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$$\longleftharp{xx}$$,
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1. Determination of Intestinal Colonization Levels
NOTE: Intestinal colonization is the most useful metric in the zebrafish model as it can be used to compare the relative fitness of various V. cholerae strains or the effects of mutations or gene knockouts.
1. Euthanasia
1. When the experiment has reached its desired endpoint, take a sample of the infection water (15 mL is usually sufficient) to allow for excreted bacterial counts and the measurement of diarrhea (such as mucin assay, protein content, and/or OD), if desired.
2. Pour off the remainder of the water through a fishnet (to collect the fish) into bleach to kill the V. cholerae in the water.
3. Place the fish into a beaker containing infection water plus 336 µg/mL of Ethyl 3-aminobenzoate methanesulfonate (tricaine) and incubate the fish in this tricaine solution for 20 min at RT.
NOTE: The fishnets were sterilized with a 10% bleach solution.
2. Dissection
1. Preparation of fish
1. Scoop a fish out of the tricaine solution with a disposable plastic spoon and place it on the dissecting surface.
2. Position the fish with its ventral side facing upward and pin it through the lower jaw, with the blunt end of the pin angled away from the center of the body. Place another pin just posterior to the anus, also angled away from the body.
2. Exposure of intestinal tract
1. Swab the ventral surface of the fish with a lint-free wipe dipped in 70% ethanol.
2. Sterilize a scalpel and Vannas scissors by dipping them in 70% ethanol and flaming them.
3. Make a small incision lengthwise in the belly just under the skin by penetrating the scales and using the scalpel. Be careful not to cut too deeply, as the intestinal tract is located just under the skin.
4. Using the scissors, extend the incision carefully along the length of the body, cutting no deeper than skin level and avoiding the anus.
5. Make two lateral cuts with the scissors toward the head of the fish to allow an opening of the incision.
6. Pin the skin on each side of the lateral incisions to the dissecting surface, angling the pins out, away from the body.
NOTE: The tips of the pins were previously sterilized by flaming them with alcohol.
3. Removal of intestinal tract
NOTE: The intestinal tract should be visible as a pale, very thin tube on top of the other organs.
1. Flame-sterilize the forceps and then use them to remove the entire intestinal tract (typically 12–15 mm in length). Place the intestine into a homogenization tube containing glass beads (see steps 1.3.1–1.3.2) and 1 mL of 1x PBS or LB, on ice.
3. Homogenization
1. Prepare the homogenization tubes in advance by pouring ~1.5 g of 1 mm glass beads into 2 mL screw cap tubes (fill them approximately half-way) and then sterilize them by autoclaving.
2. Add 1 mL of sterile LB or 1x PBS.
3. Once the zebrafish intestines have been added (step 1.2.3.1) to the tubes, screw the caps on very tightly, and then secure the tubes in the vortex homogenizer.
4. Homogenize the samples for 1 min on the maximum setting, then cool them on ice for 1–2 min. Repeat this homogenization cycle once more for a complete homogenization. Note: Several alternative methods for homogenization will also work.
4. Quantifying intestinal colonization levels
1. Prepare tubes (either small glass test tubes or 1.5 mL microcentrifuge tubes) for the serial dilution of the homogenate by adding 900 µL of LB or 1x PBS to each tube. For each fish, prepare 5–6 tubes and make 10-fold serial dilutions of the intestinal homogenate by adding 100 µL of homogenate to the first tube, vortexing it to mix, and then adding 100 µL from the first tube to the second tube.
2. Repeat this procedure until all dilutions have been prepared.
3. Plate 100–200 µL of each dilution on LB agar plates containing 100 µg/mL of streptomycin (if using Streptomycin resistant strains) and 40 µg/mL of X-gal (5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside). Incubate the plates at 30 °C for 16–18 h.
4. After the overnight incubation, count V. cholerae colonies on the plates, either using an automated colony counter or manually counting the colonies; it is helpful to mark the counted colonies with a marker tip.
5. Determine the CFU per fish intestine by multiplying the plate count by the dilution factor of the suspension, taking into account the volume that was plated.