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After overnight incubation at 37 °C, 3 mL of chloroform were added to 5 mL culture supernatant and mixed vigorously. One milliliter of 0.2 M hydrochloric acid was added to the organic layer and the absorbance was measured at 520 nm. Pyocyanin concentration (mg/L) was calculated as: P = (OD × 17.072) x (5/3) where OD is optical density at 520 nm, 17.072 is the pyocyanin extinction coefficient, and 5/3 is the dilution factor28. Three independent experiments were performed for each isolate.
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