Crystal violet assay for biofilm formation in microtiter plates

Overnight cultures of P. aeruginosa grown to stationary phase in NSLB supplemented with 30 μg/mL GEN, where appropriate, were diluted to a final OD_600nm of 0.005 in 1 mL of NSLB. Cultures containing PBADpel-derived strains were supplemented with 0.5% (w/v) L-(+)-arabinose to induce pel expression. Cultures containing strains expressing pPSV39 were supplemented with 30 μg/mL GEN for plasmid maintenance. 100 μL of the normalized cultures were added to the wells of a Corning CellBind 96-well microtiter plate (n = 5 for each strain examined) and incubated for 20 h at 25 °C statically. Non-adherent biomass was removed by washing the wells three times with water, and the remaining adherent biomass was stained with 150 μL of 0.1% (w/v) crystal violet for 10 min with agitation at room temperature. To remove excess stain, the wells were washed three times with water and the residual stain was solubilized by adding 200 μL of anhydrous ethanol to each well and left to incubate for 10 min with agitation at room temperature. 40 μL of the solubilized crystal violet solution was transferred to a plate containing 160 μL of anhydrous ethanol, and the absorbance was measured at 595 nm to quantify the biofilm biomass.