This study investigates the interactions between motile and non-motile bacteria using a coculture system. The experimental setup allows for tracking bacterial behavior and understanding the chemical signaling involved in their interactions.
Begin with a glass coverslip dish seeded with a coculture of fluorescently labeled motile bacteria and unlabelled non-motile bacteria.
Take the dish containing a nutrient-infused agarose pad and carefully remove the surrounding silicone mold.
Tilt the dish and use a bent spatula to slide under the edge of the pad. Then place it onto the lid of a Petri plate.
Transfer the pad, bottom side down, to the dish containing the bacterial cells to ensure even contact with the bacterial layer.
Gently press the pad to eliminate any air bubbles.
Place the pre-wetted wipes around the edge of the dish to maintain humidity.
Under an inverted microscope, track the bacterial behavior over a time period.
As the bacteria grow, the non-motile bacteria release chemical attractants. The motile bacteria sense these signals and move toward the non-motile bacterial colonies.
Over time, motile bacteria accumulate and invade non-motile bacterial colonies, confirming bacterial interactions.
Once the pads are dry, pipette one microliter of co-culture evenly across the bottom of a pre-warmed sterile 35 millimeter glass coverslip dish.
Remove the silicone cutout from the agarose mold using sterile tweezers, tilt it to the side, and slip a slightly bent spatula under the edge of the agarose pad to drop it onto a sterile petri plate lid.
Transfer the pad to the dish with the bacterial cells, bottom side down, by sliding a 90-degree angled spatula under the pad and placing it on top of the inoculated cover slip. Use the spatula to make the pad flush against the cover slip and gently press out any air bubbles.
Remove excess moisture from the moist wipe and place it around the edge of the dish, making sure it does not touch the pad. The sample is now ready for imaging.
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