Biofilm Formation Quantification Assay

Biofilm formation assays were performed following the methodology previously described (Domenech et al., 2015 (link)) and it was determined by the ability of cells to adhere to the walls and base of 96-well, flat-bottomed polystyrene microtiter dishes (Falcon 353072, Corning Incorporated, New York, NY, United States). Unless stated otherwise, cells were grown in C+Y medium to an A₅₅₀ of ≈0.5–0.6, sedimented by centrifugation, resuspended in an equal volume of the indicated pre-warmed medium, diluted 1/100, and then dispensed 200 μl per well. After 5 h of incubation at 34°C, the A₅₉₅ was determined using Tecan Infinite F200 (Tecan Group Ltd., Switzerland). The biofilm formed was stained with 0.2% crystal violet (Sigma-Aldrich) and rinsed three times with distilled water to remove non-adherent bacteria. Biofilm formation was quantified solubilizing the biofilm in 95% ethanol (200 μl per well) and measuring the A₅₉₅.