Drug Resistance in Serratia marcescens 235 



series of replica plates. The majority of the cells of colonies isolated 

 at increased concentrations were resistant to the same range of 

 concentrations, and the small fraction surviving on plates with 

 considerably higher concentrations represented the further-step 

 variants. 



In general, the higher the concentration to which the organism 

 was exposed, the greater was the resistance of a fraction of the 

 emergent colony; while colonies surviving concentrations of 80- 

 100 [xg. streptomycin/ml. in the repeated retests, consisted mainly 

 of fully resistant cells. In some instances, variants resistant to high 

 levels of streptomycin (100 or 1000 (i-g./ml.) were selected from large 

 populations of sensitive cells, by plating, and were supposed to have 

 originated by a single step (Newcombe and Hawirko, 1949). 



We do not believe that even a low degree of stable resistance to 

 streptomycin was acquired by physiological adaptation alone, as 

 claimed by Gibson and Gibson (1951). Adaptive processes may play 

 a role in the phenotypie manifestation of the resistant variant, and 

 unquestionably they favour colony formation of persistors. Thus, 

 the simultaneous appearance of "normal overlaps" and of resistants 

 with a range of phenotypie variability always tends to obscure the 

 stepwise discontinuity, and simulates a "continuous spectrum" 

 which could be used to support the theory of physiological adapta- 

 tion (Eagle, Fleischman and Levy, 1952). 



"Repetitive training", i.e. repeated subculturing in subthreshold 

 concentrations (0-1 and 4 \ig. streptomycin/ml.) is now in progress. 

 Results obtained so far are that, in the series where a concentration 

 of • 1 \Lg. streptomycin/ml. was used, the survival fraction remained 

 unaltered after 20 transfers; whereas in the series where 4 {xg. 

 streptomycin/ml. was used, a moderate increase in the fractions 

 surviving concentrations of 16-50 [j,g./ml. w^as obtained already after 

 20 transfers. This is in agreement with results obtained by Eagle, 

 Fleischman and Levy (1952), Gibson and Gibson (1951) and Akiba 

 (1955); (see however EngUsh and McCoy, 1951). But, as yet, we 

 can only speculate as to whether enforced phenotypie modification 

 or emergence of step variants occurred during the prolonged 

 subculture. 



The mutational origin of streptomycin-resistant variants of 

 Serratia marcescens was indirectly demonstrated by the "fluctuation 

 test" of Luria and Delbriick (1943). We are aware of the possibili- 

 ties of error in analysis by means of this test; variability between 

 independent cultures is not in itself conclusive evidence of mutation, 

 and the results should be treated with reserve (Barer, 1951 ; Hinshel- 

 wood, 1952). In repeated experiments, a highly significant variation 

 was observed in the number of resistant variants among independent 



