3. Xylol. 4. Xylol balsam. Nuclei of resting and dividing cells, green; cell-protoplasm, Saturated alcoholic solution of acid fuchsin, 6 drops; Distilled water, I. 70 per cent. alcohol. 2. Stain in the solution six hours. 3. Decolorize in dilute acetic acid. 4. Absolute alcohol. 5. Xylol. 6. Xylol balsam. 8 IO C.C. 66 Resting nuclei, red; nuclein of karyokinetic figures, yellow, cell-protoplasm, dark olive-green; "cancer-bodies," mainly olive-gray, but some or portions of them may be ruby-red. V. Light Green (Lichtgrün) and Hematoxylin.— Ehrlich's acid hematoxylin, Saturated solution of Lichtgrün in 70 per cent. Distilled water, 15 c.c. 5 66 1. Distilled water. 2. Stain in above mixture half an hour. 3. Wash thoroughly in several waters. 4. Alcohol. 5. Oil of bergamot. 6. Balsam. Nuclei, green; "cancer-bodies" take the hematoxylin stain. VI. Acid Fuchsin and Hematoxylin.- I per cent. solution of acid fuchsin in 70 per cent. alcohol, Distilled water, 15 C.C. Stain as in V. 19 2 KI 30120 19 Nuclei, red; protoplasm, brick-red; "cancer-bodies" take the hematoxylin stain. Orcein, a vegetable dye obtained from certain tinctorial lichens, is used mainly for staining elastic fibers. It is soluble in alcohol, and is employed either in a neutral or acid (HCl) alcoholic solution. Iodin is the oldest of the histological stains, but is now but little used for that purpose. The tincture of iodin, a saturated solution in alcohol, is used for getting rid of the precipitate of mercury formed in tissues fixed in corrosive sublimate or in Zenker's fluid. Lugol's solution, a solution of iodin in water containing iodid of potash, is of varying strength. Iodin in this form is much used as a test for starch, amyloid, glycogen, and corpora amylacea. In Gram's stain and its modifications iodin produces some chemical change in the coloring material employed, in consequence of which, when appropriate decolorizers are used, the stain remains fast in certain structures, while from others it is easily entirely extracted. The strength originally employed by Gram for his staining method was— Weigert in his modification of this method employed a stronger solution: Recently he has recommended the following strength both for fibrin and for neuroglia-fibers: Iodid of potash, 5 grams saturated with iodin. 100 C.C. The only difference in the action of the various solutions probably is that the strong solution acts practically instantaneously, while the weaker solutions require some little time. Acid Alcohol (Orth's Discharging Fluid).— Hydrochloric acid, 70 per cent. alcohol, I c.C.; 66 99 Aniline Water (Aniline-oil Water).-Shake together 5 parts of aniline with 95 parts of water, and filter the resulting milky fluid. It should come through perfectly clear. Carbolic-acid water is made in like manner by shaking together 5 c.c. of melted carbolic-acid crystals and 95 c.c. of water. The solution should be filtered. Mayer's glycerin-albumin mixture for attaching paraffin sections to slides is composed of equal parts of the white of egg and of glycerin. The mixture should be thoroughly beaten and then filtered, or after standing for some time can be decanted. Add 1 per cent. of sodium salicylate to prevent decomposition. Egg-albumin is dissolved by acids and alkalies, so that when such reagents are to be used the sections are best attached to the slide by some other substance. For this purpose Schällibaum's solution, of celloidin 1 part in 3 or 4 parts of oil of cloves, is often useful. Cover the slide with a thin layer of the solution. Arrange the sections in order on the slide and place it in the thermostat at 54° to 60° C. for several hours, or heat for a few seconds to half a minute over the flame until the oil of cloves runs together in drops. After cooling, remove the paraffin with xylol, pass through origanum oil to 95 per cent. alcohol, and proceed as with other paraffin sections. Clearing Reagents.-The object of clearing reagents is to render certain tissue-elements more prominent than others. This result may be brought about by dilute acetic acid (2–5 : 100), which swells up the ground substance, so that nuclei, elastic fibers, fat, myelin, and micro-organisms are more distinct, or by alkalies, which destroy the cells and ground substance and leave only elastic fibers and bacteria but little changed. This method is used almost wholly for fresh tissues. The same result is more commonly obtained by soaking the tissues in substances which by reason of their high index of refraction render the tissues more or less transparent. Any structure which it is desirable to study is usually previously stained and thus easily rendered prominent. This second method is most applicable to hardened tissues. For soaking and clearing the tissues a variety of reagents of different chemical properties are used. Glycerin and acetate of potash are not so much employed as formerly, because balsam mounts are more generally preferred. Of the other reagents (ethereal oils and coal-tar products), the the choice depends mainly on two factors-the kind of stain which has been employed, and the substance in which the sections have been imbedded. Many of the clearing reagents either dissolve celloidin or will not clear it from 95 per cent. alcohol, and nearly all of them will extract aniline colors more or less rapidly. Most of the clearing reagents can be used after hematoxylin and carmine stains. For celloidin or paraffin sections stained by either of them oleum origani cretici, oil of bergamot, or the mixture of the oils of cloves and thyme is recommended in the order given. For aniline stains the best clearing reagent is xylol, which, however, clears directly only from absolute alcohol. It can be used, however, for celloidin or other sections dehydrated in 95 per cent. alcohol by a simple method original with Welch, and lately brought into notice by Weigert. Blot the section on the slide with smooth soft filter-paper, and then pour on a few drops of xylol; repeat the blotting, followed by xylol two or three times, and the section will be found to be perfectly clear. Oleum Origani Cretici.-Colorless to light brown in color; clears readily from 95 per cent. alcohol without dissolving celloidin; affects aniline colors slowly. Ordinary origanum oil is impure oil of thyme, and should not be used. Oil of Bergamot.-Light green in color; clears quickly from 95 per cent. alcohol; does not dissolve celloidin, but after repeated use of the same lot of oil it will sometimes soften it a little. Affects aniline colors slowly, with the exception of eosin, which it extracts very quickly. Oil of Cloves.-Straw-colored; clears quickly from 95 per cent. alcohol; dissolves celloidin; extracts aniline colors, especially methylene-blue. Oil of Thyme.-Colorless; clears readily from 95 per cent. alcohol; makes sections brittle; does not dissolve celloidin; affects aniline colors. Oil of Lavender.-Clears celloidin sections readily from 95 per cent. alcohol. Oil of Cedar-wood.-Pale straw-color; clears from 95 per cent. alcohol, but, unfortunately, clears celloidin sections very slowly; does not affect aniline colors. Aniline (Aniline Oil).-Colorless when perfectly pure and fresh, but soon oxidizes and turns brown; does not dissolve celloidin; clears readily from 70 per cent. alcohol; will clear from water by Weigert's method; extracts aniline colors slowly. Xylol.-Colorless; does not dissolve celloidin; does not affect aniline colors; clears directly only from absolute alcohol; but will clear even celloidin sections from 95 per cent. alcohol if they be blotted on the slide, and the xylol be then poured over them; the process of blotting followed by xylol must be repeated two or three times. Dunham's Mixture of the Oils of Cloves and Thyme.— Excellent for sections stained in hematoxylin or carmine. Not nearly so expensive as pure origanum or bergamot oil. Oil of cloves, Oil of thyme, I part; 4 parts. Filter if cloudy; clears celloidin sections readily from 95 per cent. alcohol without dissolving the celloidin. Weigert's Mixture of Carbolic Acid and Xylol.— Carbolic-acid crystals, Xylol, I part; 3 parts. |