Development of a Zebrafish Larval Infection Model Using Microgavage

Abstract

Source: Li, J., et al. Development of a Larval Zebrafish Infection Model for Clostridioides difficile. J. Vis. Exp. (2020).

This video demonstrates the microgavage procedure, where an anesthetized zebrafish larva is immobilized in agarose and orally injected with a pathogenic bacterium using a tracer-guided needle. The non-invasive delivery mimics natural infection, enabling analysis of bacterial replication, toxin release, and intestinal epithelial damage.

Protocol

1. Gavage of Zebrafish Larvae

1. Calibrate the microgavage needle.
2. Measure the diameter of the tip of the needle by placing the needle on a calibration slide with one drop of mineral oil. Ensure that the tip is 30–40 µm in diameter, blunt, and smooth. Discard the sharp or rough needles.
   NOTE: Sharp edges of needles can be blunted by quick flaming.
3. Prepare the gavage solution.
4. Load and mount the needle onto a micromanipulator.
5. Adjust the micromanipulator to position the needle at a 45° angle.
6. Anesthetize the zebrafish larvae. When the larvae stop moving, transfer them to the groove of a microgavage mold using a Pasteur pipette.
7. 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° angles in the groove and tails against the wall of the groove. Ensure that the angles of the heads are approximately the same so that they are aligned with the angle of the gavage needles. Place the microgavage mold on ice for 30–60 s, allowing the low-melting agarose to solidify in order to stabilize the positions of the larvae.
8. Adjust the injection pressure between 200–300 hPa. Set the injection time to 0.1–0.3 s to obtain an injection volume of 3–5 nL of Clostridioides difficile.
9. 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 the bacteria. Fill the lumen of the intestine with the delivered volume. Do not let it overflow from the esophagus or cloaca. Gently withdraw the needle from the mouth of the zebrafish.
10. 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. Transfer these larvae into sterile 30% Danieau's medium. Rinse the larvae in sterile medium twice. Transfer the larvae to a fresh 10 cm Petri dish. The larvae will be maintained for up to 11 days post fertilization (dpf).

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Agarose	Sigma-Aldrich	A2576	Ultra-low gelling agarose
Agarose low-melting (LM)	Pronadisa	8050	It is used in agarose plates
Capillary Glass	Harvard Apparatus	30-0019	Injection needles
Clostridioides difficile			R20291,, a ribotype 027 strain, TcdA+/TcdB+/CDT+ production
DMSO	Carl Roth GmbH	A994
FIJI	open-source platform		Image processing
HEPES	Carl Roth GmbH	6763
Horizontal needle puller	Sutter instrument Inc	P-87
L-cysteine	Sigma-Aldrich	168149
Leica Application Suite X (LAS X)	Leica		Image processing
Micro injector	eppendorf	5253000017
Microinjection molds	Adaptive Science Tools	TU1
Leica SP8 confocal microscope	Leica
Phenol Red	Sigma-Aldrich	P0290
Potassium chloride (KCl)	Carl Roth GmbH	5346
Sodium chloride (NaCl)	Carl Roth GmbH	9265
Taurocholate	Carl Roth GmbH	8149
Tricaine	Sigma-Aldrich	E10521
Yeast extract	BD Bacto	212750