166 VICTOR BURKE 



is shown by the fact that eighty per cent alcohol decolorizes the 

 Gram negative organisms more slowly and the Gram positive 

 organisms more rapidly than 100 per cent alcohol. If the alcohol 

 is diluted sufficiently both types of organisms decolorize at the 

 same rate. With the addition of more than 50 per cent water the 

 Gram negative organisms do not decolorize over night. 



The explanation of these facts upon a physical basis is that 

 (1) the precipitate is more soluble in alcohol or acetone than in 

 water and (2) the water alters the cell wall of the Gram positive 

 organism or reduces the size of the molecules of the dye precipi- 

 tate so that the dye is more easily washed out by the decolor- 

 izer. With the addition of suflicient water to the decolorizer 

 the Gram positive organisms or the molecules of the precipitate 

 are so altered that the dye comes out of the Gram positive 

 organisms as readily as out of the Gram negative organisms. 

 Conversely the elimination of water from the decolorizer and 

 from the cell results in the Gram positive organisms retaining the 

 dye more tenaciously than the Gram negative organisms. 



Unfortunately prolonged exposure to a water free decolorizer 

 will remove the dye from the Gram positive organisms and 

 thorough drying of the precipitate delays its solution in the 

 decolorizer. It follows that if we dry the film too thoroughly 

 before adding the decolorizer the rate at which the dye precip- 

 itate goes into solution ma}'' be decreased to such an extent that 

 before the dye can be washed from the slide and the Gram nega- 

 tive organisms it ma}^, but does not always, begin to come out of 

 the Gram positive organisms. It is not all washed out of Gram 

 positive organisms by acetone in 12 hours. By taking advantage 

 of these facts it is possible by careful blotting of the film before 

 adding the decolorizer to increase the Gram positiveness of the 

 Gram positive organisms without affectmg the rate at which the 

 dye precipitate goes into solution in the decolorizer and the 

 decolorization of the Gram negative organisms. Successful 

 staining with some dyes depends upon the skill with which the 

 water is removed from the film. This constitutes perhaps the 

 most critical step in the process of staining by Gram's method. 

 Success with the poorer .\merican dyes depends upon one's 

 ability to properly gauge the effect of the blotting upon the 



