374 GROWTH OF BACTERIOPHAGE 



The results of three experiments at 37°C. are plotted in Fig. 2, and 

 confirm the suggestion of d'Herelle that phage multiplies under a 

 spatial constraint, i.e. within or upon the bacterium, and is suddenly 

 liberated in a burst. It is seen that after the initial rise (discussed 

 above) the count of infective centers remains constant up to 30 

 minutes, and then rises about 70-fold above the initial value. The 

 rise corresponds to the liberation of the phage particles which have 

 multiplied in the initial constant period. This interpretation was 

 verified by measurements of the free phage by centrifuging out the 

 infected bacteria, and determining the number of phage particles in 

 the supernatant liquid. The free phage concentration after adsorp- 

 tion was, of course, small compared to the total and remained constant 

 up to the time of the first rise. It then rose steeply and became sub- 

 stantially equal to the total phage. 



The number of bacteria lysed in this first burst is too small a fraction 

 of the total bacteria used in these experiments to be measured as a 

 change in turbidity; the ratio of uninfected bacteria to the total 

 possible number of infected bacteria before the first burst is 400 to 1, 

 the largest number of bacteria which can disappear in the first burst is 

 therefore only 0.25 per cent of the total. 



The phage particles liberated in the first burst are free to infect 

 more bacteria. These phage particles then multiply within or on the 

 newly infected bacteria; nevertheless, as before, the concentration of 

 infective centers remains constant until these bacteria are lysed and 

 release the phage which they contain into the medium. This gives 

 the second burst which begins at about 70 minutes from the start of 

 the experiment. Since the uninfected bacteria have been growing 

 during this time, the bacteria lysed in the second burst amount to less 

 than 5 per cent of the total bacteria present at this time. There is 

 again therefore, no visible lysis. 



This process is repeated, leading to a third rise of smaller magnitude 

 starting at 120 minutes. At this time, inspection of the culture, which 

 has until now been growing more turbid with the growth of the unin- 

 fected bacteria, shows a rapid lysis. The number of phage particles 

 available at the end of the second rise was sufficient to infect the re- 

 mainder of the bacteria. 



These results are typical of a large number of such experiments, at 



46 



