Minimum inhibitory concentration (MIC) determination

Minimum inhibitory concentration of the different antiseptics for the two bacteria was determined by the broth microdilution assay. In more detail, the three antiseptics were diluted via serial dilutions in fresh BHI in a flat-bottom 96-well plate (Greiner, Belgium). S. mutans and A. actinomycetemcomitans were added in a final concentration of 10⁶ cells/mL or 10⁷ cells/mL in each well. 10⁷ cells/mL was chosen for A. actinomycetemcomitans because it was found that the minimal inoculation concentration allows for growth under control (no antiseptic) conditions. Cell concentrations were previously determined by flow cytometry following the procedure that was explained previously.

The cultures were incubated at 37°C for 24 hours, with A. actinomycetemcomitans incubated under anerobic conditions. After incubation, growth was defined by measuring OD₆₀₀ with a plate reader (Tecan Infinite M200 PRO, Tecan, Belgium). The MIC was defined as the lowest concentration where no growth was observed.