NEW METHOD FOR GRAM STAIN 175 



organisms ground in a mortar were placed on the same slide, 

 fixed by heat in the usual way, treated for two minutes with 

 0.5 aqueous solution of methyl violet and then deeolorized with 

 95 per cent alcohol it was found that the dye was held much 

 more strongly by the ground up debris of the organism than by 

 the intact. According to Benians "This seems to provide almost 

 certain evidence that the dye had never really permeated the 

 intact bacilli, to get into their substance, as it had been able to 

 get into the substance of the bacilli when broken up. The dyes 

 were therefore only absorbed to the exterior of these intact 

 organisms." 



We suggest here that the difference in rate of decolorization 

 between the intact organisms and the amorphous material may 

 have been due to a greater saturation and more rapid drj'ing of 

 the amorphous material rather than to an entire lack of penetra- 

 tion of the dye into the interior of the intact cells. 



It seemed important to us, from a purely practical considera- 

 tion of the subject, to determine whether all the members of 

 the large coli-typhoid-dysentery group of organisms resisted 

 penetration of the dye as had been demonstrated for Bacterium 

 coli and to such an extent as to permit of differential staining 

 between these organisms and the gonococcus like organisms. 

 Our experiments were based on three assumptions: (1) That if 

 the cell wall of these organisms resisted penetration by the dye 

 it should resist decolorization of the cell if the dye could be 

 gotten into the cell without altering the cell wall and the decolor- 

 izer did not affect the cell wall; (2) That if the organisms were 

 stained with methyl violet, then stained with a dye such as 

 Safranin which slowly covered over the methyl violet and then 

 cleared in xylol the cells should not show a violet center and a 

 Safranin periphery unless the violet had penetrated to the interior 

 of the cells; (3) That if the organisms were stained with methyl 

 violet, partially decolorized with acetone and counter stained 

 with Safranin the center of the cells should not appear violet 

 and the margin of the cell Safranin unless the violet had pene- 

 trated to the center of the cells. 



