Multiwell 24 well

The GE1 (RIKEN Cell Bank, Tsukuba, Japan) mouse-derived gingival epithelial cell line was used (Figure 1a). GE1 cells were cultured in basal serum-free medium (SFM-101, Nissui, Tokyo, Japan) containing 1% fetal bovine serum (Biowest, Nuaillé, France) and 10 ng/mL mouse epidermal growth factor (Corning, New York, NY, USA) in a humidified atmosphere with 5% CO₂ at 33 °C. Cells were seeded within a 1 mL volume onto each Ti plate, at a density of 5 × 10⁴ cells per well in a 24 well culture plate (Multiwell 24 well, Corning).

Biofilm production for the various morphologies of ECOR57 was determined using a modified protocol from O’Toole (2011) [97 (link)]. Overnight bacterial culture was diluted 1:100 in fresh LB of which one mL was added to 24-well plates (Corning Multiwell 24 well) in duplicates. Plates were incubated at 37 °C for 24 h. After 24 h, the media was removed and washed twice with PBS to remove excess bacterial debris and media components. 250 µL of 0.1% Crystal Violet (BD Gram Crystal Violet) was added to each well and allowed to stain for 15 min at room temperature. After crystal violet removal, wells were washed approximately four times or until the washing fluid was no longer purple. Plates were allowed to dry in a 37 °C incubator for approximately five minutes. To quantify the biofilm, 250 µL of 30% acetic acid in water was added to each well to solubilize the biofilm and allowed to sit for 10 min. Fifty microliters of each sample was transferred to round bottom 96 well microtiter plates (Corning 96-well cell culture cluster round bottom) in quadruplets. Optical density at 550 nm was measured using BMG LabTech POLARstar Omega plate reader. One-way ANOVA with post hoc Tukey statistical analysis with n = 5 was performed using GraphPad Prism version 5.0.