In all of ten strains of H. influenzae examined prior to exposure to streptomycin a very small fraction of the bacteria formed colonies in the presence of 1,000 units of streptomycin per cc. These variant organisms possess the characteristics of bacterial mutants.

1. Different independent cultures of the same strain on different occasions, and even cultures seeded in a single test with the same inoculum, show marked variation in prevalence of resistant variants in populations of comparable size. This variation is just as great in genetically homogeneous culture sources as in heterogeneous sources. Evidence is presented for the continuous random occurrence of these resistant organisms.

2. The rate of occurrence of the resistant members is not significantly different for the ten strains studied; it varies from 2.6 x 10–11 to 7.0 x 10–11 per bacterium per bacterial generation. The results of five different tests on the same strain show comparable variation, 2.9 x 10–11 to 5.3 x 10–11. This low rate contributes evidence consistent with the mutation hypothesis.

3. Variants exhibiting resistance to 1,000 units of streptomycin per cc. transmit this trait unchanged in degree through many generations. The results obtained after twelve or more subcultures in streptomycin-free media suggest that in a portion of the colonies resistant to streptomycin an additional trait, differing from those exhibited by the parent culture, is associated; the nutritional requirements of these cultures are different.

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