2.7. Biofilm Formation Ability

Biofilm formation was assessed either by static culture in a 96-well polystyrene microplate or aerated culture with a plate covered by a lid with pegs. Biofilm was measured using the crystal violet method [47]. For measurement of biofilm production in static cultures, each isolate was cultured in 3 mL MHB for 16 h at 37 °C. The cultures were adjusted to OD₆₀₀ of 0.01, and 100 µL of the dilution was transferred into a 96-well polystyrene microplate (eight replicate wells per isolate) and incubated at 37 °C for 24 h without shaking. After removal of the bacterial cultures, the wells were washed with sterile water, stained with 150 µL of 0.1% (w/v) crystal violet at room temperature for 30 min, washed three times with water, and then air-dried. For measurement of biofilm formation in aerated cultures, 200 µL of diluted suspension was aliquoted into an MBEC assay^® biofilm inoculator with a 96-well base (Innovotech, Edmonton, Canada) and shaken at 110 r.p.m. for 16 h in a 37 °C incubator. Biofilm formed on the pegs was rinsed with water three times, stained in 200 µL of 0.1% (w/v) crystal violet at room temperature for 30 min, washed with water, and dried. The stained biofilm was dissolved in 33% acetic acid solution and absorbance at 550 nm was measured in a spectrophotometer. ATCC 19606 served as a positive control and MHB blank as a negative control. Statistical significances were determined by one-way ANOVA. Statistical analyses and graphs were constructed using GraphPad Prime 5.