μ slide 6 0.5 glass bottom slide

Erwinia amylovora strains expressing gfp from pMP2444 (Stuurman et al., 2000 (link)) were grown for 18 h and equilibrated to an OD₆₀₀ of 0.5. Flow channels in a μ-Slide VI 0.5 glass bottom slide (Ibidi, Martinsried, Germany) were pre-conditioned with LB for 24 h prior to conducting the assay. Normalized cultures were inoculated into individual flow channels and incubated at 24°C for 1 h, following which all inoculum from the channels was flushed out with 0.5X LB. This was followed by either a static incubation or incubation under flow (with 0.5X LB) generated using a peristaltic pump (Ismatec REGLO Digital 4-CH pump (Cole-Parmer; Vernon Hills, IL, United States) for 5 h. Biofilms developed in the flow channels were visualized using a Zeiss 510 Meta ConfoCor3 LSM confocal laser scanning microscope (Carl Zeiss Microimaging, Jena, Germany). Imaging was performed by acquiring Z-stacks of fluorescent bacterial cells in the individual flow channels. ImageJ software (Schneider et al., 2012 (link)) was used to obtain three-dimensional representations of the biofilm distributions.

To monitor initial surface interaction and attachment, E. amylovora strains expressing pMP2444::gfp [13 (link)] were grown for 18 h at 28°C and normalized to an OD₆₀₀ of 0.5. A total of 1 ml of inoculum for each strain was introduced into a flow cell chamber in a μ-Slide VI 0.5 glass bottom slide (Ibidi, Martinsried, Germany). Immediately, the base of the flow chamber was repeatedly imaged using a Olympus FlouView 1000 confocal laser scanning microscope (Olympus, MA, USA). Images were acquired for up to 1 h or until the frame was saturated with fluorescent cell signals. Following this, the flow cell chamber was flushed with 5 ml of 0.5X phosphate buffered saline (PBS). To evaluate biofilm formation, following the initial attachment incubation, the flow chamber was subjected to flow (0.5X LB) using a peristaltic pump (Ismatec REGLO Digital 4-CH pump) (Cole-Parmer IL, USA) for 5 h. Fluorescent Z-stacked images were acquired to measure overall attachment and biofilm levels in the flow cell chambers [13 (link)]. ImageJ software was used to invert the color on the images, and the RBG plugin was used to process these images and to graph the GFP signal intensity profile for the Z-stacked images [62 (link)].