INDUCED MUTATIONS IN E. COLI 



263 



99% of the exposed bacteria were killed in about 

 three hours by a 1% solution in distilled water. 

 Table 5 gives the results of two experiments with 

 methyl green, in which the survival was as low as 

 0.2%, with no increase in the proportion of mutants 

 among the treated bacteria. These results were con- 

 firmed by repeated tests. Within the limits of the 

 sensitivity of the technique, which permits the 

 detection of induced mutants under conditions re- 

 sulting in the destruction of 99.9% of the treated 

 cells, methyl green seems to show no mutagenic 

 activity. 



3. Acriflavine 

 Neutral acriflavine, or euflavine, is an acridine 

 dye used therapeutically as a bacteriostatic agent. 

 Mcllwain (1941) has shown that the inhibition of 

 bacterial division caused by acriflavine can be re- 

 versed by adding polymerized yeast or thymus 

 nucleic acids. He also showed, in vitro experi- 



Table 6. Zero Point Mutations Induced by Acriflavin 



ments, that stable complex salts are formed by 

 acriflavine and nucleates. There is again no basis 

 for assuming specificity in these effects, since a 

 number of other compounds, including certain 

 amino acids, were also found to be effective in 

 reversing the inhibition brought about by acri- 

 flavine. 



Table 6 gives a summary of results obtained with 

 acriflavine. Although reproducible results can be 

 obtained if all factors are carefully standardized, 

 acriflavine presents certain difficulties which stand 

 in the way of quantitative investigation. In the 

 presence of large numbers of bacteria, some of the 

 dye precipitates at high concentrations, and the 

 precipitate goes gradually kto solution as the tubes 

 are incubated. Thus it is likely that the effective 

 concentration changes during the exposure. This 

 difficulty can be overcome to some extent by using 

 low concentrations and longer periods of exposure. 

 Results with acriflavine consistently indicate an 

 increase in the proportion of mutants among sur- 

 vivors of treated cultures, and suggest that this com- 

 pound is active in inducing mutations. 



Demerec et al (1946) tested the action of acri- 

 flavine on Drosophila, prior to the experiments re- 

 ported here, and obtained negative results. More 

 recently, Demerec (unpub.) has retested acriflavine, 

 using an improved method of administering the 

 chemical, and has found that it is active in inducing 

 lethal mutations in Drosophila. 



Discussion 



The experiments described above have indicated 

 that exposure to three out of the four chemicals 

 tested, at concentrations sufficiently toxic to kill 

 all but a small fraction of the treated bacteria, 

 results in a heightened proportion of mutants among 

 the survivors. Since B/r/1 was found to be no more 

 resistant than the nonmutant strain B/r to each of 

 the chemicals, it has been concluded that simple 

 selection is not responsible for this effect. Sodium 

 desoxycholate, pyronin and acriflavine are there- 

 fore regarded as mutagenic chemicals, although the 

 results for the latter two compounds are only pre- 

 liminary. Methyl green is apparently unable to 

 induce mutations to phage-resistance. 



Although a single specific phenotype, resistance 

 to bacteriophage Tl, was the genetic character used 

 in these experiments, it is likely that at least two 

 separate mutations were involved, since B/r/1 mu- 

 tants are known to fall into two distinct classes, 

 differentiated by such secondary characters as 

 colony size, cross resistance to another bacterio- 

 phage and growth factor requirements. No attempt 

 was made to separate the two mutant types in these 

 experiments, and the frequencies observed are prob- 

 ably the sums of the two independent mutations. 



Because of the lack of specificity in the chemical 

 action of the compounds tested, no attempt can 

 be made at present to relate their effectiveness as 

 mutagenic agents to the properties for which they 

 were selected. The high incidence of positive results 

 obtained, in 3 out of 4 compounds examined, is also 

 difficult to interpret at the present time. Further 

 experiments are required to determine whether these 

 results are due to a particularly fruitful or fortunate 

 basis of selecting chemicals, or, as appears more 

 likely, whether mutagenic action may prove to be 

 more common among biologically active compounds 

 than has hitherto been believed. 



Although bacteria are becoming increasingly use- 

 ful as material for genetic investigations, it is still 

 necessary to be cautious in generalizing results 

 obtained exclusively in bacterial studies. Thus, the 

 fact that sodium desoxycholate and acriflavine ap- 

 pear to induce mutations in Drosophila as well as in 

 E. colt is an important contribution toward the 

 validation of the techniques used in these experi- 

 ments. In addition to confirmation provided by tests 

 on other organisms, the use of other mutations in 

 E. coli, entirely independent of the phage-resistance 

 system, would constitute a valuable check on results 

 obtained with nonspecific mutagens. Experiments to 



126 



