“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?
Focusing: Since you will be looking at specimens in a dish with water in it, it is possible
to put the objective lens into the water. Please try to avoid this. The best way to focus
on your eggs is to first look at the objective lens and dish from the side of the
microscope while you bring the objective lens as close to the water, but not touching it,
as you can. Then look through the eyepieces of the microscope and move the specimen
away from the lens (down) until the egg comes into focus. This may take some practice,
so please practice on a microscope slide (dry specimen).
SAMPLE LAB NOTEBOOK PAGE
TITLE OF EXPERIMENT:_____________________________________________
DATE: _______________
METHODS:
DESIGN OF SAMPLES:
CNTRL
RESULTS:
Control:
Test1:
Test2:
Test3:
Test4:
NOTES:
TEST1
NORMAL
TEST2
DEAD
TEST3
ABNORMAL
SAMPLE LAB NOTEBOOK PAGE
TITLE OF EXPERIMENT:_____________________________________________
DATE: _______________
METHODS:
DESIGN OF SAMPLES:
CNTRL
RESULTS:
Control:
Test1:
Test2:
Test3:
Test4:
NOTES:
TEST1
NORMAL
TEST2
DEAD
TEST3
ABNORMAL
SAMPLE LAB NOTEBOOK PAGE
TITLE OF EXPERIMENT:_____________________________________________
DATE: _______________
METHODS:
DESIGN OF SAMPLES:
CNTRL
RESULTS:
Control:
Test1:
Test2:
Test3:
Test4:
NOTES:
TEST1
NORMAL
TEST2
DEAD
TEST3
ABNORMAL
Resources:
There are literally hundreds of sites devoted to discussion of zebrafish biology, rearing,
research and education. A good one to start with is below.
Zebrafish K-12
(http://www.neuro.uoregon.edu/k12/zfk12.html)