Colony formation in solid medium by the relapsing fever spirochetes Borrelia hermsiiand Borrelia turicatae

Relapsing fever (RF) in North America is caused primarily by the spirochete Borrelia hermsiiand is associated with the bite of its tick vector Ornithodoros hermsi.Although this spirochete was known long before the discovery of the Lyme disease (LD) spirochete, Borrelia burgdorferi,basic methods to facilitate the study of B. hermsiihave lagged behind. One important technique to expedite the study of the molecular biology and pathogenesis of B. hermsiiwould be a reliable method to grow and clone these bacteria in solid medium, which we now describe. We have defined the solidifying agent, plating temperature, oxygen concentration, and pH for the efficient plating of two species of RF spirochetes, B. hermsiiand Borrelia turicatae. Importantly, this technique allowed us to successfully isolate virulent, clonal cell lines of spirochetes, and to enumerate and isolate viable B. hermsiifrom infected mouse blood and tick tissues. Our results also demonstrate the value of testing a range of several environmental variables to increase the efficiency of bacterial isolation, which may be helpful for researchers working on other prokaryotes that are intractable for in vitrogrowth.