234 THE BACTERIOPHAGE AND ITS BEHAVIOR 



obtained upon agar a separation into mixed colonies and ultrapure 

 colonies. With the fifth it was necessary for him to resort to the other 

 method which I have indicated, namely, that of growth on glucose 

 agar. ^2^ In this way he obtained ultrapure colonies of this strain when 

 isolation on ordinary agar had been unsuccessful. Having then of these 

 5 strains both mixed colonies and ultrapure colonies he showed that 

 although the first were indefinitely cultivable in the form of mixed 

 cultures from which it was possible to isolate a bacteriophage, the second, 

 on the contrary, continued indefinitely as ultrapure cultures, and from 

 these it was henceforth impossible to isolate a bacteriophage. This 

 makes it obvious that in mixed cultures the bacteriophage represents 

 an impurity. 



From two other cases of uncomplicated cystitis Tomaselli isolated 

 ultrapure cultures with no bacteriophage from the urine. 



Gildemeister and Herzberg,-^- working on old cultures, tried in vain to 

 derive a bacteriophage, although they applied very drastic treatment. 

 They subjected the bacteria to trituration and extracted the bacterial 

 juices under a pressure of 4000 atmospheres. 



Pondman^^*^ studied systematically the 13 strains of B. dysenteriae 

 found among the stock cultures of the Institute of Tropical Medicine at 

 the University of Leiden. He cultivated these different strains in 

 bouillon, and filtered them after an interval varying from 24 hours to 30 

 days. Among these 13 strains one only (Y-14) contained a bacterio- 

 phage. In additional experiments he dissolved the bacteria with trypsin 

 or with pyocyanase and in other tests he substituted heating for filtra- 

 tion through a candle. The final products obtained in these different 

 ways were combined with the bacteria. He did not succeed by any 

 of these procedures in demonstrating any "lytic" principle whatever in 

 cultures of 12 of these strains. But with strain Y-14 he was able to 

 isolate a bacteriophage by all of these methods. Twelve of these 

 strains were, therefore, ultrapure; 1 was a mixed culture. 



During a series of experiments, representing several months' work, 

 Flu2i2 systematically examined 43 different bacterial strains: 13 of 

 Vibrio cholerae, 6 of B. typhosus, 4 of B. dysenteriae Shiga, and 20 of the 

 Flexner or Hiss dysentery bacillus. 



With each strain he performed a double series of tests. A flask con- 

 taining 50 cc. of bouillon was seeded with a loopful of a fresh culture of 

 the strain under examination. After incubation at 37^C. for 10 days 

 the material was passed through a candle. To the filtrate he added an 

 equal volume of sterile bouillon and seeded it again with a loopful of a 



