4.8. Determination of Minimal Biofilm Inhibitory Concentration (MBIC) and Minimal Biofilm Eradication Concentration (MBEC)

MBIC and MBEC assays were performed as previously described with minor modifications [15]. For the MBIC assay, a suspension of broth diluted bacteria culture (5 × 10⁵ CFU/mL) was incubated with the peptide solutions at 37 °C for 24 h. For MBEC assay, 100 μL inoculum culture was seeded to a 96-well-flat-bottom plate and incubated at 37 °C for 48 h to obtain the mature biofilm. Afterwards, the plate was washed by sterile phosphate-buffered saline (PBS; Sigma-Aldrich, Gillingham, UK) twice and treated with a series peptide solution at 37 °C for 24 h. Then, all the plates were washed with PBS and stained by 100 μL 0.1% crystal violet solution (Sigma-Aldrich, Gillingham, UK), and further dissolved by 30% acetic acid (Sigma-Aldrich, Gillingham, UK). The absorbance of each well was recorded by Synergy HT plate reader (Biotech, Minneapolis, MN, USA) at 595 nm.