This article describes a method for injecting fluorescently labeled Clostridioides difficile into the intestinal lumen of anesthetized zebrafish larvae. The technique allows for the visualization of bacterial localization using fluorescence microscopy.
Take a dish containing an anesthetized zebrafish larva.
Apply a drop of low-melting agarose over the larva and gently position it laterally to allow intestinal access during injection.
Now, briefly cool the agarose to solidify it, thereby immobilizing the larva.
Add media containing an anesthetic agent to maintain the larva under anesthesia.
Next, take a suspension of fluorescently labeled Clostridioides difficile, a pathogenic bacterium, and add a tracer dye to facilitate monitoring of the injection.
Load this mixture into a microinjection needle and mount the needle onto a micromanipulator.
Under a stereomicroscope, position the needle at an angle directed toward the larval intestine.
Then, gently insert the needle tip into the intestinal lumen.
Inject the bacteria directly into the lumen and confirm successful delivery using the tracer dye. Then, retract the needle.
Using fluorescence microscopy, confirm the localization of Clostridioides difficile in the larval intestine.
First, place a drop of 0.8% low-melting agarose onto the zebrafish larvae in a Petri dish to cover. Gently adjust the larvae to a lateral position. Place the Petri dish on ice for 30 to 60 seconds to allow the low-melting agarose to solidify.
Add 30% Danieau's medium containing 0.02% to 0.04% tricaine to cover the agarose. To prepare injection solution, add one microliter of 0.5% phenol red in PBS solution into nine microliters of the dye-stained Clostridioides difficile inoculum. Load a calibrated microinjection needle with the injection solution using a Microloader.
Mount the loaded needle into a micromanipulator, and position it under a stereo microscope. Adjust the injection pressure between 600 and 900 hectopascals. Set the injection time to 0.1 to 0.3 seconds to obtain 0.5 to 1.0 nanoliters.
Set the needle in the micromanipulator at an approximately 45-degree angle, pointing toward the embedded larvae. Place the needle tip above the gastrointestinal tract, close to the urogenital pore. Pierce through the agarose, then the muscle with the needle tip.
Then insert it into the intestinal lumen, and inject 0.5 to 1.0 nanoliters of Clostridioides difficile. Use a fluorescence microscope to monitor the injected larvae, and use a flexible Microloader tip to pick up the properly injected larvae for confocal imaging.
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