Flow cell biofilm of PAO1-N growth using BioFlux
This protocol is extracted from research article:
Fluorescent nanosensors reveal dynamic pH gradients during biofilm formation
NPJ Biofilms Microbiomes, Jun 17, 2021; DOI: 10.1038/s41522-021-00221-8

Overnight cultures of P. aeruginosa grown in LB were used to set up liquid cultures in 5 mL LB using 100 µL of the overnight culture. The cultures were grown to 0.4–0.8 OD600 and normalised to an 0.05 OD600 in 1 mL of minimal medium (M9 succinate). Nanosensors were suspended at 5 mg mL−1 in selected medium and filter sterilised using 0.22 µm PES filters. The BioFlux flow cell (BioFlux 200 48-well low shear plate, 0–20 dynes cm2) was prepared according to the manufacturer’s instructions (Fluxion Biosciences, BioFlux System). For the seeding of the cells, 50 µL of the normalised cells was used with a seeding time of 45–60 min. After the seeding, 1 mL of the prepared medium with or without nanosensors was used as flow medium. The flow was set to 0.25 dyn cm−2 and the system was run for 20–24 h. Time-lapse imaging was applied using a Nikon widefield microscope with brightfield and fluorescence channels (OG-FAM excitation = 460 nm, TAMRA excitation = 550 nm). Images were taken every 15 min for 16 h.84

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