Representative strains of some species of Mycobacterium were degraded by both acid and alkaline methanolysis. Two-dimensional thin-layer chromatography was used to determine the patterns of mycolic acids and other long-chain components in these methanolysates. Patterns composed of alpha-, methoxy- and ketomycolates were found in Mycobacterium asiaticum, Mycobacterium bovis, Mycobacterium gastri, Mycobacterium gordonae, Mycobacterium kansasii, Mycobacterium marinum and Mycobacterium tuberculosis; a representative of Mycobacterium thermoresistibile also contained lower molecular weight alpha'-mycolates in addition to these three acids. In representatives of Mycobacterium avium, Mycobacterium intracellulare, Mycobacterium nonchromogenicum, "Mycobacterium novum", Mycobacterium paratuberculosis, Mycobacterium scrofulaceum, Mycobacterium terrae, Mycobacterium xenopi, and Mycobacterium sp. MNC 165 alpha- and ketomycolates were accompanied by omega-carboxymycolates and 2-eicosanol and homologous alcohols which are derived from wax-ester mycolates. Mycobacterium fortuitum and "Mycobacterium giae" contained alpha'- and epoxymycolates and both serovars of Mycobacterium simiae had a very characteristic pattern of alpha-, alpha'- and ketomycolic acids. Comparison with data for other mycobacteria showed the chemotaxonomic significance of these mycolic acid patterns.

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Representative strains of Mycobacterium fortuitum, 'Mycobacterium peregrinum' and Mycobacterium smegmatis were degraded by acid methanolysis and patterns of long-chain compounds were determined by two-dimensional thin-layer chromatography. The same general pattern of mycolic acid methyl esters was found in all 39 strains examined, the major components being socalled a-mycolates and characteristic pairs of polar mycolates. Analysis of alkaline methanolysates of selected strains confirmed that these polar mycolates were derived from epoxymycolic acids, as found previously.

Comparative immunodiffusion studies and thin-layer chromatographic analyses of whole-organism acid methanolysates were performed on 37 strains of Mycobacterium farcinogenes, Mycobacterium senegalense and Nocardia farcinica. The latter were clearly distinguished from the mycobacteria in containing a single mycolic acid methyl ester and showing more precipitinogens with nocardial than with mycobacterial and rhodococcal reference systems. The distribution of precipitinogens showed that M. farcinogenes and M. senegalense were very closely related and that both showed a greater affinity to Mycobacterium fortuitum than to any of the other established species of Mycobacterium tested. The complex pattern of alpha-mycolates and characteristic polar mycolates found in both M. farcinogenes and M. senegalense has only previously been found in M. fortuitum and Mycobacterium smegmatis.

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