Detection of Biofilm Disassembly Through a Dispersion Assay

1. Preparation of Reagents

1. Bile salts medium: To prepare tryptic soy broth (TSB) containing 0.4% bile salts (weight/volume), resuspend 200 mg of bile salts in 50 mL autoclaved TSB. Filter sterilize using a 0.22 µm filter. Make fresh medium weekly.
   NOTES: The bile salts routinely used is a 1:1 mixture of sodium cholate and sodium deoxycholate isolated from ovine and bovine gallbladders. As demonstrated previously, the presence of glucose was required for bile salt-induced biofilm formation. TSB has added glucose relative to Luria-Bertani (LB) broth; and therefore, was sufficient to induce biofilm formation in Shigella and the other enteric pathogens analyzed. Depending on the bacteria to be analyzed, different glucose concentrations or different sugar requirements might be needed.
2. 0.5% w/v crystal violet in water: Dissolve 2.5 g of crystal violet in 500 mL distilled water. Filter sterilizes using a 0.22 µm filter.
3. Phosphate-buffered saline (PBS) + Glucose: Dissolve 0.2 g glucose in 10 mL 1x PBS (2% w/v glucose final). Filter sterilizes using a 0.22 µm filter. Make fresh on the day of use.
4. PBS + Bile Salts: Dissolve 40 mg in 10 mL 1x PBS (0.4% w/v bile salts final). Filter sterilize using a 0.22 µm filter. Make fresh on the day of use.
5. PBS + glucose and bile salts: Dissolve 40 mg bile salts and 0.2 g glucose in 10 mL 1x PBS (0.4% w/v bile salts and 2% w/v glucose final). Filter sterilizes using a 0.22 µm filter. Make fresh on the day of use.
6. Prepare LB agar plates.

2. Preparation of Bacteria

1. Grow overnight cultures of the bacterial strains to be tested by inoculating 3 mL of TSB with a single, well-isolated colony in a sterile culture tube. Incubate at 37 °C with shaking at 225 rpm for overnight incubation (16 – 24 h).      
   NOTE: Strains should be re-streaked from freezer stocks every 2 to 4 weeks and maintained on plates no more than 2 weeks old.

3. Dispersion Assay

NOTE: In this assay, the disassembly of biofilm through bacterial dispersion is detected. Here, mature biofilms are established and subsequently (usually the next day), the media are replaced with PBS or supplemented PBS. The supernatant component is then assessed to quantitate the number of bacteria that have dissociated from the biofilm.

1. Set up two 1.5 mL tubes. Label with TSB or TSB + BS (bile salts).
2. Add 1 mL of TSB or TSB + BS to the respective tubes.
3. Inoculate tubes with 20 µL of overnight culture (at a 1:50 dilution).
4. In a sterile, clear, flat-bottomed, tissue culture-treated 96-well plate, add 130 µL/well of uninoculated control media to three wells to serve as the blank control. Set up three control wells for each media type to be tested.
5. Add 130 µL/well of inoculated culture to three wells and repeat until all experimental conditions are plated in triplicate.
6. Incubate the plate for 4 – 24 h at 37 °C statically.
7. Before continuing, warm the PBS, PBS + glucose, PBS + bile salts, and PBS + bile salts + glucose to 37 °C. Prepare these reagents fresh daily.
8. Using a plate reader, record the optical density, OD₆₀₀. Set the control wells as 'blank.' Confirm the medium is clear with no evidence of turbidity. If any turbidity is detected, discard the experiment. The OD₆₀₀ values can be used to normalize the data if there are significant differences in growth rate between bacterial strains.
9. Remove the culture medium using a vacuum line. Be sure to collect all the culture medium without disrupting the adherent population located on the plastic surface.
10. Gently wash the wells twice with 200 µL/well of sterile PBS. Remove the PBS wash using the vacuum line.
11. Replace the wash with 130 µL/well of the following into three wells each: PBS, PBS + glucose, PBS + bile salts, and PBS + bile salts + glucose. These reagents must be pre-warmed to 37 °C.
12. Incubate the plate for 30 min at 37 °C.
13. Carefully remove the plate from the 37 °C incubator. Transfer the supernatants to a fresh, sterile 96-well plate.
14. Using a 96-well plate or dilution block, prepare 10-fold (1:10) serial dilutions of the supernatant into sterile PBS.    
    NOTE: The dilution series should range from undiluted to 10^-6 depending on the bacterial strain to be tested. Pilot experiments can be performed to determine the optimal dilution range.
15. Spot plate 5 µL of each dilution onto LB agar using a multichannel pipette. Incubate at 37 °C overnight. Count colonies on the next day and account for the dilution factor when determining the recovery colony forming units (CFU). To calculate the percent dispersion relative to the 1x PBS control (set at 100%), divide the recovery CFU from each treatment condition by the recovery CFU from the 1x PBS control sample.
    NOTE: Routine media plates for the bacterial strain of interest can also be used. For example, Shigella can be plated on Congo red plates. Ensure the plates are dry for appropriate spot-plating techniques.