Use bc 0-20 dynes plate
If running experiment at 37°C, warm the plate and media to 37°C prior to starting the experiment
Use bacterial culture at OD600 = 0.5-0.8
Prime low-shear (0-20dyn) bcxx 48-well plate
(pre-warmed and placed on heater insert or in heated chamber)
- Load at least 40uL of warm media to the outlet well.
- Place the interface cover on the plate and ensure a snug/tight seal
- Perfuse from outlet to inlet (if using Autorun mode, set flow direction as reverse) at 20dyn/cm2 for 40 seconds.
- Direction of flow
- There should be now a small bead of media in each inlet and out let well.
Seeding the culture
- Do not allow them to dry. Add 100uL of media to the inlet well.
- Load 20uL of the bacterial culture (OD600 = 0.5-0.8) to the outlet well
- Perfuse at 2 dynes from outlet to inlet well for 5 seconds
- Incubate at 37°C for 1 hour
- Remove as much as possible bacterial culture/media from outlet well
- Rinse the outlet well with media
- Load 1mL of fresh media to the inlet well
- Perfuse at 1 dyne for 5 minutes
- Wash/rinse the outlet well with media
Acquiring time lapse data of biofilm formation
- Ensure that the inlet well have about 1 mL of fresh media
- Add 100uL of fresh media to the outlet well
- Perfuse at 0.15-0.2 dyne for 16 to 24 hours
- Acquire data every 20 minutes under 10X or higher magnification, ≥ 2 data points (positions) per channel preferred.