Isolation and Culture of Clostridioides difficile Bacteria from Larval Zebrafish Intestine

Abstract

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

This video demonstrates the isolation and culturing of Clostridioides difficile bacteria from the intestine of larval zebrafish. The infected intestine is dissected, homogenized, and incubated in selective anaerobic media containing antibiotics. These conditions suppress competing microbes and promote the growth of viable cells.

Protocol

Dissection of Larval Zebrafish Intestine to Recover Viable Clostridioides difficile

1. Isolate gastrointestinal tracts from larvae to analyze bacterial load. Start by euthanizing zebrafish larvae with 0.4 % tricaine.
2. Rinse the zebrafish briefly with sterile 1x PBS (Phosphate-Buffered Saline) and transfer them to a fresh agarose plate.
3. Dissection of zebrafish
   1. Insert a needle into the dorsal trunk of zebrafish larvae close to the head to immobilize the zebrafish. Remove the head behind the gills with a lancet.
   2. Insert the second needle into the middle of the dorsal trunk. Insert the third needle into the abdomen of the zebrafish and pull the intestine out of the body cavity.
      NOTE: Extreme care is needed to isolate the intact intestine. If it is difficult to do so, perform additional pulls to separate the rest of the intestine from the remaining internal organs.
   3. Use a microinjection needle to transfer 10–15 intestines into a 1.5 mL tube containing 200 µL sterile 1x PBS.
   4. Homogenize the intestines with a pestle to disrupt the tissue and prepare homogenates. Ensure the pestle reaches the bottom of the tube to completely disrupt all intestines.
4. Incubate the homogenates in C. difficile rearing medium containing D-cycloserine and cefoxitin, with or without taurocholate (TCA, a germinant of C. difficile spores) in an anaerobic chamber.
5. Incubate the plate anaerobically for 48 h at 37 °C.

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
Clostridioides difficile			R20291,, a ribotype 027 strain, TcdA+/TcdB+/CDT+ production
Horizontal needle puller	Sutter instrument Inc	P-87
Micro injector	Eppendorf	5253000017
Taurocholate	Carl Roth GmbH	8149
Tricaine	Sigma-Aldrich	E10521