STAGES IN PHAGE MULTIPLICATION 183 



Fisher (1952) in quantitative studies of T2-infected bacteria 

 which were prematurely lysed by the decompression technique at 

 different times during the latent period. The lysates were 

 mixed with polystyrene latex particles and sprayed on collodion 

 films for electron micrography, thus permitting counts of the 

 number of particles per infected bacterium. Doughnuts are 

 first detected at about the 9th minute of the latent period. By 

 the 12th minute, when mature phage first appears, the doughnuts 

 number about 15 per bacterium. At the 21st minute their num- 

 ber reaches a inaximum of 30 per cell, while mature phage par- 

 ticles continue to increase, numbering 60 per cell at 25 minutes. 

 It seems reasonable to conclude that the doughnut represents a 

 stage in the assembly of phage particles. The relationship of 

 phage nucleic acid to these structures is not clear. It is possible 

 that the incomplete forms contain nucleic acid that is lost during 

 the preparative manipulations. 



Relatively pure preparations of doughnuts can be obtained by 

 the use of proflavine, which specifically inhibits final steps in the 

 maturation of phage particles (DeMars, Luria, Fisher, and 

 Levinthal, 1953). The incomplete particles can be readily con- 

 centrated and purified by high speed centrifugation. On ex- 

 amination in the electron microscope they are indistinguishable 

 from doughnuts observed by other means and are essentially free 

 of tailed particles. The formation of doughnuts during early 

 stages of viral growth occurs at the same rate with or without 

 proflavine. 



The preparation of purified concentrates of doughnuts ob- 

 tained by the proflavine technique permitted a study of their 

 serological properties. They fix complement with antiphage 

 serum with about half the efficiency per particle of mature phage. 

 They are precipitated by antiphage serum but do not adsorb 

 phage-neutralizing antibodies from the serum. Purified dough- 

 nuts were used for the immunization of rabbits by Lanni and 

 Lanni (1953). The resulting sera gave very strong complement 

 fixation reactions with both doughnuts and matui^e phage par- 

 ticles, but contained very little neutralizing antibody. From 



