Detection of Biofilm Disassembly Through a Dispersion Assay

Begin with a multi-well plate containing a bacterial biofilm — bacteria encapsulated within the protective extracellular polymeric substance or EPS.

Aspirate the medium to remove non-adherent bacteria and wash.

Treat bio-film-containing wells with either buffer alone, buffer with glucose, buffer with bile salts, or buffer with glucose and bile salts.

Buffer and glucose treatments support the release of bacterial enzymes that degrade the biofilm's EPS.

This results in the disassembly of the biofilm, releasing the bacteria.

While bile salts and a combination of bile salts and glucose treatments mimic stress conditions and retain bacteria within the protective EPS.

Transfer the supernatant from each well to a fresh multi-well plate.

Spot diluted supernatants on agar plates and incubate.

Enumerate bacterial colonies and determine the bacterial dispersion for each treatment.

A higher dispersion with buffer and glucose treatments compared to bile salts treatments indicates the disassembly of the biofilm.

Record the OD₆₀₀ values from the overnight biofilm plate on the plate reader by setting the control well as blank. Next, aspirate the culture medium using a vacuum line without disturbing the adherent population on the plastic surface. Gently wash the wells twice with 200 microliters of sterile PBS.

After washing with PBS at 130 microliters of the prewarmed PBS, PBS with glucose, PBS with bile salts, and PBS with bile salts and glucose to each of the three wells. Then incubate the plate at 37 degrees Celsius for 30 minutes. After 30 minutes, remove the plate from the incubator. Pipette the supernatant out to a fresh sterile 96-well plate.

Finally, prepare 1 to 10 serial dilutions of the supernatant with PBS in the 96-well plate or in a dilution block. Then use a multichannel pipette to transfer 5 microliters of each dilution on LB agar plates. Incubate the plates at 37 degrees Celsius overnight. The next day, count the number of colonies.