Isolation and Culture of Clostridioides difficile Bacteria from Larval Zebrafish Intestine

Begin with an euthanized zebrafish larva with an intestine infected by Clostridioides difficile, a pathogenic anaerobic bacterium.

Secure the upper dorsal trunk of the larva and remove its head to expose the internal organs.

Secure the mid-dorsal trunk and gently isolate the intestine from the body cavity.

Transfer the isolated intestine into a microcentrifuge tube containing buffer.

Homogenize the intestine using a pestle to disrupt the tissue and release the intestinal microbes.

Transfer the homogenate into a tube containing selective rearing media supplemented with antibiotics.

Incubate the homogenate in an anaerobic chamber.

Anaerobic conditions inhibit the growth of aerobic bacteria, while antibiotics specifically target and suppress the growth of native anaerobic intestinal microbes.

Clostridioides difficile resists these antibiotics and proliferates under anaerobic conditions.

Nutrients and growth factors in the rearing media further promote cell division, enabling the growth of viable Clostridioides difficile.

After euthanizing the infected zebrafish larvae, 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.

Insert a second needle into the middle of the dorsal trunk. Insert a third needle into the abdomen of the zebrafish, and pull the intestine out of the body cavity. Use a microinjection needle to transfer 10 to 15 intestines into a 1.5-milliliter tube containing 200 microliters of sterile 1X PBS.

Homogenize the intestines with a pestle to disrupt the tissue and prepare homogenates. Ensure the pestle reaches the bottom of the tube to disrupt all intestines completely. Into the homogenates, add Clostridioides difficile rearing medium containing D-cycloserine and cefoxitin, with or without taurocholate, and incubate in an anaerobic chamber.