B. hominis. (i) LE medium. There are two versions of LE medium in the literature that have been used for axenic cultivation of B. hominis. Zierdt and Williams (72) used the same LE medium as described above except with the addition of 20% human or horse serum to the Locke's solution overlay. In the version of Lanuza et al. (43) the medium is modified by addition of 0.5% glucose to the Locke's solution, which is prepared at double the concentration of the recipe given before. The glucose-supplemented 2× Locke's solution for the overlay is mixed in equal volumes with fetal horse or calf serum. In both cases the medium is prereduced in an anaerobic environment for 48 h before use.

(ii) IMDM. IMDM for axenic cultivation of B. hominis was first used by Ho et al. (33) and is available commercially from a number of sources. It is prepared with 10% horse serum and is also prereduced before use.

Axenization of B. hominis. The axenization of B. hominis is also quite laborious, and all authors appear to have started from established xenic cultures. Monoxenic cultures have not been found to be necessary. Xenic cells or Ficoll-metrizoic acid gradient-purified cells (43) are inoculated into LE-based axenic culture medium with a cocktail of antibiotics. Weekly subcultures with large inocula and fresh antibiotics are performed for up to 6 weeks before sterility is achieved. It is not clear whether direct axenization of B. hominis into liquid IMDM is possible. Established axenic cultures in LE medium were transferred to IMDM in the original publication (33). However, since then the axenization of B. hominis using IMDM-soft agar with addition of antibiotics has been described (50). The B. hominis organisms form colonies in the agar which can then be isolated in liquid IMDM with little or no bacterial contamination.