This method can help answer key questions in the vibrio cholerae field, such as how the vibrio cholerae colonizes in the Quiet Coast and competes with the intestinal microbiota to establish a niche. The main advantage of this technique is that fish are a natural vibrio cholerae host so we can study the complete vibrio cholerae life cycle. Demonstrating the procedure will be Dhrubajyoti Nag, a postdoc from my laboratory and Paul Breen, a Ph.D.student from my laboratory.
After preparing the V.cholerae cultures, inoculate the zebrafish by immersion. First, place four or five zebrafish into a 400 milliliter beaker containing 200 milliliters of sterile infection water. Then add one milliliter of V.cholerae culture for the desired infection concentration.
Then, cover the beaker with a perforated lid. After preparing all the beakers, transfer them to a glass front incubator set at 28 degrees Celsius For a transitory exposure, after about six hours, pour out the beaker water through a fish net to collect the fish. The infected water must be disposed of in bleach to kill the bacteria.
Then, place the fish into 200 milliliters of sterile infection water for five minutes. After five minutes, run this water through a fishnet, disposing of the water in bleach. Now, the fish can go into a new clean beaker with 200 milliliters of sterile infection water.
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 hominization 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. If several colony morphologies are observed, patch a few colonies of each different morphology onto a different TCBS plate. On TCBS, V.cholerae produces large, slightly flattened yellow colonies with opaque centers and translucent peripheries.
To determine the mucin levels in fish diarrhea, perform an assay on a 96 well plate. In triplicate, load 100 microliters of infection water for each test condition, including a blank, into the wells. Then, mix in 50 microliters of fresh, 0.1%periodic acid solution into each test well.
Use pipetting to mix. Next, wrap the plate tightly in plastic and incubate the plate at 37 degrees Celsius for one to one and a half hours. After the incubation, cool the plate down to room temperature.
Into each well, add 100 microliters of Fuchsin solution and mix with pipetting. Now, rewrap the plate and put it on a rocker at room temperature. In about 20 minutes, read the plate at 560 nanometers using a plate reader.
To quantify the reaction caused by mucin in solution. To measure protein levels in fish diarrhea use the Bradford assay. Mix 66 microliters of one of the BSA standards or 66 microliters of infection water with 1, 000 microliters of Bradford assay reagent in a disposable cuvette.
Now, incubate the samples at room temperature for two minutes. And then read the absorbance at 660 nanometers on a spectrophotometer. A simple metric for the amount of diarrhea is the turbidity in the water measured at 600 nanometers.
Mix the water well, add one milliliter to a disposable cuvette and take a measurement. For a baseline, blank measurement, use a clean infection water. The excreted V.cholerae bacteria can also be measured.
This can be done with fish that have been moved to clean water after being inoculated. Make serial dilutions of an aliquat of tank water as previously described. Then plate the dilutions on selective media and incubate the plates at 30 degrees Celsius for 24 hours.
Later, count the colonies as previously described and then determine the CFU. Groups of zebrafish housed in 200 milliliters of water were inoculated with five million CFU of a pandemic cholerae. After six hours in the bacteria infested water, the fish move to clean water for 18 hours.
The air intestines were then harvested. Each fish harbored an estimated 100, 000 to 1, 000, 000 V.cholerae cells. The infection water was used for other assays.
Mucin levels were higher when the infectious doses were higher. The OD600 of the water was significantly higher when fish were infected with V.cholerae. Finally, the total protein level in the infected fish water was nearly twice that observed in the water of the uninfected fish.
After watching this video, you should have a good understanding of how to infect adult zebrafish with vibrio cholerae and quantify diarrhea induced by the infection. Following this procedure, other methods, like transmission assays, can be performed to assess infection of naive fish by previously infected fish.
