This study investigates the effects of bile salts on bacterial biofilm formation. The methodology involves using a multi-well plate to assess bacterial adherence and biofilm development through crystal violet staining.
Take a flat-bottom multi-well plate with wells containing bacteria in growth media or bacteria in growth media supplemented with bile salt.
The bile salts induce stress, causing cellular damage and releasing DNA and proteins. These biomolecules coat the well bottoms, enabling the surviving bacteria to adhere to the solid surface.
The attached bacteria multiply, forming micro-colonies and producing auto-inducers — or chemical signaling molecules, allowing bacterial communication.
This communication triggers bacteria to colonize and initiate the production of extracellular polymeric substances or EPS, generating a biofilm.
Post-incubation, remove the media to eliminate free-floating bacteria.
Introduce a crystal violet stain that interacts with the peptidoglycan of the bacterial cell wall, giving it a violet color.
Remove excess stain and dry. Overlay with ethanol to solubilize crystal violet from the bacterial cell wall, changing the solution color.
A more intense coloration in bile salt-treated wells than in untreated wells confirms biofilm formation through solid-phase adherence.
First, label two 1.5-milliliter tubes as TSB and TSB plus BS. Then, add 1 milliliter of TSB or TSB plus BS to the respective tubes.
Next, inoculate the tubes with 20 microliters of overnight culture at a 1:50 dilution. Then, add 130 microliters of uninoculated control media to each of three wells in a sterile, clear, flat-bottomed tissue culture-treated, 96-well plate. This will be the blank control.
Add 130 microliters of inoculated culture into each of the three wells. After adding the control and the inoculated culture in the respective wells, incubate the 96-well plate for 4 to 24 hours at 37 degrees Celsius statically. Once the incubation is over, set the control well as blank on the plate reader.
Next, aspirate the culture medium from each well. Then gently wash the wells once with 200 microliters of sterile PBS. After washing, aspirate the PBS, and then, invert the plate to dry for about 20 minutes.
It's very important to ensure gentle removal of the media and gentle washing to ensure that the EPS matrix is not disturbed.
Next, add 150 microliters of 0.5% crystal violet to each of the experimental and control wells. Then, incubate the plate for five minutes at room temperature. After five minutes, wash the wells once with 400 microliters of distilled water. Add 200 microliters of distilled water to wash the wells five times. After all the five washes, invert the plate, and let it dry completely, protected from the light.
In order to obtain consistent results, it is important that the samples are thoroughly dried before proceeding.
After the plate is completely dried, use 200 microliters of 95% ethanol to destain the wells. Then leave the plate on the shaker for 30 minutes at 4 degrees Celsius in order to avoid evaporation. After 30 minutes, record the OD540 on the plate reader.
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