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

Samenvatting

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....

Materialen

Lijst van materialen gebruikt in dit artikel

Naam	Bedrijf	Catalogusnummer	Opmerkingen
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