396 G. J. HUCKER 



Decolorizing solution 



Anilin oil (2 parts) . . _ , 



^ 1 , , , . ^f mixture 5 parts 



Xylol (1 part ) 



Alcohol (95 per cent) 95 parts 



Counter stain 



Bismarck brown 4.5 grams 



Water (boiling) 50.0 cc. 



Filter 



Alcohol (95 per cent) 30.0 cc. 



The milk smears were prepared by the usual Breed method 

 (Breed and Brew, 1916); i.e., depositing 0.01 cc. of milk on a 

 clean glass slide and spreading with a needle over an area of 

 1 sq. cm. The smears were dried and placed in xylol until the 

 fat was dissolved, removed, drained, and immersed in 95 per 

 cent alcohol for two minutes for fixing. The shdes before being 

 allowed to dry were placed in the gentian violet for forty-five 

 seconds, blotted or allowed to drain after removing from the 

 stain, and immersed in Gram's iodine solution for one minute, 

 destained in the anilin-xylol-alcohol solution until no more stain 

 could be removed; and then counterstained in Bismarck brown 

 for forty-five seconds. 



Several formulas of gentian violet solution were used but the 

 particular concentration given has yielded the most consistent 

 and satisfactory results. Satisfactory preparations could not be 

 obtained with "Method 1" (commonly known as Stirling mod- 

 ification) of the Report of the Committee on the Descriptive 

 Chart of the Society of American Bacteriologists (Conn et al., 

 1919) as hght blue and green areas were deposited on the slide 

 when such concentrated gentian violet was used. This was espe- 

 cially true of smears prepared from milk which had developed 

 any degree of acidity. This reaction was probably due to the 

 conversion of the gentian violet into closely related dyes in the 

 presence of the acid and the alcohol of the destaining solution. 

 No definite data are available at present on this point. The 

 stains used in all cases were Griibler's. 



The addition of the anilin oil and xylol to the destaining al- 

 cohol resulted in retarding the action of the solution sufficiently 



