654 INTRACELLULAR GROWTH OF BACTERIOPHAGES. I 



sufficient concentration to bacterial cultures. The very first experiments with 

 bacteria infected with T5 (3) left no doubt that lysis from without by T6 will 

 liberate T5 particles prematurely from infected bacteria. From the evidence 

 contained in the present paper it cannot be definitely established whether the 

 combined action of T6 and cyanide liberates all of the mature phage present in 

 the cells. However, the fact that, during the terminal stages of intracellular 

 development, the cyanide-lysis method yields as much phage as does spontane- 

 ous lysis, suggests that the cyanide method liberates all of the mature phage. 

 Furthermore, during the second half of the latent period, exactly the same 

 amount of phage is liberated by cyanide alone as by cyanide plus T6. This sug- 

 gests that cyanide acts promptly in arresting phage growth. Otherwise one 

 would expect to find a consistently higher number of phage particles in the 

 cyanide medium than in the medium in which cyanide and T6 are combined. 

 The experiments presented here therefore warrant the working hypothesis that 

 mature intracellular phage is effectively liberated by the treatment described, 

 and that the method gives a true picture of the intracellular phage population. 

 The validity of this working hypothesis will be conclusively demonstrated for 

 the phage T3 in the second paper of this series (10). 



The bearing of the present experiments on our concept of phage reproduction 

 might be discussed here. The finding that the original infecting particles are not 

 recoverable from the cells during the first stages of the latent period appears at 

 first sight surprising. Nevertheless some indirect evidence indicates that this is to 

 be expected. The discovery that yields from mixedly infected bacteria may con- 

 tain new combinations of the genetic material of the infecting types (11-13) 

 suggests that some alteration of the infecting particles may occur. Furthermore, 

 in mixed infections of bacteria with unrelated phages only one type is repro- 

 duced. The other type, although adsorbed on the cells, it not only prevented from 

 multiplying but the infecting particle of that type is lost (5, 14). On the basis of 

 multiple infection experiments with ultraviolet-inactivated phage particles, 

 Luria (15) has proposed that reproduction of phage occurs by reproduction of 

 subunits which are at some later stage assembled into complete virus particles. 

 The failure to find infective phage particles within the infected cell in the early 

 stages of reproduction agrees with what would have been predicted from these 

 experiments. 



The results of our experiments agree quite well with the scheme which 

 Latarjet (16) suggested on the basis of x-ray inactivation studies of phage 

 inside infected bacteria. Latarjet differentiated three segments of the latent 

 period of phage growth. Using T2 he found that during the first segment of 6 to 

 7 minutes' duration, singly infected bacteria show the same inactivation charac- 

 teristics as do unadsorbed phage particles. In the second period, from time 7 to 

 time 13 minutes, the phage in infected cells became more resistant to x-rays, 

 even during the first 2 minutes of this segment in which the inactivation curves 



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