that tissues placed in them should be especially thin. The most generally useful stain for mitosis is probably safranin. The time of staining varies with the solution used. Babes' is the quickest. The mitotic figures should be stained deeply then, when treated with alcohol slightly acidulated with hydrochloric acid, they will retain the color, while the resting nuclei will yield up most of theirs and become very pale or even colorless. In consequence of this intense stain mitotic figures can then be very readily found. Fixation in Zenker's fluid and staining in phosphotungsticacid hematoxylin can be highly recommended. Centrosomes and spindles are brought out with great distinctness. Directions for Staining Karyomitotic Figures with Safranin.-1. Stain paraffin sections five minutes to twentyfour hours, according to solution used. 2. Wash in water. 3. Wash in 95 per cent. alcohol to which are added a few drops of acid alcohol. 4. Wash in pure 95 per cent. alcohol, followed by absolute alcohol. 5. Xylol. 6. .Xylol balsam. For celloidin sections dehydrate in 95 per cent. alcohol, blot, and pour on xylol; repeat the last two steps until the specimen is clear. Safranin can be used after any of the above fixing reagents. Other useful stains are carbol-fuchsin and aniline-gentianviolet, used in the same way as the safranin. The GramWeigert method gives good results after Flemming's solution. After fixing in corrosive sublimate mitotic figures can be demonstrated by the Biondi-Heidenhain solution, which stains resting nuclei blue-violet and mitotic figures green. After Pianese's solution his special staining mixtures should be used (see page 286). His methods are said to give beautiful results. SPECIAL STAINS FOR CERTAIN TISSUE-ELEMENTS OTHER THAN NUCLEI. MASTZELLEN. MASTZELLEN are found in the connective tissue, more especially in chronic inflammatory processes. Their protoplasmic granules stain intensely like bacteria with the basic aniline dyes. Several methods of staining the granules are given. With Unna's stains for plasma-cells a differential color-stain is obtained for the granules of the mastzellen. Ehrlich's Method.-A. General Stain.-Harden in alcohol. 1. Stain with a saturated aqueous solution of dahlia. 2. Wash out with acidified water. 3. Dehydrate in alcohol; clear and mount in Canada balsam. B. Specific Stain.-Only the protoplasmic granules are stained. Harden in alcohol. 2. Wash out in alcohol, clear, and mount. C. Ehrlich-Westphal Method.-Nuclei red; granules blue. Harden at least a week in alcohol. I. Stain in the following solution twenty-four hours: 200; Saturated solution of dahlia in absolute alcohol, 200; Glacial acetic acid, 100; 20. (Stir repeatedly, then allow the mixture to stand for some time.) 2. Decolorize for twenty-four hours in absolute alcohol. 3. Oil, Canada balsam. Unna's Isolated Stains for Mastzellen.-Harden in alcohol. Nuclei blue; protoplasmic granules of "Mastzellen" red. A.-1. Stain in polychrome methylene-blue solution, plus a little alum, for three hours to over-night. 2. Wash in water. 3. Absolute alcohol, oil of bergamot, balsam. B.-1. Stain in polychrome methylene-blue solution onequarter of an hour. 2. Wash in water. 3. Decolorize in glycerin-ether mixture for five to ten minutes. 4 Wash a long time in water. 5. Absolute alcohol, oil, balsam. PLASMA-CELLS. These are certain cells, much studied by Unna, which are very abundant in subacute and chronic pathological processes, and which are characterized by protoplasm which stains quite deeply in alkaline methylene-blue solutions. The two methods best suited for their demonstration furnish at the same time a differential color-stain for mastzellen. The granules of the latter are stained red, the plasma-cells are stained blue. Unna's Differential Stains for Plasma-cells and Mastzellen.-Harden tissues in absolute alcohol. A.—1. Stain paraffin sections in polychrome methyleneblue one-quarter of an hour to over-night. 2. Decolorize in a small dish of water, to which are added a few drops of glycerin-ether mixture. 3. Wash thoroughly in water. 4. Absolute alcohol, xylol, balsam. B.-I. Stain in polychrome methylene-blue solution five to fifteen minutes. 2. Wash in water. 3. Decolorize and dehydrate in a per cent. alcoholic solution of neutral orcein (about fifteen minutes). 4. Absolute alcohol, xylol, balsam. Methyl-green-Pyronin Stain (Unna-Pappenheim). Fix in alcohol, stain five to ten minutes in incubator, wash in cold water, differentiate and dehydrate quickly in absolute alcohol, clear in oil of bergamot, and mount in balsam. THE COLLAGEN FIBRILS AND RETICULUM OF CONNECTIVE TISSUE. Several methods are available for the demonstration of collagen fibrils and reticulum. The simplest is by means of Van Gieson's picric acid and acid-fuchsin solution, but it is applicable to the coarser fibers only. The stain with aniline blue is believed to be better than any yet proposed, but is limited to tissues hardened in Zenker's fluid. A. Mallory's Aniline Blue Stain.-The following method is not absolutely differential because, besides collagen fibrils and reticulum, it also stains certain hyaline substances, but hese latter usually are so different morphologically that confusion cannot arise. The method is also useful for the study of fibrin, fibroglia fibrils, smooth and striated musclefibers, and amyloid. 1. Fix in Zenker's fluid. 2. Imbed in celloidin or paraffin. 3. Stain sections in a per cent. aqueous solution of acid fuchsin for five minutes or longer. 4. Transfer directly to the following solution and stain for twenty minutes or longer: Aniline blue soluble in water (Grübler), Orange G (Grübler), One per cent. aqueous solution of phos phomolybdic acid, 1 Mallory: The Journal of Medical Research, 1905, xiii., 0.5; 2.0; 100.00. 113-136. 5. Wash and dehydrate in several changes of 95 per cent. alcohol. 6. Clear in xylol or in oleum origani cretici. 7. Xylol balsam. The collagen fibrils and reticulum of connective tissue, amyloid, mucus, and certain other hyaline substances stain blue; nuclei, protoplasm, fibroglia fibrils, axis-cylinders, neuroglia-fibers, and fibrin red; red blood-globules and myelinsheaths yellow; elastic fibers pale pink or yellow. The various structures do not stain with equal intensity, so that certain ones are brought out with great sharpness. This is particularly true of the collagen fibrils and reticulum of connective tissue, and of fibrin and smooth and striated musclefibers. If it is desired to bring out the collagen fibrils as sharply as possible, omit the staining with acid-fuchsin. Then the nuclei and protoplasm stain yellow, and the blue fibrilla and reticulum stand out more prominently. B. Van Gieson's Stain.-The proportions given are those recommended by Freeborn. Occasionally it will be found necessary to increase the proportion of the acid fuchsin. 1. Harden in chrome salts or in corrosive sublimate. The results after alcohol are not so good. 2. Stain deeply in alum-hematoxylin. 3. Wash in water. 4. Stain for three to five minutes in I per cent. aqueous solution of acid fuchsin, 5. Dehydrate in 95 per cent. alcohol. 6. Oleum origani cretici. 7. Canada balsam. 5 c.c. 100 " C. Unna's Orcein Stain.-I. Harden in alcohol. 2. Stain in the concentrated solution of polychrome methylene-blue five minutes. 3. Wash in water. 4. Decolorize, differentiate, and stain in a 1 per cent. solution of orcein in absolute alcohol fifteen minutes. 5. Wash in absolute alcohol. |