APPENDIX 495 



Another important characteristic of an indicator strain that 

 must be determined is the generation time. As noted in Table 

 XXIV, the mutants B/3,4 and B/3,4,7 have a much longer gener- 

 ation time than do the other mutants and the parent strain. 

 This means that with these mutants, whether grown in broth or in 

 plates, the turbidity will develop more slowly. To compensate 

 for this slower growth, a larger initial inoculum must be used. 



Strain B/3,4,7 is a relatively "rough" strain in comparison 

 with the other mutants, which means that suspensions of this 

 strain in broth tend to agglutinate spontaneously and settle out of 

 solution. Also, when this mutant is grown on a slant, it is dif- 

 ficult to make a uniform suspension because the organisms tend 

 to stick together in clumps. Unless special pains are taken to 

 disperse such a suspension, by repeated blowing of the suspen- 

 sion through a fine orifice such as a pipet tip, plates inoculated 

 with B 3.4,7 by the agar layer method will present a granular 

 and uneven appearance of the bacterial growth. Another 

 method of dispersing this mutant is to wash the growth from the 

 2 per cent agar slant with broth and aerate the broth suspension 

 for 20 min. at 37 ° C. 



Also, as noted previously, certain phage-resistant mutants lack 

 the ability to synthesize certain amino acids, so that these mu- 

 tants cannot be used as indicator strains in chemically defined 

 media unless the amino acids concerned are included in the me- 

 dium. However, this nutritional deficiency in itself lends a cer 

 tain versatility to the mutant and permits the investigator to in- 

 terrupt phage synthesis during the latent period by simply dilut- 

 ing the infected bacteria in a medium free from the required 

 growth factor. 



Plating of Mixed Viruses with Mixed Indicator Strains 



This technique has led directly to important discoveries in the 

 field of bacterial viruses. A specific example of the technique 

 will first be described, then 2 applications of the technique will be 

 discussed which have resulted in new principles. 



Procedure. A mixture of equal concentrations of phages Tl and 



