A Peg Plate Biofilm Assay for Screening Antibacterial Agents

Take a fresh 96-well plate, and add 100 microliters of CRPMI equilibrated to room temperature to each well of rows B to H. Dilute up to four test compounds separately in 1.0 milliliter of CRPMI to 2x the desired final concentration. Add 200 microliters of compound one in wells A1 to A3, 200 microliters of compound two in wells A4 to A6, 200 microliters of compound three in wells A7 to A9, and 200 microliters of compound four in wells A10 to A12.

Serially dilute the test compounds using a multichannel pipette. Begin by transferring 100 microliters from row A to row B, and mix. In that manner, continue to transfer 100 microliters well to well. Mix after each transfer and stop in row F. After mixing, expel 100 microliters from row F into a waste container. Do not transfer any liquid to rows G and H. Finally, add 100 microliters of CRPMI to each well of the plate using a multichannel pipette, to bring the final volume to 200 microliters.

Add 200 microliters of phosphate-buffered saline to a fresh sterile 96-well plate that features wells with dimensions identical to those of the biofilm initiation plate. Next, remove the plastic bag and paraffin film from the incubated biofilm initiation plate. Lift the lid straight up, avoiding contact between the pegs and the plate bottom as this may damage the biofilm. Keep the bottom part for subsequent OD₆₀₀ analysis.

Rinse off planktonic cells by placing the peg lid onto the plate containing PBS. Submerge pegs for 5 seconds, lift them out of PBS, and submerge once more, being careful not to hit the pegs against the well sides. Place the rinsed peg lid into the challenge plate. Avoid unnecessary contact between pegs and the bottom plate, as this will damage the biofilm leading to inconsistent results.

Seal the plate with paraffin film, and place into a sealable plastic bag as before. Incubate the plate without shaking for 24 hours at 37 degrees Celsius in a 5% carbon dioxide incubator. Take the bottom of the biofilm initiation plate and resuspend the bacteria in each well using a multichannel pipette. Measure the OD₆₀₀ with a suitable microplate reader to confirm growth occurring in all wells, but the sterility controls in row H.

Use a plate reader equipped with an incubation chamber, and capable of taking kinetic fluorescence readings for detecting resazurin conversion. Set up a 24-hour kinetic fluorescence measurement using a 530-nanometer excitation and 590-nanometer emission. Set the chamber temperature to 37 degrees Celsius and have the plate read every 20 minutes.

Set the plate reader to measure from the bottom of the plate, according to the manufacturer's instructions. Prepare to wash the plate by adding 200 microliters of PBS to each well of a sterile 96-well plate with a multichannel pipette. Then, prepare biofilm recovery medium by adding 400 microliters of 0.8 milligram per milliliter resazurin stock solution to 20 milliliters of CRPMI medium, to a final concentration of 16 micrograms per milliliter resazurin.

Next, add 150 microliters of biofilm recovery medium with a multichannel pipette, to each well of a fresh 96-well plate. Transfer the challenge plate from the incubator into a biosafety cabinet, and carefully remove the plastic bag and sealing film. Remove the peg lid from the challenge plate and rinse off planktonic cells in PBS as before, keeping the bottom of the challenge plate for subsequent OD₆₀₀ analysis.

After rinsing, place the peg lid into the plate bottom containing the biofilm recovery medium. Wrap the side of the plate with paraffin film, being careful not to obstruct the bottom of the perimeter wells. Immediately after wrapping the plate, place it on the plate reader, and start a kinetic read over a 24-hour period by initiating the previously made resazurin kinetic protocol.