648 INTRACELLULAR GROWTH OF BACTERIOPHAGES. I 



titer of infected bacteria given by the preburst control platings, gives the average yield 

 per infected cell. As a working hypothesis, this yield was considered to be the average 

 number of intracellular phage particles per bacterium at the time of dilution into the 

 lysing medium. Dividing these numbers by the control burst size gives the fraction of 

 the control yield found in the experimental lysing medium tubes. 



The results of several typical experiments are shown in Fig. 1 in which the 

 data are plotted on semilogarithmic coordinates. The fraction of the control 

 yield found in a given experimental culture is plotted against the time at which 

 the dilution is made into lysing medium. Curve 1 shows the results from a single 

 experiment in which the bacteria were infected with an average of 7 phage 

 particles each. Curve 2 is the composite result of four experiments in which the 

 bacteria were infected with single phage particles. Curve 3 is the control one- 

 step growth curve derived from the control growth tube platings in the four 

 experiments of curve 2. 



Several striking results can be seen in these experiments. First, it is clearly 

 seen that during the early stages of the latent period the virus-host complex is 

 inactivated by the cyanide-T6 mixture, and that not even the infecting particles 

 are recovered. Even when 7 phage particles were adsorbed on each bacterium, 

 less than two are recovered per cell at the earliest stage tested, and the shape of 

 the curve suggests that if earlier stages had been tested, still fewer would have 

 been recovered. In experiments with singly infected bacteria, the earliest tests 

 indicated that less than one infected bacterium in 80 liberated any phage at all. 

 A second point to be noted is that the multiplicity of infection appears to 

 influence slightly the time at which phage particles can be recovered from the 

 cell, and it continues to affect the fraction found in the bacteria at a given time. 

 That this difference is a real one seems clear from the consistency among the 

 points of curve 2. This result has been observed in each experiment, although 

 the effect appeared to be less pronounced in some experiments made at the 

 lower temperature of 30°C. Attention should also be drawn to the fact that the 

 shape of the curves is clearly not exponential. In fact, it parallels with a delay 

 of several minutes the approximately linear DNA increase observed in this 

 system (6). 



In connection with the preceding experiments a test was made to establish 

 whether the cyanide concentration chosen was maximally effective in inhibiting 

 phage synthesis. Using the described technique, but changing the cyanide con- 

 centration of the lysing medium from 0.01 m to 0.004 m and 0.001 m in parallel 

 aliquots, no difference was detectable in the three lysing media. Thus 0.01 m 

 cyanide is well beyond the minimum concentration necessary and was con- 

 sidered to be adequate for these experiments. 



Action of Cyanide in the Absence of the Lysing Agent T6. — Cohen and Anderson 

 (7) reported a loss of infectious centers when infected bacteria were incubated 

 in the presence of the antimetabolite 5-methyl tryptophan. Although the details 



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