microdilution method, the broth was supplemented
with 0.25% (v/v) tween-20 (Sigma Chemical Co.,
USA). Two-fold serial dilutions of essential oils were
prepared in a microtitre plate. The minimal inhibitory
concentration (MIC) was lowest concentration of essential
oil inhibiting visible bacterial growth after
incubation for 20–24 h at 35 °C. Cell suspensions
(1 loop) from the wells showing no growth were
subcultured on sheep blood agar (or chocolate agar in
case of H. influenzae) to determine if the inhibition
was reversible or permanent. Minimal bactericidal
concentration (MBC) was determined as the highest
dilution (i.e., lowest concentration) at which no
growth occurred on the agar plates.