In searching and in extracting antitumor agents of microbial origin, there has been an increasing demand for a rapid and simple method for estimating antitumor entities in culture broth and processing streams. Kikuchi and Matsuzawa reported, in connection with the screening of antitumor substances, an application of in vitro contact and eosin-unstained cell count method¹⁾, and in vitro contact and TTC (2, 4, 5-triphenyltetrazolium chloride) staining method, with²⁾ or without³⁾ succinic acid as a substrate.

Method of determining antitumor-cell action by the agar plate diffusion technique was reported by Yamamoto, et al.⁴⁾ and by Miyamura⁵⁾.

The method herein presented is a simplified agar dilution technique, similar in principle to those reported previously in that the estimation is made with the dehydrogenase activity of tumor cells as the indicator. However, the present technique affords quantitative results in a short period of time, usually within three hours, and requires simpler technique than that of the previous methods.

Trichomycin is an antibiotic isolated by Hosoya, et al.¹⁾ from the Streptomyces hachijoensis, which, besides being active against fungi and yeasts, is characterized by its activity against the Trichomonas group. It has been found to be an effective chemotherapeutic agent in the treatment of trichomonasis, moniliasis, etc^2),3),4).

On its structural studies, there has been scarcely any findings other than the presence of a polyene group, which had been suggested by its ultraviolet absorption spectrum⁵⁾. In the present paper, we report on several oxidation products of trichomycin, which was purified by a modified Hosoya’s method⁶⁾.

The purified sample was acetylated and then ozonized; acetaldehyde, glyoxal, and ρ-acetaminobenzoic acid have been found amongst the oxidation products. The ρ-acetamino group of ρ-acetaminobenzoic acid is presumed to be formed in the acetylation step, because purified trichomycin shows positive reactions on diazo-coupling, carbylamine and Ehrlich’s reaction. In agreement with this, p-aminoacetophenone has been obtained by KMnO₄ oxidation of a purified non-acetylated sample. The purity of the starting material, ‘purified trichomycin’, and structural units of trichomycin will be reported in the next paper⁷⁾.

Counter-current distribution (C.C.D.) has been applied to the purification of trichomycin, and it has been found that the main fraction is composed of two similar constituents. These constituents have been separated, their properties investigated, the origin of the decomposition products from ‘purified trichomycin’ reported in the previous paper has been studied, and a partial structure for trichomycin has also been proposed.

The occurrence of the spontaneous mutants resistant to drug in bacteria has been reported by following investigators: Luria and Delbruck, 1943; Oakberg and Luria, 1947; Witkin, 1947; Vennesland, et al., 1947; Yegian, et al., 1950; Demerec, 1948, 1951; Newcombe, et al., 1949, 1951; Hsie, et al., 1950; Szybalski, et al., 1953, and others., and reviewed by Luria (1947), Catcheside (1949), Braun (1953) and others.

Naturally resistant forms of Mycobacterium tuberculosis have been isolated by Pyle (1947) directly from the sputa of patients who have not been treated with streptomycin, and the presence of naturally resistant mutants in stock cultures of H_37RV has been demonstrated by Vennesland, Ebert and Bloch (1947). A quantitative analysis of the resistance of Mycobacteria to streptomycin has been at first made by Yegian and Vanderlinde (1948), and streptomycin resistance and neomycin resistance mutation in Mycobacterium ranae have been quantitatively studied by Hsie and Bryson (1950). We reported the mutation rate to streptomycin resistance in a parent strain and in its isoniazid-resistant mutant of Mycobacterium avium (1955).

With the purpose of obtaining fundmental results for chemotherapy of tuberculosis and especially determing the order of administration of antituberculous drugs, the number of naturally-occurring drug-resistant mutants was determined in a parent sensitive strain, in its streptomycin-resistant mutant, in its isoniazidresistant mutant and in its PAS-resistant mutant Mycobacterium tuberculosis var. hominis.

The problem of the development of resistance to antituberculous drugs in Mycobacterium tuberculosis is the most important one in the chemotherapy of tuberculosis. Several basic biological considerations on the problem have been made by Yegian and Vanderlinde^(1,2), Hsie and Bryson⁽³⁾ etc. To prevent the development of resistance to streptomycin, the combined effect of PAS with streptomycin has been reported by Graessle and Pietrowski⁽⁴⁾ and the effect has been clinically proved by Tempel, et al.⁽⁵⁾ The author and his associates also have made several genetic studies^(6,7,8,9). These studies have concerned to the frequencies of appearance of mutants in strains already resistant to any antituberculous drug, to the characteristics of drug-resistant strains and to several clinical considerations derived from these in vitro results. Since the combined chemotherapy of tuberculosis has started, several years have already passed through, and now it appears that we face the problem of double or triple resistance in Mycobacterium tuberculosis. The frequency of mutation in mutants resistant to any one drug has been studied by us. However, it seems to be none of the reports concerning with the frequency of mutation to drug resistance in mutants resistant to two antituberculous drugs, that is, so-called double-resistant mutants.

The present paper is concerned with the frequency of mutation to PAS-resistance in a streptomycin- and isoniazid-resistant strain (a strain simultaneously resistant to streptomycin and isoniazid) of M. tuberculosis var. hominis.