Drug Resistance in Myco. tuberculosis 243 



microcolonies, even in tubes which originally contained up to 

 50 [xg./nil. Thus, although it was not possible to follow the 

 fate of individual cells, it was possible to study the fate of 

 individual small clones in such a population of microcolonies. 

 Single colonies appearing after 2-4 weeks in tubes to which the 

 drug was added after 3 days of incubation were found to be 

 highly resistant on subculture, whereas the pseudomutants 

 mentioned above were much less numerous. 



Thus, by incubating cultures of drug-resistant cells for 

 3 days so that microcolonies developed before exposure to 

 the drug, drug-resistant mutants were much more easily 

 obtained. This phenomenon was unlikely to be simply the 

 effect of inoculum size ; if it were it would be reasonable to 

 expect a corresponding increase in the proportion of pseudo- 

 mutants. This did not occur. It is more likely to be related 

 to the different physiological state of the cultures. When a 

 mature culture is inoculated into drug-containing medium it 

 is possible that some of the cells might survive exposure to 

 the drug because they were not metabolically active at the 

 moment of inoculation, and in the new drug-containing 

 medium they remain in a dormant state until the drug decays. 

 On the other hand, when the drug is added to actively divid- 

 ing cells without their transference to new medium it is likely 

 that more of the cells will be vulnerable to the drug and the 

 only survivors will be true drug-resistant mutants. This 

 explanation however is not entirely satisfactory, since pseudo- 

 mutants did not appear, even in tubes to which the drug was 

 added immediately, if the inoculum w^as increased five- to 

 tenfold, whereas true mutants did appear, in increased 

 numbers, as in the tubes to which drug was added after 3 days 

 of incubation. From this, it seems possible that a large 

 population of normal sensitive cells exerts a suppressive 

 effect on the development of pseudomutants. 



It might perhaps be said that by the time we have taken 

 into account factors such as inoculum size, the physiological 

 state of the culture, the composition of the medium and the 

 decay of the drug in it, we have reached a very complex 



