442 INACTIVATION OF BACTERIOPHAGES 



of radioactivity were made on dry samples by means of an end-window GM tube, 

 whose counting efficiency for P^- had been established by reference to a standard solu- 

 tion of radiophosphorus supplied by the National Bureau of Standards, United States 

 Department of Commerce. The specific radioactivity of the growth media was de- 

 termined by radioactive counting and chemical analysis of total phosphorus in the 

 case of a number of T2 lysates in order to establish the specific inactivation rate aN 

 for that phage and to confirm the value obtained by Hershey et al. To conserve the 

 supply of isotope, the specific activity of the growth medium in the case of the other 

 phages was usually estimated only by reference to the rate of inactivation of a stock 

 of T2 grown in an aliquot of the same medium. 



Bacteriophages of high specific activity were grown in the following way: A volume 

 of the radioactive stock solution containing the desired amount of P^^ was evaporated 

 to dryness in a boiling water bath and resuspended in 0.1 ml. of H medium. The radio- 

 active growth medium was then adjusted to neutral pH and inoculated with 0.01 ml. 

 of a culture of 2 X 10^ cells /ml. of B/r already in its exponential phase of growth in 

 non-radioactive H medium. The growth of the radioactive culture at 37°C. was 

 followed by microscopic counts in a Petroff-Hausser bacterial counting chamber. 

 When the bacterial density reached 5 X 10^ cells/ml., the culture was infected with 

 0.01 ml. of a stock containing 10" phages/ml. and incubated until microscopic counts 

 indicated satisfactory lysis. At this point, the remainder of the 0.1 ml. culture was 

 diluted into cold glycerol-casamino acid medium and assayed for its titer of infective 

 phage particles. 



Experimental Results 



Rate of Inactivation. — 



Hershey et al. observed that if a stock of T2 or T4 containing P^- at high 

 specific activity was assayed daily, the logarithm of the number of surviving 

 phages fell linearly with the number of P^' atoms that had decayed up to the 

 time of assay. The slope of this survival curve was found to be proportional to 

 the specific activity of the medium in which the phages had been grown, pro- 

 vided that the stock was stored in sufficiently great dilution under conditions 

 in which control lysates containing an equal amount of non-incorporated 

 P^2 were stable. This indicated that the inactivation of one phage particle was 

 not due to the radiation emitted by the radioactivity contained in other phages 

 but was the consequence of the disintegration of one of its own atoms of P'^'^ 

 The rate of change in the fraction 5 of surviving phage particles with the time 

 / in days may, therefore, be expressed as 



ds/dt = -aN*\s (1) 



in which a is the fraction of the P^- disintegrations which are lethal (hereafter 

 referred to as the "efficiency of killing"), A^* the number of radioactive phos- 

 phorus atoms per phage particle, and X the fractional decay of P^- per day. 

 Integration of (1) and substitution of more practical parameters lead to 



logio^ = -1.48 X lO-^aAoN(l - e-^') (2) 



281 



