26 



necessarily be less acid. On further incubation the Bact. coli would die off, 

 whereas the more alkaline Bact. aerogenes culture would continue to grow 

 exhausting the available sugar, after which the reaction would become 

 progressively more alkaline. This reversion is due partly to the decompo- 

 sition of the peptones with the liberation of alkali but particularly to the 

 conversion of the organic acids originally produced from the glucose into 

 carbonates and bicarbonates, as shown by Avers and Roup. As incuba- 

 tion is prolonged, the difference in reaction between a Bact. aerogenes cul- 

 ture and one of Bact. coli may be considerably increased. 



Clark and Lubs, in some very careful work, found that a concentra- 

 tion of 0.5 percent anhydrous glucose, dipotassium phosphate, and Witte's 

 peptone affords the proper combination of glucose and buffer substances 

 for the differentiation of the coli and aerogenes sections. They recommend 

 an incubation temperature of 30 degrees C. for 5 days, after which period, 

 Bact. coli is acid and E act. aerogenes will be found to be alkaline to the 

 indicator methyl red. 



TABLE XI. EFFECT OF THE TEMPERATURE AND THE PERIOD OF INCUBA- 

 TION ON THE REACTION WITH METHYL RED. 



As to the effect of temperature and period of incubation on the re- 

 action with methyl red, the indications are that the final reaction is reached 

 more quickly at body temperature than at 30 degrees C. (Table XL). 

 With an incubation period of 5 days there is little choice between 30 de- 

 grees C. and 37 degrees C., but if the time of incubation is reduced to two 

 or three days, which would be very desirable for routine water work, the 

 body temperature seems preferable. It is of course recognized that some 

 strains of the aerogenes section will not grow well at body temperature, 

 but from the point of view of the bacterial analysis of water it is felt that 

 there is little value in detecting such strains. The extra expense of main- 

 taining a 30 degree incubator for their isolation is consequently not war- 

 ranted. 



Too much emphasis can not be placed on the necessity of employing 

 Witte's peptone in preparation of the medium mentioned above. The in- 

 discriminate substitution of other peptones is bound to lead to confusion 

 and error. Owing to the great difficulty in obtaining Witte's peptone, 

 Clark and Lubs were led to devise a synthetic medium which consists of 

 the following: 



0.7% anhydrous Na 2 HP0 4 . 



0.2% acid potassium phthalate. 



0.1% aspartic acid. 



0.4% anhvdrous dextrose. 



