A. Isolation of Zebrafish Embryos

1. The night before embryo collection, vacuum the tank well with a siphon to remove debris several hours after the last feeding.
2. Deposit a layer of washed marbles to cover the entire bottom surface of the tank.
3. Do not disturb for the first hour after dawn to allow fish to mate.
4. Collect embryos and fertilized eggs from between the marbles with a siphon then and collect with a fine fry net or mesh filter.
5. Transfer the net to a glass dish full of tank water and examine the embryos with a dissecting microscope at low magnification.
6. Transfer healthy embryos to a clean glass Petri dish containing embryo media with large-bore glass Pasteur pipettes. Discard any that are cloudy or ruptured.
7. When not being used for the experiment, the embryos should be kept at 28°C (Zebrafish embryos’ rapid development makes constant temperature crucial for obtaining a reproducible group of embryos for experimentation).
*protocol adopted from DB lab Website: Isolation of Zebrafish Embryos

B. Ethanol Treatment

1. Select 40 healthy zebrafish embryos that are at 30% epiboly stage (Figure 1 below).
2. Prepare four separate petri dishes that contain each of the following four: normal
zebrafish embryo medium, 1%, 2%, and 3% ethanol zebrafish embryo medium
solutions.
3. Take embryos out of the incubator (set at 28°C) and place 10 zebrafish embryos
into each petri dish. Place all four dishes in the incubator. Then wait for 3 hours
for the ethanol to take effect.
4. After the 3 hours, remove the dishes from the incubator and move the embryos
into new petri dishes containing normal zebrafish embryo solution. Different
batches of embryos must go into different dishes. Place the dishes in the
incubator and wait for 24 hours.
5. Return after 24 hours. Observe the embryos for abnormalities and photograph
them.
6. Return 24 hours later to observe the embryos for abnormalities. Photograph them.
*protocol adapted from: The Effects of Ethanol on Zebrafish Development: Jeff Mindel, F&M.

Figure 1. Zebrafish embryo at 30% epiboly stage (4 3/2 h). Top is the animal pole.
http://cegs.stanford.edu/image/244/medium/30%20epiboly%20side.jpg

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