NEW METHOD FOR GRAM STAIN 169 



addition of water to the decolorizer slows down the rate of the 

 decolorization of the Gram negative and increases the rate of 

 decolorization of the Gram positive organisms. Seventy-five 

 per cent. alcohol decolorizes the Gram positive almost as rapidly 

 as the Gram negative organisms. Fifty per cent alcohol decolor- 

 izes both tyjies of organism at about the same rate. 



Alcohol absolute. Absolute alcohol gives better results than 

 95 per cent alcohol as there is a greater margin of time between 

 the decolorization of the Gram negative and Gram positive 

 organisms. The high cost and difficulty of obtaining absolute 

 alcohol miUtate against its use. 



Alcohol and acetone. The addition of acetone to the alcohol 

 increases the rate of decolorization of the Gram negative organ- 

 isms and slows down the rate of decolorization of the Gram 

 positive organisms. Therefore a decolorizing solution of alcohol 

 and acetone gives better results than absolute alcohol. 



Acetone. Acetone decolorizes the debris on the sUde and the 

 Gram negative organisms from 5 to 10 times as fast as absolute 

 alcohol and the Gram positive organisms much more slowly. 

 Acetone does not completely decolorize the Gram positive organ- 

 isms if they have been stained with a good dye, in 15 hours. 

 With one of the dyes used by us absolute alcohol decolorized 

 Staphylococcus aureus as much in fifteen minutes as acetone did 

 over night. The addition of water to the acetone has the same 

 effect on the decolorization of organisms as the addition of water 

 to alcohol. Eighty per cent acetone gave as good results as 

 95 per cent alcohol. 



Acetone and ether.^ With 100 per cent acetone the decolori- 

 zation of Gram negative organisms is almost instantaneous. 

 If desired this rapid decolorization can be slowed down by the 

 addition of ether to the acetone. One part of ether to 1-3 parts 

 of acetone serves as a very good decolorizer. As ether costs 

 about the same as acetone there is little or no economic advan- 



' It is a common laboratory procedure to use a mixture of alcohol and acetone 

 as the decolorizing solution. Lyon (1920) recommends the use of acetone alone 

 as the decolorizer. We have found that a mi.\ture of acetone and ether is just 

 as satisfactory as acetone. 



