234 B. Gyorffy and I. Kallay 



colonies survived the same concentrations of streptomycin, on 

 retest, in a high percentage ; the cells of some of these colonies even 

 extended their survival range beyond that of the parental popula- 

 tion, in some cases up to 50-100 [ig. streptomycin/ml. Nevertheless, 

 on subsequent retest the colonies formed at higher concentrations 

 of streptomycin showed many "normal overlaps"; and it is by no 

 means unlikely that their occurrence can be explained by the 

 physiological-biochemical heterogeneity of the cell population 

 within a colony, as suggested by Sevag (1955). 



In general, we observed that only a small fraction of the isolates 

 of colonies formed on plates containing 50 and 80 [ig. streptomycin/ 

 ml. consisted of cells which not only gave survival at above 16 and 

 24 yig. streptomycin/ml., respectively, but which also tended to 

 keep this high resistance unaltered during a number of transfers. 

 It seemed likely, however, that this small proportion of greater 

 resistance originated from the selection of second- step variants 

 which had arisen on the plates on retest (Abraham, 1953). 



In summary, variable levels of resistance were obtained in a single 

 exposure to streptomycin, and differences were observed between 

 the independent isolates from the same plate. The survival dis- 

 tributions in re tests were highly variable, and we found no adjust- 

 ment to the concentration at which colonies w^ere isolated (Barer, 

 1951; Gibson and Gibson, 1951; Eagle, Fleischman and Levy, 1952). 

 The occurrence of "normal overlaps" of an unstable, readily rever- 

 sible nature was very frequent but stable variants with low levels 

 of resistance also developed. 



Serial transfers were carried out with different strains of Serratia 

 marcescens. The discrete steps resulting in progressively higher 

 levels of survival were clearly evident. The small size of inocula 

 (10^ — 10* cells) used in the transfers excluded the presence of pre- 

 existent resistant variants. 



The average range of the first steps was less variable than that 

 of the succeeding ones. In some instances, resistance to 100 \Lg. 

 streptomycin/ml. was attained by three successive steps; in others, 

 the number of transfers separating the steps differed, and flat 

 plateaus also occurred (Oakberg and Luria, 1947). No quantitation 

 was made as proposed by Treffers (1956). 



This stepwise development of higher resistance was also apparent 

 in serial transfers on solid medium, when the isolated single colonies 

 were repeatedly retested. The average range of the single steps 

 was approximately the same as that obtained in transfers in liquid 

 medium. The outgrowth of second-step variants appearing in 

 microcolonies on a background region on plates containing strep- 

 tomycin in concentrations above 16 (i,g./ml. was very definite in the 



