GENERIC CHARACTERS IN THE COCCACEAE 165 



Coagulation depends upon the amount of acid produced, and 

 is more easily studied in sugar-broth cultures. 



Action upon carbohydrates. The characteristics usually observed 

 in sugar broth are turbidity and sediment, relation to oxygen, gas 

 production, and acid production. We have given reasons, in dis- 

 cussing nutrient broth, for considering turbidity and sediment 

 unimportant, and the relation to oxygen is most sharply denned 

 by surface growth in the agar tube. None of the cocci, so far as 

 known, produce gas, and there remains only acid production to 

 be recorded. For this purpose ordinary straight tubes were used. 

 The sugars tested were dextrose and lactose. Saccharose has been 

 omitted for the present, for lack of time. A preliminary test indi- 

 cated that this sugar is less commonly fermented by the cocci than 

 are dextrose and lactose. 



The media were made up in the usual manner with 2 per cent 

 of the sugar to be tested. The reaction was made about neutral, 

 and after tubing and sterilization it was usually between 0.5 and 

 i.o per cent. After standing for two weeks sterile blanks showed 

 a slight further rise in acidity, so control tubes were always kept 

 with each batch inoculated and titrated at the end of the experiment. 

 After considerable preliminary experimentation, 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 more by the breaking-up of unstable com- 

 pounds than it decreases it by driving off carbon dioxide. The 

 cold method was therefore used. To 5 c.c. of the sugar broth, 

 grown for two weeks at 20, was added 95 c.c. of distilled water 

 and two or three drops of phenolphthalein. This was titrated against 



_ NaOH and from the value obtained was subtracted the acidity 



20 



of the blank controls titrated at the same time. All tests were made 

 in duplicate, and the final value recorded as the acid or alkali pro- 

 duction of the organism is the difference between the average of 

 two titrations of tubes in which it had grown for two weeks and 



