An Assay to Study Bile-Salt Induced Biofilm Formation through EPS Matrix Detection

1. Preparation of Reagents

1. Bile salts (BS) 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 sterilize using a 0.22 µm filter.
3. Concanavalin A (ConA) conjugated to fluorescein isothiocyanate (FITC): Reconstitute the stock in 1x PBS. Dilute the 10 mg concentrated stock with 400 μL of 1x PBS to a final concentration of 25 µg/mL, and protect from light.
4. PBS + Glucose: Dissolve 0.2 g glucose in 10 mL 1x PBS (2% w/v glucose final). Filter sterilize using a 0.22 µm filter. Make fresh on the day of use.
5. 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.
6. 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 sterilize using a 0.22 µm filter. Make fresh on the day of use.
7. Prepare LB agar plates.
8. Formaldehyde/glutaraldehyde fix: Add 810 µL formaldehyde (37% stock solution, 3% final concentration) and 125 µL glutaraldehyde (25% stock solution, 0.25% final concentration) to 14 mL 1x PBS. Mix thoroughly and store at 4 °C. The fix should be cold for proper use.
   Caution: The fix is toxic and requires hazardous waste disposal.
9. Antifade mountant solution: Use antifade mountant solution containing 4,6-diamidino-2-phenylindole (DAPI) stain to inhibit photobleaching of immunofluorescent microscopy samples while fluorescently staining the DNA of the bacteria.

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 restreaked from freezer stocks every 2 to 4 weeks, and maintained on plates no more than 2 weeks old.

3. EPS Matrix Detection

NOTE: These complimentary assays quantify and visualize the EPS. In both, EPS is detected using a lectin to bind polysaccharides. The fluorescently-conjugated protein allows quantification.

1. Semi-quantitative detection of EPS
   1. Set up two 1.5 mL tubes. Label with TSB or TSB + BS.
   2. Add 1 mL of TSB or TSB + BS to the respective tubes.
   3. Inoculate tubes with 20 µL of overnight culture (a 1:50 dilution).
   4. In a sterile, black, 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 medium type to be tested. Add 130 µL/well of inoculated culture to the wells, and repeat until all experimental conditions are plated in triplicate.
   5. Incubate for 4 – 24 h at 37 °C statically.
   6. Transfer the culture medium to a clear 96-well plate with a pipette, ideally a multichannel pipette. Be cautious to collect all the culture medium without disrupting the adherent population and/or the EPS located on the plastic surface. Set aside.
   7. Fix the black plate for 15 min at RT using 200 µL/well of the formaldehyde/glutaraldehyde in 1x PBS.
   8. While the adherent population is fixing, evaluate the supernatant fraction from step 1.1.6. Using a plate reader, record the OD_600. Set the control wells as 'blank.' Confirm the control medium is clear with no evidence of turbidity. If any turbidity is detected, discard the experiment.
      1. Alternatively, the entire assay can be performed in a black clear bottom plate. In those circumstances, the OD₆₀₀ values can be recorded and the culture medium can be subsequently discarded prior to proceeding to step 3.1.7. The OD₆₀₀ values can be used to normalize the data in case there are significant differences in growth rate between bacterial strains.
   9. Remove the fix and dispose in the hazardous waste.
   10. Gently wash the wells twice with 200 µL/well of sterile PBS. Remove the PBS wash using the vacuum line by gently tilting the plate and slowly aspirating the wash at the lower edge of the well.
   11. Add 150 µL/well of 25 µg/mL ConA-FITC, and incubate for 15 min at RT.
   12. Gently wash twice with 200 µL of PBS.
   13. Add 150 µL of PBS to each well. Record the fluorescence at 488 nm.