In this study we compared some common Bartonella culturing methodologies using four diverse species causing human illnesses. Based on a review of the literature, we focused on three major inconsistencies between protocols: base medium, cell coculture, and temperature. Our data showed that Bartonella tamiae demonstrated temperature-dependent growth limitations between common culturing conditions only 2°C apart. Additionally, growth of B. quintana was significantly enhanced by the presence of mammalian cell coculture under mammalian cell culture conditions; however, when the medium was modified to incorporate insect cell culture-based medium, coculturing with mammalian cells was no longer needed. In this study, we were able to overcome these temperature-and cell-dependent limitations and accommodate all of the strains tested by combining mammalian cell culture-based medium with insect cell culture-based medium.Bartonella is a genus of Gram-negative, facultative intracellular bacteria that have been detected in a plethora of insect and mammalian hosts (5,14,19,25). Numerous Bartonella species have been associated with clinical illnesses ranging from mild skin lesions to more severe manifestations, including persistent fevers, neurological symptoms, and endocarditis (6, 10). These bacteria can be fastidious under current laboratory conditions, and attempts to isolate cells from pure culture of biological specimens are often unsuccessful, despite positive molecular detection (1, 7).The majority of culturing methods for Bartonella species found in the literature focus on growth requirements for the two species commonly associated with human infection, Bartonella henselae and B. quintana (3,9,22,27,35). Through a review of the literature on methods for Bartonella isolation from biological samples, we found many differences in the culturing protocols. To summarize, most laboratories use cocultivation with mammalian cells or an axenic, insect cell culture-based medium or plating onto blood-supplemented agar (15,24). The mammalian cell coculture and insect cell culturebased medium are usually used for enrichment before plating of samples on agar. Of particular interest is the variability between the cell culture systems, as this method is traditionally considered to be the most successful for the initial isolation of Bartonella species (22,23,27,31). We focused our comparison on the protocols of three prominent laboratories that commonly culture Bartonella spp. and found the major differences to be localized to three main variables: (i) medium base (RPMI, medium 199 [M199], or Dulbecco modified Eagle medium [DMEM]), (ii) mammalian cell line (bovine endothelial, human endothelial, primate epithelial), and (iii) culturing temperature (35°C, 37°C).In 2005, a cell-free, liquid medium called Bartonella alphaproteobacteria growth medium (BAPGM) was developed to detect Bartonella in veterinary and human blood samples (28). It is a modified formulation of liquid medium designed to support insect cells. This medium required sheep ...