Biofilm formation

PAO1 from −80°C frozen stocks were streaked on a trichostatin A plate. The plate was placed in an incubator (37°C) overnight until single colonies were formed. A single colony was picked and inoculated into lysogeny broth (LB) medium. The inoculated medium was incubated overnight at 37°C to stationary phase in a shaker (225 rpm). The overnight suspension was diluted 100 times in fresh LB medium and incubated at 37°C on a shaker (225 rpm) until the optical density reached 0.2. Bacterial suspension of 150 μl was then added into each well of the surface-modified 96-well microplate. The microplates were incubated (37°C) for 24 hours to make sure biofilms were mature. The liquid was then transferred into a new microplate. The relative pyoverdine concentration can be obtained by exposure of the culture to a 405-nm excitation light and recording the fluorescence intensity at 460 nm. The rest of the liquid culture and loosely attached bacteria were removed from each well by vigorously washing each well three to four times with deionized water (dH₂O). Biofilms were then stained by 175 μl of dH₂O with 0.1 weight % crystal violet for 10 min. Then, the crystal violet solution was removed by washing each well three to four times until the liquid in each well became a clear solution. The microplate was dried in the air at room temperature for 24 hours to remove residual water in each well. The biofilm formed was subsequently quantified. A total of 200 μl of acetic acid solution (30 v/v%) was added into each well to release the absorbed crystal violet and the relative amount of absorbed crystal violet was quantified spectrophotometrically by measuring the OD₅₇₀ using a microplate reader (Infinite M1000 Pro, Tecan).

To obtain SEM images of the biofilm, after incubating the microplates for 24 hours, the microplates with biofilm on the sidewall were treated with 0.05 M cacodylate buffer containing 2% glutaraldehyde and 1% osmium tetroxide for fixation. Samples were then dehydrated using critical point drying. The SEM images were obtained using Zeiss Gemini 500 with an acceleration voltage of 3 kV. Gold was sputter-coated onto all samples before imaging.