42 



and mount in balsam. This may be used alone to give a blue stain with tissue 

 fixed in Hermann's or Flemming's fluid. (See also 112, 131). 



105. Methylene Blue. This valuable stain, used particularly in the 

 histology and pathology of the blood and nervous system, and in bacteriology, 

 is represented in a large number of formulae. For the staining of nuclei, basic 

 granules in the cytoplasm, neurochromatin granules, etc., simple aqueous solu- 

 tions may be employed. A 1% solution suffices for most purposes; in some 

 cases, a concentrated solution is to be preferred. 



Stain from water for 5 minutes to as many hours, with or without heat, rinse 

 with distilled water, differentiate if desired in a l/10th% Hcl. in 95% alcohol, 

 or l/10th% alum solution. Wash, dehydrate, mount in neutral balsam. 



106. Alkaline Methylene Blue. Formula: Methylene blue, 2 grms.; 

 absolute or 95% alcohol neutralized with pure dry sodium carbonate, 50 c. c.; 

 add distilled water, 450 c. c.; 1% potassium hydroxid, 5 c. c. An excellent stain, 

 giving best results after mercuric chlorid fixers (incl. Zenker's, etc.). 



107. Eosin Methylene Blue. Stain sections l / 2 hour with a l /4% to 1% 

 aqueous solution of eosin, rinse in water, stain in alkaline methylene blue 10 

 minutes, rinse well in water. Differentiate and dehydrate rapidly with neutral 

 95% alcohol and absolute alcohol, clear in xylene, mount in neutral balsam. 

 Particularly useful for staining blood in the tissues (hemolymph) glands, etc.. 



108. Toluidin Blue. This may be used, often to advantage, in place of 

 and for the same purposes as methylene blue. It gives a somewhat darker stain. 



109. Methyl green. This is a nuclear stain of much value, besides being 

 an important ingredient of triple stains (e. g., Ehrlich's triacid mixture and 111). 

 In very dilute solutions it is serviceable in staining the nuclei of fresh tissue and 

 of isolated cells. A 1 % aqueous solution may be used with hematoxylin and picro- 

 fuchsin in differentiating the structure of the hair follicle. (Gage). 



110. Safranin. Formula (Babe's): Concentrated aqueous solution of 

 safranin, 1 part; concentrated alcoholic solution of safranin, 1 part. 



Stain sections 1 to 4 hours, or over night; wash away excess of stain with 

 95% alcohol, differentiate with acid alcohol (95% alcohol, 100 c. c., hydrochloric 

 acid, 1/10 c. c.) for a few seconds, rinse with 95% alcohol and clear in carbol- 

 xylene or bergamot oil. If a pure nuclear stain is not required, the differentiation 

 may be omitted. This gives a good stain with tissue fixed in Hermann's or 

 Flemming's fluid. It is a brilliant, transparent red. 



Other formulas may be employed (concentrated alcoholic solution, alcoholic 

 solution diluted with anilin water, equal parts concentrated solutions in alcohol 

 and anilin water, etc.). Differentiation may be accomplished with the use of 

 iodin-potassium iodid solution, or by counterstaining with an alcoholic solution 

 of light green or acid violet ( 121, 122). 



111. Ehrich-Biondi-Heidenhain Mixture. Formula: Saturated aqueous 

 solutions of Orange G. Rubin S. (Fuchsin acid) and Methyl green, 100 c. c., 20 c. c. 

 and 50 c. c. respectively. In preparing the mixture, only fully saturated solutions 

 should be taken which should be mixed in the order given slowly with constant 

 agitation. Only tissue that has been fixed in sublimate solutions (11) should 

 be used, the sections should be thin and slightly acid. This may be secured by 



