Quorum sensing inhibitory assay

This assay was conducted according to Zhu et al. (2011) with minor modifications. C. violaceum was inoculated into Miller Luria–Bertani Broth (LB) and incubated overnight at 27 °C for 24 h. The overnight culture was diluted 1:1000 with LB to obtain a concentration of 10⁶ CFU/mL. The green cardamom essential oil was dissolve in ethanol (95%) and diluted into LB to obtain a concentration of 10 mg/mL. The stock solution was serially diluted with LB using 48-wells microplate. Then 500 µL of bacterial dilution (10⁶ CFU/mL) cells were separately added to each well of microplate containing 500 µL of certain concentration of essential oil and then plate was incubated 24–28 h at 27 °C.

After incubation, 1 mL of the each well was centrifuged at 8000×g for 5 min and the supernatant was discarded. But the pellet was resuspended into 1 mL Dimethyl sulfoxide (DMSO) and vortexed for 2 min. The sample was centrifuged again at 8000×g for 5 min. Aliquots, 200 µL of the supernatants were added into a 96-wells plate and the absorbance was measured at OD₅₉₅ nm using the plate reader. In order to confirm that cardamom inhibit quorum sensing without influence on bacterial growth activity, the pellet containing bacteria cell was resuspended in 1 mL sterile water, vigorously vortexed and the optical density was measured at OD₅₉₅ nm using a plate reader. The experiments were repeated two times in duplicate.