RESISTANCE OF BACTERIA 235 



fresh culture of the same strain. The seeded medium was returned to 

 the incubator at 37°C. and allowed to remain there for 6 weeks. After 

 this time the old culture was filtered and the filtrate was tested systemati- 

 cally on different strains of susceptible bacteria, either in bouillon in 

 order to see if any "lytic" phenomena took place, or on agar in order to 

 detect the presence of plaques. This same procedure was repeated for 

 each strain examined. Furthermore, the test was repeated with each 

 strain substituting heating for 1 hour at 58°C. for the filtration. Finally 

 he made serial passages with the filtrates (or with the heated suspen- 

 sions) with different bacteria, for the purpose of increasing the virulence 

 of the bacteriophage if any was present. 



These experiments forming a double series ;^ — one with a filtration 

 between each passage and the other with heating, — were carried out for 

 each of the 43 strains. The results showed that only one was contami- 

 nated by a bacteriophage and this strain was the Y-14 strain of the Hiss 

 bacillus, the same strain that had proved to be contaminated when 

 studied by Pondman. 



Flu then purified this Y-14 strain by means of colony isolation, obtain- 

 ing ultrapure colonies. The cultures derived from these ultrapure 

 colonies were subjected to the same treatment that permitted him to 

 isolate a bacteriophage from the original strain. It was impossible for 

 him to isolate one. Cultures derived from the ultrapure colonies were 

 indefinitely ultrapure; the bacteriophage had been eliminated. It was, 

 then, an impurity. 



In another series of investigations'- ° Flu, who as has just been stated, 

 examined 13 strains of Vibrio cholera, tested the ultrapurity of 10 other 

 strains. This second series of studies, conducted as methodically as 

 was the first, showed one of the 10 strains to be a mixed culture and from 

 it he isolated a bacteriophage. 



In all, then, of 53 strains of different types of bacteria taken at 

 random from the cultures kept at the Institute of Tropical Medicine at 

 the University of Leiden, 51 were ultrapure. None of the methods 

 applied permitted the detection of a principle exerting any action in any 

 way resembling bacteriophagy. Two were contaminated by races of 

 the bacteriophage which Flu isolated. After a series of passages with 

 susceptible bacteria, the first brought about complete bacteriophagy 

 of a suspension of the Hiss bacillus, the second, complete bacteriophagy 

 of a suspension of Vibrio cholerae. 



Reichert^^" examined 11 strains of dysentery bacilli; 9 were ultrapure, 

 2 were contaminated by a bacteriophage. He purified these two strains 



