G. S. STENT AND C. R. FUERST 449 



Evidently, it is possible to effect at least a fourfold variation in a. by varying 

 the temperature of storage from the lowest to the highest practicable range. 

 It is to be noted that the increase in a per degree is greater between — 20 and 

 +4°C. than between +4 and +50°C. This, no doubt, implies that a is afifected 

 not only by the ambient thermal energy, but also by the change of phase from 

 liquid to solid state. 



P^- Decay after Injection. — 



Hershey and Chase (1952) have shown that when T2 infects a sensitive 

 bacterium, the phosphorus, and hence the DNA, of the bacteriophage particle 

 enters the host cell, whereas the bulk of the phage protein remains outside. 

 It may then be asked whether P^'^ decay can still prevent the reproduction of 

 the parental phage and the ultimate emergence of infective progeny if such 

 decay occurs only after the introduction of the DNA of a radioactive T2 

 particle into the interior of the bacterial cell. 



In order to study the effect of P^^ decay after infection, it is necessary to 

 arrest intracellular phage development reversibly for days or weeks so that 

 the slow radioactive decay may proceed at an early stage of the brief 20 minute 

 latent period. This can be achieved by quick-freezing the bacterial cells shortly 

 after infection and storing them at — 196°C. in liquid nitrogen. As in the case 

 of free phages, non-radioactive controls show that more than half of the 

 infected centers survive freezing and thawing, and that the fraction recovered 

 is independent of the length of storage at — 196°C. In those infected bacteria 

 which survive, phage development resumes upon thawing where it had left off 

 at the moment of freezing. 



A culture of strain B/r was grown in nutrient broth to a density of 10^/ml., centri- 

 fuged, and resuspended in fresh broth at one-fourth of its original volume. The sus- 

 pension was then infected with 3 X 10^/ml. radioactive T2 particles, containing P^^ 

 at a specific activity of 88 mc./mg. Phage development was again arrested 2.5 minutes 

 after infection by chilling the culture in ice. The infected bacteria were separated from 

 the small fraction of unadsorbed free phage by centrifugation and resuspended in cold 

 glycerol-casamino acid medium. Aliquots of 0.1 ml. of this final suspension were 

 frozen and stored in liquid nitrogen. From day to day, one of the aliquots was thawed 

 by addition of 1.9 ml. of warm medium and plated at once for the number of surviv- 

 ing infective centers. A control culture infected with non-radioactive T2 under other- 

 wise identical conditions was similarly frozen, stored, and assayed. Aliquots of the 

 initial radioactive stock of free T2 and a non-radioactive control stock were also stored 

 in liquid nitrogen and assayed for their survival from day to day. 



The results of this experiment are presented in Fig. 4. It is seen that in the 

 population of bacteria infected for 2.5 minutes with a multiplicity of 0.075 

 radioactive T2, per cell, the logarithm of the fraction of individuals capable of 

 giving rise to a plaque when plated after thawing decreases linearly with 

 (l — e~^0- The slope of the survival curve is about three-fourth that of the 



288 



