8 M. LEVINE 



Concentration of Peptone. The following experiment was made in Wash- 

 ington to determine the effect of the concentration of peptone on acid produc- 

 tion and reversion: 



Three batches of medium (0.2% glucose, 0.4% dipotassium phosphate, and 

 1.0%, 1.5% and 2.0% peptone respectively) were prepared as described; 7 cc 

 portions were placed in tubes, autoclaved at 10 pounds for 10 minutes and 

 incubated for 48 hours to eliminate unsterile tubes. 



Seven tubes of each medium were inoculated from 24-hour cultures of 

 organisms and incubated at 37 C. 



Acidity determinations were made by the comparator in place of the dilu- 

 tion method previously described. Two duplicate cultures were taken and to 

 one was added 0.3 c c of an appropriate indicator and the color matched with 

 standards, the duplicate tube being employed to correct the error due to the 

 color and turbidity of the culture medium in the comparator test. This tube 

 was reincubatel and employed for this purpose in acidity determinations on 

 subsequent days. 



The concentration of peptone did not influence acidity production nor rever- 

 sion of the true dysentery bacilli nor of B. ambiguus, but that with B. alkalescens 

 and B. dispar reversion was much more rapid with 1.5% peptone than when 

 1.0% peptone was employed. Increasing the concentration to 2.0% did not 

 further increase the rate of reversion. 



Comparing the results with 1.0% peptone with those previously obtained in 

 the original experiment in France, reversion was somewhat delayed in the 

 new series. Although an adequate explanation is not available, it is felt that 

 the difference is probably due to a difference in the actual concentration of 

 glucose. The glucose available overseas was probably not thoroughly anhydrous. 



Aeration seems to increase the rate of alkali production, after the primary 

 acidity, in the case of B. alkalescens and B. dispar. 



To determine whether the differentiation indicated in the quantitative obser- 

 vations could be applied qualitatively, each organism was inoculated in duplicate 

 into the peptone phosphate medium containing as indicators 1% of a 0.5% 

 phenol-red and 1% of a 0.2% brom-cresol-purple, respectively, and incubated 

 at 37 C. Records of acidity were made daily. 



With exception of (37 and 57), which have been referred to as probably mis- 

 placed in this group, and which remained alkaline throughout the experiment, 

 all other cultures were distinctly acid to both indicators after 24 hours' incuba- 

 tion. With brom-cresol-purple as the indicator, all cultures of B. alkalescens and 

 B. dispar showed reversion to distinct purple-blue color, as did also one of the 

 B. dys. Sonne after 3 days' incubation. The true dysentery strains and B. 

 ambiguus were yellow or brownish in color. On further incubation (6 days), 

 the other strain of B. dys. Sonne and one B. flexneri became distinctly alkaline 

 and a number of the true dysentery cultures began to show some reversion, 

 thus obscuring, though not eliminating, the differentiation. 



With phenol-red, on the other hand, all cultures of B. dys. Shiga and B. 

 ambiguus, and all but one of B. flexneri were distinctly acid for 6 days. The 

 12 B. alkalescens strains were distinctly alkaline. Two of the 11 B. dispar were 

 neutral, the others distinctly alkaline. One B. dys. Sonne was neutral and 

 another alkaline. 



Rate of Acid Production. It was observed that glucose was attacked more 

 rapidly by B. alkalescens and B. dispar than by the other organisms of this 

 collection. Inoculation was from 24-hour broth cultures (0.1 cc to 30 cc of 



