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95-99 % Alcohol | v Clear in Carbol-xylene Vv Mount in Balsam 1206 § 99. Staining with Safranin Flemming’s or Hermann’s fluid fixation (best) Vv Paraffin sections v *. Benzin v 95 % Alcohol Vv Safranin (Babes’ ) 2-12 hours Vv Distilled water (rinsed) Vv 95 % Alcohol Vv Acid 95 % alcohol (45 Hel.) for a few seconds Vv 95-99 % Alcohol ! Carbol- xylene v Mount in Balsam MOUNTING. § 100. Whether stained or unstained, prepared for microscop- ical examination by isolation or sectioning, and especially if it is de- sired to keep the preparation, it is necessary to moun? it in some way,—?. €., SO arrange it upon some suitable support (glass slide) and in some suitable mounting medium that it may be satisfactorily studied with the microscope. Mounting may be I. Temporary, or II. Permanent,—as A. Dry, or in air, B. Ina medium miscible with water, or C. Ina resinous medium, in which case it is neces- 121 sary first to remove all water by either (a) drying—Deszccation, or (4) a series of displacements, 7.¢., 1. Removing the water with strong alcohol—Dehydration ; 2. Removing the alcohol with clearer—Clearing ; 3. Replacing the clearer with balsam or other resinous mounting medium. § 101. Temporary mounting. Used in this course princi- pally in the examination of blood corpuscles and living ciliated cells (§ 2). Temporary examination of tissues is quite simple, though important, and for this it is only necessary to place the teased tissue or section on the slide in a drop of the fluid in which it is at the time, normal salt solution, dissociator, or alcohol, and cover. The examination of preparations intended for permanent mounts during the staining or before mounting will often serve to detect faulty treatment at a time when it may be remedied without great expend- iture of time, or discard the specimen as worthless. § 102. Permanent mounting. In this course are employed (a) mounting dry on a ring or in a cell, (4) in glycerin or glycerin jelly, media miscible with water, and (c) in xylene balsam, a resin- ous medium. § 103. Mounting dry. The preparation may be either upon the under side of the cover-glass (best if possible) or rest upon the bottom of the cell. In the first case a shallow cell made by a shellac ring will be sufficient ; in the second, a shellac ring may not give a deep enough cell and a paper, hard rubber, or metal ring may be cemented to the slide. (a) When the preparation is on the cover, Prepare a shellac cell (§ 106) on the slide of a size slightly smaller than the cover to be used, and allow it to dry for a day or so. Warm the cover bear- ing the preparation to remove the last traces of moistuie, and place it film side down upon the ring. Warm the slide until the edge of the cover may be made to adhere to the shellac ring and press the cover down until it adheres all the way round. Seal the cover with shellac and label ($§ 111, 113). : (b) Mounting in a paper or rubber cell, With a brush, cover one side of the ring with a layer of shellac and place it on the center of the slide, shellac side down; place within the cell the prepara- tion, arranging it in the manner desired, and place upon the ring a cover-glass of a suitable size, and seal it with shellac ; label. 122 § 104. Mounting in glycerin media. (a) Pure glycerin ; (6) glycerin and acetic acid, 1%; (¢) glycerin, alum carmine and eosin (§ 156). Glycerin and glycerin-jelly are most serviceable in mounting isolation preparations. For both of these mounting media the ob- ject must be mounted from water or an aqueous solution. Arrange the section or teased tissue in the center of the slide, drain off the water or aqueous solution in which the preparation is and adda small drop of glycerin. Take a clean cover in the forceps, breathe on the under side and carefully lower it upon the object ; gently press it down. It is best to use only a small drop of glycerin so as not to get it outside the cover, as it is hard to clean away sat- isfactorily. Clean carefully and seal with shellac in accordance with § 111. § 105. Mounting in glycerin-jelly. The preparation should be mounted from some aqueous solution. Warm the slide gently and put it upon the centering card; in the center of the slide place a drop of warmed (melted) glycerin-jelly. Remove the object from the water or aqueous solution and arrange it in the glycerin-jelly. Grasp a cover-glass with the fine forceps, breathe on the lower side, gradually lower it upon the object and gently press it down. Allow the glycerin-jelly to set, keeping the slide horizontal meanwhile. Scrape away the superfluous glycerin-jelly around the cover-glass and seal with shellac (§ 111). § 106. Preparation of shellac mounting cells. Place the slide upon the turn table and center it (i. e., get the center of the slide over the center of the turn table). Select a guide ring on the turn table which is a little smaller than the cover-glass to be used ; take the brush from the shellac, being sure that there is not enough cement adhering to it to drop. Whirl the turn table and hold the brush lightly on the slide just over the guide ring selected. An even ring of the cement should result. If it is uneven, the cement is too thick or too thin or too much, was on the brush. Afteraring is thus prepared, remove the slide and allow the cement to dry spontaneously, or heat the slide in some way. Before the slide is used for mounting, the cement should be so dry when it is cold that it does not dent when the finger nail is applied. A cell of con- siderable depth may be made with shellac by adding successive lay- ers as the previous one dries. 123 §$ 107. Mounting in balsam. Certain preparations may be mounted in balsam by drying, the method of desiccation, (§ 100), e. g., cover-glass preparations of bacteria, stained cover-glass prepara- tions of blood, etc. For this it is only necessary that the prepara- tion be absolutely dry, a sinall drop of balsam placed upon it or upon the under side of the cover-glass, which is carefully placed over the specimen and pressed down. Mounting in balsam by desiccation is serviceable for but few preparations in histology, and in most cases the removal of the water by a series of displacements is resorted to (§ 100). For this the following steps are necessary: Dehydration, Clearing, Mount- ing in balsam. Dehydration. The sections are entirely freed from water by the use of 95% or absolute alcohol. The slide or free section may either be placed in a jar of alcohol or alcohol from a pipette be poured over it. Treat the preparation to be mounted for 5-15 min- utes. The thicker the section the longer the time required ; collo- dion sections require a longer time than paraffin sections. In any case, be sure that the dehydration is complete, giving a longer rather than a shorter time, and then clear. § 108. Clearing. This is accomplished by putting the slide in a jar of clearer or dropping the clearer upon the section from a pipette. When the section is cleared it will be transparent. Test it by holding it against a dark background ; if it is not cleared it will be cloudy, white, and opaque. § 109. Mounting in balsam. Drain off the clearer and allow the section to stand until there appears the first sign of dullness from evaporation of the clearer from the surface. Then place a small drop of balsam upon the section or upon the cover-glass which is then inverted over the specimen. Remember that in mounting in this way you must always ‘‘ De- hydrate, Clear and Mount in Balsam,’’ and that the three steps are inseparable. SEALING THE PREPARATIONS. This is only necessary when the preparation is a glycerin or glycerin-jelly mount. It is better not to seal balsam preparations, or only quite late after the balsam has thoroughly dried out. 124 § 110. Sealing glycerin mounted specimens. Wipe away the superfluous glycerin as carefully as possible with a moist cloth or a piece of lens paper. Place four minute drops of cement care- fully at the edge of the cover at the four quarters and allow them to harden for half an hour or more ; these will anchor the cover-glass and the preparation may then be placed upon the turn-table and a ring of shellac cement put round the edge while revolving the turn- table. §$ 111. Sealing glycerin-jelly mounts. Allow the glycerin- jelly to harden for 12 hours or longer. With a knife scrape away the superfluous jelly and then carefully wipe around the cover-glass with a cloth moistened with water. Place the slide on a turn-table, carefully center the cover-glass, and with a brush seal the edge of the cover by a ring of shellac while revolving the turn-table. A second coating may be given subsequently if needed, after the first coat has dried. § 112. Sealing balsam mounts. This is necessary only with special preparations, and should in any case be done only after the preparations have dried out for several weeks. With a knife scrape off all superfluous balsam from around the cover-glass and wipe it carefully with a cloth moistened with alcohol or benzin (or xylene). Seal as with glycerin-jelly mounts. When the oil im- mersion is to be used often, it is advantageous to seal the prepara- tion with shellac (after it has dried) to facilitate cleaning away the immersion fluid. LABELING MICROSCOPIC SLIDES. § 113. Every permanent microscopic preparation should be carefully and neatly labeled in ink, the label being placed upon the right hand end of the slide. The label should furnish at least the following information : EXAMPLE. (1) The number of the preparation, | No. ©. 415: the thickness of the cover- S. to ML. glass and of the section. (2) The name, kind, and source of the preparation. (3) The date of the specimen. November, 1898. Tleum of Cat. Transection. 125 In the case of specimens with which it is advantageous to have more information at hand a second label may be placed upon the other end of the slide, and it may bear the following information : (1) Mode of fixation. (2) Imbedding method. (3) Stains employed. (4) Mounting medium (generally not necessary). (5) Special purpose of the preparation. . A catalog giving the full data of the specimen,—age, condition of the animal, mode of preparation in detail, special points illus- trated, etc., is not required for the preparations in this course, but is valuable particularly in special investigations and with standard specimens. CLEANING SLIDES AND COVER-GLASSES. $114. Cleaning cover-glasses. Fill the large glass box one-half full of cleaning mixture and put in the new covers, one at a time, being sure that they are entirely immersed and the cleaning mixture reaches all points. The one cover may be pushed under by the next. Let them remain over night (or longer) and then wash them thoroughly in running water, until all trace of the cleaning mixture is removed. Then place the covers in 50 or 67% alcohol. § 115. Wiping the cover-glasses. When ready to wipe the cover-glasses, remove. several from the alcohol and put them ona soft, dry cloth, or on some of the lens paper to let them drain. Grasp a cover-glass by its edges, cover the thumb and index finger of the other hand with a soft, clean cloth, or some of the lens paper. Grasp the cover between the thumb and index finger and rub the surfaces. In doing this it is necessary to keep the thumb and index well opposed on directly opposite faces of the cover so that no strain will come on it, otherwise the cover is liable to be broken. When a cover is well wiped, hold it up and look through it to- ward some dark object. The cover will be seen partly by transmit- ted and partly by reflected light, and any cloudiness will be easily detected. If the cover does not look clear, breathe on the faces and wipe again. If it is not possible to get a cover clear in this way it should be put again into the cleaning mixture. When the covers 126 are wiped, put them in a clean glass box. Handle them always by their edges, or use fine forceps. Do not put the fingers on the faces of the covers for that will surely cloud them. $116. Measuring the thickness of the cover-glasses. With the cover-glass measurer determine the thickness of the cover- glasses and sort them into three groups: (a) those with a thick- ness of .13-.17 mm., (6) those less than .13 mm., and (c) those thicker than .17 mm. Groups (a) and (6) only should be used ; (c) should be discarded or used only with objects for low magnification. It is advantageous to know the thickness of the cover-glass on an object for the following reasons: (a) That one do not try to use objectives in studying the preparation of a shorter working distance than the thickness of the cover-glass (Microscopical Methods, § 57) ; (6) In using adjustable objectives with the collar graduated for dif- ferent thicknesses of cover, the collar might be set at a favorable point without loss of time; (¢) For unadjustable objectives the thickness of cover may be selected corresponding to that for which the objective was corrected (see Microscopical Methods § 27 table). Furthermore if there is a variation from the standard one may rem- edy it in part at least by lengthening the tube if the cover is thinner and shortening it if the cover is thicker than the standard (Micr. Meth. § 96). § 117. Cleaning slides. Rinse new slides thoroughly in clean water and then wipe them with a soft towel. In cleaning the slides handle them by their edges to avoid soiling the face of the slide. After the slides are cleaned, to keep them free from dust store them again in the box in which they came, or in a covered glass dish or jar. § 118. Cleaning used slides and covers. If only water, glycerin or glycerin-jelly has been used on them, they may be cleaned with water, preferably warm water, and then, if necessary, wiped out of 50% alcohol. If balsam has been used, heat the slides until the balsam is soft and then remove the cover-glasses. Scrape from the slides all the balsam possible and place them (and cover-glasses) in cleaning mixture for several days. If then they cannot be readily cleaned, place them in fresh cleaning mixture for a period of several days. Wash away the cleaning mixture thoroughly with water and wipe them with a clean towel. 127 SPECIAL METHODS. THE BLOOD. § 119. Special methods in the examination of the blood include (1) Examining fresh ; (2) Technic of staining blood films; (3) Determination of the number of red and white corpuscles per cubic millimeter ; (4) Determination of the relative amount of hemoglo- bin ; (5) Spectroscopic examination of blood (hemoglobin). (1) and (2) are briefly given here ; for (5) see Microscopical Methods, §§ 201-203. § 120. Examining fresh. This consists in covering a drop on a slide and immediately sealing the cover-glass to prevent evapo- ration, observing the following cautions: (1) The drop of blood (from the finger or the lobe of the ear) should flow freely and not be obtained by pressure. The drop should be a medium-sized one, which will spread out in an even, thin layer under the cover. (2) The drop should be received upon a cover or slide, covered, and sealed at once with castor oil. Examination of fresh blood may be used in clinical examination for the detection of some abnormal conditions, and it is of value in the rough diagnosis of many others. § 121. Stained preparation of blood. (a) Preparing the blood film. 'This may be best done in one of two ways: (1) The edge of a slide is first drawn through a drop of fresh blood and then moved quickly across the surface of a clean cover-glass, in this way spreading the blood in a thin, even layer upon the cover. Success depends upon getting the right amount of blood upon the edge of the slide and the quick, even movement by which it is spread upon the cover-glass. A second, possibly better, method is the following : (2) Have ready two thin clean cover-glasses and obtain a drop of fresh blood. ‘Take one of the covers in the forceps, touch it to the drop of blood and place it upon the second cover-glass eccentric- ally, with one edge projecting slightly. Sd the two covers apart in the plane of their surfaces and dry them quickly by waving them in the air or by passing them rapidly over the tip of a flame. The lower cover-glass will have the better film. (6) Fixing the hemoglobin with (a) ether-alcohol or (b) heat. 128 § 122. Fixing with ether-alcohol. When the blood films on the covers are dry, place them in ether-alcohol (equal parts) for 4% -1 or several hours, Let them fix for a longer rather than a shorter time, as the quality of the stain (with triacid mixture) will be improved. After they have fixed a sufficient time remove and again dry them in the air. They may now be stained, immediately or at convenience. § 123. Fixing with heat. Place a gasor alcohol flame under the apex of a triangular copper table (or other similar warm table or incubator). When it is well heated determine the region that has a temperature equal to the boiling point of water (100° C) by plac- ing on it drops of water at varying distances from the flame. Just wethin the point so determined (nearer the flame) place the covers bearing the dried film of blood, film side down upon the copper plate. Leave them for 15-30 minutes or longer. When the covers have cooled they are ready to be stained. (c) Staining with (a2) Eosin and Hematoxylin, (4) Ehrlich’s Triacid Mixture, or (¢) Methylene Blue. § 124. Eosin and hematoxylin. Stain the fixed blood films for 2-3 minutes with a !4% aqueous solution of eosin, rinse with water and stain for 10-15 minutes with hematoxylin, rinse again with distilled water and allow the film to dry. When dry, warm the cover gently to remove the last traces of moisture and mount in balsam. This gives a good general stain of the blood corpuscles, both red and white. Red corpuscles will be stained red or pink, the nuclei of the white corpuscles will be blue, and their cell bodies but faintly stained ; eosinophile granules, a bright pink. § 125. Ehrlich’s triacid mixture (§ 93). Place a drop or two of the mixture upon the film side of the cover. Stain for 10-15 minutes, rinse in distilled water, dry, and mount in balsam. If the stain is successful, red corpuscles will be orange-vellow, nuclei of white corpuscles, blue, cell bodies, pale pink, eosinophile granules, copper red. This stain is used a great deal in the clinical examination of blood and is a valuable one. Should the red corpus- cles be colored red instead of orange-yellow, it means that the fixa- tion has been insufficient ;- if ether-alcohol was used for fixing, the time was too short ; if heat was employed, the degree of heat was insufficient or the time too short. The right time of fixation and staining must be, in many cases, determined by experiment. 129 § 126. Methylene blue. Formula: Methylene blue, satu- rated alcoholic solution, 1 part ; distilled water, 2 parts. This is for staining ‘‘ basophile’’ granules in mast cells. Prep- arations fixed by heat or ether-alcohol are stained 15 minutes or so, washed with water, dried, and mounted in balsam. Nuclei and bas- ophile granules stain blue ; all else is colorless, or nearly so. This stain has but little value in the study of normal blood, since baso- phile leucocytes are of rare occurrence. FINE INJECTION. For the purpose of examining microscopically the finer arteries and veins and the capillaries in a tissue, and their relation to the other parts, it is necessary to fill them with some colored injection mass, or otherwise stain or color them. The masses employed for the fine injection of tissue in this course are Carmine Gelatin Mass (red) and Berlin Blue Gelatin Mass. $127. Carmine gelatin mass. Formula - Dry gelatin, 75 grams ; carmine (No. 40), Io grams water, 90 c.c. ; ammonia, 10 c.c. ; acetic acid, g. s.; chloral hydrate, 10 grams. Soak the gelatin in water until it is soft ; pour off the superflu- ous water and melt it (in an agate or porcelain dish) over a water bath. Grind the carmine to a paste with water ; add all the am- monia and water ; filter, warm to 80° or go° C., and add to the warm gelatin. Then add slowly the acetic acid diluted with an equal vol- ume of water, while constantly stirring the mass, until the mass smells very slightly of the acid. Filter through fine flannel. If the mass is acid, the chloral hydrate may be safely added (as a preserva- tive) ; if any ammonia is present it will decompose it forming chlo- roform and a granular precipitate. If too much acid is added, the gelatin will not set. § 128. Berlin blue injection mass. Formula: Dry gela- tin, 75 grams: saturated aqueous solution of Berlin blue, 150 c.c. chloral hydrate, to grams. Prepare the gelatin in the manner given above (§ 127) ; warm the Berlin blue solution (to 80° or go° C.), and add it to the hot gelatin. Heat the mixture for 10 minutes or more, stirring it occasionally, and filter it through fine flannel and add the chloral hydrate. 130 § 129. For securing the best results in injecting the following conditions should be observed: (1) A young but nearly mature, lean animal is to be preferred. (2) Kill the animal with an anes- thetic (chloroform) and leave it in the anesthetic at least half an hour before beginning the injection; do not, however, wait until vigor mortis sets in. (3) Inject only the part desired, tying all an- astomosing vessels and all vessels to other parts. Inject into the artery of the part, leaving the vein open until nearly pure injection mass escapes, then tie it and continue the injection until the part feels hard and is the color of the injection mass. (4) When the injection is finished cool the part injected by means of cold water, ice, or snow. (5) Harden the injected tissue 1 or 2 days in 50% alcohol, 2 or 3 days in 67% and 82% alcohols. The acidity of the alcohols should be insured by adding to the 50% alcohol a few drops of acetic acid. The tissue may be stored in 82% alcohol until ready for sec- tioning. For sectioning the collodion method is usually preferable. $130. Silvering blood vessels. Silver nitrate may be used for coloring blood vessels, and thus differentiating them. See § 147. CALCIFIED STRUCTURES,—BONE AND TOOTH. (A). Decalcification. § 131. For the purpose of investigating the soft structures of tissues containing lime salts, such as bone, teeth, and calcified carti- lage, it is necessary to remove the lime salts before sections can be prepared in the usual way by a process known as decalcification, Solutions of a large number of acids, combined or uncombined with other substances, may be used as decalcifiers. Very satisfactory are: (z) nitric acid, 3 c.c.; 70% (67%) alcohol, 97 c.c., and (2) nitric acid, 5 c.c. ; saturated aqueous solution of (potash) alum, 50 c.c.; water, 50c.c. In the first formula the alcohol, in the second the alutmn acts as a restrainer of the nitric acid. The first of these formulas is, perhaps, better for bone ; the second has a more rapid action and is possibly a better decalcifier for teeth. § 132. Directions for use. The tissue to be decalcified must be first thoroughly fixed and hardened by one of the approved methods,—picric alcohol is quite satisfactory,—and should be in 131 82% alcohol. In fixing, structures not needed should be removed, — muscles trimmed away from the bone, etc. Bones or teeth should be opened with nippers or a saw, so that the fluid may reach the marrow or pulp cavity. Place the hardened tissue in the decalcifier, where it should remain until the lime salts have been entirely removed, as may be ascertained by inserting a fine needle ; if any calcified matter remains there will be a gritty feeling on using the needle. The time neces- sary for complete decalcification will depend upon the size and den- sity of the calcified tissue, and will vary from 3 to 15 days or longer. The decalcifier should be changed after the first day, and if the tis- sue is large it is best to change it subsequently two or three times at intervals of two or three days. When decalcification is complete rinse the tissue well in water for a few minutes and place it in 67% alcohol for one or two days and then in 82% alcohol for several days, or until ready to imbed. The 82% alcohol should be changed once or twice in order that the nitric acid may be well washed out. Although paraffin in many cases may be employed for imbedding, the collodion method is gen- erally more satisfactory. Hematoxylin with eosin, hematoxylin with picrofuchsin, and hematoxylin with picrocarmine afford good stains; by staining thoroughly with hématoxylin a differential staining of bone and cartilage may be obtained. (B). Sections of Dry Bone or Tooth. § 133. Though the general structure of bone and teeth is shown moderately well when the tissue has been decalcified (§ 131), the Haversian canals, canaliculi and lacunae of bone and the denti- nal tubules of the teeth are shown much better in sections of dried, non-decalcified tissue, rendered sufficiently thin for microscopic ex- amination by grinding or filing. § 134. Directions for procedure. Prepare thin transverse sections of dried bone in accordance with the directions below. Longitudinal (radial) sections and tangential (surface) sections may also be prepared in the same manner, the former to show the Haver- sian canals and their anasomoses, the latter to indicate the shape of the lacunae as seen in a different plane. 132 1. Sawing the section. Make an exact transection of a part of the shaft of a long bone. The section should be about 1 cm. long and include the thickness of the shaft from the surface to the medul- lary cavity. Make the sections about 1 mm. thick. 2. Grinding the sections. Place the piece of bone on a cork or piece of soft wood and wet it with water. File it on one side until smooth and then turn it over. Continue the filing till the piece is from .05 to .10 mm, thick, using the cover-glass measurer to deter- mine the thickness. In the beginning one can press quite hard in filing ; as the section thins, more care should be exercised and the pressure should lessen. 3. Washing and drying the section. When the section is thin enough, rinse it and dry it with lens paper. 4. Mounting the sections in hard balsam. To prepare the bal- sam, put two or three large drops on the middle of a slide and heat the slide in some way to drive off the volatile constituents. Do not heat the balsam hot enough to produce bubbles. When the balsam chips after cooling, it is ready for use. In mounting, have the section and a clean cover so placed that they may be easily and quickly grasped. A cork somewhat smaller than the cover-glass should be within reach, and also a stone or piece of glass upon which to quickly cool the specimen as soon as it is mounted. : Heat the slide until the balsam is well melted. Put the slide upon a piece of paper, grasp the piece of bone with the forceps and plunge it into the melted balsam ; put on the cover as quickly as possible and press it down with the cork ; finally put the slide on the stone or glass to cool the balsam quickly. All of this should be done as rapidly as possible, and if done rapidly, the air will be retained in the lacunae and canaliculi, and cause them to stand out as black spots and lines. If soft balsam were used it would soon drive out the air, and being of nearly the refractive index of bone, it would obliterate the lacunae and canaliculi. Further, if the hot bal- sam were not cooled quickly, the air would be driven out and balsam would take its place in the spaces. CENTRAL NERVOUS SYSTEM. § 135. There are two special groups of methods employed in the microscopical examination of the central nervous system, (a) 133 Wergert Hematoxylin Methods, for differentiating myelinic nerve tracts, and (6) the Chrome-siluver Impregnation Methods, to bring out the form of the cells and differentiate the amyelinic nerve-fibers. These have their greatest value when used in conjunction with more general staining methods, which supplement the first and give a basis for the correct interpretation of the appearances produced by the second method. In addition, there are other stains helpful in bringing out the finer structure of the cells. $136. The Weigert hematoxylin methods. These meth- ods all involve (a2) mordanting the tissue with a chromium or cop- per salt, (6) staining (over-staining) with a strong hematoxylin stain, and (c) decolorizing (differentiating) the sections in a bleacher until the myelinic fibers are blue, all else (except blood) white to brown. , § 137. The following method is serviceable: Harden the tis- sue in a 3% aqueous solution of potassium dichromate for about 2 weeks, and in a 5% aqueous solution for an equal period (§ 31). Imbed in collodion ; paraffin may be used if the piece of tissue is small. The sections should not be thicker than 254. Wash the sections in water ($§ 68-70) and (a) Mordant them 1-12 hours in a half-saturated aqueous solution of copper acetate; rinse them well in water and (4) Sfazm them for 1-24 hours (until black) in Weigert’s hematoxylin (§ 83) ; rinse well in water and (c) Differ- entiate in the Weigert’s bleacher (Formula: Water, 200 c. c.; borax, 2 grams; potassium ferricyanide, 2.5 grams). Watch the differentiation carefully and when the cinerea has become a golden brown and the myelinic fibers a rich blue, stop the action and wash the sections in water, running or changed several times, for at least % hour. Dehydrate, clear, and mount in alkaline balsam (best) ($158). Ifthe specimens are to be left for some time before mount- ing, place them in 95% alcohol with sodium carbonate added to render it alkaline. § 138. Pal’s method may be used if it is desired to stain the nerve cells subsequently. Harden the tissue thoroughly in solutions of potassium dichro- mate (3% solution, 2 weeks; 5%, 2 weeks or so). Imbed in collo- dion (paraffin, if the sections are sniall). Stain the sections in Wei- gert's hematoxylin (§ 83) until a blue black ; rinse in tap-water. 134 Treat for a short time—20-30 seconds—with a 4% aqueous solution of potassium permanganate and decolorize in Pal’s bleacher until the cinerea is grey (white), the myelinic fibers blue. ‘The action will be very rapid and must be carefully watched ; a few sec- onds will suffice. Wash the sections thoroughly (1% hour or more) in running water. If it is desired, counter-stain with a red stain,— eosin, erythrosin or carmine. This method is not as reliable as the method first given; great care must be exercised in the differentiation with the bleacher. § 139. Chrome-silver impregnation methods. In this group of methods the tissue is Aavdened a certain length of time ina fluid containing a chrome salt, especially potassium dichromate, and then placed in an aqueous solution of silver nitrate (44% and 34% solutions generally employed). If successful, the cells and their processes, amyelinic and, to a certain extent, myelinic nerve fibers, are outlined by a black precipitate (black by transmitted light ; brown by reflected light). § 140. Success depends on (a) the #zvd of animal; different parts and tissues react more satisfactorily in some animals or classes of animals than in others. (d) The age of the animal; young or fetal animals give better results than do adults. (¢) The time of hardening ; it is necessary that the tissue be hardened a certain length of time, constant (relatively) for a certain kind of tissue under the conditions above (a and 4). It is necessary that the correct de- gree of hardening be carefully regulated. (d@) Different organs and regions of the central nervous system vary greatly in the ease with which they can be made to furnish satisfactory impregnations. Al- most certain impregnations of hippocamp can be gained ; cerebral cortex is likewise quite easy to stain. With the olfactory bulb the action is not constant though fairly complete. The optic lobes and retina of birds and large reptiles are more satisfactory than those of mammals. The myel (spinal cord) of embryo birds (7-14 day chick best) is generally more satisfactory than that of mammals; in any case, fetal or new-born animals should be employed. Difficult are satisfactory impregnations of sympathetic ganglia, organs of special sense and the intrinsic nerves of the viscera. § 141. Golgi’s Rapid Method. This is the most generally serviceable of the different methods. 135 § 142. Directions for use. ‘Tissue of a (preferably) young ani- mal is placed in a mixture of 4 parts of 3% potassium dichromate and 1 part of 1% osmic acid. The amount of the fluid should be at least twenty times the bulk of the tissue and should be changed as soon as it grows turbid or loses the strong characteristic odor of the osmic acid. After the hardening has proceeded to the right degree (§ 143), rinse the tissue in water for about 5 minutes and place for 15 min- utes in a 4% solution of silver nitrate, and then for 2 or more days in a 34% solution of silver nitrate, preferably keeping it in the dark. Without washing, imbed rapidly in collodion as follows: (a) Dehydrate 2-3 hours in 95% alcohol, changed two or three times ; (4) place in thin collodion for 20 minutes, in thick collodion for 20-30 minutes ; (c) imbed in thick collodion, on a cork or block of wood (best ; § 55, 4); (@) harden the mass in chloroform for 20-30 minutes, and (¢) place the block in clarifier and cut, sections being 50-100 y thick, according to the nature of the tissue and the character of the impregnation. (/) Place the sections in 95% alcohol for a few minutes; clear in carbol-xylene and mount in balsam by placing the section on the slide, absorbing the clearer thoroughly by means of tissue paper and spreading over it thick xylene balsam. Do not cover. Later,when the balsam has hardened somewhat, it may be melted by heat and much of the superfluous balsam drained from the section and scraped away with a knife. $143. Time of hardening. The following periods will prob- ably be found approximately correct. In general: The best results are to be obtained with kittens 3-20 days old, puppies 2 weeks old, rats 8-10 days, rabbits 8 days. (a) For cerebral cortex (and hip- pocamp) : New-born kitten, 1-2 days; kitten half grown (3-4 months), 3-4 days ; new-born rabbit, 24 hours; rabbit one month old, 2-3 days, etc. (4) For spinal cord : Chick of 5-6 days’ incubation, 24 hours ; chick, 14-15 days’ incubation, 3 days; new-born kitten or puppy, 2-3 days. (c) Cerebellum : New-born kitten, 1-2 days ; kitten half grown, 4 days. § 144. Methylene blue. Imbed in paraffin tissue that has been hardened in 95% alcohol ; cut sections rather thick, 15-20 p, 136 or even thicker. Fasten the sections to the slide or carry them through in watch-glasses. Stain the sections in a .5% aqueous solu- tion of methylene blue for 5-10 minutes, heating it until it steams ; rinse in water and dehydrate, clear in oil of origanum or cajuput, and mount in balsam. ‘The nerve cells and nuclei will be stained blue, all else colorless. In the cell bodies the corpuscles of Nissl will be stained. Should the stain be not selective enough, differen- tiate for a few seconds before dehydrating with a mixture of aniline oil, 1 part, 95% alcohol, 9 parts. § 145. General methods. vom Rath's and Zenker’s fluids are recommended for the fixation of nervous tissue ; either paraffin or collodion may be used in imbedding ; Delafield’s hematoxylin with picrofuchsin (strong formula, 4) as counter-stain is recom- mended for staining, though other hematoxylins may be used as well. SILVER NITRATE IMPREGNATIONS. §$ 146. The preparations stained by means of nitrate of silver were prepared as follows: The /vesh tissue was washed well for a minute or so in distilled water to remove from the surface all album- inous substance, and then transferred for 2-5 minutes or longer to a %4% aqueous solution of silver nitrate ; it was again rinsed in water and in it exposed to direct sunlight until a light brown. When, by examination with the microscope, the stain was found to be suf- ficient it was again rinsed in water and placed in glycerin or alco- hol. Employed in this manner with fresh tissue, silver nitrate stains the cell cement, affording thus negative images of the cells. $147. Silvering Vascular Epithelium. In order that the vasular epithelium of small arteries, veins, and capillaries should be well demonstrated, silver nitrate solutions of 4% to %% strength must be injected into the vessels. $148. Procedure. Connect a canula with the artery supply- ing the alimentary canal (superior mesenteric) or the brain (carotid) and inject distilled water until the water flows out of the returning . vein colorless, Then immediately inject the silver solution until it runs from the vein. After a minute or two follow with distilled water. Place the intestines and mesentery in water and expose them to the light until they become slightly browned. Strips ofthe muscular coat of the intestines, especially of the rabbit, will show 137 capillaries well. Veins and arteries side by side may be found in the mesentery. If the brain vessels are injected one can get admir- able preparations showing nuclei as well as cell outline by staining in hematoxylin. Mount in glycerin, or, if desired, dehydrate and mount in balsam. ‘The tissue may be kept in 50% alcohol or in 50% glycerin for several months before mounting if it is kept in the dark. For large vessels and endocardial epithelium open the vessels or the heart and silver as directed above for mesentery. It may be necessary to make thin free-hand sections so that the preparation will be thin enough for high powers. FORMULAS. In addition to those given elsewhere; see also Zhe Microscope, 7th ed., 8% 297-316. 2149. Acid alcohol. 95% alcohol, 100 c.c.; hydrochloric acid, qo cc. For Hcl carmine, use 95% alcohol, roo c.c. ; hydrochloric acid, % c.c. 4150. Alcohol. (1) 67%. Take 95% alcohol, 2 parts; water, 1 part. (2) 82%. Take 95% alcohol, 5 parts; water, 1 part. 4151. Clarifier. (Castor-xylene). Castor oil, 1 part; xylene, 3 parts. 4152. Clearer. (Carbol-xylene). Melted carbolic acid crystals, 1 part (by volume) ; xylene, 3 parts. 3 153. Normal salt solution. NaCl. (common salt), .6 gram; distilled water, 100 C.c. @154. Collodion. (1) Thick collodion, 8% solution. Ether-alcohol, roo c.c.; soluble cotton, 8 grams. (2) 6% solution. Ether-alcohol, 100 c.c. ; sol- uble cotton, 6 grams. (3) Thin collodion, 1%%, ether-alcohol, 100 c.c. ; solu- ble cotton, 114 grams. 4155. Lther-alcohol. Sulphuric ether, 1 part ; 95% alcohol, 1 part. »4156. Glycerin, cosin and alum carmine. Glycerin, 85 c.c.; alum car- mine, 7% c.c.; % % aq. Sol. eosin, 7% c.c. 4157. Lampblack mixture. Lampblack, t gram; gum arabic, 1 gram; common salt, ;4; gram ; water, 20 c.c. 4158. Neutral (alkaline) balsam. Canada balsam is liable to be slightly acid. This is of advantage for mounting sections stained with carmine or with acid fuchsin (as when picro-fuchsin is used), and for injected preparations where carmine or Berlin blue is used as the coloring matter. For hematoxylin and other stains easily affected by acid media it is often advantageous to use neutral or slightly alkaline balsam as a mounting medium. To obtain this slightly alkaline balsam, add some pure sodium carbonate to the thin xylene balsam and shake thoroughly at intervals for a day or so. Allow the balsam to stand until the soda has settled, then decant and thicken by evaporation till of the desired consistency. 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