Multiple Mechanisms of Drug Resistance 157 



The existence of these was confirmed by the repHca plating 

 technique of Lederberg and Lederberg (1952), modified 

 shghtly by us. We were able to isolate two first-step mutants, 

 and from one of these two further mutants with successively 

 increased resistance. If we take as a measure of resistance 

 the concentration of proflavine which allows one cell in a 

 thousand to grow, then the increase in resistance of the first- 

 step mutants was X8 and Xl8. The two further steps from 

 the latter had further increases of resistance of x3-2 and 

 Xl-8. Thus, the increase in resistance of the third-step 

 mutant, compared with the sensitive strain, was by a factor 

 of about 100. 



From the same sensitive strain of Esch. coli, we have also 

 been able to isolate, in the same way, two first-step mutants 

 to chloramphenicol resistance. In addition, we have isolated 

 a proflavine-resistant mutant from the rough strain in which 

 we had carried out most of our induction experiments. 



There was a general relationship between resistance to 

 proflavine and resistance to chloramphenicol in all of these 

 mutant strains. In only one instance, between the first- and 

 second-step proflavine mutant, was there an increase in 

 resistance to one drug — proflavine — with no change in 

 resistance to the other drug. 



Transformation to proflavine resistance 



We attempted transformation of sensitive cells to resist- 

 ance by deoxyribonucleic acid (DNA) from resistant cells, pre- 

 pared with modifications according to the methods of Boivin 

 (1947), McCarty and Avery (1946) and Myers and Spizizen 

 (1954). 



The preparations all gave a positive Stumpf reaction, 

 indicating the presence of DNA. They were tested by dilut- 

 ing the preparation with broth and inoculating 0-1 ml. of 

 diluted 3-hour culture of the sensitive organism. After 

 24-48 hours incubation, the numbers of resistant and sensitive 

 cells were determined on plates with and without proflavine. 



Preparations made by toluene treatment according to the 



