332 BACTERIOPHAGES 



mixed growth is a general property of bacteriophages. All 

 that is necessary to study the genetics of a phage strain is to 

 isolate an adequate number of variant strains with readily 

 recognizable genetically controlled characteristics. A descrip- 

 tion of various kinds of mutants that have been observed in 

 phages is given in Chapter XVI. Those characteristics that 

 have been most useful as genetic markers are host range and 

 plaque morphology, although others such as serological speci- 

 ficity, resistance to heat and ultraviolet light, and virulence have 

 been used. 



In mixed infection experiments it is desirable to infect each 

 bacterial cell nearly simultaneously with both kinds of phage 

 particles. This is because an interval between primary infection 

 and secondary infection may result in genetic exclusion of the 

 second phage as was discussed in Chapter XVII. In the case of 

 rapidly adsorbing phages such as T2 one can readily calculate 

 from the adsorption rate constant the bacterial concentration 

 and the input ratio of each phage strain to be used in the adsorp- 

 tion mixture so that, for instance, the bacterial cells will be 

 infected with a mean multiplicity of five phage particles of each 

 type within one minute. In order to insure mixed infection of 

 essentially all of the bacterial population it is customary to use 

 conditions giving an average multiplicity of five phage particles 

 of each type. Another method of avoiding exclusion of one of 

 the infecting phage strains is to carry out the adsorption step 

 using bacteria suspended in buffer (Benzer, 1 952) or in cyanide 

 broth (Benzer and Jacob, 1953). Under these conditions simul- 

 taneous mixed infection is achieved starting at the moment of 

 addition of broth (Chapters XV and XVII). Residual un- 

 adsorbed phage is removed by centrifugation or use of antiphage 

 serum and the infected bacteria are suitably diluted in broth and 

 permitted to lyse. The proportions of parental and recombinant 

 types are then determined by suitable assays either on lysates of 

 a few hundred infected cells or in single cell burst experiments. 

 The degree of linkage between genetic loci may then be calcu- 

 lated from the relative proportions of parental and recombinant 



