242 R. Knox 



It was found that the apparent rate of development of 

 drug-resistant colonies in cultures of Myco. tuberculosis varied 

 both with the drug and with the medium. Some of these 

 differences have been described elsewhere (Knox and Wood- 

 roffe, 1957). With isoniazid in Kirchner semi-solid agar, pre- 

 sumptively resistant colonies of the drug-sensitive H 37 Rv 

 strain grew in decreasing numbers as the drug concentration 

 was increased. But colonies which had developed in tubes 

 w^hich initially contained quite high drug concentrations, up 

 to 10 [i.g./ml. or more, often appeared to be fully sensitive to 

 isoniazid when retested in subculture. Sometimes they 

 behaved as though they consisted of a "mixed" culture, 

 mainly drug-sensitive but containing more resistant cells 

 than a fully sensitive strain. This could be explained partly 

 by the fact that isoniazid decays rapidly in Kirchner medium 

 and any individual cells which might survive exposure to 

 isoniazid would be able to multiply when the drug decayed 

 below a certain level. Such individuals would clearly not be 

 true drug-resistant mutants. On the other hand, they must 

 be different in some way from the great majority of the 

 individuals in a drug-sensitive population, whether w^e seek 

 to explain them in terms of temporary adaptation to the 

 drug (Hinshelwood, 1946; Dean and Hinshelwood, 1953), 

 impermeability, "persistence" (Bigger, 1944) or clonal 

 variation (Yudkin, 1953). For brevity, such cells may be 

 described as pseudomutants. 



It was felt that more information would be obtained about 

 the distribution of resistant individuals in semi-solid medium 

 if cultures were allowed to grow for a few days before adding 

 drug to the medium. In this way, when a large inoculum was 

 used (say, 10^ cells/ml. of Dubos culture) innumerable minute 

 colonies could be seen with a hand lens. When serial dilutions 

 of isoniazid were added at this stage (say after 3 days of 

 incubation), the drug diffused rapidly through the medium 

 and these microcolonies showed no further increase in size. 

 However, after 2-4 weeks of incubation a few large isolated 

 colonies could be seen developing from among the innumerable 



