Intestinal Colonization and Diarrhea Induction by Vibrio cholerae in Adult Zebrafish

Abstract

Source: Nag, D., et.al. Quantifying Vibrio cholerae Colonization and Diarrhea in the Adult Zebrafish Model. J. Vis. Exp. (2018)

This video demonstrates the inoculation of adult zebrafish with a pathogenic Vibrio cholerae strain to study intestinal colonization and disease outcomes. It shows how bacterial toxins disrupt intestinal integrity, leading to fluid leakage, mucus secretion, and watery diarrhea before the fish are prepared for further analysis.

Protocol

1. Inoculation of zebrafish by immersion
   NOTE: This means of exposure resembles the natural course of infection.
   1. Preparation of Vibrio cholerae
      1. Inoculate 5 mL of lysogeny broth (LB) with an isolated V. cholerae colony from an LB plate and incubate it at 37 °C with shaking (180 rpm) for 6–8 h (overnight culture).
      2. Inoculate 25 mL of fresh autoclaved LB medium in a 250 mL conical flask with 50 µL of the above overnight culture. Incubate it for 16–18 h at 37 °C with shaking (150 rpm).
      3. Read the optical density at 600 nm (OD₆₀₀) of a 1/10 dilution of the overnight culture to estimate the number of bacteria per mL (the OD₆₀₀ = 1 is ~10⁹ CFU/mL.)
      4. Harvest the cells by centrifugation at 6,000 g for 10 min. Remove the media with a pipette or pour it off into a disinfectant solution. Resuspend the cells in sterile PBS to the desired concentration, typically between 1 x 10⁷ to 1 x 10¹⁰ per ml of PBS.
         NOTE: Here, we refer to autoclaved reverse-osmosis water containing 60 mg/L sea salts as sterile infection water.
   2. Inoculation and incubation
      1. Place four or five zebrafish into a 400 mL beaker containing 200 mL of sterile infection water.
      2. Add 1 mL of V. cholerae to get the desired infection concentration (typically 5 x 10⁴ to 5 x 10⁷ CFU/mL) in 200 mL of infection water.
      3. Cover the beaker with a perforated lid to prevent the fish from jumping out; the top of a 200 µL tip box works well for this.
      4. Label each beaker and place them into a glass-front incubator set at 28 °C for the duration of the experiment.
   3. Procedure for transitory exposure
      NOTE: A transitory exposure to V. cholerae (typically 6 h) followed by the removal of the inoculating bacteria is useful for studying the transmission and for the more accurate quantification of excreted bacteria.
      1. After 6 h of exposure, pour out the beaker water through a fishnet to collect the fish. Discard the infected water into a container of bleach to kill the V. cholerae.
      2. Place the removed fish in a beaker of 200 mL of sterile infection water. Allow the fish to swim in this clean water for 5 min to remove the surface bacteria from the fish.
      3. Repeat the net procedure and place the fish in a new beaker of 200 mL sterile infection water. Keep the fish in this beaker for the duration of the experiment.

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Instrument
Incubator	NUAIRE, Plymouth, MN	Auto Flow
Materials
400 ml glass beakers	Pyrex
Perforated lids	Microtip holder with holes from tip box
Fish Tank System	Aquaneering, San Diego, CA
RO Water Purifier	Aqua FX	TK001
Fish net	Marina
Fish food	Tetra fin
Petri plate	Fisher Brand, Hampton, NH	FB0875713
1.5 ml centrifuge tube	Midsci, Valley Park, MO	AVSS1700
Test tubes	Pyrex	9820
Micro pipettes	Sartorius Biohit, Göttingen, Germany	m1000/m200/m20
Tips	Genesee Scientific, San Diego, CA	24-150RS/24-412
LB medium
Trypton	BD Biosciences, San Jose, CA	211705
Yeast extract	BD Biosciences, San Jose, CA	212750
NaCl	Fisher Scientific, Hampton, NH	BP358-212
Agar	BD Biosciences, San Jose, CA	214010