Biofilm Formation Assay for E. faecalis

E. faecalis strains were inoculated in BHI with the indicated antibiotics and kept 16 hr at 37°C. The next morning, they were diluted in a 1:100 ratio in TSB-D with the indicated antibiotics, 10 ng/ml cCF10, and 50 ng/ml nisin. 200 µl fractions were dispensed into a 96-well microtiter plate (Costar) with 8 replicates per strain. 200 µl TSB-D fractions were used as blanks. The 96-well plate was then incubated aerobically at 37°C without agitation in a humidified chamber for 24 hr. The suspension was transferred to another 96-well plate to determine the optical density at 600 nm (OD₆₀₀). The plate containing the biofilm was washed with distilled water three times and then left to air dry at room temp for 2.5 hr. The biofilm was stained with 100 µl 0.1% (wt/vol) safranin (Sigma) at room temp for 20 min, then washed three times with distilled water and left to air dry at room temperature. Afterwards the absorbance was determined using a plate reader (BMG Labtech) at 450 nm. Biofilm production was calculated as an index of safranin staining of the cell biomass divided by absorbance of its optical density (OD₄₅₀/OD₆₀₀) (Willett et al., 2019 (link)).