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(Source: B. Trevarrow from Zebrafish Book 5th Edition)

This is an update of the ENU mutagenesis procedure at the fish facility of the Institute of Neuroscience, University of Oregon. It is similar to other procedures except that after the exposure to ENU, the fish are maintained in fish water containing 10 ppm tricaine for about 5 hours. This concentration of ENU has a calming effect on the fish without anesthetizing them. Without the tricaine, the fish are very sensitive to almost any stimulus and react with extremely strong startle responses. Without the tricaine step, a high percentage (50% or more) of mutagenized fish die. With the tricaine step, the death rate drops to almost zero.

Prepare:
ENU preparation buffer (fish system water, 10 mM PO4, pH 6.5):
ENU exposure water (fish system water, 1 mM PO4, pH 6.5)
Fish wash water (fish system water, 1 mM PO4, pH 6.5, 10 ppm (0.01 mg/ml or 10 mg/L)  tricaine)
Remove the fish to be mutagenized from their tank and allow them drop to room temperature (~21˚C). This is thought to calm the fish.

Clear and set-up hood, start dissolving ENU:
Wear a lab coat, gloves, goggles, and dust mask or other face protection.
Line hood with bench coat and set-up the stir plate.
Inject 50 ml of ENU preparation buffer into the closed ENU container (1 g Isopacks, Sigma N-3385).
Swirl and pour the liquid into the flask.
Repeat to remove the rest of the ENU.
Cover ENU containing flask and stir at a fairly high rate until dissolved (usually ca. 30 minutes).
Put your ENU exposure chamber in the hood, fill with ENU exposure water and add the fish to be mutagenized.

Calibrate and normalize the ENU solution:
Once the ENU is dissolved, determine its OD at 398 nm relative to a control, such as the ENU preparation solution (see Justice et al., 2000).
Determine how much of your concentrated dissolved ENU solution is required to bring the volume of your ENU exposure chamber to your target concentration (usually 3.0 to 3.5 mM).
Add proper amount of ENU and cover the exposure chamber.
Leave fish alone for an hour during exposure to ENU.

Wash out ENU:
In a quiet and non-disturbing manner, transfer the fish into fish wash water, or change the ENU containing water to fish wash water.
Replace the wash water three times (1 hour each).
Keep the fish in the last wash until the fish have been in the tricaine for a total of 5 hours. Not enough time in the Fish Wash Water solution and the fish will start to show their startle reaction and die.

Waste Treatment and Disposal:
Neutralize the ENU contaminated solutions. For each liter of liquid waste, add:
5 g NaOH
100 g Na thiosulfate
Agitate the solution.
Agitate again the next day, morning and evening.
The following day, the NaOH in the liquid waste should be neutralized before calling Environmental Health and Safety for a pick-up.

Reference

Justice et al., (2000). Effects of ENU dosage on mouse strains. Mam. Gen. 11, 484-488.

4 Comments

  1. Anonymous

    "This concentration of ENU has a calming effect" should read "This concentration of tricaine has a calming effect".  Thanks again for this great advice.

    1. Done.  Thanks for the correction.

  2. Good to have this posted. There's a key inconsistency though: the first paragraph says 10 mM tricaine, but under "Prepare", the fish wash water says 10 ppm tricaine. Surely these aren't equivalent? FW of tricaine is 261.

    Thanks for clearing this up.

    1. Thanks very much, Chi-Bin!  "10 mM " is the tricaine concentration shown in the print version of The Zebrafish Book 5th Ed., but it's just gotta be a typo.  It's weirdly high - the recipe for tricaine anesthesia calls for a working solution of (if you trust my calculations) 0.16 mg/ml = .016 % by mass ~ 0.6 mM.  The correct value just has to be 10 ppm = .01 mg/ml ~ 37.5 micro-molar, which is the value shown in the fish wash water recipe in this protocol.  Considering the potential consequences of someone leaving mutagenized, stressed fish in 10 mM tricaine for 5 hours, I'm going to change 10 mM to 10 ppm right now.