The microdilution technique using 96-well microtitre plates, as described by Eloff [5 (link)], was used to obtain the MIC values of the positive drug controls against the various microorganisms. The positive controls for oral bacteria, chlorhexidine gluconate (CHX) (Dental Warehouse, Sandton, South Africa) at 12.5 µg/mL, and Tetracycline (Sigma-Aldrich, 3050 Spruce Street, St. Louis) at 200 μg/mL for P. acnes were serially diluted in relevant broth medium adding 48 h old oral bacteria and 72 h old P. acnes and were incubated at 37°C in anaerobic conditions. The final concentration of CHX, ranged 3.13 µg/mL–2.44 × 10−2
µg/mL and Tetracycline ranged 100 μg/mL–0.781 μg/mL. S. mutans and P. intermedia were incubated for 24 hrs and P. acnes for 72 hrs at 37°C.
To indicate bacterial growth, 40 μL of (0.2 mg/mL) INT, 20 μL PrestoBlue, and 20 μL Alamar Blue was added to microplate wells and reincubated until a colour change occurred. The MIC was defined as the lowest concentration that inhibited the growth of the bacteria [6 (link)].
µg/mL and Tetracycline ranged 100 μg/mL–0.781 μg/mL. S. mutans and P. intermedia were incubated for 24 hrs and P. acnes for 72 hrs at 37°C.
To indicate bacterial growth, 40 μL of (0.2 mg/mL) INT, 20 μL PrestoBlue, and 20 μL Alamar Blue was added to microplate wells and reincubated until a colour change occurred. The MIC was defined as the lowest concentration that inhibited the growth of the bacteria [6 (link)].
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