166 The Philippine Journal of Science i»i6 



medium under small parts of the growth. That this localized 

 acid production was temporary is indicated by the fact that 

 when sufficient time had elapsed for the reaction of the medium 

 to become uniform throughout by diffusion the entire slant again 

 became blue. 



With 1 per cent agars the strains inactive in series I showed 

 fermentation, Arabinose cultures also showed more marked 

 acidity, but the galactose reactions were still irregular and not 

 satisfactory. With salicin there was complete inactivity with 

 three strains, while the glycerin cultures, instead of reacting 

 more rapidly, were somewhat delayed in attaining complete 

 change. These results emphasize the fact that litmus-sugar 

 agars, even of 1 per cent concentration, are not satisfactory for 

 the demonstration of acid production by organisms such as B. 

 pestis, the fermentation activities of which are of low degree. 



SUGAR BOUILLONS, PHENOLPHTHALEIN TITRATIONS 



In order to establish quantitatively the fermentative power of 

 the different strains, and also to determine the relation between 

 actual acid production by B. pestis and the indication of such 

 activity given by the media previously employed, a complete 

 series of broth cultures was made in bouillons containing the 

 eighteen carbohydrates previously used. 



Each tube contained from 15 to 20 cubic centimeters of 0.5 per 

 cent sugar bouillon, made with beef extract (Liebig's) in the 

 ordinary manner. This was used because the pest organism 

 grows fairly well in it and because preliminary tests had shown 

 that no change in reaction detectable by titration occurs from 

 growth of B. pestis in broth so made. The tubes of the first 

 series contained small, inverted "fermentation tubes." After 

 three days, no gas having appeared, these were removed. After 

 ten days of total incubation they were heated in an Arnold 

 sterilizer for twenty minutes before being titrated. The esti- 

 mations were made, using N/20 and N/50 sodium hydroxide, 

 respectively, in the two series tested. Hot titration was used 

 by choice, in spite of the fact that it gives somewhat higher 

 readings than are obtained in the cold. Accuracy of comparison 

 was considered of first importance rather than the exact de- 

 termination of absolute acid increase. 



Two series were titrated, as upon titration the first lot of 

 media was found to have been between 1.5 and 2.2 per cent acid. 

 The results obtained in series I agree in the main with those of 

 the second series, which was adjusted to about 0.3 per cent acid 

 before sterilization. 



