Henry Viscardi Harlandale
School High School
Albertson, NY San Antonio, TX
Notes to the Teacher: to top
2 to 3 hour or older zebrafish embryos
Compound and/ or Dissecting Microscope
0.3M Lithium Chloride
Egg water (see teacher instructions)
Small Petri dishes
Pasteur Pipettes or medicine droppers
Zebrafish need dechlorinated water which can be created by aging the
water for 24 hours before use or you can buy bottled water. If your
area puts chloramine in the water you need to buy bottled water because
aging will not remove this substance. (To find out if your water contains
chloramine you need to call your local water authority.) To keep
producing embryos for future experiments you will need at least 2 - 10
gallon tanks. A ten gallon tank can hold anywhere from 25 to 50 fish.
One tank for breeding adults, and one for embryos/fry. Zebrafish
will lay eggs but they will cannibalize them. A dish of marbles should
be placed on the floor of the tank. Fertilized eggs that fall between
these marbles will not be eaten. The eggs can be siphoned or
pipetted from the marbles and put into petri dishes of embryo media.
(This can be obtained from a pet store or made, see recipe in teacher information.
For further info, see fish net website
To lay eggs the fish need to be on a 14 hour light cycle (14 hrs of light/ 10 hours of dark). They will lay eggs in the morning when the light is first presented to them. If you keep them covered you can get them to spawn when you want them to but the photoperiod must remain constant. The eggs have an 85-100% fertilization rate so once you get them your on your way. If you feed the fish 2-3 times a day with fish food obtained from your local pet store you can keep them happy and producing eggs every second or third day. The fry need to eat baby fish food that can be obtained from your local pet store (you are going to get to know the guy at the pet store really well) or you can feed them paramecium until they are 9 days old. At 9 days you can begin to wean them to brine shrimp. They should remain separated from the adults until they are 3 months old. At this point they are ready to begin breeding and will not be eaten by the older fish.
Many chemicals can interfere with normal embryological development. These chemicals are called teratogens because they cause a variety of developmental anomalies. Some chemicals we cannot avoid, such as environmental pollutants. Others can be avoided, such as drugs and alcohol. Fetal Alcohol Syndrome causes a wide range of developmental problems including growth retardation, facial abnormalities, and neurological disorders. Alcohol has the most dramatic effect if taken during organogenisis which occurs during the first trimester in humans. Zebrafish are also affected by alcohol. Zebrafish are an excellent organism for using to study teratogens in development because they have a clear egg casing.
Your job is to find out what effects alcohol and lithium chloride have on developing embryos.
3. Place 5 embryos in a petri dish of embryo media. Mark
4. Place 10 embryos in a petri dish of 2.8% ethanol solution. Mark this--ALCOHOL.
5. Seal each dish with parafilm or tape. Place in incubator at 28oC.
PART 2: EFFECTS OF LITHIUM CHLORIDE
1. Place 5 zebrafish embryos into a petri dish of embryo media. Mark this--CONTROL.
2. Place 10 embryos in a petri dish of 0.3 M LiCl for 9 minutes.
3. Remove the embryos after 9 minutes and rinse them in fresh embryo media. Place the embryos in a dish of embryo media marked Lithium Chloride.
4. Seal the dishes with tape or parafilm and place them in the incubator set at 28oC overnight. (If an incubator is not available a warm moist place will be sufficient.)
Look at the control embryos and then look at the treated embryos.
Describe what you see. Draw pictures of both control and treated embryos.
1. What effect does ethanol have on the development of zebrafish embryos?
2. What effect does LiCl have on the development of zebrafish embryos?
3. When you added the LiCl what observations can you make?
4. Predict what might happen to a human embryo if the mother drank during the pregnancy?
1. Look up Fetal Alcohol Syndrome in a text book or on the internet. What are some of the effects listed? How does this compare to your zebrafish embryos?
2. Look up a drug you think may affect the development of a human embryo. How would you set up an experiment to test the effects of this drug on zebrafish? Some drugs you may want to consider are aspirin, Retin A, vitamins, caffeine and nicotine.
Teacher: You may wish to test the effects of Retin A in zebrafish development.
Retin A is classified as a retinoid along with all other vitamin A derivatives.
They come from eggs, liver, meat, fruits and vegetables. Vitamin
A is important for many things such as vision, spermatogenesis, conception
and normal development of the fetus (Gregor, 1993). Retin A is commonly
used to treat troublesome acne and cancers such as promyelocitic leukemia,
breast and ovarian cancer and carcinomas of the respiratory tract.
In rats Retin A causes cleft lip and palate, brachygnathia and eye disorders.
In humans and chickens high doses have lead to abnormal development in
limbs, brain and spinal cord (Soprano, 1995). Zebrafish also show
a high toxicity for Retin A. In concentrations as low as 10-7
M zebrafish have very severe side effects. They can develop
without a head or eyes, microcephalic, micro-opthalmic, abnormal somites
or abnormal heart (Zhang, 1996).