66 M. DEMEREC [VOL. 56 



mechanism of this origin: (1) that resistance was induced by some interaction 

 between the antibiotic and the bacteria when they were together on the plate; 

 and (2) that it originated independently of the antibiotic, by mutation, the anti- 

 biotic acting only as a selective agent in the isolation of mutants by destruction 

 of sensitive bacteria. 



A relatively simple method was available for distinguishing experimentally 

 between these two possibilities. It was devised by Luria and Delbruck (1943) 

 in a study of the origin of bacterial resistance to bacteriophages, and was adapted 

 for work with antibiotics in our study of the origin of resistance to penicillin 

 (Demerec, 1945a). Following is a brief description of the method as used in 

 our experiments. 



From a single culture of bacteria, small inocula (50 to 300 bacteria) were taken 

 and used to start 21 or more independent broth cultures. These were incubated 

 for 24 hours, or until growth had reached the saturation point. During incu- 

 bation the number of bacteria (in experiments using E. coli or S. aureus) in- 

 creased to about 2 X 10 8 per ml. From the same culture that served as the 

 source of the inocula, 10 samples of bacteria of the same size as the inocula were 

 plated on a culture medium containing the same concentration of antibiotic as 

 was used in the tests, in order to determine if any resistant bacteria were present 

 in these samples. The concentration of the antibiotic had to be high enough so 

 that there were no survivors among the small number of bacteria plated — in 

 other words, that there were no bacteria resistant to that concentration in the 

 inocula used to start cultures. It followed, then, that all resistant bacteria found 

 in full-grown cultures had necessarily originated in these cultures during the 

 period when the number of bacteria increased from 50 to 300 to about 2 X 10 8 

 per ml. 



Next, from 20 of the broth cultures, samples of 0.1 ml were taken and plated 

 on petri dishes containing the same concentration of the antibiotic (0.064 units 

 of penicillin, or 5 units of streptomycin per ml of medium). Fifteen 0.1 -ml samples 

 were plated from the twenty-first tube. Thus two sets of plates were obtained: 

 one set of 20 in which each plate had bacteria from a different culture, and another 

 set of 15 all having bacteria from the same culture. The plates were incubated 

 for 24 hours, or for a longer period if the growth of colonies was slow. After in- 

 cubation the number of colonies on each plate was determined. These colonies 

 represented resistant bacteria that had been present in the sample plated. 



On both sets of plates in this test the experimental conditions were similar, 

 like numbers of bacteria (about 2 X 10 7 ) having been plated onto nutrient agar 

 containing identical concentrations of antibiotic. Therefore, if resistance were 

 induced through interaction between the bacteria and the antibiotic when they 

 were in contact with each other, approximately similar numbers of resistant 

 bacteria would presumably be obtained on all the plates, regardless of the origin 

 of the bacterial samples ; the variation between plates should not exceed random 

 variability. In the event that the origin of resistance is mutational, on the other 

 hand, similar numbers of resistant colonies would be obtained only among the 

 platings taken from the same culture, since these represented repeated tests of 



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