2.6.1 Pyocyanin Assay

Pyocyanin quantification was performed by using King’s A broth media. Overnight culture of P. aeruginosa strains was inoculated into 5 mL of King’s A media. Both untreated and treated cultures with sub-MICs (1/2 and 1/4 MICs) of CFP, CFPC, and CFPF were incubated at 37°C for 48 hours with shaking. Pyocyanin was extracted from the supernatant with 3 mL chloroform. The mixture was centrifuged at 3000 rpm for 10 min. Chloroform fractions were transferred to a new tube, then 1 mL of 0.2 M HCL was added, and the mixture was re-centrifuged for 5 min at 3000 rpm. The absorbance of the aqueous layer was measured at OD 520 nm. Pyocyanin concentration was calculated from the equation: pyocyanin concentration (µg/mL)= OD 520 nm × 17.072 (He et al., 2014; Saurav et al., 2016).