APPENDIX 487 



average burst size of the infected bacteria; however, it would 

 mean that a few plates would have 1 or 2 plaques due to free 

 phage particles This can be prevented by diluting the adsorption 

 tube into diluted anti-T2 serum at a concentration which would 

 neutralize 90 per cent of the free phage in 5 min. Then es- 

 sentially all plaques on the plates would represent phage liberated 

 from infected bacteria. 



a. Reagents. 1. Culture of strain B grown with aeration in 

 broth to 5 X 10^ cells/ml. 



2. Stock of T2 phage diluted in broth to 2 X 10^ /ml. 



3. Anti-T2 serum diluted 1 : 50 in broth. 

 All reagents are to be prewarmed to 37 °C. 



b. Protocol. 



The broth suspension containing about 0.7 bacterium/ 

 ml. is distributed in 0.5 ml. samples into 50 tubes; dis- 

 tribution must be completed before the end of the latent period. 

 The samples and remainder of the suspension in the sample flask 

 are incubated at 37 °C. until well after the end of the rise period to 

 insure that all bursts have taken place ; in the case of T2 for 40-50 

 min. from the beginning of the adsorption. A flask of 0.7 per 

 cent agar is melted in a boiling water bath and cooled to 45 °C., 

 then inoculated with plating bacteria washed from the surface of 

 a slant with a few milliliters of broth. With a warmed pipet, 

 2.5 ml. of inoculated 0.7 per cent agar is added to a sample tube 



