Quantifying Biofilm Formation with Crystal Violet

Quantification of biofilm formation by MS2507, MS2507ΔSA0701 and PAO1 was performed using a crystal violet staining method (Karaolis et al., 2005 ) with some modifications. Bacteria were precultured on LB agar plates at 37 °C overnight. Culture conditions for biofilm formation were LB for PAO1 and TSB for MS2507 and MS2507ΔSA0701 at 30 °C 12-h static culture. The media for biofilm formation were supplemented with various concentrations of synthetic cyclic-di-GMP and its analogs. A single colony was inoculated into 4 mL of LB or TSB and incubated at 37 °C with constant shaking at 160 r.p.m. until late logarithmic stage. The culture was then diluted 1 : 10⁴ with fresh LB or TSB supplemented with various concentrations of cyclic-di-GMP and its analogs and a 200 μL of the diluted cultures was transferred into round-bottom wells of a polystyrene microtiter plate (Iwaki, Tokyo, Japan). The microtiter plate was incubated statically at 30 °C for 12 h. The supernatant was then discarded and the wells were washed three times with 200 μL of phosphate-buffered saline. After the wells were dried, 200 μL of 0.1% crystal violet was added and the wells were stained at room temperature for 15 min. The wells were then washed three times with distilled water to remove the extra dye and dried. The dye staining the biofilm in each well was extracted with 200 μL of dimethyl sulfoxide and the OD_{570 nm} was measured with a plate reader (Tecan Austria GmbH, Austria). These experiments were repeated at least three times, and the results were expressed as the mean values ± SD. P-values for significance were calculated by the Student t-test.