2.5. Minimum inhibitory concentration (MIC) determinations
The in vitro antibacterial activity of WE and EE were determined against Streptococcus mutans and Porphyromonas gingivalis
according to the Clinical and Laboratory Standards Institute (CLSI)
recommended minimum inhibitory concentration (MIC) protocol
with modifications (Cai and Wu, 1996; CLSI, 2012). Briefly, 2-fold
dilution series were made from all tested antibacterial agents starting from 2000 g/mL in a 96-well plate. An aliquot of 20 L of
bacterial suspension was added to 180 L of antibacterial dilution.
Each well in the microtiter plate contained Streptococcus mutans
[final concentration of 5 × 10
5
colony forming units (CFU)/mL]
or Porphyromonas gingivalis (5 × 10
6
CFU/mL). The crude extracts
were dissolved in 10% DMSO. The final concentration of DMSO was
5% (v/v) and this solution was used as a negative control. Chlorhexidine gluconate (Sigma) and sanguinarine (Sigma) were used as a
positive control. The MIC was defined as the lowest concentration of
the test agent that had restricted growth to a level