A reference system for the soluble antigens of Mycobacterium bovis, strain BCG is described. The system is based on crossed immunoelectrophoresis with intermediate gel. A commercially available immunoglobulin preparation made from rabbit anti-BCG hyperimmune serum was used as reference antibodies, while a concentrated BCG culture filtrate was used as reference antigen. The pattern obtained was highly reproducible, and most of the components were stable when the fiftyfold-concentrated culture filtrate was stored at -20 degrees C. About thirty different antigenic components were selected as reference antigens and numbered. The majority of the reference antigens were present in extracts prepared from BCG by ultrasonication or bacterial press extraction. Use of the system for studies of antigenic relationship between mycobacteria, identification and quantification of antigens, and characterization of antimycobacterial antibodies are illustrated by examples. The antigens of two preparations of tuberculin purified protein derivative (PPD) were identified. The antigen designated BCG60 was found to be a najor constituent of tuberculin PPD. Evidence is presented that this antigen is cell wall associated.

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.

Characteristic waxes, based on methoxy and keto long-chain diols, members of the phthiocerol family, have been isolated from representatives of Mycobacterium bovis, M. kansasii, M. marinum, M. microti and M. tuberculosis. M. kansasii produced essentially di-esters of the ketodiol phthiodiolone A, but the remaining species also had waxes based on the methoxy-diols phthiocerol A and phthiocerol B. Gas chromatography of derivatives of the components of the waxes showed that the phthiocerol A components from M. bovis, M. microti and M. microti and M. tuberculosis were qualitatively similar, being mainly C34 and C36, but potentially significant differences were seen in the proportions of the components from M. bovis. The phthiocerols A from M. marinum were C28 and C30 and the phthiodiolones A from M. kansasii were C25 and C27. The multimethyl-branched acids from the waxes of M. bovis were quantitatively different from those of M. microti and M. tuberculosis but all these mycocerosic acids ranged in size from C23 or C24 to C32, with C29 or C30 being the major component in most cases. M. marinum and M. kansasii strains had mainly C26 or C27 and C29 or C30 multimethyl-branched acids, respectively.

A co-operative taxonomic study has been performed on slowly growing nonpigmented mycobacteria (Run yon's group 111). Phenetic data on 89 strains, studied in 18 laboratories, were collected and analysed by a numerical taxonomic method. A variety of immunological properties, lipid analyses and measures of pathogenicity were analysed independently to establish correlation with numerical classification. Mycobacterium gastri, M . nonchromogenicum, M . terrae, M . avium and M . xenopi were recognized by almost all participants as distinct species. Mycobacterium novum was considered to be synonymous with M . terrae. A clearcut distinction could not be made between M . avium and M. intracellulare; the majority of participants in the study recommend that M . intracellulare be reduced to a synonym of M . avium. A minority of authors cannot agree with this proposal.