2.2. Crystal Violet Biofilm Assay

Biofilm production in microtiter plates was measured in the crystal violet biofilm assay and performed as described previously [16] using 96-well Nunc^TM Nunclon^TM microtiter plates (Nunc A/S, Roskilde, Denmark) with few modifications. For each strain, 30 μL of an undiluted overnight culture and 100 μL LB ^wo/NaCl were added in three parallel wells of a microtiter plate. Three wells with 130 μL LB ^wo/NaCl were used as negative controls on each plate. The microtiter plates were incubated statically for two days at 20.0 ± 1.0 °C or 37.0 ± 1.0 °C. After incubation, optical density at 595 nm (OD₅₉₅) of the planktonic solution was measured as an indication of planktonic growth (Multiscan MS; Thermo Fisher Scientific, Inc., Waltham, MA, USA). Then, the wells were washed with saline, stained with 1% crystal violet solution (Sigma-Aldrich, St. Louis, MO, USA) for 30 min at room temperature, and washed at least three times to remove dye not bound to the biofilm. The biofilm bound dye was dissolved in ethanol-acetone (70:30, v/v) for 10 min at room temperature before the OD₅₉₅ was measured. The results were calculated by subtracting the median OD₅₉₅ of the three parallel control wells from the median OD₅₉₅ of the three parallel sample wells. The median was used to avoid the influence of accidental outlier data. All samples were tested three times and the mean for each strain was calculated. OD₅₉₅ values higher than mean of the negative controls plus three standard deviations were classified as positive for biofilm production [29]. As the results of the negative control had already been subtracted from the results before this evaluation, the cut-off was set to three SDs of the negative controls. All strains with biofilm formation above the set cut-off were considered positive for biofilm formation in the crystal violet biofilm assay in microtiter plates.