THE BACTERIOPHAGE AND THE BACTERIUM 77 



For example, if we place a small quantity of a secondary culture 

 in normal saline, or preferably in 30 per cent glycerine bouillon, 

 that is to say, in a medium which interferes with the reproduction 

 of the bacteria and which exerts no destructive action on the 

 bacteriophage, the equilibrium is disturbed in favor of the 

 bacteriophage. 



The ultramicrobe is very sensitive to the action of acids, and if 

 transfers from a secondary culture are made upon glucose agar it is 

 found that the bacterium reacts upon the sugar, acidifies the 

 medium, and breaks the equilibrium in favor of the bacterium. 

 The bacteriophage, not being able to exert its parasitizing action, 

 will be eliminated after a few transfers. 



Still another method of separation, the most practical of all, 

 consists in employing the method used by Eliava and Pozerski 

 (described further on) for obtaining cultures of resistant bacteria 

 free from the bacteriophagous ultramicrobe. It is only necessary 

 to make a few passages on agar slants, culturing each time from 

 the extreme upper margin of the agar layer where the medium is 

 somewhat desiccated. 



An ultrapure bacterial culture can also be obtained by the use of 

 quinine, since this substance has a higher antiseptic activity for 

 the bacteriophage than for the bacterium. 



We have seen that in the case of a bacteriophage but slightly 

 virulent the addition of the filtrate in which it is present to some 

 bouillon does not impair the development of the inoculated 

 bacteria; it is only by spreading cultures on agar that we can 

 detect the presence of the bacteriophage through the plaques which 

 develop there. To increase the virulence of such an inactive strain 

 we have seen that it is necessary to make several successive passages 

 of the bacteriophage along with the bacterium. Between each 

 passage it is essential either to filter the mixed culture through 

 a bougie to separate bacteria and ultramicrobes or to heat the 

 mixture to 60°C. to destroy the bacteria, while leaving the bac- 

 teriophage unharmed. What is the basis for this technic? The 

 elimination of bacteria which, because of contact with the bac- 

 teriophage, are defending themselves and are acquiring a certain 

 degree of resistance, a resistance which permits them to subsist in 

 spite of the progressive increase in virulence of the bacteriophage. 



