66 
 Monobrome-naphthalin, 14, 15 
 Mordants, 53 
 Morphine, 27 
 Mounting, 45, 71, 76, 77, 78 
 
 media, 77, 78 
 
 of impregnated specimens, 71 
 Mueller's fluid, 31 
 Multiple staining, 52, 65 
 
 Nerve-sheath, staining of, 65 
 Nitric acid, 28, 31, 35, 48 
 Nosepiece, 11, 12 
 Nuclear dyes, 58 
 Nuclear staining, 60 
 Numerical aperture, 14 
 
 Object distance, 13 
 
 Object glass. See Objective 
 
 Objective, 9, 12 
 
 achromatic, 13, 15 
 
 apochromatic, 13 
 
 cleaning of, 22 
 
 illustration (Figs. 10, II, 12), 16 
 
 marking of, 15 
 Ocular (eyepiece), 9, 17 
 Ocular micrometer, 79 
 Oil immersion objective, 13, 14, 15, 23 
 Oil of cloves, 45 
 Optic axis, 12, 13 
 Optic system, 8 
 Optimum of staining, 52 
 Orange G., 64 
 Orange G. M. P., 67 
 Orthochromatic staining, 54 
 Osmic acid, 29, 30, 31 
 Osmium tetroxide, 29 
 
 Paracarmine, 55 
 Paraffin, 36, 41, 42 
 
 embedding, 43 
 
 melting-point, 42 
 
 preparatory media, 41 
 
 section method, 41, 47 
 
 staining of sections, 52 
 Penetrating power, 27 
 Picric acid, 29, 64, 65 
 Picrocarmin, 66 
 
 Picrofuehsin, 66 
 
 Picro-indigcarmin, 66 
 
 Plctet, 37 
 
 Planatic image, 15 
 
 Plane mirror, 19 
 
 Platinum chloride, 28, 32 
 
 Platinum chloride-osmio-acetic acid, 32 
 
 Post-mortem specimens, 27 
 
 Potassium bichromate, 30, 31 
 
 Potassium bichromate-osmic acid, 31 
 
 Potassium cyanide, 29 
 
 Potassium hydroxide, 35 
 
 Power of objective, 13 
 
 Preservation, media for, 25 
 
 method of, 24, 25, 46 
 Primary lens, 8 
 Prism, metal, 11 
 Progressive staining, 52 
 Protection of hands, 61 
 Pyrogallol method, 71 
 
 Ramsden eyepiece, 17 
 Rays, course of, 8 
 Razor, 37 
 Red glue mass, 74 
 Reduction, 61, 68 
 Reflected light, 18 
 Refraction, 5 
 
 index of, 7, 13 
 Refractive errors, 12 
 Regressive staining, 52 
 Reichert, 21 
 Resorcin fuchsin, 63 
 Retrogressive. See Regressive 
 Ringer's fluid, 25, 51, 59, 60, 61 
 
 Safranine, 63 
 
 Saline, solution of, 25 
 
 Saturation with celloidin, 46 
 
 Scarlet R., 65 
 
 Schanze, 38 
 
 Schoenfeld & Co., 81 
 
 Section, 24 
 
 method of, 24 
 
 preparation of, 34, 47 
 
 staining of, 51 
 
 thickness of, 40 
 
 treatment of, 40 
 Sedimentation, 35 
 Seibert, 21 
 
 Silver-ammonia method, 71 
 Silvering, 69 
 
 Silver nitrate, for impregnation, 69 
 Simple staining, 52 
 Simultaneous mordant dyeing, 53 
 Simultaneous staining, 52 
 Size of specimen, 27 
 Sketching, 80 
 Slide, 24 
 
 Sodium sulphate, 31, 32 
 Soluble Berlin blue, 74 
 Solvents of dye, 49 
 Spherical aberration, 7, 12, 15 
 Stabilit, 47 
 
 Stage of microscope, 10 
 Staining, 24 
 
 adjective, 53 
 
 diffuse, 53 
 
 elective, 53 
 
 method of, 24, 48 
 
 mixtures, 65 
 
86 
 
 Staining of celloidin, 52 
 of frozen specimen, 51 
 of living specimen, 50 
 of paraffin specimen, 52 
 of preserved specimen, 51 
 of surviving specimen, 50 
 progressive, 52 
 regressive, 52 
 simultaneous, 52 
 substantive, 53 
 succedaneous, 52 
 Stains, 49 
 
 artificial, 58 
 concentration of, 49 
 indifferent, 65 
 in mixture, 65 
 of animal origin, 54 
 of vegetable origin, 55 
 Sublimate-nitric acid, 48 
 Substantive staining, 53 
 Succedaneous staining, 52 
 Succedaneous mordant dyeing, 53 
 Successive. See Succedaneous 
 Sudan III. 65 
 
 Tannic acid, 26, 58 
 Tar-dyes, 58 
 Thermos bottle, 37, 38 
 Thermostat, 42 
 Thionine, 59 
 
 Transmitted light, 19, 24 
 Trichlor-acetic acid, 29, 31 
 Tube length, 9 
 
 Vital staining, 50 
 
 Vital staining of nerves, 60 
 
 Voigtliinder, 21 
 
 Wash-glass, 28 
 
 Water as immersion medium, 14, 15 
 
 WirikeJ, 21 
 
 Wooden slide, 71 
 
 Xylol, 52 
 
 Zeiss. 21 
 Zenker's fluid, 32 
 
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