Crystal violet biofilm formation assay.

The biofilms formed by the C. ochracea strains were examined in 96-well polystyrene plates (Sumitomo Bakelite, Tokyo, Japan) as described previously, with minor modifications (20). Stationary-phase cultures of C. ochracea were adjusted to an OD₆₆₀ of 0.1 with fresh tryptic soy broth. Cells were then added to 96-well plates (100 μl/well) and incubated for 48 h at 37°C under anaerobic conditions. Following incubation, the wells were washed three times with 200 μl of distilled water and stained with 50 μl of 0.1% crystal violet for 15 min at room temperature. After the crystal violet solution was removed and each well was washed twice with distilled water, the biomass-associated crystal violet was extracted with 200 μl of 99.5% ethanol. The extracted biomass-associated crystal violet (100 μl/well) was transferred to a new microtiter plate, and the OD₅₉₅ was measured with a microplate reader (Spectra MAX M5e; Molecular Device, Sunnyvale, CA). Total biomass was calculated by using the following equation, which was described previously (36): total biomass = absorbance of crystal violet-stained biofilm at 595 nm/absorbance of total growth (including biofilm and planktonic cells) at 660 nm.