Crystal Violet Biofilm Assay

The Crystal Violet Biofilm Assay is a widely used method to quantify bacterial biofilm formation in vitro. This assay measures the adherence and accumulation of bacterial cells on a surface by staining the biofilm with crystal violet dye and then quantifying the dye retained by the biofilm.

 

Here's a basic protocol for performing a Crystal Violet Biofilm Assay:

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Materials Needed:

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Bacterial culture (liquid culture of the strain you want to test)
Growth medium appropriate for your bacteria
Sterile 96-well microtiter plates
Crystal violet stain solution (0.1% w/v in water)
Ethanol or isopropanol
Microplate reader or spectrophotometer
Pipettes and tips
Shaker or incubator
Sterile saline solution or phosphate-buffered saline (PBS)
Sterile water

 

Procedure:

 

Bacterial Culture:
a. Prepare an overnight culture of the bacterial strain you want to test in a suitable growth medium.
b. Adjust the bacterial density to a known optical density (OD) or concentration.

 

Inoculation of Biofilm Formation:
a. Dilute the overnight culture in fresh growth medium to the desired concentration.
b. Add the diluted culture to the wells of a sterile 96-well microtiter plate. Each well should contain a uniform volume of culture.

 

Biofilm Growth:
a. Incubate the microtiter plate at the appropriate temperature for the bacterial strain (usually 37°C) for a suitable period to allow biofilm formation (e.g., 24-48 hours). You can use a shaker or static incubation, depending on the experimental setup.

 

Removal of Non-Adherent Cells:
a. Carefully remove the culture from the wells, making sure not to disturb the biofilm.
b. Wash the wells gently with sterile saline solution or PBS to remove any non-adherent cells.

 

Fixation of Biofilm:
a. Air-dry the plate or use a gentle heat source to evaporate any remaining liquid.
b. Fix the biofilm by gently adding ethanol or isopropanol to each well to cover the biofilm. Incubate for around 15 minutes.

 

Staining with Crystal Violet:
a. Pour off the ethanol or isopropanol and gently rinse the wells with sterile water to remove excess fixative.
b. Add crystal violet stain solution to each well, covering the biofilm completely. Incubate for around 15-20 minutes.

 

Washing and Drying:
a. Gently pour off the crystal violet solution and rinse the wells under gentle running water or using a wash bottle.
b. Allow the plate to air-dry completely.

 

Quantification:
a. Add a solvent such as acetic acid or dimethyl sulfoxide (DMSO) to solubilize the crystal violet dye from the biofilm.
b. Measure the absorbance of the solubilized dye in each well using a microplate reader or spectrophotometer at an appropriate wavelength (usually around 570 nm).

 

Data Analysis:
a. The absorbance values are proportional to the amount of biofilm formed. You can compare biofilm

formation between different strains or experimental conditions.

 

Always follow safety guidelines, sterile techniques, and your lab's protocols while conducting experiments. The protocol can be adapted to your specific bacterial strain, growth conditions, and experimental goals.