48 A. D. HERSHEY AND RAQUEL ROTMAN 



of this "mixed indicator effect," similar to that observed by Delbruck and 

 Bailey (1946), remains obscure. It does not appear to be the result of segre- 

 gation of multiple h factors, because no intermediate genetic types can be 

 found in crosses between h and wild type. In h)>ir crosses, it affects equally 

 counts of parental and recombinant virus. 



The validity of the mixed indicator count itself rests on three lines of evi- 

 dence. First, plaques sampled and retested always conform to the genetic type 

 deduced from inspection. Second, the ratio of // to k^ virus in the yield following 

 mixed infection, measured by the mixed indicator count, is the same as the 

 corresponding ratio of infecting viruses, with minor exceptions to be mentioned 

 below. Third, the yield of the two recombinants in either /fX?* or hr X wild type 

 crosses is very nearly equal, and the slight bias (actually of doubtful signifi- 

 cance) correlates not with the h, but with the r pair of alleles. 



For these reasons, and because of its statistical efficiency, only the mixed 

 indicator plating is used for counting single bursts. The reproducibility of the 

 counts so obtained may be judged from the examples shown in table 1. 



Table 1 



Mixed Indicator Counh of Viral Types in the First Eight 

 Bursts Examined from the Cross hXr7. 



The counts shown are for 3 aliquots of 0.3 ml from each tube. The volumes are not measured 

 very accurately, owing to the effort made to plate the entire sample. In computing results, it was 

 assumed that totals of counts for each tube represented 90 percent of the actual virus content, 

 10 percent of the fluid being lost mechanically. 



TUBE /z+r"*" hr'^ h^r hr tube h'^r'^ hr^ h'^r hr 



35 



10 18 17 95 13 36 



26 9 84 51 8 38 



34 2 20 58 17 40 



The nutrient broth referred to above is composed of Bacto-peptone 10 g, 

 Bacto-beef extract 3 g, NaCl 5 g, glucose 1 g, per liter distilled water. The 

 pH (unadjusted) is about 6.8. An occasional batch of broth prepared according 

 to this formula proves unfavorable to the adsorption of the virus. 



Nutrient agar plates are poured with a minimum of 35 ml per 9 cm Petri 

 dish of Bacto-agar 10 g, Bacto-Tryptose 10 g, NaCl 8 g, sodium citrate crys- 

 tals 2 g, glucose 1 g, per liter distilled water. The pH of the agar is adjusted 

 to 6.8 to 7.0. Contrary to an early experience, we have recently found that 

 Bacto-Tryptone can be substituted for Tryptose. Poured plates are stored in 



155 



