P. gingivalis FDC 381, T. denticola ATCC 35404, and T. forsythia ATCC 43037 were used in this study and were routinely cultured anaerobically at 37°C as described previously [33] (link), [35] (link). Bacterial concentration was determined and cells were resuspended in reduced transport fluid (RTF) at 1010 cells per mL [35] (link). For topical oral polymicrobial infection, P. gingivalis was mixed with an equal quantity of T. denticola for 5 min; subsequently, T. forsythia was added to the culture tubes containing P. gingivalis and T. denticola, and cells were mixed thoroughly and allowed to interact for an additional 5 min. P. gingivalis, T. denticola, and T. forsythia were then mixed with an equal volume of 4% (w/v) sterile carboxymethylcellulose (CMC; Sigma-Aldrich, St. Louis, MO) in phosphate buffered saline (PBS), and this mixture was used for oral infection (5×109 bacteria per mL) in ApoEnull mice as described previously [35] (link).
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