6/25/14 SUE - CORE LAB

web.stanford.edu/group/Urchin/first.htm 1/6



| OVERHEADS | GLOSSARY | REFERENCES | SKILLS | CLASSIC | TROUBLE |
CORE LAB
SUMMARY: This lab is designed to provide students with a laboratory
experience with sea urchins in which they will fertilize gametes and observe
early developmental stages. In this investigation we will:
1. Induce spawning of gametes by injecting potassium chloride solution.
2. Collect the gametes released.
3. Fertilize the eggs with sperm.
4. Observe development from early cleavage to the pluteus larval stage.
Medium
Difficulty
| TIMING | BACKGROUND | MATERIALS | PROCEDURE | OBSERVATIONS | IMPLICATIONS |
EVALUATION |
TIMING
The initial lab exercise requires a minimum of one 50-minute period. [Good idea to
start on a Monday]
The second day's activities require one 50-minute period to observe cultures, to
analyze observations, and to discuss.
The observations will require approximately 15 minutes on subsequent days for
about 5 days total.
BACKGROUND
Studies of sea urchins provide information on fertilization and development that apply to
all organisms from jellies to humans. These eggs, then, provide a model embryo for
understanding development in all forms. It is important to observe early embryonic
development because it is during this time that the patterns of development of the
organism originate.
Sea urchin gametes are the same size as human gametes and development is very
similar through the gastrula stage. Both sea urchins and humans are deuterostomes,
meaning that patterns of cleavage are radial and the mouth arises at a site distant from
the site of gastrulation. The name deuterostomes means second mouth. In contrast
protostomes follow a spiral cleavage pattern and the mouth forms near the blastopore,
the origin of gastrulation. See Classification tree. Notice the relationship between
humans and sea urchins.
For a visual overview of events in this lab see PATH OF DEVELOPMENT
SETUP
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IMPORTANT - to maximize observations the first day, cultures of embryos should be
started 1 1/2 and 3 hours before the class period. This will allow viewing of early
cleavage stages. If you are teaching multiple periods remember to start cultures each
period for subsequent periods to observe. See DETAILED timing and check-off list
MATERIALS
0.5M potassium chloride solution (3.73g of KCL in 100ml of distilled water)
sea water (or Instant Ocean, or artificial sea water)
fertile sea urchins, see also keeping adults
1-5cc syringe
plastic or glass pasteur pipets or eyedroppers
small tubes to store sperm (micro-centrifuge tubes work great) (1-5ml)
beakers a little smaller in diameter than the diameter of female urchins
microscopes (at least 10x and 40x objectives)
PROCEDURE
A. Spawning Animals:
To insure that there is at least one male and one female sea urchin it is best to have
approximately 10 sea urchins on hand. (If sea urchins are being shared with multiple
classes then save eggs and sperm and inject only one sea urchin for demonstration
purposes.)
Inject 0.5 M potassium chloride into the sea urchin.
WARNING - ONLY TEACHERS should handle the syringe with the potassium chloride.
Potassium chloride itself is not particularly hazardous, being the main ingredient in
substitute salt formulations, but injection into a vein or artery can cause an electrical
imbalance in the heart or in the brain resulting in death.
>>> see animation
Note how the needle goes over the
top of the teeth to inject KCl into the
body cavity and not into the mouth..
Side View /\ and Top View \/
Inject about 0.1-0.2 ml
per inch of urchin width in
each side of the urchin. A
total of two injections are
made. It is best to use as
small a needle as possible
as a larger needle leaves
a larger wound, subjecting
the urchin to infection.
(#25 - #30).
Gently shake the urchin for
a few seconds to mix the
KCl solution inside the sea
urchin. (shaking too hard
can kill the urchin by
ripping apart the delicate
internal membranes).
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Males: see animation. sperm are a milky white color
After injection, place the male urchin mouth side down onto a small dish, like a
petri dish. (NOT UNDER WATER).
In a minute or two you will start to see the white sperm appearing on the surface
of the urchin.
Collect the sperm "dry" in a pipet or eyedropper and place it into a small test tube.
[Seawater "activates" the sperm reducing their life span from days to minutes!
Keep concentrated until just before use.]
After you have collected all that you can, place the tube into a refrigerator (not
freezer) or an ice bucket with ice. Sperm will keep at 4 C for 2-5 days.
Females: see animation eggs are pale yellow to
orange to dark maroon, depending on species.
After injection place the female urchin mouth
side up onto a beaker FULL OF SEAWATER.
Eggs will not shed unless in contact with the
seawater. Beaker should be slightly smaller
than the diameter of the sea urchin.
The eggs will be shed into the seawater and
collected at the bottom of the beaker. This
can take from 10-30 minutes to finish
shedding the eggs, although, within a few
minutes you will have enough eggs to begin
work.
It is best to keep the eggs and the urchins at
the same temperature. (This is species
specific.)
WHEN THINGS GO WRONG AND YOU GET EITHER [NO EGGS OR NO
SPERM!]
B. Fertilization:
Appropriate sperm dilution is essential for successful fertilization. Too low a sperm
concentration results in fewer eggs being fertilized (safer than too high). Too high a
sperm concentration will result in polyspermy (more than one sperm per egg) and
abnormal development of the embryo. There are a number of built in mechanisms to
help prevent polyspermy, but they are not full proof and at very high sperm
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concentrations they can be overwhelmed.
see animations Just right sperm, Too many sperm, Too few sperm
Sperm Dilution:
A sperm dilution series is a useful way of showing just how few sperm are needed to
fertilize eggs. For comparison purposes, it is important to use the same sperm
concentration for each fertilization. Use a pasteur pipet to make the initial dilution .
Mark a line part way up the narrow end of the pipet. see illustration below
Always use this quantity of sperm as the starting point for dilutions. Add "one notch" of
sperm (2mm?) to 100ml of sea water (adjust this volume up or down if necessary to get
proper fertilization levels without polyspermy). This is referred to as the STOCK SPERM
SUSPENSION. A single drop of this suspension will be enough to fertilize 5 ml of a dilute
egg suspension (1-2%). A single drop can be used to demo fertilization under a
microscope. Although this is too many sperm for proper development, the embryos are
unlikely to develop under a microscope for a variety of reasons (heat from lamp will
make them too warm, the slide will dry out, etc.). This does, however, allow the student
to witness, first hand, the raising of the fertilization membrane.
Eggs:
1. Pour the egg suspension gathered from spawning into a 100 ml graduated cylinder
2. Fill to 100ml with seawater, and let the eggs settle. Without centrifugation the
eggs will swell (egg jelly swells) to about 2x their actual size. 4ml of eggs at the
bottom of a 100ml cylinder is really a 2% egg concentration.
3. Dilute the 2% eggs 1:20 (10 ml of 2% eggs into 180 ml of seawater) to a 0.1% egg
concentration. This concentration is better for long term storage of unfertilized
eggs (a few hours to one day without antibiotics) or development (3-5 days).
4. Alternatively suspend the eggs in a large flask no greater than 1 cm in depth to
insure adequate exchange of gases. (concentrations as high as 1% can be used in
this way to make it easier for students to find the eggs in early stages of
development)
Eggs can be re-concentrated to allow for easier viewing by letting them settle in a
beaker or test tube and pouring off the excess seawater.
To fertilize:
1. Have the students place a drop of egg suspension on a glass depression slide
under their microscope (a less that 1% egg concentration will allow students to see
individual eggs).
2. Let them see and focus on the eggs.
3. Next, while the student focuses on the eggs, add a small drop of stock sperm
suspension to the drop of eggs and add a cover slip. Quickly, let the students focus
on the eggs and watch the fertilization membranes rise.
see animations:
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Normal Development 94K
A Time Lapse Video from Sea Studios of early events, 241K
C. Development:
Keep dilute cultures of embryos, at less than 1% concentration, in sea water no more
than 1 cm deep or in slowly stirring cultures.
Timing:
This is different for each species and is HIGHLY temperature dependent (at warmer
temperatures under a microscope, division is faster). examples:
species
1st
division
2 nd
division
blastula gastrula pluteus
L. pictus
18C
90' 2.5 hrs 24 hrs 2 days 5 days
S. purp
12C
120' 3 hrs 24 hrs 2 days 5 days
What you usually have to do is set cultures up at different times.
See STORAGE for suggestions on how to store gametes for long periods of time.
See DETAILS for timing suggestions on how to set up cultures.
OBSERVATIONS
This lesson is often done when mitosis and meiosis are discussed. Students will
definitely be able to observe the first division, if they are present when it occurs, but
may not see "centering", "streak" and "metaphase".
Students should keep careful records of when each of the recognizable development
steps occurs. Detailed drawings are very helpful. If you are lucky enough to have two
species or two different temperature environments, students can compare stages and
rates of development.
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See URCHIN animation (55K)
IMPLICATIONS
1. What would happen if the sperm could not get into the egg?
2. How do organisms develop from the size of a sperm and egg to an adult? What
directs these changes?
3. Why is there such a difference in the size of the egg and the sperm?
4. Water currents in the ocean are much stronger than any sperm. How do sperm and
egg find each other? [Remind students that water currents can bring together as
well as separate and not all eggs will be fertilized, that is why so many are
produced.]
5. Why do sea urchins have external fertilization? [Discuss the life style of the
developing sea urchin embryo in the water column compared to a very different
ecological niche of the adults on the ocean floor.] What are the adaptive
advantages of external fertilization? What are the adaptive advantages of internal
fertilization?
6. What would happen if one cell was removed or damaged at the 2 cell, 4 cell, 8 cell
stage?
7. What would happen if the two cells were separated after the first cleavage and
allowed to develop on their own?
8. Why do cells with multiple chromosomes need the complex steps of mitosis to
divide in comparison to the steps of binary fission in bacteria?
EVALUATION
Active participation. This is a group assignment. Make sure everyone is allowed to
and does participate.
Lab reports - layout, tables, drawings, etc.
Answers to IMPLICATION questions.
Self evaluated: "Check Your Understanding"