This article details a method for live-cell imaging of Myxococcus xanthus, a slow-growing soil bacterium. The procedure involves culturing the bacteria in a medium with antibiotics and using agarose pads for long-term imaging.
Begin with a culture of Myxococcus xanthus, a slow-growing soil bacterium.
Inoculate a colony into a medium supplemented with an antibiotic.
The cells have been engineered to resist the antibiotic, ensuring that only the desired bacteria survive.
Transfer the suspension into a larger flask containing the medium and incubate with shaking to promote optimal growth.
Take a coverslip mounted on a metal frame, which provides mechanical support during imaging.
Dispense the bacterial culture onto it, then add fluorescent microspheres to serve as reference points for image alignment while acquiring multiple images.
Place a pre-warmed agarose pad containing the medium, then overlay it with a coverslip to maintain hydration.
Incubate to allow the cells to adhere to the agarose pad.
The pad supplies nutrients to the cells, enabling long-term live-cell imaging of cellular processes during proliferation in the slow-growing bacterium.
To begin, re-suspend a single M. xanthus colony in 500 microliters of 1% CTT, supplemented with antibiotics, in a sterile tube and transfer the entire suspension to a 50-milliliter Erlenmeyer flask containing five milliliters of 1% CTT. Prepare 1% agarose microscopy solution containing 0.2% CTT by mixing one gram of agarose with 80 milliliters of TPM buffer and 20 milliliters of 1% CTT medium. Microwave the solution until the agarose is molten.
Fill a Petri dish with approximately 60 milliliters of molten agarose and let it cool down to room temperature. Pre-warm the agarose pad at 32 degrees Celsius for at least 15 minutes prior to use. Next, place a sterile glass coverslip on a plastic or metal frame that has a hole in the middle. Then use tape to fix the coverslip to the frame.
Add 10 to 20 microliters of exponentially grown M. xanthus cells onto the coverslip. To add fluorescent microspheres as fiducial markers of the cells, use TPM buffer to dilute the microspheres to 1:100. Shake the bead suspension thoroughly and add five to 10 microliters to the cells.
From the large, pre-warmed 1% agarose pad, cut out a small pad approximately the size of the cover slip and place it on top of the cells. Then place a cover slip on top of the pad to prevent evaporation and to maintain cells in a humid environment. Incubate the microscopy sample at 32 degrees Celsius for 15 to 20 minutes to let the cells attach to the bottom of the agarose pad before time-lapse microscopy recordings.
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