174 PLASMA STAINS WITH COAL-TAR DYES. 



tion of the methyl green, which easily comes away in the alcohol. 

 DRUNER (Jena Zeit., xxix, 1894, p. 276) stains for ten minutes in 

 the concentrated solution, treats for one minute with alcohol con- 

 taining 0-1 per cent, of hydrochloric acid, and then with neutral 



alcohol. 



The best results are obtained with sublimate material', chrom- 

 osmium material, and the like, give a much inferior stain. Prepara- 

 tions made with the usual mixture, as given above, are liable to 

 fade ; by acidifying the mixture a stronger and more sharply 

 selective stain is obtained, which does not fade. But too much acid 

 must not be added, as this would cause a staining of the interfilar 

 substances. According to the Encycl. mik. Technik, you may add 

 15 to 24 drops of 0-2 per cent, acetic acid to 100 c.c. of the diluted 

 solution. 



Another process of acidification is given by M. HEIDENHAIN (Ueber 

 Kern und Protoplasma, p. 116) ; for this see fourth edition. See also 

 ISRAEL (PraUikum Path. Hist., 2 Aufl., Berlin, 1893, p. 69) ; TRAMBUSTI 

 (Eicerche Lab. Anat. Roma, v, 1896, p. 82 ; Zeit. wiss. Mik., xiii, 1896, 

 p. 357) ; and THOME (op. cit. supra). EISEN (Proc. Calif. Acad. (3), i, 

 1897, p. 8) acidifies with oxalic acid. 



After acidification the solution must not be filtered, and if it has 

 been kept for some time a little more acid must be added. 



Before staining (M. HEIDENHAIN, loc. cit.), sections should be 

 treated for a couple of hours with 0-1 per cent, acetic acid, then for 

 ten to fifteen minutes with officinal tincture of iodine, and be rinsed 

 with alcohol before bringing into the stain. The treatment with 

 acid is necessary in order to ensure having the sections acid on 

 mounting in balsam. The primary object of the iodine is to remove 

 any sublimate from the preparations, but it also is said to enhance 

 the power of staining of the chromatin with methyl green, and to 

 produce a more selective staining of protoplasmic elements. 



The stain is a very fine one when successful. But it is very 

 capricious. The correct result should be a precise chromatin stain 

 combined with a precise stain of the plastin element of cytoplasm 

 by the Saurefuchsin. Now the least defect or excess of acidity 

 causes the plasma stain of the Saurefuchsin to become a diffuse one, 

 instead of being sharply limited to the plastin element. It is 

 difficult to dehydrate the sections without losing the methyl green. 

 For this reason the stain will only work with very thin sections ; to 

 be quite sure of good results, the sections should be of not more 

 than 3 /x in thickness, and if they are over 5 the desired results are 

 almost hopeless. The stain keeps very badly. I admit that the 



