ENUMERATION OF BACTERIOPHAGE PARTICLES 33 



has been exploited for the analysis of vegetative phage within 

 infected bacteria. The nucleic acid of this serological group of 

 phages contains the unique base hydroxymethylcytosine (Wyatt 

 and Cohen, 1953). This base may be readily distinguished 

 from the other bases present in bacterial or phage nucleic acids 

 by paper chromatography of acid hydrolyzates of the nucleic acid 

 fraction, and may be determined quantitatively. It is there- 

 fore possible to distinguish the DNA of phage T2 from host cell 

 DNA and to determine quantitatively the total amount of 

 phage DNA present per infected cell. The amount of mature 

 phage DNA per cell can be calculated from plaque counts on 

 prematurely lysed infected bacteria. The amount of phage 

 DNA present as vegetative phage in the infected bacteria can be 

 calculated by subtracting the mature phage DNA from the total 

 phage DNA. This method has been used by Hershcy, Dixon, 

 and Chase (1953) to study the intracellular multiplication of 

 phage T2. 



Another method for assay of intracellular phage substances is 

 based on the high specificity of phage antigens. The antibody 

 that neutralizes phage infectivity reacts with a protein situated 

 on the phage tail but fails to react with any substances present 

 in uninfected bacteria. The relative amount of neutralizing 

 antibody present in a sample of serum can be accurately deter- 

 mined (see Chapter VIII). Phage tail antigen will combine 

 with phage neutralizing antibodies and so decrease the neutraliz- 

 ing ability of the serum. This "serum-blocking power" of 

 phage antigens can be calibrated in terms of phage particle 

 equivalents and used as a measure of the total amount of phage 

 tail antigen present in infected bacteria when they are prema- 

 turely lysed at various times during the latent period. The 

 amount of phage tail antigen already included in mature phage 

 particles can be calculated from the mean number of mature 

 phage particles present and the known antigen content per 

 phage particle. The difference between the content of total tail 

 antigen and mature phage tail antigen gives the phage tail 

 antigen not yet built into mature phage at the time of prema- 



