METHODS FOR THE MICROSCOPIC STUDY OF BACTERIA 179 



kept in stock as saturated aqueous or alcoholic (96 per cent.) solutions. 

 The solubility of stains in water and in alcohol respectively varies, 

 but, as a rule, the solubility in alcohol is greater than that in water. 

 Saturated solutions of anilin dyes are unsuited for the staining of 

 microorganisms, but they are more stable than diluted solutions 

 provided they are kept in tightly-stoppered bottles away from the 

 light. Dilutions of saturated solutions are prepared as they are 

 needed for current use. 



C. Technic of Staining Bacteria. 1. Preparation of a film of bac- 

 teria for staining: A drop of a culture of bacteria from a fluid medium 

 as broth is removed with a platinum loop and spread upon a clean 

 cover-glass or glass slide. Bacteria from a solid medium are emul- 

 sified in a small drop of water on the slide. 1 



2. The film of bacteria is allowed to dry in the air; evaporation 

 may be hastened in the incubator at 37 C. 



3. The air-dried film is next fixed by passing it once slowly through 

 the flame of a Bunsen burner, film side upward; about one-half second's 

 exposure to the flame suffices; a longer exposure destroys or changes 

 the staining properties of the organisms. 



4. Staining: A 5 per cent, aqueous solution of methylene blue, 

 fuchsin, or gentian violet, prepared by adding 5 c.c. of filtered satu- 

 rated stock solution to 95 c.c. of distilled water, is used. The slide 

 or cover-glass is flooded with the desired stain, and after one to five 

 minutes, depending upon the intensity of the stain used, the excess 

 is poured off and the preparation is washed thoroughly with water. 

 The residual moisture is removed with filter paper or by air-drying, 

 and a small drop of Canada balsam (dissolved in xylol) is placed in 

 the center of the stained area. The film is finally enclosed between a 

 slide and a cover-glass. 



D. Intensive Stains for Bacteria. Simple aqueous or alcoholic solu- 

 tions of anilin dyes are frequently inefficient for staining bacteria and 

 resort is made to intensified stains. One of the most useful of the 

 intensified stains is Loffler's alkaline methylene blue, prepared in the 

 following manner: 



1 to 10,000 aqueous solution of potassium hydroxide 2 70 c.c. 



Saturated alcoholic solution methylene blue 30 c.c. 



1 It is essential that the emulsion shall be but faintly opalescent when viewed by 

 reflected light; a distinct clouding indicates that too many organisms have been added, 

 in which event the preparation will be found to be unsatisfactory. 



2 Conveniently prepared by dissolving 1 gram of KOH in 100 c.c. distilled water 

 and adding 1 c.c. of this solution to 99 c.c. of distilled water. 



