A. D. HERSHEY AND MARTHA CHASE 49 



cent of the phage phosphorus, half of which is given up without any mechan- 

 ical agitation. 



3. The treatment does ngt cause any appreciable inactivation of intracellu- 

 lar phage. 



4. These facts show that the bulk of the phage sulfur remains at the cell 

 surface during infection, and takes no part in the multiplication of intracellu- 

 lar phage. The bulk of the phage DNA, on the other hand, enters the cell soon 

 after adsorption of phage to bacteria. 



Transfer of Sulfur and Phosphorus from Parental Phage to Progeny. — We 

 have concluded above that the bulk of the sulfur-containing protein of the 

 resting phage particle takes no part in the multiplication of phage, and in 

 fact does not enter the cell. It follows that little or no sulfur should be trans- 

 ferred from parental phage to progeny. The experiments described below show 

 that this expectation is correct, and that the maximal transfer is of the order 

 1 per cent 



Bacteria were grown in glycerol-lactate medium overnight and subcultured 

 in the same medium for 2 hours at 37°C. with aeration, the size of seeding 

 being adjusted nephelometrically to yield 2 X 10^ cells per ml. in the sub- 

 culture. These bacteria were sedimented, resuspended in adsorption medium 

 at a concentration of 10^ cells per ml., and infected with S^^-labeled phage 

 T2. After 5 minutes at 37°C., the suspension was diluted with 2 volumes of 

 water and resedimented to remove unadsorbed phage (5 to 10 per cent by 

 titer) and S^^ (about 15 per cent). The cells were next suspended in glycerol- 

 lactate medium at a concentration of 2 X 10^ per ml. and aerated at 37°C. 

 Growth of phage was terminated at the desired time by adding in rapid suc- 

 cession 0.02 mM HCN and 2 X 10" UV-killed phage per ml. of culture. The 

 cyanide stops the maturation of intracellular phage (Doermann, 1948), and 

 the UV-killed phage minimizes losses of phage progeny by adsorption to bac- 

 terial debris, and promotes the lysis of bacteria (Maal0e and Watson, 1951). 

 As mentioned in another connection, and also noted in these experiments, 

 the lysing phage must be closely related to the phage undergoing multiplica- 

 tion {e.g., T2H, its h mutant, or T2L, but not T4 or T6, in this instance) in 

 order to prevent inactivation of progeny by adsorption to bacterial debris. 



To obtain what we shall call the maximal yield of phage, the lysing phage 

 was added 25 minutes after placing the infected cells in the culture medium, 

 and the cyanide was added at the end of the 2nd hour. Under these condi- 

 tions, lysis of infected cells occurs rather slowly. 



Aeration was interrupted when the cyanide was added, and the cultures 

 were left overnight at 37°C. The lysates were then fractionated by centrifuga- 

 tion into an initial low speed sediment (2500 G for 20 minutes), a high speed 

 supernatant (12,000 G for 30 minutes), a second low speed sediment obtained 

 by recentrifuging in adsorption medium the resuspended high speed sediment, 

 and the clarified high speed sediment. 



97 



