THE BACTERIOPHAGE AND THE BACTERIUM 85 



change in morphology. We have seen that certain colonies on 

 agar are composed of bacteria presenting the coccoid form, other 

 colonies presenting the refractile appearance and a mucous con- 

 sistency. 



Coccoid form 



The following experiments are interesting for they allow of the 

 appearance of the coccus form with a later return to a normal 

 morphology — the first in the colony itself at a few days interval, 

 the second in the course of successive passages. 



Experiment XXIV. A Petri dish is heavily inoculated from an agar cul- 

 ture of B. dysenteriae and is placed in the incubator at 37°C. for about four 

 hours. A drop of the bacteriophage culture is then placed in the centre of the 

 plate. The strain of bacteriophage should be one of average activity, that 

 is, one capable of regularly causing complete lysis of a bacterial suspension 

 but with which secondary cultures usually develop. (With too virulent 

 a strain the area where the drop was placed remains sterile indefinitely.) 

 The plate is returned to the incubator. After eighteen to twenty-four 

 hours a layer of culture composed of normal dysentery bacilli develops, 

 showing in the centre a spot devoid of growth, apparently sterile. After 

 thirty-six to forty-eight hours, the spot becomes covered with extremely^ 

 fine colonies, which, when examined microscopically are composed of ' 

 cocci only. These cocci are of different sizes, from 1 to 4 p in diameter, 

 arranged in irregular forms, — in diplo- and in tetrad groupings. Two 

 days later microscopic examination still shows cocci, but among them are 

 bacillary forms in great number. Subcultures on to agar always give iso- 

 lated colonies, each colony always reproducing with the same appearance 

 and with the same sequence of forms, — first a coccoid culture, then a mix- 

 ture of cocci and bacilli. These cultures always contain, moreover, bacter- 

 iophagous ultramicrobes. 



Eliava and Pozerski have indicated a method for obtaining the 

 coccoid, resistant bacteria free from admixture with the bacterio- 

 phage. 5 These bacteria perpetuate themselves under this form 

 for a certain number of generations. Return to the bacillary form 

 occurs gradually and after about fifteen transplants the culture 

 acts as a normal dysentery organism sensitive to the bacteriophage. 



6 The method permits the purification of a mixed culture by the elimina- 

 tion of the bacteriophage. To accomplish this it is only necessary to make 

 transfers on agar with the mixed culture, taking for inoculum in each 

 passage material from the top margin on the agar, as near the edge as 

 possible. 



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