1956 Volume 9 Issue 4 Pages 135-140
Soil actinomycetes have been screened for their production of antitumor substances in our laboratory by using experimental animal tumors and HeLa cells in the tissue culture. The test meterial, the culture liquid, was daily injected to animals which had been inoculated with an appropriate number of tumor cells, or added to the tissue culture medium of HeLa cells, and the inhibition on the development of tumors or on the growth of HeLa cells was examined. By these studies, it was confirmed that miscellaneous antitumor substances are produced by soil actinomycetes. For instance, culture liquids of about 10% of soil actinomycetes screened exhibited antitumor effect and prolonged the survival period of mice bearing Ehrlich carcinoma. However, in the further studies isolating the active agents, there were many difficulties owing to the fact that there was not a suitable rapid quantitative method of testing the antitumor activity. The authors presented a cylinder plate method in this paper. This method is not applicable to all antitumor substances, depending on their speed of diffusion in the agar or their modus of the antitumor action. However, in the case of the substance to which this method is applicable, this method is very helpful for the isolation. This method can also be used for the screening of microorganisms for the production of antitumor substances.
Schrek(1) has indicated that the following four principles can be used for the in vitro test of anti-cell effect: (1) counting the number of eosin-unstained cells; (2) testing the capacity of excised viable tissues to react histologically to reagents; (3) testing the capacity of cells to ferment substrates; (4) testing the capacity of redoxy activity of cells. He showed dilution methods of testing the anti-cell activity depending on these principles. As it has been known in the antibiotic studies, the cylinder plate method is more quantitative than the dilution method. If the conditions to keep the cells alive in the agar medium is found, according to these principles a suitable cylinder plate method can be devised.
Among these principles, that of testing the redoxy activity has been utilized in the rapid cylinder plate method of testing the antibacterial activity(2). In the case when on the plate, where the bacteria had not grown enough to exhibit visible turbidity, methylene blue was placed, then the clear blue inhibition zone could be observed. The principle of testing the capacity of redoxy activity of cells was considered to be most easily applicable to the cylinder plate method.
The authors’ preliminary experiments indicated that the cells of Ehrlich carcinoma of mice, when they were placed in Hanks salt agar or mouse Ringer agar, maintained the dye-reducing activity at least for 48 hours at 37°C. This time lemz;th has been known to be enough to permit the diffusion of the antibiotics. Among retloxy dyes, Schrek used 2, 6-dichlorophenolindophenol and Kikuchi(3) used 2, 3, 5-triphenyl tetrazolium chloride. The author’s preliminary tests indicated that these dyes and also methylene blue could be used for the detection of the reducing activity of cells of Ehrlich carcinoma. In the case of methylene blue, some devices were necessary to prevent the reoxidation by air. 2, 3, 5-Triphenyl tetrazolium chloride was expensive. Therefore, the authors used...Please see the PDF file for the full Abstract.
