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