Localized Delivery of Bacteria into the Intestinal Lumen of a Zebrafish Larva Using Microinjection

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-melti