APPENDIX 455 



B of E. coli, when grown in nutrient broth with aeration, will 

 reach a final concentration of about 5 X 10'' cells/ml. but the 

 logarithmic phase of growth will cease at about 10'' cells/ml. 

 If such a culture is grown to a concentration of 5 X 10^-10^ 

 cells/ml. and is then inoculated with enough phage to reach a 

 final concentration of 2-4 X 10^ phage particles/ml. the bacteria 

 will all be infected nearly simultaneously, will all lyse within a 

 few hours, and a final titer of phage of between 10^° and 10^^ 

 infectious particles/ml. should be achieved. Since average 

 yield per infected bacterium is 100-300 phage particles, it might 

 be expected that lO'* infected bacteria/ml. should liberate at 

 least 10^1 phage particles/ml. This maximal yield is seldom 

 achieved in practice since large numbers of liberated phage 

 particles are permanently lost by readsorption to infected but 

 not yet lysed bacteria. [Phage will also adsorb to bacterial 

 debris. Bacterial lysates should therefore be centrifuged at 

 low speed to remove the debris and thereby prevent loss of 

 titer on standing. ] 



Similar results may be achieved with coliphages in the chemi- 

 cally defined media described on p. 446. It should be noted 

 that the medium may have to be reinforced with other sub- 

 stances such as salts, amino acids or other growth factors in 

 special instances. For example, coliphage T5 will not be 

 produced in this medium unless 0.001 A^ calcium ion is 

 added. ... In all instances better bacterial growth and higher 

 phage yields are obtained when air is actively bubbled through 

 the medium during the growth period. 



[A method of preparing genetically homogeneous phage 

 stocks has been described by Doermann and Hill (1953). 

 Young plaques (4-6 hr of incubation for the T even phages) 

 are used to inoculate young bacterial cultures (about 10^ 

 cells/ml.).] 



Another method of obtaining very high titer phage stocks has 

 been described by Hershey, Kalmanson, and Bronfenbrenner 

 (1943a). It appears simple, but actually requires consider- 

 able experience to obtain consistently good yields of phage. 



