148 M. J. Thornley, J. Sinai and J. Yudkin 



of high resistance. These cells were cross-resistant to the other 

 drugs. We believe that they arise not by division of pre- 

 existing mutants but either by clonal variation or by direct 

 (Lamarckian) induction. 



We now investigated whether a variation in this phenotypic 

 adaptability might explain the continuous distribution of 

 resistance in a culture. We tested the possibility that the 

 variation might be a function of the various stages of the 

 division cycle. Synchronization of division was attempted by 

 cooling for a few minutes and then returning to 37°. The 

 degree of synchronization depended on the time from inocula- 

 tion of the culture, and the temperature and period of cooling. 

 In appropriate conditions, it was possible to obtain a reason- 

 able degree of synchronization, in which a burst of divisions 

 occurred during the first 10 minutes after cooling, followed 

 by cycles of 30 minutes with half of the divisions occurring 

 in the first 20 minutes and half in the next 10 minutes 

 (Fig. 4). 



The cultures with synchronized division cycles also showed 

 cyles of fluctuation in resistance. These cycles lasted for about 

 20 minutes (Fig. 5). The variation in resistance during the 

 cycles was demonstrated by taking samples at intervals and 

 testing on plates with varying concentrations of proflavine. 

 The variation showed itself in three ways. First, with appro- 

 priate concentrations of about 6 jxg./ml. of drug, there was a 

 great difference at different times in the number of cells able 

 to produce colonies; this difference was as high as 1,000-fold, 

 compared with the maximum of less than tenfold in samples 

 from a non-synchronized culture. Second, the differences at 

 different times became progressively less with lower concen- 

 trations of drug on the testing plates, so that when the con- 

 centration was about 4 [xg./ml. proflavine, the same number 

 of colonies grew throughout the cycles. This concentration 

 thus appears to be the inherent resistance of the cells. Third, 

 the period out of the cycles during which the cells were 

 able to grow on proflavine plates was longer with the lower 

 concentrations of drug. 



