Pyoverdine Inhibition Assay for P. aeruginosa

P. aeruginosa inoculum was incubated in the absence (growth control) and presence of sub-MIC (1/2 MIC, 1/4 MIC, 1/8 MIC, 1/16 MIC, and 1/32 MIC) doses of gentamicin and peptide extract at 37°C for 48 hours. Culture media were then centrifuged at 4,000 rpm for 45 minutes. One hundred microliters of cell-free supernatant was dispensed into a 96-well microtiter plate for pyoverdine measurement. The relative concentration of pyoverdine in all treated supernatants with respect to control (no drug) was measured by fluorescence (BioTeK® Synergy H1 Multimode Microplate Reader, Germany) at an excitation wavelength of 405 nm and an emission wavelength of 465 nm [27 (link), 38 (link)]. Percentage inhibition was determined relative to untreated culture (control) by the expression in (1).