This study investigates the swarming behavior of Pseudomonas aeruginosa in response to antibiotic exposure. By using agar plates, the research demonstrates how bacteria can sense and avoid areas with antibiotic stress.
Place an agar plate onto a printed template to align the center and six evenly spaced satellite spots.
Spot a healthy Pseudomonas aeruginosa culture at the center of the plate.
Place a mixture of the same culture with an antibiotic onto the satellite spots.
Cover the plate with a black lid to reduce light reflections.
Place it on a scanner inside an incubator with a constant temperature and humidity to maintain bacterial growth.
Healthy bacteria at the center grow and spread outward in tendril-like swarming patterns that radiate across the agar surface.
Bacteria mixed with the antibiotic undergo stress and release chemical signals that diffuse through the agar.
As the swarm reaches these zones, it senses the stress signals.
This triggers an avoidance response, redirecting bacterial growth away from the antibiotic-exposed regions.
Time-lapse imaging shows bacterial growth and changes in swarm expansion under antibiotic stress.
For swarming agar plating, hold a P20 pipette tip loaded with five microliters from an overnight P. aeruginosa culture at a 10-45 degree angle 2-1/2 centimeters above the center of one swarming plate and depress the plunger to the first stop touching the agar with only the liquid drop. Use a template to position the spot consistently across the different swarming agar plates.
To plate antibiotic treatments, mix 30 microliters of overnight cultured P. aeruginosa with the appropriate concentration and volume of the antibiotic of interest and immediately add six microliters of the antibiotic-treated bacteria at six equidistant satellite positions on a 2.8 centimeter radius concentric circle about the center of the dish. Use a template to position the spot consistently across the different swarming agar plates.
When all of the cultures have been plated, replace the clear Petri dish lids with custom-made black lids. Carefully place the swarming agar plates on a scanner in an incubator set at 37 degrees Celsius with a 10 liter water bath to maintain the humidity at 75%.
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