This study investigates the progression of bacterial infection in zebrafish larvae after ingesting pathogenic E. coli. By utilizing fluorescent imaging techniques, researchers can visualize the colonization of bacteria in the intestinal tract.
Begin with a multiwell plate containing paramecia pre-infected with pathogenic E. coli expressing fluorescent proteins.
Add anesthetized zebrafish larvae and co-incubate them under daylight conditions to mimic the natural feeding environment.
During incubation, the larvae regain consciousness and prey on infected paramecia.
Once ingested, the paramecia are digested, releasing E. coli into the intestinal tract.
Bacterial colonization progresses from the anterior intestine to the posterior intestine, mimicking a foodborne infection.
Next, wash the larvae in a medium supplemented with an anesthetic agent to re-anesthetize them.
Then, place the larvae in low-melting-point agarose within a fluorescent imaging plate.
Using a stereomicroscope, align the zebrafish and incubate to allow the agarose to solidify.
Overlay with a nutrient-rich medium supplemented with an anesthetic agent.
Using a fluorescence microscope, observe the larvae for the distribution of fluorescent E. coli to evaluate the progression of bacterial infection
For food-borne infection of the zebrafish, dilute the paramecia samples to a two times ten to the five paramecia per milliliter of E3 medium concentration and add 3 milliliters of each paramecia culture to one well of a 6-well plate per condition.
Next, transfer 10 anesthetized zebrafish to each well in a minimal volume of liquid and incubate the co-cultures for two hours at 30 degrees Celsius in a diurnal incubator under daylight conditions.
At the end of the incubation, wash the zebrafish in 5 different wells containing 3 milliliters of fresh E3 medium supplemented with 100 milligrams per liter of tricaine per well and embed each zebrafish in 3 milliliters of 1 percent low-melt agarose in a black-walled 6-well plate.
When all of the fish have been embedded, place the plate under a stereo microscope and use a clipped gel loading tip to make sure that the heads are on the left and the tails are on the right in each viewing field.
Wait for 5 minutes for the agarose to set, then overlay the embedded fish with fresh E3 medium supplemented with tricaine, and image the zebrafish on a fluorescent microscope to evaluate the progress of the bacterial infections.
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