This study investigates the impact of a signaling-disrupting enzyme on biofilm formation by a biofilm-forming bacterium. The research employs confocal microscopy to visualize the effects of the enzyme on biofilm architecture.
Begin with two glass-bottom microdishes containing suspensions of a biofilm-forming bacterium.
The treatment dish contains an active signaling-disrupting enzyme, while the control includes the inactivated enzyme.
Cover the dishes and incubate to allow bacterial adhesion to the microdish surface.
Wash to remove non-adherent cells, then introduce fresh medium containing the enzymes and incubate.
In the control dish, the bacteria communicate through signaling molecules and produce extracellular polymeric substances (EPS), forming a biofilm.
The active enzyme degrades the signaling molecules, disrupting their function and reducing biofilm formation.
Replenish with fresh medium and incubate further to reinforce the enzyme's biofilm-disrupting effects.
Remove the medium, add fluorescently labeled lectins, and incubate to allow lectin binding to the EPS.
Wash to remove any unbound lectin.
Incubate with a fixative to preserve biofilm architecture, then rinse the dish.
Acquire confocal microscopy images to assess the enzyme-mediated suppression of biofilm formation.
To carry out confocal laser scanning microscopy, or CLSM, of A. baumannii S1 biofilms, 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 of A. baumannii S1 to an OD600 of 0.8.
Then use fresh LB containing 1.2 milligrams per milliliter of purified GKL enzyme to dilute the culture, 1 to 100, and add 1 milliliter to a 35 milliliter glass-bottomed microdish. Cover the microdish with a lid and place it in a 10 liter plastic container and seal it before incubating the dish at 30 degrees Celsius for three hours. After gently removing the medium, add 1 milliliter of fresh medium containing 30 microliters of purified GKL enzyme, then incubate at 30 degrees Celsius for another 21 hours.
Gently remove the medium a second time and replace with another 1 milliliter of GKL enzyme in fresh medium. Incubate at 30 degrees Celsius for 24 hours. After the incubation, gently remove the medium and add 500 microliters of 5 micrograms per milliliter of Alexa Fluor 488 conjugated wheat germ agglutinin dissolved in HBSS.
Incubate the microdish at 37 degrees Celsius for 30 minutes to stain the biofilms. To fix the biofilms, add 500 microliters of 3.7% formaldehyde dissolved in HBSS and incubate at 37 degrees Celsius for 30 minutes. Use 2 milliliters of HBSS to wash the microdish once, then remove the solution completely.
Store the dish in the dark at 4 degrees Celsius prior to CLSM imaging. For CLSM imaging and analysis, use a 63 X objective to generate 97 stacks per image with an interval of 0.21 microns per stack.
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