3.7. Checkerboard Synergy Assay

Synergistic effects of plant extract combinations were evaluated with a checkerboard synergy assay to obtain FIC values. One extract was serially diluted along the abscissa, while another extract was serially diluted along the ordinate. The total volume of the combination was 100 μL per well. Then 100 μL of each bacterial suspension (1 × 10⁶ CFU/mL) was added to each well of a 96-well microplate. The microplate was incubated at 37 °C for 18 h. The OD was measured at 595 nm with a spectrophotometer. FICI was calculated using the following equation: FICI = FIC_A + FIC_B, where FIC_A was MIC of extract A in combination/MIC of extract A alone, and FIC_B was MIC of extract B in combination/MIC of extract B alone. Results were interpreted as synergistic interaction (FICI ≤ 0.5), partial synergy (0.5 < FICI ≤ 0.75), additive interaction (0.75 < FICI ≤ 1.0), indifferent (1.0 < FICI ≤ 4.0), or antagonistic interaction (FICI > 4.0) [68].