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Boy Yo ah tee on } iil ave ce ae 2 r ima) ae any Oe ae eae ener om m oh a ae phe; a ah: rae oy e)) i ms Spit j wt e Rie ele bith ah. beh dada - 8 ANIMAL MICROLOGY THE UNIVERSITY OF CHICAGO PRESS CHICAGO, ILLINOIS gents THE BAKER & TAYLOR COMPANY NEW YORK CAMBRIDGE UNIVERSITY PRESS LONDON AND EDINBURGH “ANIMAL MICROLOGY PRACTICAL EXERCISES IN MICROSCOPICAL METHODS BY _i MICHAEL*F, GUYER, Px.D. Professor of Zoélogy in the University of Cincinnati OCT 25 1962 LIBRARIES THE UNIVERSITY OF CHICAGO PRESS CHICAGO, ILLINOIS ‘CopyRIGHT 1906 By THE UNIVERSITY OF CHICAGO All rights reserved Published November 1906 Second Impression November 1910 ’ Composed and Printed By The University of Chicago Press Chicago, Illinois, U.S.A. PREFACE For the past ten years it has been a part of the writer’s duties to give instruction in microscopical technique, and it has seemed to him that there is need for a series of practical exercises which will serve to guide the beginner through the maze of present-day methods, with the greatest economy of time, by drilling him in a few which are thoroughly fundamental and standard. The book is intended primarily for the beginner and gives more attention to the details of procedure than to discriminations between reagents or the review of special processes. The student is told what to do with his material, step by step, and why he does it; at what stages he is likely to encounter difficulties and how to avoid them; if his preparation is defective, what the probable cause is and the remedy. In short, the book attempts to famil- iarize the student with the little ‘‘tricks” of technique which are commonly left out of books on methods but which mean everything in securing good results. A very brief, non-technical account of the principles of the microscope is inserted (Appendix = ann ly 4 ol i : Th : aay a.) ae hy it. hah vs hy, uy i seal BIA he 7 pile vag eat Bats MA oe: Din! a fo ) a rib ve (ote Pt one, - ron Lier 7 Ree ita es an a } = ee i ~v } >< INTRODUCTORY APPARATUS AND SUPPLIES REQUIRED The student should provide himself with the following sup- plies: One half-gross box best grade glass slides, standard size (25x75 mm.). One-half ounce, 18 mm. or 3 in., round cover-glasses, medium thickness, (o. 18 mm.). Thirty, 25x50 mm. cover-glasses, medium thickness. Two or three Pillsbury slide boxes (Fig. 1). One box of labels for slides. Three to six camel’s hair brushes (Fig. 2). Six pipettes (Fig. 3). One set of dissecting instruments as follows: One large scalpel or cartilage knife (Fig. 4). One small scalpel (Fig. 5). Two needles (Fig. 6). One fine straight scissors (Fig. 7). One fine straight dissecting forceps, file-cut points (Fig. 8). One blow-pipe (Fig. 9). One section lifter (Fig. 10). To which may well be added: One heavy scissors (Fig. 11). One curved scissors (Fig. 12). One heavy forceps (Fig. 13). One fine forceps, curved tips (Fig. 14). One horn spoon. One desk memorandum calendar. Blank cards (about 75100 mm.) for keeping records of experiments. The kind of card used for library card catalogue will do. One section razor (Fig. 15). A piece of moderately heavy copper wire with one end hammered out to a width of 7 to 10 mm. Towels. A glass-marking pencil (wax) or writing diamond will be found useful. See, however, Memorandum 21, chap. vi. 1 2 Animal Micrology TD SF. SS = == ==) SSS ll. | ii i Fia.9 Fic.10 |W Apparatus and Supplies Required =a. WS SS Ae =i Fi i) Uh RAL or On ae vi os 4 Animal Micrology Apparatus ordinarily supplied by the laboratory: Desk with drawers. Locker for microscope. Compound microscope and accessories (Appendix A). Dissecting microscope (Fig. 66). Microtomes (Figs. 27, 28, 29, 32, 33). Paraffin oven (Figs. 24, 25, 26). Tall stenders (about 85 mm. deep). Each student should have at least eight (Fig. 16). Coplin staining jars (Fig. 17). Tall stenders may be used instead. About eight are needed for each student. Flat stenders (Fig. 18); half a dozen for each student. Syracuse watch-glasses (Fig. 19); eight to each student Balsam bottle (Fig. 20). Graduated cylinders for measuring liquids (Fig. 21). Wash-bottle (Fig. 22). Celloidin bottle (Fig. 23). Turntable (Fig. 36). Injecting apparatus (Fig. 35). Reagent bottles and vials. Other apparatus and supplies such as bone-forceps, bone-saws, glass tubing, glass rods, beakers, burners, filter paper, funnels, evapo- rating dishes, sand bath, dropping-bottles, balances, mortar and pestle, ete. For apparatus or supplies not listed in this book the student is referred to the illustrated catalogues of dealers and manufacturers such as: The Bausch and Lomb Optical Co., Rochester, N. Y.; The Ernst Leitz Opti- cal Works, Wetzlar, Germany (American branch, 30 E. 18th St., New York City; or, 32 Clark St., Chicago); The Spencer Lens Co., Buffalo, N. Y.; Carl Zeiss Optical Works, Jena, Germany; R. & J. Beck, 68, Corn- hill, London; The Kny-Scherer Co., New York City; Eimer and Amend, New York City; Whitall, Tatum and Co. (especially for glassware), New York City. IMPORTANT GENERAL RULES 1. Keep everything clean! 2. Have a definite place in your desk for each piece of appa-. ratus and arrange reagents in order on top of it. 3. Use cards for keeping records of materials. Each card should have a number corresponding to that of each special object or piece of tissue, and should show the name of the preparation, date, reagents used, time left in each reagent, in short, all data concerning the manipulation of the material. 4. Jot down in a blank calendar the various things to be done at future dates, such as changing of reagent on tissues, etc., and then go over this memorandum carefully each day when you first come into the laboratory. 5. Use only clean vessels in preparing reagents, and clean up all glassware while it is yet moist. 6. Reserve and mark a separate pipette for each of the chief reagents (absolute alcohol, oils, acids, etc.). 7. In making up solutions, 1 gram of a salt in 100 cc. of liquid is reckoned ordinarily as a 1 per cent. solution, 3 grams as a 3 per cent. solution, etc. A saturated solution contains all of a given substance that the liquid will take up. When a solution is called for without specifying the solvent an aqueous solution is meant. 8. In weighing salts, always first put paper in the scale pans to protect them. 9. In making solutions or mixtures in which only a small amount of one reagent is used, after mixing, pour back some of the mixture into the small vessel and rinse it thoroughly in order to get all of the original contents out. 10. When pouring liquids from bottles keep the label of the bottle turned toward the palm of the hand. Do not lay down stoppers but hold them by their tops between the knuckles. 11. Before leaving the laboratory put away your instruments and clean and put in its place whatever laboratory apparatus you may have been using. ia ary i: ps ca ae ‘ : ' by: _ a pean : , a 7 eg | ee : i itn i Gn" eee, Oe Path rhe a ar ; ; Bi eye’ Le “tote, wi er at ‘ oe, ; ‘ ae 7 t, sh a. uy Bea: es a rs fe’ 4 n ’ Tr, US a ae SRNL Loe ar ig ed am ar Paiva Le OTT ot Re eo. ed | na) we : oP 7 par, he Pd 4 eel, us a ie ae.< haan el Ae geht ae ane, rg ow ' kitts) A) 5 Aisa : ia ” aa i a ¥ rr oF in Ronda ae ag AF 2 on - a we a VAG: : i 20) Bada) Me & r +, os , ey ee mi oe oie - eee Pa i WG (i ‘aie ie eam Le ' erie Puy os ae) 7 Re hes a boa my oy fit . aa 7 ae - 7 ‘er Food ur a te ee | a sy ace ma yieg a Mint _ at hs “ee i ae ie Fs > Ve eee fe : é 7 ; . "9 we Sieif i" oon) ny vo re eae ae a E 1 es na a cue Ra ire. Paes’: yy if er, Var oe ie Ln a, Ts, UP, ‘ce eC aa erm dw kt ee a =a bs o all ; S elo rs ee a 4, e+e > ly he ~ Le ay fet C ae | ea ot se moe, ae a Brats ar 2 hs he. ; ES et oa eae ead ye eS el sig are ee ae > eee 7 a | % ¥ Py ; a) fl = 7 ne od ‘e at i min re, nic 6 Oy te oi 7 at itive mati! . : ; Ty - < i pes. ie ate > ih :"# o i Aa i @ : q r + in ol Acie Ae a a More Viva, Ae yan ee ey: ee eV Mees te Gt 7 ia ef oy ug Srey Roy fg x Sal mi I i oe, eae Sire on i. ee", - , - Aa Pry ts, eee ae Ti. oe a zt aS Aer ie Ss Ly ae: vival hae an Ma Avs, ns ih, ee ls ae a ey as ,) bl ee ot ak oo Cw ure ea . Ean? ee. 1 ar, Aer ee ’ ha a 7 A. a. Pay oom 3 |, Ai uF a a or | > aie a ws Rey Pe at brite ws Bip a Aire pate 7 ens y) 7 a = : = ay. ca ed ge eee er ; oe oe ey Dr, rst hogs at ie i a a ee: v ra fs "AY 4 (ae or roa ih gh a #) Bipt fra ee . ae an a wi oath ie eae CHIE - cue i : a ae a6 Yai yo ay ne - oe ies / ie uy ghee axa Pye ae ~ i. ed je 4 a er ete bei, i oa PY ae ts ay a1 Re tN se 4 te a a Gage ae ap. . i ‘ : i is a Ao. ; aan ar ny a , a ve Ras oi . ee 7 ree ae - "7 Lice a ay ‘i am ye ie fetes as) rs ” eo tire (yn —— i. 2) ie a a OO eres The hematoxylin should ripen (see 13) for some three or four weeks. 19. Safranin.— Safranina SO v2 4 vs) aes es hone eel OMeenr Absolutealcohol®. 7: > ie Wey ae ee ee OL eres Amilimywater- (6,224, 600.6 Gee lee eens Loy Oa ee OO ner Make the “anilin water” by shaking up 5 ¢.e. of anilin oil in 90 c.c. of distilled water. Filter through a wet filter. Dis- solve the safranin in the anilin water, then add the alcohol. Chapter I: Preparation of Reagents iL 20. Canada Balsam.—Dry 2 grams of Canada balsam on a sand bath, or in a warm chamber until it becomes hard (1 to 2 hours at 65°C.). Do not overheat. When cool add enough xylol to make a thin, syrupy fluid. Roll a sheet of paper into a cone to serve as a funnel, and filter the fluid through absorbent cotton. ma sil Fia. 24.—The Lillie Water-Bath. The bath consists of a large chamber containing a series of drawers of equal size, 250mm. long, 100mm. wide, 80 mm. deep. Each drawer has copper front and bottom ; the sides and back are perforated zinc, thus securing free circulation of warmair. The drawers are separated by perforated cross partitions and run onslides free from the lateral supports, thus permitting sufficient circulation of warm air to secure equal temperature in the top and bottom of the bath. Water gauge and tubulatures for gas regulator and thermometer are provided. This bath is especially adapted to class work, since each student may carry on his work in a separate drawer. Thicken the solution slightly by leaving the cap off the bottle in a place free from dust, and allowing some of the xylol to evaporate. Or, fill your balsam bottle one-third full of the liquid xylol- balsam now on the market, and dilute to the proper consistency. 12 .21. Mayer’s Albumen Fixative. with scissors and filter it through moist filter paper. Fie. 25.—Simple Water-Bath. This is a useful bath for individual workers. Itis provided with imbedding- cups, infiltration vials, a shelf for watch- glass imbedding or for warming instru- ments, and tubulatures for gas regulator and thermometer. ing it with its own volume Animal Micrology Chop the white of an egg It filters Add an equal volume of glycerin, and a bit of salycilate of soda (1 gram to 50 ¢.c.) or thymol to prevent putrefaction. 22. Celloidin.—Put 5 grams of Schering’s shredded or granular celloidin into a celloidin bottle (a bottle with glass stopper and ground glass cap) and dissolve it in equal parts of absolute alcohol and ether (see 4). Add only suf- ficient fluid (about 100 c.c.) to make a thick, syrupy mass. through very slowly. In a second celloidin bottle make a thin solution by taking about one-third of the original solution and dilut- Label the of the ether-alcohol. bottles thick and thin celloidin, respectively. 23. Paraffn.—In one of the cups of a warm paraffin oven (Fig. 24, 25, or 26), put 75 grams of paraffin, melting at about 53° C. The bath should be kept at a temperature of some two degrees above the melting-point of the paraffin. A supply of softer and of harder paraffin (e. g., melt- ing at 483° and 60° C.) should also be at hand. Other Reagents.— Provide your- self with 200 c.c. of xylol, 25 c.c. of clove oil, 25 c.c, of glacial acetic acid, 50 c.c. of cedar-wood oil, 75 c.c. of chloroform, 30 c.c. of gly- ecerin and 250 «.c. of absolute Fia. 26.—Imbedding-Table. There should be two rectangular boxes (about 3% 3X 16cm.) to contain paraflin. When in use the boxes are so placed on the imbedding-table that the paraffin in one end remains melted; in the other, solid. Regulate the tempera- ture by placing the flame at the proper distance under the acute angle of the table. It is best, when gas is used, al- ways to turn on the gas completely and then regulate the height of the flame by means of a clamp on the rubber tubing which conducts gas to the burner. Chapter I: Preparation of Reagents 13 alcohol if it has not already been prepared. Keep the absolute alcohol and the xylol carefully corked to exclude moisture. Before measuring out any of these reagents, see that both the graduate and bottle are perfectly clean and dry. MEMORANDA 1. Ethyl Alcohol is the kind commonly used in histological labora- tories. Upon presentation of the proper credentials to the internal revenue officers, it may be purchased by the barrel from distillers, tax free, by educational institutions. Such commercial alcohol is of about 96 per cent. strength. When the strength is unknown it should be tested by means of an alcoholometer (see 2, below). Methyl Alcohol (called also wood alcohol or wood spirits) is cheaper than ethyl alcohol in case the latter cannot be had tax free, and is fairly satisfactory in most cases. It is poisonous and must be carefully hand- led. It is of about 90 per cent. strength. Synthol is a manufactured product now on the market which seems to answer the purposes of ordinary absolute alcohol. It is designated as a synthetic alcohol by its manufacturers and is cheaper than absolute alcohol. ; Rectified Spirit is a 91 per cent. alcohol (84 per cent. in England). 2. The Alcoholometer is a convenient instrument for determining the strength of alcohol, or the percentage of absolute alcohol in a spirituous mixture. It is a kind of hydrometer with a scale marked to indicate the percentages of alcohol. Different strengths of alcohol have different specific gravities, consequently, the instrument will float higher or lower in the liquid depending upon the percentage of alcohol present. The number on the scale just at the surface of the liquid indicates its strength. 3. Rule for Dilution of a given strength of a solution with a lower per cent. of the same solution. (For where the diluent is water, i. e., zero per cent., see rule under reagent 1.) Subtract the per cent. required from the per cent. of the solution to be diluted; also subtract the per cent. of the diluent from that of the strength required. The differences are the relative proportions of the diluent and the solution to be diluted that must be used. Thus, to prepare a 35 per cent. solution from 95 and 20 per cent. solutions: 95—385 = 60; 35—20=15; hence, 60 to 15, or 4 to 1 are the proportions desired. That is, 4 parts of the 20 per cent. and 1 part of the 95 per cent. solution must be used to obtain a 35 per cent. solution. 4, “To Remove Fixed Stoppers, take the bottle in the left hand with the forefinger applied to one side of the stopper, then tap the other side of. 14 Animal Micrology the stopper with some heavy instrument, such as the handle of a pocket- knife, pressing the forefinger against the direction of the tap. Turn the bottle round, gradually tapping until the stopper loosens. Should this device prove of no avail (which is very rarely), hold the neck of the bottle in a spirit flame, and quickly withdraw the stopper as the glass of the neck expands. This is a somewhat risky procedure, but is very | effectual if done smartly.” (Journal of Applied Microscopy, Vol. VI, p. 2116.) The glass of the neck may be more safely heated by looping a heavy cord about it and sawing the cord back and forth until the fric- tion warms the glass. CHART EES) DL GENERAL STATEMENT OF METHODS Each of the reagents which has been prepared is used for one or more of the purposes to be discussed in this chapter. All methods of preparation in microscopy are to enable us to learn more of the structure and functions of objects than would otherwise be apparent. We endeavor to study them in as near their natural condition as possible. While the study of living or of fresh material is desirable it can be carried on only to a very limited extent. Most structures of the animal body, though opaque, must be examined largely by transmitted light, hence, special preparation is necessary to put them into suitable condition. This is accomplished— 1. By cutting them into thin slices (section method). 2. By separating them into their elements (isolation )— a) Mechanically (teasing), or b) With the aid of fluids which remove the cement sub- stance (dissociation or maceration). In most instances, the minute structure of a tissue or of an organism can be studied to the best advantage only after the appli- cation of certain agents which serve to emphasize the various struc- tural elements. A tissue so prepared is an artificial product in that it is not exactly the same as it was in the living organism, but recent studies of protoplasm in the living condition by competent investigators strengthen the belief that many reagents preserve very faithfully the actual structure of the cell contents. The liquid albuminoids are apparently the materials which suffer the greatest modifications. Since alterations do occur, however, it is clear that in our interpretations of prepared material we must reckon carefully with both the original nature of the object and with the factors introduced by ourselves. 15 16 Animal Micrology KILLING, FIXING, AND HARDENING The first step in the preparation of tissues ordinarily is the employment of some reagent which will kill the tissues and fix: their various components in the characteristic stages of their activities. Such material may then be preserved indefinitely for future use. It is customary to discriminate between killing, fixing, and hardening, although the same reagent may fulfil all three require- ments. Killing refers particularly to the destruction of the life of the tissue, a process which may be either slow or instantaneous. In slow killing it is usual to employ narcotics such as ether, chloroform, chloral hydrate, chloretone, carbon dioxide, nicotin, cocain, or weak alcohol. Ice is also used sometimes. Such methods are of particular value with highly contractile animals which are desired in the extended condition. Such forms are narcotized completely or until they are unable to contract and then frequently fixed and hardened in other or stronger fluids. Where practicable, instantaneous killing and fixing is preferable because tissues have then no time to undergo postmortem changes. The same fluid ordinarily is employed for killing and fixing. The purpose of fixation is— a) To preserve the actual form of tissue elements. b) To produce optical differences in structure, or so to affect the tissues that such differences will be brought out through sub- sequent treatment with stains or other reagents. To accomplish this the fixing agent must possess the following qualities: 1. It should kill the tissue so quickly that few structural changes can occur. 2. It should neither shrink nor distend the tissue. 3. It should be a good preservative; that is, it must render the tissue elements insoluble and prevent postmortem changes. 4. It should penetrate all parts equally well. 5. It should put the tissue in condition to take stains unless it of itself produces sufficient optical differences in the various parts of the tissue. Unupter IT: General Statement of Methods 17 No ideal single reagent has been discovered which meets all of these requirements, hence it is customary to combine two or more reagents which individually possess certain of these desirable qualities. All of the best fixing reagents are mixtures. For example, acetic acid is very generally used in fixing mixtures because it penetrates well, produces good optical differentiation, and counteracts the tendency of some reagents (e. g., corrosive sublimate ) to shrink tissues. Again, osmic acid, which is an excel- lent fixing agent for very small pieces of tissue, penetrates very poorly; consequently for most objects it must be mixed with reagents which penetrate rapidly and thoroughly. Some fixing agents (corrosive sublimate, chromic acid, osmic acid, etc.) enter into chemical combination with certain of the tissue elements, others (alcohol, picric acid, nitric acid, hot water, etc.) act by coagulating or precipitating certain constituents of tissues, The chief object of hardening is to bring tissues to the proper consistency for cutting sections. The process, although begun ordinarily by the fixing agent, is usually completed in alcohol. Some objects are not sufficiently hardened until they have remained in alcohol for many hours, or even days. Asa rule, tissues should remain in alcohol of at least 70 per cent. strength for a minimum of 24 hours after the preliminary operations of fixing, washing, etc., before they are subjected to further treatment. WASHING Fixing agents ordinarily, with the exception of alcohol, must be washed out thoroughly or they are likely to interfere with sub- sequent processes. Aqueous solutions are washed out usually in water or a low per cent. of alcohol; alcoholic solutions, with alco- hol of about the same strength as that of the fixing agent. Wash- ing usually requires from 10 to 24 hours, with several changes of the liquid. If water is the washing agent it is best where prac- ticable to use running water. Chromic acid and its compounds should be washed out in run- ning water. This should be done in the dark in order that pre- cipitation may be avoided. 18 Animal Micrology Picric acid, or solutions containing it, must be washed in strong alcohol (70 per cent.), never in water because the latter seems to undo the work of fixation. Corrosive sublimate and mixtures containing it are washed out in water or alcohol. A little tincture of iodine should be added to the wash from time to time to insure the removal of all corro- sive sublimate crystals. Sufficient iodine has been added when it no longer loses its reddish color after being in contact with the preparation for a short time. Osmic acid and mixtures containing it should be washed in running water. DEHYDRATING While under certain circumstances objects may be mounted in aqueous media for examination, in the majority of cases, especially where the preparation is to be a permanent one, it has been found best to remove all water from the tissues, that is, to dehydrate them. This renders preservation more certain, and it is a neces- sity, moreover, if the object is to be imbedded later in paraffin or celloidin, for neither of these substances is miscible with water. Because of its strong affinity for water and the ease with which it may be manipulated, alcohol has come to be used universally for this purpose. It completes the process of hardening at the same time. The dehydration must be gradual. In tissues transferred from water or aqueous solutions directly to strong alcohol (or vice versa) violent diffusion currents are set up which produce serious distortion of the tissue elements. For this reason a series of alcohols of gradually increasing strength (e. g., 35-50—-70-83-95 per cent.) is used. The more delicate the object, the closer should be the grades of alcohol. PRESERVING After fixing and washing, the process of dehydration is begun ordinarily and tissues are carried as far as 70 per cent. alcohol. It is customary to leave them in alcohol of from 70 to 83 per cent. strength until they are needed. They may remain here indefinitely. If they are to be preserved for a long time (for Cuapter Il: General Statement of Methods 19 months), however, it is better to keep them in a mixture of equal parts of glycerin, distilled water, and strong (commercial) alcohol. STAINING A few fixing agents produce sufficient optical differentiation in tissues, but as a rule this must be accomplished through the addi- tion of certain stains. Most of the stains used have more or less of a selective action; that is, they pick out certain elements of the tissue, and thus enable one to see details of structure that would otherwise be invisible. Their action, however, depends largely upon the nature of the fixing agent which has previously been used. The secret of good staining, indeed, lies largely in proper fixation. There are large numbers of stains of very different chemical constitution (acid, neutral, and alkali), and they may act in very different ways upon the material to be stained. For example, some show affinity only for certain elements of the nucleus, others for the cytoplasm of cells, and some are present in tissues only physically as deposits, while others enter into chemical combina tion with certain of the cell constituents. A few, such as borax- carmine, are general stains, and affect to a greater or less degree practically all the tissue elements. It is not the purpose of the present book to enter into a pro- longed discussion of the theory of staining or to undertake a description and classification of stains. For this the reader is referred to the excellent compendium of Lee (The Microtomist’s Vade-Mecum). The stains of widest application are (1) the Carmines, (2) the Hematoxylins, (3) the Anilins, and (4) Metallic substances. Carmine is a brilliant scarlet or purplish coloring matter made from the bodies of the cochineal and kermes scale insects. The carmine stains, including cochineal, have been largely used in the past for all kinds of work, but at present they are used more par- ticularly for staining objects in bulk before sectioning, or objects which are not to be sectioned. They are easy to use, and will follow almost any fixing agent. In case of over-staining, weak 20 Animal Micrology hydrochloric acid (0.1 to 1 per cent.) is used to decolorize the tissues. For formulae see Appendix B. Hematoxylin is a compound containing the coloring matter of logwood. The hematoxylins follow well almost any of the fixing agents; they are especially recommended after fluids containing chromic acid or its salts. According to Mayer, the active agent in these stains is a compound of hematein with alumina. The hematein is produced by the oxidation of hematoxylin. The so- called “ripening” is simply this change, which is brought about by exposing the hematoxylin solution to air. If the pure hematein is used in making the stain, therefore, the latter will be ready for use immediately, because it need not undergo the ripening process (see reagent 47, Appendix B). For formulae see Appendix B. Anilin is a colorless coal-tar derivative, and is the base from which many of the numerous coal-tar dyes are made. The anilins are brilliant stains of all colors. They are used almost exclusively for staining sections or thin membranes, and are of great service to the microscopist, although, as a rule, they fade in time. The basic anilin stains, such as methyl green, methyl violet, gentian violet, methylen blue, safranin, Bismarck brown, toluidin blue, and thionin are usually nuclear stains. On the other hand, the acid anilin stains, such as acid fuchsin, eosin, erythrosin, light green, orange G, bleu de Lyon, nigrosin, benzopurpurin, and aurantia are ranked as cytoplasmic stains. These stains must be made up fresh every two or three weeks, as they frequently spoil if kept much longer. The metallic substances used for color differentiation operate principally as «mpregnations rather than as stains. The coloring matter is held physically as a precipitate or reduction product in certain of the tissue elements. The commonest reagents of this class in use are silver nitrate and gold chloride. The different tissue elements frequently show affinity for different stains, consequently it is a common practice to use more than one stain. Very decided contrasts may thus be produced, such as red and blue, red and green, green and orange, etc. It Chapter II: General Statement of Methods 21 is not uncommon, in fact, to have triple and even multiple staining. In such staining, the stains are sometimes applied consecutively ; in other cases, at different points in the process of general manipu- lation. Sometimes all the stains may be mixed together, so that immersion of the sections in one liquid is all that is required for double or multiple staining. A general rule in staining, especially for entire or bulky objects, is that the specimen should be transferred to the stain from a reagent in which the percentage of water is approximately the same as that of the stain. The same is true when the object is removed from the stain. For example, if the stain to be used is an aqueous solution, the object should enter it from an aqueous solution; if the stain is made up in 95 per cent. alcohol, the object should enter from 95 per cent. alcohol, etc. For reasons see “dehydrating.” CLEARING In the vast majority of cases tissues are too opaque for satis- factory examination until they have been treated with certain clarifying reagents or clearers which render them more trans- parent. Such reagents as glycerin, glycerin-jelly, etc., are used when the object is to be cleared, without alcoholic dehydration, directly from water. Usually, for permanent preparations, the alcoholic dehydration method is employed and it then becomes necessary to use a clarifying reagent which will replace the alcohol and facilitate the penetration of the final mounting-medium (balsam or damar). Perhaps the most useful and rapid clearer is xylol. Xylol, however, is very sensitive to moisture and if the preparation has not been thoroughly dehydrated the final mount will appear milky. For this reason the beginner is recommended to use a carbol-xylol mixture (see reagent 9, chap. i). Carbolic acid has a great affinity for water, and the mixture will therefore clear preparations that are not fully dehydrated. Cedar-wood oil, though somewhat slower than xylol, is one of the best clearers. It is also one of the safest, because tissues may be left in it 22 Animal Micrology indefinitely. Other good clearers after alcohol are oil of origanum, sandal-wood oil, oil of cloves, toluol, oil of bergamot, anilin oil (for watery specimens), carbolic acid (for watery specimens ), and beechwood creasote. Clove oil should not be used for celloidin sections because it dissolves celloidin. It is also inapplicable ordinarily after most anilin dyes because of its tendency to extract them. Among the best reagents for celloidin sections are cedar-wood oil, carbol-xylol, oil of origanum, creasote, and Kycleshymer’s clearer (memorandum 4, chap. vii). While; -*clearing,” refers especially to the rendering trans- parent of tissue elements, and dealcoholization to the removal of alcohol previous to imbedding in paraffin, very frequently the same reagent is used for either purpose and the term “clearing” has come to be used in either sense. MOUNTING After tissues have been cleared the final step is to mount them in some suitable medium for preservation and inspection. If tissues are to be mounted directly from water or aqueous media, glycerin, glycerin-jelly, or Farrant’s solution is used ordi- narily. If the alcoholic dehydration method is employed, balsam or gum damar is the final mounting medium. The balsam or damar is dissolved commonly in xylol, although turpentine, chlo- roform, or benzol may be used as the solvent. Xylol-balsam is the most satisfactory for ordinary purposes. IMBEDDING In order to section tissues or objects satisfactorily it is fre- quently necessary to imbed them in a suitable matrix. Simple imbedding consists in merely surrounding the object by an appropriate medium to hold it in place while it is being cut. In tterstitial imbedding the object is saturated (infiltrated) with the imbedding substance which, when all cavities and inter- sticies are filled, is caused to set; thus it supports all parts of the tissue and holds the components in place when sections are made. Infiltration imbedding is of great importance to micros- copists and much of the space of the present book is given up to Chapter II: General Statement of Methods 23 drilling the student in the details of the two chief infiltration methods, viz., the paraffin method and the celloidin method. Infiltration with gum is also not infrequently resorted to, espe- cially for tissues which would be injured by alcohol, or for sectioning by the freezing method. Paraffin is a translucent, waxy material derived from various sources, one of the commonest of which is crude petroleum. Paraffins of low and of high melting-points, termed respectively soft and hard paraffin, should be kept on hand so that mixtures of different degrees of hardness may be made up as necessity demands. Celloidin is a form of pyroxilin (gun cotton or collodion cotton) specially prepared for interstitial imbedding. It is dis- solved in a mixture of ether and alcohol (chap. i, reagent 4) and solutions of two or three strengths are used for infiltration. For details see the method, chap. vii. Collodion instead of celloidin is used by some workers (see memorandum 11, chap. vii). AFFIXING SECTIONS When mounting sections upon a slide, especially if they are yet to be stained, it is usually necessary to affix them firmly to the slide to prevent later displacement. For paraffin sections Mayer’s albumen fixative (reagent 21, chap. i), or a combination of this method with the water method, is most widely used. The water method alone often proves adequate, particularly with thin sections. The slide is flooded with water and the sections are floated upon its surface. As the layer of water evaporates the sections are slowly drawn down into close contact with the slide. When perfectly dry they are usually so firmly affixed that they will not become detached even after the removal of paraffin from them. It is common, however, and safer to use a thin film of albumen fixative as a cementing substance between the water and the surface of the slide. In the case of celloidin sections, if only one or a few sections are to be mounted on one slide, it is a common practice to stain the sections and transfer them through the various reagents, even 24 Animal Micrology to clearing, before mounting them on the slide. In such cases the sections need not be fixed to the slide. With serial sections, however, the sections must be held in place some way during their transition through the reagents (see memorandum 12, chap. vii). Unlike paraffin, the celloidin is not ordinarily removed from the tissues. DECOLORIZING Not infrequently in staining the tissue becomes overstained and requires that some of the color be extracted from certain of the elements to bring about a proper differentiation. The fact that certain tissue elements retain stain more tenaciously than others is sometimes taken advantage of and overstaining followed by decolorization is practiced intentionally. Alcohol slightly acidulated with hydrochloric acid (0.1 to 1 per cent.) is commonly used for the extraction of surplus color. In special cases other decolorizers are used: for example, iron-alum in the iron-hema- toxylin method (reagent 18, chap. i). BLEACHING In some cases, tissues are obscured because of the presence of natural pigments or on account of blackening caused by the fixing reagent. Such tissues must be bleached. Chlorine, peroxide of hydrogen, or sulphurous acid are commonly employed. A method is given in chap. v, memorandum 12. CORROSION To obtain skeletal structures, as for example the spicules of sponges or the hard parts of insects, various methods of corrosion are employed. Nitric acid, caustic potash, caustic soda, eau de Javelle are reagents often used for this purpose. Corrosive prep- arations of injected vessels and cavities may also be made, DECALCIFICATION AND DESILICIDATION Tissues impregnated with lime salts or with silica must have such hard parts removed usually before they can be sectioned. For decalcification, one of several acids may be used. The details are given in the chapter on bone, tooth, etc. (chap. xi). For de- calcifying reagents, see Appendix B, v. Chapter II: General Statement of Methods 25 Where desilicidation is necessary hydrofluoric acid may be em- ployed, although, because of its property of attacking mucous membranes, its use is attended with more or less danger for the operator. It is added drop by drop to the tissue which has pre- viously been placed in a paraffin-coated vessel (the acid attacks glass). If the tissue is not too heavily impregnated with silica, it is safer to use an old section razor and try to cut sections with- out previously treating them with hydrofluoric acid. INJECTION METHODS The injection of colored masses into the blood vessels and other vessels of the body is frequently practiced to aid in deter- mining their distribution and their relation to the surrounding tissues. The dye is termed the coloring mass and the substance to which it is added, the vehicle. ISOLATION OF HISTOLOGICAL ELEMENTS Isolation is one of the most valuable means of forming a cor- rect conception of cells and fibers. It has the advantage over sections that the elements may be inspected in their entirety and from all sides. The separation is accomplished, as already noted, by (1) reagents which dissolve or soften cell cement and inter- stitial material without seriously affecting the cells (maceration or dissociation), or (2) mechanically by means of dissecting needles (teasing), or both. Hardening and fixing reagents in general if diluted to about one-tenth are efficient for dissociation. Gage recommends normal saline as preferable to water for dilution. The dissecting microscope or some kind of lens-holder and lens are valuable aids in isolating tissue elements. For practical methods consult chap. x; for reagents, Appendix B, iv. NORMAL OR INDIFFERENT FLUIDS FOR EXAMINING FRESH TISSUES It is desirable frequently to examine fresh material in as near a natural condition as possible, hence recourse is had to the so- called indifferent fluids. While not wholly indifferent, they ordi- narily produce but slight changes in tissues and their elements from the view-point of the microscopist. The liquids most com- monly used for this purpose are discussed in Appendix B, iii. 26 Animal Micrology GENERAL SCHEME FOR MOUNTING WHOLE OBJECTS (IN TOTO PREP- ARATIONS) OR SECTIONS Whole Objects (for balsam mounts) Killing and fixing | Washing | Staining (Decolorizing if necessary) Section Methods (paraffin and celloidin) Killing and fixing Washing (Staining, if to be stained in bulk) Hardening and dehydrating | Absolute alcohol Dehydrating Clearing Paraffin Method Celloidin Method Mounting Dealcoholization (xylol) Ether-alcohol | Melted paraffin Imbedding If not stained in bulk Sectioning Through alco- hols to stain J Affixing sections Staining Removal of paraffin | | Washing Absolute alcohol | | Dehydrating | Clearing (and decol- orizing if / Mounting necessary) | Thin celloidin | Thick celloidin bulk | | If not stained in \ Imbedding Staining Sectioning * | | Washing (and de-\ Dehydrating to colorizing if 95 per cent. al- necessary ) cohol Clearing | Mounting * If sections are to be arranged serially they must be affixed to the slide as soon as cut. CHAPTER III KILLING AND FIXING Cautions.—l. Use only fresh tissues and work rapidly so that the tissue elements will not have time to undergo postmortem changes. 2. Remove organs carefully, and avoid crushing or pressing the parts to be prepared. 3. Tissues should never be allowed to dry from the time they leave the animal until they are finally mounted for microscopical examination except at one point in the paraffin method. 4. Use only small pieces (2 to 6 mm. cube) of tissue whenever possible, or penetration of the reagent will be insufficient. Embryos and small objects up to 4 cm. in size may be placed entire in certain of the fixing fluids. 5. For fixing and hardening, the bulk of the fluid should be from 10 to 50 times that of the object. Too many pieces should not be placed in the same vial. 6. Use only clean reagents. It is well to let the object rest on a bit of cotton in the bottom of the vial or have tt suspended from the vial mouth so that the reagent may penetrate equally from all sides. Penetration is aided by heat. 7. When necessary to wash fresh tissue, it is usually best to use normal saline, and not water. Let it flow gently over the surface of the object or slowly twirl the latter in the fluid. Do not scrape off foreign matter. 8. In many cases the killing and fixing reagent does not harden the tissue sufficiently and the hardening process must be completed in alcohol. 9. Keep the reagents and preparations from direct sunlight. 10. Carefully label each vessel containing tissue. State the contents, the fluid used, and the date. Label on the side. 11. Keep a careful record on cards of the reagents used, and the time when changed, for each separate piece of tissue. 27 28 Animal Micrology PRACTICAL EXERCISE Kill a frog by placing it under a bell jar which contains a bit of cotton saturated with chloroform. Open the body as soon as possible after death and secure the tissues specified below. 1. Alcohol Fixation.—Remove the dorsal aorta and small pieces of the liver and harden in absolute alcohol (at least, not less than 95 per cent.) in a vial or small bottle. The tissue will be ready for further treatment in two days. Larger pieces of tissue require longer time. The pieces should be thin. Change the alcohol every day for the first three days. Alcohol is in many instances an unsatisfactory fixing reagent, but it is frequently employed because it is usually at hand and is easily manipulated. Hot absolute alcohol is very often used for insects. If absolute alcohol is used, the fixation may be fairly good, but because of the expense attached to the best absolute alcohol, the lower percentages are more frequently used. They shrink protoplasm, however, and are not to be recommended for the finer histological work. Ninety-five per cent. alcohol is as low as should be used for fixing, although 70 per cent. is sufficient to preserve specimens for other than microscopical work. Acetic acid (Appendix B, 2) is used with alcohol sometimes to increase penetration and to counteract its tendency to shrink tissues. The mixture is usually preferable to alcohol alone. 2. Fixing with Gilson’s Mercuro-Nitric Mixture.— Place small pieces of liver, kidney, pancreas, esophagus, cardiac and pylo- ric ends of the stomach, apex of the heart, bladder, testis or ovary, and tongue in Gilson for from two to six hours. Remove a piece of intestine about 12 mm. long, and after washing it thoroughly in normal saline place it in a small vial containing about fifty times its bulk of fixing mixture and leave it for two hours. After fixation wash the objects thoroughly in water fol- lowed by 35 and 50 per cent. alcohol (15 minutes each), and pre- serve them in 70 per cent. alcohol. Read remarks on washing out corrosive sublimate, Appendix B, reagent 13, caution 1. Gilson’s is an excellent general reagent and gives a very deli- cate fixation. It is perhaps the most satisfactory killing and fix- Chapter III: Killing and Fixing 29 ing reagent that the beginner can use. The time which objects should be left in the fluid varies from ten or fifteen minutes for very delicate objects to six hours for larger or denser tissues, although many objects may be left for thirty-six hours without injury. When an object becomes opaque throughout it is suffi- ciently fixed. This holds true of other corrosive sublimate fixing fluids. Corrosive sublimate alone is also widely used as a general reagent. See caution 2 under reagent 13 in Appendix B. 83. Fixing with Erlicki’s Fluid.— Remove a small piece of the spinal cord 1 cm. in length and place it in about one hundred times its volume of Erlicki’s fluid. Likewise place the brain in this fluid. The spinal cord must remain about five days and the brain a week or ten days in the liquid. At the end of this time transfer the object to 35 per cent. alcohol, keeping it in the dark for two hours to avoid precipitation. The alcohol should be changed occasionally during this time. Repeat the process using 50 per cent. alcohol, and finally preserve the material in 70 per cent. alcohol. Erlicki’s fluid is an excellent reagent for general use, and is especially valuable for voluminous objects such as advanced embryos. Its principal drawback is the length of time required properly to harden objects (ten days to three weeks for objects larger than the above tissues). The process may be hastened by keeping the fluid containing the tissue at the temperature of an incubator (39° C.). 4. Formalin as a Fixing Reagent.—Place a piece of spinal cord, liver, and fragments of muscle in which nerves terminate in 10 per cent. formalin and leave until needed for work later. Formalin in varying percentages is widely used for the preserva- tion and fixation of specimens for dissection. It is especially serviceable for the central nervous system. Most specimens may remain in it indefinitely without injury. For simple preservation, solutions ranging from 2 to 5 per cent. are adequate, but for fixa- tion, it should be stronger (10 per cent.). Entire human brains may be fixed and hardened in a 10 per cent. solution with fairly good results. 30 Animal Micrology MEMORANDA 1. Tissues Are Preserved in Alcohol of from 70 to 85 per cent. strength, but if they are to remain several months it is better to preserve them in a mixture of equal parts of glycerin, distilled water, and 95 per cent. alcohol. 2. Hardening.— Read carefully the remarks on hardening in chap. ii. 3. Tissues Should Not Be Left in the Fixing Agent longer, ordinarily, than is necessary to get results. Some, however, require a long time to bring out the optical differences of their elements. Experience alone can teach the time required in a given case. Such a reagent as formalin kills, fixes, hardens, and preserves, all at the same time. 4. For Transferring Small Objects through reagents the method of Walton is an excellent one. For the several reagents, he uses shell vials which measure about 10 em. in height by 3 cm. in diameter. Through the center of a flat cork which fits the vials, a hole is made and a glass tube (about 9 em. by 1.5 cm.) is inserted so that its lower end dips well into the reagents in the vials. The lower end of the tube is closed with fine-meshed cloth and the objects are placed within the tube. To transfer the objects one simply removes the cork bearing the tube, and inserts it in the vial containing the desired reagent. The upper end of the tube may be closed with a cork of the proper size. To avoid disturb- ance from changes in air pressure a small hole should be bored in the side of the tube just below the lower level of the larger cork. The vials are supported as indicated in memorandum 5. 5. Sheil Vials, Small Bottles, etc., when in use are best supported in shallow auger holes of proper size in thick blocks of wood. 6. Material Which Is To Be Kept Indefinitely should be put in tightly stoppered vials in a place away from strong light. It is best to pack the vials in a museum jar on cotton and then seal the jar securely to prevent evaporation. Material is even more secure if the museum jar is partly filled with aleohol; in such a case each small vial should have a label of the contents placed within it. Another way to prevent evaporation from vials or bottles is to “‘cap” them with a suitable varnish (see 7). 7. To Seal Bottles and Preparation Jars (“‘bottle-capping”) dip the stopper and part of the neck in collodion varnish made as follows: Pyroxylimy fe. acu! ot aa ee ee eee OZ Ether eee rr TS GMS) out! tor a OCA AN CONO]G: 2 ac hws yes. Sa og ec ne ORO Zed When the pyroxylin has completely dissolved add 2.5 drams of camphor. (From Pharmaceutical Era, Vol. XXX, p. 528.) Chapter IIT: Killing and Fixing 31 8. For the Preservation of Anatomical Specimens for other than histo- logical purposes, Galt (The Lancet, Nov. 16, 1901, p. 1334) recommends the following fluid as superior to the well-known Kaiserling’s fluid. Serchinim calomel: 66 6 6 «© o o 5 0 6 6 0 OD joeumls IPotassiumenitratew) ween nme os ell pant Chulorllonsbem 5 sb Gove o& 6 6 & @ seo Jhjoniee WVUC tania eum msl eit kel ascicst ate ra scete st. p LOO parts Wash fresh tissues for several hours in running water, then “set” in an excess of methyl alcohol to which 0.5 per cent. formalin has been added (time required: six hours to a week according to nature and size of spe- cimen). Next transfer the specimen directly to the preserving fluid, changing the latter after two or three weeks if necessary. In case the preparation is not sealed, sufficient water to make up loss by evaporation must be added occasionally. Specimens are said to retain their natural colors. The following mixture, recommended to the author by Professor Kineaid of the Washington State University, has given most excellent results. To a mixture of equal parts of glycerin and strong alcohol sufficient formalin is added to make the whole about a 2 per cent. forma- lin. Specimens remain perfectly flexible in this mixture, and, indeed, after they have become thoroughly saturated, many forms (crustacea, insects, etc.) may be removed and kept as dry specimens which still retain their flexibility. ial ry “i iy meh rf see 7. a ° : Yi wa ie aia nia z a A see ae a BIN a ii or Pei A aay A a ae as) iy ma ss ie we re ay he es a is , My oe i " leet a ms Mh vee ae : ie ai = Hi te bat a. a nea bee a) 7 pie aby, eae: : Tuy: oo tan : a if ‘ a 4 7 = : 7 al ie ai b he my, a Taha Bea, (ag res Ais Oia akan ae : ) 2a , ' Fy a 7 a a a ! WR oe ; eet, Vie m | ; pee a a hes ft ey id a) ’ ma a ae es Tah oa _ % at iss iV ian L ‘ oT] Lited pie a pine - a 4 “a = a » a .8 PW | - 70 c 4 XO é 7 a : Rite doe nar #4 ry it mare oe hk : a mre cee ee at Aa: nak meer p tego 7! 6 . oO ‘ ae a 7 ‘ tN aN aD 2 - BY Wr Wied i, aN - ; Up ‘af : ¥ ihe 7 a ii : we) 7 wee wh : > La 7 ; r see wa a ’ “Téa 7 BF, ' 7 » 4 : ai : > in a nl 7 eal. 7 «4 iy) > At te OUR oe J oe 7 Li rine Te pis es the gd Ds. @). » See ire i is J* gO CHAPTER IV SIMPLE SECTION METHODS FREE HAND SECTION CUTTING This method is important because it requires no costly appli- ances; although the sections are not as accurately cut as when mechanical aids are used, the method is simple, rapid, and adequate for the more general histological and pathological work. 1. The section razor is flat on one side (the lower), and hollow ground on the other (Fig. 15). It must be sharp. 2. A shallow glass dish or watch-glass partly filled with water is also necessary. Before making a section dip the razor flatwise into the liquid, or use a camel’s hair brush; see that the upper surface is well flooded. 3. Sit in such a way that the fore-arm may be steadied against the edge of the table. 4, Use a piece of liver which was fixed in formalin, first rinsing it in water. Take the tissue between the thumb and forefinger of the left hand, and hold it in such a way that a thin slice may be cut by drawing the knife along the surface of the forefinger. 5. Rest the flat surface of the knife upon the forefinger, and, beginning at the heel of the knife, carefully draw the blade toward you diagonally through the tissue, slicing off a thin section of as uniform thickness as possible. 6. As each section is cut, float it off into the water; if it adheres to the blade, remove it by means of a wet camel’s hair brush. 7. Practice until very thin sections are obtained, then place the dish upon a black surface, and with a needle or section lifter transfer the thinnest and best sections, if only fragments, to a watch-glass containing water. Note.—In case the tissue has been preserved in alcohol, cut the sec- tions under 70 per cent. alcohol instead of water, then transfer them to 50 and 35 per cent. alcohol successively and finally to water, leaving them in each liquid from 3 to 5 minutes. 33 34 Animal Micrology 8. Next, place the sections in about 3 c.c. of Delafield’s hema- toxylin diluted with an equal volume of water, and leave them for various lengths of time (3, 7, 12 minutes) to determine the time for successful staining. 9. Transfer the sections from the stain to tap water, and gently move them about for from 5 to 10 minutes to wash out the excess of the stain. If the sections are still overstained, place them in 5 c.c. of distilled water to which 3 drops of acetic acid have been added. Leave for 5 minutes, or until they become lighter in color, then wash in several changes of tap water until they have again become blue. 10. Remove the sections from the water and transfer them through 35, 50, 70, 85, and 95 per cent. alcohol successively, leaving them from 3 to 5 minutes in each, and lastly transfer them to absolute alcohol for 10 minutes, and finally to carbol- xylol for 10 minutes, or until clear. 11. Select one or two of the best sections and transfer them to the center of a clean glass slide. After straightening them out properly, drain off the excess of the carbol-xylol, and before the sections can become dry, add a drop of Canada balsam. Carefully lower a clean cover-glass (for cleaning see memorandum 14, chap. vi) on to the balsam. There should be just sufficient balsam to spread evenly under the cover without exuding around the edges. 12. Label, stating card number, name of the preparation, and other data that it is desired to add (see chap. vi, i, step 10). 13. Carry one of the pieces of stomach prepared in Gilson through the same treatment. The sections should be transverse sections of the stomach wall. 14. Clean up all dirty glassware immediately. MEMORANDA 1. The Thinnest Sections are not always the best. For a general view of an organ, large, comparatively thick sections are usually better; for details of structure, thin sections. 2. Small Pieces of Tissue may be cemented to a cork if too small to hold conveniently between thumb and forefinger. A piece of stout copper wire is heated for a moment in the flame and touched to a bit Chapter IV: Simple Section Methods 35 of paraffin. As the paraffin melts transfer drops of it.to the edge of the tissue, which has been previously placed on the cork. The paraffin cools and holds the tissue fast. Another and better method of handling a small object is to imbed it in a piece of hardened liver. In sectioning, the liver as well as the object is sliced, but they readily separate when placed in alcohol. Beef liver or dog liver is prepared for such purposes by hardening pieces about 5x22 cm. in size in 95 per cent. alcohol for 24 hours, and then trans- ferring to fresh 95 per cent. alcohol until needed. When much hand sectioning is to be done, a supply of hardened liver should be kept on hand. Many small objects may be held between pieces of pith, and successfully sectioned. 3. Well Microtomes (Fig. 27) are inexpensive instruments which are used for simple sectioning. Such a microtome consists of a tube in which the object is placed, and at one end of which is a plate to guide the razor. The other end is provided with a screw, which, when turned, pushes the contents of the tube above the plate, thus making it possible to cut sections of a uni- form thickness. The object to be cut must be firmly fixed in the well. Such tissues as kidney, liver, spleen, hard tumors, cartilage, etc., may be held sufficiently rigid by wedging small slabs of carrot, turnip, pith, or hardened liver in about them. These supporting substances must, of course, rest squarely against the bottom of the well. Soft tissues, such as soft tumors or brain, must be imbedded. Three parts of paraffin and one part of vaselin melted together and thoroughly mixed makes a very good imbedding-mass for a well microtome. To imbed, warm the microtome slightly and fill the well with the imbedding mix- ture. Remove all liquid from the surface of the tissue, and pass it below the surface of the mixture just as it begins to harden around the edges. When the imbedding mass has become cold the sections are cut in the ordinary way. 4, Temporary Mounts may be made directly from water after staining by using glycerin as a mounting-medium. Transfer the section to the slide, add a drop or two of glycerin, and a clean cover-glass. Fic. 27.—Well Microtome. a PX *¥P a F sy 7K 0 i oe oe OE pee Ce ate A i Li WL SO oli re M » Hae as i ro; ij ne on wae mi, ay! Ay Stith mate, fon Tou rm Hai Cm me, ae fe eee init a ride ial LD abil i in Qa : ee ee 1 on ae a atin Oe eyo een ale a a a a 7 ie aay a? katt ee - aie ign? i = " m Ata i Pi? : x ee - na a -—- Pine . eT folk i) ; At . hen " Ngee : ae ie P. Ne ny i mie Aa Aas os ae My a ; ata Saha Te ie Lee ae ie 299 pr — ey 7 GIS ETS ES Na ee a a CR bar ve ; Pa ’ a Cos ae " ‘ us nie 1 cee Vie Baie s Sei qr ke a7 ve Mere : VO OE a ae a [ dy wn te ‘Ne ie iat oa - ta Rank _ ey \ ae +~s is Ths ry Wf) as feat “ltt Sred / ace © nie: ary _ eae : Temi sve a a ri DL a ey ayer aa ve Eee | wig es ar, wen : as r x a Wines Row ate ha part 4 ’ Noe: - hate ee yt iy iw. Uae if bs aon y ee wae ae i oueli Apu tare ay _ yet See a ia a tie ir, a i 4 : - i) ial i. oh. 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Fin ea) iia’ Sh On Pin) Ea an sul ia b. ip is oy : aay Cee : CHAPTER V THE PARAFFIN METHOD: IMBEDDING AND SECTIONING 1. From 70 per cent. alcohol take a small piece of intestine (6 mm. long) fixed in Gilson, and also pieces of kidney and tongue, and proceed according to the following schedule. Keep accurate records on your cards. 2. Ninety-five per cent. alcohol, 30 to 45 minutes. A longer time will do no harm. 3. Absolute alcohol, 45 minutes. Before transferring to absolute, remove the excess of 95 per cent. alcohol from the object by touching it with a piece of blotting paper or a clean cloth. 4, Xylol, 2 hours or until the object looks clear. It may be left several hours. Rapidly remove all excess of xylol before proceeding with step 5, but do not allow the tissue to become dry or dull looking. 5. Melted paraffin (melting-point about 53° C.), 2 hours. The object may be left an hour or two longer, but it is best to avoid as much as possible subjecting tissues to an elevated temperature. Shift its position in the paraffin once or twice to facilitate penetration of the latter. Cautions.—a) Do not have the bath too hot. Cooked tissues are worse than useless. b) To keep material clean, it is well to have a false bottom of paper in the vessel containing paraffin. Make this by swinging a strip of white paper into the cup so that the loop of the paper is submerged in paraffin and the ends attached on either side to the mouth of the cup. 6. Prepare paper boxes according to the following instructions: A small rectangular block of wood or a stick with a flat end measuring approximately 15x20 mm. is used. Cut a strip of stiff paper so that it measures about 4x7 cm. Place the flat end of the block in the center of the paper with its long diameter coinciding with the long diameter of the 37 38 Animal Micrology paper. Fold the narrow side margins of the paper up along the sides of the block first, then do likewise with the ends of the paper. Turn the ears which have been formed at each corner back over what is to be the end of the box, and then fold the long end of the paper back to hold the ears in place, and also to make the end of the box of the same height as the sides. Manifestly, any size of box may be made by varying the size of the block. With a little practice, the same kind of box may be: folded without the use of a wooden block. 7. With a warm, wide-mouthed pipette transfer sufficient melted paraffin to a paper box to cover the bottom, then, with warm forceps, remove the tissue to the box. Next, fill the box with melted paraffin. Orient the object with heated needles if necessary. As soon as the paraffin has congealed sufficiently for the surface to become opaque, cool it rapidly by plunging it into cold water; otherwise, the paraffin will crystallize and become unsuited for sectioning. Cautions.—a) Tissues must be oriented (i. e., placed in proper position for cutting) while the paraffin is still in liquid condition. Arrange the tissue so that it will be cut at right angles (trans- verse) or parallel to the surface of the organ. Avoid oblique sections as they are very puzzling. For present purposes of practice cut transverse sections. b) If whitish-looking patches are present in the block after imbedding they are due to xylol which has been carried over into the paraflin. If they occur in the immediate vicinity of the ob- ject, the block should be placed in the bath again until melted, and the object be reimbedded. c) Be sure that every piece of tissue is marked after it is im- bedded. Tissues are sometimes kept in paraffin for months or even years before they are finally sectioned. To mark, scratch the number of the record card in the paraffin, or better, write it on the paper box and leave the box in place. CUTTING SECTIONS 8. Study the paraffin microtome (e. g., Fig. 28); identify the parts and learn how the thickness of sections is controlled. 9. Proceed with the block of paraffin containing the intestine. Make it fast to the carrying disk of the microtome in the follow- Chapter V: The Paraffin Method 39 ing manner: Remove the disk from the machine and by means of a heated steel spatula or copper wire flattened at one end, melt a small chip of paraffin on to it. Likewise warm the end of the paraffin block and quickly press it into the melted paraffin on MM Lr J PP Smee = iii Tt il ff : ff ) | ! | H 1 | = at =a Fig. 28.—Minot Automatic Rotary Microtome. The object carrier is adjustable in three planes and is perfectly rigid. The knife carrier is also adjustable and extra heavy and solid. The feed is controlled by an adjustable cam, giving cuts of any number of microns in thickness from 1 to 25. By means of an automati- cally closing split-nut the carriage is returned to the beginning position after the screw is fed out the entire length. the disk. Cement it firmly in place by means of the heated wire or spatula and cool in water. 10. With a sharp scalpel trim the free end of the block so that it presents a perfectly rectangular outline (however, see caution c). The length should exceed the breadth by at least one-fourth. Cautions.—a) In trimming do not cut farther back than the base of the object. This leaves a wide shoulder for support. b) Leave a margin of about 2 mm. around the object. 40 Animal Micrology c) To avoid reversing sections in mounting, it is frequently advantageous to have the imbedding mass trimmed unsymmetri- cally. The edge which first comes in contact with the knife is left longer than the opposite edge. One may thus readily dis- cover when a section or part of a series has been turned over. 11. Mount the object firmly in the microtome. It should just clear the knife. The flat end-surface of the paraffin block should Fie. 29.—Minot-Blake Microtome, designed especially for cutting thin sections. Manu- factured by Buff & Buff My’y Co., of Boston, Mass. be parallel to the edge of the knife, and the block so oriented that in cutting, the long edge will meet the edge of the knife squarely. (12. Place the knife in position with the handle to the side away from the wheel (if a rotary microtome is used). By means of the adjusting screws tilt the cutting edge slightly toward the object so that the side of the knife will not remain in contact with Chapter V: The Paraffin Method 41 the paraffin block after a section has been cut. If the knife has a flat under surface it requires more tilt than if the surface is hollow ground. Fora flat under surface the tilt should be about 9 degrees from the perpendicular. See that the knife is held firmly in place. Caution.—The knife should be kept in its case when not in the machine. The edge is very easily injured. 13. Set the regulator so that the microtome will cut sections about 10 microns thick. A micron is one-thousandth of a milli- meter. 14. Unloose the catch which locks the wheel and revolve the wheel with the right hand. agree 1“ de T CHAPTER XV BACTERIA No attempt is made here to give even an elementary account of bacteriological technique. Only such phases of the work as are concerned with the immediate microscopical examination of bacteria are touched upon, and these chiefly to afford some practice in this kind of manipula- tion. For special technique, identification, or descriptions of apparatus and accessories, the student is referred to standard textbooks. BACTERIAL EXAMINATION Bacteria when prepared for microscopical examination are in the form of A. Cover-glass preparations, B. Bacteria in tissues (section method), or C. Hanging-drop preparations. A. Cover-Glass Preparations I. Killing and fixing. 1. From Fluid Media (e.g., bouillon, milk, water, saliva, blood, pus, etc.).—Sterilize a platinum wire loop by heating it red hot in a flame. When cool, touch the loop to the culture and spread the adherent bacteria in a thin film over the surface of a cover-glass which has been sterilized in a flame. After the film has dried in the air, kill and fix the bacteria to the cover by passing it three times, film side uppermost, through the apex of a flame. Each time should not exceed half a second. Prepare several films from a given material. Coronet or similar forceps (Figs. 38, 39) should be used for handling such films, because the cover- glass can be left in them through the entire operation of fixing and staining. If a platinum loop is not at hand a second cover-glass may be used to spread the smear. The first cover-glass is held in a pair of cover-glass forceps and the second cover-glass is dropped on to it. The glasses are then rapidly drawn apart with a sliding motion by means of forceps. The glasses should not be pressed tightly together. Proficiency in making such preparations is gained only after considerable practice. The chief secret in making a good preparation is to get the films extremely thin and evenly distributed. 2. From Solid Media (gelatin, agar, meat, potato, animal tissues and organs, etc.).—The procedure is the same as for 1, except that a drop of 105 106 Animal Micrology sterilized water or bouillon is put on the cover-glass to facilitate the spreading of the bacteria in a film over the cover. Il. Staining and Mounting. 1. Gentian violet (memorandum 8a) 5 minutes. The cover-glass is left in the forceps, film side up, and the film flooded with the staining fluid. | i Fia. 38.—Cornet’s Cover-Class Forceps. 2. Rinse in water. 3. Gram’s solution (memorandum 38f) until the color becomes black (2 to 83 minutes). 4, Ninety-five per cent. alcohol until the violet color has almost com- pletely disappeared. 5. Rinse in water and examine by placing the cover-glass film side downward on a slide. Only a thin film of water should remain between the slide and the cover. Remove surplus water by means of blotting paper. Ifa prolonged examination is to be made, water lost by evapora- tion must be replaced by occasionally placing a small drop of water at Fie. 39.—Stewart’s Steel Wire Cover-Glass Forceps. the edge of the cover. In ordinary work the final inspection is frequently made at this stage. If a permanent preparation is desired, however, proceed with the following steps: 6. If the bacteria are well stained, a counterstain of Bismarck brown (memorandum 34d, sol. 2) may be added (5 to 10 seconds). This step may be omitted. 7. Absolute alcohol, 10 to 15 seconds, 8. Xylol. 9. Xylol-balsam. Norte.—In staining, if the cover-glass is warmed over a flame some 15 or 20 seconds until the stain steams, the action of the stain is usually more intense and more rapid. Boiling, however, must be avoided. Chapter XV: Bacteria 107 ’ B. Bacteria in Tissues Tissues may be fixed and hardened (e. g., Gilson’s fiuid, Appendix B, reagent 15; or Zenker’s, reagent 6; or formalin, reagent 17) in the ordinary way, and sections made by the usual methods. Where practi- cable paraffin sections are preferable to celloidin sections, because the celloidin tends to hold the stain and thus obscure the bacteria. Sections should be fixed to the slide (paraffin by albumen fixative, celloidin by ether vapor). Bacteria which do not stain by the Gram method (memorandum 3/) or the tubercle bacillus method (memorandum 38e) are difficult to demon- strate, because it is hard to stain them so as to differentiate them from the tissues in which they lie; furthermore, most of them easily lose what- ever stain they may have taken up. Lé6ffler’s alkaline methylen blue (memorandum 30) is, perhaps, the most useful stain for these organisms. Methylen Blue Stain for Bacteria in Tissues.—1. Stain sections (paraffin) 30 minutes to 24 hours. 2. Acetic acid (1 to 1,000 of water) 10 to 20 seconds. 3. Rinse in absolute alcohol 20 to 30 seconds. 4. Xylol. 5. Xylol-balsam. With celloidin sections substitute 95 per cent. alcohol for absolute (step 3), then treat with xylol or, better, carbol-xylol until sections are clear. Mount in xylol-balsam. Anilin gentian violet, methyl blue, methyl violet, or fuchsin (memo- randum 8a), also carbol-fuchsin (memorandum 3c) may be used in the same way. Gram’s Method for Bacteria in Tissues (Weigert’s modification).— 1. Stain sections (any kind) in lithium carmine 2 to 5 minutes. Lithium Carmine (Orth’s) : CarminOw teats cis n eaed sik Gehdu dl vote. Mauna, dee aah 2.080 OD Prams, Carbonate of lithium, saturated aqueous solution 100 c.c. aithya01@ Garey eet en es) wan ee CE Y Stallor two: Filter. 2. Anilin gentian violet 5 to 20 minutes (celloidin sections should first be dehydrated in 95 per cent. aleohol and affixed to the slide with ether vapor). 8. Rinse in normal saline. 4, Gram’s solution (memorandum 3f) 1 to 2 minutes. 5. Rinse in water. 6. Blot sections with filter paper to remove as much water as possible. 7. Anilin oil, several changes. The oil dehydrates, and at the same time decolorizes the celloidin. 8. Xylol, several changes. 9. Xylol-balsam. 108 Animal Micrology C. Hanging-Drop Preparations 1. Aslide with a concave center is used (Fig. 40). With a fine-pointed brush paint a narrow strip of vaselin around the margin of the concavity. The vaselin makes the cover-glass stick to the slide and also prevents evaporation. 2. Place a small drop of the fluid containing bacteria in the center of the cover-glass. If the bacteria to be examined are on a solid medium the “drop” should be made by aS] ~=mixing a small portion of the growth with a drop of bouillon, normal saline, or serum. Place the cover-glass, drop downward, over the depression in the slide and press it down well into the vaselin. 3. Use only a small opening in the diaphragm when examining the bacteria, in order to get as much contrast by refraction as possible. Focus first with a medium-power dry objective on the edge of the drop, then employ the oil immersion. Such unstained organisms are fre- quently difficult to find and there is great danger of breaking the cover- glass with the objective. Hanging-drop preparations are used mainly in determining the motility of bacteria, or in the study of spore formation. For the latter purpose, the slide and cover-glass must be carefully sterilized and the sealing with vaselin complete. The preparation may then be placed on a warm stage or in an incubator and examined from time to time. Fia. 40.—Culture Slide. MEMORANDA 1. The Main Points to Be Observed in the Microscopical Examination of Bacteria are as follows: (1) form of the individual, whether spherical (coccus), spiral (spirillum), or rodlike (bacillus) with end square, pointed, or rounded; (2) uniformity in size; (3) the arrangements of individuals whether single (micrococci, etc.), in pairs (e. g., diplococci), in chains (e. g., streptococci), groups of four (e. g., tetracocci), cubical groups of eight or more (sarcinae), or small grape-like bunches of various-sized cocci (staphylococci); (4) presence or absence of cell-wall, gelatinous capsule, ete.; (5) motility in living forms (do not confuse with Brownian movement); (6) reaction to stains; (7) presence of spores which are rec- ognizable as bright, highly refractive rounded bodies. 2. Material for the Demonstration of Bacteria (coccus, bacillus, spirillum, and beggiatoa forms) will be found in abundance in foul water, espe- cially when contaminated with sewage. By scraping the inside of the cheek such forms as Leptothrix may often be found. Make a cover- glass preparation; kill and fix in the flame in the ordinary way; stain in Chapter XV: Bacteria 109 methyl violet, gentian violet, or fuchsin (basic) and, if desired, counter- stain lightly with Bismarck brown; examine in water or dehydrate in absolute alcohol, clear in xylol and mount in balsam. To demonstrate bacteria in tissues, a mouse may be inoculated with anthrax, and paraffin sections of the spleen prepared. Stain by the gentian violet method. 3. Some of the Most Important Stains for Bacteria are as follows: a) Anilin water solution of gentian violet (Koch-Ehrlich’s).— Gentian violet, saturated alcoholic solution. . . 10c.c. Anilin water (see Appendix B, reagent 29) . . . 100c.c. After shaking it, the mixture should be set aside for 24 hours because of the precipitation which takes place soon after making. Solutions of fuchsin (basic) and methyl blue are made in the same way. These solu- tions begin to decompose after about 10 days and must then be freshly prepared. They yield good results with many species of bacteria. The gentian violet, particularly, is widely used in connection with Gram’s method (see /). b) Alkaline meth, len blue (Loeffler’s).— Methylen blue, saturated alcoholic solution . . 30c.. Caustic potash, aqueous solution (1: 10,000). . . 100c.c. This stain keeps well and is one of the most widely used of the general stains. It is especially serviceable in staining the bacillus of diphtheria or of glanders. c) Carbol-fuchsin (Ziehl-Neelson’s).— Fuchsin, saturated alcoholic solution . . . . . 10c.e. Carbolic acid, 5 per cent. aqueous solution . . . 90c.c. This stain keeps well, stains powerfully, and can be used on many forms of bacteria. d) Neisser’s method for the diagnosis of Diphtheria.— Solution I. Methylenwolwe:(Gritlblers)s vse eeeeenn nnn 1 gram Alcohol, 96 per cent. . . ssh LOLCIC. Distilled water (add after he tasthvlcn blue has @issolivedsinatheyalcoholl)i) as -enente ncn OOOlG:c: GiR Orel EGNMO AGG 6 5 ois «6 6 6 6 6 o o of BOO Solution IT. Bismarck brown . . : 1 gram Distilled water (should ie poiline wheal fhe Big: Mmarcksbrowns/ad Ged) else el mene DOOLGC! Cover-glass preparations are stained for from 2 to 3 seconds in Solution J, rinsed in distilled water, placed in Solution IT for from 3 to 5 seconds, 110 Animal Micrology rinsed again in water, and examined in the ordinary way. The bacteria of virulent diphtheria should appear as pale-brown rods, some of which show at one or both ends bluish-black oval bodies of greater diameter than the rod. Such dark bodies will not be seen in the pseudo-diph- theria bacilli. The bacilli must have been grown for from 12 to 18 hours on Léff- ler’s blood-serum which is a mixture of glucose bouillon 1 part and beef- blood serum 3 parts. The mixture is run into test-tubes and coagulated at 100° C.; the tube should be tilted to one side to give a slanting surface for culture purposes. The formula for glucose bouillon is as follows: dry glucose, 10 grams; Liebig’s extract of beef, 3 grams; peptone, 10 grams; sodium chloride, 5 grams; water 1,000 c.c. e) Gabbet’s solution for demonstrating tubercle bacilli.— Methylen blue? ..8 <4. ihn 8 tO prams Distilled water ... Wah we eh haa eey Immersion Objective.—A kind of objective in which a liquid is used between the front lens and the cover-glass. Cedar oil is the most widely used medium. In as much as the optical properties of cedar oil (refrac- tion and dispersion) are almost the same as crown glass it is often termed a homogeneous immersion fluid. A homogeneous immersion lens, therefore, would be one intended for use with such a fluid. The advan- tage of an immersion over a dry lens lies in the fact that, other things being equal, after leaving the cover-glass rays which would be so refracted in a rarer medium like air as to miss the front end of the objec- tive, reach this lens in the case of immersions and traverse the objective. With homogeneous immersions the rays of light are carried without deflection through cover-glass and fluid and into the glass of the front lens. Water has a greater density than air and less than glass, hence, with a water immersion more rays of light reach the front lens than with a dry lens, and less than with a homogeneous immersion lens (Fig. 67). The effect of an immersion is practically to widen the angle of the lens (see angular aperture). AIR WATER. ue Magnifying Power.—The power of a lens to multiply the apparent dimensions of an object viewed through it. It should be expressed in diameters not in areas. While magnifying power is very important it is only so in connection with resolving power. If high power were the only essential, a series of single lenses might be used. The impossibility of using such a series for high Fic. 67.—(From Bausch, “‘Manipu- aoniheati is . nie at pr 7 jntion of theMuseoseope.”) magnification is due to the fact that proper OIL. m1 Appendix A: The Microscope and Its Optical Principles 153 correction of aberrations cannot be made, and consequently, a distinct image cannot be obtained. For determination of magnification see micrometer. Mechanical Stage.—A stage attachment (Fig. 68) for the more accurate manipulation of an object or a series of objects which must be moved about under the objective. The best mechanical stages are provided Fia. 68.—Attachable Mechanical Stage. with scales and verniers so that an object once recorded may be easily found again. They are often very serviceable, especially with high powers. Micrometer.—A scale for measuring objects under the microscope. The stage micrometer consists of a finely divided scale (45 and 7}5 mm.) ruled on glass or metal. It is commonly mounted on a glass slide of standard size. To determine the actual size of an object with the stage micrometer, it is most convenient to use a camera lucida. The outline of the object to be measured is projected on to a sheet of drawing- paper and marked off. The object is then replaced under the micro- scope by the micrometer and the micrometer scale is projected on to the paper. Knowing the actual distance between the lines on the microm: 154 Animal Micrology eter scale, the magnification as well as the real size of the object is readily calculated. The size of the image projected onto a piece of drawing paper at the level of the table, however, does not represent the true magnifying power of the microscope. The latter is really considerably smaller if the micro- scope is ina vertical position because the magnification of a lens or a system of lenses is calculated in terms of the conventional distance of vision (250 mm., see page 144) while the distance from the ocular to the table is considerably more than 250 mm. Since the rays of light diverge j ausch 8: Aomb Gptical Ev. ROCHESTER, N.Y, Fic. 69.—Filar Micrometer. after leaving the ocular, manifestly, the projected image will be larger at the level of the table than at a level just 250 mm. from the point of emer- gence of the rays from the ocular. To determine the actual magnifica- tion of the microscope, therefore, one would have to bring the drawing surface to within 250 mm. of this point of emergence, sketch the pro- jected scale of the stage micrometer on the paper, and then, by means of an ordinary metric rule, compute the number of times the divisions of the micrometer scale have been magnified. The standard distance of 250 mm., if the Abbe camera lucida is used (with camera mirror at 45°), includes the distance along the mirror-bar from the optical axis of the ocular to the mirror, plus the distance from the mirror to the drawing surface. In practical work it is not necessary to make drawings or measure- ments exactly at this standard distance; one needs only to have a scale made out for the distance from the camera lucida at which the drawings are actually to be made, although it must be carefully borne in mind that any variation in the elevation of the drawing surface will alter the size of the projected image. A series of carefully prepared scales for Appendix A: The Microscope and Its Optical Principles 155 various combinations of objectives and oculars should be made and kept for future use. On each should be recorded the tube-length used, the number of the objective and of the ocular, the length of the camera mir- ror-bar, and the angle of the mirror, for if any one of these is changed the scale is no longer accurate. When much measuring is to be done an ocular micrometer is used. It consists of a circular glass disk with a scale ruled on it and is inserted in the ocular between the eye-lens and the field-lens. By means of a stage micrometer the value of the divisions of the ocular micrometer is determined for a known tube-length and every combination of lenses it is desired to use in the work of measurement. Suppose that it takes four divisions of the ocular micrometer to correspond to one of the finer divi- sions of the stage micrometer, then since the divisions of the latter are equal to 7+> mm., each space in the ocular micrometer must be equal to a}omm., that is 0.0025 mm. A filar or screw micrometer is a more conven- ient form of ocular micrometer which is provided with delicate movable spider lines that can be adjusted to the space to be measured by means of a fine screw with very accurately cut threads (Fig. 69). At the end of the screw is a graduated disk which gives the value of the distance between the spider lines. The pitch of the screw is either sy inch or 0.5 mm. Micron.—The one-thousandth part of a millimeter; expressed briefly by the Greek letter ». It is the unit of measurement in microscopy. Mirror.—The compound microscope is usually provided with both concave and plane mirrors, which may be rotated or swung in any direc- tion. The plane mirror is used with the condenser, the concave, when- ever it is of advantage to have light concentrated upon the object with the condenser out. The mirror should be capable of being moved up or down the mirror-bar so that it can be accurately focused upon the object. See also tllumination. Muscae Volitantes.—Small filaments or specks which float across the . field of vision. They are really small opacities in the vitreous humor of the eye. Numerical Aperture.—A system which expresses the efficiency of an objective by indicating the relative proportion of light rays which trav- erse it to form animage. With the introduction of immersion objectives, it became evident that angular aperture alone is not sufficient to indicate the real capacity of an objective. For instance, an immersion and a dry lens may be of precisely the same angular aperture and yet the immer- sion lens is more efficient because it sends more rays of light through the objective (see immersion lens). It was found necessary to take cogniz- ance of the medium which intervenes between the cover-glass and the front lens of the objective. 156 Animal Micrology Professor Abbe, in 1873, proposed the name numerical aperture and introduced the formula N. A.=n sin u in which vn signifies the refractive index of the medium between cover-glass and objective, and w equals half the angle of aperture. That is, by multiplying the refractive index of the medium by the sine of half the angle of aperture, the numerical aperture is obtained. For example, suppose that one had an oil-immer- sion lens of 90 degrees angular aperture, then half the angle of aper- ture is 45 degrees, and by turning to a table of natural sines, the sine of 45 degrees is found to be 0.707. The refractive index of cedar oil is 1.52. Then N. A.=1.52<0.707=1.075. Suppose that the lens were a dry instead of an immersion lens; then since the refractive index of air is 1, the for- mula would read N. A.=1x0.707=0.707. Thus the two products 1.075 and 0.707 respectively, represent the relative capacities of an oil immer- sion and a dry objective of 90 degrees angular aperture. Parfocal._—_A term ordinarily applied to eyepieces of different powers that may be exchanged in the microscope without very materially affect- ing the focus of the instrument. The term is also applied to objectives attached to a revolving nosepiece if each is approximately in focus when turned into place. Pedesis. Same as Brownian movement. Penetration.—The quality of an objective that permits of “looking into” an object having sensible thickness. It is greatest with low powers and narrow angles and is antagonistic to resolving power. It is the nat- ural consequence of certain conditions in the making of lenses and is reckoned of secondary importance, because practically the same results are obtained by manipulating the fine adjustment. Polariscope.—As used in microscopy the polariscope consists of two parts, each composed of a Nicol prism of Iceland spar; one, the polar- izer, fits into the substage, and the other, the analyzer, is inserted between the objective and the tube of the microscope or, in some forms, just above the ocular. The polariscope is used more in chemical and in geological than in histological work. Some of the uses are as follows: determining whether an object is singly or doubly refractive; detecting the presence of minute crystals; determining the composition of rocks; examining sections of bone, hoof and horn, hairs and fibers of animals and plants, starch, ete., for certain characteristic and striking effects. Resolving Power.—The quality of an objective which enables the observer to make out fine details of structure. It is the most essential property for precision in observation, and determines largely the excellence of an objective. Resolving power depends upon careful correction of aberrations, general accuracy in the mechanical construction of the microscope, and upon the aperture of the objective (see angular aperture, Appendix A: The Microscope and Its Optical Principles 157 numerical aperture). Resolving power is tested by the resolution of fine parallel lines ruled on glass or the striae on the surface of diatoms. The test is to determine how many lines to the inch or centimeter may be distinguished, and whether the objective simply glimpses the markings or whether it resolves them clearly. The wider the angle of aperture, the better the resolving power, provided the width is not so great as to inter- fere with the correction of the lenses. The increased resolution of immer- sion lenses is due to the fact that the immersion fluid practically widens the angle of aperture (see immersion objective). Tube-Length.—The distance between the places of insertion of ocular and objective into the tube of the microscope. There are two standard tube-lengths; the short standard is 160 mm. (6;% inches), the long standard, 216 mm. (8;°5 inches). Many makers, however, do not adhere to the standards. The optical efficiency of the instrument is the same in either case. The short length is more advantageous in that it is more compact. The lenses must be corrected for the length of tube. with which they are to be used. The short standard is in use in most American laboratories. Overcorrection and Undercorrection.—In correcting for chromatic aberra- tion, if the coneave lens is stronger than is necessary to neutralize the aberration of the convex lens, the blue rays are brought to focus beyond the true principal focus of the objective, and the latter is said to be over- corrected; if the concave lens is not strong enough, the result is what is known as undercorrection. In case of overcorrection, the object takes on an orange tint if, after focusing, the distance between object and objective is slightly increased; or it becomes of bluish color if the distance is decreased. In case of undercorrection just the reverse is true. In some instances the objective is purposely undercorrected, and the eyepiece (e. g., compensating ocular) is equally overcorrected. Working-Distance.—The distance between the front lens of the objective and the object when the latter is in focus. With high powers it is very small, so that with some oil-immersion objectives if a thick cover is used it is impossible to focus upon the object. For this reason thin cover- glasses (No. 1) should be used on preparations which are to be used with high-power immersion lenses. MANIPULATION OF THE COMPOUND MICROSCOPE 1. Always handle the instrument cautiously; it is a delicate mechanism. Lift it by the base, not by the tube or the arm. 2. The work-table should be of such a height that the observer can sit at it comfortably without compressing the chest or tiring 158 Animal Micrology the neck. Sit as upright as possible. If the instrument is inclined it should set farther in on the table than if it is in the upright position. 3. With a piece’ of old linen, a chamois skin, or a bit of lens paper, carefully clean the eyepiece to be used and put it in place. Always use the low-power eyepiece first. 4. Likewise clean and attach the objective (low-power first) after elevating the tube far enough above the stage for this pur- pose. Guard particularly against screwing the objective in crooked, as this will injure the threads. It is best to swing the objective between the first and second fingers of one hand and bring the screw squarely into contact with the screw of the tube (or nosepiece); with the thumb and forefinger of the other hand it is then screwed into place. 5. Bring the draw-tube to the standard length (see tube-length) for which the lenses are corrected. If a nosepiece is used, allow- ance must be made for its height. 6. Place the slide which bears the object on the stage with the object over the central opening of the latter, and clamp it in place by means of the spring clips. While looking at the object from one side, turn the mirror until a flood of light shines up through the center of the stage. 7. Lower the tube until the objective nearly touches the cover- glass, then look through the eyepiece and slowly raise the tube by means of the coarse adjustment until the specimen to be examined is plainly visible. Focus accurately by means of the fine adjustment. If a high-power objective is being used, since it must come very near the cover, the operator should lower his head to the level of the stage, and look toward the light between objective and cover-glass in order to prevent actual contact. This is of great importance, for otherwise the objective or the object is liable to injury. Remember that in focussing wp the lowest part of the object comes into view first, the highest part last. 8. The higher the power, the more difficult it is to find an object or a particular part of it. For this reason the finding is usually done by means of a low-power objective, or a low-power Appendix A: The Microscope and Its Optical Principles 159 ocular, or both, and after accurately centering the object in the field, the high power is attached. In case a revolving nosepiece is used, great care should be used in turning in the high power not to strike the slide with the objective. This is very likely to happen if the objectives are not parfocal. 9. After the object is in focus give any further attention to the illumination that is necessary (see illumination and mirror). If intensified illumination is desired, use the concave mirror, or use the substage condenser and the plane-mirror. For ordinary purposes the field should be evenly illuminated, although oblique light is frequently useful. Manipulate the diaphragm until the structure to be studied shows with the greatest distinctness. Too much light ‘“‘drowns”’ the object, and is hard on the eyes. (To determine the proper distance at which the concave mirror should stand below the stage, let direct sunlight shine upon the mirror, and then adjust the latter so that the apex of the cone of light comes just at the top of the stage where the object will rest.) 10. In using oil-immersion objectives, a small drop of cedar oil (specially prepared by the maker of the lens) is applied to the front lens by means of a small rod or brush. It is very important to keep the oil free from dust, and to see that it does not contain air bubbles when applied to the lens. Carefully lower the tube until the oil on the objective comes in contact with the cover-glass. The operator should lower his head to the level of the stage to observe this properly. Focus up as with a dry objective. With a piece of lens paper or a soft cloth, clean the immersion lens immediately after you have finished using it. Likewise remove the oil from the cover-glass. 11. The range of the fine adjustment is limited. Keep it as near the middle point as possible. If the tube does not respond to the movement of the screw you have probably gone beyond the range of the fine adjustment. 12. In working with the microscope keep both eyes open. The eye which is not in use soon becomes accustomed to ignoring objects in the field of vision. To avoid fatigue it is well to use first one eye and then the other for observation. The eye should be placed 160 Animal Micrology at the eyepoint (see above) of the lens. This is some distance from the eye-lens in low-power eyepieces, close to it in high- power eyepieces. 13. Put the microscope in its case when you have finished using it, or at least cover it with a cloth or cone of paper. For further details regarding the use or care of the microscope consult one of the following books: The Microscope, by Gage; Manipu- lation of the Microscope, by Bausch; The Microscope and its Revelations (1,200 pages), by Carpenter and Dallinger. 14. Do not apply alcohol to any part of the instrument. The lenses may be cleaned ordinarily by breathing upon them and wiping them with a rotary motion on lens paper or a piece of soft old linen. In case a solvent must be used for balsam or oil, ben- zene is the one commonly recommended. It must be quickly wiped away so that it will not affect the setting of the lens. Bits of dust may be flecked off the surface of a lens by means of a camel’s hair brush. The beginner in microscopy should acquaint himself with various common objects that are liable to get into his preparations in the form of dust, etc., so that he may not mistake them for es~ - sential parts of his specimen. Such objects are hairs, fibers of silk, wool, linen, cotton, and the like, and particularly air-bubbles. Air-bubbles are usually circular with black borders and bright centers; they may show tinges of color. Examine a drop of saliva for examples. APPENDIX B SOME STANDARD REAGENTS AND THEIR USES I. FIXING AND HARDENING AGENTS 1. Acetic Acid.—Acetic acid is more commonly used in mixtures or in diluted form than pure. It is valuable because it tends to produce good optical differentiation and facilitates penetration. When employed alone it causes some tissues to swell and disinte- grate. Inasmuch as most fixing agents give the best results when they have an acid reaction, from 1 to 5 per cent. of acetic acid is generally added to acidify them in case they are not nat- turally acid. Acetic acid is also of great value in mixtures be- cause it counteracts the shrinking action of certain reagents. Ordinary acetic acid is of about 36 per cent. strength; glacial acetic, of about 99.5 per cent. strength. A strength of from 0.2 to 1 per cent. is recommended by Flemming for work on cell nuclei. Strong glacial acetic acid is ‘sometimes used for highly contractile animals, such as Coelenter- ata, Mollusca, and Vermes. The animal is rapidly flooded with the acid and remains immersed until it is thoroughly penetrated (6 to 10 minutes). It is then washed in repeated changes of 50 or 70 per cent. alcohol and left to harden in 70 to 83 per cent. alcohol. The pure acid, if allowed to act for more than a few minutes, swells and softens the tissues. Acetic acid should not be used when connective tissue or delicate calcareous structures are to be preserved. 2. Acetic Alcohol.—Carnoy recommends each of the following formulae: a) Glacial acetic acid Fi ake mca ae tN a Sook Dart Nibsoluteralcohol es cc 4 el clei seest «. Oparts baGilacialktacetieweid’ — yeni. Ys: se) seme err part Abseoluteralcohol)s he. oe wee aye -O paris Chiloroformia ey. th.5.20 13 ke Feu. ah Oo pacts The chloroform is said to hasten the action of the mixture. Either of these reagents penetrates well and acts rapidly. Almost 161 162 Animal Micrology any stain will follow them. Even such difficult objects as the eggs of Ascaris may be fixed by the second mixture. The reagent should be washed out in absolute or at least in strong alcohol. A mixture of absolute alcohol, glacial acetic acid and chloro- form, equal parts, saturated with corrosive sublimate (formula of Carnoy and Lebrun) becomes even more valuable for the fixation of difficult objects. According to Lee, isolated ova of Ascaris are fixed in 30 seconds, entire oviducts in 10 minutes, in this liquid. 3. Alcohol.—Alcohol is used especially for gland cells and for preserving the brain and spinal cord for Nissl’s method of staining nerve cells, See chap. iii, alcohol fixation; also chap. i, reagents I and 2. Alcohol and Chloroform.—See 2b. Bichloride of Mercury.—See corrosive sublimate. 4. Bichromate of Potassium.— Bichromate of potash is one of the oldest and best-known fixing reagents. At present it is more commonly used in mixtures than alone. It is widely used in hardening nervous tissue. Its fixation of nuclei is unsatisfactory unless it is properly corrected through the addition of acetic acid. It acts very slowly, about three weeks being necessary to harden properly a sheep’s eye, and from three to six months for a good- sized brain. A weak solution (2 per cent.) should be used at first, to be replaced gradually by stronger solutions up to 5 per cent. When hardening is completed the object should be thor- oughly washed in running water and then put into alcohol; begin with low percentages of alcohol and gradually increase the strength up to 70 or 80 per cent. Change the alcohol as often as it becomes yellow. After the object has been placed in alcohol, keep it in the dark in order to prevent a precipitate forming on the surface. Hither carmine or hematoxylin may be used as a stain after bichromate of potash. In case carmine is used, the staining is best done before the object is placed in alcohol. Tissues which do not stain well should be placed for 3 hours in acid alcohol and then washed in alcohol before staining. Appendix B: Some Standard Reagents and Their Uses 163 5. Bichromate of Potassium and Acetic Acid (Tellyesnicky’s fluid). — Bichromate of potassium: 7.0.5. ~. , : >. dgrams Glacwiacehic@aeid.* Mec tic. tt 2.) OPEL, Waters. %. . SE eames 0.0 ren cs It is best not to det ane neste acid until fae before using. This is a good general reagent. It is valuable for embryos. Objects should remain in some 20 volumes of the fluid from 24 to 48 hours, according to size. It is well to change the fluid once, after a few hours. After fixation, tissues should be washed thoroughly in running water (6 to 12 hours) and passed through alcohols of increasing strength beginning with 15 per cent. 6. Bichromate of Potassium and Corrosive Sublimate (Zenker’s fluid ).— Cormosiversublimates.-.:0.. 9... Gs te Yo grams Potassium bichromate 7.0. .-:* 2720 a" 2erams Soqiumsulphatotie: 24 0 sae fe) 0 eee era Glacialeacciie acid) ae oe es. Tee ie Di G:c: Waterers net eins paren 100c e! It is best to add fhe Rete: el nanrmadhonels before using. Zenker’s is a valuable reagent for both histological and embryo- logical material (embryos up to 25 mm.). Several hours are required for fixation: 2 to 4 hours for a 2 day chick; 8 to 10 hours for objects or embryos of 6 to 8 mm.; 24 hours for embryos of 12 to 14 mm., ete. For washing, running water is employed for from 12 to 24 hours. The object is then transferred to gradually increasing strengths of alcohol up to 70 per cent., leaving it according to size from 1 to 3 hours in each alcohol. To remove the excess of corrosive sublimate, see 13, caution 1. Almost any stain follows this reagent well. Both nuclear and cytoplasmic structures are properly fixed. 7. Bichromate of Potassium and Cupric eunhat ( Erlicki’s fluid ).—See chap. i, reagent 8 and chap.-iii, 3. 8. Bichromate of Potassium and Sodium Sulphate (Miiller’s fluid ).— Bichromate of potassium . . . . . 20 to 25 grams Sodium sprbhate geisie ous s,s ae ee (90h ele. mlcahol, Oayper Cemts a aol. ap sues cee Once: Filter before using. Gribler’s ‘‘Safranin O” is the most reli- able dye. Sections of tissues fixed in Hermann’s or Flemming’s 186 Animal Micrology solution are left in the stains for from 24 to 48 hours. Decolorize as directed under 29. 66. Safranin and Gentian Violet.—This is a combination that is almost indispensable in the study of cell problems, especially spermatogenesis. For formulae of stains see 44 and 65. ‘Tissues are best fixed in Flemming’s or Hermann’s solutions. Stain thin sections for 36 to 48 hours in the safranin; differentiate in alco- hol very slightly acidulated (see 29), then stain for 5 to 10 min- utes in the gentian solution and transfer the sections to Gram’s solution (see under 44) for 1 to 3hours. Finally differentiate in absolute alcohol. As soon as purple clouds have ceased to come from the sections in absolute alcohol, they should be transferred to clove oil for a few minutes and thence to xylol. The clove oil seems to intensify the safranin in the chromatic granules, but too prolonged an immersion in clove oil extracts the gentian violet. 67. Silver Nitrate.—The nitrate-of-silver method is used largely as an impregnation method for work on nerve tissue and for dem- onstrating intercellular substances and outlining boundaries of cells in the epithelial coverings of membranes, etc. Wash the fresh tissue in distilled water, then place it for 2 to 5 minutes in 0.5 to 1 per cent. aqueous solution of silver nitrate. Rinse in distilled water, then expose the tissue to bright sunlight in water or glycerin (or in 70 per cent. alcohol, if it be mounted in balsam) until a brown coloration appears. Temporary mounts should be made in glycerin. For application to nerve see chap. ix. 68. Sudan III.—This is a specific stain for fat. A saturated alcoholic solution is used (5 to 10 minutes). Wash rapidly in alcohol. Since alcohol is a solvent of fat, too long an immersion will destroy the preparation. Mountin glycerin. With this dye large fat drops stain orange, small ones yellow. The tissue should have been fixed previously in Millers fluid (8) or other medium which does not dissolve fat. Van Giesen’s Stain.—See 43. 69. Wright’s Stain (for blood and for the malarial parasite ).— See chap. xiv, memoranda 5 and 6. Appendix B: Some Standard Reagents and Their Uses 187 III. NORMAL OR INDIFFERENT FLUIDS (For fresh tissues) 70. Aqueous Humor.—Obtained by puncturing the cornea of a freshly excised beef’s eye. A small amount may readily be obtained by means of a capillary pipette from the eye of a freshly killed frog. 71. Blood Serum.—Blood is allowed to clot and after 24 hours the serum is poured off. If necessary it may be further freed of blood cells by means of a centrifuge. The serum will keep for only a day or two. Schultze’s iodized serum made by saturating blood serum with iodine is sometimes classed as an indifferent fluid, but it is really a dissociating fluid. 72. Fluid of Ripart and Petit.— Camphorated water)... 5"... 7 5 Ibe: Neetate ORCOppen so. as ay tans) Oo era Ghiorndeiot-coppeniy ("5 (2... 5" i. ge ae Old eram DistilleGiwater © a.6 6 sb isek se ie a eee eb Onere: Glacial acetic acid . .. . Tider omen IFOIESC: After the solution becomes ee (a few hours) it should be filtered. It is especially useful for examining fresh animal cells. Methyl green is an excellent stain to follow this fixing fluid. 73. Kronecker’s Fluid.— Distilled’ watere «....) & «Jai. & «2 L000 ce: SOCiumCHIOLIGeG 22 ss. us le 0.60 gram Sodium carbonate <« . . . 7) Nia 0.06 gram 74. 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PROTOZOA a) Cultures.—Amebae, etc., may usually be obtained in quan- tities sufficient for class use by the following method recommended by H. 8S. Jennings: A number of glass dishes measuring 8 or 9 inches in diameter by 8 inches deep are crowded full of water plants (especially Ceratophyllum and Elodea), filled with water, and the plants allowed to decay. Keep the dishes in warm, light places. In two or three weeks the layers of plants at the surface of the water will be covered with a brown slime which should be examined occasionally under the microscope for the desired forms. The scum that appears on the surface of the water consists mainly of bacteria upon which amebae largely feed. They will be found most frequently in the slime that immediately surrounds the plant tissue. Since they frequently last only two or three days in a culture, to insure material for class work, a number of cultures must be made at different dates and from different localities. Other protozoa such as Arcella, Difflugia, Carchesium, Stentor, etc., will also be found in the cultures. Paramoecium may be kept from dying out by keeping bits of stale bread in cultures. Euglena will be found in some of the cultures, but usually not in any quantities before the end of four or five weeks. They appear along the side of the dish toward the light. Carchesium and Vorticella are frequently found on decaying duckweed (Lemna) and hornwort (Ceratophyllum). To secure a culture, have a more plentiful supply of water than for ameba. Professor Walton tells me that he always finds a supply of EHpistylis on the shells of fresh water snails. 215 216 Animal Micrology Opalina may be obtained readily by killing a frog wich chlo- roform and slitting open the large intestine. Examine scrapings of the epithelial wall in normal saline (reagent 74, Appendix B). Sporozoa. Gaegarina may be found in the alimentary canal of the cockroach and Monocystis, in the male reproductive organs of the earthworm. They are best studied in normal saline. If it is desired to stain and mount specimens they may be fixed in corro- sive-acetic (reagent 14, Appendix B) for 5 minutes, washed thor- oughly in 35 per cent. alcohol to which a little tincture of iodine has been added, and stained with Ehrlich’s triple stain (reagent 39), or hematoxylin and acid fuchsin (reagents 49 and 42). b) Quieting infusoria.—1. Let sufficient water evaporate from under the cover to permit the latter to press lightly upon the ani- mals. Guard against too great evaporation of water or the infu- soria will be crushed. 2. Entanglement in fibers of cotton, etc., sometimes proves efficacious. 3. A small amount of gelatin or better, cherry-tree gum, dis- solved in water makes a viscous mass which is often useful in retarding their motions. A bit of white of egg may be used in the same way. 4. Animals may be narcotized by means of a small drop of very dilute alcohol (preferably methyl alcohol) or chloretone (about one drop of a 1 per cent. solution to 10 drops of water). (Chloretone is manufactured by Park, Davis & Co., of Detroit, Mich. For its use as an anaesthetic in biological work see Jour- nal of Applied Microscopy, Vol. V, p, 2051.) c) Feeding.—Place finely pulverized carmine or indigo under the cover-glass. The colored powder rapidly accumulates in the food vacuoles. In such a preparation the action of the cilia of infusoria is also indicated by the rapid movement of the particles in the vicinity of the animal. See also chap. xiv, memorandum 4. d) Staining.—For intra vitam staining see Appendix B, rea- gents 55a, 31, and 58. To see cilia of infusoria treat the animal with very dilute iodine solution or a drop of a dilute solution of tannin. Appendix D: Preparation of Microscopical Material 217 To see the macronucleus and the micronucleus use a drop of a 2 per cent. solution of acetic acid or, better, methyl green (Appen- dix B, reagent 56). e) Permanent mounted preparations.—Benedict’s method is as follows: “Smear a glass slide with albumen fixative, as in preparing for the mounting of paraffin sections. Then place on the surface of the film of fixative a drop or two of water containing the forms which it is desired to stain. Let nearly all the water evaporate by exposure to the air of the room until only the film of fixative remains moist. The slide can now be immersed in Gilson or any other fixing reagent, and then passed through the alcohols, stains, etc., in the same way that mounted sections are handled. ‘“‘T have had no difficulty in getting preparations of Paramoe- cium by this method, with very little distortion of the body, and any kind of staining desired. By this method students can pre- pare in ten minutes very satisfactory preparations of protozoa for demonstration of nuclei, etc.””—Journal of Applied Microscopy, Vol. VI, p. 2647. SPONGES To isolate the spicules of calcareous sponges boil a bit of the sponge in 5 per cent. solution of caustic potash for a few minutes. Fairly thick transverse, longitudinal, and tangential sections of Grantia showing spicules in the tissues are useful. Make these with an old razor or sharp scalpel. To hold the object while sectioning, place it between two pieces of pith or cork. For a careful study of the relations of the two systems of canals in the body-wall, thinner sections are necessary. To prepare these it is best to decalcify (2 per cent. chromic acid, 24 to 36 hours) the sponge and cut celloidin or paraffin sections on the microtome although fairly good sections may be made by hand. They should be dehydrated and mounted in balsam if permanent prep- arations are desired; if not, they may be examined in glycerin. To color the collar cells use an aqueous solution of anilin blue. Spicules of szliciows sponges are isolated by treating bits of 218 Animal Micrology the sponge with strong nitric acid or a mixture of nitric and hydrochloric acid. COELENTERATES Hydra should be sought for in spring-fed pools. In the autumn they are found most frequently on smooth dead leaves which are completely submerged. Material should be collected and placed in battery jars or larger glass jars, which are then filled with fresh, clear water and placed in a fairly light place, but not too near a window. Put a small amount of hornwort or Chara ineach jar. Ina few hours (12-36) the hydra will be found attached to the sides of the vessel and to the plants. They may readily be kept in the laboratory throughout the winter if glass plates are placed over the jars to prevent excessive evaporation and the temperature is not allowed to go below freezing. Fresh water should be added from time to time to make up for evaporation. In case their supply of food (Cyclops, Daphnia, and other small crustacea) is exhausted it should be renewed by skimming out from other aquaria the small forms upon which the animal feeds and putting them in the hydra jars. For staining and mounting entire see chap. xiii TI, B. Killin the same way for sectioning. The most instructive sections are (1) transverse sections, (2) longitudinal sections through the mouth and a bud, and (3) sections showing the sexual organs. Stain in bulk with hematoxylin (reagent 49, Appendix B), imbed in paraffin using the method for delicate objects (chap. vi, VII), and after the paraffin has been removed from the sections, stain them for a few seconds in acid fuchsin, Dehydrate and mount in the usual way. The sections are much more satisfactory if the hydra have been placed in small stender dishes filled with filtered water (not dis- tilled) and kept from food for a week or ten days before killing. This eliminates the metabolic products and oil globules which ordinarily obscure the details of structure. To Stain the Nematocysts of Living Hydra, place several of the animals in a small stender dish of water which has been tinted a Appendix D: Preparation of Microscopical Material 219 sky blue through the addition of methylen blue solution made up as follows: Methylenttblues 2295, 5. fet Mime. 91) 1.0: gram Craciilewoupre. motte. Lenk Go a 0D Sram Wistere mente, cr teit 6 a ae ieee ts bh OOOO EEC, After two hours the hydra may be transferred to fresh water; the nematocyst cells are stained a deep blue. (Method of Little, Journal of Applied Microscopy, Vol. VI, p. 2116.) To Discharge Nematocysts drum on the cover-glass gently with a pencil. By using a very small opening to the diaphragm they are usually sufficiently distinct without staining. For Other Polypoid Forms, the methods given for hydra will answer in most cases. For Collecting Free-Swimming Medusoid Forms full directions will be found in Brook’s Invertebrate Zoology. Compound Hydrozoa should be placed alive into the cells which they are to occupy when mounted. One per cent. formic acid is then added drop by drop to the sea-water. After the animals have been killed, the fluid is replaced by glycerin-jelly and the cover-glass is put in place. Another method is to kill the animals slowly by adding a few crystals of chloral hydrate, from time to time, to the small vessel of sea-water containing them. Small Jelly-Fish may be fixed and hardened in 1 per cent. osmic acid and, stained or unstained, mounted in cells. PLANARIA Look for planarians on the under sides of stones in small streams of running water. They are usually examined alive. To see them thrust out the proboscis, keep them from food for a few days and then feed them on dead flies. Planaria which have been kept in the laboratory for months display the internal organs much more clearly than freshly captured ones. If it is desired to study stained specimens, for preparation see chap. xili, iv, A. To Kill Planaria with Pharynx Protruded Cole (Journal of Ap- plied Microscopy, Vol. VI, p. 2125) recommends covering 220 Ammal Micrology them in a watch-glass with a 1 per cent. aqueous solution of chloretone until they are immobilized and then rapidly transfer- ring them to 5 per cent. formalin. Other fixing agents than formalin can be used. DISTOMES Perhaps the most easily obtained form is the one which is found in the liver of the cat. Search for it in the bile passages. Fix it in hot corrosive sublimate, wash out with alcohol to which tincture of iodine has been added, and stain for 24 hours in alum cochineal (reagent 27, Appendix B), or hemalum (reagent 47). As with Planaria, they should be compressed between two glass slides (see chap. xiii, iv, A, 7). If the large liver fluke of the sheep (Fasciola hepatica) can be obtained, both the alimentary canal and the excretory system may be injected with finely powdered carmine in water. A sepa- rate fluke should be used in each case. For injection, a very fine- pointed cannula with rubber cap is used, or the manipulator may operate the cannula by simply blowing through it. The excretory system is injected through an incision made with a sharp-pointed scalpel in the median line near the hinder end of the animal. For the alimentary canal, the incision should be made about 1 mm. to one side of the median line. When the injection is completed, flatten the animal somewhat between two slides (see chap. xiii, iv, A, 7), harden in 95 per cent. alcohol for 12 to 24 hours, then dehydrate, clear, and mount in balsam. CESTODES Near large cities an unlimited supply of the sheep tapeworm (Monieza) can usually be secured from slaughter houses. Ample supplies can ordinarily be obtained from dogs, or, less frequently, from cats. Tapeworms can be kept alive for considerable length of time in tepid normal saline. The most instructive portions to mount are scolex, and sexually mature proglottids. For fixing and staining use the same methods as for distomes with the excep- tion that cold instead of hot corrosive sublimate should be used. The scolex should not be compressed. Appendix D:; Preparation of Microscopical Material 221 To Find Cysticerci, open the body cavity of a rabbit and look for large whitish bodies imbedded in the peritoneum or liver (the cysticercus of T. serrata). Likewise, the cysticercus of T. cras- Fre. 70.—Compressor. sicollis may be found in the liver of the mouse. If a cysticercus is found, its outer wall should be slit open in order to show the reversed scolex. ASCARIS See chap. xvi, memorandum 11. TRICHINA The simplest way to obtain it is to apply for infected pork to the government inspector whose headquarters are to be found near all large slaughter houses in cities. Bits of the infected Fic. 71.—Compressor Used by the Government Bureaus for Meat Inspection. muscle should be teased and flattened out in a compressor (Figs. 70 and 71) until a favorable area hasbeen found. The flattened tissue may then be dehydrated and mounted unstained or it may be stained in hematoxylin (reagent 49, appendix B). Better results will be obtained if the material is fixed for from 4 to 6 hours in 222 Animal Micrology Carnoy’s fluid (reagent 2) before dehydrating or staining. If desired, the tissue may be sectioned in celloidin or paraffin. To Demonstrate Living Trichinae Barnes (American Monthly Microscopical Journal, Vol. XIV, p. 104) subjects small bits of trichinized muscle to a mixture of 3 grains of pepsin, 2 drams of water, and 2 minims of hydrochloric acid, for about three hours at body temperature with occasional shaking. | When the flesh and cysts are dissolved, the liquid is poured into a narrow glass vessel and allowed to settle. The live trichinae may be withdrawn with a pipette from the bottom of the fluid and examined on a warm stage. ' ROTIFERS Rotifers will usually be found in abundance in some of the laboratory aquaria on the lighted side of the vessel. | For ordinary class work they are best studied alive. They are difficult to preserve properly. Full directions for killing and preserving will be found in Jenning’s paper, ‘“‘Rotatoria of the United States,” U.S. Fish Commission Bulletin, 1902, p. 277. To Quiet Rotifers, Cole (Journal of Applied Microscopy, Vol. VI, p. 2179) anaesthetizes them by adding from time to time a drop of 1 per cent. aqueous solution of chloretone to the water on the slide in which the animals are being examined. BRYOZOA They may be treated in the same way as compound hydrozoa. Plumatella may frequently be found in shallow fresh-water streams on the under side of flat rocks; Pectinatella, in rivers and streams on the upper surface of mussel shells, ete. EARTHWORM EKarthworms are best collected on warm, rainy nights when they may be found extended on the surface of the ground near their burrows. They are most plentiful in old gardens or rich lawns. Cua re . at ip are * Mite < 7 ene: i 7 a! _ a is ® ” rece " Ms nya’ “hy Te 1s inal . a > : mn ale rie ve sad a a oe “475 eae - ‘= a. Cae Crd he oe See ae " BPR yor! Ve aie : tie h a be Sigh | - INDEX Abbe, 156 ; camera lucida, 147. Abbot’s method for staining spores of bac- teria, 111. Aberration, spherical, 138 ; correction of, 139. Absolute alcohol, testing for water, 56. Absorption of fat, 198. Accessory chromosome, 191. Acetic acid, 161. Acetic alcohol, 161 ; and chloroform, 123, 125, 161. Achromatic objective, 145. Achromatism, 145. Acid fuchsin, see fuchsin. Acidophil granules, 190. Action of liquids, to hasten, 56. Adenoid connective tissue, 197. Adipose tissue, 194. Affixing sections, 23, 41, 64. Air bubbles, 159, 160. Albumen fixative, 12, 23, 41. Albumenoids, 15. Albuminous coats of eggs, to remove, 118. Alcohol, absolute, 7, 13, 49, 84; acid, 8, 24, 50, 162; alkaline, 49, 50; and chloroform 162; ether, 8, 23; ethyl, 13; fixation, 28, 162; methyl, 13; replenishing, 56. Alcoholometer, 13. Alimentary canal, 197, 198; of cockroach, 77. Alum carmine, 171, 181; dahlia, 191. Alum cochineal, 126, 170, 181. Aluminium chloride, 184. Altmann, 192 Ameba, 215. Ameboid movement in leucocytes, 102. Amphibia, material for the embryology of, 118; fecundation and early embryonic stages, 122; to study eggs of, 118. Amphibian eggs, 167. Amphioxus, 226, Amphophil granules, 190. Amitosis, 191. Ammonia copper sulphate solution, 152. Ammonium chromate, 214. Ammonium picrate, 181, 183. Amyloid, 172, 199. Anatomical specimens, to preserve, 31. Andrews, 117. Angular aperture, 145. Anilin blue, 217; and orange G, 172. Anilin, alcohol, 182; dyes, 19, 20, 57, 63, 171; formulae, 171; water, 171. Antennae of insects, 93. Anthrax, 109, 111. Aorta, 193. 231 Apertometer, 145. Aphid, 91. Aplanatism, 145. Apochromatic objective, 146. Apparatus, |; dealers in, 5. Appliances, microscopical, 145. Aqueous humor, 187. Arcella, 215. Areas of Conheim, 204. Areolar tissue, 194. a eueoment of apparatus and reagents, 6, Artery, 193. Arthropods, 224. Ascaris, 162; maturation, fertilization, and cleavage, 123. Asphalt-paraffin-rubber method of imbed- ding, 44, 119. Aurantia, 20. Axial illumination, 150. Axis cylinder, 206. Axone, 206. Bacillus, 108; aerogenes capsulatus, 111; of anthrax, 109, 111; of bubonic plague, 111; of chancroid, 111; coli communis, 111; diph- theriae, 109, 111; of dysentery, 111; of glan- ders, 111; of influenza, 111; of malignant edema, 111; mucosus capsulatus, 111; pro- teus, 111; pyanocyaneus, 111; of tetanus, 111; of tuberculosis, 111; of typhoid, 111. Bacteria, cover-glass preparations of, 105; features to be observed in studying, 108; Gram’s method of staining, 107; hanging- drop preparations of, 105, 108; in tissues, 105, 107; material for demonstrating, 108; methylen blue stain for, 107; mounting from fluid media, 105; mounting from solid media, 105; staining and mounting films, 105; spores, 108; staining flagella, 111; stains for, 109. Bacterial examination, 105. Balsam, 11, 22, 34, 80, 90; bottle, 4; exudation, to remove, 58. Bardeen, 128, 129; microtome, 67. Basophil granules, 190. Basophil substance in nerve-cells, 182. Bausch, 152, 160. Bausch and Lomb microscope, 143. Beale’s carmine, 173. Bee, 93. Beetle, 92. Beggiatoa, 108. Bell’s cement, 79. Benedict, 217. Benzopurpurin, 20. Berlin blue, 81. 232 Bethe’s fluid, 181. Bichloride of mercury, 165; see also corrosive sublimate. Bichromate of potassium, 162, 187; and acetic acid, 163; and corrosive sublimate, 163; and cupric sulphate, 163; and sodium sulphate, 163. Binocular microscope, 146. Bioblasts, 192. Bismarck brown, 20, 106, 173. Bladder, 213. Blastoderm of chick, 113, 114. Blastoderms, to orient colorless, 117. Bleaching, 24, 45. Bleu de Lyon, 20; see also Lyons blue. Blocks for celloidin imbedding, 59, 62. Blood, 97, 190; cover-glass preparations, 98; clinical examination of, 99; corpuscles, 172, 176; corpuscles, living, 97; crystals, 97, 190; currents, to observe, 101; dry prep- arations of, 98; effects of reagents on, 97; enumeration of corpuscles, 99; exami- nation of fresh, 97; platelets, 97; rapid method, 99; serum, 187; LoefHler’s serum, 110; Schultze’s iodized serum, 187; to study in sections, 101; test for, 98; Wright’s stain for, 102. Blood forming organs, 190. Blow pipe, 1. Bone, 79; corpuscles and their processes, 194; decalcified, 79, 189, 194; endochondral development of, 194; fibers of Sharpey, 195; grinding, 80; Haversian canals and lamellae, 195; intra-membranous_ devel- opment of, 195; isolation of corpuscles and of lamellae, 195; sec.ioning, 79, 80; young, to decalcify, 189. Borax-carmine, 9, 49, 51, 54, 61, 77, 89, 91, 119, 121, 169; formula, 9. Berdeaus red, 10, 49, 53, 55, 117, 173; formula, Born, 127. Bottle-capping, 30. Bouillon, glucose, 110. Box, slide, 1. Brain, 206, 207. Brain cells, 206. Brain sand, 206. Brittle objects, sectioning, 43, 44, 63, Bronchi, 211. Brownian movement, 146. Brushes, camel’s hair, 1. Bryozoa, 222. Bubonic plague, bacillus of, 111. Bull's eye, 151. Bunge Lockie staining flagella of bacteria, Biitschli, 192. Butterfly, wings of, 92; eggs of, 93. Calendar, 1; record, 6. Calleja’s staining fluid, 174. Camel’s hair brushes, 1. Camera, photographic, 155, Camera lucida, 146, 147, 148. Canada balsam, 11, Animal Micrology Cannulae, 82, 85. Capillaries, blood, 193; lymph, 193. Carbol-fuchsin for bacteria, 109. Carbolic acid, 21, 22. Carbol-xylol clearer, 9, 21, 34. Carbon dioxide, for freezing, 67; for killing, Carchesium, 215. Card records, 1, 6. Carmalum ;76: formula, 173. Carmine, 19, 57, 162; acid, 76, 123, 174; alum, 168; and Lyons blue, 51; Beale’s, 173; injec- tion mass, 81; picric acid and indigo car- mine, 174; picro-, 10, 184. Carnoy, 161. Carpenter and Dallinger, 160. Carrier for paraffin ribbon, 41. Cartilage, 195; capsule of, 195; connective tissue and elastic fibers in, 195; elastic (yellow-fibro-), 195; glycogen in, 195; hya- line, 196; white fibro-, 196. Caustic potash, 187. Cell, animal, 191; living or fresh, 192; of Paneth, 197; pigment, 193; prickle, 213; reduction division in, 193. Cell-making, 87, 95. Celloidin, 12, 22; bottle, 4; clearing before sectioning, 64; hardening, 61, 62; imbed- ding, 59; Gilson’s rapid method, 64 ;method, 26, 59; preparation of material for imbed- ding in, 53; sections, to transfer from the knite, 64; time required for the method, 62. Coed and paraffin methods compared, Celloidin-paraffin infiltration, 63. Cells, to lessen evaporation from, 95. Cement, Bell’s, 79, 89. Cement substance, 188. Center of slide, to find, 43. Centering an object in a cell, 93. Centigrade to Fahrenheit scale, 228. Central illumination, 150. Central nervous system, 188, 206. Centrosome, 53. Cerebellar cortex, 206. Cerebral cortex, 206. Ceruminous glands, 201. Cestodes, 220. Chancroid, bacillus of, 111. Chick, embryology of, 113; stages necessary for a course in, 115, 116. Chick embryos, fixing, staining, and mount- ing, 114, 115; freeing blastoderm from yolk in, 117; orienting, 114, 116; reconstruction of heart, 126; removing blastoderm, 114; sectioning, 116. Child, 117, 118. Chironomous larva, gland cells of, 192. Chloretone, 216. Chloride and acetate of copper, 164. Cholera, spirillum of, 111. Choroid, 203. Choroid plexus, 206. Chromatic aberration, 189. Index Chromic acid, 118, 164, 189, 225; and its com- pounds, 17, 164, 165. Chromic acid material, bleaching, 45. Chromo-aceto-osmic acid, 164. Chromo-platinic mixture, 165. Chromosome, 53. Chrom-silver method, 71. Cicatricula, 113. Cilia of infusoria, 216. Ciliated epithelium, 75. Circulation, in foot of frog, 101; in mesen- tery, 101. Circulatory system, 193. Citric acid, 177. Cleaning lenses, 158, 159, 160. Cleaning slides and covers, 57. Cleanliness, 6, 44. Clearer, 21, 62; carbol-xylol, 9, 21, 30; Eycle- shymer’s, 22, 62. Clearing, 21. Cleavage, in Ascaris, 123; in echinoderms, amphibia, and teleosts, 122; in living material (snails), 123; in mammals, 171. Clinging of paraffin sections to the knife, 48. Clitoris, 210. Clove oil for minute dissections, 78. Coal-tar dyes, 20. Coccus, 108. Cochlea, 201; nerve fibers and nerve endings of, 201. Cole, 219, 222, 224, 225. Collar cells of sponge, 217. Collodion, 23, 44, 64. Colostrum, 213. Columnar epithelium, 75. Compensating ocular, 145, 148, Compressor, 221. d Concave or diverging lens, 134, Condenser, 148, 151. Conheim, areas of, 204. Conjugate foci, 134. Conklin, 117, 122, 170; picro-hematoxylin, 177. Connective tissue, 172, 174, 185, 194, 196. Contractile animals, 16, 161. Conventional distance of vision, 144. Convex or converging lens, 134. Coplin staining jars, 4, 49. Copper sulphate method of preparing abso- lute alcohol, 7. Cornea, 203. Corneal corpuscles and nerves, 203; spaces and canaliculi, 203. Corpora lutea, 119, 210. Correction collar, 148. Corrosion, 24, 86, Corrosive sublimate, 9, 18, 165; handling, 166; and acetic acid, 166; and nitric acid, 166. Cover-glass, 1; supports for, 78; forceps, 106; correction, 148; to clean, 57. Creasote, beechwood, 22. 62. Crescents of Gianuzzi, 198. Crooked paraffin ribbons, 46. 233 Crown glass, 140. Crumbling of tissues in paraffin, 48, 44, 46, 47, 66 Crustacea, small, 90, 224. Crystals, blood, 97. Culture slide, 108. Curare, 101. Cyanin, 211. Cyclops, 90, 218. Cylindrical end bulbs, 206. Cypris, 90. Cysticerci, 221. Gyte logical work, reagents for, 162, 164, 170, ips Cytoplasmic granules, 183. Damar, 22. Daphnia, 90, 218. Dealcoholization, 22. Dealers and manufacturers, 5. Decalcification, 24, 79; fixing before, 80. Decalcifying fluid, 9,79; formulae, 189. Defective mounts, 57, 58. Definition, 148, Dehydration, 18. Delicate tissues, 43; paraffin method for, 54; dehydrating apparatus for, 55; freezing method for, 70. Demilunes of Heidenhain, 198. Descemet, membrane of, 204. Desilicidation, 24. Desk, 4, 157. Desmids, 94. Determination of elements that will be im- pregnated in Golgi method, 73. Diamond, writing, 1. Diapedesis, 101. Diaphragms, 149, 159. Difficulties in sectioning of, 46. Difflugia, 215. Digestive organs, 197; blood vessels of, 197. Dilution, rules for, 7, 13. Diphtheria, 109, 111. Diplococci, 108. Diptocorchs intra cellularis meningitidis, paraffin, table Dipping-tube, 94. Dirty paraffin, 43. Dirty sections, 42. Dispersion, 139. Dissecting instruments, 1. Dissections, minute, 77. Dissociation, 15. 25, 75, 78; general rule for, Dissociators, formaldehyde, 9, 75; formulae for others, 187. Distomes, 220, Dogiel, 181. Double staining, 20, 51, 53, 62. Doublet lens, 138. Dragon-fly nymphs, 225; heart beat in, 225. 234 Drawing-board, with camera lucida, 147. Drawing-table, Bardeen’s 129. Drawing with camera lucida, 146, 147. Dropping-bottle, 58. Dropping out of object from paraffin sec- tion, 47. Dry or dull-looking areas in mounts, 58. Duodenun, 198. Duval, orientation of young chick embryos, 116. Dysentery, bacillus of, 111. Dityscus, fore-leg of, 92. Ear, 201. Earthworm, collecting, 222; coelomic cor- puscles of, 224; to immobilize, 224; to keep alive in winter, 224; nephridia of, 223; ovary or testis of, 224; setae of, 224; sec- tioning, 222. Eau de Javelle, 24. Echinoderms, fertilization and early embry- onic stages, 122. Eggs, of butterfly, 93; of chicken, 113; of amphibia, 118; fish, hatching-box for, 123. Ehrlich-Biondi (Heidenhain) triple stain, 174. Ehrlich, hematoxylin, 178 ; method for blood, 198 ; methylen blue method for nerve tissue, 180; triple stain, 99, 175. Beet fibers, 184, 185, 193; fine, 196; coarse, 196. Embyrograph, 149. Embryological methods, 113. Embryology, of amphibia, 118; of the chick, 113; of mammals, 119, 120, 121; of the mouse, 121; of the pig, 120; of the rabbit, 119; of teleosts, 117. Embyronic membranes, 121. Embryos, human, 126; measuring, 116; re- agents for, 126, 163, 166, 167, 169, 170, 178. Encircling fibers, 196. Endothelial cells, 202. Endothelium of blood vessels, 193. Eosin, 10, 20, 49, 51, 55, 62, 97, 175; formula, 10. Epidermis, 212. Epididymus, 210. Epistylis, 215. Epithelia, 165, 174, 176, 186, 187, 188, 201; isola- tion of, 202. Epithelium, ciliated, 201; columnar and glandular, 201; cubical, 201; of mouth, 198; of small intestine and villi, 198; of stomach, 198; of lungs, 211; of uriniferous tubules, 214; pigmented, 202; stratified, 202; squamous or pavement, 202; transi- tional, 202. Erlicki’s solution, 9, 29, 53, 59, 79, 163; form- ula, 9. Erythrocytes, 190. Erythrosin, 20, 176. Esophagus, 198. Ether alcohol, 8, 23, 166. Ether freezing attachment, 68, 69. Eustachian tube, 201. Animal Micrology Exner, 123. Eycleshymer’s clearer, 22, 62; methods of orientation, 125, Eye, 203; of sheep, 162. Eyeball, 203; blood vessels of, 203. Eyelid, 203. Eyepiece, 137. Eyepoint, 149, 160. Faded preparations, restaining, 58. Fading of blue color in injection mass, 86, Fahrenheit to Centigrade scale, 228. Fallopian tube, 210. Farrant’s solution, 22. Fasciola, 220. Fat, 186, 196. Fatigue of eyes, 169. Feathers, 93. Fecundation, see fertilization. Femoral artery, injection through, 85. Fenestrated membrane, 196, Fertilization, in Ascaris, 123; artificial, 122; in amphibia, echinoderms, and teleosts, 122; in mammals, 121. Fetuses, 126. Fibrillae in striated muscle, 205. Fibrillar (white fibrous) connective tissue, 197; cells of, 196. Fibrin, stained preparation of, 97. Fibrous tissue, 176. Fine adjustment, 159. Fixing, 16, 27, 30; purpose of, 16. Fixing agents, necessary qualities of, 16. Fixing and hardening agents, formulae, 161. Fixing sections to slide, 23, 41, 64. Flagella of bacteria, staining, 111. Flat worms, 90, 170. Flatness of field, 150. Flemming, 161; solution of, 164, 171, 185, 186. Flint glass, 140. Fluid mounts, 87, 88, 94. Flukes, 94. Foam structure, 192. Focal point, 134. Focus, principal, 134; real, 135; virtual, 135. Formalin, 8, 29, 53, 71, 84, 118, 121,126; asa reducing agent, 166; with alcohol and ace- tic acid, 166. Formic acid, 74, 177. Formol-sublimate, 167; and acetic acid, 167. Free-hand sectioning, 33. Freezing method, 67; carbon dioxide, 67; ether or rhigolene, 70. Fresh tissnes, examination of, 25, 187; fixing and washing after sectioning, 70; section- ing by the freezing method, 69. Friable objects, sectioning, 43, 44, 63- Frog, embryology of, 118. Fromman, lines of, 74. Fuchsin, acid, 20, 176, 189; and picric acid, 176; basic, 109, 176. Index Gabbet’s method for tubercle bacilli, 110. Gage, 9, 160, 169; carbol-xylol clearer, 9; formaldehyde dissociator, 9. Gall bladder, 198. Galt, 31. Ganglia, 182, 207; canaliculi in, 207; periph- eral, 73 Gastric glands, 198. Gelatin for injection mass, 81. General rules, 6. Gentian violet, 20, 106, 109, 176, 186. Germinal disc of chick, 113. Gianuzzi, crescents of, 198. Gilson’s mercuro-nitric fixing fiuid. 8, 77, 118, 166; formula, 8 Gilson’s rapid celloidin process, 64. Gizzard of cricket or katydid, 77. Gland cells, 162, 165, 175; of chironomous larva, 192. Glanders, 109, 111. Glomerulus of kidney, 214. Glycerin, 21, 22, 87, 88. Glycerin jelly, 21, 22, 79, 89, 90; formula, 90. Glycerin-picrate mixture, 162, Gnat, 91. Goblet cells, 198. Gold chloride, 20, 177; method for nerve- endings, 74. Gold size, 87. Golgi method, 71, 72; mounting Golgi prep- arations, 72, 73. Gonococcus, 111. Graafian follicle, 119, 210. Grades of alcohol, 7. Graduated cylinder, 4. Gram’s solution, 110, 177; method, 107, 110, 111. : Grandry’s corpuscles, 207. Grantia, 217. Great water beetle, foreleg of, 92. Gregarina, 216. Grenacher’s borax-carmine, 9; see also borax- carmine. Gritty feeling in paraffin sectioning, 46. Griibler and Hollborn, address, 174. Gum and syrup mass for freezing, 67. Gun cotton, 23. Guyer, 101. Hair, 212; development of, 212; follicle, 213; renewal of, 213. Hanging-drop preparations, 108. Hardening, 15, 17, 27. Harder’s glands, 203. Hardesty, 72, 73. Hard objects, to section, 47, 80. Hatching-box for fish eggs, 123. Hazy mounts, 57. Heart, 193. Heart-beat in nymph of dragonfly, 225. Heidenhain, 10, 52, 174, 178; demilunes of. 198, Hemalum, 84, 177. Hematein, 20, 177. Hematoidin crystals, 98. Hematoxylin, 19, 20, 162, 165, 168; ripening of, 9, 20. 57; Delafield’s, 9, 34, 49, 54, 55, 56, 57, 68, 84, 89, 91; iron, 10, 52, 53, 55, 123, 178; Ehrlich’s, 178; Weigert’s, 178. Hematoxylin and eosin, 51, 62. Hemin crystals, 98. Hemocytometer, 99. Hemoglobin crystals, 97. Herbst’s corpuscles, 12. Hermann’s fluid, 117, 120, 170, 171, 186; form- ula, 170. Hertwig’s macerating fluid, 76. Heterotypical mitosis, 192. His, 116, 129. Histological elements, isolation of, 75. Homogeneous immersion lens, 150, 152. Honing microtome, 44. Horn spoon, 1. Huber, 73, 128. Human embryos, 126. Hydra, 76, 89, 218; to kill expanded, 89. Hydrochloric acid, 8, 214. Hydrofluoric acid, 25. Hydrozoa, compound, 219, Tilumination, 150, 159. Images, 135; defects in, 138. Imbedding, 22; paraffin, 37; celloidin, 59; mass for well microtome, 35; a number of minute objects, 63; Ls, 45. Immersion objective, 152. Impregnations, 20. Incubator, 113. Indifferent fluids, 25, 187. Indulin, 190. Infiltration, 22. Inflammation, 101. Influenza, bacillus of, 111. Infusoria, 168, 173; quieting, 216. Initial magnifying power, 144. Injected vessels, corrosion of, 86. Injecting, with a syringe, 81; blood and lymph. vessels, 81; lymphatics, 85; liver fluke, 220; through femoral artery, 85. Injection, test for complete, 83; double, 84 85; continuous air pressure, 84; syringe, b) Injection masses, 81; to keep, 85; fading of blue in, 86; cold fluid gelatin, 86. Injection methods, 25, 81. Ink for writing on glass, 58. Insects, alimentary canal of, 77; antennae of, 93; having hard coverings, 93; delicate, 93; legs of, 93; mounting entire, 225; mouth parts of, 77, 93; muscles of, 90; nervous system of, 77; salivary glands of, 77, 192; seales of, 93; small or soft, 93; stings of, 77; wings of, 93. Intercellular bridges, 202. Intercellular substance, 186. 236 Interstitial imbedding, 22. Intestinal absorption of fat, 198. Intestine, large, 199; small, 199. In toto preparations, 26, 87. Intra vitam staining, 173, 179, 183, 216. Iodine, for washing after corrosive subli- mate, 166; Gram’s solution, 110, 177. Tris, 203. Iris diaphragm, 149. Iron hematoxylin, 10, 49, 178. Irrigation, 97. Isolation of histological elements, 15, 25, 75. Jamming together of paraffin sections, 46. Jelly fish, 219. Jelly of Wharton, 197. Jennings, 215, 222, 224, Johnson, 44, 119, 167. Joris, 86. Julin, 120. Kaiserling’s fiuid, 31. Karyokinesis, 165, 171, 192, 193. Keratin, 172, 185. Kidney, blood vessels of, 214; cortex and medulla of, 214; glomeruli of, 214; injec- tion of, 85; isolatica of tubules, 214; me- dullary rays of, 214; nerves of, 214. Killing, 16, 27. Kincaid, 31. Kleinenberg’s picro-sulphuric, 169. Koch-Ehrlich gentian violet, 109. Kronecker’s fluid, 187. Labelling vessels, 27; slides, 50. Labels, 1. Lacrymal glands, 293. Landois’ solution, 188. Large intestine, 199. Large objects, sectioning in paraffin, 45. Larvae, transparent, 88; small or soft, 93. Larynx, 211. Lavdowsky’'s mixture, 166, Lebrun, 161. Lee, 72, 171, 161, 185. Leech, 224. Length of time for staining tissues, 56. ee capsule and epithelium of, 203; fibers, 203. Lenses, 134; cleaning, 158; systems of, 137. Leprosy, bacillus of, 110. Leptothrix, 108. Leucocytes, 191; feeding, 102; granules of, 190, 191; to demonstrate movement in, 102. Ligament, 197. Ligamentum nuchae, 179. Light, for microscopical work, 151, 152; re- flected, 150; transmitted, 150. Light green, 20, 179. Lillie, 170. Ammal Micrology Lingual ribbon of snail, 84, Lip, 199. Lithium carmine, 107. Liver, amyloid infiltration of, 199; bile capillaries of, 199; blood vessels of, 199; cells, 199; flukes, 220; hepatic lobules of, 199; injection of, 85: interlobular connect- ive tissue of, 199. Locker, 4. Loeffler’s alkaline methylen blue, 109; blood serum, 110. Logwood, 20. ‘Lugol’s solution, see Gram’s solution. Lung, 212; blood vessels of, 212; elastic tis- sue of, 212; epithelium of, 211; foetal, 211; injection of, 85. Lymph, canals, 183; capillaries, 193; glands, 191; spaces, 183. Lymphatics, injection of, 85. Lyons blue, 10, 20, 49, 51, 179; formula, 10. MacCallum’s macerating fluid. 188. Macerated tissue, fixation of, 78. Maceration, 15, 25, 75, 76. . Magnification, determination of, 153, 154, Magnifiers, 136. Magnifying power, 144, 152. Majenta, 176. Malarial parasite, 103. Mallory and Wright, 102. Mallory’s connective tissue stain, 172. Mammal, maturation, fertilization, and seg- mentation in, 121. Mammalian embryos, older stages, 120. Mammary gland, 213. Manufacturers, 4. Mark, 43. Marking imbedded specimens, 38, Marchi, 207. Marrow, 191. Mast cells, 191. Material for a course in zodlogy, 215. Material, storing, 30. . Maturation, in amphibia, echinoderms, and be leorte: 122; in Ascaris, 123; in mammals, 121. early stages, 119; Mayer, 20, 45, 173, 177. Mayer’s albumen fixative, 12, 23; paracar- mine, 184; hemalum, 177; carmalum. 173. Measurement of microscopic objects, 153. Mechanical stage, 153. Medulla oblongata, 207. Medullary sheath, 270. Medullated fibers, of cord and tract, 207; tracts of, 178. Medusoid forms, 217, Meissner’s corpuscles, 208. Membrane of Descemet, 204. Mercuro-nitric fixing fluid, 8, 28, 53, 77; form ula, 8. Merkel’s fluid, 165. Mesothelial cells, 202. Index Metagelatin, 90, Metallic substances for color differentia- tion, 20, 71. Methods, general statement of, 15. Methylen blue, 20, 179; for bacteria, 107, 109; for impregnation of epithelia, 183; for nerves and nerve-terminations, 180, 181; for non-striated muscle, 182; for ordinary sections, 182; immersion method, 181; in- tra vitam stain, 180; Loeffler’s alkaline, 109; polychromatic, 180. Motty! green, 20, 76, 168. 183, 187; formula, Methyl violet, 20,57; formula, 183. Metric weights and measures, 227. Micrococci, 108. Micrococcus tetragenus, 111. Micro rater, stage, 153; ocular, 155; filar, Micrometry, 153-55, Micron, 44, 155. Microscope, 133; simple, 136; compound, 137, 139, 140; makers, 141-44; binocular, 146; dissecting, 4, 149; manipulation of, 157. Microscopical terms and appliances, 145. Microtome, well, 35; for paraflin, 39, 40, 60; Minot, 39; Minot-Blake, 40; oil for, 44; celloidin, 60; freezing, 67, 68. Microtome knife, tilt of, 40; sharpening, 44. _ Milk, 213. Milky looking mounts, 57. Minot, 116, 121, 174; microtome, 39, 40. Minute dissection, 77, 78. Mirror, 155. Mites, 88. Mitosis, 165, 171, 192, 198; heterotypical, 192. Mollusk, 225. Monieza, 220. Monocystis, 216. Moth, wings of, 92. Mounting, 22. Mouse, embryology of, 121, 122. Mouth, epithelium of, 198. Mucin, 172, 199. Mucoid connective tissue, 197. Miiller’s fluid, 70, 163, 188, 189; formula, 163. Muscae volitantes, 155. Muscle, 172, 176, 204; cardiac, 75, 187, 188, 204; to tendon, 205; smooth, 187; voluntary, 75; of insect, 90. Muscle fiber, branched-striated, 204; non- striated, 182, 205; striated, 75, 204; fibrillae of, 205; end of striated, 205; cardiac, 75. Mussel, gills of, 225; cross-section of, 225, Myelin, 207. Nail, 213. Naphthylamin yellow, 190. “Neck length” of embryos, 116. Needle, 1. Negative eyepiece, 137. Notseer: method of diagnosis of diphtheria, Nematocysts, 218, 219. 237 Nerve, 73; endings, 74, 177, 180; tissue, 162, 163, 176, 180, 186; degenerated fibers, 179, 207; plexuses in alimentary canal, 199; in- tra epithelial fibers, 207 ; medullated fibers, 207, 208; non-medullated fibers, 208; fiber bundles, 208. Herve cells and their ramifications, 71, 120, Nerve cells, Nissl’s method for, 162, 182. Nervous system, of grasshopper, 77; of ver- tebrates, 206. Neurokeratin, 208. Neuroglia, 73, 208. Neutral red, 183. Neutrophil granules, 175, 191. Nitric acid, 9, 79, 189; see also mercuro- nitric fluid. Nissl, method for nerve cells, 162, 182% gran- ules, 182, 183, 208, Nodes of Ranvier, 208. Nomenclature of objectives and oculars, 144, Normal! or indifferent fluids, 125, 187. Normal saline, 8, 187. Nose, 209; mucous membrane of, 209, Nosepiece, 139, 158. Numerical aperture, 155. Objects which will not stain, 56. Objects which alcohol would injure, section- ing, 70. Objects of generalinterest, mounting, 87. Objective, 137, 138; immersion, 152, 159; apo- chromatic, 146; using high power, 158. Oblique illumination, 150, Ocular, 137; compensating, 145, 148; search- ing, 148; working, 148, Odontoblasts, 201. Oil, anilin, 22; of bergamot, 22; cedar-wood, 21, 43, 48, 62; of cloves, 22, 78; of origanum, 22, 62; of sandal-wood, 22; of thyme and castor oil, 62. Oil immersion lens, 152, 159. Olfactory cells, 209; nerve processes of, 209, Opalina, 216. Opaque mounts, 92. Opaque objects, 150. Optical center of lens, 134. Optical principles, 133. Orange G, 20, 183. Orcein, 184. Organs and tissues with methods of prepara- tion, 190 Orientation, 38. Orienting chick embryos, 113. Orienting objects in the imbedding mass, 124; Patton’s method, 125; Eycleshymer’s methods, 125. Orienting serial sections, 124. Orth’s lithium carmine, 107. Osmic acid, 18, 78, 79, 116, 120, 167, 168, 183, 185; discussion of, 167; vapor, 168. Osmic material, bleaching, 45. Osmium-bichromate mixture, 72. Otoliths, 201. 238 Ova, 210. Ovary, 210. . Over-correction, 157. Oviduct, 210. Ovogenesis, 210. Oxalic acid, 87, 177. Oxyphil granules, 175. Pacinian corpuscles, 209. Pancreas, 199; granules of, 198. Paneth, cells of, 198. Paper box for paraffin imbedding, 37. Papillae of tongue, £00. Paracarmine, 169, 184. Paraffin, 12, 23; oven, 12; dirty, 43. Paraffin-asphalt-rubber method, 44. Paraffin block, trimming, 39. Paraffin method, 26; imbedding and section- ing, 37; staining and mounting, 49; for delicate objects, 54; compared with cel- loidin method, 63. Paramoecium, 215, 217. Parfocal, 156. Parotid gland, 199. Pathogenic bacteria, 111. Patton, 125. Pearl, 167. Pectinatella, 222. Pedesis, 146. Pencil, glass-marking, 1, 42. Penetration, 156. Penis, 210. Peroxide of hydrogen, 9, 24. Peyer’s patches, 199. Pfliiger’s egg tubes, 210. Phloroglucin method, 189. Picric acid, 18, 168, 184, 189. Picric alcohol, 169. Picro-acetic, 117, 122, 123, 169. Picro-carmine, 76, 79, 168. Picro-hematoxylin, 177. Picro-sublimate, Rabl’s formula, 169; vom Rath’s formula, 169. Picro-sulphuric acid, 117, 121, 169. Pig embryos, to obtain and prepare, 120, 121; stages necessary for study, 120; placenta- tion of, 121; orientation of, 121, Pigment cells, 193. Pigments, to remove, 40. Placenta, 174, 260. Placentation, 121. Planaria, 90, 219. Platino-aceto-osmic mixture, 170. Plumatella, 222. Pneumococcus, 111. Polariscope, 156. Polypoid forms, 219. Positive eye-piece, 137. Potassium bichromate, 162. Pouring liquids, 6. Preparation of reagents, 7. Anmal M icrology Preservation of anatomical specimens, 31. Preserving, 18, 30; mixture, 30; objects im- bedded in paraffin, 43; sections cut by the freezing method, 70. Prickle cells, 213. Principal axis of lens, 134. Protococcus, 94. Plotoplasmic currents, 193. Protozoa, 95, 167; cultures, 215; feeding, 216; ~ permanent mounted preparation, 217; quieting, 216; staining; 216. Purkinje cells, 73, 209; fibers, 194. Pyrogallol, 170, 185. Pyroxilin, 23. Rabbit, embryology of, 119; dissection of, to obtain embryos, 119, 120; ova, time to ob- tain various stages, 119. Rabl, 169. Radula of snail, 94. Ranvier, one-third alcohol, 188; picro-car- mine, 185; cross of, 74; nodes of, 208. Rath, O. vom, 169. Rating of objectives and oculars, 144. Rays of light, 133. Razor, section, 1, 33. Reagent bottles, 4. Reagents and their preparation, 7; Appendix B. Reconstruction of objects from sections, 127; in wax, 127; geometrical, 129. Reconstruction points, 125. Records, card, 6, 28; calendar, 6. Rectified spirit, 13. Reduction division, 193. Refraction of light, 133. Remak’s fibers, 208. Removal of liquid from slide, 55. Reproductive organs, 210. Resolving power, 156. Respiratory organs, 211. Restaining old mounts, 58. Reticular connective tissue, 197. Retina, 165, 168, 188, 204. Reversing sections, to avoid, 40. Rhigolene freezing attachment, 68, 70. Ripart and Petit, 164, 187. Rolling of paraffin sections, 46. Romanowsky stain, 103. Rosin, 183. Rotifers, 222. Rubbing sections off the slide, to avoid, 56. Rubin §, 176. Rules, general, 6. also Safranin, 10, 20, 165,185; formula, 185; and gentian violet, 186. Salivary gland, 199, 200;. granules of, 198; of cockroach or cricket, 77. Salycilic acid, 170. Sarcinae, 108. Sarcolemma, 205. Index Scales from wings of insects, 93. Scalpel, 1. Scissors, 1. Scheme for mounting whole objects or sec- tions, 26. Schiefferdecker’s fluid, 188. Schneider’s acid carmine, 174. Schultz’s dehydrating apparatus, 55. Schultze’s iodized serum, 187. Sclera, 204. Scolex of tapeworm, 220. Scraping of microtome knife, 47, Scratches across paraffin sections, 47. Sealing bottles and preparation jars, 30. Sealing mounts, 89. Sebaceous glands, 213. Secondary axis of lens, 134. Section lifter, 1. Section method, 15; simple, 33. Sectioning in paraffin, 37, 38; in gum, 67; in celloidin, 61; free hand, 33. Sections, affixing, 23; drying of, 55; milky or hazy, 57; plane of, 123, 124; scheme for mounting, 26; to stain by flooding, 58; washing off of, 58. Semicircular canals, 201. Seminal vesicle, 210. Seminiferous tubules, 211. Serial sections, orienting, 123; see paraffin or celloidin method. Sharpey, fibers of, 195. Shell vials, support for, 30. Silver, nitrate, 20, 120, 186; formula, 186; for nerve, 73. Size of microscopic objects, to measure, 153. Skin and its appendages, 212, 213; blood ves- sels of, 213. Slide box, 1. Slides, 1; passing through reagents, 56; to clean, 57 Small intestine, 198; epithelium of, 198. Small objects, to transfer through reagents, 30; to section free hand, 34; to orient in paraffin, 43. Smear preparations, blood, 98; bacteria, 105. Smegma bacilli, 110. Snail, 225; or slug, 84; to obtain eggs, 123. Sobotta, 122. Sodium chloride, dissociating fluid, 188. Solutions, rules for making, 6. Spawning of fish, 122. Spectrum, 146; tertiary, 146. Spencer microscope, 142. Spermatogenesis, 211. Spermatozoa, 211. Spherical aberration, 138. Spicules of sponges, 217. Spinal cord, 73, 209. Spinal ganglia, 209. Spirillum, 108; of Asiatic cholera, 111. Spirogyra, 94. Spexges, 217. 239 Spores of bacteria, staining, 111. Sporozoa, 216. Sputum, to examine for tubercle bacilli, 110. Staining, 19-21, 49; double or multiple, 20, 21, 51; in bulk, 54; causes of failure in, 56, 60. Staining jars, Coplin, 4. Stains, formulae, 170; classification of, 19; cytoplasmic, 20; nuclear, 20; replenishing, 56; precise with hematoxylin, 56; for bac- teria, 109. Standard tube-length, 157, 158. Staphylococci, 108. Staphylococcus pyogenes aureus, 111; al- bus, 111 Stenders, 4, 49. Sting of wasp or bee, 77. Stomach, 200; epithelium of, 198, Stoppers, to remove, 13. Storing material, 30. Streptococci, 108. Streptococcus pyogenes, Stropping, 45. Sublingual gland, 200. Submaxillary gland, 200. Sudan IIT, 186. Supplies, 1. Supporting tissue, 194. Suprarenal gland, 214. Sweat glands, 213. Sympathetic ganglia, 209. Synovial villi, 197. Syphilis, bacillus of, 110. Syracuse watch-glass, 4. Syringe, injection, 82, 85. 111; capsulatus, Tables of equivalent weights and measures, 99 ais Tactile corpuscles, 209. Tactile menisci, 2U9, Taenia, 220, 221. Tandler, 86. Tannic acid, 97. Tapeworm, 94, 22 Tastebuds, 200. Teasing, 15, 25, 75, 76. Teeth and other hard objects, grinding, 89. Teichmann’s crystals, 98. Teleosts, embryology of, 117; to orient blas- toderms of, 117; manipulation of embry- onic material, 117, 167; fertilization and early embryonic stages, 122. Tellyesnicky’s fluid, 163. Temperature of laboratory, 438. Temporary mounts, 35. Tendon, 197; cells of, 197; to muscle, 197. Terminal bars, 202. Testis, 76, 211. Tetanus, bacillus of, 111. Tetracocci, 108. Thermometers, 228. Thin sections, 34, 43, 56. 0, 221. 240 Thionin, 20. Thymol, 170, 177. Thymus gland, 191, 212. Thyroid gland, 212. Tigroid substance, 182, 208. Tilt of microtome knife, 40. Tissues and organs with methods of prepa- ration, 190. Tissues, killing and fixing, 27; which crum- ble in paraffin, 43; length of time for staining, 56. Toluidin blue, 20. Toluol, 22. Toisson’s solution, 100. menene: 200; papillae and folliculi linguales, Tonsil, 200. Tooth, sectioning decalcified, 79, 200; devel- opment of, 200; odontoblasts, 201. Tough objects, sectioning, 47. Trachea, 212. Tracts of medullated nerve fibers, 178. Transparent aquatic organisms, 180. Transparent larvae, 88. Trichina, 221, 222; examining alive, 222 Triplet lens, 138. Tube-length, 157. Tubercle bacilli, 110, 111. Turn-table, 4, 88. Typhoid, bacillus of, 111. Umbilicus, 211. Under-correction, 157. Unna, 182, 184. Ureter, 214. Urethra, 211, 214. Urinary organs, 213. Uriniferous tubules, epithelial cells of, 214. Uterus, 211; and placentation, 121. Vagina, 211. Vas deferens, 211. Valves of heart, 194. Van Beneden, 120. Van Giesen’s stain, 176. Variation in thickness of paraffin sections, Animal Micrology Vascular system, double injection of, 84. Vein, 194. Vertebrata, 226. Vials, 4 Vision, conventional distance of, 144. Visual purple, 204. Volvox, 94. Von Ebner’s fluid, 189. Vorticella, 215. Vulcanized fiber for celloidin mounts, 62. Walton, 30, 117, 21 Wash bottle, 4. Washing, 17. Watch-glass, Syracuse, 4. Water immersion lens, 152. Water method for affixing sections, 23. Water mites, 88. este plates for reconstruction, preparation of, 127. Weigert, hematoxylin, 178; method for bac- teria, 107. Weights and measures, table of equivalent, Well microtomes, 35. Wharton, jelly of, 197. White objects, orientation of, 43. White spots in paraffin, 38. Whitman, 118. Whole objects, mounting, 26, 87. Wire, copper, 1. Wollaston’s camera lucida, 146. Worcester, 119, 167. Working distance, 157. Work-table, 157. Wright’s stain for blood and for malarial parasite, 102. Wrinkles in paraffin sections, 42, 46. Writing on glass, ink for, 58; pencil for, 1, 42. Xylol, 13, 21, 38, 49; for removing paraffin, 56, Zenker’s fluid, 120, 126; formula, 163. Zeiss microscopes, 141, 144. Ziehl-Neelson, carbol-fuchsin, 109. 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