The overall goal of this procedure is to establish and study biofilms in a microtiter dish. This is accomplished by first adding bacterial culture to a 96 well plate.Next. The culture is incubated at the appropriate temperature and time, depending on the microbe to stain the biofilm.
Crystal violet is added to each well of bacteria, and then the bacteria are washed. Finally, the wells are photographed and the bacteria are quantified. Ultimately, results can be obtained that show the accumulation and formation of biofilms over time through the measurement of crystal violet.
The main advantage of this method is it allows investigators to assess biofilm formation rapidly in bacterial and fungal organisms. It can also be configured for high throughput genetic screens, or to identify novel therapeutic compounds that inhibit biofilm formation. Prepare a five milliliter overnight culture of the wild type pseudomonas, aerogen, or mutant strain.
In LB medium, dilute 10 microliters of culture into one milliliter of fresh M 63, minimal medium supplemented with magnesium, sulfate, glucose, and cassino acids. Next, using a pipette transfer, 100 microliters of diluted culture into separate wells of a 96 well round bottom plate. Incubate the plate at 37 degrees Celsius for four to 24 hours to allow for biofilm growth.
Once the biofilms have formed, remove the plate from the incubator and discard the culture supernatants by inverting the plate and shaking out the liquid. Then gently submerge the plate in a small tub of water to wash off any remaining unbound cells or medium components that may cause background staining. Shake out the water, blot the plate, and repeat the wash.
Once the plate is clean, add 125 microliters per well. A 0.1%crystal violet solution. To stain the cells, incubate the plate at room temperature for 10 to 15 minutes.
Then wash the plate three or four times as demonstrated earlier. Vigorously blotting the plate dry on a stack of paper towels to remove excess liquid after the final wash, set the plate upside down and let it dry upside down for a few hours. Once the plate is dry, the wells can be photographed with a digital camera to assess the quality of the biofilms.
As shown here, motile bacteria such as pseudomonas aerogen on the left and pseudomonas fluorescence on the right tend to form biofilms at the location of the air liquid interface during growth of the biofilm. In contrast, staphylococcus ora bacteria, which are non motile form biofilms at the bottom of the wells. In order to quantify the biofilms first, add 125 microliters of 30%acetic acid to each well, including a blank well containing just acetic acid, and incubate the plate at room temperature for 10 to 15 minutes to allow the crystal violet retained by the biofilms to solubilize.
Next, transfer the 125 microliters of Solubilized stain from each well to a new flat bottomed 96 well microtiter plate. Using a plate reader quantify the absorbance from each well at a wavelength of 550 nanometers. The concentration of crystal violet in each well is proportional to the number of cells in the biofilm.
This graph shows a quantification of biofilm formation and accumulation by pseudomonas fluorescence over time during incubation at 37 degrees Celsius. After watching this video, you should have a good understanding of how to perform a 96 well Dish biofilm assay used in conjunction with other methods like microscopy. It can contribute significantly to our understanding of biofilm formation.
