THE MICROSCOPE AND MICROSCOPIC METHODS 43 



II is this substance mixed with an equal amount of methylene 

 blue. His Azur II-eosin is the compound precipitated when 

 aqueous solutions of Azur II and eosin are mixed. The Giemsa 

 solution is made according to the following formula : 



Azur II-eosin 3.0 grams. 



Azur II 0.8 gram. 



Glycerin 250 . o grams. 



Methyl alcohol 250.0 grams. 



Dissolve the powdered dyes in the glycerin at 60 C.; then add 

 the methyl alcohol previously heated to the same temperature. 

 After mixing, let it stand 24 hours at room temperature, and 

 filter. To stain, mix one drop of this solution with i c.c. of water 

 and immerse the film, previously fixed, for 15 minutes to 24 hours. 

 Direct preparation of Romanowsky Stains. In a study of the 

 essential constituents of the Romanowsky stain, MacNeal 1 

 found both methylene azure and methylene violet to be present 

 and participating in the nuclear staining. The preparation of 

 solutions directly from the pure dyes, methylene azure, methy- 

 lene violet, methylene blue and eosin, has been recommended 

 as the best manner of preparing these staining solutions, as the 

 proportion of the various constituents may be varied at will to 

 obtain various kinds of differentiation. As a routine blood 

 stain for study of leukocytes and staining of hematozoa, the fol- 

 lowing is recommended : 



Solution A . 



Methylene azure 0.3 



Methylene violet (Bernthsen's, insoluble in water) . o. i 



Methylene blue 2.4 



Methyl alcohol, pure 500. o 



Solution B. 



Eosin, yellowish, water soluble 2.5 



Methyl alcohol, pure 500 . o 



These solutions keep for at least a year. They are mixed in equal 

 parts and diluted by the addition of 25 c.c. of methyl alcohol to 



1 Journ. Infectious Diseases, Vol. Ill, 1906, pp. 412-433. 



