Live/dead staining and confocal microscopy.

Glass and SBS-functionalized devices subjected to rinsing challenge are stained by a Live/Dead BacLight bacterial viability kit (Thermo Fischer). A staining solution containing a final concentration of 3.34μM Syto9 and 20μM propidium iodide are prepared in PBS and flowed through the devices at a 10μL/min flow rate for 40min in the dark. Devices are imaged with a Stellaris confocal microscope (Leica) for green (500-550nm) and red (590-650nm) signals corresponding to live and dead populations at the bottom surface. The resulting single channel grey-scale images are quantified for the number of fluorescent cells using FIJI “find maxima”, which identifies and quantifies pixels with a local maximal intensity, signifying a fluorescently stained cell. The threshold is set at 25. The percentage of live bacteria is calculated as the percentage of live-stained cells divided by the total-stained cells that are at the surface at the time of imaging. Data collected from the widest zone of the microfluidics device (shear rate at 10⁴ s⁻¹) from three experiments.