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

Abstract

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

This video demonstrates the microinjection-based delivery of fluorescently labeled Clostridioides difficile into the intestinal lumen of an anesthetized zebrafish larva. It outlines the steps involved in larval preparation, bacterial injection, and fluorescence microscopy–based confirmation of bacterial localization within the intestine.

Protocol

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.

1. Injection of Stained Clostridioides difficile into Zebrafish Larvae

1. Anesthetize 20–30 zebrafish larvae at 5 days post-fertilization (referred to here as 5 dpf) with 0.02%–0.04% tricaine (tricaine powder is dissolved in double-distilled water and adjusted to pH = 7 with 1 M Tris-HCL solution) in 30% Danieau's medium ~10 min before injection. Transfer the anesthetized larvae to a fresh 10 cm Petri dish and remove any excess 30% Danieau's medium.
2. Place a drop of 0.8% low-melting agarose onto the zebrafish larvae to cover. Gently adjust the larvae to a lateral position. Place the Petri dish on ice for 30–60 s to allow the low-melting agarose to solidify. Add 30% Danieau's medium containing 0.02%–0.04% tricaine to cover the agarose.
3. Prepare the injection solution. Add 1 µL of 0.5% phenol red in phosphate-buffered saline (PBS) solution into 9 µL of the dye-stained Clostridioides difficile inoculum to visualize the injection process.
4. Load a calibrated microinjection needle with the injection solution using a microloader. Mount the loaded needle onto a micromanipulator and position it under a stereomicroscope.
5. Adjust the injection pressure between 600–900 hPa. Set the injection time to 0.1–0.3 s to obtain 0.5–1.0 nL. Set the needle in the micromanipulator at a ~45° angle pointing toward the embedded larvae.
6. Place the needle tip above the gastrointestinal tract, close to the urogenital pore. Pierce through agarose, then the muscle with the needle tip, then insert it into the intestinal lumen and inject 0.5–1.0 nL of Clostridioides difficile. Use a fluorescence microscope to monitor the injected larvae and pick up the properly injected larvae for confocal imaging.

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Agarose low-melting (LM)	Pronadisa	8050	It is used in agarose plates
BacLight Red Bacterial Stain	Thermo Fisher Scientific	B35001	Fluorescent dye
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
Micro injector	eppendorf	5253000017
Leica SP8 confocal microscope	Leica
Phenol Red	Sigma-Aldrich	P0290
Tricaine	Sigma-Aldrich	E10521