This article details a method for preparing agarose slides to visualize swarmer and swimmer cells of Vibrio parahaemolyticus using fluorescence microscopy. The cells are engineered to express a fluorophore-tagged protein, allowing for their identification based on their morphology and protein localization.
Take a dish containing a colony of Vibrio parahaemolyticus bacteria.
The bacteria exhibit two forms: short rod-shaped swimmer cells with a single polar flagellum located at the colony center and elongated swarmer cells with lateral flagella that migrate outward.
The cells are engineered to express a fluorophore-tagged protein, which localizes unipolarly in swimmer and bipolarly in swarmer cells, aiding in their identification.
Next, pour molten agarose between tape guides on a microscope slide and press it with a second slide to spread evenly.
Allow it to solidify, trim the excess, and remove the top slide to expose the agarose pad.
Cut a section from the colony edge and invert it onto the pad.
Allow the cells to adhere and remove the section.
Place a coverslip to prepare the cells for imaging.
Elongated cells with bipolarly distributed fluorescent protein indicate the presence of swarmer cells.
The first step in this procedure is to prepare agarose slides for microscopy.
Add one gram of agarose to 100 milliliters of a 20% PBS and a 10% LB broth solution. Carefully boil the solution in the microwave to dissolve the agarose, and then let it cool down to 65 to 70 degrees Celsius while mixing with a magnetic stir bar. Place a clean microscope slide on to a perfectly level section of a working bench.
Fix the slide to the bench by covering both ends of the slide with two layers of general purpose laboratory labeling tape. The distance between the two pieces of tape should be around three centimeters. Pipette about 250 microliters of the previously prepared agarose solution onto the non-covered glass surface.
Place a second microscope slide in the same orientation as the bottom slide on top and slightly press down so that the residual agarose can flow out to the sides. Let the agarose solidify for one to two minutes. Use a scalpel to cut off any residual agarose that is attached to the sides of the microscope slide.
When the agarose has solidified, slowly pull the top glass slide off of the bottom one in a horizontal movement. Slowly remove the tape from the ends of the microscope slide. The agarose slide should be used as soon as possible, as it will slowly begin to dry out.
Immediately before the transfer of swarmer cells to the microscope slide, Use a scalpel to cut the plastic tape off the previously prepared swarmer plate. During the excision of the swarming agar, it is important to direct the cut from the outside to the inside of the swarming colony to avoid contaminating the swarming flares with cells that originate from the middle of the colony. Use a fresh scalpel to cut out a three millimeter by 10 millimeter piece of swarming agar from the most outer edge of a swarming colony, making sure to direct the cuts from the outside of the swarming colony edge to the inside of the colony.
Transfer the piece of swarming agar to the microscope agarose slide with the cells facing the agarose pad to imprint the swarming cells onto the agarose pad. After waiting for about 30 seconds, remove the agar piece carefully from the agarose pad. Place a glass coverslip on the agarose pad where the cells were imprinted.
The cells are now ready for fluorescence microscopy.
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