32 H. J. Sears 



ber of cases. More careful investigation of this subject was considered 

 important, and the following experiment was therefore carried out : 



A large amount of 2% peptone solution containing 0.5% NaCl was divided in 

 half and to one portion 1% glucose was added. The two portions were placed 

 in sterile flasks, 100 c.c. to each, and sterilized in the autoclave for 10 minutes. 

 The flasks were then inoculated and placed in the incubator at Zl C. Each organ- 

 ism to be tested was inoculated into a flask containing glucose and into one not 

 containing glucose. 



Folin" claimed accuracy for his methods for the determination of creatin 

 and creatinin in the presence of considerable quantities of glucose. Neverthe- 

 less, it was considered essential to maintain two sterile controls, one with, and 

 one without, glucose. Samples were withdrawn from each of the cultures and 

 from the controls at definite intervals and after sterilization in steam at 100 C, 

 they were subjected to analysis for creatinin and creatin, by Folin's methods. 



Tables 20 and 21 give the results of these analyses. The values 

 are in milligrams per 100 c.c. The fact that neither of the sterile con- 

 trols gave at any time a color reaction seems sufficient to establish at 

 once the noninterference of glucose with the determinations. We may 

 assume, therefore, that the figures given indicate with reasonable 

 accuracy the comparative creatinin- and creatin-forming power of the 

 micro-organisms in question. 



It will be observed that only B. proteus and Sp. cholerae form both 

 creatin and creatinin in the absence of sugar. B. subtilis forms creatin 

 but not creatinin. Of the cultures on the glucose-containing solutions, 

 only B. faecalis-alkaligenes fails to give the reaction for creatinin. This 

 culture shows on the second day a considerable quantity of creatin. 

 Tests for this substance thereafter, however, are all negative. 



Examination of the tables will suggest that there are several possi- 

 bilities for the diflferentiation of species by this method. For exam- 

 ple, on the second day the amount of creatinin in the culture of B. 

 typhosus is 5.9 mgm. per 100 c.c, an amount which is easily detectable 

 in a 5-c.c. portion. The corresponding cultures of B. coli and B. 

 faecalis-alkaligenes both are negative in the test for creatinin. Like- 

 wise, Sp. cholerae gives 6.6 mgm. of creatinin on the second day, while 

 the morphologically and culturally similar organism, Sp. metchnikovii, 

 gives only a trace. It is interesting to note that in the point of their 

 creatin-production all five of the organisms mentioned, show just the 

 reverse characteristics; the cultures of B. coli, B. faecalis-alkaligenes, 

 and Sp. metchnikovii show high concentration, while those of B. 

 typhosus and Sp. cholerae give low values for this compound. 



In most of the cultures the maximal concentration of both creatin 

 and creatinin seemed to be reached from about the 5th to the 8th days. 



*' Jour. Biol. Chem., 1914, 17, p. 475. 



