Quantitative Biofilm Assay in Microplates

Biofilm formation was determined in flat-bottom 96-well polystyrene microplates (Corning, New York, NY, USA) according to Stepanović et al. with some modifications [56 (link)]. A total of 200 μL of bacterial suspension 0.5 McFarland (1.5 × 10⁸ CFU/mL) in Tryptic Soy Broth medium (Becton Dickinson, Franklin Lakes, NJ, USA) with 1% (w/v) D-(+)-glucose (TSBG) was inoculated in wells and incubated at 37 °C for 24 h. After the incubation period, the wells were discharged and washed three times with 200 μL of phosphate-buffered saline (PBS). 200 μL of 1% (v/v) crystal violet (CV) (Merck, Damm, Germany) was added per well for 15 min and after the wells were discharged and washed three times with 200 μL of PBS. The microplates were air-dried and the biofilm-bound CV was dissolved adding 200 μL of 96% (v/v) ethanol per well. O.D. was determined at λ = 570 nm [56 (link),57 (link),58 (link)]. Bacterial strains were categorized as non-adherent (O.D. ≤ 0.120), weakly adherent (O.D. > 0.120) and strongly adherent (O.D. > 0.240) [30 (link)]. Results are expressed as the mean of three experiments.