“Fish eggs”
Cougar Quest July 13-24
INSTRUCTOR: Eric Shelden, Ph.D.
Live animals will be used as a necessary part of the instruction in this course to increase your understanding of the concepts and practical skills. This is to be done in an appropriate manner with care and humane treatment exercised at all times. The federal policies for the use of animals in instruction and research are set forth in the Animal Welfare Act and administered locally by the WSU Institutional Animal Care and Use Committee (IACUC). These polices, often different from customary guidelines used on private farms or in agriculture, must be strictly followed.
If you have any questions regarding live animal use, please contact your instructor. For general questions regarding the regulation of animal use, contact the IACUC office (335-7951) or the Office of the Campus Veterinarian (335-6245).”
“Fish eggs” HANDOUT I
SCHEDULE:
Monday:
Brief introduction to zebrafish research, instructors and the “fish eggs” workshop
Lab Safety Issues
Animal Research Issues
Set up of breeding tanks
Experimental Design
Introduction to Microscopy
Tuesday:
Harvest and cleaning of eggs
Set-up of experiments
Use of microscopes
Preliminary study of early embryos
Wednesday:
Observation of 24 hour old embryos
Data collection and logging
Treatment of 24 hour old embryos with test reagents
Thursday:
Observation of 48 hour old embryos
Data collection and logging
Analysis of results
Presentation of final results by students
Discussion
INTRODUCTION:
The Zebrafish: a versatile model vertebrate for research and education.
Zebrafish Biology: The zebrafish is a small (adults are about 1 inch long) tropical freshwater fish widely available in pet stores and from biological supply companies. Zebrafish are indigenous to eastern India, where they live in shallow pools and rice paddies. Zebrafish are hardy omnivores, and easy to maintain in a freshwater aquarium. The use of zebrafish in scientific research was largely initiated by Dr. George Streisinger at the University of Oregon. Zebrafish have a high reproductive capacity; a single female may produce over 200 eggs in a single day. The zebrafish also has a relatively short generation time, reaching maturity within three months of hatching. Zebrafish egg and embryo are nearly transparent, and much of the major development of the organism occurs within three days.
Zebrafish as a research animal: The features above have led large numbers of educators and scientists to adopt the zebrafish as a model of choice. Increasingly sophisticated knowledge on the embryogenesis of vertebrates, including humans, and disease processes has been gained from the study of the zebrafish, and entire businesses have arisen devoted to producing, housing and maintaining zebrafish. In 2005, the National Institute of Health spent an estimated $10 million dollars on a dedicated zebrafish facility housing half a million animals in 25,000 aquaria. Similar government and privately funded installations have been opened in a number of countries around the world, including Japan, Singapore, and Germany.
Zebrafish care: Zebrafish are easy to care for. They grow fairly well at room temperature and can be fed commercial fish flakes. Their most critical requirement is good quality water. We use distilled water supplemented with, and change about ¼ of the water in our tanks twice a week.
Zebrafish Breeding: The best production of fertile eggs will be obtained from fish maintained at about 28oC (82oF) that have been fed twice a day with a high protein food source (conventionally, fish are fed live brine shrimp once a day and commercial flake food once a day) for several days. Ideally males and females will be maintained in separate enclosures until the day of breeding. Males are streamlined and slightly golden/orange in color, while females with eggs have rounder bellies and are more silvery in color than the males. Males and females should be placed in a breeding tank the night before eggs are needed, and eggs will generally be produced at “dawn” on the following day. One minor problem with zebrafish is their exuberance in eating their own eggs and larval offspring. To prevent this, zebrafish breeders often put one or two layers of clean glass marbles at the bottom of their breeding tanks. This allows eggs to settle between the marbles and out of reach of the breeding adults. We also use nylon mesh, sometimes weighted with small stainless steel nuts and bolts. The mesh can be found at art supply stores and is used for making “hooked” rugs, and the nuts and bolts can be found at most hardware stores. It is essential to use only stainless steel hardware (they are a little more expensive, but you don’t need many). It is also important to cut the mesh carefully so that there are no gaps between the mesh edges and the aquarium tank walls. Otherwise, adults may sneak below the mesh and either fail to breed or eat the eggs.
LAB SAFETY
Zebrafish are harmless and although they can become diseased, no disease of zebrafish is known to infect humans. However, some safety precautions must be observed at all time in the workshop.
1) EYE PROTECTION MUST BE WORN WHENEVER YOU ARE PIPETTING ANY CHEMICAL SOLUTION, NO MATTER WHAT THE CONCENTRATION.
2) NO ACCESS TO FISH TANKS WHEN THE PUMPS ARE RUNNING.
3) SMALL SPILLS OF AQUARIUM/EGG WATER MUST BE CLEANED UP IMMEDIATELY. LARGE SPILLS OF AQUARIUM/EGG WATER MAY CREATE AN ELECTRICAL HAZARD IN THE CLASSROOM. DO NOT GO NEAR THEM.
4) WASH YOUR HANDS AFTER LEAVING THE LAB. Bathrooms are found on the east end of the building. Men’s rooms are on the 5th floor, Women’s one the 4th.
5) NO EATING OR DRINKING IN THE LABORATORY.
6. Wash your hands before leaving the laboratory.
REMEMBER: DO NOT WALK THROUGH, STAND IN, OR TOUCH PUDDLES OF WATER ON THE FLOOR!!!
SPECIFIC PROCEDURES
BREEDING (Monday):
BREEDING CHAMBER:
You need to construct a simple breeding chamber. We will use a 4 liter food storage container. To prevent the fish from eating the eggs they lay, you need to create a physical barrier that will let eggs through but not the fish. We will do this by adding a layer of washed glass marbles to the bottom of the tank. Fill the tank with fish water until it is about 2 inches above the level of the marbles.
ADDING FISH:
You need to put two males and two female fish in the chamber. They will already be in separate tanks for you, but you should try to see the differences. Males are skinnier and a little orange, females are rounder (if they have eggs inside) and they are more silvery. The glow fish are much harder to tell apart, because they don’t have natural colors. You can usually tell the difference by the shape of the fish, especially if the females are ready to lay eggs.
The easiest way to do this is to use a large net to catch a fish, but don’t take it out of the water with the large net. You should move the net to “corral” the fish in a corner of the tank, and then move the net from the bottom of the tank toward the top. The movement is sort of a “scooping” action. Pull the large net with a fish partially out of the water – so that the fish ends up in the net in a small amount of water. Now take a small net and scoop the fish out of the larger net. VERY IMPORTANT-> Move the net with the fish in it with your hand over the top of the net. The fish can jump out of the net if you are not careful about this. Don’t take too long to move the fish, but don’t rush so much that you knock it against something. They can often survive out of the water (if damp) for over 10 minutes with no harm. BE ESPECIALLY CAREFUL WITH FEMALES. Their big bellies put a strain on their bodies, and they can break their backs if they are handled roughly or even try too hard to escape the net when they are out of water. Surprisingly, they will survive and even breed with a broken back.
If you have trouble with any of this, ask for help. It is best not to stress the fish with too much chasing if you want them to breed well.
Write your name on the tank to identify it. When everyone has gotten their chambers set up, put your tank with the fish back in the styrafoam enclosure at the back of the room.
COLLECTING EGGS (Tuesday).
If things go well, you should have live fish and lots of eggs on Tuesday. Scoop the fish back out of the tanks with a small net, and put them into the aquaria. Don’t worry about which one you put the fish in – we will sort them out back at the Shelden lab. Now you must separate the eggs from the debris at the bottom of the chamber. The eggs are tiny and nearly transparent, so they are hard to see. Sometime, you can see them better if you place the container they are in over something that is dark in color and smooth (a piece of black construction paper would be fine).
Remove the marbles.. Now, carefully pour about half of the chamber water into a waste bucket. Do this slowly, so the eggs stay on the bottom of the chamber. Now, swirl the chamber and then pour the eggs briskly into a tripour beaker and then into a 50 ml blue capped falcon tube. Try to tip both the chamber and the beaker toward each other so the transfer is smooth – you don’t want to slam the eggs into the bottom of the beaker using a waterfall-like action.
Allow the eggs to settle to the bottom and then slowly pour out as much of the water you can, without pouring out the eggs. You may want to do this into a second plastic beaker, rather than your waste bucket, just in case. Add enough new water to cover the eggs with about an inch of water and swirl them around again. Repeat this three times to get rid of anything lighter than the eggs. If there is debris that you cannot remove with this method, use a transfer pipette to suck out and discard the debris. If there are any cloudy white eggs, discard those too.
Cut the end of your plastic transfer pipette off to create a larger opening (the opening the pipettes come with is too small to move eggs with – it will damage them if you try. Now, transfer your eggs to a 6 cm petri dish. Use your pipette to remove any last debris or dead eggs.
EXPERIMENTAL SET-UP
We will design experimental protocols during the workshop. The basic set up is to place about 10 eggs in each 6 cm Petri dish. Add enough buffer to cover the eggs, but don’t fill the Petri dish to the top. Overfilling will increase the risk of spills and reduce oxygen available to the embryos. It will also make it difficult to see the embryos with your microscope.
Seal the Petri dish with a ½ inch wide strip of parafilm.
Write your name and the test condition on the top of lid of the dish.
Place the dish in the incubator. You can stack up to four them on top of each other.
USING YOUR MICROSCOPES
You will use a dissecting microscope for most of your studies. All of the scopes have enough power to see most of the structures of interest in the developing embryo. Use the focus knob to bring the eggs into focus. If this is not possible, the microscope may need its height adjusted. I can help if you with this. There are two lights on the microscope – see which way makes the embryos easier to look at for you.
You will also have access to a high quality college-level compound microscope. Unlike microscopes you may have had access to in the past, there are several parts that must be properly adjusted for optimal use.
Eyepieces: There are two adjustments that can be made: the width between pupils, and the focus for each eye. You should adjust these to match you own eyes. If you don’t do this, you will have trouble seeing things well, and you may develop eyestrain/headaches with long term use. To adjust the microscope for the distance between your pupils, look into the microscope with the light on. Push the eye pieces together or pull them apart until you see a uniformly bright field with both eyes. Your microscope has one “fixed” eyepiece and one that can be focused. To adjust the focus of each eyepiece for your own eyes, you must first find a specimen and then focus the microscope for the eye that looks through the “fixed” eyepiece. Then adjust the other eyepiece until it is also perfectly in focus.
Condenser lens: Below the specimen platform, there is a separate lens that focuses light on your specimen. The housing that holds the lens also has an adjustable diaphragm that can be used to increase and decrease the amount of light that comes through it. A small lever is moved from side to side to do this. First find the knob on the lower left of the microscope that moves the condenser lens upward. Move the lens to the top of its range (all the way up). Next close the diaphragm. As an exercise, find a specimen and adjust the height of the condenser lens and the diaphragm opening. How does the image of the specimen change?