Determination of dehydrogenases in atypical strains and in other Mycobacteria.

The dehydrogenase activity in mycobacteria is a promising field of research for the following reasons. Youmans et al. (1955, 1957) showed that small particles, containing dehydrogenases and possessing characteristics of mitochondria, stimulate in mice the production of a degree of immunity which is equal to the immunity produced by a 1-mg quantity of whole living cells of H37Ra. On the other hand, Martin et al. (1955) demonstrated that dehydrogenase activity is diminished in the kidney and other organs of guinea pigs in which tuberculosis develops. Therefore, any study of dehydrogenase activity in mycobacteria mav contribute to our knowledge of the problem of immunity in tuberculosis. The striking difference in behavior of atypical strains and human strains in the presence of neotetrazolium is certainlv another reason to investigate the field of dehydrogenase activity more closely (GastambideOdier and Smith, 1958). Some data on the dehydrogenase activity of mycobacteria can be found in the review by Bonicke (1956/1957) in which the results obtained with 508 mycobacterial strains, using methylene blue as H acceptor, are given. In recent years, other workers used tetrazolium salts for these measurements. Hanks (1951, 1956) showed differences between the hydrogen transfer capacity of JMycobacterium lepraemurium and Mycobacterium phlei under anaerobic conditions. Vandiviere et al. (1952) established a rapid test of viability based upon triphenyltetrazolium chloride reduction. He worked with RlRv and 1 Presented before the Medical Session, as part of Section 2B, at the annual meeting of the National Tuberculosis Association, Kansas City, Missouri, May 6, 1957. This study is part of the cooperative study on atypical mycobacteria sponsored by the Laboratory Subcommittee of the American Trudeau Society, and has been aided by grants from the Wisconsin Anti-Tuberculosis Association, Milwaukee, Wisconsin. 2 Present address: Institut de Biologie PhysicoChimique, 13 rue Pierre Curie, Paris Ve, France. BCG strains and used 1.2 to 3.2 mg of bacilli. De Goes et al. (1952-1953) compared the reducing activity of saprophyte strains and human strains. Winling (1953) reviewed many data on tetrazolium salts and described a few applications of these compounds to the study of mycobacteria. Kanai and Yanagisawa (1954) studied in detail the technique of triphenyltetrazolium chloride reduction as a test of viability. They worked with strain H37Rv, whereas Arima et al. (1954) performed the same type of experiments on a BCG strain. The inconvenience of the techniques of the Japanese authors is that they grew the bacilli on Sauton's medium where only surface growth is obtained. They used very large quantities of bacteria, from 3 to 40 mg, in some instances even more. Koch-Weser and Ebert (1955) studied the reduction of triphenyltetrazolium chloride by the H37Rv strain in Dubos Tweenalbumin medium. Segal and Bloch (1956) demonstrated differences in dehydrogenase activity between strains of H37Rv grown in vivo and in vitro. D'Arcy-Hart and Rees (1956) showed differences in dehydrogenase activity between virulent and avirulent strains. Some observations made by Ling et al. (1957) and by Sourkes and Lagnado (1957) suggest that the mechanisms of reduction of methylene blue and tetrazolium salts are different but still incompletely understood. To our knowledge, neotetrazolium has not been used previously in quantitative measurements of dehydrogenase activity in mycobacteria. This substance has numerous advantages over methylene blue. In the reduced state, it is colored purple, the reduction proceeds irreversibly at pH 7, and the reduced acceptor is not sensitive to oxygen. It has also advantages over triphenyltetrazolium chloride. It is much less readily reduced by light (Smith, 1951; Glock and Jensen, 1953; Mustakallio et al., 1955) and the dehydrogenase activity of such small quantities of bacteria as 0.5 to 1 mg can be measured with this compound. If the dehydrogenase level of the strain is high, as it is the case for llyco-