296 BACTERIAL VIRUSES. II 



appreciable amounts of complex products, radioactive and otherwise, liber- 

 ated by lysed cells. Some of the chemical phenomena and basic biological 

 aspects of this system have been described in Paper I (1). 



The yield of virus from infected cells under conditions of multiple infec- 

 tion in the aerated F medium at 37° was studied at concentrations of 5 X 

 10^ 10^ 2 X 10^ and 5 X 10^ bacteria per cc. It was found that with both 

 T2 and T4 maximal titers were obtained with 2 X lO'^ bacteria per cc. in 

 about 4 to 6 hours. After this time, titers generally decreased markedly, 

 and to a greater extent in T2 lysates than in T4 lysates. This was due 

 probably to a combination of several factors, readsorption to cellular debris, 

 spontaneous thermal inactivation, surface denaturation, etc. In view of 

 this marked loss of titer, it was considered desirable to prepare virus lysates 

 by a single generation in infected cells for a 6 hour period, to minimize the 

 destruction of virus activity. 



Distribution of DMA and Protein-Bound P in To and Ti Lysates — Two 

 types of experiments were done. (1) Bacteria were grown in media contain- 

 ing radioactive P, washed several times, and infected in media containing 

 non-radioactive P; (2) bacteria were grown in media free of P^^ and in- 

 fected in the presence of P^-. 



The following typical control experiments were performed to indicate 

 whether (1) the experimental conditions employed would permit the isola- 

 tion of sufficient virus for analytical purposes, (2) virus isolated by these 

 procedures would be very low in inorganic P and otherwise possess the 

 proper chemical properties, (3) the newly synthesized DNA appeared in the 

 virus fraction. Bacteria were grown to 2 X 10* per cc. in F medium. Two 

 125 cc. ahquots were sedimented and the bacteria washed twice with 0.85 

 per cent NaCl. Each aliquot was resuspended in 125 cc. portions of F me- 

 dium to which were added an adsorption cofactor, 12 cc. of 5 X 10~^ m tryp- 

 tophan ill F (2). To these bacterial suspensions (A and C) were added 

 small volumes of purified concentrates of T4r+-F and T2r+-F to give cultures 

 T4A and T2C containing 6 X 10* virus particles per cc. The virus concen- 

 trates were also added to unwashed bacterial cultures (B and D) in F me- 

 dium at the same concentrations to yield T4B and T2D; B contained 5 X 

 10"^ M tryptophan. The four infected cultures were assayed periodically; 

 the titers followed the course described previously. 



After 5 hours at 37°, the T2 and T4 lysates were stored for 13 hours at 4°. 

 Very little inactivation occurred; the final titers were T4A 2.0 X 10^", 

 T4B 4.0 X 1010, T2C 1.5 X 101", T2D 1.5 X 1010 active virus particles per 

 cc. The lysates were analyzed for total protein-bound P and DNA (1). 

 They were sedimented at 4000 r.p.m. for 30 minutes and the sediments were 

 washed twice with cold 0.85 per cent NaCl. The supernatant fluids were 

 sedimented at 10,000 r.p.m. for 2 hours, and the supernatant fluids from 



117 



