1. The examination of 1100 sera by both the Wassermann reaction and the complement-fixation test with spirochaetes revealed a superior sensitivity of the latter reaction and practically equal specificity of the two tests.2. Syphilitic serum contains two different antibodies: one reacting with the lipoid antigen of the Wassermann reaction, the other with a specific antigen in the spirochaete.3. The spirochaetal antibody of syphilitic serum has a complex serological structure, corresponding to spirochaete strains of different antigenic make-up.4. The existence of this antibody and its specific absorption by the homologous antigen can also be demonstrated by agglutination.5. The difference between agglutinin titres found in normal and syphilitic sera is not pronounced enough to render this method satisfactory for the practical diagnosis of syphilis.6. The spirochaete contains, apart from its specific antigen, the ubiquitous lipoid substance representing the Wassermann antigen.7. A fraction was obtained from spirochaetes by Raistrick and Topley's method which in complement-fixation and precipitation tests reacted actively with spirochaete antisera from rabbits, but which so far failed to react with syphilitic sera.This work was carried out with the aid of a grant from the Rockefeller Foundation. I wish to thank Prof. Golla, Director of the Central Pathological Laboratory, L.C.C. Mental Hospitals, who rendered this work possible, and Dr Arthur Davies, Director of the Devonport Laboratory, for the hospitality afforded me at his laboratory and for the patients’ sera used in this work. I am indebted to Prof. R. T. Hewlett for his revision of the manuscript, to Prof. Raistrick for advice in chemical matters, and to Dr Amies, of the Lister Institute, for his help with the Sharples centrifuge.

THE genus Mycobacterium contains a number of fairly clearly defined species, which may be obligatory pathogens, opportunistic pathogens or saprophytes, together with an ill-defined group of anonymous strains. So far, the definition of species has relied largely on morphology, cultural appearances, biochemical activity and temperature requirements. The grouping of strains by Runyon (1959) and Marks and Richards (1962) relies on these and related criteria. Information about the antigenic components of a number of species and unclassified strains has been accumulating over the past few years (Parlett and Youmans, 1958; Castelnuovo et al., 1960; Lind, 1961 ; Sushida and Hirano, 1963; Schaefer, 1965; Jensen, Klaer and Lundberg, 1964, 1966a, b andc). This paper records an antigenic analysis of Mycobacterium fortuitum, Myco. kansasii, Myco. phlei, Myco. smegmatis and Myco. tuberculosis made by Ouchterlony's double diffusion-in-agar precipitation technique. MATERIALS AND METRODS OrganismsAntigen extracts were prepared from 25 strains of mycobacteria that included some obtained from the National Collection of Type Cultures (NCTC) and some isolated by us from patients and soil (PGH). They were: Mycobacterium fortuitum strains NCTC 8573t, 946, 1542,2006,2291,2891,3631 and 8697, and PGH strains " Hoey "t, '' Ugoji " and " Soil "t; Myco. kanrarii strains PGH " Brown "t, " Walker ", " Birk " and " Sheppard "; Myco. phlei strains NCTC 8156t, 525, 8151, 8157, 10008 and 10266; Myco. smegmatis strains NCTC 3337, 8159 and 10265; and Myco. tuberculosis strain H37 Rat. Strains marked with an obelus were also used for preparation of antisera. Owing to the risk involved in the work with living bacteria only one representative of Myco. tuberculosis was used.All the strains of Myco. kansasii and the Hoey strain of Myco. fortuitum were isolated from the sputum of patients. Myco. fortuitum strain Ugoji was recovered from a patient's abscess and Myco. fortuitum strain Soil from a sample of garden soil. All strains of Myco. kansasiiexcept strain Walker, which was a single isolation, were clinically signiscant in that they had repeatedly been found in the patient's sputum. Their identification, and that of the Myco. fortuitum cultures isolated by Present address: (1959, 1965). Preparation of antigensCultures were grown at 37°C in I-litre volumes of modified Sauton's medium (Boyden and Sorkin, 1955) in large Carrel or Thompson flasks, and their pellicles were harvested when no further growth appeared to be taking place. The age of the cultures depended on the species used and varied from 1 to 8 wk. Parts of the harvested pellicles were extracted at -20°C in an " X " press (Edebo, 1961).Microscopical examination of the product obtained after three pressings revealed that the cell walls were almost totally broken up. The crushed material was allowed to stand at 4°C and the cleared fluid layer between the surface flocculate and the sediment was then pipetted off. This extract served as antigen and was stored in small volumes at -20°C.

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