APPENDIX 479 



Example: Concentration of strain B in the adsorption tube was 5 X 10''/ 

 ml. Phage input resulted in an initial concentration of T2 in the adsorption 

 tube of 20 X lOVml-j or an input ratio of 4 T2 :B. Adsorption was for 5 min. 

 at 37° C. The total infective centers as determined by assay of the adsorption 

 tube at the end of adsorption period was 9 X lOVml. The number of infected 

 bacteria as determined by assay of the serum tube was 5 X 10'^, which agrees 

 with the input of bacteria, as it should with such a high multiplicity of infection. 

 Free phage is then the difference: total infective centers, 9 X 10^, less in- 

 fected bacteria. 5 X 10'^, equals free phage, 4 X 10''. Direct determination 

 of free phage in the supernatant of a centrifuge tube from a dilution of the ad- 

 sorption tube gave 4 X 10'', agreeing with the calculated value of free phage. 

 Total phage input, 20 X 10^, minus the unadsorbed phage, 4 X 10'^, equals 

 adsorbed phage, 16 X 10^. Since the 16 X 10^ phage particles were actually 

 adsorbed on 5X10^ bacteria, average multiplicity of infection was 16/5 = 3.2 

 phage particles per bacterium. 



Since 16 X 10^ phage particles of a total of 20 X 10^ were actually adsorbed, 

 per cent adsorption was 16/20 X 100 = 80 per cent during the 5 min. ad- 

 sorption period. By means of the Poisson formula, the proportion of bacteria 

 not infected for a multiplicity of 3.2 is P(0) = e~'^ = e"^'- = 0.041, which 

 means that 4 per cent of the bacteria should not have been infected. Hence 

 the count of infected bacteria as determined by assay of the serum tube should 

 have been 4 per cent less than the assay of the bacterial input. This difTerence 

 is within the experimental errors of the assays and was not detected. 



The actual number of uninfected bacteria could be deter- 

 mined by plating an appropriate dilution of the serum tube by 

 the spreading technique on an agar plate coated with anti-T2 

 serum (described in method 3, p. 470) and counting the bacterial 

 colonies. The actual number as determined by this method 

 will be slightly higher than that calculated on the basis of the 

 Poisson formula for the reason discussed on p. 472. 



The dilution factor in going from the adsorption tube to F.G.T. 

 is chosen so that a reasonable number of plaques, usually 100- 

 200, will be present on the plates during the latent period. The 

 dilution factor in going from F.G.T. to S.G.T. is chosen on the 

 basis of the expected increase in plaque count at the end of the 

 rise period so that the plates from S.G.T. will also contain 

 100-200 plaques. Considerable latitude in this respect can be 

 obtained by varying the volume of the sample taken from 

 F.G.T. and S.G.T. for plating. When the agar layer technique 



