This study investigates the swarming motility of Pseudomonas aeruginosa on agar plates and its response to antibiotic stress. The methodology includes automated imaging to visualize and quantify swarm behavior over time.
Take a swarming agar plate that supports swarming motility- a coordinated bacterial movement across a semisolid surface that forms tendril-like patterns.
Inoculate the plate’s center with a Pseudomonas aeruginosa culture and place a mixture of the same culture with an antibiotic at evenly spaced satellite positions.
Cover the plate with a black-painted lid to minimize light reflections.
Place the plate on a flatbed scanner inside an incubator maintained at optimal humidity.
During incubation, the central population begins to multiply and swarm.
As the swarm approaches the antibiotic-treated colonies, it detects stress signals secreted by the bacteria and redirects to avoid these regions.
The scanner, controlled by automated software, captures high-resolution images at regular intervals.
Compile these images into a video to visualize swarm progression and avoidance over time.
Quantify swarm avoidance by measuring the distance from each satellite colony to the edge of the central swarm in a late-stage image.
After placing the plates into the scanner, open file and save settings to set the saving path for the images and click scan and OK at 30-minute intervals. To automate the image acquisition, use the scripting software.
Select both idle scanning and single scan and right-click on idle scanning, then left-click on enabled. At the end of the incubation, import all of the scanned images into ImageJ. In the sequence options window, number of images will indicate the number of images selected.
Keep starting image at one to start from the first picture in the folder and the scale images at 100%to conserve the original size of the images. Click convert to RGB to keep the images in color. Leave use virtual stack unchecked and click OK to load the images.
Click file, save as and AVI to merge the images into an AVI file. Adjust the compression to JPEG and the frame rate to five frames per second. Then save the AVI time-lapse in the appropriate storage folder.
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