ELIMINATION OF SPURIOUS TESTS FOR B. COLI 333 



cultures are free from aerobic contamination, but in some cases 

 we feel certain two or more species of anaerobes are present in 

 a single culture. For the purpose of this paper we have not 

 yet undertaken to purify them — hence our hesitancy to empha- 

 size the question of identity. 



The first tests were made in deep agar according to the technic 

 used by one of us (I. C. H.) with Mr. Taber (1914) in connection 

 with the tetanus bacillus, but this proved not to be feasible in 

 studying a larger number of cultures, owing to the care neces- 

 sary in preparing and inoculating the tubes; it was replaced by 

 the simpler method of tubing the media, after the desired con- 

 centration of dye had been secured by addition of the proper 

 amount of a 1 per cent solution followed by sterilization in the 

 Arnold sterilizer. In some of the earlier tests, crude glucose 

 was used by oversight, resulting in decolorization which we attrib- 

 ute to residual SO3 used as a bleaching agent in the preparation 

 of the glucose and which is known to reduce dyes of the gentian 

 violet group to their leuco-bases. A medium made with chemi- 

 cally pure glucose does not decolorize gentian violet. The reac- 

 tion of the media is no doubt important, although stronger 

 acids are required to decolorize gentian violet than is the case 

 with alkalis; the reaction was adjusted to +1- Heavy seed 

 cultures were prepared in the constricted tube described by Hall 

 for the cultivation of anaerobes in glucose broth (1915) After 

 twenty-four hours incubation at 37°C., 1 cc. was transferred to 

 the tubes of melted glucose agar with the dye cooled to 40 to 

 45°C — rolled to mix thoroughly and incubated at 37°C. Con- 

 trol tests were always made in similar media without dye. Con- 

 trol tests for aerobic contamination were also made from each 

 seed tube on agar plates in each experiment. Further, all posi- 

 tive growths were subcultured on agar slants or plates to detect 

 possible aerobic contamination. All of the organisms produce 

 gas even in the absence of fermentable carbohydrates; this fact 

 and the appearance of definite colonies in the depths served as 

 the criteria of growth. In all cases also, growth is accompanied 

 by decolorization of the dye. The results of the tests are tabu- 

 lated in table 1. 



