3.3. Biofilm Disruption Assay

Bacterial cultures (S. aureus and E. coli) were grown in Muller Hinton broth (MHB; Oxoid, Basingstoke, UK) media overnight at 37 °C with shaking at 120 rpm. Cultures were diluted (1:20) in MHB medium and 200 µL aliquots were dispensed to wells in a flat bottom 96-well plate (Costar; Sigma-Aldrich, St Louis, MO, USA). Biofilm was then grown in the 96-well plate at 37 °C overnight. After that, loosely bound cells were washed away with 1x phosphate-buffered saline (PBS) and cultures were then supplemented with different concentrations of test compounds dissolved in DMSO and incubated for a further 24 h with shaking at 120 rpm. Biofilms adhered on the plate substratum were quantified using crystal violet staining as described previously [18,63]. The experiment was performed in triplicate.