Take an anesthetized zebrafish larva in the groove of an agarose mold. Its head is positioned upward at an angle for intestinal access during injection.
Cover it with low-melting agarose, then allow the agarose to solidify and immobilize the larva.
Take a microinjection needle containing pathogenic bacteria suspended in a tracer dye solution.
Under a stereomicroscope, position the needle at an angle and guide the needle through the agarose into the larval mouth.
Advance it through the food pipe into the intestine.
Inject the bacteria into the intestinal lumen and confirm delivery using the tracer dye. This non-invasive method, called microgavage, mimics the natural infection route.
Withdraw the needle. Cut the agarose and transfer the larva into fresh medium.
The bacteria replicate and release toxins that damage the intestinal epithelium, promoting bacterial invasion and colonization in deeper tissues.
The zebrafish infection model is ready for analysis.
In a 1.5% agarose plate with grooves, place a drop of 0.8% low-melting agarose onto the zebrafish larvae to cover.
Gently adjust the larvae with heads facing upright at 45-degree angles in the groove and tails against the wall of the groove. Gently operate the needle through the agarose, then into the mouth of zebrafish larvae, through the esophagus. Once the tip of the needle is inside the anterior intestinal bulb, press the injection pedal to release 0.5 to 1 nanoliters of bacteria culture.
Fill the lumen of the intestine. Do not let it overflow from the esophagus or cloaca. Gently withdraw the needle from the mouth of the zebrafish. Following gavage, rescue the infected zebrafish larvae from the agarose with a flexible microloader tip by first cutting the agarose away, then by lifting the larvae.
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