A Solid Phase Adherence Assay to Assess Biofilm Formation

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 sterilizes 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.

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. Solid-phase Adherence Assay

NOTE: This assay quantifies adherent bacteria using a 96-well plate method. Bacteria are grown statically in flat bottom plates. Washing is performed to remove non-adherent bacteria and adherent bacteria are stained with crystal violet. The crystal violet stain binds peptidoglycan in the bacterial cell wall and can be solubilized using ethanol. The number of adherent bacteria is determined based on crystal violet retention.

1. Set up two 1.5 mL tubes. Label with TSB or TSB + bile salts (BS).
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 (TSB and TSB + BS) to be tested.
5. Add 130 µL/well of inoculated culture into three wells and repeat until all experimental conditions are plated in triplicate.
6. Incubate for 4 – 24 h at 37 °C statically.
7. Using a plate reader, record the OD₆₀₀. 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. The OD₆₀₀ values can be used to normalize the data if there are significant differences in growth rate between bacterial strains.
8. Remove the culture medium using a vacuum line by gently tilting the plate and slowly aspirating the medium at the lower edge of the well. Be sure to collect all the culture medium without disrupting the adherent bacterial population located on the plastic surface. If extracellular polysaccharide (EPS) matrix was produced during the incubation time, the matrix will be visualized as a white precipitate. Do not disturb the EPS matrix.
9. Gently wash the wells once with 200 µL sterile phosphate-buffered saline (PBS). Remove the PBS wash using the vacuum line.
10. Invert the plate to dry. Allow a minimum of 20 min to dry.      
    NOTE: Since the biofilm must be thoroughly dried before staining, the protocol can be paused at this step for a few hours or even overnight. The added drying time will not alter the staining procedure while incomplete drying will affect quantification of the results and reproducibility.
11. Add 150 µL of 0.5% crystal violet to each experimental and control well.
12. Incubate for 5 min at room temperature (RT).
13. Wash the wells once with 400 µL of distilled water. The added volume helps to remove residual crystal violet stain from the sides of the wells. Remove the wash with the vacuum line.
14. Wash the wells five times with 200 µL of distilled water. Remove the wash with the vacuum line.
    NOTE: Thorough washing is important for quantification. When the blank wells are clear from the distilled water and do not contain any residual crystal violet stain, proceed to the next step.
15. Invert the plate to dry, protected from light. Ensure complete drying of the plate as noted above.
16. Destain the wells with 200 µL of 95% ethanol. Incubate the plate on the shaker for 30 min. To avoid evaporation, particularly at higher temperatures, perform this step at 4 °C.
17. Using a plate reader, record the optical density at 540 (OD₅₄₀).       
    NOTE: The wavelength for maximum absorbance of crystal violet is near 590 nm. The literature reports a range from OD₅₄₀ – OD₅₉₅ for crystal violet absorbance; thus choose a wavelength available based on the plate reader.