258 



EVELYN M. WITK1N 



tube in buffer or distilled water to serve as the 

 control. The volume of liquid in each tube was the 

 same. The tubes were incubated for the arbitrarily 

 chosen time, usually 2 or 3 hours, at 37° C, and 

 then assayed to determine the number of bacteria 

 per ml. in each of the tubes. Sometimes a second 

 toxicity test was required, using dilutions between 

 two of the original steps, to determine the proper 

 concentration more precisely. 



(3) Test for mutagenic activity 

 Knowing the concentration of the chemical that 

 will kill 99% of the bacteria in 2 or 3 hours, a pre- 

 liminary test is run to determine the effectiveness 

 of the chemical under these conditions in inducing 

 zero point mutations. Since sample experiments 

 will be described in detail in the experimental sec- 

 tion, only a general outline of the procedure will be 

 given here: 



A sample of a stock low-background culture is 

 divided into two equal parts, centrifuged, and the 

 nutrient medium decanted. One pellet is resuspended 

 in the proper concentration of the chemical, and the 

 other in distilled water or buffer as a control. The 

 tubes are incubated for the length of time required 

 to kill 99% of the bacteria in the experimental tube, 

 and then the cultures are assayed to determine the 

 number of viable bacteria per ml., and the number 

 of B/r/1 mutants per 10 8 bacteria. The number of 

 mutants per 10 8 bacteria in the control, the back- 

 ground number, is subtracted from the number of 

 mutants per 10 8 survivors in the treated culture. As 

 the background number of a given culture is ex- 

 tremely constant in independent determinations, 

 seldom differing by more than 2 or 3 mutants per 

 10 8 , an increase of 10 mutants per 10 8 over the 

 background number is considered to be an indica- 

 tion of a positive effect. 



Since the effect of chemicals on zero point muta- 

 tions is under consideration, it is important to 

 establish conditions under which division of bac- 

 teria during treatment will not occur. The use of a 

 non-nutrient medium, and a concentration of bac- 

 teria at least as great as, and often 10 to 20 times 

 greater than, the concentration reached after maxi- 

 mal growth, were among the precautions taken to 

 prevent division during treatment. In addition, the 

 exposure time was usually well below the normal 

 lag, even for small inocula in fresh nutrient medium. 

 Microscopic total counts revealed no measurable 

 increase in cell number under the conditions de- 

 scribed, over periods as long as 72 hours. 



(4) Differential survival test 

 Whether the preliminary test for mutagenic 

 activity is positive or negative, it is important to 

 eliminate the possibility of a selective action of the 

 chemical. If B/r/1 mutants are, for some reason, 

 less sensitive to the toxic effects of the chemical, the 

 proportion of mutants among the survivors of the 



treated culture will be higher than in the control, 

 and a mutagenic action might be erroneously 

 ascribed to the compound. If the mutants should be 

 more sensitive than the nonmutants, it is possible 

 that a positive mutagenic effect could be masked by 

 the differential killing of mutants. Thus, to be cer- 

 tain that selection for or against the mutants is not 

 responsible for an apparent positive or negative 

 effect, it is necessary to compare the sensitivity of 

 mutants and nonmutants to each of the chemicals 

 tested. The standard procedure used in these experi- 

 ments involved the following steps: 



A stock of B/r/1 was established by inoculating a 

 small amount of growth from about ten representa- 

 tive B/r/1 colonies on a control plate into a tube of 

 M-9. A new stock was made up for each experiment, 

 and the colonies which were isolated always came 

 from a plating of the control tube of an experi- 

 ment designed to test the mutagenic action of the 

 chemical in question. Thus, the B/r/1 stock iso- 

 lated in this way was representative of the mutants 

 present in an experimental culture at the start of 

 the exposure to the chemical. The culture derived 

 from these colonies was streaked on agar to elimi- 

 nate contaminating particles of bacteriophage, and 

 another culture was started with an inoculum from 

 at least ten colonies. This culture was used as a 

 source of B/r/1 for the selective killing test. 



To compare the sensitivity of B/r and B/r/1 to a 

 given chemical, 48-hour aerated M-9 cultures of 

 the two strains were grown, and equal volumes of 

 the two cultures were mixed together, to give a cul- 

 ture containing approximately half mutants and 

 half nonmutants. Two tubes were set up, each con- 

 taining the same volume of the mixed culture, cen- 

 trifuged, and the nutrient medium poured off. One 

 pellet was resuspended in the standard concentra- 

 tion of the chemical, and the other in buffer or dis- 

 tilled water. The tubes were incubated for the 

 standard time, and assayed to determine the pro- 

 portion of mutants in each culture. If mutants and 

 nonmutants are killed at the same rate, there 

 should be no difference in the proportion of mutants 

 between the control and experimental tubes. A sam- 

 ple experiment of this type will be described below. 



(5) Test of mutant colonies 

 In all cases where a positive effect was obtained, 

 samples of the mutant colonies obtained after treat- 

 ment with the chemical were isolated, and tested 

 carefully to establish the fact that they were true 

 B/r/1 mutants. In experiments where the total 

 number of colonies obtained was small, and where 

 a few contaminants could distort the results, every 

 colony was isolated and tested. The colonies were 

 examined for resistance to Tl, and also to another 

 phage, T2, to which strain B/r is sensitive. Colonies 

 showing resistance to Tl and sensitivity to T2 were 

 regarded as B/r/1 mutants. Colonies showing re- 

 sistance to phages were regarded as contaminants. 



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