268 



EVELYN M. WITKIN 



selected for resistance to its toxic effects and there- 

 fore capable of surviving in relatively high concen- 

 trations. 



No attempt has been made to approach a compa- 

 rable killing value with another mutagenic agent 

 (Zephiran chloride) because this quaternary am- 

 monium compound shows a selective toxic effect 

 against B/l at high concentrations, a process which 

 incidentally would tend to make the detection of 

 mutations a more difficult process. An experiment 

 using 1:14,000 aq. Zephiran chloride (a non-selec- 

 tive concentration) is shown in Table 1. A definite 

 mutagenic effect is observed if periodic sampling of 

 the same culture is performed. 



Table 1. Periodic Assay of Cells Incubated with 



Zephiran Chloride at 37° C. and Tested for 



Point Mutations to Phage Resistance 



Time of Bacteria B/l per 10" Per cent 

 Assay per cc. bacteria survival 



Zero 

 point 

 mutants 



In another experiment yielding 19% survival the 

 zero point mutations numbered 24 per 10 8 . 



The intergeneric variation in sensitivity of micro- 

 organisms to toxic agents is perhaps of more gen- 

 eral importance than experimentally induced inter- 

 strain differences as a factor to be kept in mind 

 when screening of possible mutagens is contem- 

 plated. Unless one adopts the relatively extreme 

 position that cell death in the presence of chemicals 

 is the consequence of induced lethals it is not un- 

 duly speculative to assume that cells highly re- 

 sistant to deleterious chemical effects on extragenic 

 processes will be best suited for a demonstration of 

 mutagenesis. If analysis of toxicity is to be per- 

 formed as a differential survival test it may also be 

 necessary with some chemicals to extend the tests 

 over the killing range included within limits of the 

 experiment. 



As Dr. Witkin has observed, the span of our ex- 

 perience with induced phage resistance as a genetic 

 tool has not been of sufficient length to evaluate its 

 place in the general scheme of experimentally in- 

 duced mutations in more complex biological sys- 

 tems. Investigators using the method may find 

 themselves in the paradoxical situation that each 

 new success contributes to a failure, that is, to the 

 widening of a gap between mutagenesis of micro- 

 organisms and of higher forms of life. This would 

 inevitably decrease the value of bacterial studies in 

 interpreting mutation among higher organisms. Un- 



til other mutations in bacteria have been studied 

 by similar methods and until the growing list of 

 chemical mutagens has been used on more familiar 

 genetic material and placed within some kind of 

 quantitative and qualitative limits, it will be im- 

 possible to judge the real significance of Dr. Wit- 

 kin's most stimulating and capable study. 



Herskowitz: The work of Dr. Witkin demon- 

 strates a facile bacterial method for identifying cer- 

 tain groups of chemicals as mutagens. She has al- 

 ready pointed out that by using this technique nega- 

 tive mutagenic action by a chemical is not con- 

 clusively demonstrated. However, it may well be 

 that those chemicals screened out by the bacterial 

 technique as being non-mutagenic will be im- 

 portant in the long run. Therefore, it seems ad- 

 visable to supplement this technique with others 

 which may permit a more sensitive test for mu- 

 tagenic action. 



For the detection of the action of a chemical on 

 nucleic acids and nucleoproteins the use of Droso- 

 phila sperm seems most suitable. There are several 

 reasons for this. In spermatozoa there are a mini- 

 mum of cytoplasmic substances which might inter- 

 fere with a chemical affecting genie nucleoprotein. 

 Moreover, even though a chemical is not specific 

 for nucleoprotein it may yield positive results more 

 readily in sperm than in other cells. There is ex- 

 cellent evidence from genetic and irradiation experi- 

 ments that genie nucleoprotein may be drastically 

 changed without killing the sperm cell; this permits 

 the utilization of such sperm for fertilization with 

 the subsequent detection of lethal genes and 

 chromosome rearrangements in addition to other 

 types of inherited changes. In Drosophila it is also 

 possible accurately to localize inherited changes in 

 the chromosomes. Dr. Demerec no doubt had these 

 advantages in mind when he developed the aerosol 

 technique. 



As Dr. Witkin has mentioned, there is no con- 

 clusive proof that aerosols always reach the sper- 

 matozoa in the testes. Again, negative mutagenic 

 results with a chemical may be misleading. There- 

 fore, a technique which would directly treat 

 Drosophila sperm with chemicals is highly desirable. 

 Accordingly, an investigation was initiated to dis- 

 cover if chemicals could be injected into the vagina 

 of adult females, which would then copulate, and 

 thereby expose the sperm to the respective 

 chemicals. 



A vaginal douche technic was successfully worked 

 out, and is described in detail in a recent published 

 note (Herskowitz, Evolution 1: 111-112, 1947). 

 The method was tried with methyl bis amine hydro- 

 chloride at concentrations of 0.2% and 10.0%, with 

 positive results. 



The data establish the practicality of the vaginal 

 douche technique for the detection of chemical 

 mutagens. There are two advantages of this method. 

 Low concentrations of mutagens may be effective 



131 



