Figure 1. Systematic approach to developing media supporting host cell–free growth of obligate intracellular bacteria.

(A) Predictions based on niche characteristics, metabolic pathway reconstructions, and known physiology of purified organisms can be used to establish initial media pH and compositions. Oxygen tension should be tested empirically. (B) Stepwise testing of media formulations and oxygen tension to find conditions that support increased metabolic fitness using informative indicators of metabolic activity such as SDS-PAGE/autoradiography and transcriptional microarrays. Titrations of all media constituents should be conducted since high concentrations of some can be inhibitory [23]. (C) Growth assays to determine if increasing metabolic fitness correlates with increasing bacterial numbers. The infectivity, optical density, and/or genome equivalents of input bacteria can be compared to output bacteria following incubation.