Agglutination Assay with Chromone Derivatives

The agglutination assay was studied using chromone derivatives as the earlier procedure (Sethupathy et al., 2020 (link)). V. parahaemolyticus overnight grown cultures (1:100) dilution were treated with chromone derivatives for 24 h at 30°C kept at 250 rpm. The cell pellets were adjusted to an approximate optical density of 0.5 OD, and 400 µL of the mixture was progressed to 14 mL tubes containing 1500 µL phosphate buffer solution and 500 µL concentration of 2% newly prepared S. cerevisiae (Sigma–Aldrich, St. Louis, USA). After gentle vortexing of the mixture for 5 s, the initial OD was measured at 600 nm using a UV-spectrophotometer (Optizen 2120UV, Korea). Subsequently, 25 min of incubation was kept at ambient temperature, 100 µL of the translucent supernatant was transferred into 96-well plates, and OD600 were measured. The following formula calculation showed agglutination as percentage: 100 × (1 − (OD_600before/(OD_600after)).