A biofilm is a collective of microorganisms, such as bacteria, that adhere to a surface and to each other. These communities are encased in a self-produced slimy, protective layer. The crystal violet biofilm assay is a foundational laboratory method used to determine the amount of biofilm formed on a surface. It is a straightforward and widely adopted technique for quantifying the total biomass of these microbial structures.
The Principle Behind the Assay
The primary goal of the crystal violet assay is to measure the total biomass of a biofilm. This biomass includes not only the microbial cells but also the extracellular polymeric substance (EPS) they secrete. This EPS is a complex matrix of sugars, proteins, and DNA that holds the community together and attaches it to a surface. Crystal violet is a simple, dark purple dye that works by adhering to negatively charged molecules.
Since components of both bacterial cell walls and the EPS matrix carry a negative charge, the dye effectively stains the entire biofilm structure. A larger, denser biofilm will retain more crystal violet. Consequently, the intensity of the purple color is directly proportional to the total amount of biofilm biomass present.
This method offers an indirect way to quantify the biofilm’s overall size. It provides a snapshot of the total structure, not just the number of living cells. The ease and low cost of this technique make it a common tool in microbiology.
The Step-by-Step Laboratory Procedure
The assay begins with growing the biofilm in a 96-well microtiter plate, a tray with many small wells. A liquid nutrient broth with bacteria is added to the wells, and the plate is incubated at 37°C for 24 to 48 hours. During this incubation, bacteria attach to the plastic wells and form a biofilm.
After incubation, unattached, free-floating (planktonic) cells are removed. This step ensures the measurement reflects only the adhered biofilm. The wells are washed with a phosphate-buffered saline (PBS) solution or sterile water. This washing process is repeated to eliminate any cells not part of the biofilm.
With only the attached biofilm remaining, the staining process begins. A 0.1% crystal violet solution is added to each well, submerging the biofilm. The plate incubates at room temperature for 15 to 30 minutes, allowing the dye to bind to the negatively charged components of the biofilm and stain the entire structure.
After staining, excess crystal violet solution is washed away to reduce background noise for an accurate measurement. The wells are rinsed with water until the runoff is clear, indicating all unbound dye is gone. The plate is then left to air dry completely.
The final stage releases the trapped dye for measurement. A solvent, like 30% acetic acid or pure ethanol, is added to each well. This dissolves the crystal violet, releasing the purple color into the liquid. After incubating for 10 to 15 minutes for the dye to dissolve, the colored solution is transferred to a new plate for analysis.
Quantifying and Interpreting the Data
Once the solubilized crystal violet is in a new plate, its color is measured with a spectrophotometer. This device passes a beam of light through the sample and measures its absorption. The measurement is given as an absorbance or optical density (OD) value at a wavelength between 550 and 595 nanometers.
A higher absorbance value signifies a more intense purple color, which directly correlates to a greater amount of biofilm biomass. For instance, a sample with an absorbance reading twice as high as another is interpreted as having double the biofilm mass. This quantitative data allows for direct comparisons between samples.
To ensure accuracy, controls are necessary. A “blank” control well contains only the nutrient broth and solvent, with no bacteria. The absorbance reading from this blank represents the background from the plastic and solvent. This value is subtracted from all experimental wells to correct for baseline noise.
Common Applications and Key Considerations
The assay is frequently used to screen for new anti-biofilm agents. Researchers grow biofilms with various compounds to quantify how effectively each substance prevents or destroys a biofilm. A significant reduction in the absorbance reading compared to an untreated control indicates the compound has anti-biofilm activity. Its format is ideal for testing many substances at once.
Another application is comparing the biofilm-forming abilities of different microbial strains. Researchers can investigate how genetic mutations affect biofilm development by comparing a mutant strain to its parent. The assay is also used to study how environmental conditions, like nutrient availability or temperature, influence biofilm growth.
When interpreting results, it is important to consider the assay’s nature as an indirect measurement of biomass. The stain binds to living and dead cells, as well as the EPS matrix. This means the assay cannot distinguish between viable and non-viable cells. It provides a measure of the total physical structure, which may not correlate with the number of living bacteria.