APPENDIX 469 



2. Deter mination of Number of Infected Bacteria 

 This method depends on the assumption that each adsorbed 

 phage particle resuhs in an infected bacterium which will 

 produce a plaque when plated. The free phage is eliminated 

 by diluting the adsorption mixture at appropriate intervals into 

 enough antiphage serum to inactivate at least 99 per cent of the 

 free phage during the period of antiserum action. In the case 

 of the coliphages of the T system, antiphage serum has no effect 

 on the ability of infected bacteria to produce plaques provided 

 the antiserum is so diluted that its concentration on the plate 

 does not affect plaque formation (Delbriick, 1945b). In the 

 case of other phages it is well to check this before using the 

 method. The ratio of phage to bacteria in the adsorption 

 mixture must be so low that the probability of a single bacterium 

 adsorbing more than 1 phage particle is negligible. For prac- 

 tical purposes the ratio of adsorbed phage to total bacterial count, 

 the multiplicity of infection, should be 0.1 or less. Also, the pro- 

 portion of bacteria which are dead or otherwise incapable of 

 forming a plaque after adsorption of a phage particle should be 

 less than 5 per cent. If these qualifications are met the number 

 of infected bacteria present at any time should give directly the 

 number of phage particles which have been adsorbed. 



Procedure. Bring the bacterial suspension to temperature 

 equilibrium and remove a sample for bacterial assay. Add 

 the required amount of phage stock, mix well, remove a sample, 

 dilute, and assay for phage. If the various qualifications listed 

 in the preceding paragraph are met, this assay will give directly 

 the total number of phage particles put into the adsorption tube 

 since free phage and infected bacteria will both produce plaques. 

 At intervals during adsorption, samples are removed and diluted 

 1 : 10 in antiphage serum at an appropriate dilution, as calculated 

 from the A' value, to inactivate 99 per cent of the free phage in a 

 convenient time, e.g., 5 min. After this time the sample is 

 further diluted to dilute out the antiserum and plated to de- 

 termine the number of infected bacteria. The entire procedure 

 must be completed within the latent period of phage growth 



