168 VICTOR BXJRKE 



in "Lugol's" solution or increasing the period of exposure to 

 "Lugol's" solution he was able to make some Gram negative 

 organisms retain the violet dye. He also claimed that heating 

 the iodine solution on the slide tends to make the Gram negative 

 organisms retain the stain . Benians studied the effect of steam- 

 ing the iodine solution for five minutes and found that after this 

 treatment of the Bacterium coli organisms either intact or crushed 

 resisted decolorization with 100 per cent alcohol for five minutes. 

 He assumes that the heat causes a chemical change in the dye- 

 iodine precipitate in the presence of the bacterial cell substance 

 and that the precipitate ceases to be soluble in alcohol. 



If increasing the exposure to, or increasing the strength of, 

 or steaming, the iodine solution causes the Gram negative organ- 

 isms to retain the dye then these are factors to be controlled in 

 making a Gram stain. However, in our o^vti experiments we 

 were unable to see that increasing the period of application to 

 two hours or doubling the strength of the iodine solution (2^- 

 100) had any effect on the decolorization of Gram negative 

 organisms. Likewise the steaming of the iodine solution on the 

 slide for five minutes did not effect the decolorization of typhoid 

 organisms. Films so treated decolorized as rapidly as when 

 exposed to the iodine solution for one minute. If, however, the 

 film became dry during the process the Gram negative organisms 

 retained the dye much longer. In our experiments acetone was 

 used as the decolorizer. The former workers used absolute 

 alcohol which may account for the differences in results. 



Our experiments convince us that if acetone or acetone and 

 ether is used as the decolorizer and the film is not allowed to 

 dry steaming or prolonged exposure to the iodine solution will 

 not materially affect the decolorization of the Gram negative 

 organism. 



VALUE OF DIFFERENT DECOLORIZERS 



Alcohol 95 per cent Commercial 95 per cent alcohol is used 

 in many laboratories as the decolorizing solution. It gives 

 satisfactory results with some of the better dyes but it can not 

 be used with the poorer dyes. As we have already shown the 



