Biofilm Formation Assay for A. butzleri Strains

To determine the ability of A. butzleri strains to form the biofilm, a previously described protocol was followed with few modifications [15 (link)]. The suspensions of the two strains under study were prepared by diluting an overnight culture to an OD_{620 nm} of 0.2, which was used to inoculate 24-well polystyrene plates (VWR, Belgium) with 1 mL of each suspension. Wells with only a medium were used as the negative control. After 48 h of incubation at 30 °C in microaerophilic conditions, the medium was removed, and the wells were dried for 1 h at 55 °C. After that, the biofilm was stained with 1 mL of crystal violet (AMRESCO, Leuven, Belgium) at 0.1% (w/v) in deionized water and incubated for 15 min at room temperature, followed by removing the unbound crystal violet by the washing of the wells three times with distilled water. The wells were dried at 55 °C for 15 min and then the bound crystal violet was solubilized with a 30% methanol and 10% acetic acid solution. Finally, to quantify the biofilm formation, the absorbance at 570 nm was determined using a microplate reader (Biorad, xMark) [15 (link)]. The assay was performed with eight replicates in at least three independent assays.