44 ' BACTERIOLOGY 



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, MacNeaP 

 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, methylene 

 violet, methylene blue and eosin, has been recommended as the 

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

 tion 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 following is 

 recommended : 



Solution A. 



Methylene azure 0.3 



Methylene ^olet (Bernthsen's, insoluble in water) o.-^ 



Methylene blue, medicinal i . o 



Methyl alcohol, pure 500.0 



Solution B. 



Eosin, yellowish, water soluble i .0 



Methyl alcohol, pure 500 . o 



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

 parts and filtered to prepare the actual staining solution, which 

 keeps for a few weeks. This final mixture is employed in the same 

 manner as Leishman's stain. It keeps for a few months. 



Method of Staining Cover-glass Preparations.— :(a) A Em.ear 

 preparation of bacteria having been made and fixed in the manner 

 above described, and a watery solution of either fuchsin, gentian 

 violet or methylene blue having been prepared, the cover-glass 

 is to be dropped into a dish containing the dye, or the dye may 

 be dropped upon the cover glass held in the forceps. 



(b) Allow the stain to act for about thirty seconds. 



(c) Wash in water. 



(d) Examine with the microscope in water directly or after , 

 drying and mounting in Canadian balsam. 



1 Journ. Infectious Diseases, Vol. Ill, igo6, pp. 412-433. 



