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J. D. WATSON, O. MAAL0E VOL. 10 (1953) 



that each virus particle contained an average of 0.25 ^zp atoms. Since the inactivation efficiency 

 of nuclear decay is only 1/12 (Hershey, Kamen, Kennedy and Gest^^), the fraction of labelled 

 particles which will lose infectivity during an experiment is negligible. 



With minor variation indicated by adsorption requirements, etc.. the procedure just described 

 was also used to prepare ^^p-iabelled stocks of the phages T2r+, T^, and T4r+. Phage labelled with 

 i*C adenine was grown on a purine requiring mutant of E. coli, strain B, which was obtained from 

 Dr A. H. Doermann of the Oak Ridge National Laboratory. For this purpose the g-mediura was 

 supplemented with 5 y "C adenine per ml. The specific activity of phages grown on the purine 

 requiring strain was ten times that of phages grown similarly on B/i. 



EXPERIMENTAL 



The basic experimental procedure is unchanged throughout this study. It will, 

 therefore, suffice to describe one typical and simple experiment in detail ; more complex 

 experiments can then be introduced briefly and the results summarized in tables. 



Distribution of ^^P following infection of unlabelled Bfi with labelled T4r : 



Exponentially growing bacteria from an unlabelled broth culture were collected 

 by centrifugation and resuspensed in unlabelled broth at 37° C at a concentration of 

 2-io9 cells per ml. ^ap-labelled T4r phage was added at a ratio of 4.5 particles per bac- 

 terium and one and one-half minutes allowed for adsorption. The culture was then 

 chilled and centrifuged at 5000 g for 5 minutes. The supernatant was carefully siphoned 

 off and samples for phage assay and radioactivity measurement were taken. The pellet 

 was resuspended in broth at 37° C to give a suspension of infected bacteria with about 

 108 cells/ml; this figure was determined by a separate assay. Aeration was then started, 

 and 25 minutes after infection one volume of undiluted antibacterial serum was added 

 to 19 volumes of culture to prevent adsorption of progeny phage particles to bacterial 

 debris. The cooling and centrifugation retarded phage growth by about 8 minutes. 

 The assays and radioactivity measurements showed that in this experiment over 99.5% 

 of the phages and 96% of the input radioactivity were adsorbed on the bacteria. 



About 30 minutes after lysis, the culture was centrifuged at 5000 g for 5 minutes 

 to remove bacterial debris and then at 12,000 g for one hour to sediment the progeny 

 phage. The material collected during these centrifugations will be referred to as the 

 "low speed pellet" and the "high speed pellet", respectively. The latter was resuspended 

 in broth and again centrifuged at low speed to remove remaining bacterial debris. The 



TABLE I 



distribution of 32p AFTER INFECTION OF B/l WITH LABELLED T4r 



Growing bacteria were concentrated to 2-108 cells per ml and infected with labelled T4r at 

 a concentration of 9- 10* particles per ml. Following an adsorption period of 1 1/2 minutes the bacteria 

 were centrifuged for 4 minutes at 5000 g to remove unadsorbed phage and then resuspended m 

 nutrient broth at a concentration of li-io^ cells per ml. Approximately 99-5% of the phage and 

 96% of ^2P adsorbed to the bacteria. 



107 



