APPENDIX 521 



determine the amount of T4 phage produced/infected bacterium 

 up to the time of sampHng. 



Using this technique, there is no detectable intracellular T4 

 phage present from the earliest samples taken at about 9 min. 

 after infection until about 14 min. after infection, at which time 

 about 1 phage particle/bacterium is demonstrable. The num- 

 ber of intracellular phage particles that can be liberated per in- 

 fected cell then increases at an approximately linear rate until 

 the normal burst size is reached at about 28-30 min. after infec- 

 tion. This maximal yield is reached several minutes before the 

 end of the normal rise period. 



It is obvious that the effect of many other cell poisons on the 

 growth of phage could be studied in this manner. For instance, 

 5-methyltryptophan, shown by Cohen and Anderson (1946) 

 to prevent phage growth, was used by Doermann with results 

 very similar to those with KCN. This inhibitor, however, does 

 not stop synthesis promptly in host cells able to synthesize their 

 own tryptophan so that the mutant B/l,tr was used as the host 

 cell in experiments involving 5-methyltryptophan. 



[2. Premature lysis of infected cells by chloroform. A simple 

 technique for obtaining premature lysis of phage infected cells 

 has been described by Sechaud and Kellenberger (1956). They 

 found that chloroform eflfects a rapid lysis of infected cells when 

 they contain at least one infectious unit per cell. Lysis of such 

 cells is complete two minutes after exposure to chloroform. To 

 follow the intracellular development of infectious phage the 

 infected culture was diluted to contain around 3X10"* infected 

 bacteria per ml. At intervals during the latent period, samples 

 were taken and diluted further in a diluent containing chloro- 

 form (3-6 drops of chloroform per 5 ml. of diluent). These sus- 

 pensions were shaken vigorously for several seconds and 15-30 

 min. later assayed for phage. The action of chloroform was found 

 to be independent of the media used (i.e., tryptone, M-9, phos- 

 phate buffer). It was noted, however, that in the case of T2 



