11 



value, the fact that gas is produced at all may, nevertheless be of con- 

 siderable significance. If a culture is inoculated into sugar broth and 

 gas is formed, while no gas is produced in plain broth, the organism would 

 most certainly be regarded as a fermenter of the test sugar irrespective of 

 whether more or less than 1.5 percent acid is formed. 



The low liter might be due to a secondary alkali-production which 

 masks the acid, as suggested by Rogers. It has been repeatedly observed 

 by the author that Bact. aerogenes in peptone dipotassium-phosphate so- 

 lution, containing one percent or two percent glucose, may be acid to 

 methyl red after 24 hours' incubation but alkaline after a period of 48 to 

 96 hours at 37 degrees C. 



Rogers, Clark, and Evans also determined titratable acid and selected 

 one percent normal acid as the point of demarcation between fermenters 

 and non-fermenters but they point out the possible errors in acid-deter- 

 mination and give precedence to gas-formation, if positive. 



The author's observations are that with peptone water as a base and 

 one percent of the test carbohvdrates, nonfermenters rarely produced as 

 much as 0.2 percent normal acid. 



At what point on the acid scale are fermenters to be differentiated 

 from nonfermenters? There is considerable disagreement as to the max- 

 imal amount of acid formed by Bact. coli. Kligler, using meat infusion 

 media, often obtained titers of four percent normal acid or more and sim- 

 ilar results have been recorded by Rogers and others. Browne, however, 

 using Liebig's meat-extract media, states that the limiting acidity for Bact. 

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

 Winslow and Walker determined acid-production in 12 substances by Bact. 

 coli. The maximal acidity observed was 0.45 c. c. N/20 NaOH to the 

 cubic centimeter of culture medium, or 2.25 percent normal acid. 



The writer's experience, with peptone water as the basic medium, is 

 in entire accord with Winslow and Walker, and with Browne. Of more 

 than 2500 titrations, none showed over 2.4 per cent normal acid. 



The difference in acid-production observed by various investigators is 

 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. 



Acid-production should not be given precedence over gas-formation. 

 They may be independent characters. If, however, after careful studies, 

 it appears that there is a marked correlation between quantitative acid-pro- 

 duction 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 

 demarkation between fermenters and nonfermenters would have 

 to be determined for the medium employed. 



Table III. shows the relation of gas-production to the amount of acid 

 formed from sucrose, raffinose, ducitol, glycerol, and salicin in peptone 

 water. Other test substances were observed but are not indicated because 

 they were invariably fermented with production of gas. 



