A maintenance medium for Treponema pallidum was designed to hold its Eh at the optimum for that organism, -10 to -110 mV. After 100% motile (freshly harvested) T. pallidum was inoculated into the medium, the motility of the treponemes decreased to 80% after 2 days, 50% after 3.5 days, and 0% after 9 days during incubation at 34 C. Full virulence was retained for 2 days, but it dropped rapidly thereafter, and the treponemes became avirulent by day 5.

Cultures of brucella-sensitized mouse spleen cells exposed to Brucella abortus antigens in vitro release macrophage migration inhibition factor (MIF) and macrophage spreading factor. Subjecting the supernatants from such cultures to preparative scale electrophoresis in acrylamide gel yields several fractions, one of which contains both MIF and macrophage spreading factor. This material has properties attributable to guinea pig MIF: it is nondialyzable, heat stable, nontoxic to macrophages from heterologous murine donors, and has a greater anodal electrophoretic mobility than guinea pig serum albumin. Another fraction from the gel column inhibits macrophage spreading; its electrophoretic mobility is similar to that of guinea pig gamma globulin. Neither brucella antigen nor skin reactive substances were detectable in any acrylamide gel column fraction when tested by the mouse footpad induration assay technique.

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Mice are suitable donors of peritoneal macrophages for the capillary migration inhibition test, provided a period of preincubation with antigen is used A correlation has been shown between the development of cellular immunity and the presence of delayed-type hypersensitivity (3, 8, 9). A popular in vitro test for delayed-type hypersensitivity is macrophage migration inhibition (2, 4, 6, 7; J. R. David, Fed. Proc. 27:6-12). This technique has been used effectively in guinea pig and human systems but has had only limited success, except with pooled alveolar macrophages, in the mouse system (1, 10, 11). By employing only peritoneal exudate cells in our capillary migration tests, we have demonstrated delayedtype hypersensitivity in a genetically heterogeneous population of 6to 8-week old female (Swiss-Webster, code ROR) mice infected with Brucella abortus.

Treponema pallidum (Nichols virulent strain) was incubated with or without oxygen, using a modified medium supplemented with reduced glutathione and a variety of nutrients (PRNFjo-B). Twoto fourfold increases in treponemal numbers were observed in cultures without mammalian cells within 96 h of incubation under 5 to 6% oxygen. Treponemal motility and multiplication were maintained more satisfactorily in cultures that were diluted and transferred daily, using an equal volume of fresh medium. Treponemes incubated without oxygen did not significantly increase in number. Virulent microorganisms were detected for at least 96 h in the cell-free system. In the presence of 3 to 4% oxygen, two-to fivefold increases in treponemal numbers were observed in the supernatant fluids of cultures containing human prepuce cells after 48 to 120 h at 35°C. Without oxygen, treponemal numbers rarely approached a threefold increase. Virulent treponemes were detected by the rabbit skin lesion test after at least 120 h in vitro. Regardless of the system of incubation, increases in treponemal numbers could not be sustained for longer than 120 h, and treponemal virulence decreased as a function of time in vitro.