1142 PATHOGENIC PROTOZOA 



allowed to dry thereon ; when thoroughly dry, dissolve this precipitate 

 in pure methyl alcohol in the proportion of three-tenths of a gram to 

 one hundred cubic centimeters of alcohol. This alcoholic solution is 

 the staining fluid. 



MacNeal 1 has shown that both methylene azure and methylene 

 violet participate in the nuclear staining, and that an excellent stain, 

 equal to any, may be made directly by mixing the pure dyes accord- 

 ing to the following formula: 



Solution A. Methylene azure 0.3 gm. 



Methylene violet (Bernthsen's, insoluble in water) 0.1 



Methylene blue 2.4 



Itt ethyl alcohol, pure (Merck's reagent) 500.0 



Solution B. Eosin, yellowish, water soluble 2.5 



Methyl alcohol, pure 500.0 



These stock solutions will keep for at least a year. They are mixed 

 as needed in equal parts, and diluted by the addition of 25 c.c. of 

 methyl alcohol to each 100 c.c. of the mixture. The mixture will 

 keep for several months. 



The method of staining is the same, whatever methyl alcohol stain 

 be used. The cover glass or slide is flooded with the stain, which is 

 allowed to act for one minute ; next, as much distilled water is added, 

 drop by drop, as the slide will hold, or until a yellowish metallic 

 scum, is formed on the surface, and the mixture is allowed to act 

 from three to five minutes. A convenient staining dish is made by 

 laying a pair of glass rods on top of a flat oblong dish ; the rods may 

 be held in place with adhesive plaster or perforated strips of wood. 

 Fixation is accomplished by the undiluted solution, but the actual 

 staining does not occur until water is added ; by repeated washing in 

 distilled water, any desired differentiation may be made. 



It is sometimes convenient, when searching for malarial parasites 

 in clinical cases, to stain as follows : 2 



Use four bottles or Coplin jars; in the first, put the undiluted 

 stain, and in it immerse the slide for one minute ; in the second, put 

 distilled water and transfer the slide to it for four or five minutes; 

 in the third jar put diluted Manson's stain, about 0.5 c.c. to 50 c.c. 

 water, and in this the smear remains one-half minute; in the fourth 



1 MacNeal, Jour. Inf. Dis., Chicago, 1906, iii, 412. 



* Russell, F. F., Jour. Amer. Med. Ass., Chicago, 1915, Ixiv, 2131. 



