INDUCED MUTATIONS IN E. COLI 



257 



sensitive bacteria are quickly lysed, while mutants 

 resistant to the particular bacteriophage applied ap- 

 pear as distinct colonies after suitable incubation. 

 Demerec (1946) and Demerec and Latarjet 

 (1946) studied the effect of ultraviolet and X-rays 

 in inducing mutations to resistance to one bacterio- 

 phage, Tl, and the techniques used by them have 

 been adapted in these experiments to the investiga- 

 tion of chemical mutagens. These authors showed 

 that a certain proportion of the mutations induced 

 by radiations are expressed immediately, before the 

 treated bacteria undergo division. These mutations 

 were called "zero points," as opposed to the "end- 

 point" mutations which are also induced by the 

 radiation, but which are expressed phenotypically 

 only after a number of cell divisions. In the in- 

 vestigations to be discussed here, the effect of four 

 chemicals on the induction of zero point mutations 

 to resistance to bacteriophage Tl was studied. 



Material 



Strain B/r of E. coli, a radiation-resistant mutant 

 of the B strain (Witkin, 1947), was used exclu- 

 sively throughout these experiments. This strain 

 was employed by Demerec (1946) and by Demerec 

 and Latarjet (1946) in their studies of radiation- 

 induced mutations, and it seemed desirable to use 

 the same material in these investigations, in order 

 to simplify comparison of the mutagenic action of 

 radiation and chemicals. 



The bacteriophage used to isolate resistant mu- 

 tants was Tl, sometimes known also as alpha, or 

 P28. Mutants of strain B/r resistant to phage Tl 

 are referred to as B/r/1. 



Difco nutrient agar was used for all platings, 

 and cultures were grown initially in a synthetic 

 medium known as M-9, having the following com- 

 position per 1,000 ml. of distilled water: 



KH 2 P0 4 3 g. NH 4 C1 1 g. 



MgS0 4 0.2 g. Na 2 HP0 4 -12H 2 IS g. 



NaCl 0.5 g. Dextrose 4 g. 



Methods 



The method used to determine the number of 

 mutants resistant to bacteriophage Tl, in both con- 

 trol and experimental cultures, was the standard 

 procedure of coating the surface of agar plates with 

 a suspension containing about 10 9 particles of Tl, 

 and then spreading 0.1 ml. of the undiluted bac- 

 terial culture on the phage-coated plate with a 

 sterile glass rod. The plates are incubated for 48 

 hours at 37° C, after which time the colonies of 

 resistant mutants are counted. The total number of 

 bacteria per ml. of the culture is determined by 

 plating suitable dilutions on agar, and making 

 colony counts. The frequency of B/r/1 mutants is 

 expressed throughout as the number per 10 8 

 bacteria. 



The procedure for testing the mutagenic activity 



of chemicals has been standardized as far as pos- 

 sible. Variations in technique are required to allow 

 for the peculiar properties of each chemical, and 

 will be described in connection with the specific 

 experiments. The basic procedure used in prelimi- 

 nary tests may be outlined as follows: 



(1) The preparation of suitable bacterial cultures 

 Ten to 20 cultures are usually started at one time, 

 each with a small inoculum (about 100 cells) from 

 a stock slant of strain B/r. The cultures are grown 

 for 48 hours at 37° C, with aeration, in a volume 

 of 40 ml. of the synthetic medium, M-9, described 

 above. Each culture is assayed to determine the 

 number of bacteria per ml., and the number of 

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

 mutants per 10 8 bacteria in a fully grown, untreated 

 culture is called the "background" number, and 

 must be subtracted from the number of mutants 

 per 10 8 survivors in the same culture after treat- 

 ment with a possible mutagenic chemical, to deter- 

 mine the number of induced mutations. It is desir- 

 able, therefore, to use cultures having the lowest 

 possible background number, since a high back- 

 ground may obscure a positive effect, particularly 

 where the mutagenic activity is weak. Cultures 

 found to contain more than 10 background mutants 

 per 10 8 bacteria are discarded, and the remaining 

 cultures are combined to form a stock pool, which 

 is stored at 5° C, and serves to provide samples for 

 experiments for a period of about a week. 



(2) Toxicity tests 



The optimum concentration of a chemical to be 

 tested depends largely upon its toxicity for the 

 bacteria. In general, for preliminary tests, a com- 

 bination of concentration and time of exposure was 

 used so as to kill about 99% of the bacteria. The 

 high killing is an indication that the substance is 

 penetrating the cell and reacting with its com- 

 ponents, and 1% survival is usually just enough to 

 permit the detection of mutants among the survivors 

 even if no mutations are induced. The concentra- 

 tion or time of exposure may be adjusted later, de- 

 pending upon the results of the preliminary tests. 

 For convenience, an arbitrary time, usually 2 or 3 

 hours, was chosen, and the concentration of the 

 chemical that would kill 99% of the bacteria in 

 this period of time was determined. The following 

 method was used. 



A concentrated solution of the chemical in dis- 

 tilled water or a suitable buffer was prepared, and 

 a series of widely spaced dilutions of the stock solu- 

 tion were made. A number of centrifuge tubes, each 

 containing the same volume of bacterial culture, was 

 set up and centrifuged for 20 minutes at 4 y 000 RPM. 

 The clear supernatant was decanted from each tube 

 to eliminate the nutrient medium. The bacterial 

 pellets were then resuspended, so as to have one 

 tube for each dilution of the chemical, and one 



