238 Journal of Agricultural Research voi. xm, no. 4 



useful medium for the study of the streptococci. The hydrogen-ion 

 concentration of such medium is about Ph = 6.0. 



Cultural characteristics were determined in litmus skim milk, and in 

 litmus skim milk to which 0.5 per cent of peptone had been added. 



Another very useful medium was trypsin-digested milk. One per 

 cent of commercial trypsin was added to the milk, and it was kept at 

 about 40° C. for two or three hours. Sodium hydroxid was added to 

 bring the reaction to about Ph = 7.2 at the beginning of the digestion 

 and several times afterward to neutralize the liberated amino acids. 

 The final hydrogen-ion concentration of the medium was about Ph==6.2. 

 The medium was filtered, heated in the autoclave as for sterilization, 

 refiltered, put into tubes or flasks and sterilized for use. 



Volatile-acid production was determined in milk cultures. Most of 

 the cultures were grown in 500 c. c. of skim milk, but a few of the first 

 determinations were made in cultures grown in 200 c. c. of skim milk. 

 For these the data have been calculated for 500 c. c. Duplicate cultures 

 were analyzed after a varying length of incubation, and the results 

 showed that after the first week there is Httle or no increase in volatile 

 acids. The subsequent determinations were made after from 7 to 15 

 days' incubation. Dilute phosphoric acid was added to the culture 

 until a blue color was given with Congo red, to release any volatile acid 

 that might be in combination. In some preliminary determinations 

 sulphuric acid had been employed for the purpose, but it was decided that 

 small quantities of formic acid resulted during the distillation from the 

 action of sulphuric acid on some constituent of the milk, possibly casein. 

 The cultures were distilled with steam until 2,000 c. c. of distillate were 

 collected. At first an effort was made to distill under reduced pressure 

 to hasten the process, but it had to be abandoned because the cultures 

 foamed so freely. 



The distillate was neutralized with barium hydroxid [Ba(0H)2], 

 evaporated to a small volume, decomposed with sulphuric acid (H2SO4) 

 and distilled by the method of Duclaux (j). This method serves both 

 to identify and to estimate the volatile acids present. Although some of 

 the data indicated traces of acids of lower molecular weight than acetic, 

 probably formic, and also traces of acids higher than acetic, possibly 

 butyric, which might arise from a slight hydrolysis of the fat in the milk, 

 it seems justifiable to conclude that acetic acid is the only volatile acid 

 produced by either 5". lacticus or the cheese streptococci in their normal 

 processes of metabolism in milk culture. 



STREPTOCOCCUS LACTICUS 



This organism has been known in the literature under the names 

 "Bacterium giintheri," "Bacterium lactis acidi," and various other names, 

 but is now generally designated as "Streptococcus lacticus (Kruse)." 

 Its close relationship to the other varieties of streptococci argues forcibly 



