262 



EVELYN M. WITKIN 



curve obtained by Demerec and Latarjet, and dif- 

 fers fundamentally from their analogous ultraviolet 

 curve. The upper abscissa in Fig. 3 represents X-ray 

 dose in r units, on a scale required to make the 

 desoxycholate curve coincide with the X-ray curve 

 of Demerec and Latarjet. It will be noted that 

 100,000 r units correspond, in terms of the number 

 of induced zero point mutations, to an 8-hour ex- 

 posure to 5% sodium desoxycholate. 



On the basis of these positive results obtained 

 with desoxycholate as a mutagenic agent in bacteria, 

 Demerec (unpub.) has tested the ability of this 

 compound to induce lethal mutations in Drosophila. 

 The proportion of lethals among the tested sperm 

 was about 1.5%, as compared with 0.24% in con- 

 trols, an effect of the same order of magnitude as 

 that obtained with nitrogen mustard by the same 

 technique. These results with Drosophila suggest 

 that sodium desoxycholate may prove to be a non- 

 specific mutagen, like radiations and mustard. No 

 tests have been made to investigate the possibility 

 of specific effects on the particular mutation used 

 as an index of mutagenic activity, namely, re- 

 sistance to bacteriophage Tl. For this purpose it 

 will be necessary to investigate the action of de- 

 soxycholate on other bacterial mutations. 



2. Pyronin and Methyl Green 



Basic dyes, which stain chromatin by virtue of 

 their affinity for nucleic acids, were considered as a 

 class of compounds worthy of investigation for 

 possible mutagenic activity. Pyronin and methyl 

 green, the components of the Unna-Pappenheim 

 mixture currently of interest in cytochemical re- 

 search, were the first dyes to be tested. Brachet 

 (1940) has described the usefulness of this mixture 

 as a means of differentiating cytochemically be- 

 tween ribose and desoxyribose nucleic acids, by 

 virtue of the selective affinity of pyronin for the 

 ribose type of nucleic acid. Although there is good 

 evidence for the specificity of the pyronin-methyl 

 green mixture under certain conditions, it must be 

 pointed out that the treatment of living bacteria, 

 without recourse to the procedures of fixation and 

 differentiation which are standard in cytological 

 work, may very well present entirely different con- 

 ditions. Thus pyronin may be taken up by fatty 

 acids, or other basophilic elements ordinarily re- 

 moved in cytological preparations. There is no 

 basis, therefore, for assuming that these dyes are 

 specific, under the conditions of these experiments, 

 in acting exclusively upon nucleic acids, or in dif- 

 ferentiating between the two types of nucleic acid. 



Pyronin was tested by essentially the same tech- 

 niques described in connection with desoxycholate. 

 One difficulty encountered, however, was the great 

 variability of the toxic effects of a given concentra- 

 tion in different experiments, or in different tubes 

 in the same experiment. One possible source of the 

 variability was thought to be the precipitate which 



forms in the presence of bacteria at high concentra- 

 tions of the dye. Low concentrations, in which 

 there is no precipitate, were tried, and in some 

 experiments bacteria were spread on the surface of 



Table 4. Zero Point Mutations Induced by Pyronin Y 



Cone, of Time Method 



Pyronin Y of of 



(%) Exposure Treatment 



No. In- 

 Survival duced Mu- 

 (%) tations/10 8 

 Survivors 



0.5 20min. in agar 43.0 10.6 



0.01 5hr. in liquid 32.2 22.4 



0.075 1 hr. in liquid 18.6 14.0 



0.75 lhr. in agar 18.1 22.8 



0.5 2§hr. in agar 12.5 33.7 



0.05 3hr. in liquid 3.9 36.0 



2.0 30min. in agar 2.5 41.3 



0.5 15min. in agar 0.16 62.7 



0.75 2hr. in agar 0.11 150.0 



0.5 2hr. in agar 0.03 100.7 



0.025 4hr. in liquid 0.01 1080 



agar containing pyronin, were washed from the 

 surface, and a concentrate of the wash was assayed. 

 The variability of the results was not overcome by 

 these modifications in technique, and the basis of 

 the difficulty is not yet understood. The results 

 obtained with pyronin, at various concentrations, 

 and under various conditions of treatment, are sum- 



Table 5. Number of B/r/1 Mutants in Cultures 

 Exposed to 1% Methyl Green 



Time of Experiment #1 



Cul- Ex- Sur- No. 



ture posure vival Mut. 



to 1% (%) per 10" 



Experiment #2 

 Sur- No. 



vival Mut. 



(%) per 10* 



Control — — 



Exp. 1 hr. 40 



Exp. 2hr. 11 



Exp. 3hr. 1.1 



Exp. 4hr. 0.2 



7.7 



10.2 



5.8 34 10.1 



8.1 7 8.6 



7.2 1.1 11.2 



6.9 0.1 9.9 



marized in Table 4. All results obtained were con- 

 sistent in indicating a higher proportion of mutants 

 among the survivors of treated bacteria than among 

 controls. Tests for selective killing of mutants 

 showed no difference in the sensitivity of B/r and 

 B/r/1 to pyronin. Although these results are to be 

 regarded as preliminary and tentative, they suggest 

 that pyronin is active as a mutagenic agent. The 

 problem of variability will have to be solved before 

 any detailed quantitative analysis can be made. 



Pyronin has not yet been tested on Drosophila, 

 and there is no evidence as to its specificity in 

 inducing mutations. 



Toxicity tests with methyl green showed that 



:2 5 



