Isolation and Cultivation of Bacteria from Zebrafish Embryos to Assess Infection Load

Take a zebrafish embryo infected with Mycobacterium abscessus, a pathogenic bacterium that targets the central nervous system and forms abscesses containing bacteria and host immune cells.

Incubate the embryo on ice to immobilize it, then expose it to an overdose of anesthetic to stop cardiac and neural activity, resulting in death.

Wash the embryo to remove residual anesthetic.

Add a detergent solution to disrupt cellular membranes and lyse the embryo.

Shear the lysate through a needle to homogenize the tissue and release the bacteria.

Centrifuge, discard the supernatant, and resuspend the pellet in a surfactant solution to prevent bacterial clumping.

Serially dilute the bacterial suspension and spread it onto agar plates.

The medium is supplemented with antibiotics to inhibit the growth of other microbes while the Mycobacterium cells engineered for antibiotic resistance proliferate.

Incubate to promote colony formation, enabling quantification of the embryo's bacterial load.

To enumerate colony-forming units or CFUs at the desired time point, collect five embryos per infection condition and transfer each embryo into a 1.5 milliliter microcentrifuge tube. Cryoanesthetize the embryos by incubation on ice for 10 minutes. After euthanizing the embryos with 300 to 500 milligrams per liter tricaine, use sterile water to wash the embryos twice in a new tube.

Next, after removing water at 2% Triton X-100 in 1 X PBS to each embryo, and use a 26-gauge needle to homogenize the tissue for complete lysis.

After centrifuging the suspension, use 1 X PBST to resuspend the pellet. Then, plate serial dilution of the homogenates on Middlebrook 7H10O^ADC supplemented with BBL MGIT PANTA.

Incubate the plates at 30 degrees Celsius for 4 days before counting the colonies.