O'Toole-Kolter Biofilm Adhesion Assay

Contact: Mary Jo Kirisits

N.B. Crystal violet is a mutagen. Therefore, always wear gloves when using it, and minimize your exposure to it. Dispose of crystal violet waste through EHS.

1. Add 100 μL of medium to the microtitre well. It is good to use outside wells for the assay, but do not use the corner wells.  These tend to dry out faster.
2. Add 1 μL of a late log culture (OD_600nm = 0.7) to the well.
3. Run the samples in triplicate.
4. Run two types of controls.
   1. Control #1: Run 100 μL of medium, without inoculation.
   2. Control #2: Run the wild type strain to have a basis of comparison for other strains.
5. Incubate statically at the desired temperature and time. Standard incubation time is around 10 hours; 24-hour incubations are on the long side.  Putting a paper towel underneath the plate seems to aid in maintaining humidity in the wells.
6. Remove the plate from the incubator, and pour out the medium.
7. Immerse the plate in tap water (in a small autoclave tray), and then pour the water out.
8. Dry the plate by tapping it upside down on a paper towel.
9. Add 125 μL of 1% (w/v) crystal violet to each well. Stain for 15 minutes. Pour out the stain into a plastic container from EHS.  Rinse with water 3-4 times.  The rinse water should also be poured into the crystal violet waste container. 
10. Add 200 μL of 95% ethanol to solubilize the crystal violet in the cells. Aspirate gently 3 times.
11. Take 100 μL of this, and move it to a new well. Read the absorbance of the 100-μL aliquot at 600 nm.
12. Subtract the blank (no inoculum) from each sample absorbance.