Bacterial Biofilm Protocol

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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)

  1. Load at least 40uL of warm media to the outlet well.
  2. Place the interface cover on the plate and ensure a snug/tight seal
  3. Perfuse from outlet to inlet (if using Autorun mode, set flow direction as reverse) at 20dyn/cm2 for 40 seconds.
  4. Direction of flow
  5. There should be now a small bead of media in each inlet and out let well.

Seeding the culture

  1. Do not allow them to dry. Add 100uL of media to the inlet well.
  2. Load 20uL of the bacterial culture (OD600 = 0.5-0.8) to the outlet well
  3. Perfuse at 2 dynes from outlet to inlet well for 5 seconds
  4. Incubate at 37°C for 1 hour
  5. Remove as much as possible bacterial culture/media from outlet well
  6. Rinse the outlet well with media
  7. Load 1mL of fresh media to the inlet well
  8. Perfuse at 1 dyne for 5 minutes
  9. Wash/rinse the outlet well with media

Acquiring time lapse data of biofilm formation

  1. Ensure that the inlet well have about 1 mL of fresh media
  2. Add 100uL of fresh media to the outlet well
  3. Perfuse at 0.15-0.2 dyne for 16 to 24 hours
  4. Acquire data every 20 minutes under 10X or higher magnification, ≥ 2 data points (positions) per channel preferred.