2.5. Minimum Biofilm Inhibitory Concentration (MBIC)

The MBIC was calculated as the lowest concentration that inhibited at least 80% of the biofilm biomass growth. The standard safranin colorimetric assay was used as reported previously [20]. E. faecalis ATCC 47077 strain was used. Serial dilutions of _L-Chg₁₀-teixobactin were carried out with BHI broth to produce final concentrations of 0.8 (MIC), 0.4 (MIC/2), 0.2 (MIC/4), and 0.1 μg/mL (MIC/8). We transferred 20 μL of the _L-Chg₁₀-teixobactin from each concentration into the microtiter 96-well plate, followed by the addition of 200 μL of bacterial inoculum suspension (10⁶ CFU/mL), to obtain a total volume of 220 μL. The plates were then aerobically incubated at 37 °C for 24 h. The supernatants from all wells were discarded at the end of the culture period and washed three times with sterile phosphate-buffered saline (PBS) to remove the planktonic cells. Biofilms were stained with 200 μL of 0.1% safranin solution and were subjected to further incubation for 30 min at room temperature. After incubation, the excess stain was removed by washing three times with PBS, followed by 10 min of air drying. Finally, wells were incubated for 15 min with 33% acetic acid per well to dissolve the stain, and 100 μL was pipetted from each well into a new microtiter plate. The OD was measured at a wavelength of 492 nm in a microplate reader (SoftMax pro-6.3.1, SpectraMax M2, Molecular Devices Corp., Sunnyvale, CA, USA) to quantify the biomass.