ACTIVE FROM INACTIVATED BACTERIOPHAGE 103 



Comparison of theory with experiment 



Quantitative experiments consisted of testing mixtures of bacteria and ir- 

 radiated bacteriophage for the number of bacteria that liberate active phage. 

 Only phages T2^ T4, and T6 were studied in detail. 



In a typical experiment, such as the one described in detail in table 4, a 

 standard culture of bacteria, grown to a titer of either 10** cells per ml or 10^ 

 cells per ml, was chilled by immersion in a water bath at 5-6°C. This treatment 

 interrupts multiplication without changing the ability to resume immediate 

 multiplication and to support normal growth of phage upon return to 37°C. 

 In experiments with bacteria grown to a titer of 10^ (latest part of logarithmic 

 growth phase), immersion in ice-water is not necessary, since multiplication 

 stops almost immediately upon interruption of aeration and transfer to room 

 temperature. 



Ten minutes after interrupting multiplication, a sample of the culture is 

 diluted and assayed for viable count. If several mixtures of bacteria and phage 

 are to be prepared, the culture may have to be used for one or two hours, in 

 which case at least one other similar assay is made at the end of the experiment 

 to make sure that no proliferation has occurred. At intervals, undiluted samples 

 of the culture are placed into test tubes, and a constant volume of irradiated 

 (or control) phage variously diluted is added. In this way, we know for each 

 mixture the input of bacteria and of phage per ml. For irradiated samples, the 

 input of active phage is determined from one or more assays done at very high 

 dilution. When this is impossible — for high doses of radiation — the amount of 

 active phage is determined by extrapolation from the first part of the inacti- 

 vation curve. 



Adsorption is interrupted by heavy dilution after a short time (generally five 

 or ten minutes). The amount of adsorption is determined either by determi- 

 nation of free phage in the supernatant of a centrifuged sample from a mixture 

 containing active phage, assuming similar adsorption in all other mixtures 

 (see table 1), or by determining the bacterial survival in samples from various 

 mixtures. With the T-even phages, 80 to 95 percent of the phage is adsorbed 

 in ten minutes. The value for the multiplicity of infection, x, for each mixture 

 is used in calculating the fraction m of bacteria with two or more phage par- 

 ticles: m=l — (x-\-l)e~'. 



Before lysis begins, a suitably diluted sample of the mixture is plated with an 

 excess of sensitive bacteria for plaque count. The plaque count — corrected, 

 when necessary, for the active phage by subtracting the value corresponding 

 to the latter — is divided by m to obtain the experimental value w for that 

 mixture (see table 4). 



A large number of experiments, yielding a total of over 1000 values of w, 

 for various doses of radiation and for different multiplicities, were done with 

 the T-even phages. Phage T5 was only partially investigated, because of diffi- 

 culties in obtaining reproducible results in view of the low and irregular adsorp- 

 tion rate for this phage. 



The data for phages T-even include those for several of their r mutants, 



257 



