This study investigates the impact of an enzyme on biofilm formation by pathogenic bacteria. The enzyme disrupts biofilm formation by degrading bacterial signaling molecules, leading to reduced attachment and proliferation.
Take a microplate containing control and treatment wells, each inoculated with a biofilm-forming, pathogenic bacterium.
The treatment wells contain an enzyme that disrupts biofilm formation.
Incubate under micro-anaerobic conditions to create a low-oxygen environment. This promotes the secretion of bacterial signaling molecules that mediate surface adhesion. In the treatment wells, the enzyme degrades these molecules, thereby reducing bacterial attachment.
Wash the wells to remove non-adherent cells.
Introduce fresh medium and incubate. The adhered bacteria proliferate and secrete extracellular polymeric substances, which form the biofilm matrix.
Remove the medium and wash the wells.
Add a cationic dye and incubate to allow binding to negatively charged biofilm components.
Wash to remove unbound dye.
Incubate with a solvent under agitation to release the bound dye into the solution.
Measure the optical density or OD to quantify the biofilm biomass.
Lower OD in the treatment wells reflects reduced biofilm formation, resulting from impaired initial cell adhesion.
To begin, grow a 5 milliliter culture of A.baumannii S1 in LB at 30 degrees Celsius in a shaking incubator for 16 hours.
Adjust the culture to an OD600 of 0.8. Then use fresh LB containing 4 milligrams per milliliter of purified GKL enzyme to dilute the culture 1 to 100. And add 100 microliters to the wells of a 96-well plate.
Use the lids to cover the plates, and place them into a 10 liter plastic container. Seal the container and incubate at 30 degrees Celsius for three hours before gently removing the medium. Add another 100 microliters of fresh LB medium to the wells, and incubate the plate at 30 degrees Celsius for 21 hours.
After gently removing the medium, use 200 microliters of sterile water to gently wash the planktonic bacteria cells. Position the pipette tip at the bottom of the well and carefully remove the liquid medium to ensure minimal perturbations on the biofilm foreign between the air and liquid interface. Then add 100 microliters of 1% crystal violet solution to each well, and incubate for 15 minutes at room temperature.
Remove the crystal violet solution by using 200 microliters of sterile water to wash the wells three times. Add 100 microliters of 33% acetic acid to each well and incubate with gentle shaking for 15 minutes to dissolve the dye. Quantitate the amount of biofilm by measuring the absorbance of crystal violet at 600 nanometers. The amount of crystal violet is proportional to the amount of biofilm formed.
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