Crystal violet biofilm eradication assay

The ability of the peptides (2Abz¹⁴S²⁹, 2Abz²³S²⁹, and HNP1ΔC18A), or antibiotics (amoxicillin and cefixime) to eradicate preformed biofilm of MDR E. coli isolate and P. aeruginosa PAO1 was also investigated. Briefly, bacteria grown overnight were diluted 1.100 in MHB and incubated in 96-well microtiter plates at 37°C for 24 h. The 24 h-old biofilms in 96-well plates were washed and air-dried. Serial two-fold dilutions of antimicrobial agents in MHB were prepared. Antimicrobial agents (100 μl individual antimicrobial agent) or (50 μl peptide plus 50 μl antibiotic) were added to each well. Plates were then incubated for further 24 h at 37°C. After incubation, the crystal violet assay was used to measure biofilm reduction according to the formula above.