4.4. Evaluation of Biofilm Biomass Level Using the Crystal Violet Dying Method [CV] in 96-Well Microtiter Plate

Bacterial inoculum at a density of 0.5 McF (McFarland turbidity scale) (established using densitometer Densitomat II, BioMerieux, Warsaw, Poland) was prepared from the 24 h planktonic cultures in TSB, TSB + G, and DMEM. The inoculum was diluted to obtain ca. 1 × 10⁵ CFU/mL (colony-forming unit, CFU) and added to the six wells in the 96-well microtiter plate (VWR, Radnor, PA, USA), with 100 μL to each well. The plate was incubated for 24 h at 37 °C under static conditions. After a time, the medium above the biofilm was discarded, and the plate was moved for 10 min drying at 37 °C. Next, 100 μL of 20% water solution of crystal violet (v/v) (Aqua-med, Lodz, Poland) was poured into each well and incubated for 10 min at room temperature. Next, the solution was discarded, and the attached biofilm was gently rinsed twice with 100 μL of 0.9% NaCl (Chempur, Piekary Slaskie, Poland). The plate was dried for 10 min at 37 °C, and afterward, 100 μL of 30% aqueous solution of acetic acid (v/v) (Chempur, Piekary Slaskie, Poland) was added to the wells. The plate was set to a mechanical shaking at 450 rpm for 30 min (Schuttler MTS-4, IKA, Königswinter, Germany). Next, the colour solution was transferred to a new plate to measure the absorbance of the extracted CV at a 550 nm wavelength (MultiScan Go Spectrophotometer, Thermo Fischer Scientific, Waltham, MA, USA). The crystal violet staining method was performed in six repetitions in three independent experiments for each strain.