62 RELATIONSHIPS OF THE COCCACE^ 



experiment. After considerable preliminary experimen- 

 tation, it was decided to titrate with phenolphthalein as 

 an indicator, in the cold. Methyl orange is not sensitive 

 to the organic acids and gives a poor end-point. With 

 phenolphthalein a comparative series of titrations, made 

 on the same tubes, first cold and then boiling, showed 

 slightly higher results by the latter method. Evidently 

 heating increases the apparent acidity by the breaking 

 up of unstable compounds more than it decreases it by 

 driving off carbon dioxid. The cold method was therefore 

 used. To 5 c.c. of the sugar broth, incubated for two 

 weeks at 20 degrees, were added 95 c.c. of distilled water 

 and two or three drops of phenolphthalein. This was 



N 

 titrated against — NaOH, and from the value obtained 

 20 



was subtracted the acidity of blank controls titrated at 

 the same time. All tests were made in duplicate, and 

 the final value recorded as the acid or alkali production 

 of the organism is the difference between the average of 

 two titrations of tubes in which it had grown for two 

 weeks and the average of two blank controls. No 

 determination was made of the rate of acid production, 

 as distinguished from this total final acidity, tho such 

 observations might be of much interest. 



We did not attempt a study of the specific products 

 formed, altho their nature might yield important assist- 

 ance in distinguishing various species now confused. 

 For example, Nencki (1891) showed that the production 



