Biofilm Formation Analysis of P. aeruginosa

Biofilm formation was analyzed on 96-well polystyrene microtiter plates by the modified crystal violet method described by Ramos-Vivas et al. (2019) (link). P. aeruginosa isolates were grown on LB agar (Neogen, USA) at 37°C for 24 h. Then, 180 µL of LB broth (Neogen, USA) and 20 µL of P. aeruginosa suspension (initial concentration of 1.5 × 10⁶ CFU/mL) were added to each well and incubated at 37°C for 24 h in the presence and absence (control) of ½ MIC bio-AgNPs. After incubation, the supernatants were discarded, and the wells were washed three times with phosphate-buffered saline (pH 7.2), fixed with 250 µL of methanol (Merck, Germany) for 10 min, and stained with a 1.0% crystal violet aqueous solution (Merck, Germany) for 15 min. Subsequently, the crystal violet was discarded and the wells washed three times with purified water. Adhered cells were resuspended in 250 µL of 33% (v/v) glacial acetic acid (Merck, Germany). Absorbance was read spectrophotometrically (Multiskan™ FC, Thermo Fisher Scientific, USA) at 620 nm.