Prepare the cells of an overnight cultures grown at 30°C in YPD medium to a final OD600 of 0.5
Priming the Plate
- Slowly add to the center of the outlet wells 100uL pre-warmed PBS or media (for 48-well plates, these are the even number columns) avoid forming bubbles.
- Apply pressure to the outlet wells at 10 dyne/ cm2 for 30 seconds. Check for the beading of liquid in the inlet wells (roughly about 5uL in size).
- If needed, repeat the prime process. Check under microscope for formation of bubbles in the channel viewing areas and also at the channel openings at the inlet and outlet.
- Remove excess media from the outlet wells, leaving a small amount remaining at the inner centers. DO NOT allow them to dry or bubbles will form. If bubbles form at the openings of the channels, use a pipet and gently dislodge them with GENTLE drawing up and down of the media, going around the edge of the inner circles. Check under microscope to make sure they are completely dislodged.
- Slowly add to the center of the inlet wells 100uL pre-warmed PBS or media (for 48-well plates, these are the odd number columns) avoid forming bubbles.
Seeding the Channels
- Add 5-10uL of the prepared cells to the outlet wells
- Apply pressure to the outlet wells at 2 dyne/cm2 for 3 seconds and allow cells to adhere at 37°C for 20 minutes.
- Apply pressure at 1 dyne/cm2 for 5 minutes to the inlet wells to wash off away non-adhering cells.
- Wash outlet wells with 100uL of media. Remove the wash, leaving a small amount remaining at the inner centers. DO NOT allow them to dry or bubbles will form.
- If necessary also remove the media from the inlet wells and add to the center of the inlet wells 1mL of fresh media.
- Apply pressure to the inlet wells at 0.5 dyne/cm2 and allow the biofilm to form at 37°C for 12 hours.
- Remove media from the outlet and replenish with fresh media to the inlet wells every 12 hours