IRLF 
 
c / ^ 
 
 
ELEMENTARY 
 
 MICROSCOPICAL TECHNOLOGY. 
 
 A MANUAL FOE 
 
 STUDENTS O.F MICROSCOPY. 
 
 IN THRKE FARTS. 
 
 PART I. 
 
 THE TECHNICAL HISTORY OF A SLIDE 
 
 FROM THE CRUDE MATERIALS TO THE FINISHED MOUNT. 
 
 BY 
 
 FRANK L. JAMES, PH. D., M. D. 
 
 President St. Louis Society of Microscopists, Member of American Society 
 of Microscopists, Ed. St. Louis Medical and Surgical Journal. 
 
 ST. LOUIS, MO.: 
 ST. Louis MEDICAL AND SURGICAL JOURNAL COMPANY, 
 
 1887. 
 
PREFACE. 
 
 IN all existing text-books of microscopical technology 
 with which I am acquainted, not only in the English, 
 but in the French, German and Italian tongues, the 
 technology proper or the manipulations and processes 
 incident to the preparation of material for microscopical 
 examination, is so mixed up with the micrography histo- 
 logical, pathological or biological, that it is an almost 
 hopeless task for the student, especially the beginner, 
 working without a master, to separate them. In the mul- 
 titude of details and the interruptions to continuity caused 
 by the attempt at teaching simultaneously such widely 
 divergent subjects, the elementary student fails to grasp a 
 clear idea of either the one or the other. 
 
 Another difficulty, incident to and inseparable from in- 
 struction attempted to be conveyed in this manner, is that 
 the author must, at many points anticipate details or pre- 
 suppose some acquaintance with the subject on the part 
 of the student. 
 
 Realizing these difficulties, first as a student remote 
 from skilled teachers, slowly working out by experiment 
 each problem as it arose, and afterwards as a teacher, 
 searching for a text-book to put into the hands of my 
 students, I finally undertook the" preparation of a manual 
 modelled after an ideal in which nothing should be taken 
 for granted, no previous acquaintance, on the part of the 
 student, with the subject-matter presupposed, and in which 
 
IV 
 
 each step of the work, each process and manipulation 
 should be explained in orderly sequence. 
 
 This little manual is the result of this idea ; how well 
 I have succeeded in its execution is for the reader to^say, 
 I will only add that I have endeavored to make it strictly 
 practical, having embodied in it the actual experiences of 
 many years of almost incessant labor in this direction. 
 
 The present volume is devoted entirely to Elementary 
 Technology, and details the Technical History of a Slide 
 from the crude materials up to the finished mount. It con- 
 stitutes Part I of a work on General Microscopical Tech- 
 nology constructed upon the same scheme and plan, the 
 other parts of which will appear in due^time. 
 
 No. 615 Locust St., | FRANK L. JAMES. 
 
 St. Louis, Mo. J 
 
ELEMENTARY MICROSCOPICAL TECHNOLOGY, 
 
 i. 
 
 Microscopical technology is a description of those processes 
 and appliances by means of which objects are prepared for ex- 
 amination under the microscope and permanently preserved for 
 future reference and study. In microscopy, as in every other 
 pursuit which involves the use of tools or instruments, the num- 
 ber and' nature of the apparatus and appliances deemed necessary 
 for the performance of good work will vary very greatly accord- 
 ing to the taste, ingenuity, and above all, the pecuniary ability of 
 the individual. While some men will be content and do good 
 work with a few simple instruments, costing but a few dollars, 
 others will require the most elaborate outfit, costing as many hun- 
 dreds. In the present series of articles I shall describe only those 
 instruments which experience has taught me to be absolutely 
 essential, leaving the student to learn from more elaborate text- 
 books and the catalogues of the instrument makers, those more 
 complicated appliances which, while they are frequently of great 
 assistance, are not absolutely essential in doing good work and 
 hence must be considered as articles de luxe. 
 
 I. The processes through which an object passes from its 
 crude or natural condition up to the finished slide, ready for the 
 cabinet, vary according to the nature of the material. They may 
 be grouped under six general headings, viz; 
 
 a. Preserving in the mass. 
 
 6. Hardening those substances which are too soft, and soften- 
 ing those which are too hard to be cut with the section knife. 
 
 c. Embedding. 
 
 d. Section cutting. 
 
 e. Staining. 
 
 /. Mounting on slips. 
 Some objects have to pass through all of these processes, 
 
8 ELEMENTARY MICROSCOPICAL TECHNOLOGY. 
 
 while others have to undergo only a portion of the manipulations 
 indicated, 
 
 . II. The instruments and apparatus required for the per- 
 formance of each step will be described under its proper 
 heading. There are, however, certain tools which are required 
 in almost every stage of our progress, and it will be well for the 
 student to provide himself with them at the outset. They are as 
 follows: 
 
 a. A half dozen needles mounted in wooden handles. Four 
 of these should be straight aud the others curved. Surgeons 
 needles are the best for this purpose, though ordinary sewing 
 needles will answer for the straight ones. The handles should 
 be five or six inches long and large enough to give a good 'grip' 
 to the fingers. Tapering cedar penholders are excellent for the 
 purpose. The needles should be inserted into the small end and 
 carried deep enough to be perfectly firm. They should not pro- 
 ject more than three-quarters of an inch from the handle, as 
 otherwise they are too springy for delicate dissections. 
 
 b. Small forceps, straight and curved. The ordinary iris for,-, 
 ceps are excellent for this purpose. If dentated, the teeth should 
 be ground or filed out and the ends of the blades brought to a 
 point. 
 
 c. Small scissors straight and curved. 
 
 d. A few delicate scalpels. Old tenotomes, or cornea- 
 knives, well sharpened are the very things. Almost every oc- 
 ulist has some such knives which have become useless for his 
 work but which answer admirably for microscopical dissections. 
 
 e. A dozen watch crystals. The old fashioned <bulls-e3'e ; 
 cystals are the best. They may be obtained from any large 
 jewelry establishment for a trifle. Two or three "individual 
 salts" will also be found useful. 
 
 /. Three flat saucers, like those used for shirred eggs. 
 
 g. A wash-bottle holding 10 or 12 fluid ounces of distilled 
 water. 
 
 h. A half dozen medicine droppers or pipettes. 
 
 i. A half dozen camel's-hair pencils, assorted. 
 
 j. Small spirit lamp and stand. 
 
 k. Salt mouths for preserving crude material. Sulphate of 
 morphia bottles fitted with good soft corks are very useful for 
 
ELEMENTARY MICROSCOPICAL TECHNOLOGY. 9 
 
 containing small bits of material in process of hardening, sec- 
 tions, etc. 
 
 . III. The following chemicals are needed at the outset; 
 
 Alcohol, 95 and absolute; glycerin, pure; benzol, pure, and 
 commercial; oil of turpentine and benzin, of each one pound. 
 The two latter are used for cleansing purposes, as explained 
 hereafter. Chloroform and sulphuric ether, of each 6 or 8 fluid 
 ounces. Picric acid and bichromate of potassium, of each an 
 ounce. Distilled water. 
 
 Other chemicals will be described when needed and under 
 the proper headings. 
 
 . IY. Since all organic matter is prone to decay when 
 left to the influences of the atmosphere, our first care must be to 
 properly preserve the material which we are hereafter to prepare 
 for examination. This is best done by means of certain preserv- 
 ing fluids which are modified according to the substances upon 
 which they are required to act. These fluids maybe divided into 
 "th'tee classes, viz: 
 
 a. Those which simply arrest decay. 
 
 b. Those which harden or soften as well as preserve. 
 
 c. Those which, in addition to other functions, stain the 
 material. 
 
 In the first class we have solutions and combinations of the 
 various antiseptics, such as common salt; boracic, carbolic and 
 salicylic acids, chloral hydrate, glycerin, corrosive sublimate, etc. 
 
 In the second class we have alcohol, Mueller's fluid, solutions 
 of the mineral acids etc. 
 
 The third class embraces picric, chromic and osmic acids, 
 chloride of gold, etc. 
 
 Each of these agents has its special use, which must be devel- 
 oped as we proceed in the study of microscopy, since it is man- 
 ifest that the treatment which is proper for delicate tissues 
 (foetal tissues, larvae of insects, desmids or algae, for instance) 
 would not suit for a tumor, a piece of muscle or objects of like 
 description. There are two fluids however which are of such 
 universal use and applicability that to a certain extent they sup- 
 ply the places of all the balance viz; alcohol and glycerin. It 
 is recommended that the elementary student confine himself as 
 much as possible to the use of these fluids in preseving material 
 
1 ELEMENTARY MICROSCOPICAL TECHNOL OGl . 
 
 for his future work always bearing in mind the following rule, 
 viz; 
 
 Fresh organic matter should never be put into strong alco- 
 hol, but treated as follows; 
 
 Provide three saltmouths holding a pint each, and having 
 well ground glass stoppers or closely fitting velvet corks. In 
 lieu of these, preserve-jars will answer. They should have a 
 mouth large enough to receive the object intended for preserva- 
 tion without squeezing or forcing it in. In the first of these jars 
 put a mixture of 1 part of alcohol and 2 parts of distilled or 
 rain water. In the second reverse the proportions, making the 
 mixture two parts of alcohol to one of water, and in the third 
 put strong alcohol (95). The material (which we are supposing 
 to be animal matter, is placed first in jar No. I where it should be 
 left for a period vary ing according lo the size of the object, from 
 24 to 48 hours. It is then removed to the second jar and left an 
 equal length of time, and finally transferred to the third jar. 
 In the course of time the fluid in the first jar will be so weaken- 
 ed by the fluids abstracted from the material placed in it that it 
 must be thrown away. Jar No. 2 will also have become weaken- 
 ed, but it can now be made to serve for the first bath, and No. 
 3, for the same reason will act as No. 2. The third jar will thus 
 be the only one that it is necessary to replace from time to time. 
 
 Small objects immersed in glycerin will keep for a long time 
 without any special precautions. Delicate objects containing 
 much aqueous matter should not be put directly into pure anhy- 
 drous glycerine for the same reason that they should* not go at 
 once into strong alcohol the affinity of the preserving fluid 
 causes it to abstract water from the object too rapidly, thus 
 shriveling and injuring delicate tissues. 
 
 Where a number of specimens are put into the same jar at the 
 same time, each one should be marked in such a manner that it 
 may be known subsequently. Colored or white threads may be 
 attached to it and knotted in such a way as to serve as a distin- 
 guishing marks. Where the student has plenty of laboratory 
 room and other conveniences those precautions are not necess- 
 ary, as each object may be kept in a separate receiver. 
 
 . Y. The following are formulae for those preserving fluids 
 most commonly in use. We shall hereafter refer to them only 
 by the title here given: 
 
ELEMENTARY MICROSCOPICAL TECHNOLOGY. 11 
 
 GOADBY'S FLUID. Chloride of sodium 2 ounces, alum 1 ounce, 
 corrosive sublimate 1 grain, distilled water 1 pint. For tender 
 tissues add a grain of sublimate and double the quantity of water. 
 For substances containing carbonate of calcium omit the alum 
 and use double the quantity of common salt. 
 
 MUELLER'S FLUID. Bichromate of potassium 25 grains; sul- 
 phate of sodium 10 grains; distilled water 2 ounces. 
 
 WICKERSHEIMER'S FLUID, as modified by Hager. Salicylic 
 acid 4 drams, boracic acid 5 drams, carbonate of potassium 1 
 dram, mix and dissolve, by the aid of heat, in a mixture consisting 
 of 12 ounces of water and 5 ounces of glycerin. When solution 
 is complete add 12 ounces of alcohol at 95 in which 3 drams, 
 each, of oil of cinnamon and oil of cloves have been previously 
 dissolved. 
 
 MEYER'S FLUIDS. Make a stock fluid consisting of 1 part of 
 salicylic acid dissolved in 100 parts of pyroligneous acid C. P 
 For larvce, hydrce and nematoidce, take 3 parts of the fluid 
 and add 10 parts c. p. glycerin and 20 parts distilled water. 
 
 For infusoria ; glycerin 2 parts, distilled water 8 parts, mix. 
 and add 1 part of the stock fluid. 
 
 For algce, desmids etc., take 1 part of the stock fluid, 1 part 
 glycerin, and 20 parts of water. 
 
 In addition to the above the following are the proportions 
 usually observed in making hardening and preserving fluids. 
 Chromic acid solution 1 per cent. 
 Picric acid solution Saturated. 
 Osmic acid solution One-half of one per cent. 
 Chloride of gold solution One-half of one per cent. 
 Bichromate of patassium solution 2 per cent. 
 
A TYPICAL MOUNT. 
 
 II. 
 
 In order to make plain the plan and scope of the present work, 
 and that the student may understand the rationale of the proces- 
 ses through which an object must be carried in its transition from 
 crude material to the finished slide, wo w;ll in this present chap- 
 ter briefly describe the making of a suppositious mount. Each 
 step in the manipulations will be described in detail in the chap- 
 ters which follow, and will be taken up as nearly as possible in 
 the order in which it oomes in the actual progress of the work" 
 
 VI. We will suppose the object to be a pathological speci- 
 men, a tumor for instance, recently removed. It is plain that 
 the direct examination of ..uch an object can only be made with 
 very low magnifying powers, such for instance, as may be ob- 
 tained by the use of a pocket magnifyer or Coddington lens. 
 In order to reach the histological elements we must use high 
 powers, and these can only be used by transmitted light that is 
 light sent through the object to be examined. We must there- 
 fore contrive a method by which the object, or a representative- 
 portion of it, may be made translucent. This may bo done by 
 taking a small portion of the material and mashing it out very 
 thin between two pieces of glass. In former times this method 
 was frequently resorted to, but as it could manifestly yield but 
 very distorted results it has long since been abandoned by those 
 who use the microscope as an instrument of precision. The 
 other alternative at our disposal is the cutting of a section from 
 the object with a very keen knife ; and hero we meet with an- 
 other difficulty viz.; the object is (usually) too soft to offer such 
 resistance to the passage of the blade as will enable us to cut a 
 section of sufficient and uniform thinness. It is true that formerly 
 such sections were cut with a Valentine's knife and which were 
 supposed to be thin enough to yield practical results, but the de- 
 
ELEMENTARY MICROSCOPICAL TECHNOLOGY. 13 
 
 vice is now very rarely resorted to. The object must therefore be 
 submitted to a process which will harden it ana acme same tirno 
 preserve it. If we are in a great hurry to arrive at results we 
 may attain the desired end by freezing our object, but we will 
 suppose that in the present instance resort is had to one of the 
 hardening and preserving fluids already mentioned or yet to be 
 described. Having hardened the material, our next step is to 
 cut it into thin sections. Formerly this was accomplished by 
 holding the hardened object in the hand and slicing off a section 
 with a razor. This process ic no longer used in exact and scien- 
 tific work. Free-hand cutting has given place to the microtome 
 or section cutter just as in exact work free-hand drawing has 
 yielded to photography. The object is therefore transferred to 
 a section cutter to be sliced into sections, and as these sec- 
 tions must be made extremely thin and uniform, it must be 
 securely held in the microtome. It must be arranged so that it 
 can be fed to the knife and at the same time have no lost lateral 
 motion. This necessitates embedding it in some liquid material 
 that will harden around it and hold it firmly in place. This 
 done, the section knife is brought into play and the object is 
 sliced to the requisite degree of thinness. Here we must 
 digress a little in order to explain subsequent operations. 
 
 If we take any very thin substance say a piece of paper, 
 and place it under the microscope in a dry state, we will find on 
 examination that we get a very insufficient idea of its intimate 
 structure. If we moisten the object with water we find that 
 many details of the structure are brought out and shown us 
 which were invisible under the former examination. If instead 
 of water, we use glycerin or Canada balsam, the structure is 
 rendered still more distinct, and if the specimen be only thin 
 enough the minutest detail is thus finally brought into view. 
 
 Let us suppose, further, that this bit of paper consists of two 
 or three, or more, kinds of fibres say silk, cotton and linen, all 
 of the same color and so interwoven with each other that it is 
 impossible for the eye to follow the ramifications of either mat- 
 erial. It is plain that if we can find a dye or stain which will 
 attack the cotton and not the silk or linen, or vice versa j or that 
 stains cotton one shade or hue, silk another, and linen another, 
 the problem of differentiating the elements which enter into the 
 structure is a very simple one. 
 
14 ELEMENTARY MICROSCOPICAL TECHNOLOGY. 
 
 With these two hints as to the reason why the sections are 
 put through the next two processes, and leaving the philosophy 
 of the same to be explained hereafter, we will resume the pro- 
 gress of our slide toward completion. 
 
 The sections as they fall from the knife are received in a ves- 
 sel filled with fluid water, glycerin or alcohol, according to cir- 
 cumstances, and when a sufficient number has been cut we pick 
 out one of the thinnest and best, and place it in the staining fluid, 
 where we will leave it while we prepare the glass slip upon 
 vhich it is to be mounted. 
 
 We take for this purpose a piece of clear glass 3 inches long 
 and 1 inch wide, the edges of which have been ground and pol- 
 ished, and placing it on an instrument called a turn-table, with 
 a pencil dipped in cement, (the nature of which depends upon 
 the fluid which we shall use as a mounting medium), we spin a 
 ring in the center of it. This ring is large enough and deep 
 enough to receive the object to be mounted, and should be al- 
 lowed to get quite dry before the slip is used. The object, in the 
 mean time, has been removed from the staining fluid and put 
 through a number of little details to fix the stain, clear away 
 the embedding material, etc., and is now soaking in the fluid which 
 is to serve as a mounting medium, the functions of which are to 
 render the object transparent and preserve it against decay. In 
 this instance we will suppose that glycerin has been chosen as 
 a medium. The ringed slip, thoroughly cleaned, is now placed on 
 the mounting box (a framo with a glass top and provided with 
 a mirror so arranged as to throw the light upwards through the 
 top and objects laid on it) and a drop of pure glycerin is allow- 
 ed to fall in the center of the ring j the object is quickly trans- 
 ferred from the glycerin bath in which it has been lying, placed 
 on the drop of glycerin already on the slip, and arranged in the 
 position it is henceforth to occupy; air bubbles are gotten rid of 
 as hereafter explained, the cover glass is applied, and clamped 
 in position j surplus glycerin is washed away and the slip and 
 cover glass carefully and thoroughly dried with prepared blot- 
 ting paper. The clamp holding the cover glass in place is now 
 removed and the slip transferred back to the turn-table where a 
 ring of cement is spun around the edges of the cover glass. 
 This ring is allowed to dry, and the slip is again washed and 
 carefully dried before a second layer of cement is applied. The 
 final touches which vary according to the taste, skill etc., of 
 
ELEMENT AR Y MICROSCOPICAL TECHNOLOGY. 1 5 
 
 operator are then given to the slide ; it is labelled and if the 
 job is properly done, is good for an indefinite number of years. 
 
 VII. Such, in brief, is the ordinary routine of processes 
 usually employed in making a mount of a pathological or histo- 
 logical specimen of the soft tissues. There are many minor oper- 
 ations, matters of detail, entirely omitted or barely alluded to in 
 the foregoing sketch, while many of the manipulations there 
 described are varied according to the nature of the mounting 
 medium finally chosen ; but an analysis of the processes enables 
 us to divide them into three principle groups, as follows, viz; 
 
 1. Those pertaining to the preparation of the material. 
 
 2. The preparation of the slide to receive the object. 
 
 3. The mounting of the object on the slide, including finish- 
 ing. 
 
 VJII. The operations belonging to the first group include 
 all of those enumerated in I except the last (f). We will re- 
 peat them, subdividing them as we go along, as follows; 
 
 1. Preservation of the material in mass. 
 
 2. Hardening soft structures and softening hard ones. 
 
 3. Embedding. The details of this series of processes vary 
 greatly, according to the nature of the structure of the material 
 (i. e. solid matter, such as muscle, requiring a different treat- 
 ment from vacuolar or spongy matter, like lung tissue), the kind 
 of microtome used, etc. They may be roughly subdivided into 
 prepartory manipulations, (such as freeing from the preserving 
 or hardening fluid, dessication of the surface of solid matter, dip- 
 ping into a fluid which prevents adhesion of embedding material, 
 dehydration of spongy tissues, soaking in liquid embedding ma- 
 terial to fill areoles etc.), and the actual operation of embedding 
 which consists of a series of several manipulations which we 
 need not enumerate. 
 
 4. Section cutting including the treatment of the sections, 
 preparatory to staining, such as freeing from embedding material, 
 etc. 
 
 5. Staining, including the fixation of stains, dehydration, 
 clearing with essential oils, etc., and saturating with the fluid 
 which is to be used in the final mounting medium. 
 
16 ELEMENTARY MICROSCOPICAL TECHNOLOGY. 
 
 . IX. The second group embraces the following manipula- 
 tions and processes 
 
 1. Cleaning the slips, and cover glasses. 
 
 2. Preparation of cements, etc. 
 
 3. Making cells, the use of the turntable, etc. 
 
 X. The third group of operations bring together the fin- 
 ished results of the other two. In it the finished section is placed 
 upon the prepared slide. It consists of 
 
 1. The arrangement of the section or object within the cell. 
 
 2. Filling the cell with the mounting medium. 
 
 3. Removal of air bubbles and foreign matter. 
 
 4. Putting on the cover glass and arranging it in place. 
 
 5. Washing off surplus mounting medium, and drying the 
 slide. 
 
 6. Returning the slide to the turntable, centering, etc. 
 
 7. Final closing of the cell by cementing the cover glass to 
 the cell wall. 
 
 In the chapters which are to follow each process will bo taken 
 up in the order in which it occurs in this schedule or scheme of 
 the technology of mounting, and with the explanations given in 
 this chapter before him the student will be enabled to follow 
 understandingly each step that is taken. 
 
HARDENING PROCESSES. 
 
 III. 
 
 X. HARDENING. Most of the preserving agents men- 
 tioned in our opening chapter, have also hardening powers, 
 the method of action being about the same in all of them, 
 the variation being in the degree of hardness attained and 
 the length of time required to reach the requisite point. 
 Thus alcohol is by far the best arid most useful of the harden- 
 ing agents for most animal tissues, but when applied to the 
 softer tissues of the lower organisms, by withdrawing the 
 aqueous matter of which they are largely composed, it shrivels 
 arid shrinks them so that they are practically worthless for 
 subsequent observation arid study. Osrnic acid, which in 
 weak solution is a perfect hardening agent for the latter class 
 of tissues, is too slow in its action for the former. No abso- 
 lute rule can therefore be given as to the choice of a harden- 
 ing agent. In the great majority of cases, however, alcohol, 
 used as already suggested (i. e. in gradually increased 
 strength; will answer every requirement. When a more 
 rapid effect than that produced by absolute alcohol is desired, 
 the latter may be supplemented by picric acid which, however, 
 stains as well as hardens. The same may be said of chromic 
 acid, bichromate of potassium and their combinations (as in 
 Mueller's fluid). The materials and methods used in hard- 
 ening being so diverse and numerous, we can, at this stage in 
 our work, give only the philosophy and outline of the pro- 
 cess, leaving for future explanation the suggestion as to the 
 proper medium to be used for various tissues. 
 
 XI. The hardening agents usually resorted to in animal 
 histological researches are : 
 
 a. Heat. 
 
 b. Cold. 
 
 c. Desnication. 
 
1 8 EL EMENTA R Y MICE OSC OPICAL TE CBNOL OGY 
 
 d. Chemicals, viz.: alcohol, chromic, picric, and osmic 
 acids, etc. 
 
 HEAT Boiling: There are certain tissues the structure 
 of which is rendered visible by submitting them to the action 
 of heat. Such, for instance, are the branched muscular fibers 
 of the frog's tongue, the fibers of the crystalline lens, etc. 
 The object to be examined is put into a large test tube, cov- 
 ered with distilled water and boiled for a few moments over 
 the flame of an alcohol lamp, or in a water bath. This 
 method was formerly much in vogue, especially among 
 English microscopists, but it is now rarely resorted to. If 
 however, one is pressed for time and desires merely to differ- 
 entiate certain elements in the structure of material such as 
 that referred to, it affords a quick and ready means of so 
 doing the word differentiate being used in a limited sense, 
 as no one, even a novice, can believe that the true histologi- 
 cal features of any structure can remain unaltered after being 
 submitted to an agent or process so destructive to life and 
 living matter. 
 
 COLD Freezing : It is but a, very few years since German 
 histologists and investigators maintained that material which 
 had undergone the processes of -hardening by heat or chem- 
 icals, gave results, when examined under the microscope, 
 that were altogether unreliable and misleading. These 
 teachers advocated the process of hardening by freezing and 
 declared that frozen sections of fresh material were the only 
 ones in which the true histological elements and conditions 
 could be observed and studied. Of late, however, those who 
 advocated the freezing process most strenuously, have found 
 that it produces abnormalities quite as numerous and as 
 serious as does hardening by chemicals, and that it has little 
 to offer in return for the many advantages of which it 
 deprives us. There are, however, circumstances under which 
 it may be advantageously resorted to, and most of the modern 
 microtomes (section* cutters) are provided with freezing 
 devices. In the absence of special contrivances the material 
 may be frozen sufficiently hard for all practical purposes by 
 immersing it (contained in a test tube) in any of the well, 
 known freezing mixtures, of which the following are the best: 
 
 1. Snow or powdered ice. 12 parts; nitrate of ammonium 
 and chloride of sodium . each. 5 parts. This will lower the 
 
ELEMENTARY MICROSCOPICAL TECHNOLOGY. 19 
 
 temperature from any point to 25 below zero, F. 
 
 2. Snow or powdered ice, 4 parts; crystalized chloride of 
 calcium, 6 parts. This will cause a depression of the tem- 
 perature 82 F. from any point above zero (the actual depres- 
 sion below this point being not quite so much, though very 
 nearly so.) 
 
 3. Equal parts of uitrate of ammonium, carbonate of 
 sodium and water. This lowers the temperature 57 F. from 
 any point. 
 
 Dr. Beale suggests a method of freezing which is very con- 
 venient for dentists and others having nitrous oxide at com- 
 mand. Tissues may be instantly frozen by imbedding them in 
 albumen or gelatine and placing them in a test tube, submit- 
 ing it to the action of a jet of that gas. It requires but a few 
 moments to render the mass as solid as rock. After freezing 
 the material, very thin sections may be cut from it and after- 
 ward prepared and examined at leisure. The advantages to 
 hardening by freezing are that perfectly fresh material may 
 be used and much time thus saved- when time is an element 
 to be considered. 
 
 DESSICATION. Skin, mucous membrane, and material of 
 this kind may be hardened by simply drying, by exposure to 
 the atmosphere at ordinary temperatures, or by the careful 
 application of a very low degree of heat. To quote Dr. 
 Beale again (whose book "How to work with the Micro- 
 scope " is one of the very best text-books ever published), 
 tissues that we wish to harden in this manner may be 
 stretched on boards, fastened in place with pins and thus 
 allowed to dry. Sections cut from material thus prepared 
 when placed in water or diluted glycerin, resume, to a great 
 extent, their fresh appearance. When an air-pump is con- 
 venient a most rapid and excellent method of dessication is 
 to suspend the article in the receiver over a vessel containing 
 anhydrous sulphuric acid. On exhausting the air the mois- 
 ture is rapidly withdrawn from the object and taken up by 
 the acid. 
 
 CHEMICAL MEDIA. By far the most useful and generally 
 applicable of these is ALCOHOL, used as described in Chapter 
 I, for preserving purposes. The range of animal textures to 
 which this agent is applicable is limited only by the fact that 
 it renders albuminous tissues, otherwise transparent, opaque 
 
20 ELEMENTARY MICROSCOPICAL TECHNOLOGY. 
 
 and granular, and that it precipitates albumen from solutions. 
 Its avidity for water and consequent shriveling action on del- 
 icate tissues has already been noted. 
 
 ANHYDROUS GLYCERIN will harden many tissues very 
 nicely, but its action is comparatively slow, and to obtain n 
 satisfactory result the glycerin must be kept in an air-tight 
 vessel and changed frequently. 
 
 The brain, spinal cord and such tissues are hardened very 
 beautifully by immersion in solutions of CHROMIC ACID, the 
 chromate and bichromate of potassium, and mixtures con- 
 taining these chemicals, such as Mueller's and other 
 solutions. 
 
 PICRIC ACID is another hardening agent of a wide range of 
 usefulness. It is used in either alcoholic or aqueous solu- 
 tion or as an ingredient in complex hardening fluids, such as 
 Kleinenberg's. If used in water the solution should be sat- 
 urated, since it is but sparingly soluble in that medium. 
 For the same reason the bulk of the solution should be large 
 in proportion to that of the -material. When dissolved in 
 alcohol the percentage may be varied according to the rapid- 
 ity of the desired effect. 
 
 OSMIC ACID. To those whose studies lie among the lower 
 forms of animal and vegetable life this chemical is one of the 
 most valuable in the whole list of hardening agents. Its 
 application not only kills those organisms but it does 
 instantly and without distortion. It preserves the tissues 
 indefinitely, and by its selective staining property (which 
 will hereafter be alluded to in its proper place) it brings out 
 features that would otherwise escape observation. It should 
 be used in solution not stronger than one per cent., and this 
 solution should be kept absolutely protected from the action 
 of the light. I use for this purpose a glass stoppered bottle, 
 thickly painted with drop black and afterward covered with 
 heavy tin or lead foil. The whole should be placed in a 
 closely covered metallic box (a yeast powder can is the very 
 thing.) In preparing this solution the following precautions 
 should be observed : Clean the bottle which is to hold it, in 
 the most perfect manner, rinsing frequently with distilled 
 water. Clean also in the same manner the outside of the 
 glass tube in which the osmic acid is found in commerce. 
 Weigh the distilled water or measure it carefully, pour it 
 
ELEMENTARY MICROSCOPICAL TECHNOLOGY. 21 
 
 into the prepared bottle, and then drop the tube of osmic 
 acid into it. Put in the stopper and shake the vessel sharply 
 until the glass tube is fractured and the water allowed to 
 reach the acid. Even when thus prepared the solution is 
 prone to decompose and must be made afresh quite frequently. 
 Specimens to be hardened in this agent should be cut -up 
 quite small a rule by the way, that holds good with all 
 other agents, and put into a vessel prepared as suggested 
 above for the exclusion of light. The period required for 
 hardening varies according to the object. 
 
SOFTENING AND DECALCIFICATION. . 
 IV. 
 
 . XII. HARDENING VEGETABLE TISSUES. The remarks on 
 hardening hitherto made are supposed to apply to tissues of ani- 
 mal origin only, but there are certain vegetable tissues which 
 are also too soft and friable to stand cutting while in their nat- 
 ural condition. In such cases where it is not desired to preserve 
 the shape and relative positions of the parts, the objects may be 
 hardened by simple drying. As this process almost invariably 
 shrivels and distorts a specimen it is better to resort to other 
 and less objectionable methods. 
 
 Here again, alcohol is the best medium with which we are 
 acquainted, and it may be used exactly in the manner described 
 for hardening objects of animal origin. Sometimes, however, the 
 specimens have colors which it is desirable to retain and which 
 would be extracted or changed by this hardening medium. When 
 such is the case the ordinary processes of hardening must be 
 avoided, and resort had to a device which answers the purpose 
 by mechanical methods. This process, which consists of satura- 
 ting the object with some inert material which melts at a low 
 heat but is solid at ordinary temperatures, will be described un- 
 der the head of embedding. 
 
 . XIII. SOFTENING. The process of softening tissues to fit 
 them for the section knife is a very simple one and is applied, as 
 a general thing, only to the stems and roots of plants, seeds with 
 a hard coat or shell, etc. The articles to be treated are placed in 
 rain or distilled water and left to macerate for several days. In 
 summer time the normal- temperature of the atmosphere will 
 be sufficient, but in cold weather the process is hastened by 
 placing the vessel containing the material close to the fire or 
 in some warm place. If the object be very dense (a bit of co- 
 coanut shell, or vegetable ivory, for instance) a gentle artifi- 
 cial heat may be applied. 
 
ELEMENTAMYMICROSCOPICAL TECHNOLOGY. 23 
 
 After macerating from 24 to 48 hours, the objects, if not suf- 
 ficiently soft, should be transferred to dilute alcohol (50) or 
 methylated spirit of the same strength, where they should be left 
 for a similar length of time. If on trial they are still found to 
 be too hard to cut, the process should be repeated from the begin- 
 ning. There are very few vegetable substances which will not 
 yeild to this treatment. 
 
 Roots or stems containing gummy or resinous matter insolu- 
 ble in water or dilute alcohol, should be soaked in fluids in 
 which their resins are soluble. Benzin is a good general solvent 
 for gums and resins, and is much cheaper than benzol, chloroform, 
 ether and other fluids usually recommended for this purpose. 
 Where benzin is inefficacious the other solvents should be tried. 
 
 The lower forms of vegetable life, the fungi and parasitic 
 plants and the tender shoots of the higher forms, require but little 
 preparation for the knife. 
 
 . XIV. DECALCIFICATION. Certain forms of organic mattei 
 of both animal and vegetable origin consist of soft tissues cover- 
 ed by, or permeated with a harder material of mineral or earthy 
 nature. The greater part of this hard material, in both animals 
 and plants, consists of the carbonate or phosphate of lime, or 
 both these salts, (though some plants carry a large amount of 
 silex). It is frequently desirable to rid such material of its 
 mineral constituents in order that the softer parts may be inti- 
 mately examined. When such is the case recourse is had to the 
 process known as decalcification. The agent used for this pur- 
 pose is a very dilute solution of one of the mineral acids, either 
 nitric or muriatic. In his work on the microscope the late Dr. 
 Carpenter gives the following directions for the employment 
 of these erodent materials : 
 
 "When the lime is in the state of a carbonate, as in the skel- 
 eton of the ecchinoderms, the body to be decalcified should be 
 placed in a glass jar or wide mouthed bottle holding from 4 to 5 
 ounces of water, and the acid added gradually, drop by drop, 
 until the disengagement.of bubbles shows that it is taking effect. 
 The solvent process should be allowed to take place very grad- 
 ually, more acid being added as required. When, on the other 
 hand, much of the lime is in the shape of phosphate, as in bones 
 and teeth, the strength of the acid solvent must be increased. 
 For the hardening of the softer parts of the organic matrix 
 
24 ELEMENTARY MICROSCOPICAL TECHNOLOGY. 
 
 ^after being freed from lime) chromic acid should be used. In the 
 case of small bones, such as the cochlea of the ear, a one-half of 
 one per cent solution of chromic acid will not only dissolve the 
 lime but at the same time harden the matrix. Larger masses of 
 bones require the addition of nitric or hydrochloric acid to the 
 chromic, the proportion being 2 per cent nitric or 5 per cent of 
 hydrochloric. By some investigators the acids are mixed at the 
 beginning of the process, but others recommend that the bone 
 should lie in the chromic acid solution for a week or ten days be- 
 fore the second acid is added. If the softening be not complete 
 in a month, more acid must be added. When thoroughly decalci- 
 fied the matrix must be transferred to alcohol and kept in it un- 
 til needed for section cutting." 
 
 . XV. Much discretion must necessarily be used in the se- 
 lection of the proper agents and methods to be employed in 
 preparing material for the knife as indeed in every other 
 department of microscopical technology, and the student with- 
 out a teacher will frequently be at a loss how to proceed. He 
 must rely upon himself, and must expect to make many failures 
 and mistakes; but by following the ways and means suggested 
 by us, he will eventually succeed, and will be all the better post- 
 ed by the experience gained in overcoming difficulties. Exper- 
 ience, after all, is the great teacher, and the knowledge that is to 
 guide one in doubtful cases is rarely to be obtained from text- 
 books and manuals, no matter how elaborate, practical and com- 
 plete they be; They can only point the way, but individual ex- 
 periment alone can make the successful worker. 
 
EMBEDDING PROCESSES. 
 V 
 
 XVI. EMBEDDING. Our material having passed through 
 the various operations of hardening, softening, or decalcification, 
 as the ease may be, its next step in its progress toward a com- 
 pleted slide, is its preparation for the section cutter or microtome, 
 as the instrument in which it is sliced into thin sections is tech- 
 nically called. 
 
 The various forms of microtomes will be described in the 
 ensuing chapter, with illustrations showing the best patterns 
 for histological work. At present it is sufficient to say that 
 in its simplest form the section cutter consists of a receptacle 
 for holding the material to be cut, a screw or other apparatus for 
 feeding it to the knife, and a razor or knife with a very keen 
 edge. Formerly the razor alone was employed for this purpose, 
 the material being held in the left hand and the cutting instrument 
 used free handed. This method is still adhered to by some Eu- 
 ropean microscopists, but as it bears the same relation, in regard 
 to exact work, as the photograph does to free sketching, or the 
 old fashioned jtwo-foot rule does to a micrometer scale, it has no 
 place in modern exact science. 
 
 Whatever form of microtome be used, some method must be 
 devised for holding the material to be cut, so that it shall be pro- 
 perly supported on all sides and that it shall have no motion, 
 lateral or vertical', save that given to it by the feeding device. 
 Where the object is dense and the sections not required to b'e of 
 any great degree of thinness, this may be secured by wedging 
 it into the. section cutter by means of some substance itself 
 easily cut without damage to the knife. Formerly, this plan 
 was much resorted to, carrots, potatoes, alder-pith and such like 
 material being used for the wedge. 
 
 This method is still available for many operations and it is 
 well for the student to bear it in mind; but for all purposes of ex- 
 
26 ELEMENTARY MICROSCOPICAL TECHNOLO G Y. 
 
 act histological, pathological or biological research of to-day, the 
 method of embedding is by means of a material which is solid 
 under ordinary circumstances but which may be rendered fluid by 
 the application of a low degree of heat or by dissolving it in 
 some rapidly evaporating medium, such as ether, benzol, chloro- 
 form, etc. The object is immersed in the fluid and the latter is 
 allowed to harden around it, thus fitting itself to the minutest 
 portion of its exposed surface, and holding it firmly within its 
 embrace. 
 
 XVII. The choice of an embedding medium depends upon 
 three things, viz; 
 
 1. The nature of the object to be cut. 
 
 2. The temperature at which the cutting is accomplished. 
 
 3. The form of microtome used. 
 
 It is plain, for instance, that a very hard substance, giving 
 considerable resistance to the knife, should not be embedded in 
 a very yielding material; or that an embedding material which 
 is sufficiently soft and tenacious at 60 F. might be too hard 
 and brittle for a freezing microtome, or too soft and fluid to 
 be used when the thermometer ranges in the summer heats; 
 or, again, that an embedding material which can be poured into 
 the well of an ordinary microtome (of the 'army medical' pat- 
 tern for instance), would not do for fixing the object to the cork 
 holder of a machine microtome of the Thoma or Schanze pat- 
 tern (see illustrations in next number). 
 
 For these reasons microscopists have devised quite a number 
 of ingenious embedding mixtures suitable to every class of work, 
 the best of which only need be described here. In this, as in 
 every other department of an elementary work, the multiplica- 
 tion of formulae serves merely to confound the student with an 
 embarras de richesse. 
 
 XVIII. For the general run of histological or patholog- 
 ical work undertaken by physicians, and for use in the form of 
 section cutter most commonly employed, there is no embedding 
 material with which I am acquainted that is any better than 
 PARAFFIN used either in its natural state, or mingled with other 
 fatty or oleaginous matter. By the judicious selection of the 
 various grades of paraffin, or mingling any one grade of it, as a 
 basis, with solid fats or oils, as the case may be, an embedding 
 
ELEMENTARY MICROSCOPICAL TECHNOLOGY. 27 
 
 mixture may be made which will suit almost any required emer- 
 gency. 
 
 XIX. The materials most generally used as adjuncts or 
 corrigerents of paraffin, are cacao butter, lard, wax, spermaceti, 
 vaselin, tallow, suet, and olive or refined cotton-seed oil. For 
 ordinary work, in a room where the temperature averages 65 or 
 70 F. during the winter months, I have found a mixture of 
 equal parts of paraffin (hard), cacao butter and mutton suet, to 
 work very well, especially if a few drops of sweet oil be added 
 to the stock occasionally, after frequent meltings and remeltings. 
 Prof. Stowell, of the University of Michigan, in his "Manual of 
 Histology" (an excellent text-book, by the way, for those who de- 
 sire to pursue this line of microscopical study) gives the follow- 
 ing formulae for the embedding mixtures used in his laboratory: 
 
 Soft. Solid paraffin, three parts; cacao butter, one part; lard, 
 three parts. 
 
 Hard. Solid paraffin, three parts; cacao butter, two parts; 
 spermaceti, one part. 
 
 Harder. Solid paraffin, two parts; cacao butter and sperma- 
 ceti, of each, one part. 
 
 For a very soft and transparent medium, easy to cut, he re- 
 commends a mixture consisting of two parts of paraffin, and one 
 part of vaselin. 
 
 Among other solids which may take the place of paraffin, 
 none is better than a vegetable wax, known in commerce as bay- 
 berry tallow (or myrtle wax, as it is called in the Southern States, 
 where it grows in great plenty). It may be used alone or in 
 mixtures, as in the case of paraffin. It is brittle at low temper- 
 atures and hence had best be cut with a little olive oil, if the 
 weather be very cold. It is insoluble in cold alcohol but readily 
 soluble in that medium at a temperature of from 110 to 130 F. 
 This latter quality makes it quite valuable as an embedding ma- 
 terial for certain kinds of work, of which, however, more will be 
 said when special technology is taken up. 
 
 Albumin, from hen's eggs, is also a valuble embedding medi- 
 um. It is best when used, as suggested by Runge, in combina- 
 tion with tallow, according to the following formula; 
 
 Take fresh white-of-egg and, after removing the chalazse, add 
 to each one hundred parts of albumin, fifteen parts of the offici- 
 nal liquor sodii, and shake them together in a large test tube. 
 
28 ELEMENTARY MICROSCOPICAL TECHNOLOGY. 
 
 When well mixed add forty parts of melted beef tallow and 
 again agitate until a homogeneous mass is obtained. One ad- 
 vantage of this mixture is that it is unaffected by alcohol, and an 
 object once embedded in it, the entire mass may be removed 
 from the section cutter and placed in that fluid for indefinite 
 keeping. 
 
 In the appendix or supplementary chapters of this series, 
 other embedding materials will be described in detail. At pre- 
 sent we must content ourselves with those given above, which 
 the student using the ordinary microtomes will find amply suf- 
 ficient for all practical purposes. 
 
 XX. PREPARATION OF EMBEDDING MIXTURES. The ingre- 
 dients should be melted together in a porcelain or glazed earth- 
 en-ware vessel, provided with a cover fitting sufficiently closely 
 to prevent the ingress of dust and foreign matter. The melting 
 should be done in a water-bath, as otherwise there is danger of 
 burning or scorching the fats. If heated directly over a lamp 
 scorching may be avoided, however, by watchfulness and con- 
 stant stirring. Enough of the material to last for many months 
 maybe made up at one time, and when required for use a portion 
 sufficient for the work in hand may be withdrawn and re 
 melted. The surplus material removed from the microtome may 
 be used over and over again, if the precaution is taken of strain- 
 ing it occasionally. Even this precaution may be avoided if the 
 remelting is done with a gentle heat and the vessel containing 
 the liquid material be allowed to stand in hot water until all for- 
 eign matter settles to the bottom. 
 
 XXI. METHODS OF USING. The preparatory treatment of 
 material to be embedded varies according to its nature and struct- 
 ure matter which is sufficiently dense throughout to need no 
 internal support (a bit of liver or of muscle, for instance) requir- 
 ing a different treatment from one which is areolated or spongy 
 (as a bit of lung tissue). 
 
 1. In the first instance the material is removed from the pre- 
 serving or hardening fluid, the adherent surplus of which is al- 
 lowed to drain back into the bottle, and it is then placed on a 
 piece of heavy bibulous paper or of porous tile (a bit of a bro- 
 ken flower pot is excellent for this purpose)and allowed to remain 
 there until the surface is quite free from moisture. This exter- 
 
ELEMENTARY MICROSCOPICAL TECHNOLOGY. 29 
 
 nal dessication may be hastened by frequently turning the mat- 
 erial, or by the application of soft bibulous paper or cloth, which 
 application should be made with as little pressure as possible, in 
 order to avoid distortion of the relations of the histological or 
 anatomical elements. Care must also be taken not to allow the 
 drying process to proceed too far, as otherwise the material may 
 become distorted by shriveling. When the surface is sufficiently 
 dry the object is taken fti the point of a dissecting needle and 
 dipped into a weak solution of gumarabic or gelatin, withdrawn 
 and again allowed to dry. This process should be repeated a 
 second or even a third time, the object being to give a coating of 
 a substance easily gotten rid of by washing in water, and which 
 will prevent the adherence of the embedding material to the 
 edges of the sections. 
 
 I wish to impress upon my readers the importance of this 
 preliminary, though it will require but a single trial to convince 
 one of its utility as a time saver. When it is neglected the sec- 
 tions must frequently in fact, almost invariably, be placed in 
 some fluid solvent of the embedding material, which itself is often 
 very hard to get rid of, and which sometimes injures delicate 
 structures submitted to its action. 
 
 While the coating of gum or gelatin is drying the embedding 
 material may be put into the water-bath and melted. As soon 
 as it becomes entirely fluid the source of heat should be with- 
 drawn, as it is not desirable to heat it much beyond the melting 
 point. The object still remaining on the point of the needle, 
 is dipped into the melted material two or three times, the coat- 
 ing recived each time being allowed to harden before a second 
 is given. This ensures its complete envelopment at all points, 
 with the embedding matter. The well of the microtome, being 
 clean and freed from dust, is now filled with the fluid embed- 
 ding material, the object is lowered into it and arranged in the 
 desired position and held there, still on the point of the needle, 
 until the material sets around it, when the needle should be 
 withdrawn. The well should be filled quite full and the surplus 
 displaced by the object allowed to run over on the surface of the 
 microtome. The material, shrinking in cooling, would otherwise 
 leave a depression in the center, thus failing to support a part 
 of the object. For the same reason it is well to warm the mic- 
 rotome somewhat before using it, as when the process of cooling 
 is too rapid the material shrinks away from the side of the well, 
 
30 ELEMENTAEY MICROSCOPICAL TECHNOLOGY. 
 
 thus occasioning lateral motion in the whole mass and prevent- 
 ing very accurate section cutting. The method of avoiding the 
 results of such an accident will be further treated of in the suc- 
 ceeding chapter. 
 
 XXII. EMBEDDING AREOLATED OB SPONGY TISSUE When 
 it becomes necessary to cut material which is full of minute 
 cavities, like a bit of sponge, lung, or^Dther areolated tissue, 
 the process of embedding must be modified somewhat ; since 
 not only the external surface, but much of the internal struc- 
 ture, needs support. In such cases we proceed as follows : 
 The object is removed from the hardening or preserving 
 fluid, freed of its excess and dried exactly as in the foregoing 
 cases. If it be dipped in the gum-arabic solution, all excess 
 of this material must also be squeezed out most carefully. 
 In very delicate tissues which will not stand much manipula- 
 tion it is, therefore, best to dispense with the preliminary dip- 
 ping in gum-arabic water, and proceed to place the object 
 directly into some fluid embedding material which will enter 
 into the areoles and there harden. This embedding material 
 may either be identical with that which it is proposed to use 
 for the external support, or it may essentially differ from it. 
 In most cases the same material will answer for both external 
 and internal support ; but sometimes, from the nature of the 
 internal structure, this is not advisable. A most excellent 
 material for filling the areoles of delicate tissues is gelatin 
 melted in a very small amount of water, and with the addi- 
 tion of a few drops of glycerin to each ounce of the fluid. 
 Gelatin, when used for this purpose, should be put into cold 
 water and allowed to swell for ten or twelve hours at a tem- 
 perature of not over 75 F. The excess of water is then 
 carefully drained off and the vessel containing the gelatin 
 submerged in the water-bath until melted. While still hot 
 add 15 to 20 minims of glycerin to each ounce of the liquid, 
 stir until homogeneous and remove from the water-bath. 
 When the solution has cooled to a point at which it will no 
 longer injure the delicate tissues, but is still fluid, the object 
 may be immersed in it and left there until the solution jel- 
 lies. A knife or spoon is then passed around it and it is lifted 
 from the surrounding gelatin, the excess of which, adherent 
 to the outside, must be carefully removed before placing it 
 
ELEMENTARY MICROSCOPICAL TECHNOLOGY. 31 
 
 into an oleaginous embedding mixture. The latter must be 
 of such composition that it will remain fluid at a compara- 
 tively low temperature, as otherwise the gelatin filling the 
 areoles will again become fluid and some of it escape. 
 
 For the great majority of areolated tissues it will suffice 
 to place them into the melted embedding material, after it has 
 somewhat cooled off but before it begins to solidify, and 
 leave them there until they become saturated. Where 
 even this amount of heat will be hurtful the filling must 
 be effected by immersion in a saturated solution of par- 
 affin in oil of turpentine. Where resort is had to this device 
 the material should be placed first in absolute alcohol, and 
 thence transferred to oil of turpentine, before its final immer- 
 sion in the paraffin solution. More will be said on this sub- 
 ject when special technology is taken up. 
 
 XXIII. EMBEDDING FOR MACHINE MICROTOMES. In this 
 class of microtomes (See plate I, Fig. 3) the object to be cut is 
 fastened to a holder, usually of cork, the embedding material 
 having been previously poured around it or applied after- 
 ward, layer by layer. Formerly the paraffin and other mix- 
 tures already given, were used for this purpose, and, indeed, 
 are still adhered toby a great many investigators, the object, 
 after preliminary preparation, being placed in a small box 
 made of writing paper and the embedding material poured 
 around it. Very small objects are easily embedded by laying 
 them on a piece of solid paraffin (cold) in which a cavity is 
 made by touching it with a bit of heated wire. The object 
 is dropped into the cavity and the liquefied paraffin hardens 
 around it almost instantly, leaving it ready for the knife in a 
 very few minutes. 
 
 Within the past two years a new embedding material called 
 celloidin has come into favor with users of the machine mi- 
 crotomes, especially with those who cut serial sections (that 
 is, sections each of which bears a definite relation to the one 
 preceding and succeeding it). This material, which is a pat- 
 ented article, made in Germany, is simply pure pyroxylin or 
 gun cotton. It is found in commerce in the shape of shreds 
 which resemble very closely those of gelatin or isinglass, it 
 is soluble in equal parts of sulphuric ether and alcohol (97 C ) 
 and when used for embedding, the solution should be saturated, 
 
32 ELEMENTABY MICROSCOPICAL TECHNOLOGY. 
 
 to make which requires from 24 to 30 hours digestion, with 
 frequent agitation. The object to be embedded should in, the 
 meantime be put to soak in a similar mixture of ether and 
 alcohol and left until thoroughly saturated; or it may be soaked 
 (as advised by Prof. Libby, in a paper read before Sect. G of the 
 American Association, 1884), first in absolute alcohol for one 
 or two hours ; next in strong ether for six hours ; and lastly, 
 in a weak solution of celloidin in alcohol and ether for an equal 
 length of time. When ready to embed, a good cork, which has 
 previously been soaked in absolute alcohol, is chosen and a 
 small quantity of the saturated solution of celloidin is placed 
 on the end of it, and allowed to dry until it is well stiffened. 
 Then the specimen, with as much of the embedding solution 
 as will adhere to it, is placed on this and allowed to dry par- 
 tially, when another coating is placed over it. The proceed- 
 ing should be repeated until the specimen has a sufficient 
 quantity of the embedding material around it to hold it firmly. 
 It is then allowed to dry, and when stiff is placed in alcohol 
 of 80 Q for at least 12 hours before cutting. (Prof. W. Libby 
 in the paper referred to.) 
 
 In the laboratory of the N. Y. College of Physicians and 
 Surgeons the above method is sometimes used, but more gen- 
 erally the proceedure is as follows : " The specimens are em- 
 bedded in paper boxes, in the usual way; or a cork is wrapped 
 with one or two layers of thick writing paper, allowing it to 
 project an inch or an inch and a half above the surface of the 
 cork, thus making a round box of paper with the cork for a 
 bottom. Into this box pour a small quantity of the embed- 
 ding fluid and let it dry. The specimen, having been previ- 
 ously soaked, as directed above, is now placed in the box, 
 adjusted to position and allowed to dry for 10 minutes, when 
 the balance of the box is filled with the embedding fluid. 
 The box is exposed to the air until the entire mass has be- 
 come semi-solid and then immersed in weak alcohol (alcohol 
 1 part, water 2 parts) where it is left for 24 hours, when it is 
 ready for cutting." 
 
 The method of staining and clearing sections of material 
 embedded in this way will be explained under the proper 
 heading. 
 
ELEMENTARY MICROSCOPICAL TECHNOLOGY. 
 
 33 
 
 XXIV. THE SECTION CUTTER OR MICROTOME. As ex- 
 plained before, the earlier investigators contented themselves 
 with a simple razor or sharp knife and cut their sections free 
 handed. The first attempt at improvement on this method 
 was Valentine's knife, an instrument consisting of two very 
 thin, sharp blades, placed parallel to each other and so ar- 
 ranged that the distance between them can be regulated by 
 a set-screw. This knife is of no service whatever in exact 
 work ; and as to the old free-hand method, while one who is 
 skillful may occasionally, or even quite frequently, make sec- 
 tions of small surfaces of great thinness and evenness, it is 
 not to be relied upon in those operations where every section 
 ]ias a definite value (as in serial sections.) 
 
 Fig. 1. Army Medical Museum Microtome. 
 
 XXV. Fig 1 represents the form of microtome most 
 commonly used in this country, and known as the Army Med- 
 ical Museum pattern. It consists of a bed-plate of brass 
 into one end of which is set a round plate of thick glass per- 
 forated in the centre with a hole one inch in diameter. To 
 the under side of the plate is screwed a brass cylinder hav- 
 ing an inside diameter exactly the same as that of the hole in 
 the glass surface plate. This cylinder, which is technically 
 
34 ELEMENTARY MICROSCOPICAL TECHNOLOGY. 
 
 termed 4C the well," is provided with a closely fitting piston 
 and follower, worked by a screw with a milled head, which 
 constitutes the feed apparatus. The object embedded in the 
 well, as described in Chapter V, is moved upward to the 
 knife by a turn of the feed screw, the threads of which are 
 usually 64 to the inch ; consequently one full turn would give 
 a vertical movement of -g^" , a half turn y|y, a tenth of a turn 
 5^75-", etc. The apparatus is provided with a clamping ar- 
 rangement for attaching it firmly to the work table. When 
 desired this pattern is also furnished with a pan which fits 
 closely under the bed-plate and around the feed cylinder, and 
 which can be filled with a freezing mixture, so that it can be 
 used as a freezing microtome. With a little practice very thin 
 and even sections of small objects can be made with this form 
 of microtome. 
 
 A modification of this form of instrument is made by W. 
 H. Bullock, of Chicago, The general principles are the same 
 in both microtomes, though I think that Mr. Bullock's device 
 has the merit of greater convenience and that the feed-screw 
 in his pattern, being supported in the base of the instrument, 
 works more evenly and surely than in the older Army Medi- 
 cal Museum instrument. 
 
 For cuttirg hard substances the same general forms of 
 microtome are used, the only difference being in the fact that 
 the well is provided with a clamp and set-screw, working 
 from the side, the function of which is to hold the object to 
 be cut more firmly than it could be held by embedding mix- 
 tures. 
 
 A very clever device for use with the Army Medical 
 Microtome and instruments of that description, is shown in 
 Fig. 2. It is the invention of Dr. Seiler of Philadelphia, and 
 is known as his knife carrier. Its method of construction and 
 use is so well shown in the engraving and is so obvious that it 
 needs no description. The knife may be made with a mova- 
 ble handle as shown in the cut, or the arms may be made to 
 carry an ordinarj 7 " section knife. 
 
 XXVI. MACHINE MICROTOMES. Although the idea of an 
 instrument in which the cutting apparatus forms an integral 
 part, is not a new one microtomes of this description having 
 been made and used a half century ago, it is only within the 
 
ELEMENTARY MICBOSCOPICAL TECHNOLOGY. 
 
 35 
 
 last five or six years that any great attention has been paid to 
 them, either in Europe or America. The very rapid strides 
 made in other departments of biological research, and es- 
 pecially in the improvement of microscopic objectives, ren- 
 dered necessary the perfection of instruments by which ex- 
 ceedingly thin and uniform sections could be prepared with 
 certainty and rapidity. The demand was met by the appear- 
 ance in Europe of the microtomes of Ranvier, Schanze, Thoma 
 
 Fig. 2. Seller's Microtome Attachment (from W. H. Walmsley 
 & Co., Philadelphia.) 
 
 and others, several of which were immediately imported into 
 this country by institutes of learning and private individuals. 
 It was not long, however, before American workmanship 
 evolved from the best features of the imported articles, an 
 instrument surpassing them in solidity, lightness, beauty of 
 finish and accuracy of workmanship. Prominent among 
 those who tackled the problem in this country was Mr. W. 
 H. Bullock, of Chicago, W. H. Walmsley, of Philadelphia, 
 and the Bausch & Lomb Optical Company, of Rochester, 
 N. Y. The cutting and feeding parts of all are upon the same 
 general principles and Mr. Bullock has added to his instru- 
 ment a device for removing the cut sections in series upon 
 an endless band, which is a very great convenience to stu- 
 dents of biology and histology. 
 
 The plate on the next page represents the Bausch & 
 Lomb large laboratory microtome. The engraving shows the 
 parts of the instrument so clearly that a description of it is 
 
36 ELEMENTARY MICROSCOPICAL TECHNOLOGY. 
 
ELEMENTAEY MICROSCOPICAL TECHNOLOGY. 37 
 
 scarcely necessary. The base, arm and bed of the knife and 
 object holder are made of a single casting, which insures the 
 maximum of strength and rigidity with the minimum of 
 weight. The knife is fitted with a curved and slotted handle 
 which enables the operator to use any part of the blade, and 
 within the block which carries it is a spring which so regu- 
 lates the resistance that it passes through objects of varying 
 hardness without deviating from its plane or requiring a 
 change of pressure on the part of the operator. The object 
 holder is attached to a carriage which is adjustable, so that any 
 degree of obliquity may be given to the material to be cut. 
 The feed-screw is provided with an exceedingly delicate and 
 accurate rnicrometric arrangement which insures absolute ac- 
 curacy in gauging the thickness of the sections. The same 
 firm makes a smaller instrument which is a simple modifica- 
 tion of the one here pictured, for the use of students. 
 
 Fig. 4. Walmsley's Microtome. 
 
 Fig. 4. represents a modification of Rivet's Microtome, 
 manufactured by Mr. W. H. Walmsley, of Philadelphia, 
 The principle upon which the feeding device is built, as 
 shown in the engraving, is that of the inclined plane ; the 
 screw giving great delicacy and accuracy to the movement of 
 the object carrier, thus enabling the operator to secure sec- 
 tions of great and uniform thinness. This microtome is 
 exceedingly simple in construction and very serviceable. 
 
38 ELEMENTARY MICROSCOPICAL TECHNOLOGY. 
 
 XXVII. THE SECTION KNIFE. Whatever form of microtome 
 be used, the knife is the most important mechanical factor in suc- 
 cessful section cutting; and the more accurate the feeding devices 
 and other accessories, the greater the necessity for a perfect cut- 
 ting edge. In the machine microtome the knife is essentially a 
 part of the instrument, and hence need not be considered here, 
 save to remark that the blades are usually much larger and 
 heavier than those used with the hand cutting-instruments. As 
 in other matters of detail, there has been much difference of opin- 
 ion among experts as to the best form of blade, both as to shape 
 and size. Some have recommended a short blade with a bellying 
 edge, like a razor; while others, myself among the number, think 
 that a longer blade is preferable, with an edge perfectly straight 
 from point to heel. Whatever shape be adopted, however, all 
 agree that one side of the blade (that which is to lie next to the 
 surface of the microtome) should be ground perfectly flat, while 
 the other should be hollow-ground, as shown in Fig. 7, which is 
 a section through the blade of the section knife used by myself. 
 
 Fig. 5. Section through blade. 
 
 The blade should be from 5" to 7" in length, from " to !-" 
 in width, and from -f^" to i*-"in thickness at the back. The mat- 
 erial should be the very best razor steel, tempered to a hardness 
 but little, if any, short of that of the razor. Not one in a dozen 
 of the blades ordinarily in use are properly and homogeneously 
 tempered; and since, as remarked, the beauty and thinness of the 
 sections depend largely upon the evenness and fineness of the 
 cutting edge, this is a most important point to be considered in 
 the selection of a knife. It is almost needless to add that the 
 blade should be kept as keen as hone and strap can make it; and 
 hence a few words on the subject of sharpening will not be inap- 
 propriate here especially as the remarks apply to surgical cut- 
 ting instruments as well. 
 
 THE BEST HONES are the Turkish razor or barber hones, and 
 those made from the finer grades of the Hot Springs or Ouachita 
 stone. The former is the quickest cutter, but the latter makes 
 an edge that is improved but little by strapping. The stone 
 should be sufficiently wide and long to allow the blade to rest 
 entirely upon it. The surface should be kept as true as possib e 
 
ELEMENTARY MICROSCOPICAL TECHNOLOGY. 39 
 
 and in honing a section knife most of the attrition should be up- 
 on the hollow-ground side. The reason for this latter precaution 
 will be apparent to anyone on glancing at Fig. 5, and is, namely, 
 that there being much less bearing surface on the hollow side the 
 process of honing progresses much more rapidly and evenly than 
 it could upon the flat side. After getting the blade nearly to an 
 edge the flat side should be passed a few times over the hone, 
 holding it so that every point is in contact with the surface of 
 the stone. The best strap is a piece of soft French calf-skin, 
 thoroughly freed from dust and dirt and tightly stretched on a 
 smooth, flat piece of wood. A knife should never be put upon 
 a hone or a strap until both have been carefully wiped. A sin- 
 gle grain of the fine, sharp sand or dust with which the atmos- 
 phere is so frequently laden may, by nicking the delicate edge, 
 cost one an hour's hard honing. 
 
 Fig. 6. Section Knife. 
 
 THE HANDLE of the section knife should be large enough to 
 afford a good grip to the fingers and should be roughened after 
 the fashion of those of surgical instruments. The section knife 
 should never be used for any purpose other than for cutting sect- 
 ions, and it should never be put away after use until it has been 
 thoroughly cleaned, wiped. dry and passed once or twice lightly 
 over the strap. A soft rag or bit of chamois, charged with putz 
 pomade, or the finest jeweler's rouge, is most excellent for preser- 
 ving the polish, not only of knives but of other instruments and 
 apparatus.. 
 
 XXVIII. THE OTHER ACCESSORIES for section cutting, be- 
 side the knife and microtome, are as follows; 
 
 A small vessel of alcohol for wetting the knife blade; 
 
 A saucer for receiving the cutsections; 
 
 Two camel's hair pencils, rather large; one of these is for use 
 in the alcohol, and the other for removing the cut sections from 
 the blade; 
 
 A pair of delicate forceps; 
 
 Alcohol, glycerin and distilled water; 
 
 Wash bottle, cloths for wiping the blades, etc. 
 
40 ELEMENTAEYMICEOSCOPICAL TECHNOLOGY. 
 
 . XXIX. ARRANGEMENT OF THE WORK TABLE. The object 
 being embedded, and the microtome being placed in a convenient 
 position (if of the 'Army Medical' pattern it is clamped to the 
 edge of the table or board), we are at last ready to commence 
 section cutting. The little vessel of alcohol (an acetate of mor- 
 phine bottle answers admirably), with its camel's hair pencil, 
 should be placed in front and slightly to the left of the microtome. 
 The saucer for receiving the sections should be placed con- 
 veniently, to the front and right of the microtome. It should be 
 about half-filled with clean distilled water, water and alcohol 
 or even alcohol alone, as the case may be the fluid vary- 
 ing according to the nature of the material to be cut, and upon 
 the character of the subsequent mount. This is a discretion 
 which is to be gained only by experience; though it will be man- 
 ifest to anyone, for instance, that a section of a bit of material 
 injected with gelatin carmine, should not go into water or gly- 
 cerin. The camel's hair pencil for removing the sections from the 
 knife should lie in this saucer. 
 
 Near by and within convenient reach, should be a receptacle 
 for the surplus embedding material from time to time removed 
 from the microtome. The instruments likely to be needed dur- 
 ing the course of the work in hand should also be placed within 
 easy reach a rule which applies to all kinds of technical work, 
 since time and annoyance are thus saved, and nothing is such a 
 foe to good, even work as constant interruption from any source 
 whatever. A peice of soft linen or cotton cloth, for wiping the 
 knife after each cut, completes the arrangement of the working 
 apparatus. 
 
 XXX. CUTTING. We are now ready for the first cut. 
 We turn up the milled head of the feed screw until the object to 
 be made into sections is brought to a point where it will engage 
 the edge of the blade passed along the surface of the microtome. 
 If much surplus embedding material has run over the surface we 
 remove it with a scalpel or pen-knife and give the glass plate a 
 thorough wiping to remove any adherent dust or grit. This is 
 an operation that must be repeated frequently during the pro- 
 gress of our work. The section knife is then siezed in the right 
 hand and, after being well flooded with alcohol, is placed, flat side 
 down, upon the surface of the microtome and with an even and 
 
ELEMENTARY MICROSCOPICAL TECHNOLOGY. 41 
 
 uniform motion carried entirely through the object and the sur- 
 rounding embedding mass. 
 
 Here is another point in section-cutting upon which experts 
 differ. Some prefer to make the cut from the person, outward; 
 while others claim to achieve better results by drawing the knife 
 toward the person. While this is entirely a matter of habit and 
 opinion, it has an importance from the fact that in ordering a 
 section knife it is necessary to state which method is pursued; 
 since the knife, which in cutting toward one would have the flat- 
 ground side of the blade next the microtome plate, would present 
 that side upward in cutting from the person. I prefer to cut 
 toward the body, though sometimes as in cutting very hard sub- 
 stances, the other way is the better, since it enables one to steady 
 the end of the blade with the left hand, which also aids in forc- 
 ing it through the resistant tissues. 
 
 After making the first incision, the object should be exam- 
 ined closely for the purpose of ascertaining whether the embed- 
 ding material has surrounded it perfectly at all parts. If, as fre- 
 quently happens, from imperfect dessication or other causes, air 
 bubbles should have formed around it, these cavities must be 
 filled up with freshly melted material. Sometimes also, as men- 
 tioned before (C. Y. 21), the embedding material shrinks so 
 much in cooling that an appreciable space is left between it and 
 the walls of the well of the microtome, thus giving rise to a 
 slight lateral motion in the entire mass. When this space is very 
 minute it may be remedied by leaving a portion of the embed- 
 ding material uncut by the section knife after traversing the ob- 
 ject, the projection afterward serving as a rest for the forefinger 
 of the left hand, which thus steadies the mass as the knife passes 
 through it. A better plan, however, is to pass a heated knife 
 blade into the embedding material, close enough to the sides to 
 cause sufficient of the melted material to flow into the crevice 
 and fill it up. Any vertical motion of the mass, other than that 
 given it by the feed screw, is prevented by a button attached to 
 the top of the piston. 
 
 Having assured ourselves of the absence of faults in the em- 
 bedding mass, or having remedied them, we next proceed with 
 a scalpel or pen-knife blade to remove, for a limited depth, all 
 embedding material from in front of the object, the presence of 
 which, while lending no support, serves as an obstacle to the 
 edge of the knife and must otherwise be traversed before the 
 
42 ELEMENTARY MICROSCOPICAL TECHNOLOGY. 
 
 latter can engage the object to be cut. This done we are pre- 
 pared to make the first section. 
 
 The blade is again drenched with alcohol and laid flat upon 
 the top of the microtome in such a manner that its heel will be 
 the first part to come in contact with the object. The milled 
 head of the feed screw is manipulated with the fingers of the 
 left hand and the object thus brought upwards through a dis- 
 tance equal to the thickness of the proposed section. The knife 
 is then brought toward the person (or shoved from it, as the 
 case may be), with an even and steady motion, drawing it at the 
 same time so that it traverses the object from the heel toward the 
 point of the blade. Care must be taken to keep the knife flat 
 upon the microtome. This is another most important point in 
 successful section cutting, the failure to preserve the contact at 
 all points being the most fruitful source of failure with begin- 
 ners in this part of the work. 
 
 When, at length, the knife has traversed the object and the 
 section is lying upon the blade, the latter is carried over to the 
 saucer and the point dipped into the fluid therein ; the camel's hair 
 pencil, laden with the liquid, is applied to the blade just above 
 the section and the latter is floated off into the receptacle designed 
 to receive it. The knife is wiped, again flooded with alcohol, 
 and the operation of cutting repeated until the object is cut up, 
 or until a sufficient number of sections are made. 
 
 The feed screws of many of the microtomes in general use 
 are cut 50 threads to the inch, so that one entire turn of the 
 milled head gives a section of -fa", one fourth of a turn -^" , 
 etc., thus enabling the operator to gauge the thickness ot his 
 sections very accurately. The beginner is prone to try to cut 
 his sections very thin and, as a consequence, makes many fail- 
 ures, sometimes spoiling a good deal of valuable material with- 
 out getting a single passable section. He should make his first 
 attempts on comparatively thick sections, and after he has 
 learned to hold his knife perfectly steady, gradually decrease 
 the thickness of his cuts. 
 
 XXXI. CARE OF INSTRUMENTS. When a sufficient num- 
 ber of sections have been obtained, the first care of the operator 
 should be to clean and put away the instruments that he has been 
 using. The knife should be dried and strapped, as described; 
 and the balance of the embedding material removed from the 
 
ELEMENTARY MICROSCOPICAL TECHNOLOGY. 43 
 
 microtome. If any portion of the object remain and it be 
 something which one desires to keep, the embedding material is 
 cut away from around it and the fragment returned to the 
 preserving fluid from which it was taken. When celloidin 01 
 albumin is used as embedders the entire mass may be dropped 
 back into alcohol. 
 
 The microtome should be thoroughly cleaned and dried be- 
 fore it is put away. Care should be taken, especially, to clean 
 the feed screw, as a very slight amount of erosion here may de- 
 stroy the exactness of fit upon which so much of the accuracy and 
 delicacy of section cutting depends. 
 
 XXXII. If from any cause it becomes necessary to sus- 
 pend the operation of section cutting before it is completed, the 
 object may be hermetically sealed into the embedding mass by 
 resorting to the method described for filling the space left by 
 shrinkage, viz.: by passing a heated knife blade into the mass and 
 smearing a portion of the melted material over the exposed surface 
 of the object. If this or some similar precaution be neglected, the 
 object soon dries up and is shriveled and distorted until it no 
 longer has, any histological or anatomical value. 
 
STAINING AND STAINS. 
 
 VII. 
 
 XXXIII. PRELIMINARY TREATMENT OF THE SECTIONS. 
 The sections are now ready to undergo the varied manipulations 
 through which they must pass prior to the final act of mounting, 
 viz.; staining, dehydration, freeing from still adherent embed- 
 ding material, etc. If the object which we have been cutting 
 was solid, the embedding material will give us little or no trouble, 
 as it will have separated from the section spontaneously, owing 
 to our having taken the precaution of dipping the object ill the 
 gum solution prior to embedding it. If, on the contrary, it was 
 porous, spongy or areolated, like a piece of lung tissue, and we 
 have been compelled to fill its cavities with embedding material, 
 the process is a more tedious one, involving two or more steps, 
 according to the nature of the material, the embedder and the 
 subsequent mount. The jirst is dehydration, or removal of the 
 water which has been absorbed by the sections. This is done 
 by removing the latter into alcohol of 95, if they have been 
 lying in water, and thence into absolute alcohol. If they were 
 deposited in dilute or commercial alcohol when they were cut, 
 they should be removed thence directly into absolute alcohol. The 
 proportion of the fluid should be large in comparison with the 
 mass of the cut sections, for reasons that are obvious. From the 
 alcohol they should be removed into one. of the essential oils 
 oil of turpentine, bergamot, etc., or into benzol, to dissolve out 
 the embedding material. If the subsequent mount is to remain 
 unstained and to be made in balsam, the section may be removed 
 from the solvent oil directly into a solution of balsam in benzol, 
 chloroform or turpentine, and there left until ready to be put on 
 the slip. If, however, the mounting medium is to be glycerin, 
 glycerin jelly, or any of the aqueous media, the steps must be 
 retraced exactly in reverse order to that in which they were 
 
ELEMENTARY MICROSCOPICAL TECHNOLOGY. 45 
 
 from alcohol to water or glycerin. But before any of the above 
 described steps be taken there is another process for which the 
 Motions are already prepared, as they lie in the saucer of water, 
 or alcohol, and which can be performed better at this than at any 
 subsequent stage of progress, viz., STAINING. 
 
 XXXIV. As suggested in one of the earlier chapters of 
 this work ( Chap. II. YI. ) the process of staining serves a 
 three-fold purpose in microscopical technology, viz.: 
 
 1. By giving a tint to very delicate, transparent membranes, 
 tissues, or parts of objects, it enables the eye to detect and fol- 
 low details which would be otherwise invisible. 
 
 2. By the selective properties of some stains, which readily 
 impart color to certain elements, tissues or substances and are 
 inert upon others; or by the comparative readiness with which 
 different tissues, or different elements of the same tissue, absorb 
 the same stain, but with varying degrees of intensity, staining 
 enables a differentiation of elements or parts thereof that would 
 otherwise have the same general appearance under the micros- 
 cope. Thus, osmic acid stains fat-cells black and protoplasmic 
 matter gray; picro- carmine imparts to certain tissues a yellow 
 or orange, and to others a reddish or carmine hue; applied to 
 epithelium, haematoxylon stains the nucleus deeper and quicker 
 than it does the balance of the cell. To differential staining, as 
 applied to bacteria and the lowlier forms of life, we owe some 
 of the latest and most important discoveries in biological and 
 pathological research. 
 
 3. In addition to these advantages in differentiation, cer- 
 tain stains facilitate the study of tissues by imparting cool and 
 grateful tints to them, thus enabling the eye to dwell upon them 
 for a long time without fatigue or injury to the organ. 
 
 Aside from the practical advantages enumerated, the beauty 
 and general appearance of mounts of most animal and vegeta- 
 
 , ble tissues are greatly enhanced by judicious staining. 
 'ar 
 
 XXXY. From what has been said it will be plain to the 
 student that the subject of stains and staining is a very impor- 
 tant one to the microscopist in whatever branch of the science 
 be may elect to pursue his investigations, and one which, in the 
 light of comparatively recent advances in the study of the germ 
 or parasitical origin of infectious diseases, daily acquires more 
 
46 ELEMENTARY MICROSCOPICAL TECHNOLOGY. 
 
 weight and significance. Of what far-reaching consequence, for 
 instance, was Koch's discovery that the bacilli of tubercle will 
 absorb certain of the aniline colors and cling to them with such 
 tenacity that not even nitric acid will bleach them ? The judi- 
 cious application of stains becomes, therefore, of paramount im- 
 portance to the worker in micro-botany, no less than to the bio- 
 logist, the histologist and pathologist. So great a field can be 
 but barely touched in the present work, where we must confine 
 ourselves to general principles, leaving for special treatises the 
 technology of bacterial and similar investigations.* 
 
 XXXYI. While the processes of staining animal and veg- 
 etable tissues for examination under the microscope are not a 
 " mystery past finding out ", confined to a few professionals in 
 France and Germany, as was thought in this country but a few 
 years ago; and while everything about them is rational and sim- 
 ple, the art is, nevertheless, one requiring considerable judge- 
 ment, patience and attention to detail. Exactly what stain should 
 be applied to a given tissue in order to bring out all its hidden de- 
 tails, how long to leave any object in contact with the stain, etc., 
 are points to be learned from experience and practice only, aid 
 ed by the experience of others in similar cases. 
 
 XXXVII. From the multitude of formulae met with in 
 text books on microscopy and the current literature of the day, 
 I have selected those which from my own experience, as a prac- 
 tical microscopist and teacher, I have found the most generally 
 useful and meritorious stains to place in the hands of the student 
 and practitioner. There are many others which in certain depart- 
 ments of the study are useful and even indispensable, which I will 
 not mention at this stage of our progress, reserving them for 
 chapters on special methods. In the arrangement of the stains 
 I have followed the old plan of grouping them into simple or 
 non-selective, and selective, though, as a matter of fact, there are 
 no stains but which may not under proper manipulation, be made 
 to do selective tinting. 
 
 * For the technology of the bacteria investigation I would recommend 
 the following text books, viz.: Methods of Bacteriological Investigation, 
 by F. Hueppe, X. Y., D. Appleton & Co., and The Technology of the 
 Bacteria Investigation, by Chas. S. Dolley, Boston, Cassino & Co. 
 
ELEMENTARY MICROSCOPICAL TECHNOLOGY. 47 
 
 XXXVIII. THE CARMINES. The most generally useful 
 of all staining materials at the command of the microscopist, 
 whether he be a worker in animal or vegetable histology, is 
 CARMINE. Its diffusibility, high staining power, brilliancy and 
 persistency, combine to make it a stain of almost universal 
 applicability. Among the best of the carmine fluids are the 
 following, viz.: 
 
 1. Ammonia Carmine or Beale's Carmine: Carmine (No. 40V 
 20 grains; stronger water of ammonia, 1 dram; alcohol 1 ounce; 
 glycerin and distilled water, of each, 4 ounces. Bub the carm- 
 ine into a paste with a small portion of the distilled water, being 
 careful to break down all lumps: put into a flask and add the 
 ammonia; agitate for a few moments and then bring to a boil 
 in the water bath, and continue to boil until solution is complete. 
 The flask must now be set aside to cool, leaving the mouth open 
 so that the residual ammonia may escape. Mix the glycerin 
 and the balance of the water and add to the carmine as soon as 
 the latter is cold; agitate, and finally, add the alcohol. Filter 
 and nick off into small bottles with well ground glass stoppers. 
 Properly prepared this solution will keep a n indefinite period 
 without thickening or precipitation. If, however, it should 
 show any signs of separation this process may be corrected by 
 the addition of a lew drops of ammonia. This stain may be 
 "used either full strength or diluted, as explained hereafter. 
 
 2. Borax Carmine. Put 60 grains of carmine in a porcelain 
 capsule; pour over it 2 drams of stronger water of ammonia and 
 triturate well until rubbed smooth. Set aside for 24 hours, or 
 until the free ammoniacal gas has escaped. Make a saturated 
 solution of borax in distilled water and of this add, very gradu- 
 ally and with constant rubbing, 4 ounces to the carmine in the 
 capsule. Pour into a bottle of sufficient size and agitate until 
 every particle of carmine is dissolved. Let stand for a few 
 hours, filter and rack off into small glass-stoppered vials. 
 
 3. Woodward's Carmine, or Violet Carmine. To one part 
 of borax carmine, made as in No. 2 above, add 4 parts of dis- 
 tilled water and 8 parts of alcohol of 95. A crystaline precipi- 
 tate will be thrown down, which may be separated on the filter 
 or by decantation ; wash in alcohol of 95 and redissolve in 
 distilled water ( 4 parts of water to 1 part of the carmine crys- 
 tals). This solution is of a bright lilac color, which is changed 
 
 by the fixing solution (see below) into roses or red. 
 
48 ELEMENTARY MICBO&COPICAL TECHNOLOGY. 
 
 4. Thiersch's Carmine. Carmine, 60 grains ; stronger am- 
 monia water, 1 dram ; distilled water, 3 drams: mix. In a sepa 
 rate vessel dissolve 24 grains of oxalic acid in 1 ounce of distil- 
 led water, and add to it 12 drams of alcohol of 95. Mix the 
 two solutions. This gives a dark red stain that is exceedingly 
 permanent, and which requires no fixation. 
 
 5. Czokofs Carmine. Dissolve in 6 ounces of distilled 
 water, 8 grains of burnt alum, and in the solution triturate thor 
 oughly 50 grains of cochineal. Boil for a half hour in the 
 water bath ; remove, filter and add distilled water sufficient to 
 bring the solution up to 6 ounces. This stain is a deep cherry 
 red and possesses many advantages as a staining agent, chief 
 of which is the fact that tissues which have been hardened in 
 chromic acid, or potassium bichromate solution, take it as well 
 as do those hardened in plain alcohol. 
 
 FIXING SOLUTIONS. All of the carmine stains, with the ex- 
 ception of Thiersch's, require to be l fixed ' or ' set ', and several 
 agents have been suggested for this purpose. Dr. Beale recom- 
 mends a 1 per cent, solution of acetic acid in alcohol or glycerin. 
 For fixing Woodward's carmine and borax carmine, a solution of 
 hydrochloric acid in alcohol ( acid 1 part, alcohol 7 parts ) is re- 
 commended. When this fixer is used it must be watched very 
 closely, as it bleaches rapidly and destroys very thin sections 
 in a short time. I am in the habit of using for all carmine 
 stains a fixer consisting of 24 grains of oxalic acid dissolved in 
 1 ounce of distilled water. It acts remarkably well with tho 
 stronger ammoniacal solutions. 
 
 The method of using the 'fixer' will be explained hereafter. 
 
 XXXIX. ELEMATOXYLON, OR LOG-WOOD STAINS. These 
 are also very useful to the student of histology, whether em- 
 ployed in plain or selective staining. Log-wood has the prop- 
 erty of staining the nuclei more deeply and rapidly than 
 the remainder of the cell material. It requires no fixing or set- 
 ting, but as usually made, it has the great disadvantage of not 
 being permanent, either as an applied stain or in solution. 
 
 1. Aqueous Solution of Log-wood. Dissolve 1 part of alum 
 in 8 parts of distilled water and add to the solution a sufficient 
 quantity of the aqueous extract of log-wood to give the requis- 
 ite tint, or a deep violet color. (Frey. ) [&*'* 
 
ELEMENTAEYMICROSCOPICAL TECHNOLOGY. 49 
 
 2. Another and Better Aqueous Solution is made by the fol- 
 lowing process : rub together in a mortar 1 part of the ordinary 
 extract of logwood and -3 parts of alum. When reduced to a very 
 fine powder and thoro ugly mixed add a small quantity of water 
 and continue the trituration, gradually adding more water, until 
 the solution, when examined by transmitted light assumes a 
 dark violet hue. The amount of water depends upon the fresh- 
 ness, etc., of the extract used. When the requisite color is 
 obtained, to each ounce of the solution add two drams of 
 alcohol of 75. 
 
 3. Alcoholic Log-wood Stain. While the aqueous extracts 
 of log-wood are prone to decompose, even when the ntmost care 
 has been expended in preparing them, the following alcoholic 
 solution will be found to improve with age : Make a saturated 
 solution of calcium chloride in alcohol of 70, and another of 
 alum in alcohol of the same strength. Mix the two solutions in 
 the proportion of 1 part of the former to 7 parts of the latter. 
 Make a saturated solution of extract of log-wood in absolute 
 alcohol and add it, drop by drop, to the mixture, until the latter 
 assumes a deep purple color. Filter and preserve in glass 
 stoppered vials. 
 
STAINING ANIMAL TISSUES. 
 
 XL. SELECTIVE STAINS. While the stains enumerated and 
 described in the foregoing chapter do not by any means embrace 
 all of the simple, or what are usually termed non- selective, tinting 
 solutions, those there given are sufficient for all practical pur- 
 poses. The student of elementary histology and the practitioner 
 will rarely or never need any others. We may, however, men- 
 tion that all, or nearly all, of the aniline colors may be used for 
 this purpose ; but as they are usually employed in double or mul- 
 tiple staining we have relegated them to a place under this heading. 
 
 Of the selective stains, properly so called, none is so generally 
 useful to workers in every branch of microscopy as picro-carmi- 
 nate of ammonium or as it is usually termed, picro-carmine, which 
 is prepared as follows : 
 
 1. Picro-carmine crystals : With the aid of heat dissolve in 
 the smallest possible amount of the stronger water of ammonia, 
 lf> grains of best carmine (No. 40) and to the solution add suf- 
 ficient distilled water to make one fluid ounce. In another vessel 
 dissolve 75 grains of picric acid in boiling water sufficient to 
 make, when cold, a saturated solution. When cool mix the two 
 solutions, put into a bottle with a well ground glass stopper, agitate 
 vigorously and set aside to stand (with an occasional shaking) for 
 several days. At the expiration of four or five* days all of the 
 uncombined picric acid will have subsided to the bottom, and the 
 clear supernatant fluid may be decanted or filtered off. The 
 filtrate should now be poured into shallow flat dishes, loosely 
 covered to keep out the dust, etc., set aside in a moderately 
 warm place, and allowed to evaporate spontaneous!}' . The pro- 
 cess may be hastened by the very careful application of a low 
 heat, on the water bath, but any attempt at rapid evaporation 
 will injure the brilliancy of the product. The result of evapora- 
 
ELEMENTARY MICROSCOPICAL TECHNOLOGY. 51 
 
 tion will be a crop of minute, brick-dust red crystals, 10 grains of 
 which, dissolved in an ounce of distilled water, makes the picro- 
 carmine solution, so useful to students of microscopy. Since 
 more or less dust must get into the solution in process of prepa- 
 ration, it should always be filtered before final bottling, and the 
 filtrate should be kept in well ground glass stoppered vials. 
 
 An al oholic pier o- car mine solution may be made extemporan- 
 eously, as follows: dissolve 2 grains of carmine in 30 minims of 
 stronger ammonia water and add sufficient distilled water to make 
 one ounce. Dissolve 8 grains of picric acid in 1 ounce of alcohol 
 and mix the two solutions. After filtration the stain is read}" 
 for use. [TVie Microscope, September, 1884. 
 
 2. Indigo Carmine, or sulph-indigotate of sodium solution. 
 Take of the best indigo, in lump, 30 grains, powder in a large 
 capsule and dry thoroughly in a water bath. When dry add drop 
 by drop, 2 drams of fuming sulphuric acid (the Nordhausen 
 acid) stirring constantly with a glass rod. The indigo swells en- 
 ormously under this treatment, and hence the necessity for a large 
 capsule. The whole of the acid being added, cover closely and 
 let stand for 24 hours. At the end of this time add o ounces of 
 distilled water, transfer the mixture to a tall flask, and let stand 
 for 4 days, giving the flask an occasional shake in the interim. 
 A magnificent blue color is now attained, but its acidity is so 
 great that it can not be used in this shape, and therefore a strong- 
 solution of sodium carbonate must be added. This addition 
 must be made very cautiously, a little at a time and with frequent 
 testing, as an excess of the alkali causes the indigo to separate 
 in a doughy mass. Filter the neutralized solution and evapo- 
 rate to dry ness in a water bath, with a low heat. The resultant 
 powder is sulph-indigotate of sodium. To make the staining 
 solution, dissolve 1 part of the powder in 50 parts of distilled 
 water. 
 
 3. Chloride of Gold. Dissolve 10 grains of gold chloride in 
 1 ounce of distilled water. Keep in a glass-stoppered, dark bot- 
 tle, covered with tin foil to keep out the light. 
 
 4. Nitrate of Silver. This solution should be made of the 
 strength of 5 grains to the ounce of distilled water. If both in- 
 gredients be chemically pure the light has no effect on the solu- 
 tion. It should be kept in glass-stoppered bottles. 
 
 5. Osmic Acid. This chemical comes in little sealed glass 
 tubes, each containing 1 gram (15>s grains). It possesses a very 
 
52 ELEMENTARY MICROSCOPICAL TECHNOLOGY. 
 
 disagreeable and powerfully irritant odor, and is at the same time 
 exceedingly sensitive, when in solution, to the chemical action of 
 light. Great care must therefore be exercised in preparing this 
 reagent. To prepare a one-per-cent solution choose a 4 ounce 
 vial of dark glass, with a well-fitting, ground-glass stopper. 
 Make this thoroughly and chemically clean, by washing it first 
 with hot liquor potassae and rinsing with warm water. Wash 
 a second time with nitro-muriatic acid and rinse, first w th 
 warm water, then with alcohol, and lastly with distilled water. 
 The stopper, which should also be similarly cleansed, is then put 
 in and the bottle closely enveloped in heavy tin or lead foil, 
 which should extend up to the very rim of the mouth, and under 
 the bottom. The sealed glass tube containing the acid should 
 next be made chemically clean externally, by a similar process. 
 When this is done, weigh into the bottle 1543 grains of freshly 
 distilled, chemically pure, water ; sieze the cleaned tube with 
 a pair of clean wooden forceps and drop it into the bottle, replace 
 the stopper, and then, by a violent and sudden motion of the 
 bottle, break the tube. The fragments of glass, being clean, may 
 be left in the bottle, which should be kept in a baking powder 
 box, or some similar case with a closely fitting cover. 
 
 These details may see> i> needlessly exacting ; but the fact is 
 that the solution of osmic acid is so prone to decomposition that 
 even with these precautions it can rarely be kept for any length 
 of time. As the chemical is costly ($2.25 to $2.50 per tube of 1 
 gram) it becomes a serious tax upon one who desires to make 
 frequent use of its most valuable properties. 
 
 XLI. THE ANILIN COLORS. These stains have of late ac- 
 quired an importance in microscopical research second to that of 
 no class of reagents in its entire technology. Their value has 
 been especially shown in bacteriological investigations, and in 
 the domain of biology. Those principally used in histological re- 
 search are as follows : 
 
 1 Mayenta (or rose-anilin) ; solution, 1 grain to the ounce of 
 alcohol (95). 
 
 2 Blues. There are several of these, the most useful being 
 -(a) methyl blue, in alcohol to saturation: (b) acetate ofmauvem. 
 
 For the uses to which osmic acid may be advantageously appled in 
 histological research I would refer the reader to the flies of the ST. Louis 
 MEDICAL AND SURGICAL JOURNAL for 1885 more especially to page 320 
 (Osmie acid in histology) . 
 
EL EME KTAE Y MICRO 8COPICAL TE CHNOL G Y. 
 
 53 
 
 4 grains to the ounce of alcohol: (c) Nicholson's blue, 1-6 grain ; 
 nitric acid 2 minims ; alcohol, 1 ounce. 
 
 3 Greens. There are also several of these that are useful, 
 viz : fa} iodine green, 1 grain to 6 drams of distilled water : (bjt 
 methyl green, 5 grains to 1 ounce of distilled water : (c) a beauti- 
 ful green is made by mixing the saturated solution of methyl blue 
 (above) with a saturated solution of picric acid in alcohol. 
 
 4 Eosine. 4 grains to the ounce of distilled water and alco. 
 hoi in equal parts. 
 
 XLII. The student of histology will find the foregoing list 
 of stains amply sufficient for all ordinary purposes. Any more 
 at the present time would simply serve to embarrass and confuse 
 him. For the convenience of the reader I have grouped the 
 stains into a tabular diagram by means of which he may see at 
 a glance the function of each stain, and especially those stains 
 which are supplementary to each other. 
 
 ( Picric Acid, 
 
 General Stains. < Ammonia Carmine, 
 ( Eosine. 
 
 f Beale's carmine, 
 Borax carmine, 
 Woodward's carmine, 
 Logwood, 
 Indigo-carmine , 
 Anilin blue, J 
 
 Gold chloride, i . -, , 
 , Silver nitrate, Sl "?P le ' but requ.rmg the 
 
 -Selective stains <J Ogmic ^^ j action of light. 
 
 Picro carmine, 
 
 Carmine and indigo carmine, 
 
 Logwood and anilin blue, 
 
 Logwood and gold chloride, 
 
 Logwood and silver nitrate, 
 i Gold chloride and silver nitrate. 
 ^Methyl blue and picric acid. 
 
 f-Osmic acid, 
 
 Picric acid, 
 
 Stain in bulk and I Gold chloride, 
 harden at t h e <J Borax carmine and alcohol, 
 same time. Logwood and alcohol, 
 
 I Eosine and alcohol, 
 
 ^Chromic acid, or bichromate of potassium. 
 [This table is a modification of one published by J. W. Groves in the 
 American Journal of Microscopy, Feb., 1880.] 
 
 1 
 
 j Simple stains, not 
 [> requiring the action 
 ! of light or reagents 
 
 Double 
 Stains. 
 
54 ELEMENTAL Y MICRO SCOPICAL TE CHNOL OGY. 
 
 XLIII. APPARATUS ETC. The apparatus for staining are 
 few and simple and nothing is required beyond those things al- 
 ready enumerated, viz: an assortment of watch-glasses, some 
 pipettes for transferring stains (each stain should have its own 
 pipette), a wash bottle full of distilled water, a couple of deep 
 saucers with camel's hair pencils, and a pair of small wooden for- 
 ceps, fill the list. A few goblets or wine glasses to cover the 
 watch-glasses are alwa3 7 s in order. 
 
 XLIV. THE APPLICATION OF THE STAINS. The older histolo- 
 gists held in high esteem the processes for staining in the mass, 
 the parenchymatous injection of coloring matter, etc., while the 
 object was not yet cut in sections. For this reason they affected 
 the various hardening fluids that at the same time imparted a tint 
 to the whole material or to certain parts therof . While there are 
 occasions in which this method is yet useful, it has, as a general 
 thing, been replaced by staining the cut sections. Sometimes 
 both processes may be resorted to with advantage in one and the 
 same substance, the latter process being supplemental to the 
 earlier, or mass staining. 
 
 XLV. Since a stain begins to operate the moment that it 
 is brought into contact with a substance, and the operation of 
 tinting progresses as the agent penetrates the tissues ; and s4nce 
 the depth of the color depends upon the amount of the agent that 
 is absorbed, which in turn depends upon the length of time that 
 the staining material remains in contact with the tissues, we may 
 propound the following as AN AXIOM. 
 
 Whatever agent be employed, the best results are attained 
 when it is used in a diluted form and allowed to operate slowly. 
 
 There are, of course, exceptions to this rule, very good results 
 being sometimes obtained from the use of concentrated stains 
 acting with great rapidity. Such is the case for instance, in stain- 
 ing exceedingly thin sections, or delicate and tenuous membranes. 
 
 There are other reasons beside the uniformity in the distribu- 
 tion of color attained by the slower process, why an evenly tinted 
 section, less vividly stained, is preferable to one more deeply col- 
 ored, chief of which is that in the examination of such a prepara- 
 tion with the higher powers of the microscope, less light is re- 
 quired for its penetration; the eye is not so readily fatigued, 
 nor is it attracted to the more vivid points to the neglect of 
 
ELEMENTARY MICROSCOPICAL TECHNOLOGY. 55 
 
 equally important, though less highly colored, portions of the 
 mount. 
 
 XLVI. THE AMOUNT OF DILUTION which the various stains 
 that we have described will stand, and the length of time required 
 by them for the proper tinting of material , are largely dependent 
 upon the nature of the latter. No general rule can therefore be 
 given, but the following suggestions will be found useful : 
 
 Beale's carmine and borax carmine may be diluted with from 
 8 to 10 times their bulk of distilled water, and a writer in the 
 Royal Journal of Microscopy recommends for certain purposes 
 the addition of 20 volumes of water. 
 
 Indigo-carmine will stand a very large dilution. The best 
 plan is to take about enough distilled water to thorougly immerse 
 the object and to add to it indigo-carmine sufficient to produce 
 the shade known as sky blue. 
 
 Picro-carmine, in the strength already given ( 10 grains to the 
 ounce) is quite weak enough for all ordinary purposes. 
 
 The anilin colors, chloride of gold, nitrate of silver, etc., may 
 be used in the strength indicated in the solutions, or they may be 
 reduced by from 2 to 5 volumes of water or alcohol, as the men- 
 struum may be. 
 
 These dilutions should be made in the watch-glasses before 
 the sections are placed in them, for obvious reasons. 
 
 XLVII. SIMPLE STAINING. If the sections were floated 
 when cut, into water or glycerin, or even in dilute alcohol, they 
 may be moved thence into any of the aqueous stains without any 
 intermediary ; but, if an alcoholic stain is to be used they should 
 first be dehydrated by immersion in absolute alcohol. A neglect 
 of this precaution will sometimes result in the precipitation of the 
 stain and the ruin of the sections. This is especially true with 
 regard to some of the anilin blues. 
 
 1 Carmine Staining. Dr. Beale gives the following direc- 
 tions for the use of his carmine: "After the section has been 
 stained it is to be washed in a solution consisting of strong gly- 
 cerin 2 parts and water 1 part. After a lapse of 5 or (> hours or 
 more it should be transferred to the following fluid, viz: strong 
 glycerin 1 oz., strong acetic acid 5 drops. After having remained 
 in this fluid 3 or 4 days it will be found that the portions of even 
 soft and pulpy textures will have regained the volume which they 
 
56 ELEMENTARY MICROSCOPICAL TECHNOLOGY. 
 
 occupied when fresh." (See Microscope in Medicine, p. 66). 
 
 Excellent results may be obtained with any of the carmines 
 without so long a delay by resort to either of the 'fixing solutions, 
 given in XXXVIII. After the requisite depth of color has been 
 acquired the sections should be removed from the stain directly 
 into the fixer and immersed for a few moments, care being taken 
 not to allow them to remain long enough for the acids to bleach 
 them superficially. From the fixer, remove to distilled water, 
 rinse and return to glycerin or alcohol as the case may be. 
 
 Dr. Stowell, of Ann Arbor, in his admirable Manual of Histol- 
 ogy, and again in the Microscope for July, 1887, p. 181, says 
 "all carmine stained preparations are improved by removing the 
 excess of staining with water and placing them in a one per cent 
 solution of acetic acid. The latter takes out superfluous staining, 
 fixes the staining in the nuclei and brightens it everywhere." 
 
 2. Logwood Stained Sections do not require the acid fixing 
 agents. If the} 7 have been overstained, however, any of the 
 "fixers" will remove the superfluous color. After the sections 
 have acquired the requisite depth of tint (which can only be as- 
 certained by removing one occasionally and examining it), they 
 should be rinsed in distilled water and placed directly into al- 
 cohol. 
 
 5. The Anilin Stains may \)?. fixed so that they will retain 
 their brilliancy for a long time by immersing them in a fluid con- 
 sisting of equal volumes of a saturated aqueous solution of tannic 
 acid and distilled water. This 'fixer' will decompose if made up 
 in bulk, unless some anti-ferment, such as carbolic acid, be added 
 to it. After immersion in this fluid the sections should be rinsed 
 and placed in a one and a half per cent, solution of tartrate of 
 antimony and potassium in distilled water.. 
 
 XL VIII. MULTIPLE STAINING. By referring to the table 
 given above (XLII) it will be seen that very few of the stains 
 used in microscopy act with uniformity on all forms of matter, 
 and that the great body of them are in one way or another classed 
 as selective. This selective property is either absolute or modi- 
 lied, and may consist (a) in choosing certain tissues or elements 
 upon which to act, while leaving others unaffected; (6) in con- 
 ferring a certain tint to one element or set of elements 'and a dif- 
 ferent one to others; (<.) or, finally, in acting w ith greater energy 
 
ELEMENTARY MICROSCOPICAL TECHNOLOGY. 57 
 
 and rapidity upon an element or part thereof than it does upon 
 the balance of the substance to be stained. 
 
 Upon this selective property in its various modifications, de- 
 pend the processes known as double or multiple staining, and 
 which have been of such incalculable service in all departments 
 of scientific microscopy, but more especially in biology and his- 
 tology. It is to works on these branches and to future chapters 
 on special methods that I must now refer those who are seeking 
 information upon the selective preferences of individual stains, 
 since here we can only indicate general methods and results. 
 
 1. Pier o- car mine. As before stated, this valuable stain pos- 
 sesses the property of coloring certain tissues red and others or- 
 ange, so that in itself it is a veritable double stain. By taking 
 advantage of this fact it is made the basis of several multiple 
 staining combinations. Thus, by immersing a section from the 
 base of the tongue (say of a dog or cat) first in thepicro-carmine, 
 and successively thereafter in magenta (rose anilin) and iodine 
 green, the result will be that the muscles are stained by the first, 
 the connective tissue and protoplasmic cells by the second, while 
 the third will color the nuclei in the superficial epithelium, the ser- 
 ous glands and nonstriated muscular fibers in the vessels. Nor is 
 this all the mucous glands will show a purple from the combined 
 action of the red and green. Picro-carmine acts well with logwood, 
 iodine green and anilin-blue the method being the same in each 
 *. e: stain with picro-carmine, rinse and apply the second color. 
 
 2. Eosin is another most excellent base or ground stain in 
 multiple staining. When used for this purpose the standard 
 solution shoul'd be dilute.d with 8 or 10 volumes of water and the 
 object left in it but a few seconds. After withdrawal it should 
 be immersed in water containing 20 minims of acetic acid to the 
 ounce, rinsed, and the second color applied. Any of the blues 
 combine well with it. 
 
 #. Logwood. Hsematoxylon combines well with picro-car- 
 mine, the carmines, anilin blue and the metallic stains. An object 
 first stained witu it may be successively stained with any of the 
 others. 
 
 4. The carmines. Objects that have been stained with any 
 of this series may subsequently be stained with indigo-carmine 
 by 'removing them from the fixing bath into dilute alcohol, letting 
 them remain there four or live hours and thence transferring 
 
58 ELEMENTARY MICROSCOPICAL TECHNOLOGY. 
 
 them to the dilute sulph-indigotate of sodium solution. The 
 length of time necessary to obtain the best effects varies from 2 
 to 10 hours, according to the nature of the section. 
 
 XLIX. There are many other combinations beside those 
 suggested and, as the subject is a fascinating one, the student will 
 no doubt find them out for himself. I have gone into the matter at 
 present as fully as is necessary, or as space will admit, and will 
 conclude by quoting two axioms from Dr. Carpenter : 
 
 1. The effects produced by using one stain after another are 
 generally much better than those obtained by simultaneous 
 staining. 
 
 2. The selective action of a second stain is not prevented by 
 previous staining ; for the dye which made the latter seems to be 
 more weakly held by the parts which take the former, so as to be 
 displaced by it. 
 
 Thus, if a section of the stem of a plant be stained through- 
 out with anilin red and then placed (after washing in alcohol) 
 in a solution of Nicholson's blue, % grain to the ounce, the 
 blue will in a short time drive out the red. But by carefully 
 watching the progress, it will be seen that the different tissues 
 change color at different times, the softer tissues being first to 
 give up the red and to absorb the blue. Thus, by stopping the 
 process at the right point, the two kind of cells are beautifully 
 differentiated by their coloring matter. (Journal of the Royal 
 Mic. Soc. 1880, p. 694). 
 
 BIBLIOGRAPHY. Among the more valuable treatises on stains 
 and staining, I would refer the readers who desire to pursue the 
 matter further to Poulsen's 'Botanical Micro-chemistry,' Frei's 
 'Microscopical Technology,' Carpenter's 'The Microscope and 
 its Revelations,' Beale's 'Microscope in Medicine' and 'How to 
 Work with the Microscope. ' A most exhaustive treatise on the sub- 
 ject appeared in 1884 and 1885 in the Zeitschriftfur wissenschaft- 
 liche Mikroscopie from the pen of Dr. Hans Gierke. A good 
 translation of these articles appeared in monthly parts, in the 
 American Monthly Microscopical Journal, commencing in April 
 1885, p. 65 (published by Dr. Romyn Hitchcock, Washington 
 D. C.). 
 
PREPARATION OF THE SLIP. CEMENTS. 
 IX. 
 
 We have now arrived at a point in our progress toward a com- 
 pleted slide where we must leave, for awhile, the material to be 
 mounted, and turn our attention to the preparation of the glass 
 slip which is to receive the same and preserve it for future ex- 
 amination and reference. 
 
 LI. Every perfect mount consists, or should consist, of five 
 parts, viz: The slip, or glass plate; the cell wall, or wall of 
 cement surrounding the object and retaining the mounting ma- 
 terial ; the object; the mounting medium; and the cover-glass. 
 The proper union of these elements constitutes the slide. The 
 materials requisite for a slide, beside the object to be mounted, 
 therefore are : glass slips, cover glasses, cements, and mounting 
 media. We will consider them in detail : 
 
 LII. GLASS SLIPS. Formerly there was no uniformity in 
 the size or shape of these, every microscopist having them cut to 
 suit the stage of his instrument, the size of his cabinet, or as fancy 
 dictated. Every manufacturer of microscopical accessories and 
 materials followed his own inclination in the matter. The Ger- 
 mans, generally, affected a nearly square rectangle, some two 
 inches long by one and three-quarters broad. The French sent 
 three sizes into the market, the smallest 0.5 " wide by 2.25 ' ' long, 
 and the largest 1.10" wide by 3.10" long. Of late years, how- 
 ever, since the study of microscopy has become so widely ex- 
 tended, and especially since the establishment of postal micro- 
 scopical clubs and exchanges, the profession has settled upon an 
 uniform slip, one inch wide by three inches long. This should 
 be of the best and clearest glass, entirely free from bubbles 
 and flaws. The edges should be ground, not merely for the sake 
 of appearance, but because when left unground they scratch and 
 disfigure the stage of the microscope and sometimes cut the fing- 
 
60 ELEMENTARY MICROSCOPICAL TECHNOLOGY. 
 
 ers of those who handle them. Such slips were formerly quite 
 costly, but the increased demand for them has induced manu- 
 facturers to devise methods for their rapid production, and the 
 best ground and polished slips of Chance's glass can now be 
 bought for the price charged for plain (unground) ones five or 
 six years ago. 
 
 LIII. These slips, as they usually come from the dealer, are 
 greasy and unfit for immediate use. The most expeditious 
 method of cleaning them, where a fire is convenient, is as follows: 
 Put them in the bottom of a clean saucepan and pour over them 
 some concentrated lye or liquor potassse, cover with cold water 
 and set on the fire to boil After they have boiled for a short 
 time, remove from the fire, let cool, pour off the dirty water, rinse 
 with clear water and wipe dry with a clean cloth. After the slips 
 are wiped they should be wrapped in packages of one dozen, in 
 clean, soft paper and kept in a tight box, where dust cannot get to 
 them. This latter precaution is necessary in cities where the air 
 is constantly full of minute particles of silicious sand and dust, 
 which soon dull the polish of the surface of the slips. 
 
 Another method of cleaning is to immerse the slides for twenty- 
 four to forty-eight hours in a solution consisting of four ounces 
 of bichromate of potassium dissolved in a pint of water, to which 
 has been added four ounces of commercial sulphuric acid. This 
 solution (which is the ordinary battery fluid) is also most excel- 
 lent for cleaning cover-glasses. Many workers immerse their 
 slips and cover-glasses in this fluid and leave them there until 
 they are wanted for use. 
 
 LIV. COVER-GLASSES These are circular or square pieces 
 of very thin, clear glass purchasable of dealers in microscopical 
 supplies, in four grades and six sizes, running from K up to 1 inch 
 in diameter . The grades are determined by the thickness of the 
 glass, No. 0, the thinnest, being from ^' to 200" i n thickness ; No. 
 1 from 200" to jJo" ; No. 2, from ^" to JQO" , and No. 3, from J^'' 
 to '. 
 
 Square cover-glasses, while convenient for temporary balsam 
 mounts, are neither so sightly nor convenient for permanent ones, 
 and though formerly much affected are now but rarely used, 
 even in Germany, whose microscopists have until very recenth' 
 refused to avail themselves of those time and labor saving acces- 
 sories, the microtome and turntable. 
 
ELEMENTARY MICROSCOPICAL TECHNOLOGY. 61 
 
 The cover-glass may also be bought ID plates and cut by the 
 microscopist to suit himself. The labor of cutting is very much 
 lightened and simplified by an ingenious little device for the pur- 
 pose sold by all dealers in accessories. The price of the cut 
 covers is, however, so reasonable (from $1.50 to $3.00 per ounce, 
 according to thinness) that it will scarcely pay anyone to cut 
 them himself, unless he uses a very large number, or wishes odd 
 sizes. 
 
 Cover-glasses should be cleaned, as suggested pbove, by im- 
 mersing them for two or three days in the chromate solution, 
 after which they should be rinsed and then deposited in absolute 
 alcohol, where they should remain until required for use. 
 
 LV. THE CELL. In mounting very thin sections or very 
 minute objects, when Canada balsam, damar or a similar preserva- 
 tive medium is used, a cell wall is not an absolute necessity ; nor 
 is it essential even, that the edges of the cover-glass should be 
 sealed and finished with any cementing material, since the mount- 
 ing medium itself dries hard and impervious at all points ex- 
 posed to the atmosphere. The appearance, however, and the 
 absolute durability of the mount are always improved by such a 
 finish. It is therefore best to use it in all cases where the mount 
 is intended to be permanent. 
 
 When the object to be mounted is of any appreciable thickness, 
 or when it is excessively fragile and liable to be crushed 
 by the pressure exerted by the natural contraction of the mount- 
 ing medium in drying, the case is different, and whatsoever 
 medium be employed, a cell wall, for the support of the edges of 
 the cover-glass, becomes an absolute necessity. The reasons for 
 this must be patent to anyone who has ever given the subject a 
 moment's thought. When a supporting wall is not used the ob- 
 ject itself must sustain the cover-glass until the mounting medium 
 dries sufficiently hard to perform this function. As the medium 
 shrinks in drying the amount of shrinkage depending upon the 
 amount of volatile matter contained in it undue pressure is 
 gradually brought to bear upon both the object and the cover- 
 glass. The result of this pressure is, frequently, distortion of the 
 histological elements, or even crushing of the object (as in the 
 case of delicate frustules of the larger diatoms) and a curving of 
 the cover-glass, which renders it peculiarly liable to fracture by 
 the slightest external pressure. So marked is this distortion of 
 the cover-glass, in many instances, that it absolutely assumes a 
 
62 ELEMENTARY MICROSCOPICAL TECHNOLOGY. 
 
 lenticular shape. In the older text books on microscopical 
 technique various devices were suggested for the avoidance of 
 such contretemps though, strange to say, the true reason for 
 the distortion seems to have escaped all writers upon the subject. 
 Chief of these ancient devices formerly in vogue, were cells 
 made of different thicknesses of paper, cut or punched out by 
 hand. The introduction of round cover-glasses and the turntable 
 now enable the microscopist to make perfectly uniform cell walls 
 of any desired depth ; though as we shall see later on, cells made 
 of wax, india rubber, celuloid and of metal, still play an im- 
 portant part in mounting objects of any considerable thickness, 
 especially for direct examination. 
 
 LVI. CEMENTS A good cement should have the following 
 qualities, the importance of which is in the order named : 
 
 1. It should not be soluble in the mounting medium, even in 
 the slightest degree. 
 
 2. It should attach itself firmly to glass and should dry hard 
 and quickly. 
 
 3. It should be tough and not become brittle with age. 
 
 4. It should flow freely from the pencil, and not have a 
 tendency to become ' ridg}' ' or to run. 
 
 5. It should shrink but slightly, or not at all, in drying. 
 
 To these we may add that it should keep well in a liquid form, 
 and not alter or deteriorate with age. 
 
 From these points it is plain that the ideal cement, one which 
 will fulfill all the requirements enumerated, and under all circum- 
 stances, is a desideratum much longed for by microscopists, but 
 one which, I do riot hesitate to say, has not yet been discovered. 
 Cements, for instance, which answer perfectly well for glycerin 
 or aqueous mounting media, will dissolve and rapidly succumb 
 when brought in contact with balsam or prepared damar. On 
 the contrary, those which are unaffected by balsam and its sol- 
 vents (benzol, turpentine, chloroform, etc.) will soften when used 
 with glycerin or watery fluids. No one cement, therefore, with 
 which we are at present acquainted, will answer under all circum- 
 stances. The following are the best cements in use at present : 
 
 (a). ASPHALT CEMENT This is one of the best and earliest of 
 the cements employed in cell making, and up to very recently it was 
 used, either alone or in combination with caoutchouc, drop black, 
 
EL EMENTAH Y MICROSCOPICAL TECHNOL OGY. 63 
 
 or other ingredients, by the best foreign mounters, almost to the 
 exclusion of all other cements. It is known in commerce as 
 Brunswick black and, as purchased from dealers in painters sup- 
 plies, consists of the best foreign asphalt dissolved, with the aid of 
 heat, in linseed oil. Litharge and gum amber are added during 
 the boiling, which is continued until all fluids are driven off and a 
 nearly solid mass is obtained. This mass, dissolved in oil of 
 turpentine, constitutes the asphalt varnish of commerce. The 
 varnish may be used alone for cell making, but a far better 
 cement results when the solid asphalt, prepared as above, is dis- 
 solved in twice its weight of benzol, in every ounce of which four 
 grains of caoutchouc have been previously dissolved. Black 
 Japan varnish is sometimes used in place of Brunswick black. 
 One of the best articles of this sort that has ever come under my 
 notice, and one which presents when finished an appearance ident- 
 ical with the cell material used by the celebrated French mounter, 
 Bourcogne, of Paris, is a black enamel leather dressing used by 
 carriage finishers. It comes in square tin flasks, holding one 
 pound, and may be obtained from any dealer in coach makers' 
 supplies. 
 
 (6). GOLD SIZE. This is another most excellent cell building 
 and finishing cement. The kind used is that technically known 
 among gilders as oil size, and used for gilding outside work which 
 has to stand the action of the weather. It may be obtained ready- 
 made from any dealer in artists' or painters' supplies. There is 
 much difference, however, in the qualit}" of the material as sold 
 by dealers in microscopical accessories a great deal of it being 
 nothing but boiled linseed oil. The following is the process for 
 preparing the best : Grind together twenty -five parts of strained 
 or filtered linseed oil, one part calcined red ochre and one part 
 red lead, and boil for three hours. Let stand until the solid 
 matter sinks to the bottom and decant the clear fluid. Mix the 
 latter with an equal bulk of white lead, boil for an hour and 
 again let stand and decant. The clear liquid is a size that at- 
 taches to glass with wonderful tenacity and dries as hard as flint. 
 It will stand much rough usage and is altogether one of the very 
 best of cements. 
 
 (c). SHELLAC CEMENT. Break white shellac into small pieces 
 and soak in sulphuric ether over night. In the morning pour off 
 the surplus ether, drain completely and dissolve in absolute 
 alcohol, Bleached shellac dissolves very slowly, even with the 
 
64 EL EMENTA Z? Y MICROSCOPICAL TECHNOL OGY. 
 
 previous softening in ether, and it requires a long maceration to 
 obtain a good solution. A considerable amount will remain un- 
 dissolved after weeks of contact with absolute alcohol. To obtain 
 a limpid product, add to the solution about one half its volume of 
 benzol, agitate and let stand. In a few hours the liquids will 
 separate, the benzol retaining the detritus and leaving a layer of 
 perfectly clear alcoholic solution of shellac. This makes a very 
 beautiful and durable cell. It may be colored to the taste by the 
 addition of any of the anilin dyes that are soluble in alcohol. 
 
 (d). OXIDE OF ZINC CEMENT. To prepare this cement well 
 requires some labor and care, which, however, are in my opinion 
 amply repaid by the result, since for excellence and general 
 utility this cement is inapproachable by any other yet invented. 
 The materials are gum damar, benzol, oxide of zinc, and a small 
 amount of some drying oil, preferably nut or poppy oil. The 
 damar is dissolved in benzol to the consistency of a thin syrup, 
 and the solution carefully filtered through absorbent cotton. The 
 zinc oxide should be white and perfectly free from moisture, to 
 ensure which I am in the habit of drying it thoroughly by heating 
 in a muffle. The zinc changes color, becoming yellow under heat, 
 but soon regains its whiteness. In preparing the cement only a 
 small portion of the zinc should be put into the mortar at once. 
 This should first be wet with a few drops of benzol, the solu- 
 tion of damar then added, and the whole carefully rubbed until a 
 smooth and intimate incorporation of the ingredients is effected. 
 The mixture is then poured into a stock bottle and the operation 
 repeated until a sufficient amount of the cement has been made. 
 In despite of the most careful and continued use of the pestle or 
 muller, some portion of the zinc will remain uncrushed, and to 
 get rid of these particles the fluid must be filtered through ab- 
 sorbent cotton or allowed to stand awhile and then decanted. 
 
 The latter is far the best and most economical process. The 
 larger particles sink more rapidly than the finer ones, and in the 
 course of two or three decantations an exquisitely smooth and 
 homogeneous fluid is obtained. 
 
 To get the best results in practice, the proportions of zinc 
 and damar solution, in the finished cement, should be about 
 equal. To secure this result let the decanted fluid stand until 
 all of the zinc has separated and fallen to the bottom of the 
 vessel. If there is not enough of the damar solution, more can 
 be added from the stock bottle. If too much be present, the 
 
ELEMENTARY MICROSCOPICAL TECHNOLOGY. 65 
 
 surplus may be drawn off with a pipette. The cement is com- 
 pleted by the addition of from fifteen to twenty minims of 
 drying oil to each ounce of cement. Without the latter ingre- 
 dient the product becomes brittle on drying. 
 
 As thus made, and used with the precautions hereafter indi - 
 cated, there is in my opinion no cement which can compare with 
 it, either in the smoothness with which it flows from the pencil, 
 rapidity of drying, tenacity and evenness of attachment, or the 
 hardness and toughness of the product. In addition to these 
 practical recommendations, there is nothing in the way of ce- 
 ments that is more beautiful in finish. In saying this much 
 about the cement I am quite aware that it has been decried 
 and abused by certain parties, belonging to that class of work- 
 men who invariably lay their own lack of skill upon the tools 
 they use. I have .used it constantly for fifteen years and 
 whenever it has failed to make a good and durable mount the 
 failure could be traced directly to my own negligence or care- 
 lessness. 
 
 (e). MARINE GLUE. Dissolve one part of caoutchouc in 32 
 parts of coal tar naphtha, and when solution is complete (in the 
 course of 8 or 10 days) add "2% parts of dark shellac in fine 
 powder and let the vessel, closely stoppered, stand for another 
 week or ten days. At the expiration of this time put it into a 
 sand-bath and heat carefully until the undissolved portions of 
 shellac and caoutchouc are liquefied and a homogeneous fluid is 
 obtained. This should be poured upon a cold plate or slab and 
 allowed to solidify, after whic;> it may be broken into pieces and 
 preserved for use. It requires a temperature of 240 F. to 
 liquefy this cement. It is the most tenacious of all substances 
 yet devised for this purpose. 
 
 A liquid cement, resembling the foregoing in general charac- 
 teristics, may be made by dissolving 1 part of india rubber in 12 
 parts of benzol and adding to the solution 20 parts of shellac. 
 Heat in a water bath until solution is complete. This cement 
 may be applied with a pencil which that made by the former 
 process cannot be, unless redissolvecl in benzol or naphtha. 
 
 (/). BELL'S CEMENT is the proprietary name of the shellac 
 solution given above (c). 
 
 ({/). DIAMOND CEMENT. Dissolve one ounce of best Russian 
 isinglass in 5/2 ounces of distilled vinegar. In a separate vessel 
 dissolve a half ounce each of gum ammoniac and gum mastic in 
 
66 ELEMENTARY MICROSCOPICAL TECHNOLOGY. 
 
 two ounces of alcohol and mix the two solutions. This cement is 
 very tenacious and is used by Oriental jewelers in setting precioui 
 stones. 
 
 SEILER'S CEMENT is a modification of the above. It is made 
 by dissolving two drams of isinglass in one ounce of acetic acid in 
 which ten grains of gum ammoniac have been previously dissolved. 
 
 A similar cement may be made by dissolving one ounce of 
 isinglass in four ounces of hot skimmed milk. 
 
 (7i). CASEIN CEMENT is made b} r dissolving curd or pot cheese 
 made from skimmed milk, in a hot saturated solution of borax. 
 It makes a hard and brilliant cement, useful in certain cases. 
 
 ("). ARABICIN CEMENT. To a thick solution of gum arabic add 
 sufficient alcohol to throw down the arabicin in the form of a white 
 flaky deposit. Throw on a filter and wash with alcohol, dry and 
 redissolve in distilled water to the consistenc}^ of an officinal syrup. 
 To each ounce of the solution add twenty -four grains of sulphate 
 of aluminium and dissolve. With this as an excipient, rub in 
 sufficient finely pulverized talc to make a fluid of about the same 
 consistency as good zinc cement. This cement may be made 
 directly from a solution of gum arabic, but it does not keep so 
 well as when the arabicin alone is used. It is useful for building 
 cells for balsam mounts, answering the same purpose in these 
 cases as ziuc cement does wnen glycerin or aqueous mounting 
 media are used. 
 
 As in the case of stains, formula* for cements might be multi 
 plied almost ad infinitum were any good purpose subserved by it. 
 With those given above the student can meet all the exigencies of 
 microscopical technology, and an}- more would be superfluous. 
 
THE CELL WALL 
 X 
 
 . LVII. As hitherto intimated, the choice of a cement for 
 building the supporting wall of, and closing the completed cell, 
 depends very much upon the nature of the mounting medium 
 to be used. To be permanent it is manifest that the container 
 must be absolutely insoluble in the fluid which it is to hold and 
 with which it remains in contact. Hence, for technical purposes, 
 we may divide mounting media into two great classes, viz : the 
 aqueous and the gummy or resinous. In the first we place glycer- 
 in, glycerin jelly, camphor w T ater, camphorated gelatin, carbolized 
 gelatin, serum, etc., soluble in water. The second comprises 
 Canada balsam, damar, the oils, etc., insoluble in water, but solu- 
 ble in benzol, chloroform, oil of turpentine, ether, alcohol, etc., 
 fluids which are used in preparing most of the cements used in 
 microscopy. At first sight it would seem that no great difficulty 
 should arise on this score and that the trouble would be obviated 
 by using aqueous cements for gummy or resinous media, and vice 
 versa. But in practice we find that the aqueous cements are at- 
 tacked by the moisture of the atmosphere, by the water used in 
 cleansing slides (which will occasionally get soiled) and finally, by 
 the fluids used with immersion lenses. This difficulty is overcome 
 by covering the aqueous cements, after they have set and dried 
 thoroughly, by a resinous cement, impervious to and unaffected 
 by the atmosphere. An analogous operation enables us to protect 
 a resinous cell wall from being attacked by similar mounting med- 
 ia. In such cases all that is necessary is to cover the wall with an 
 aqueous cement which, when dry, acts as a complete protection to 
 it. This enables us to use one and the same cement for the foun- 
 dation of all cells, whether as containers for balsam or glycerin, 
 damar or gelatin, leaving subsequent manipulations to be deter- 
 mined by the mounting medium to be employed. The importance 
 cf this apparently trivial point is that it enables the microscopist, 
 to keep en hand at all times a stock of thoroughly seasoned cells 
 
68 
 
 ELEMENTARY MICROSCOPICAL TECHNOLOGY. 
 
 ready to be filled when wanted a point which will be recurred tc 
 la-^er on. 
 
 . LVIII. THE TURNTABLE. Of all the instruments which of 
 late years have been introduced as aids in microscopical technola 
 gy, not one has conduced so much to ecomomy of time, perma- 
 nence of mount and elegance of finish as has the turn-table. In 
 j'ts simplest form this is a disc of heavy wood or metal, three and 
 a half or four inches in diameter, mounted so as to rotate easily upoD 
 a perpendicular pivot or axis, and provided with clips to hold the 
 slide in place. A little table, somewhat larger than the disc, acts as 
 a hand-rest in operating. Fig. 7, represents such a turn-table. 
 
 FIG. 7. TURN-TABLE. 
 
 vJpon this simple instrument there have been constructed quit* 
 a number of improved turn-tables the improvements consisting 
 in devices for the automatic centering of the slip, improved meth- 
 ods of holding the same, devices for decentering, etc. Nearly every 
 manufacturer of microscopical accessories has some such improved 
 turn-table, and many of them are very meritorious. Among the 
 best of the self-centering devices I may refer to those of Griffith. 
 Bausch & Lomb, Bullock, Walmsley, and Queen. 
 
 FIG. 8. GRIFFITH'S TURN-TABLE. 
 
ELEMENTARY MICROSCOPICAL TECHNOLOGY. 69 
 
 Fig. 8 represents the self-centering and improved turn-table 
 of Mr. E. H. Griffith, of Fairport, N. Y., manufactured for him 
 by the Bausch & Lomb Optical Company, Rochester N. Y. 
 
 . LIX. METHODS OF USING. Having placed the slip, thorough- 
 jy cleaned and dried, upon the turn-table, and centered it by the 
 eye (if the instrument is not automatic), the disc is given a rapid 
 rotary motion by laying the forefinger of the left hand upon the 
 milled edge of the disc itself or the button underneath it, and 
 drawing it quickly toward the person. In the meantime, the right 
 hand, holding a camel's hair pencil which has been dipped in ce- 
 ment, is rested upon the "table" and the point of the pencil is 
 brought into light contact with the surface of the rotating slip. 
 The brush should be held exactly as a pen in writing, and care 
 must be .taken that the contact of the point shall be tangential 
 to the diameter of the circle that it is desired to make, and at 
 right angles to an imaginary line drawn from the body of the oper- 
 ator through the center of the rotating disc. This is in order that 
 the friction of contact shall draw the hairs composing the pencil in 
 a straight line, away from the hand and parallel with the handle of 
 the brush. If held otherwise the point will be twisted and the 
 circle made smaller or larger than is desired. A very little prac- 
 tice will give the 'knack' and will teach the student just how much 
 cement to take up with the pencil. 
 
 . LX. The cell wall, as usually built, should have a width of 
 about one-sixth of an inch, and the outer diameter should project 
 about half that space beyond the edge of the cover-glass, all 
 around. The depth of the cell depends upon the thickness of the 
 object that it is desired to mount in it. For ordinary sections of 
 pathological or histological material, the depth should not be over 
 one five-hundredth part of an inch- which is obtained by putting on 
 from two to three layers of oxide of zinc cement. When more 
 layers are required the first two or three should be allowed to dry 
 quite thoroughly before adding others. When the cells are to be 
 very deep other devices and precautions will be necessary, and 
 will be described under a special heading. 
 
 . LXI. Many microscopists are in the habit of making Cheir 
 ells as they need them, allowing the rings to dry only so oiuch 
 that the cover-glass will not stick fast when it is applied. k')me 
 -do this from ignorance and thoughtlessness, while others, who 
 have never experimented upon the relative durability of a cell 
 made all-at-once and one constructed upon a thoroughly dried and 
 
70 r ELEMENTARY MICROSCOPICAL TECHNOLOG 1'. 
 
 seasoned wall, claim actual advantages foi the former procedure. 
 They say when the cover-glass is applied to the walls while they 
 are yet plastic, a more accurate coaptation of surface is obtained, 
 and a more homogeneous mass is made with the cement that is 
 afterwards applied in closing the cell. These advantages, if they 
 be such, are more than counterbalanced, in all except dry mounts, 
 by a radical defect inherent in all such hastily prepared mounts, 
 whether made of zinc white cement, Brunswick black, or any other 
 material with which we are acquainted, viz : due allowance cannot 
 be made for the shrinkage of the cell in drying. This is the secret 
 of most of the failures and disappointments which produce the bit- 
 ter complaints that we find in the technical journals, from corres- 
 pondents denouncing this or that cement or mounting material. 
 
 All the cements described in the foregoing chapter, with the 
 exception of gold size, consist of a solid material or materials 
 euspended or held in solution in some more or less volatile mediurr 
 the evaporation of which again leaves a solid mass. The exception 
 (gold size) hardens partly, though very slightly, by evaporation, 
 its solidification depending principally upon a chemical change 
 rrought by the oxygen of the atmosphere. But even this change 
 is accompanied by a diminution of volume ; and as to the cements 
 composed partly of volatile material, it is plain that there must 
 be a very large decrease of volume in the process of solidifica- 
 tion. In asphalt, zinc-white and shellac cements and marine glue 
 among the resinous cements, aiid arabicin and gelatin cements 
 among the aqueous, this shrinkage amounts to a diminution of vol- 
 ume of at least 30 per cent. When a cell is properly finished it 
 must be entirely filled with the mounting medium. If it is not so 
 filled we are bound to have air bubbles, which are not only un- 
 sightly but which ultimately destroy the mount. It is plain that 
 if we entirely fill a cell with any mounting medium and this cell 
 afterwards loses one-fourth or one-third of its volume, something 
 must give way. The fluid (air excepted) is practically incompres- 
 sible, yet great pressure is brought upon it. It has no space with- 
 in the cell into which it can retreat, and consequently it must force 
 its way out of it. The pressure is slow, and gradual, but contin- 
 uous, and finally the cell gives way at its weakest point ; the med- 
 ium exudes or 'creeps out' and is discovered. It is washed off 
 and more cement applied. In a few months the process is repeat- 
 ed the fluid gradually infiltrating and disintegrating the cement, 
 until finally the slide is a total wreck. The builder of it, mean- 
 while, not suspecting that he has undertaken the old, old problem 
 of opposing an unyielding body to an irresistable force, damns the 
 
ELEMENTARY MICROSCOPICAL TECHNOLOGY. 71 
 
 cements or the mounting medium, or both, and forthwith indites a 
 communication to some technical journal, the editor of which 'sym- 
 pathizes' with him, and the twain unite in solemnly warning the 
 profession not to use the offending materials "unless they are pre- 
 pared to have a certain percentage of their mounts destroyed.'* 
 This is another example of the workman blaming his tools for his 
 own lack of skill. If a 'certain percentage,' only, of mounts 
 made with a cement or mounting medium are ruined by any means 
 whatever, it is proof positive that a certain percentage remains 
 which is not thus affected. And since the operations of nature are 
 carried on by fixed laws, and not by caprice of inanimate matter, 
 it is self-evident that those which were destroyed lacked the skill 
 and care that was expended on the balance. All of which induces 
 us to formulate and propound the following : 
 
 AXIOM. Never finish a cell t&e walls of which are not thor- 
 oughly dry and seasoned. 
 
 . LXII. ANOTHER PRECAUTION which I would impress upon 
 the beginner is the absolute necessity of having his glass slips 
 clean and, above all, free from moisture, especially when balsamic, 
 oleaginous or resinous cements or mounting media are to be em- 
 ployed. It is really wonderful how small an amount of moisture 
 will destroy the tenacity of the one and the transparency of the 
 other. And the same precaution against moisture should be ob- 
 served in making the solutions of damar and other resins and gums, 
 in preparing the cements. The few drops of water adherent to 
 the sides of a bottle will render milky and opaque a pint of the 
 solution of damar in benzol. 
 
 . LXIII. CELLS FOR DRY MOUNTS, or mounts in which atmos- 
 pheric air is the medium, and which are almost always intended 
 for direct examination, are usually required to be much deeper than 
 those for examination with transmitted light. Instead of resort 
 ing to the slow process of building them with cement, layer by 
 layer, they may be quickly prepared by cementing to the slide 
 rings made of glass, metal, vulcanite, ivory, mother of pearl, in 
 fact of almost any solid material of a proper size and shape. 
 Brass curtain rings, a pearl button with the center scooped out, the 
 eyelet of a copper rivet, all make excellent cell building material. 
 They may be attached to the glass by any of the cements given 
 above, but I prefer marine glue for the purpose. Beautiful cells 
 for this class of mounts may be made of sheet wax, as suggested 
 by the Rev. Dr. J. T. Brownell at the Rochester meeting of tho 
 American Society of Microscopists (1884). The wax used for 
 
72 ELEMENTARY MICROSCOPICAL TECHNOLOGY. 
 
 the purpose is that made for flower workers. Several layers may 
 be superimposed, and the whole can then be turned into shape by 
 placing the slip upon the turn-table and applying the edge of a 
 sharp knife or chisel to it as it revolves. 
 
 At the Chicago meeting of the Society in 1883, Prof. A. H. 
 Chester exhibited some ver} 7 handsome cells made by punching 
 annular discs from thick tinfoil and cementing them to the slip 
 with marine glue. 
 
 A very beautiful cell is made of shellac in the manner describ- 
 ed by Mr. Rebaz before the same societj 7 at the Rochester meet : 
 ing. The details of these three processes are too long to be de- 
 scribed here, but may be found in full in the reports of the Pro- 
 ceedings of the A. S. M. for 1883 and 1884. 
 
 Finally, a most excellent cell may be made from a paste of 
 litharge and glycerin. The paste is applied to the center of the 
 slide and as it commences to set the slip is placed upon the turn- 
 table and the mass turned down to a proper size and shape. A 
 skillful use of the turn-table, after the fashion of a lathe, enables 
 one to make cells of almost any plastic material. 
 
MOUNTING MEDIA. 
 XI. 
 
 The series of operations preliminary to the final act of mount- 
 ing have now been completed. They have thus far progressed 
 in separate but parallel lines, the one having to do with the prep- 
 aration of the object to be mounted, and the other with making- 
 ready the glass slip which is to receive it. We have now arrived at 
 a point where the manipulations will be with both, for nothing 
 now remains but to place the object within the cell- wall/ arrange 
 it as we wish to have it remain, surround it with the proper pre- 
 serving medium, adjust the cover glass and cement it to its prop- 
 er place and our task is completed we have a finished slide. But 
 in this series of manipulations, simple as they seem, are embraced 
 some of the most difficult a nd important points in microscopical 
 technique. Our slide being ringed and clean, and our object 
 ready, the first question that demands our attention is the choice 
 of a mounting medium. 
 
 LXIV. MOUNTING MEDIA. By this technical term we desig- 
 nate the fluids or materials with which the objects are surrounded 
 and permeated and the cell filled. This medium has a two-fold 
 function, viz : preservative and optical. The first is self explana- 
 tory and the second has already been explained in a previous 
 chapter (VI), to which we must now make reference. 
 
 LXV. LIKE cements, mounting media may be broadly divid- 
 ed into two classes, viz : the resinous and the aqueous. The first 
 embraces Canada balsam, gum-damar, liquidambar, the drying 
 oils, etc. ; the second, glycerin, gelatin, camphor-water, glycerin 
 jelly, etc. The choice of a medium will depend in most instan- 
 ces upon the nature of the object to be mounted and the charac- 
 ter of the investigations to be made. These problems belong to 
 special technology and hence need not be discussed here, except 
 
74 ELEMENTARY MICROSCOPICAL TECHNOLOGY. 
 
 in a general way. Leaving out those cases where optical prob- 
 lems must be worked out (as, for instance, where a medium of a 
 certain definite refractive index is required), or those cases where 
 certain peculiar preservative properties are demanded, the choice 
 of a medium is practically limited to two or three substances, 
 viz : Canada balsam, glycerin or gum-damar, and the student wil^ 
 rarely go wrong when he chooses one of these media. They are 
 commonly, I might say almost universally, used among micro- 
 scopists for histological and pathological investigations. 
 
 The methods of preparing and using the balsam and damar are 
 essentially the same, while the manipulations of glycerin may stand 
 as a type of mounting with aqueous media. 
 
 (a). CANADA BALSAM. This is the oldest and best known of 
 all the mounting media, having been used for this purpose from 
 an early period in the history of the science. At first it was used 
 in its crude state that of a semi-fluid terebinthinous resin, the 
 exudation of a tree belonging to the family of firs (Abies Bal- 
 samea) and the mounts thus made, owing to the large proportion 
 of oil of turpentine and other fluids contained in the resin, took a 
 very long time to set and harden. For many months after prep- 
 aration the slightest touch would cause a displacement of the 
 mount. This defect is remedied by driving off the turpentine 
 and other volatile matters by exposing the crude balsam to pro- 
 longed heating in the water-bath, a process which converts it into 
 a solid, brittle, resinous mass, closely resembling ordinary resin 
 or colophony. This mass, dissolved in chloroform or benzol to a 
 proper consistency, constitutes the "balsam" now almost univer- 
 sally used by microscopists. It may be purchased ready pre- 
 pared from the dealers in microscopical supplies. 
 
 (6). GUM-DAMAR. This gum in its crude commercial state 
 consists of hard, transparent, brittle nodules varying in size from 
 that of a pea, up to a hickory nut. It is also an exudation from 
 a tree belonging to the fir family, and known as the damar or 
 damara pine. It is prepared for use by dissolving in chloroform 
 or benzol. As the crude gum alwa3 r s contains more or less dirt, 
 fragments of bark, etc., the solution must be clarified for use. 
 The larger particles of foreign matter may be removed by running 
 the solution through absorbent cotton. To get rid of the finer 
 dust is a more difficult matter. A number of plans for clarifying 
 it have been suggested but about the best and most convenient 
 
ELEMENTARY MICROSCOPICAL TECHNOLOGY. 75 
 
 (though not the cheapest) is to make the solution so thin that it 
 will pass through filter-paper. This makes it perfectly limpid r 
 and the surplus benzol or chloroform may be -afterwards driven 
 off by heat. Damar by itself soon becomes very brittle and 
 somewhat opaque : these faults are remedied by adding to the 
 solution prior to filtration, a few drops of nut, poppy, or linseed 
 oil. 
 
 A perfectly limpid and colorless solution of damar, of high 
 refractive index and great beauty may be made as follows : To- 
 the clarified solution in benzol add alcohol of 90 until a precipi- 
 tate is no longer formed. Remove the precipitated gum and 
 wash with distilled water and afterward with alcohol ; let dry 
 thoroughly, and redissolve in pure benzol. This resin, when dry 
 is exceedingly brittle, falling into an impalpable white powder 
 upon the slightest pressure. The addition of 20 drops of poppy 
 or nut oil, while imparting a faint yellowish tinge, corrects the 
 brittleness. Mounts made with this medium never become dis- 
 colored or opaque or at least some that I have kept four years 
 and have constantly exposed to the light, still remain absolutely 
 colorless and transparent, while balsam mounts made at the same 
 time and submitted to the same influences, are quite yellow. If 
 the student has facilities for so doing, it will repay him to recover 
 the excess of benzol, alcohol, chloroform, etc., used in making 
 these preparations, by the usual processes of distillation at low 
 temperature. 
 
 (c). LIQUIDAMBAR, styrax, or 'sweet gum' is another resin of 
 high refractive power and though somewhat high colored, is val- 
 uable as a mounting medium. It is prepared and used like Cana- 
 da balsam. It has been found especially valuable as a medium 
 for mounting diatoms. 
 
 (d). MASTIC. This is a resinous exudation from a shrubby 
 plant of the pine family, the well-known pistacia lentiscus, of the 
 Grecian Archipelago. It is freely soluble in chloroform, ether, 
 benzol and turpentine. Alcohol dissolves about four-fifths of it, 
 leaving a friable, brittle mass called masticin. Although appar- 
 ently well-fitted for a mounting medium mastic is but little used 
 for this purpose. Some mounts made with it four years ago are 
 now in excellent condition. Its refractive index is about the same 
 as that of Canada balsam. 
 
 (e). COLOPHONY, or common pine resin, is another substance 
 
76 ELEMENTARY MICROSCOPICAL TECHNOLOGY. 
 
 that is apparently well fitted for use as a mounting medium, and 
 which is scarcely ever used probably because it is so plentiful 
 and common. Thiersch and Frei are both loud in their praises of 
 this material, the former preferring it to Canada balsam. To pre- 
 pare for use, pick out a lump of the clearest virgin resin and dis- 
 solve it in sulphuric ether, filter and heat the filtrate in a water- 
 bath until the ether and the originally contained oil of turpentine 
 are driven off, and the residual resin when cold breaks with a 
 lean, conchoidal fracture. This should be redissolyed in abso- 
 lute alcohol, and the solution used in the same manner as Canada 
 balsam. 
 
 This completes the resinous media commonly in use. The 
 method of applying them will be explained further on. 
 
 LXVI. AQUEOUS MEDIA. Under this term I include all me- 
 dia that are soluble to any extent in water, or which readily mingle 
 with the same and give clear resultant fluids. A very large num- 
 ber of such media have been suggested from time to time, but we 
 need now consider only those which have been found to answer 
 all general purposes in histological and pathological work. My 
 own experience with the great mass of the suggested materials 
 has convinced me that except in certain rare cases all except 
 those that I mention below can be dispensed with without any 
 serious inconvenience. 
 
 (a). GLYCERIN. This fluid comes at the very head of the list 
 of those media which may be used under almost all circumstances 
 When pure it is a colorless, odorless fluid, of a syrupy consistence, 
 oily to the touch, and very hygroscopic absorbing water with 
 great avidity from everything with which it conies in contact and 
 which contains that fluid. It is neutral in its reactions, soluble 
 in water and alcohol in all proportions, but insoluble in ether, 
 chloroform, turpentine, or benzol, or the fixed oils. Its specific 
 gravity is 1.225, and its refractive index 1.475. Its high refrac- 
 tive power, its affinity for water, its preservative properties, and 
 its stability combine to make it the most valuable of all the media 
 for mounting animal and vegetable tissues containing water. As 
 remarked by Frei, "what balsam is to dry tissues, glycerin is to 
 moist ones." Possessing antiseptic properties of no mean order, 
 it may be used alone or its preservative properties may be rein- 
 forced with carbolic, acetic, or formic acid. Since pure glycerin 
 
ELEMENTARY MICROSCOPICAL TECHNOLOGY. 77 
 
 renders some delicate tissues too transparent for accurate exami- 
 nation, it sometimes becomes necessary to reduce this property 
 by the addition of distilled water or alcohol. No definite rule 
 can be given for this dilution, but since it is advisable that those 
 objects destined to be mounted in it, should remain in glycerin 
 for several days before they are finally transferred to the slide, 
 the student may easily satisfy himself on this score before finally 
 finishing the mount. Where only a slight amount of dilution is 
 necessary the operator may safely use the pure glycerin, relying 
 on sufficient water entering the cell beneath the cover-glass in the 
 act of washing the unfinished cell. 
 
 Dr. Beale recommends that a small amount of acetic acid be 
 always added to the glycerin, especially in mounting objects that 
 have been injected with his blue or carmine fluids. Frei suggests 
 hydrochloric acid, and Ranvier formic acid for this purpose. One 
 part of acid to 100 of glycerin is abundant in any case, and where 
 hydrochloric acid is used the proportion should not be over one 
 part to 500. or even weaker. For objects not injected or stained 
 Professor Bastian strongly recommends G parts of crystalized 
 carbolic acid to 100 of glycerin. 
 
 (6). GLYCEKIX JELLY. Soak a good article of gelatin in cold 
 water until it has taken up as much of the fluid as it will absorb. 
 Throw on a coarse cloth or seive and drain off all superfluous 
 water. Transfer to a. water bath and heat until the gelatin melts 
 into a homogeneous fluid. To this add an equal volume of pure 
 glycerin, and continue the heat until the water bath boils. Strain 
 the mixture through a clean piece of white canton flannel and 
 preserve for future use. This jelly will keep indefinitely and 
 when required for use needs only to be fluidified by immersing 
 the container in moderately warm water. It is to be used in 
 mounting the same class of materials as pure glycerin. 
 
 (c). CAMPHOR WATER is made by placing a lump of camphor 
 is. distilled water and letting it remain until the fluid has a strong 
 camphoric odor and taste. Though much praised by some (es- 
 pecially English) workers. I have rarely found any use for this 
 medium. Creosote water, used for the same purposes as cam- 
 phor water, is made by dissolving three parts of creosote in a 
 fluid consisting of 3 parts alcohol and 94 parts of distilled water. 
 
 Methylated spirits, alcohol and glycerin, alcohol and carbolic 
 acid, glycerin and honey, serum, and a thousand other mixtures 
 
78 ELEMENTARY MICROSCOPICAL TECHNOLOGY. 
 
 and simples have been suggested as media possessing real or fan- 
 cied advantages for general or special mounting purposes. An 
 experience of nearly twenty years has taught me, however, that 
 balsam, damar and glycerin (alone or in combination as above) 
 are practically sufficient for all emergencies in mounting, except, 
 of course, where a medium of exceptionally high refractive index 
 is required in some special investigations such, for instance, as 
 Prof. Hamilton Smith's high index media for mounting the diato- 
 maceye. These special media will be given in another part^of 
 this work. 
 
MOUNTING IN BALSAMIC MEDIA. 
 XII. 
 
 . LXVII. The student who has followed this series will re- 
 member that when we finished section cutting our sections 
 were left in a watery medium, viz ; plain water, alcohol and wat- 
 er or gtycerin and water, as the case happened to be. Since the 
 sections are therefore permeated with a medium which will not min- 
 gle with any of the balsamic mounting media, our first care must be 
 to get rid of it, and to supplant it with a fluid that is soluble in 
 the balsamic medium of whi.chwe intend to make use. This pro- 
 cess is called dhydation, and since there is no known fluid, that 
 can be used for this purpose, which is at once soluble in water and 
 In the balsams, the operation must be performed in two or more 
 stages, or by the aid of two intermediaries, instead of one, viz ; 
 
 (a) Alcohol, soluble in water and essential oils ; 
 
 (6) An essential oil, soluble in alcohol and in the balsamic 
 medium. 
 
 . LXVIII. THE MANIPULATIONS are sufficiently simple. 
 The section, or object to be mounted, is removed from the watery 
 medium in which it has hitherto rested, and transferred to a 
 watch-glass or other vessel containing alcohol of 95 per cent. 
 After letting it remain in this bath long enough for the alcohol to 
 permeate the tissues, the fluid is drained off and the vessel filled 
 w r ith absolute alcohol. In order tLat the strength of the alcohol 
 shall not be perceptibly reduced by the aqueous matter extracted 
 ^rom the material introduced therein, the volume of the fluid 
 should be large in proportion to that of the material. When the 
 
80 EL EMEN TAR Y MICE OSCOPICAL TE CHNOL OGY. 
 
 sections are large or comparatively bulky, it is better that they 
 pass through three or even four washings with alcohol before be- 
 ing transferred to the essential oil. The length of time requisite 
 to this end depends upon the nature of the material, but need 
 rarely occupy more than five or ten minutes. 
 
 . LXIX. When the object is permeated with alcohol it 
 should be thoroughly drained and then placed in some essential 
 oil, which as remarked above, is equally soluble in alcohol and the 
 balsamic mounting medium. One of the best known oils and that 
 most frequently used for this purpose, is oil of cloves, although 
 there are a number equally as good and some of them free from 
 the objectionable characteristics of clove oil (such as shriveling 
 of tissues, pungency of odor etc.). Among the agents w r hich I 
 have successfully used are oil of cade, oil of turpentine, creasote 
 and carbolic acid. Whatever oil be used, the object must be left 
 in it until thoroughly permeated, a fact of which the student must 
 assure himself by examining his specimen under the microscope 
 before its final removal into the mounting medium. 
 
 . LXX. When at length the alcohol is completely supplant- 
 ed, the last step may be taken, and the object placed into the re- 
 sinous medium in which it is to be permanently mounted. For 
 this purpose 1 keep on hand a solution of balsam or damar in 
 benzol or chloroform, made much thinner than the balsam used for 
 mounting, and into this the specimens are transferred from the 
 essential oil and left to soak until ready to be transferred to the 
 slide. 
 
 . LXXI. IN MOUNTING WITH BALSAM the operator has the 
 choice of mounting with or without a cell wall. If the object be 
 very thin the latter method is preferable, inasmuch as it requires 
 less manipulation ; but since the balsamic mounting medium is 
 liable to shrink very considerably in the process of drying and 
 hardening, I would not advise the student to dispense with the 
 cell-wall whenever the object to be mounted is delicate and friable 
 (as, for instance, the delicate frustules of the larger diatoms). 
 In this method of mounting the manipulations are ver} r simple. 
 The object is removed from the thinner balsamic solution (as lit- 
 tle of which as possible should be carried with it) and is arrang- 
 ed in the position which it is to occupy upon the slide. A drop of 
 
ELEMENTABY MICROSCOPICAL TECHNOLOGY. 82 
 
 the mounting balsam is next placed upon the centre of a cover- 
 glass which has previously been cleaned and slightly warmed. 
 With a quick movement the cover-glass is turned over and ap- 
 plied to the object, being carried to its place with a very gentle 
 pressure. The slide is now placed on its edge in a warm place 
 and allowed to stand until sufficiently hardened for subsequent 
 manipulation. Air bubbles, the source of so much trouble in 
 other kinds of mounting, soon dispose of themselves by gradual- 
 ly making their way to the edges and there breaking. 
 
 .. LXXII. MOUNTING IN BALSAM WITH A CELL. The meth- 
 od of spinning the cell wall has already been described, and the 
 student has been cautioned as to the choice of a cell material. 
 If an} 7 ' of the balsamic cements (like white zinc, asphalt, etc)have 
 been used for the cell it will be necessary to protectit thorough- 
 ly by a coating of some aqueous cement (such as gelatin or arabi- 
 cin) applied with a camels-hair pencil. If the arabicin cement 
 (described in LYI, e.) be used for the cell wall no further pre- 
 cautions are necessary and the method of procedure is identical 
 with that described in the foregoing paragraph. The object is 
 arranged within the ring, the balsam applied to the cover-glass 
 and the latter deftly turned and shoved home. 
 
 For applying balsam to the cover-glass or object, nothing is 
 better than a bit of glass tubing drawn out to a somewhat coarse 
 point, or a little medicine dropper with a vulcanized bulb. 
 
 The beginner is prone to get too much balsam on his slide, an 
 error which causes some loss of time, to say nothing of its being 
 wasteful and slovenly. It is better, however, for him to err in 
 this direction than in the opposite, or in getting too little. In the 
 former case the excess may easily be scraped off after it is dry, 
 while in the latter, considerable manipulation is sometimes neces- 
 sary to remedy the default. The best plan is to place a drop of 
 balsam at the edge of the cover-glass on the side opposite to the 
 deficit. A needle is then inserted under the cover-glass and the 
 latter slightly raised. As it raises the added balsam is drawn 
 under it and a little manoeuvering suffices to distribute the fluid 
 evenly over the field. 
 
 . LXXIII. HARD BALSAM was formerly much used for 
 mounting, but of late it has given way, in a very large measure, to 
 
82 ELEMENTARY MICROSCOPICAL TECHNOLOGY. 
 
 balsam dissolved in benzol or chloroform. There are, however, 
 instances in which it may be used advantageously, and when such 
 is the case it is softened by the application of gentle heat, applied 
 by means of a water-bath. The slides and cover-glasses are also 
 warmed. The manipulations otherwise are the same as in mount- 
 ing with soft balsam. 
 
 When, on the c ntrary, the natural balsam, or balsam from 
 which the turpentine has not been thoroughly eliminated, is used 
 the process of drying becomes a ver} r tedious one and may oc- 
 cupy many weeks or even months. It may be hastened by the 
 application of artificial heat, and a number of devices have been 
 contrived for this purpose. The simplest and best is a rack, 
 made of tin or wood, in which the slide may be packed and hung 
 ap in proximity to the domestic heating apparatus, but not near 
 enough to the fire to injure the specimens. 
 
 LXXIV. FINISHING BALSAM MOUNTS. When the surplus 
 balsam around the edge of the cover-glass has become hard and 
 brittle, the slides are ready for finishing. The first step is the 
 removal of this surplus, by scraping with a penknife or other 
 suitable instrument. Care must be taken, in doing this, not to 
 get the point of the blade under the edge of the cover-glass, or 
 in any way to disturb the same ; for while the exuded resin may 
 be dry and hard, that which is under the cover is probably still 
 soft and fluid. For the same reason the operator must be careful 
 about making pressure on the cover-glass, as in this manner a 
 portion of the soft balsam may be forced out and its place taken, 
 too frequently, by an air bubble which it is almost impossible to 
 get rid of. If such an accident should happen it may sometimes 
 be remedied by proceeding as suggested in LXXII ; but before 
 fresh balsam is added in such a case, the slide in that neighbor- 
 hood must be made clean as possible, since the balsam in enter- 
 ing will carry along with it any particles of dust or dirt with which 
 it may come in contact. After refilling, the slide must be again 
 laid away to recommence the process of hardening. After 
 scraping away as much of the dry balsam, etc., as possible, place 
 the slide on the turn-table and spin a ring of arabicin or 
 gelatin cement around the edge of the cover-glass. Let dry, and 
 as soon as this occurs, clean the slides thoroughly with a linen 
 rag moistened with benzol or turpentine. The ring of arabicin 
 
ELEMENTARY MICEO&COPICAL TECHNOLOGY. 83 
 
 prevents the cleansing fluid from invading the cell. After the 
 entire slide and the cover-glass are cleaned, the ring of arabi- 
 cin cement may be rinsed off with clear water and the slide 
 labelled and put away, or it may be* finished as hereafter de- 
 scribed in the manipulations of aqueous mounts. 
 
MOUNTING IN AQUEOUS MEDIA. 
 XIII. 
 
 LXXV. Before going into the details of mounting with gly- 
 cerin, which may be taken as the type of the aqueous media, it 
 will facilitate matters to describe here certain simple forms of ap- 
 paratus which the student can make for himself and which will 
 greatly assist him in subsequent operations. 
 
 (a) THE GLYCERIN BOTTLE. This should hold about 2 ounces 
 and should be provided with mouth and delivery tubes fash- 
 ioned after those of a wash bottle. The mouth or blow tube 
 should be curved in a complete loop before entering the cork, in 
 order to prevent the entrance of dust, etc. A vulcanized rubbei 
 bulb, such as is used on atomizers, attached to the mouth tube is 
 a great convenience, but not an absolute necessity. When thus 
 arranged the apparatus forms a little pump which delivers a drop 
 of glycerin at any desired point, and the fluid is in the mean-time 
 kept free from dirt and extraneous matters. 
 
 FIG. 9. GLYCERIN BOTTLE. 
 
 (6). Section lifter. Take a strip of tin or thin sheet copper, 
 five or six inches long and three quarters of an inch wide, round 
 the ends and bend it as shown in Fig. 10. 
 
ELEMENTAE Y MICRO SCOPICAL TE CHNOL OGY. 
 
 85 
 
 This little instrument will be found useful in lifting fragile 
 sections from the saucer to the slip. When very fragile, the sec- 
 tion must be floated from the lifter to the slide by putting a few 
 drops of glycerin on the lifter just behind the section and tip- 
 ping the former so that its point just touches the drop of glycerin 
 on the slide. 
 
 FIG. 10. SECTION LIFTER. 
 
 (c). Reflecting box. Take a cigar box, seven or eight inches 
 long, six inches wide and two or two and a half inches deep, and 
 remove the top and one of the sides ( leaving one side, the bot- 
 tom and the two end pieces). Into this fit a piece of looking- 
 glass, extending diagonally from the top on the closed side to the 
 bottom on the open side. Finish the apparatus by covering with 
 a plate of good clear glass. The apparatus is almost self-ex- 
 planatory and will be found invaluable in making minute dissect- 
 ions, arranging specimens on the slide, and in fact any kind of 
 work requiring reflected light. In use the box is placed so that 
 the light, impinging on the surface of the mirror, is reflected up- 
 
 FIG. 11. REFLECTING Box. 
 
 ward through the glass cover plate, thus enabling the operator to 
 see the slightest defect or fold in the arrangement of his speci- 
 men. The box is greatly improved and enhanced in valuoby the 
 
86 ELEMENTABYMICBOSCOPICAL TECHNOLOGY. 
 
 following additional apparatus, which converts it into a true 
 mounting microscope. Take a piece of wood eight inches wide, 
 one foot long and one inch thick and in one end insert an up- 
 right iron or brass rod, one quarter of an inch in diameter and 
 ten inches high. Twist a piece of stout copper wire so that at 
 one end it will form a loop capable of receiving and holding a 
 jeweler's eye-glass, and at the other end attach it to the upright 
 rod in such a manner that it forms an arm four or five inches long 
 which can be slid up or down upon the upright rod. The jewel^- 
 er's eye-glasses or loupes are cheap and generally made of excel- 
 lent lenses. I find it convenient to have four of them, of focal 
 lengths ranging from a half inch to two inches, constantly at hand. 
 FIG. 12. WIRE CLAMP. 
 
 (d). Clamps. These may be bought ready made for 75cts. 
 per dozen, or they can be fashioned out of good steel hair pins. 
 Fig. 12 represents the best form of clamp for almost any kind 
 of mounting work. It is self explanatory 
 
 (e). Cover-glass holder. Take a piece of brass or copper 
 spiral spring wire, such as is used on bells, and wind it lightly 
 
 FIG. 13. COVER-GLASS HOLDER. 
 
 over a bit of cork Or other round substance not over an inch in 
 diameter. Fasten it to its place by sticking the ends into the 
 
ELEMENTARY MICROSCOPICAL TECHNOLOGY. 87 
 
 cork and attach the latter to some heavy base which will keep it 
 upright. The cover-glasses, rubbed and cleaned, are inserted be- 
 tween the spiral coils much as pen holders are inserted in the 
 racks made of similar wire more loosely twisted. When the covers 
 are inserted the whole should be protected by inverting a goblet 
 over it. 
 
 (f). Drying papers. These should be made of the best Swed- 
 ish filter or the best linen blotting paper, cut into a strips half 
 inch wide by two or three inches long. They should be kept in a 
 tightly covered box, protected from the dust. 
 
 The other instruments used in mounting have already been 
 described in our earlier chapters. They consist of straight and 
 curved needles, firmly set in good strong handles, camels-hair 
 or sable pencils, watch glasses, etc. All of them should be clean 
 and free from dust. 
 
 LXXVI. ARRANGEMENT OF WORK TABLE. The work-table 
 should be placed in front of a window giving a good light, and re 
 moved as much as possible from jar, dust, soot, etc. In St. Louis 
 and in most large cities where soft coal is largely used, these pre- 
 cautions are more easily prescribed than followed. Rapidity and 
 excellence of workmanship in mounting, as in all other mechani- 
 cal operations, depend a good deal upon a natural aptitude for 
 such work, but more on a proper and convenient arrangement of 
 instruments and facilities, so that no time is lost in putting the 
 hand directly upon the desired instrument or object. These, it is 
 presupposed, are always kept in a proper condition the knives, 
 needles, scissors, etc., sharp and clean, the cover-glasses and slips 
 thoroughly clean and conveniently arranged, the wash-bottle filled 
 with distilled water, etc. Directly in front of the operator is his 
 reflecting box, to the left of which and close by, is the bottle of 
 cement. In front of the reflecting box, but placed so that it will 
 not interfere with the light, is the vessel containing the objects to 
 be mounted. To the right is the case containing the glass slips, 
 ringed and ready for use, the cement hard and dry and the glass 
 clean. Near by, in convenient reaching distance, are the glycerin 
 bottle, the wash-bottle, a saucer of clean distilled water in which 
 rests a camels-hair pencil, the little box of drying papers and other 
 conveniences, among which I would especially mention a clean soft 
 linen cloth for wiping slides, and a bit of chamois for giving them 
 a final polish. 
 
SS ELEMENTARY MICROSCOPICAL TECHNOLOGY. 
 
 LXXVII. THE OBJECT TO BE MOUNTED having been remov- 
 ed from the fluid in which it was left after section-cutting, is 
 placed in pure glycerin and allowed to remain -there until it is 
 permeated. It should be carefully examined with a Coddington 
 lens or magnifying glass to see that it is clean and free from ad- 
 herent embedding material and other extraneous matter. A glass 
 slip is chosen, with a cell of proper diameter and depth and after 
 being wiped with a moist cloth, is dried and polished with the 
 chamois. Care must be taken that no particles of lint or fibre re- 
 main on the surface, either of the slip or cover glass. 
 
 The slip is now brought under the delivery tube of the glycerin 
 bottle and receives a drop of glycerin, which should be placed in 
 the center of the cell ring. In doing this it is well to expel a drop 
 or two of glycerin from the point of the lube before allowing any 
 of the fluid to touch the slip, since there is always more or less 
 dust settling around the tube, and which is thus avoided. There 
 is also less likelihood of air bubbles when this precaution is taken. 
 Only a small amount of glycerin is needed, and beginners invari- 
 ably put many times too much upon the slip. 
 
 The object to be mounted is now taken on the section lifter or 
 with the forceps and deposited on the drop of glycerin on the 
 slide ; the latter is placed on the reflecting box and here the object 
 is smoothed out and arranged exactly as it is desired it should 
 remain in the permanent mount. If the object be one having 
 one side or edge solid and the other ragged or broken (like a sec- 
 tion of certain tumors), it should be placed so that the unbroken 
 side is next the left end of the slip, for the following reason : 
 when the cover glass is adapted the lower edge thereof is usually 
 carried to the left edge of the ring and let fall toward the right. 
 It thus has a tendency to straighten out and drive toward the 
 i ight any filaments or parts of the mount that are easily moved. 
 
 LXXVIII. When the object is one consisting of several 
 parts which must maintain certain definite positions toward each 
 other, but which when in section are held together very slightly 
 or not at all, other precautions must be taken in the preliminary 
 arrangement of the slide. If celloidin has been used as an em- 
 bedding material the described end is attained by leaving the sec- 
 tion in the embedding mass and mounting it all together. The 
 celloidin becomes entirely transparent when brought into contact 
 with the glycerin, and doc s not interfere with the examination of 
 
ELEMENTARY MICROSCOPICAL TECHNOLOGY. 89 
 
 the object with the highest powers. Under other circumstances 
 the best device that I have yet found for fixing the objects in po- 
 sition consists in giving the slide a very thin coating of gelatin 
 before placing the object upon it, and adding the mounting fluid 
 only after the object has attached itself lightly to the slide. In 
 such cases the cover-glass must be applied very soon after the 
 glycerin is added or the latter will re-dissolve the gelatin and the 
 work must be done over again. 
 
 Air bubbles are gotten rid of. by slightly warming the slide and 
 puncturing each one with a hot needle. With a little practice, 
 however, the glycerin may be delivered from the bottle without a 
 particle of free air. 
 
 LXXIX CLOSING THE CELL. Take the cover-glass by the 
 edge between the thumb and forefinger of the right hand, and 
 bring it down at an angle of 45 until the lower edge touches the 
 drop of glycerin* in the ring, then carry it (still held at the angle 
 mentioned) horizontally toward the left until the lower edge reaches 
 the center of the cell wall, when the hold should be suddenly re- 
 laxed and the cover-glass allowed to fall upon the surface of the 
 glycerin. It will fall gently and if properly done will cause the 
 glycerin to fill the cell smoothly and without a bubble. It can 
 now be shoved gently home, expelling the surplus glycerin as it 
 goes. If when it is in place an air bubble is found within the cell, 
 do not waste time trying to get lid of it. Long experience has 
 taught me that one saves time, temper and material by lifting off 
 the cover-glass, rinsing and wiping it and going through the 
 operation of replacing it de noro t after adclirg fiesh glycerin to 
 the mount. 
 
 LXXX. WASHJKG THE Si IDE. "When the cover is safely 
 home apply the wire clamp[(Fig. 12) to hold it to its place. The 
 slide is now ready for washing, to free it from surplus glycerin. 
 This may be done by a stream from the wash-bottle, directed so 
 that the water will not be forced up under the cover ; or better 
 still, the glycerin may be washed off with a large camel's-hair 
 pencil plentifully supplied with water. This must be done most 
 thoroughly, for upon the absolute removal of the surplus glycerin 
 depends in a great measure the success of the mount. When the 
 last traces are removed the slide is drained and wiped with a 
 clean cloth, care being taken not to touch the cover-glass or clip. 
 
90 ELEMENTARY MICROSCOPICAL TECHNOLOGY. 
 
 The slide (the clip remaining in its place) is now laid flat on the 
 table or mounting box, and with the little drying papers, every 
 particle of moisture is removed from around the edges of the 
 cover-glass, the paper being passed lightly but firmly around the 
 line of juncture between the cover-glass and cement. To make 
 assurance doubly sure, the slide should be allowed to stand a few 
 minutes before applying the cement which is to seal the cell. 
 
FINISHING THE SLIDE. 
 XIV. 
 
 LXXXI. CAUTION AGAINST FRESHENING. Some workers are in 
 the habit of freshening the surface of the cell wall with a small 
 amount of cement before applying the cover glass, claiming there- 
 by to make a stronger and better cell. The plan is not a good 
 one, and for the following reasons : 
 
 1. By restoring the volatile 'matter or a portion thereof, to 
 the dry cell wall, the mass of the latter is sensibly augmented 
 (to be diminished again in drying), thereby producing the very 
 condition of things which I have shown ( LV ) to be the prime 
 cause of leakage and "creeping" in glycerin mounts. 
 
 2. If for any reason the attempt at adapting the cover glass 
 is a failure (as for instance, in case of the imprisonment of air bub- 
 bles or foreign matter, the displacement of the object, etc.) , and it 
 becomes necessary to remove it, it will be more or less smeared and 
 soiled by the cement. The brightness of the glycerin will be 
 affected by the agent, and the object itself is not unfrequently 
 spoiled in the same way. 
 
 3. It is altogether useless and unnecessary. It was based upon 
 the idea that when cement is applied around the edges of the 
 coverglass none, or only a small portion of it, passed under the 
 rim and that consequently a joint thus made is essentially weak 
 and unreliable. This is a mistake. The fresh cement softens 
 not merely the hard and dry surface of the cell wall with which 
 it is brought into immediate contact by the pencil of the operator, 
 but the softening process extends, by capillarity, some distance 
 under the coverglass. This is easily proven by attempting to 
 remove the cover from a well-made glycerin mount. In many 
 mounts, some of them fifteen years old, examined by me recently 
 the coverglasses Were so firmly adherent that in most instances 
 they were shivered to pieces by the effort to raise them. 
 
9'2 ELEMENTARY MICROSCOPICAL TECHNOLOGY. 
 
 LXXXII. CEMENTING THE CELL. The slide, washed and 
 thoroughly dried, is now freed from the clip which has hitherto held 
 the coverglass in place, and is put upon the turn-table ( LVIII). 
 The coverglass must be carefully and properly centered, so that its 
 .rim is equidistant from and concentric with the inner edge of the 
 cell wall. It is held to its place by atmospheric pressure, but may 
 be moved from side to side by pressure made upon the rim hori- 
 zontally and in a line exactly parallel with the surface of the slip. 
 If properly done, there is no danger of air bubbles or other 
 accidents. When the centering is accomplished, examine around 
 the edges of the coverglass to see if any glycerin or fluid has 
 escaped. If any be found it must be removed with great care 
 and thoroughness by the use of the absorbent slips. 
 
 LXXXIII. The pencil is now dipped into the cement, the 
 turn-table set in rapid motion, and a ring is spun around the 
 edges of the coverglass. For glycerin, gelatin and other aqueous 
 media, the white zinc cement described in LVI is the best that 
 I have ever used, though asphalt, shellac, and gold size, each bave 
 their admirers and advocates. 
 
 The first ring should be a light one, and as soon as it has been 
 applied the slip should be removed from the turn-table and put 
 aside until the cement dries or sets. The slip should lie flat 
 while tMs process is taking place, for the reason that, otherwise, 
 the softened cement of the cell wall would have a tendency to 
 'run' and to collect at its lowest point, thus distorting and injur- 
 ing the cell. 
 
 The object of this light ring is simply to soften the cell wall 
 and thus allow the coverglass to make an absolutely accurate co- 
 adaption and joint. It is my habit to give the slide a thorough 
 rinsing with a camel's hair pencil dipped in clean water, and dry 
 it carefully before proceeding to apply the second and sub- 
 sequent coatings of cement. The object of this is to remove the 
 last traces of glycerin that might possibly be lurking around . 
 
 LXXXIV. THE NUMBER OF LAYERS of cement necessary to 
 make a good, durable mount will depend on the depth of the 
 cell, the thickness of the coverglass and upon the quality and 
 character of the cement used. In the great majority of instances 
 where zinc cement is used, from three to four layers will be suffi- 
 cient. I am in the habit of applying sufficient cement to entirely 
 
ELEMENTARY MICROSCOPICAL TECHNOLOGY. 93 
 
 hide the angle made by the coverglass and the cell wall- in other 
 words, of using sufficient cement to make the line from the upper 
 edge of the coverglass to the surface of the slip, straight or 
 nearly so. The cements, enamels or varnishes used for finishing 
 ( XCI et seq.) will give the cell the appearance shown in Fig. 14. 
 
 FIG. 14. SECTION OF CELL. 
 
 This figure also serves to illustrate the point made above in 
 regard to the softening of the cell wall (LXXXI, 3). The 
 darker part represents the old and dried cement, while the un- 
 shaded portion shows the manner in which the fresh and softened 
 cement embraces the edge of the coverglass. Figure 15 illus- 
 trates a method by which a very strong cell may be made, when 
 for any especial reason extra strength and solidity is required. 
 
 FIG. 15. SECTION OF CELL. 
 
 The result is attained by using a coverglass somewhat larger 
 than the cell wall, so that a portion of the former projects all 
 around, as shown in the figure. In using this form of cell, great 
 care must be exercised in the process of drying after the pre- 
 liminary washing. The drying paper must be passed under the 
 projecting edge again and again , until every particle of moisture 
 is removed. 
 
 LXXXV. A RAPID METHOD OF MOUNTING IN GLYCERIN, and 
 one which answers very well for temporary mounts, is as follows: 
 The object is arranged on the slip with a very small quantity of 
 glycerin, so that when the coverglass is applied the fluid will not 
 reach quite to the edges of it. The cement is applied in the 
 usual way and runs under the cover until it meets the mounting 
 fluid. Slides can be prepared very rapidly in this manner, and 
 when carefully executed the mount will last a good long time. 
 
 LXXXVI. BALSAM MOUNTS are ^closed or cemented with 
 balsamic cements, by first coating the edges of the coverglass 
 with an aqueous cement one made with gelatin, gum arabic on 
 arabicin and insoluble in the balsam and its solvents. When 
 this intermediary coating is thoroughly dry and hard, they can be 
 treated exactly as glycerin mounts and be finished off with zinc 
 white or any of the balsamic cements. 
 
94: ELEMENTARY MICROSCOPICAL TECHNOLOGY. 
 
 LXXXVII. THE PENCIL used for cementing the cells should 
 be of camel's hair and not too small. The great mistake with many 
 workers is the use of little, finnicky, fine-pointed sable pencils. 
 The amount of cement taken up should be sufficient to sweep well 
 around the cell in one motion. Another point which must be 
 guarded against, if you would obtain a good smooth finish, is al- 
 lowing the pencil to remain too long in contact with the ring of 
 cement after it is spun. The benzol used as a solvent in most of 
 the cements evaporates from the surface almost instantly, and if 
 the pencil be held in contact with the ring a moment too long, 
 the exquisite glazed surface formed by the 'setting* of the gum is 
 dragged and broken. 
 
 LXXXVIII. LAY THE SLIDE FLAT. Whatever cement be used 
 for closing and finishing a cell, to get the best results the slide must 
 be allowed to dry in a flat position. This is a most important point, 
 and one, the neglect of which is, I am sure, the cause of a great 
 many failures in making handsome and lasting mounts. The 
 reasons for this precaution are given above ( LXXXIII) and 
 on this account I prefer for the permanent preservation of slides, 
 cabinets in which they maintain a horizontal position. 
 
 LXXXIX. THF, SLIDE is NOW PRACTICALLY FINISHED and if due 
 attention has been paid to detail in its construction, it will last for 
 a great many years though just how long the best work will re- 
 main unaltered is an open question. No mounting medium with 
 which we are acquainted will make a preparation that will last in- 
 definitely and keep bright and fresh always. The chemical effects 
 of light and heat ; the physical alterations produced by variations 
 of temperature ( shrinkage and expansion ), although exercised 
 within exceedingly minute limits ; molecular changes in the glass 
 itself and in the cements, all tend, sooner or later, to destroy the 
 labor of the cunningest hand and the most consciencious of work- 
 men. One thing, however, is certain ; the better the materials used 
 and the more careful the workmanship, the longer will be the per- 
 iod of usefulness of the slide. I say 'period of usefulness' be- 
 cause a slide or preparatidn that may appear good and fresh to the 
 unaided eye may have long since become valueless as a microscop- 
 ical specimen. This is particularly true of balsam mounts whose 
 edges have been left unprotected by a cell finish. In process of 
 time the balsam becomes red 'or orange yellow, and semi-opaque 
 or it*so thoroughly permeates the object that the more delicate 
 
ELEMENTARY MICROSCOPICAL TECHNOLOGY. 96 
 
 portions of the latter are lost to microscopical vision. Sometimes 
 after many years it becomes so thoroughly dry that it loses its 
 cohesiveness, and separates from the slip or cover-glass. The 
 balsam mount, however, has one great advantage over one made 
 with glycerin it can be cleaned and handled with much less care, 
 the cover-glass being supported at all points, instead of around 
 the edges alone. 
 
 XC. STRIPING AND VARNISHING. Cells finished with asphalt 
 or any dark, shining cement, need nothing more, and may now be 
 labelled and placed in the cabinet for future study or reference. 
 Those mounted with zinc cement, however, even when the latter 
 is highly glazed, will easily become soiled and the cells will show 
 finger or dirt marks. For this reason they should be covered, at 
 least partially, with a colored varnish or cement, which serves a 
 twofold purpose, viz : protection and ornamentation. Where a col- 
 orless or transparent covering is desired, resort may be had to any 
 of the quick-drying varnishes used by artists, or to damar dissolv- 
 ed in benzol. As these, as they are found in commerce, are usu- 
 ally too thick to flow freely from the pencil they should be thin- 
 ned down with benzol before using. 
 
 XCI. COLORED VARNISHES. The striping or ornamenting 
 varnishes that have succeeded best in my hands are those made by 
 rubbing up a dry coloring-matter in the solution of damar in ben- 
 zol used for making zinc cement. They are very brilliant in finish, 
 dry rapidly, and being to a great extent homogeneous with the 
 cement of the cell, they attach themselves so firmly thereto as to 
 become virtually a part thereof. The colors are those used by wax- 
 workers and come in little vials costing from five to fifteen cents 
 each. There are a vast number of shades of these colors, the most 
 useful and brilliant however, being the blues and reds, ultramarine 
 for the former and scarlet or crimson for the latter. In prepar- 
 ing the varnishes follow the directions given in LXI for making 
 zinc cement, using the coloring matter instead of the zinc oxide. 
 
 XCII. ENAMEL CEMENTS. I have given this name to a se- ' 
 ries of finishing cements and varnishes made by myself after the 
 following method : Good boiled linseed oil is cut with sufficient 
 benzol to make it thin enough to pass through filtering paper. 
 After filtration the benzol is driven off by heating in a water bath, 
 
9 6 ELEMEN^An Y tildRO SCO PICAL TE CHtfO LOG r. 
 
 and a stream of oxygen gas is conducted through the residue, be- 
 ing continued so long as it will pass, the oil being rapidly oxidized 
 and converted into a semi-solid, viscid mass, somewhat resembling 
 india-rubber in appearance and many of its characteristics. This 
 mass is soluble in pure benzol, making a varnish which dries rap- 
 idly and is of extraordinary tenacity and brilliancy. Into this I 
 grind the colors exactly as in making the zinc cement before re- 
 ferred to. The "strippings" from painters' oil and paint buck- 
 ets, dissolved in benzol, make a cement very similar to, if not 
 identical with that made by oxidation of boiled oil. In this case 
 the process of oxidation is accomplished by the atmospheric air, 
 only more slowly than by the use of a stream of gas. 
 
 XCIII. THE PENCIL for striping should be much sma 
 than that used for applying cement, and should come to a fine 
 point. It should be passed through the stopper of the recepta- 
 cle, and be kept in the fluid when not in use. This rule applies 
 to all pencils used in cements. This aperture in the cork or stop- 
 per should be bored to receive the handle and the latter should fit 
 tightly. 
 
 XCIV. APPLICATION OF THE COLORS may be made after the 
 cement of the cell has become thoroughly dry, or they may be 
 used as soon as the surface has become glazed. The slip should 
 be carefully centered upon the turn-table, as the least deviation 
 will be doubled by rotation and the result will be marked eccen- 
 tricity. 
 
 Some workers cover the whole cell-wall with a transparent var- 
 nish after application of the striping, but I do not find that this 
 conduces to either beanty of finish or strength, and consequently 
 the operation of stripmg finishes the slide, which is now ready to 
 be labeled and put away. 
 
 END OF PART I. 
 
 We have followed the object from the crude materials to the 
 finished preparation, taking up each step in its progress and treat- 
 ing it in extenso and in so plain a manner that anyone may, by 
 following directions, make as good a slide as those coming from 
 Cole or Bourcogne. Part II will deal with special methods of 
 work and research. 
 
APPENDIX. 
 
 PARENCHYMATOUS EMBEDDING, AND DRY SECTION 
 
 CUTTING. 
 
 In the ordinary practice of microscopy, whether for clinical 
 purposes or for the study of tissues, it has hitherto been customary 
 to cut quite a large number of sections, from which only the best 
 were selected for use and the balance thrown away. The exigencies 
 of modern biological study, however, are such that this method 
 cannot be pursued, since every section of the material is required 
 for examination and must be mounted in definite order with rela- 
 tion to the preceding and succeeding section. These sections 
 must also be of an uniform and definite thickness throughout. 
 The demands of the biologists were promptly met by the instru- 
 ment makers, and any of the machine microtomes described in 
 the foregoing chapters will cut the sections as required, providing 
 that the material has been properly prepared and embedded. 
 
 PREPARATION AND EMBEDDING. Various methods have been 
 described in the journals of microscopy and the natural sciences 
 for thus preparing material for the microtome, all of them modifi- 
 cations, to a greater or less extent, of the method first suggested 
 by Professor Lunge of Heidelberg in 1874, and known as the 
 PARAFFIN METHOD. The steps in this method embrace 1, dehy- 
 dration, first with 95 and afterward with absolute alcohol ; 2, 
 removal of alcohol by chloroform, turpentine, or benzol ; 3, 
 infiltration with paraffin dissolved in one of the media last 
 named; 4, infiltration with pure paraffin; 5, embedding. 
 
 1. Dehydration. If the object has been hardened in absolute 
 alcohol this step will not usually be necessary ; but if any of the 
 aqueous hardening mixtures or solutions has been used, the object 
 must be transferred to alcohol of 95 and there left for a length 
 of time dependent upon its mass, the nature of the tissues, etc. 
 From 95 alcohol it should be removed to absolute alcohol, in 
 
98 APPENDIX. 
 
 both instances sufficient fluid being used to insure absolute dehy- 
 dration. On this first step, and the thoroughness with which it is 
 executed depends the success of each succeeding process. 
 
 2. Removal of Alcohol. The object is now removed into what- 
 ever fluid it is intended to use subsequently as a solvent of the 
 paraffin. Oil of turpentine, chloroform and benzol have each 
 been recommended at different times by competent authority. 
 Turpentine offers the advantage of cheapness, but it is the least 
 volatile, which is a grave defect. Probably chloroform is the 
 most available. Whatever fluid be chosen, it should be abundant 
 not less than 15 to 20 times the volume of the object. In this 
 the latter should be left for 24 hours, or long enough for the fluid 
 to permeate the tissues thoroughly. 
 
 3 and 4. Infiltration with paraffin. This progresses in two 
 stages or steps. In the first the paraffin is cut with from one- 
 fourth to one-third of its volume with one of the above mentioned 
 solvents. This should be done with the aid of a very gentle heat, 
 applied cautiously. The temperature should never be allowed to 
 go above 140 F., and if pure benzol be used 130 to loo will 
 be sufficient. The paraffin should be FFF, or the hardest grade. 
 The temperature mentioned will keep it just at the point of 
 fluidity. The object, removed from the liquid (No. 2) is placed 
 in this and there left from 12 to 24 hours, or until thoroughly 
 infiltrated, when it may be removed into pure paraffin of the 
 grade above mentioned, heated just to the point of fluidity. Her e 
 it is to be kept for another day or day and night, when it will be 
 thoroughly permeated with paraffin. 
 
 5. Embedding. If a well-microtome be used, the object may 
 be removed from the melted paraffin directly into the well of the 
 instrument, arranged in position for cutting, as described in the 
 chapter on section cutting ( XXI, XXII and XXIII), and the 
 well filled with the melted paraffin. In the more modern ma- 
 chine microtomes the well is, however, discarded, and a different 
 procedure must be adopted, as follows : A box is made of strong 
 paper, of a size sufficiently large and deep to receive the object 
 and to leave from' a quarter to a half inch of paraffin around and 
 below it. This box is nearly or quite filled with the paraffin, and 
 the object is arranged in it exactly as in the well of the micro- 
 tome (see ante), and the whole is left to harden. This may be 
 hastened by standing the box in cold water. When perfectly 
 
APPENDIX. 99 
 
 solid arid hard the embedded mass is hermetically sealed, and 
 may be put away for an almost indefinite period, for future use, 
 or it is ready to go to the holder of the microtome. Before trans- 
 ferring to this, however, the paper should be removed, and also 
 enough of the paraffin trimmed away from around the object to 
 leave it almost bare at the point of contact with the knife, and 
 give the upper portion of the mass the appearance of a trun- 
 cated pyramid. 
 
 SECTIONING. This differs in no respect from the process as 
 heretofore described. Those who have achieved the highest suc- 
 cess in this line of work (Dr. Reeves, of Wheeling, W. Va. , Dr. Si- 
 mon H. Gage and others) say that the more direct and rapid the 
 cut the better the results. This is also my own experience. 
 
 FIXING THE SECTIONS ON THE SLIDE. In section-cutting, as 
 hitherto described, the sections were removed from the knife 
 to a common receptacle in which they were afterward freed 
 from embedding material, stained, etc., and made ready 
 for mounting. In dry section-cutting, especially for serial 
 mounts, this plan is manifestly impracticable, and has been su- 
 perseded by a very ingenious one of transferring each section, as 
 it is cut, directly to the lide and fastening it in place by a trans- 
 parent cement which, while resisting the cleansing and staining 
 fluids used in subsequent manipulations, does not in any manner 
 interfere with the optical and other qualities of the mount. This 
 cement is simply a solution of pyroxylin or gun-cotton, or celloi- 
 din (which is a very pure pyroxylin) in alcohol and ether, and 
 the solution mixed with clove oil, after the following formula, 
 which is known as Schallibauni's dove oil collodion: 
 
 Pure pyroxylin, 2 parts 
 
 Sulphuric ether, 15 " 
 
 Alcohol absolute, 10 " 
 
 Clove oil 100 " 
 
 A drop of this solution is placed on the centre of the slide and 
 allowed to partially evaporate. The section is then removed di- 
 rectly from the knife to the slide, placed in position on the collo- 
 dionized spot and gently pressed to place. 
 
 Prof. S. H. Gage, of Cornell, recommends ( Histological 
 method*. 41, p. -25) instead of Schallibaum's clove oil collo- 
 dion, the use of plain collodion, which he allows to dry on the 
 
100 APPENDIX. 
 
 slide and afterwards softens up with ether and alcohol when re- 
 quired for use. 
 
 I have no experience with this method, but from what I know 
 of Prof. Gage's skill in technique, have no doubt but that it yields 
 excellent results. 
 
 FREFING FROM PARAFFIN. The slide is now transferred to a 
 widemouthed jar containing xylol. benzol, or oil of turpentine. The 
 former is preferable, as it is not so inflammable as the other fluids. 
 In this it is left until freed from all traces of paraffin. This will 
 be in from 20 minutes to half an hour, though the sections may 
 be left in either of the fluids indefinitely without injury. When 
 removed, after allowing the fluid to drain off as much as possible, 
 the slides should be placed for a few moments in absolute alco- 
 hol to remove the last traces of the former bath, and then rinsed 
 with pure, cold water. This must be done immediately, as the 
 sections, unprotected by the coating of paraffin, dry and shrivel 
 almost instantly. They are now ready for staining. 
 
 STAINING AND SUBSEQUENT MANIPULATION. If the stain be a rapid 
 one and in aqueous solution, it may be dropped directly upon the 
 section, and the slip placed in a horizontal position until the tis- 
 sue is colored. This can only be done, however, with those col- 
 oring mediums which act rapidly, or in ten or fifteen minutes. 
 With the slower stains it is necessary to immerse the slide in a 
 vessel containing the coloring matter, and leave it there until the 
 tissues are sufficiently stained. This latter plan is the best under 
 all circumstances. 
 
 The subsequent processes of dehydration, clearing, etc., are 
 identical with those hitherto described in the body of this work, 
 the only difference being that the whole slide is immersed in the 
 fluids used for dehydration, instead of the sections alone. 
 
LIST OF ILLUSTRATIONS. 
 
 FIG. PAGE. 
 
 1. Army Medical Museum Microtome 88 
 
 2. Seller's Microtome Attachment 85 
 
 8. Bausch & Lomb Microtome (plate.) 86 
 
 4. Walmsley's Microtome 87 
 
 5. Section through Blade of Knife 88 
 
 6. Section Knife 89 
 
 7. Turn-table 68 
 
 8. Griffith's Turn-table 68 
 
 i). Glycerin Bottle 88 
 
 10. Section Lifter 85 
 
 11 . Reflecting Box 85 
 
 12. Wire Clamp 86 
 
 18. Cover-glass Holder 86 
 
 14. Section of Cell 98 
 
 15. Section of Cell ... ... 1)8 
 
INDEX; 
 
 PAGE. PARAGRAPH. 
 
 Acid, Hardening Solutions 11 V 
 
 Acid, Osmic See Osmic Acid 
 
 Algae, preserving fluid for 11 V 
 
 Anilin Stains, Red 52 XLI 
 
 Blue 52 XLI 
 
 Green 52 XLI 
 
 Eosin. 52 XLI 
 
 Fixing 56 (5) 
 
 Animal Tissues, Hardening 17 
 
 Staining 46 
 
 Apparatus for Mounting, General 8 II 
 
 " Staining 54 XLIII 
 
 Aqueous Mounting Media 83 LXXV 
 
 Application of Stains 54 XLIV 
 
 Colors 96 XCLV 
 
 Arabicin Cement 66 (i) 
 
 Areolated Tissue, Embedding 30 XXII 
 
 Asphalt Cement 62 (a) 
 
 Axiom, in Staining 54 XLV 
 
 in Cell-making 71 XLI 
 
 Balsam, Canada, see Canada Balsam 
 
 Balsam, Hard, Mounting in 81 LXXXIII 
 
 Balsam Mounts, Finishing. . 82 LXXIV 
 
 Closing 93 LXXXVI 
 
 Balsamic Mounting Media "... 74 
 
 Bausch & Lomb Microtome 36 
 
 Bell's Cement 65 (f) 
 
 Bichromate of Potassium, Cleaning Fluid 60 LIII 
 
 Hardening Fluid 11 V 
 
 Staining with 53 
 
 Bottle, Glycerin 84 (a) 
 
 Box, Reflecting 85 (c) 
 
 Camphor Water 77 (c) 
 
 Canada Balsam, Preparation of 74 (a) 
 
 Mounting in 80 LXXI 
 
 Mounting in, with cell 81 LXXII 
 
 Carmine, Ammonia 47 (1) 
 
 Beale's 47 (1) 
 
 Borax 47 (2) 
 
 Czokor's 47 (5) 
 
 Indigo. 51 (2) 
 
 Picro .. . 50 (1) 
 
 Thiersch's . 48 (4) 
 
 Woodward's 47 (3) 
 
 *The Arabic Numerals refer to the pages; the Roman to the paragraph. 
 Small numerals or lette-s in parenthesis refer to sub-sections. 
 
INDEX. 
 
 103 
 
 Carmines, 'The, Simple Staining with 
 
 Fixing Solution for 
 
 Multiple Staining with ... 
 
 Casein Cement 
 
 Cement, Arabicin 
 
 Asphalt 
 
 Bell's 
 
 Casein 
 
 Diamond 
 
 Gold-size 
 
 Marine Glue H. 
 
 Oxide of Zinc 
 
 Seller's 
 
 Shellac 
 
 Cements, in General 
 
 Qualities Desirable in 
 
 Enamel 
 
 Cell, The 
 
 Distortion of 
 
 Mounting in Balsam with 
 
 Closing the, in Aqueous Mounts \ 
 
 Cementing the, in Aqueous Mounts 
 
 Section of 
 
 Celloidin, Embedding in 
 
 N Y. College of Physicians' Method 
 
 Cells, for Dry Mounts 
 
 Of Lithage and Glycerin 
 
 Rebaz's 
 
 Tin-foil 
 
 Wax : 
 
 For Aqueous Mounts 
 
 Cell-Wall, The 
 
 Dimensions of .. ..: 
 
 Importance of Seasoning 
 
 Chemicals, Required in Mounting 
 
 Hardening by 
 
 Chloride of Gold, Hardening Solution 
 
 Staining 
 
 Cleaning Slides and Cover-glasses 
 
 Cleanliness, Importance of 
 
 Clamps, Wire, for Mounting 
 
 Cold, Hardening by. 
 
 Colophony, as Mounting Medium 
 
 Cover Glasses, Dimensons of 
 
 Cleansing 
 
 Holder for 
 
 Cutting Sections : 
 
 Damar, Gum, for Mounting 
 
 Colorless Solution of. 
 
 To Filter 
 
 Decalcification. 
 
 Deficiency of Balsam, to Remedy 
 
 Desmids, Preserving Fluid for 
 
 Dehydration .... 
 
 Dessication, Hardening by 
 
 Diamond Cement 
 
 Dry Sectioning (Appendix) 
 
 Embedding 
 
 47 XXXVII 
 
 48 
 
 .. 57 
 . 66 
 .. 66 
 
 .. 65 
 .. 66 
 . 65 
 
 (5(5 
 
 62 
 
 1)5 
 61 
 61 
 81 
 89 
 1)2 
 1)3 
 
 72 
 
 71 
 
 88 
 
 67 
 
 69 
 
 69 
 
 1) 
 
 19 
 
 11 
 
 51 
 
 60 
 
 71 
 
 86 
 
 18 
 
 75 
 
 60 
 
 60 
 
 86 
 
 40 
 
 74 
 
 75 
 
 74 
 
 22 
 
 81 
 
 11 
 
 97 
 
 19 
 
 65 
 
 97 
 
 25 
 
 (4) 
 (h) 
 (I) 
 
 (a) 
 
 (f) 
 
 (h) 
 
 (g) 
 (b) 
 
 (e) 
 (d) 
 
 (h) 
 (c) 
 
 LVI 
 
 XCIII 
 
 LV 
 
 LXXII 
 
 LXXIX 
 
 LXXXII 
 
 LXXXIV 
 
 XXIII 
 
 LXIII 
 LXIII 
 LXIII 
 LXIII 
 LXIII 
 LXXVII 
 LV1I 
 
 LX 
 
 LXI 
 
 III 
 
 V 
 
 XL 
 
 LIII 
 
 LXIII 
 
 (d) 
 
 LIV 
 
 (e) 
 
 XXIX 
 
 (b 
 
 XIV 
 
 LXXII 
 
 V 
 
 (1) 
 
 Cg) 
 
104 
 
 INDEX 
 
 Embedding Areolated Tissues 30 XXIII 
 
 Celloidin 31 XXIII 
 
 Choice of Material 26 XVII 
 
 Materials in General 26 XIX 
 
 Mixtures, Preparation of. 28 XX 
 
 Mixtures, Methods of Using 28 XXI 
 
 In Machine Microtomes 31 XXIII 
 
 Processes in General 25 XVI 
 
 Parenchymatous 97 
 
 Enamel Cements 95 XCII 
 
 Eosin, Staining Solution 53 XLI 
 
 In Multiple Staining 57 XLVI1I 
 
 Fixing Solution, Anilin Stains 56 (5) 
 
 Carmines 48 XXXVIII 
 
 Fluid, Goadby's. 11 V 
 
 Mueller's 11 V 
 
 Myer's 11 V 
 
 Wickersheimer's 11 V 
 
 Fluid, Mounting, see Mounting Media 
 
 Preserving, Formulae for 11 V 
 
 Freezing Mixtures 18 
 
 Freshening, Caution Against 91 LXXXl 
 
 Glycerin, as a Mounting Medium 76 (a) 
 
 Jelly, as a Mounting Medium 77 (b) 
 
 Bottle 84 (a) 
 
 Rapid Method of Mounting in 93 V 
 
 Goadby's Fluid 11 V 
 
 Gold Chloride, for Staining 51 (3) 
 
 Solution of 11 V 
 
 Gold Size, Cement 63 (b) 
 
 Gum Damar, see Damar 
 
 Hsematoxylon, as a Stain 48 XXXIX 
 
 Aqueous Solution of 48 (1) (2) 
 
 Alcoholic Solution of 49 (3) 
 
 Hardening, Agents 17 X 
 
 Processes 17 Xf 
 
 Vegetable Tissues 18 XII 
 
 Heat, Hardening by 18 
 
 Holder, Cover-glass 86 
 
 Hydrae, Preserving Fluid for 11 V 
 
 Hones, for Section Knives 38 (e) XXVII 
 
 Indigo Carmine 42 XXXI 
 
 Infusorise, Preserving Fluid for 11 V 
 
 Instruments and Apparatus 8 II 
 
 Care of : 42 XXXI 
 
 Knife, Section 38 XXV11 
 
 Shape of 39 
 
 Honing 38 
 
 Handle of 39 
 
 Valentine's 35 
 
 Larvae, Preserving Fluid for ^ 11 V 
 
 Liquidambar for Mounting 75 (c) 
 
 Logwood Stains 48 XXXIX 
 
 Simple Staining with 50 XLV1I 
 
 Multiple Staining with.. 57 (3) 
 
 Marine Glue 65 (<-) 
 
 Mastic, Mounting Medium 75 (d) 
 
INDEX. 
 
 T05 
 
 Machine Microtome, Bauch & Lomb's 36 
 
 Description of 34 XXVI 
 
 Embeddingin 31 XXVII 
 
 Seiler's . 35 XXVI 
 
 Walmsley's 37 
 
 Media, Embedding, Choice of 26 XVII 
 
 Embedding, Materials Used for... 27 XIX 
 
 Aqueous, Mounting in 84 LXXV 
 
 Mounting, see Mounting Media 
 
 Meyer's Preserving Fluids 11 V 
 
 Microtomes, see Section Cutters 
 
 Mixtures, Embedding, see Embedding 
 
 Mount, Anatomy of 59 LI 
 
 A typical 12 . VI 
 
 Balsam, Finishing 82 LXXIV 
 
 Mounting, Operations in 15 VII 
 
 InAqueous Media 84 LXXV 
 
 In Canada Balsam 80 LXXI 
 
 Mounting Media 73 LXIV 
 
 Classes of 73 LXV 
 
 Resinous 74 
 
 Aqueous 76 
 
 Mounting in Aqueous 84 
 
 Mueller's Preserving Fluids 11 V 
 
 Multiple Staining 56 XLVIII 
 
 with Carmine 57 (4) 
 
 Eosin 57 (2\ 
 
 Logwood 57 (3) 
 
 Picrocarmine 57 (1) 
 
 Example of : 58 XL1X 
 
 Neniatodae, Preserving Fluid for 11 V 
 
 Nitrate of Silver, Staining Solution 51 XL 
 
 Osmic Acid, Hardening Solution 11 V 
 
 Solution of 20 
 
 To Prepare Solution of 51 XL 
 
 Oxide of Zinc, Cement 64 (d) 
 
 Parenchymatons Embedding, Appendix.. 97 
 
 Paraffin, Embedding in, Appendix 98 
 
 Papers, Drying 87 (f) 
 
 Pencils, for Cements 94 LXXXVII 
 
 For Striping 96 XCIII 
 
 Preservation of Materials ; 9 IV 
 
 Preserving Fluids 10 IV- VI 
 
 Picrocarmine, to Prepare 50 XL 
 
 Processes, in Preparation of Slide 7 I 
 
 Hardening 17 
 
 Rapid Method of Mounting in Glycerin 93 LXXXV 
 
 Reflecting Box 85 (c) 
 
 Ringing Slides 92 LXXXIII 
 
 Section Cutter.... : ... 33 XXIV 
 
 Army Medical 33 XXV 
 
 Bausch & Lomb's 36 
 
 Bullock's 35 
 
 Machine 34 XXVI 
 
 Seiler's Attachment 35 XXVI 
 
 Walmsley's 37 XXVII 
 
 Accessories for.... ... 39 XXVIII 
 
106 
 
 INDEX. 
 
 Section Knife 38 
 
 Knife to Sharpen 38 
 
 Lifter to Make 84 
 
 Sections, Cutting'. 40 
 
 Staining, see Staining 
 
 Serial, Cutting (Appendix) 99 
 
 (Dry Appendix) 97 
 
 Seller's Cement 66 
 
 Microtome Attachment 35 
 
 Shellac Cement 63 
 
 Sharpening Section Knives 40 
 
 Silver, Nitrate, Solution for Staining 51 
 
 Simple Staining, see Staining 
 
 Slide, Definition of 59 
 
 Washing the 89 
 
 Finishing the 91 
 
 Slips, Glass, Cleaning 60 
 
 Dimensions of 59 
 
 Preparation of 59 
 
 Solutions, Hardening, see Hardening 
 
 Staining, see Staining 
 
 Cleansing 60 
 
 Fixing, for Carmines 48 
 
 Anilins 56 
 
 Softening Animal Tissues 22 
 
 Spongy Tissues, Embedding 30 
 
 Staining, an Axiom in 54 
 
 AndStains 44 
 
 Animal Tissues : 44 
 
 Apparatus for 54 
 
 Bibliography of 58 
 
 Carpenter's Rules for 58 
 
 Object of 45 
 
 Multiple 56 
 
 Simple. 55 
 
 In the Mass 54 
 
 On the Slide (Appendix) 100 
 
 Stains and Staining .- .. 44 
 
 Anilin r>0 
 
 Application of 53 
 
 Dilution of. 54 
 
 Formula for, see Individual Stains 
 
 General Table of 53 
 
 Selective 50 
 
 Striping and Varnishing Cells : 95 
 
 Styrax, as a Mounting Medium 75 
 
 Table, Work, Arrangement of in Sectioning 40 
 
 Arrangement of in Mounting 87 
 
 Table of Stains 53 
 
 Technology, Definition of 7 
 
 Tissues, Hardening Animal 17 
 
 Hardening Vegetable 22 
 
 Decalcification of 23 
 
 Turn-Table, Type of. 68 
 
 Griffith's 68 
 
 Method of Using 69 
 
 Valentines Knife ... ... 86 
 
 XXVII 
 
 (b) 
 XXX 
 
 00 
 
 XXVI 
 
 (c) 
 
 XXVI 
 XL 
 
 LI 
 
 LXXX 
 LXXXL 
 
 LIII 
 
 LII 
 
 LI 
 
 LIII 
 XXXVIII 
 
 (5) 
 XIJI 
 XXV 
 XLV 
 
 XXXIII 
 XLIII 
 XLIX 
 XLIX 
 XXXIV 
 XLVIII 
 
 XLIV 
 
 XLI 
 XLIV 
 XLVI 
 
 XLII 
 
 XL 
 
 XC 
 
 C 
 
 XXIX 
 
 LXXVI 
 
 XLII 
 
 X 
 
 XIII 
 
 XIV 
 LVIII 
 LVIII 
 
INDEX. 
 
 107 
 
 Varnishing and Striping 95 
 
 Varnishes, Colored 95 
 
 Vegetable Tissues, Hardening 22 
 
 Softening 
 
 Walmsley's Microtome 
 
 Washing Slide :... 
 
 Wickersheimer's Fluid 
 
 Work Table, Arrangement of for Sectioning. 
 
 Arrangement of in Mounting 
 
 Zinc Oxide Cement 
 
 22 
 37 
 
 11 
 40 
 
 87 
 04 
 
 XC 
 
 XCI 
 
 XII 
 
 XIII 
 
 XXVII 
 
 LXXX 
 
 V 
 
 XXIX 
 
 LXXVI 
 
 (d) 
 
*HIS BOOK IS DUE 
 STAMPED 
 
 JNi // *m^ 
 
 OCT 18 1936 
 
 NOV5 1948 
 
 MAR 17 1955 
 
 5 1955