162 VICTOR BURKE 



washing between the different solutions, the effect of heat on 

 the iodine solution, a comparison of different decolorizers and 

 the effect of water on decolorization. Certain of the experi- 

 ments have a bearing on the nature of the Gram reaction and the 

 characteristics of the Gram precipitate. 



Organisms used. Unless otherwise stated the smears used in 

 these experiments were made from pure cultures of Staphylococcus 

 aureus; Bacterium typhosum; Neisseria catarrhalis and Neisseria 

 gonorrheae; grown for approximately twenty-four hours on peptic 

 digest agar slants to which 33 per cent hydrocele fluid had been 

 added or Loeffler's blood seium tubes. 



Films. The films were made in tap water or physiological 

 salt solution. A few preliminary experiments convinced us 

 that no difference resulted from the making of the films in dis- 

 tilled water, tap water or salt solution. It was assumed that if 

 a difference did result this would be a factor that would have to 

 be taken into consideration in staining pus and body fluids. 

 Mounting the films in an acid or an alkali does influence the 

 results as will be described later. 



DYES USED IN PRIMARY STAINING SOLUTION 



One per cent aqueous solutions of six different samples of methyl 

 violet, three of gentian violet and one of crj^stal violet were used 

 and compared. The dye was added to the distilled water, shaken 

 thoroughly, allowed to stand several hours and filtered as used. 

 Such solutions remain stable for a considerable time. Some of 

 our solutions were kept two months and showed no deterioration, 

 in fact there seemed to be some improvement with age. 



Results obtained. According to the method of .Jensen, satis- 

 factory results can be obtained with aqueous solutions of methyl 

 violet 6b, only when using a strong iodine solution* and absolute 

 alcohol as the decolorizer. With our dyes we compared the 

 results obtained with the following decolorizers, 95 per cent 

 alcohol, 100 per cent alcohol, 100 per cent acetone and acetone 



• One gram of iodine, 2 grams of potassium iodide, 100 cc. of distilled water. 



