50 RELATIONSHIPS OF THE COCCACE^ 



by one minute's treatment with alcohol, while B. mega- 

 therium constantly fails to decolorize after three hours. 

 On the other hand, with B. fluorescens, B. diphtheria 

 and certain cocci the result is affected by the time 

 of decolorization, as well as by the age of the cul- 

 tures. Between the fixed points at the extreme, prepa- 

 rations will yield varying results, showing some cells 

 stained and others decolorized. As a rule, a large 

 majority of cells in a given preparation will show one 

 reaction or the other; but a second slide made from 

 the same culture may yield a different result. 



The time chosen for decolorization is, of course, an 

 arbitrary factor which will affect the proportion of 

 positive results obtained. In our work, as a result of 

 Mr. Brant's experiments, we fixed on three minutes, 

 altho we are not certain that this is preferable to the five- 

 minute period recommended by the Committee on Stand- 

 ard Methods. We have applied the anilin-oil-gentian- 

 violet for one and a half minutes and the Gram solution 

 for one and a half minutes, instead of the one- and two- 

 minute periods of the committee. 



In all cases we made the stain on young 2O-degree 

 agar cultures (not over five days old), and in each 

 case the test was made in duplicate at different times. 

 When the result' of the two tests coincided, the cul- 

 ture was recorded as positive or negative. Cultures 

 which gave one positive and one negative test, or in 

 which the stained and decolorized cells appeared in 



