Cultivation of Bacteroides fragilis in tissue culture

The capacity of animal cells to support the growth of obligate anaerobes was shown by inoculating Bacteroides fragilis and Bacteroides thetaiotaomicron into cultures of Vero, HeLa, Chinese hamster ovary, and Chinese hamster lung cells. From an extremely small inoculum (nearly 1 cfu), B. fragilis multiplied up to 10(8) cfu/ml during aerobic cultivation in L-15 medium containing kanamycin sulfate (50 micrograms/ml) and 5% fetal bovine serum (total depth, 3-5 mm). Infecting bacteria formed a cluster on the cell monolayer and permitted the concomitant growth of infected Vero cells. Treatment of Vero cells with an inhibitor of respiration (sodium azide) arrested the bacterial growth reversibly. In contrast, an uncoupler of oxidative phosphorylation, 2,4-dinitrophenol, enhanced the growth of B.fragilis. By use of the thin oxygen electrode, low oxygen pressure was detected in the vicinity of the cultured Vero cells. Thus, respirating animal cells seem to provide a microenvironment with low enough oxygen pressure for the growth of some anaerobes.