2.2. Determination of Minimum Inhibitory Concentrations (MIC)

The antibiotics sodium penicillin G and tetracycline hydrochloride were purchased from Sigma-Aldrich (St. Louis, MO), and clindamycin phosphate and vancomycin hydrochloride from Pharmacia (Stockholm, Sweden). The MIC of these antibiotics were determined for the C. septicum strains by microbroth dilution assay according to the Clinical & Laboratory Standards Institute (CLSI) guidelines for such testing in anaerobes [14]. In brief, 200 μL of a stationary overnight culture was used to inoculate 20 mL of pre-reduced defined liquid media. Cultures were grown anaerobically for approximately 2 hrs at 37°C until a turbidity equal to the 0.5 McFarland standard (OD₆₃₀ of 0.300; ~2 × 10⁶ CFU/mL) was achieved. The prepared C. septicum (50 μL) was then added to duplicate wells of a 96-well plate containing 50 μL of 2-fold serially diluted (2 – 2,000 ng/mL) penicillin, tetracycline, clindamycin or vancomycin. Plates were incubated anaerobically at 37°C for 48 hrs and growth (turbidity) was assessed by a microplate reader (OD₆₃₀). MICs were defined as the lowest antibiotic concentration that inhibited measurable bacterial growth (i.e., OD630 equal to the negative control). MICs were performed 3 times in triplicate.