BACILLUS-COLI-LlKE BACTERIA FROM FECES AND SEWAGE 27 



mation. In one small group of this collection, the lactose broth tubes at the 

 end of seven days were only slightly more acid than the blank, altho all of 

 the cultures gave gas in lactose bile. In no case was the titration of the cul- 

 ture less than that of the blank, altho this was usually the case with broths 

 in which there was no fermentation. Where positive evidence of the fer- 

 mentation of a sugar was obtained in another way, the negative evidence of 

 the titration was disregarded and in the correlation tables the culture was 

 included with the positive reactions. If, for instance, the titration of lactose 

 broth was negative, while the lactose bile fermentation tubes showed gas, the 

 cultures were considered to be lactose positive." 



Acid-production should not be given precedence over gas-forma- 

 tion. They may be independent characters. If however, after care- 

 ful studies, it appears that there is a marked correlation between quan- 

 titative acid-production and qualitative gas-formation, then it may be 

 feasible to supplement, if not substitute, the gas test by the acid test. 

 In that event, the line of demarcation between fermenters and non- 

 fermenters would have to be determined for the medium employed. 

 In this study, with peptone water containing 1% carbohydrate, non- 

 fermenters rarely produced as much as 0.2% normal acid. 



Another point of disagreement as to acid-production by B. coli is 

 the maximal amount of acid formed. Kligler, 6 using meat-infusion 

 media, often obtained titers of 4% normal acid or more, and similar 

 results have been recorded by Rogers. 7 Browne, 1 however, using 

 Liebig's meat-extract media, states that the limiting acidity for B. coli 

 is 2.4% normal acid as determined by titration with phenolphthalein. 

 Winslow and Walker 8 determined the acid-production in 12 substances 

 by B. coli. The maximal acidity observed was 0.45 c.c. N/20 NaOH 

 to the cubic centimeter of culture medium, or 2.25% normal acid. 



In the study recorded here, with peptone water as the basic medium, 

 the results are in entire accord with Winslow and Walker's, and with 

 Browne's. Of more than 2500 titrations, none showed more than 

 2.4% normal acid. 



The difference in acid-production observed by various investigators 

 is probably due to differences in the composition of the media 

 employed. It is now well established that more acid is formed in meat- 

 infusion broth than in beef-extract broth. In media containing much 

 phosphates, as yeast water, even more acid is formed than in meat- 

 infusion broth. Within certain limits the amount of acid formed, as 

 determined by titration with phenolphthalein, is a function of the 

 amount of buffer substances (as K 2 HPO 4 amino-acids, extractives, 

 etc.) present in the culture medium. Acid is formed until a certain 



8 Science, 1907, 26, p. 797. 



