This study investigates bacterial surface motility using motile bacteria expressing green fluorescent protein. The methodology includes time-lapse imaging to observe the migration patterns of bacteria on nutrient agar plates.
Begin with a tube containing motile bacteria that express green fluorescent protein.
Using a needle, spot the bacteria onto a soft nutrient agar plate to study bacterial surface motility.
Next, invert the plate to prevent water condensation on the agar surface.
Incubate the plate to allow the bacteria to grow.
As incubation progresses, the bacteria use their flagella to initiate migration outward from the inoculation point.
Once incubation is complete, open the lid and place the inverted plate into an imaging system, with the agar side facing the optical detector, to ensure accurate imaging.
Place the water-filled lid above the plate. Seal it to maintain a humid environment.
Close the imaging system and initiate time-lapse image acquisition.
Over time, the bacterial migration generates green finger-like projections called tendrils. These bacterial structures extend outward from the central inoculation point, confirming bacterial surface motility.
Inoculate one to five microliters of the overnight culture over the dried swarm agar plates by poking the agar surface with a sterile toothpick or wire inoculation needle, depending on the bacteria strain, transfer the swarm assay plates into an incubator with a temperature set to either 30 degrees Celsius, 37 degrees Celsius, or 42 degrees Celsius. Invert the plates such that excess moisture condenses on the plate lid and not on the agar, equilibrate the bacteria at strain specific temperatures for either two or four hours just prior to time-lapse imaging.
Next, transfer the plates into the in vivo imaging unit such that the bacteria on the agar surface are facing down towards the inverted camera of the unit. Fill all Petri dish lids with a small amount of water. Then place each lid with waterside facing up on top of the inverted agar plates.
Inside the imaging unit, seal the imaging enclosure to maintain a constant humidity level, adjust the imaging settings of the imaging unit and commence time-lapse imaging of swarming activity. After real-time imaging, the swarming dynamics of the bacteria can be analyzed using standard image analysis such as Image J.
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