MAX LEVINE 



decomposition of fermentable substances, and if they do, are the dif- 

 ferences in acid-formation sufficiently great to warrant quantitative 

 acid-production as a reliable differential index? 



2. Is quantitative acid-production correlated with (a) the Mac- 

 Conkey types, 3 (b) the Voges-Proskauer reaction, or (c) gas-forma- 

 tion? 



3. Are the morphologic and physiologic characteristics correlated 

 with the source? 



CULTURES STUDIED 



Altogether 167 organisms were studied; 156 were obtained from 

 sewage and from the feces of horse, cow, sheep, pig, and man, and 

 11 were from the collection of the American Museum of Natural 

 History. 



The method of isolation has been described in a previous paper. 4 

 They were all of the colon-bacillus group; that is, gram-negative, 

 usually short rods, which formed gas from glucose and lactose, coagu- 

 lated milk, and did not liquefy gelatin in 20 days. 



PREPARATION OF MEDIA 



T*he medium employed for tests of acid-production consisted of 1% peptone 

 water to which was added 1% of the test substance. Peptone water, rather 

 than nutrient broth, was used, to eliminate the formation of acid from traces 

 of any other fermentable substance which might be present in beef extract 

 or meat infusion. The reaction of the medium was neutral to phenolphthalein. 



Sterilisation. The medium was tubed (10 c.c. in Durham fermentation 

 tubes) and sterilized in the autoclave for 10 minutes at 10 pounds pressure, 

 which is a shorter period than is recommended in the Standard Methods for 

 Water Analysis (1912). Immediately on removal from the autoclave the medium 

 was rapidly cooled by immersion in cold water, then incubated for 2 or 3 days 

 at 37 C. in order to eliminate tubes which had escaped proper sterilization. 

 Nonsterile tubes were rarely found. Sufficient medium was prepared at one 

 time to permit a test of all the cultures on one substance. Variations in the 

 composition' of the medium were reduced to a minimum by using distilled 

 water and the same bottle of Witte's peptone throughout the work. 



DETERMINATION OF ACID-PRODUCTION 



Acid-production was determined in the following manner. A tube of 

 peptone water was inoculated from an agar-slant stock culture and incubated 

 at the body temperature (37 C.) for 24 hours. Two standard 4-mm. loops 

 of this 24-hour peptone culture were then inoculated into each of 2 tubes of 

 peptone medium containing the test substance and incubated for 36 hours at 

 37 C. Acid-production in duplicate tubes varied so little that duplicates were 

 not employed with dulcitol, galactose, maltose, glycerol, and salicin. 



3 Jour. Hyg., 1905, 5, p. 333; 1909, 9, p. 86. 



4 Levine: Jour. Infect. Dis., 1916, 18, p. 358. 



