Quantifying Intestinal Bacterial Load in Zebrafish Infected with Vibrio cholerae

Begin with an adult zebrafish infected with an antibiotic-resistant strain of Vibrio cholerae. This pathogenic bacterium expresses β-galactosidase enzyme and colonizes the intestinal tract.

Collect the fish and transfer it into a solution containing an overdose of anesthetic to euthanize it.

Place the fish ventral side up and secure it with pins.

Disinfect the abdomen, make a longitudinal skin incision and two lateral incisions toward the head, then pin the skin to expose the intestinal tract.

Transfer the tract into a homogenization tube containing a cold buffer with glass beads.

Incubate with agitation, allowing the glass beads to disrupt the tissue and release the bacteria.

Serially dilute the homogenate, plate it onto the medium, and incubate. The medium contains a chromogenic β-galactosidase substrate and an antibiotic to eliminate contaminants.

The bacteria express β-galactosidase enzyme, which hydrolyzes the chromogenic substrate leading to formation of blue-pigmented colonies.

Count the colonies to estimate the intestinal bacterial load.

When the experiment has reached its desired endpoint, take a 15 milliliter sample of the infection water for diagnostic purposes. Then collect the fish and dispose of the infected water appropriately. Orient the ventral side up and secure the body with a pin through the lower jaw and another pin just posterior to the anus.

Angle both pins away from the body. Next, wipe off the ventral surface of the fish with 70% ethanol. Then, sterilize a scalpel and Vannas scissors by using 70% ethanol and a flame.

Now, using the scalpel, make a small lengthwise incision in the belly that penetrates the scales, but only cut as deep as the skin. Next, with the scissors, extend the incision carefully along the length of the body, cutting no deeper than skin level. Avoid cutting the anus.

Then make two lateral cuts towards the head to expand the opening. Then, pin the skin on each side of the lateral incisions to the dissecting surface, angling the pins out, away from the body. The intestinal tract should now be visible as a very thin, pale tube.

Now, use two pair of flame sterilized forceps to carefully remove the entire intestinal tract which can be quite fragile. Transfer the tissue into a homogenization tube containing glass beads and one milliliter of cold buffer. Next, homogenize the tissues as described in the text protocol and quantify the intestinal colonization levels.

Next, make serial dilutions of the intestinal homogenate and buffer. For each fish, prepare five or six ten fold dilutions in one milliliter volumes. Vortex the tubes for mixing.

Then, plate 100 to 200 microliters of each dilution on LB agar plates containing streptomycin and X-gal. Incubate the plates at 30 degrees Celsius for 16 to 18 hours. After the overnight incubation, count the V. cholerae colonies on the plates.

These bacteria typically produce pale blue colonies on LB medium with X-gal.