2.3. Biofilm Quantification Using the Crystal Violet Assay

The microtiter plate crystal violet assay was performed, as previously described [39], with slight modifications. Briefly, each strain was grown in BHI broth at 25 °C under shaking (150 rpm) overnight. The overnight culture was diluted 1/100, and 200 µL of this cell culture were transferred (8 wells per strain) in a 96-well polystyrene microplate (Thermo-scientific^® nunclon delta surface, Paris, France). Sterile BHI medium was used as a negative control. The microplates were incubated at 25 °C in static conditions. After 24 h, the medium and the suspended planktonic cells were discarded. To remove loosely attached bacteria, each well was washed three times with 200 µL of sterile water. For staining bacterial biomass, 200 µL of 0.1% crystal violet (CV) solution (Sigma Aldrich, 1% crystal violet) were added to each well, and then the microplate was incubated at room temperature for 20 min. Then, the CV solution was discarded and the wells were washed three times with 200 µL of sterile water. All the wells were filled with 200 µL of 96% ethanol and their content was homogenized by pipetting to completely dissolve biofilm-bound CV. The amount of destained CV was assayed by reading optical density (OD) at 570 nm in a microplate reader (TECAN Spark, Paris, France). Each experiment was performed in triplicate with independent B. thermosphacta cultures (24 measurements per strain).