This study explores the differentiation of Vibrio parahaemolyticus into swarmer cells under specific environmental conditions. The process involves the manipulation of agar medium components and incubation conditions to promote bacterial growth and movement.
Begin with a molten agar medium that supports bacterial growth.
Add an iron chelator to deplete free iron, and supplement the medium with calcium.
Pour the medium into a dish, allow it to solidify, and then dry it to eliminate residual moisture.
Take a broth culture of Vibrio parahaemolyticus, a flagellated bacterium.
In liquid media, the bacteria exist in a short, rod-shaped swimmer form with a flagellum.
Spot the culture onto the plate and allow it to dry to promote bacterial contact with the surface.
Seal the dish and incubate, allowing volatile metabolic by-products from the bacteria to accumulate.
Surface contact, iron limitation, calcium availability, and accumulated by-products induce gene expression, which triggers the differentiation into elongated swarmer cells containing lateral flagella, enabling movement across solid surfaces.
The swarmer cells expand outward and form swarm flares composed of fully differentiated cells, indicating successful differentiation.
To prepare the agar solution for making swarming plates, suspend four grams of heart infusion, or H. I. agar powder, and 100 milliliters of double-distilled water.
Carefully boil the H. I. agar solution in a microwave oven, shaking the bottle from time to time until the powder is dissolved. Autoclave the agar solution at 121 degrees Celsius for 20 minutes, cool down the agar solution to 60 to 65 degrees Celsius with constant stirring. The next step involves handling 2, 2'-Bipyridyl, which is highly toxic, so gloves and safety goggles must be worn.
Add 2, 2'-Bipyridyl to a final concentration of 50 micromolar. Add calcium chloride to a final concentration of four millimolar. Inoculate a small amount of V. parahaemolyticus cells from the edge of a single colony into 5 milliliters of LB broth.
Incubate the culture at 37 degrees Celsius in a shaker incubator until it reaches an optical density at 600 nanometers of 0.8. This usually takes around two to three hours. While the cell culture is growing, pipette 30 to 35 milliliters of the final H. I. agar solution into a round 150 millimeter petri dish, and let the agar solidify.
Right before spotting the cell culture, dry the agar plate upright with an open lid at 37 degrees Celsius for at least 10 minutes, or until any liquid residues have disappeared, but no longer. Spot one microliter of cell culture at the center of the H. I. agar swarming plate and let the spot dry. Seal the plate with clear plastic tape, taking care to avoid the formation of air bubbles and folds to the tape.
Incubate the plate at 24 degrees Celsius overnight. This will result in the formation of a V. parahaemolyticus swarm colony with swarm flares extending from the center of the colony, as shown in this time lapse video clip.
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