Biofilm inhibition assay

Biofilm inhibition assay was performed as described previously to investigate the biofilm inhibition capability of Chol-37(F34-R) [42]. The assay was conducted in TSB medium supplemented with 0.5% glucose against S. aureus ATCC25923, TSB medium against S. typhimurium SL1344, and BHI medium against P. aeruginosa GIM1.551. Briefly, 200 μL 1 × 10⁶ CFU/mL bacterial cells was cultured at 37 °C for 24 h, in 96-well plates, with or without PMAP-37, PMAP-37(F34-R), and Chol-37(F34-R) (0.25–128 μg/mL). Planktonic bacteria were removed, and the wells containing biofilm were washed thrice with sterile PBS solution. Subsequently, 200 μL methanol (99%) was added per well, and the wells were fixed for 20 min. After aspiration, the plates were allowed to dry. The biofilm was stained with crystal violet (0.1%) for 10 min, and the excess colorant was gently eliminated by three successive washings with sterile PBS. The stain was resolubilized in 95% ethanol, and the absorbance was measured at 620 nm. Biofilm formation inhibition rate = [1 − OD_620nm (peptides)/OD_620nm (control)] × 100%.