staining myelin sheaths

Weigert's elastic ti ue 8.: Aqueous solutions of resorcin and fuchsin are boiled together, liquor ferri sesquichloridi is added, and the precipitate di olved in alcohol, to which has been added 2 per cent. of hydrochloric acid. Weigert’s iron-hematoxylin (for nuclei): Prepare two solutions: (a) Hematoxylin, τ gm.; 96 per cent. alcohol, roo ε.ς. (b) Liquor ferri sesquichloridi, 4.C.c.; water, oe C.0.5 hydrochloric acid, 1 c.c. For use, mix parts. Weigert’s method (for neuroglia fibers): (a) Fix thin pieces of ti ue, not over 4 cm. thick, in a 4 per cent. solution of formaldehyd for at least four days. (b) Mordant in the following solution for four or five days in an incubator, or for eight days at room-temperature: Acetate of copper, 5 gm.; acetic acid, 36 per cent. solution, 5 c.c.; chrome alum, 2.5 gm.; water, to 100 c.c. Boil the chrome alum ina covered dish (it turns to a green color); remove the gas, add the acetic acid, and then the copper acetate; stir until the latter is di olved, then cool. The solution should remain clear and without a green precipitate. (c) Wash in water; dehydrate in alcohol; embed in celloidin. id) Reduction of copper salt in sections: 1. Place the sections, which must not be over 0.02 mm. thick, in a 0.33 per cent. aqueous’ solution of pota ium permanganate for ten minutes. 2. Wash with water. 3. Decolorize and reduce for two to four hours in the following solution: | Chromogen formic acid (specific gravity, 1.20), 5 c.c.; water, to roo c.c. Add to go per cent. of this, just before using, 10 c.c. of a 10 per cent. solution of sodium sulphite. The sections can now be stained in the manner to be described, but the color of the fibers will be more intense if the following steps are observed, and a slight yellowish contrast-stain is obtained for the ganglion and ependymal cells and for the larger nerve-fibers. (e) Further reduction of copper salt: Wash twice in water. 2. Place sections in a carefully filtered saturated 5 per. aqueous solution of chromogen overnight. Wash in water. 4. The sections are now ready for staining, or may be preserved until wanted in go c.c. of 80 per cent. alcohol and 10 c.c. of 5 per cent. oxalic acid. (/) Staining of ncuroglia fibers: Lift. section from_ large dish of water on to "slide freshly cleansed with alcohol; blot with filter-paper (method recommended by Weigert for attaching sections to slide). 2. Stain in the following mixture: Saturated solution of methyl-violet in 70 to 80 yer cent. alcohol, 100 c.c. (saturated with aid of heat: decanted when cold), and 5 per cent. aqueous solution of oxalic acid, 5 ¢.c. 3. Wash with normal salt solution. 4. Iodin solution: ς per cent. pota ium iodid solution saturated with iodin. This is poured on and then off. 5. Wash with water and blot with filter-paper. 6. Decolorize thoroughly in equal parts of xylol and anilin-oil. 7. Wash repeatedly with xylol, or the stain will not keep. 8. Canada balsam. The sections keep better if exposed for from two to three days to diffuse light before being put away. Weigert’s method: See also Gram-Weige! staining method. Weigert’s mixture: 1. A clearing agent for thick sections of the central nervous system after cermin ind ematoxytin stains: carbolic acid in crys 1 part; xylol parts. 2. Anilin, 2; xylol, 1 part. Used like the preceding. Weigert’s mixture of anilin and xylol: Anilin, 2 parts; xylol, τ part. Weigert’s myelin sheath s.: 1. Fix and mordant the ti ues in Weigert’s quick mordant or in Miiller’s fluid. 2. Dehydrate in alcohol without washing in water. 3. Embed in celloidin. 4. Place sections for twenty-four hours in the following solution: Copper acetate, 5 parts; acetic acid, 36 per cent. solution, 5 parts; chrome alum, 2 to 5 parts. 5. Stain fifteen minutes to twenty-four ‘hours in hematoxylin, 1 part; absolute alcohol, 10 parts; saturated aqueous solution of lithium carbonate, 7 parts; water, go parts. The hematoxylin and alcohol must be kept on hand for some time, in order to ripen. 6. Wash thoroughly in water. 7. Decolorize in borax, 4 parts; pota ium ferrocyanid, 5 parts; water, 200 parts, until the gra: substance becomes distinctly yellow. 8. Wash thoroughly in water. 9. Dehydrate in 95 per cent. alcohol. 10. Clear in anilin oil, 2 parts; xylol, 1 part. 11. Finish in Canada balsam. Weigert’s quick method (for mordanting myelin sheaths): 1. Fix the ti ues in a 4 per cent. solution of formaldehyd (10 per cent. solution of formalin) for four days to several weeks. Four days are enough for pieces not over 1 cm. thick. The solution is changed at the end of twenty-four hours. 2. Mordant the myelin sheaths in a solution of pota ium dichromate, 5 parts; chrome alum, 2 parts; water, 100 parts, for four to eight days. The ti ues should not be left in it more than eight days, lest they become too brittle. 3. Transfer directly to 80 per cent. alcohol, and keep in the dark until wanted for embedding, changing the alcohol occasionally as it becomes colored. Weigert’s pic roc arm in: A stain prepared by stirring 2 gm. of carmin in 4 c.c. of ammonia and allowing it to remain standing in a well-corked bottle for twenty-four hours. This is mixed with 200 c.c. of a concentrated aqueous solution of picric acid, to which a few drops of acetic acid are added after a further twenty-four hours. Filter after twenty-four hours. Should the precipitate also pa through the filter, a little ammonia is added to di olve it. Weigert's 8s. (for fibrin): Harden in alcohol. 2. Stain celloidin sections in lithium-carmin. 3. Fasten sections to slide with ether vapor. 4. Stain in anilin-gentian-violet five to twenty minutes. 5. Wash with normal salt solution. 6. Lugol's iodin solution one minute. 7. Wash with water. 8. Decolorize in anilin, 2 parts; xylol, 2 parts. 9. Wash with three changes of xylol. 10. Canada balsam. Fibrin and Gram stainable bacteria are colored blue; nuclei become red. Weigert-Ehrlich s. (for tubercle bacilli): Alcoholic solution of fuchsin or of methyl-violet is added to an aqueous solution of anilin oil. The specimens are stained, washed in water, treated with dilute hydrochloric or nitric acid, and again washed. Weigert-Pal method. See Pal’s method. Wei ’ double s.: (Yor tubercle bacilli): Mix 1 part of methyl-violet solution with 3 parts of carbolfuchsin. Mixture keeps a week. Otherwise as in Much’s method. In both methods a counter-stain is desirable—either a much-diluted solution of fuchsin or a 1 per cent. watery solution of safranin. The results are much better if the slides are left from twenty-four to forty-cight hours in the staining solution at room-temperature. Welch’s method (of staining the capsule of the pmeumococcus): 1. Cover the preparation with glacial acetic acid for a few seconds. 2. Drain off and replace (without washing) with anilin gentian-violet solution, repeatedly added. 3. Wash in a 2 per cent. solution of sodium chlorid and mount in the same. Wendriner’s mixture: 8 gm. of borax are di olved-in hot water, 12 gm. of boric acid are added, and then 4 gm. of borax; after crystallization it is filtered. Williams’ method (for flagella): Cover the cover-