Masterflex c l tubing pump

Biofilm formation patterns under flow conditions were observed by operating a drip-flow reactor (DFR-110; BioSurface Technologies, Bozeman, MT, USA) using glass slides. Overnight cultured PA14 strain (OD at 595 nm = 0.05) diluted in AB medium with either compound 30 (1 μM) or tobramycin (0.63 μM) treatments (no treatment, single treatments of compound 30 or tobramycin, and combined treatment of compound 30 and tobramycin) was continuously fed into the reactor using a peristaltic pump (Masterflex C/L tubing pumps; Cole-Parmer, Vernon Hills, IL, USA) at 0.3 ml/min, and the reactor was operated at 37°C for 48 h. The biofilm cells formed on the slides were washed with PBS and stained with 2 μg/ml 4′,6-diamidino-2-phenylindole (DAPI) solution (Carl Roth) for 10 min. After washing the biofilm cells with DW, the stained cells were observed via confocal laser scanning microscopy (CLSM) (Carl Zeiss LSM700, Jena, Germany) using a 20× objective lens (W N-Achroplan × 20/0.5W [DIC] M27) under blue fluorescence light (excitation wavelength, 350 nm; emission wavelength, 470 nm). Biofilm volume and thickness were analyzed using Zen 2011 software (Carl Zeiss) and quantified using Comstat2 in the ImageJ program (51 (link)) based on the CLSM images.

Biofilm formation patterns under flow conditions were observed by operating a drip-flow reactor (DFR-110; BioSurface Technologies, Bozeman, MT, USA) using glass slides. Overnight cultured PA14 strain (OD at 595 nm = 0.05) diluted in AB medium with either compound 30 (1 μM) or tobramycin (0.63 μM) treatments (no treatment, single treatments of compound 30 or tobramycin, and combined treatment of compound 30 and tobramycin) was continuously fed into the reactor using a peristaltic pump (Masterflex C/L tubing pumps; Cole-Parmer, Vernon Hills, IL, USA) at 0.3 ml/min, and the reactor was operated at 37°C for 48 h. The biofilm cells formed on the slides were washed with PBS and stained with 2 μg/ml 4′,6-diamidino-2-phenylindole (DAPI) solution (Carl Roth) for 10 min. After washing the biofilm cells with DW, the stained cells were observed via confocal laser scanning microscopy (CLSM) (Carl Zeiss LSM700, Jena, Germany) using a 20× objective lens (W N-Achroplan × 20/0.5W [DIC] M27) under blue fluorescence light (excitation wavelength, 350 nm; emission wavelength, 470 nm). Biofilm volume and thickness were analyzed using Zen 2011 software (Carl Zeiss) and quantified using Comstat2 in the ImageJ program (51 (link)) based on the CLSM images.

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).