Evaluating Pseudomonas Biofilm Formation

P. aeruginosa biofilms were formed in a drip-flow biofilm reactor (DFR-110, BioSurface, MT, USA). Glass slides were dipped into a petri dish containing 2 ml of P. aeruginosa culture (OD at 595 nm = 1.5) and 18 ml of fresh AB medium to attach cells onto the slides and were incubated at 37°C for 24 h. The slides were then inserted into the drip-flow reactor system. Fresh AB medium with either 0 or 10 μM 6-gingerol was continuously fed into the reactor using a peristaltic pump (Masterflex C/L tubing pumps, Cole-Parmer, IL, USA) at 50 ml·h⁻¹. The reactor operated at 37°C for 24 h. After stopping the feed, the suspended cells on the slide were carefully removed with phosphate-buffered saline (pH 7.2). The biofilm cells were stained with DAPI solution (Carl Roth) for 20 min. The biofilm cells were then observed using CLSM (Carl Zeiss LSM700, Jena, Germany) based on a previous study23 (link). Confocal images of DAPI-stained biofilm cells were observed under blue fluorescence light (excitation wavelength: 350 nm, emission wavelength: 470 nm) using a 40× objective lens to evaluate the height and density of the biofilms (C-Apochromat 40×/1.20 W Korr M27, Carl Zeiss). The observed CLSM images were analyzed by the Zen 2011 program (Carl Zeiss).