236 THE BACTERIOPHAGE AND ITS BEHAVIOR 



by colony isolations and the new strains yielded henceforth ultrapure 

 colonies free of the bacteriophage. 



Burgers and Bachmann^^i carried out 200 tests on strains of Gram- 

 positive organisms and succeeded once in isolating a bacteriophage from 

 an old culture of the organism of swine fever. In spite of the fact that 

 they were only once successful these authors certainly employed a 

 method which should have enabled them to detect a bacteriophage had 

 any been present, for they employed the procedure of Buchner for the 

 extraction of zymase. The bacteria were triturated with sand and 

 then subjected to a pressure of 400 atmospheres. 



From all of these experiments it appears definitely that there are 

 bacterial strains containing bacteriophage corpuscles, but that these 

 strains, naturally contaminated, are relatively rare; more rare even 

 than one would have a priori supposed in view of the ubiquity of the 

 bacteriophage and the frequency under natural conditions of the 

 bacteriophage-bacteria symbiosis in natural mixed cultures. 



As I have shown,^^^ in accord with the present state of our knowledge, 

 if bacteriological studies are to be conducted under properly controlled 

 conditions, it must first be shown that the bacterial strains involved in 

 the study are, not only pure, but also ultrapure. 



Strains contaminated by a bacteriophage may be recognized in that 

 they give mutant colonies, while all the colonies of an ultrapure strain 

 are identical. The bacteria of contaminated strains are but slightly or 

 not at all agglutinable by a specific antiserum. The bacteria are 

 resistant to the action of bacteriophages which attack homologous 

 strains. Every time that one or the other of these characters is encoun- 

 tered there is reason to suspect contamination by a bacteriophage. 



13. THE PURIFICATION OP NATURAL MIXED CULTURES 



Purification of natural mixed cultures is the more readily accompHshed 

 when the symbiosis between the bacteriophage and the bacterium has 

 not been of long duration. In ordinary cases the usual method of 

 colony isolation upon agar permits the selection of ultrapure colonies. 

 It may be added that it is always well to repeat this purification proce- 

 dure through 2 or 3 consecutive platings. The method of Roux, in 

 connection with the purification of artificial mixed cultures is particu- 

 larly well adapted to this purpose. 



Bruynoghe^i- has discovered that mixed cultures may be purified more 

 readily if the mixed condition is of recent origin, and that when the 

 sjnnbiosis is of longer duration the simple procedure of colony isolation 



