was desirable for isolation of the B. acr- 

 o genes types. 



As to B. coli, a temperature of 40 C. 

 has of!'T 'x>','n recuiii'iei.'d'.'d for its iso- 

 lation and in the Eijkman test 46 C. is 

 employed for the isolation of the organ- 

 ism from water. In fact, it has been 

 observed that the maximum rate of mul- 

 tiplication of B. coli is at about 45 C. 



The author observed that in peptone 

 lactose media at 43 C. (in a water bath) 

 all the cultures of B. coli (16) grew 

 luxuriantly as evidenced by strong tur- 

 bidity in 24 hours, but 69 percent showed 

 no gas or only a bubble in 24 hours. Of 

 20 cultures of B. aerogenes, on the other 

 hand, 16 showed no growth, 2 slight, and 

 2 grew luxuriantly. 



Boric Acid. In agar of the follow- 

 ing composition: peptone 1.0 percent, 

 agar 1.5 percent, dipotassium phosphate 

 0.3 percent, and glucose .05 percent with 

 0.63 percent of boric acid, B. aerogenes 

 failed to grow, whereas B. coli grew lux- 

 uriantly. In liquid media, 1.0 percent 

 peptone with 0.63 percent boric acid, B. 

 coli multiplied slowly, while B. aerogenes 

 died off as evidenced by the following 

 figures : 



Culture 19b, B. coli increased from 

 65,000 per c.c. to 1,500,000 per c.c. in 48 

 hours, while B. aerogenes was reduced 

 from 2,300 per cc. to 20 in 24 hours and 

 to in 48 hours. It was found in subse- 

 quent studies, however, that the differ- 

 ence in concentration of boric acid, which 

 did not inhibit B. coli and which did in- 

 hibit B. aerogenes, was so close that it 

 could not be safely employed as a se- 

 lective agent. 



Crystal Violet. One percent pep- 

 tone water containing */2 percent lactose 

 and varying concentrations of crystal 

 violet were inoculated from 48-hour pep- 

 tone cultures of B. coli and B. aerogenes. 

 Five different strains of each species 

 were employed. A concentration of 

 1-100,000 of crystal violet prevented the 

 growth of all the cultures of B. coli, 

 whereas all of the B. aerogenes grew 

 heavily. One culture of B. coli failed to 



grow in a dilution of 1-250,000 of crystal 

 violet. 



Decreasing the concentration of pep- 

 tone to y> percent increased markedly the 

 inhibitory action of the dye. Thus in y 2 

 percent peptone lactose solution none of 

 the B. aerogenes grew with a dye con- 

 centration of 1-100,000, but all grew 

 luxuriantly in 1-250,000 crystal violet. 

 Among the B. coli cultures, all were in- 

 hibited in 1-250,000 dilution of the dye 

 and two failed to grow in a dilution of 

 1-500,000. 



Brilliant Green. Some time ago, the 

 author was informed that growths of B. 

 coli are rarely encountered in the isola- 

 tion of B. typhosns from stools by the 

 use of cosine brilliant green agar, and 

 that if a growth other than B. typhosus 

 was present, it was very likely to be B. 

 aerogenes. This suggested that the in- 

 hibitory action of brilliant green was 

 much greater for B. coli than for B. 

 aerogenes. 



In a medium consisting of 1.0 percent 

 peptone and 0.5 percent lactose with vari- 

 ous concentrations of brilliant green, 

 four cultures of B. aerogenes grew very 

 luxuriantly in a concentration of 1-750,- 

 000, whereas one failed to grow in this 

 concentration, but grew very well in 

 1-1,000,000 dilution of the dye. The 5 

 cultures of B. coli, on the other hand, all 

 failed to grow in 1-750,000 of the dye, 

 3 did not grow in a dilution of 1-1,000,- 

 000 and 2 failed to grow even in a dilu- 

 tion of 1-1,500,000. 



Reducing the concentration of peptone 

 to J^ percent increased very markedly 

 the antiseptic action of brilliant green. 

 The five B. coli cultures now failed to 

 grow even in a dilution of 1-3,000,000. 

 The B. aerogenes cultures grew luxuri- 

 antly in a dilution of 1-2,000,000. Four 

 grew in a dilution of 1-1,500,000, but 

 only 1 grew in more concentrated solu- 

 tions of the dye. 



The selective action of brilliant green 

 was even more strikingly shown by the 

 use of a plate medium consisting of the 

 simplified eosine methylene blue agar 



