4 6 diamidino 2 phenylindole dapi solution

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.