260 



EVELYN M. WITKIN 



eliminate the nutrient buffered medium. The bac- 

 terial pellets in two of the tubes were resuspended 

 in 2 ml. of a 5% solution of sodium desoxycholate, 

 and the bacteria in the remaining two tubes were 

 resuspended in 2 ml. of distilled water to serve as 

 controls. In some experiments, a phosphate buffer 

 of pH 7.7 was used for the control, but distilled 

 water was found to be equally satisfactory. In 

 neither case was there any change in the number of 

 viable bacteria or in the background number during 

 the course of any experiment. The initial centrifu- 

 gation, in addition to eliminating the nutrient 



mutants and 50% nonmutants. Ten ml. of the 

 mixed culture was pipetted into each of two centri- 

 fuge tubes, centrifuged in the usual way, and the 

 pellets resuspended in 2 ml. of liquid, 5% desoxy- 

 cholate for the experimental tube and distilled water 

 for the control. The tubes were incubated for three 

 hours at 37° C. Thus, these cultures received the 

 same treatment throughout as the cultures used to 

 test for mutagenic activity. At the end of the 3-hour 

 incubation, the tubes were assayed to determine the 

 proportion of mutants in the control and experi- 

 mental cultures. This was done by plating the final 



Table 2. Zero-Point Mutations Induced by 3-Hour Exposure to 5% Sodium Desoxycholate 

 1 and 2 are duplicate control tubes. 3 and 4 are duplicate experimental tubes, a and b are independent dilutions of each tube. 



Experimental 



Assay 

 Sample per plate 

 Colony Counts 



No. bacteria per ml. 



No. B/r/1 mutants in samples of 0.1 ml. of 

 undiluted culture (8 samples per tube) 



No. B/r/1 mutants per 10 s living bacteria 



.05 ml. of 10 7 dilution 

 #1 #2 



122 a. 101 



86 89 



1.69X10" 



medium, served to concentrate the bacteria by a 

 factor of 5 (in some experiments by as much as a 

 factor of 20), thus facilitating the detection of 

 mutants among the survivors. 



The control and experimental tubes were in- 

 cubated for 3 hours at 37° C, and were then as- 

 sayed to determine the number of viable bacteria 

 per ml., and the number of B/r/1 mutants per 10 8 . 

 The results are shown in Table 2. These results 

 indicate the proportion of mutants among the sur- 

 vivors of the exposure to desoxycholate was in- 

 creased from 3.8 per 10 8 , the background number, 

 to 60.2 per 10 8 . Subtracting the background, the 

 increase in mutants per 10 s survivors is 56.4. 



Differential Survival test. In order to test the 

 possibility that the observed increase in the propor- 

 tion of mutants among the survivors of desoxycho- 

 late-treated bacteria is due to simple selection, the 

 following experiment was done. 



Cultures of B/r and B/r/1 were grown with 

 aeration in M-9 for 48 hours, and equal volumes of 

 the two cultures were combined in a single tube, to 

 give a mixed culture containing approximately 50% 



dilution of each culture on two series of plates, one 

 consisting of ordinary agar plates, and one consist- 

 ing of plates which had been coated previously with 

 a heavy suspension of bacteriophage Tl. The ratio 

 of the colony counts on phaged plates to that on 

 unphaged plates gives the proportion of mutants in 

 the mixture. The results are given in Table 3. In 

 the control culture, the number of colonies develop- 

 ing on phaged plates was 48.9% of the number 

 appearing on unphaged plates. In the experimental 

 culture, in which about 99% of the bacteria were 

 killed, the proportion of mutants among the sur- 

 vivors was 51.6%. Thus, it appears that mutants 

 and nonmutants are equally sensitive to the toxic 

 effects of sodium desoxycholate. These tests were 

 made on mixed cultures rather than on pure cultures 

 of the two strains in view of the possibility that 

 competition phenomena might be involved in selec- 

 tive killing. 



Tests of Mutant colonies. Ninety-four colonies 

 appearing on phaged plates after treatment with 

 desoxycholate were isolated and tested for re- 

 sistance to Tl and T2. All but three proved to be 



123 



