Quantifying Bacterial Biofilm Formation

The biofilm of WT, Δhfq, and hfq complementing strains of P. ananatis was quantified as previously described by Santander and Biosca (2017) (link) with slight modifications. An aliquot of 160 μl broth culture diluted to an OD_600nm of 0.5 in half-strength LB [0.5% (w/v) NaCl, 0.5% (w/v) tryptone, and 0.25% (w/v) yeast extract; pH 7.2] was made into each well of a polystyrene 96-well microplate (Nunc^TM MicroWell^TM, Thermo Scientific, Waltham, MA, United States) and incubated for 24 h under static conditions. Eight replicates per P. ananatis strain were included in each experiment with sterile half-strength LB broth serving as a negative control. Thereafter, the inoculated 96-well plates were inverted to remove the excess LB broth, air-dried, and incubated at 60°C for 40 min to heat-fix the biofilms. The biofilms were stained with 1% crystal violet (220 μl) for 15 min before being rinsed with distilled water. After rinsing and invert-air-drying the microplate, 220 μl of ethanol:acetone in 8:2 ratio was added to the wells to solubilize the crystal violet dye for 20 min at room temperature. The solubilized biofilm was measured at OD₆₀₀ using Safire Microplate Reader (Tecan, Research Triangle Park, NC, United States), and this assay was repeated three times.