Three strains of Treponema dentium were isolated in a veal heart infusion medium enriched with 10% ascitic fluid prepared according to the method of R. J. Fitzgerald and E. G. Hampp (J. Dental Res. 21:20, 1952). These organisms had a "24-2" axial fibril relationship by electron microscopy (M. A. Listgarten and S. S. Socransky, Arch. Oral Biol. 10:127, 1965) and did not ferment carbohydrates or utilize glucose or lactate. The organisms produced acetic acid and ammonia as major end products in

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... veal heart infusion-ascitic fluid medium. A number of commercially available culture media were used in an attempt to replace the veal heart infusion base. The best basal medium was a medium used for the cultivation of another oral spirochete, T. microdentium. This medium consisted of P P L 0 Enrichment Broth (BBL) without crystal violet or serum, supplemented with (per milliliter): 1 mg of glucose, 400 ,ug of nicotinamide, 150 ,ug of spermine tetrahydrochloride, and 20 Mg of sodium isobutyrate. The pH was adjusted to 7.5 with sodium hydroxide and autoclaved at 121 C for 15 min. To this medium, filter-sterilized L-cysteine and cocarboxylase were added aseptically to a final concentration of 1 mg/ml and 5 ,ug/ml, respectively. When the above medium was used to replace veal heart infusion, it was found that T. dentium still had an absolute requirement for ascitic fluid or rabbit serum. Maximal growth occurred when the fluid enrichments were present in a 10 to 20% concentration. To determine the active components present in ascitic fluid or rabbit serum, the supplemented P P L 0 medium was used as a base, and the various fractions to be assayed were added aseptically after filter sterilization. The organisms were grown for 2 to 3 days at 35 C in an atmosphere of 95% H2 and 5% CO2. Turbidity was determined by nephelometry on the sixth serial transfer of the organisms in the test medium. Preliminary characterization of the active factor indicated that it could not be ashed, and it was inactivated by heating at 80 C for 30 mn; it was stable in visible, infrared and in ultraviolet light; it was not dialyzable; it was not volatile or steam-distillable at acidic, neutral, or basic pH, it would not partition from water into ether at acidic, neutral or basic pH, and it was excluded TABLE 1. Ability of certain serum protein fractions to support growth of Treponema dentium Growth Proten fration Source" Range of relative Protein fraction Sourc concn ( to ascluid mediumb Albumin (rabbit) 3191 0.05-2.0 8 Albumin, Fraction V 2117 0.05-2.0 9 (rabbit) B3-Globulin, Fraction 767 0.05-0.4 20 III (rabbit)