INDUCED MUTATIONS IN E. COLI 



259 



Experimental Results 

 Since the effect of chemicals in inducing zero 

 point mutations is to be investigated, it is important 

 to know to what extent mutations occur spon- 

 taneously, if at all, in resting cells. Luria and 

 Delbriick (1943) have shown that mutations to 

 resistance to Tl do not occur spontaneously in 



other compounds tested in these experiments. It is 

 a highly reactive compound, forming addition com- 

 pounds known as choleates with a wide variety of 

 organic substances, including fatty acids, ether, 

 xylol and certain carcinogenic hydrocarbons ( Wie- 

 land and Sorge, 1916; Fieser and Newman, 1935). 

 Alloway (1933) used sodium desoxycholate in his 



Table 1. Number of B/r/1 Mutants in Resting Culture or B/r Suspended in Distilled Water for 24 Hours at 37° C. 



resting bacteria, and our own observations have 

 confirmed this. Table 1 shows the results of an 

 experiment in which resting bacteria, suspended in 

 distilled water and incubated for a period of 24 

 hours at 37° C, were periodically assayed to deter- 

 mine the number of B/r/1 mutants per 10 8 cells. 

 The number of mutants per 10 8 bacteria remains 

 extremely constant under these conditions, and any 

 mutations induced in resting cells must be compared 

 with a baseline of zero. 



1. Sodium Desoxycholate 



Sodium desoxycholate is a salt of the bile acid 

 desoxycholic acid, the molecular structure of which 

 is shown in Fig. 1, along with the structures of the 



^ H ,CH e CM 2 -COONa 





(I) SODIUM DESOXYCHOLATE 



:h/ 



,-o 



o 



l/l H 



(3) METHYL GREEN 



(2) PYRONIN Y 



CH 3 .C\ 



(4) NEUTRAL ACRIFLAVINE 



Fig. 1. Molecular structure of Sodium Desoxycholate, 

 Pyronin; Methyl Green and Acriflavine. 



early work on type transformation, and it is used 

 by Avery and his coworkers in the preparation of 

 transforming principle (1944), where its function is 

 to dissolve the desoxyribose nucleoprotein complex 

 of the pneumococcus. Mirsky and Pollister (1946) 

 have shown that a .5% solution of sodium desoxy- 

 cholate dissolves their preparations of thymus 

 chromosin fibers, and can be used to extract the 

 desoxyribose nucleoprotein complex from minced 

 thymus. While this solvent action of desoxycholate 

 on nucleoproteins served as a basis for selecting 

 this compound for a test of its mutagenic activity, 

 there is no doubt that it is a nonspecific effect. 



Sodium desoxycholate dissolves easily in water 

 above pH 6.5, but solutions at pH 6.5 to 7.5 form a 

 gel at high concentrations. Since the reaction of 

 desoxycholate in distilled water is about 7.7, un- 

 buffered solutions of the compound in distilled 

 water were used. Fresh solutions were made up for 

 each experiment, and sterilized by immersion in a 

 boiling water bath for 15 minutes. 



Toxicity. Preliminary toxicity tests showed that 

 99% of the exposed bacteria were killed in 3 hours 

 by a 5% solution, or in 48 hours by a 0.3% solu- 

 tion. All of the experiments reported here were 

 done using the 5% solution. 



Test for mutagenic activity. To illustrate in de- 

 tail the method used in testing chemicals for ability 

 to induce mutations, a typical experiment done with 

 sodium desoxycholate will be described. 



Ten-ml. aliquots of a stock low-background cul- 

 ture of B/r were pipetted into each of 4 centrifuge 

 tubes, which were centrifuged for 20 minutes at 

 4,000 RPM. The supernatant was then decanted to 



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