Welcome to my
series documenting my trials and tribulations as I foray into academia.
I’ve recently been put in charge of a college-level course in developmental
biology. I do have a colleague handling the corresponding lecture, but
ultimately the responsibility for the labs falls on me. Labs are three
hours long and are held once a week for 13 weeks total.
Designing this
course has been fun, but challenging. The course
hasn’t really been run before and most of the labs have to either be created
from scratch or modified from protocols found within the Bio department or by
searching the internet. It's also hard to think of an appropriate lab
that’s specific to developmental biology, but doesn't overlap with typical labs
in a molecular biology, genetics, or anatomy course and fits within three
hours. I haven't been able to find many resources online for creating
this type of course so I thought I'd post my labs on SteemSTEM after I make
them.
In past weeks, the students have experimented on human placenta and living chick embryos. This week we've moved onto a new organism: The sea urchin.
Developing this Lab
Sea Urchins are one of the more unique organisms found in a
developmental biology lab. They’re
primarily used as a model organism for egg fertilization experiments and not
much else (https://askabiologist.asu.edu/explore/sea-urchins-do-research). Fortunately, they’re very well suited for such
experiments and it’s quite easy for students to learn to work with them. A simple fertilization can be done in under
an hour and variations to the fertilization experiment can be easily
implemented.
This was one of the last labs my students performed before
the end of the semester. Previous labs
had mostly stripped away the hand-holding aspects and I wanted to make the
students do as much as the experimental design as they could handle. Every student in lab was issued a digital lab
manual of Developmental Biology: A Guide for Experimental Study by Mary S.
Tyler (https://onlinelibrary.wiley.com/doi/abs/10.1002/ajmg.1320590229).
This book contains a chapter on sea urchins that should have all the
information needed to design a lab around them.
If this book is unavailable, Swarthmore has an online lab manual for sea
urchins that can do in a pinch (https://www.swarthmore.edu/NatSci/sgilber1/DB_lab/DB_lab.html).
These lab manuals were used because they don’t clearly spell
out a protocol. Instead, students spent
half of one lab reading through the manual to find certain variables like the
components of sea water, normal fertilization temperature, and how to induce
urchins to release gametes. To make sure
every student ended up with the same protocol, I created a power point that
ultimately laid out the protocol as students successfully described each step. They then spent the other half of the lab
making necessary reagents (artificial sea water and 0.5 M KCl) and preparing
glassware and other equipment. By then
end of this lab, students were expected to be fully prepared to begin working
with sea urchins at the start of the second lab.
For my part, I had to make sure that at least 2 sea urchins
(Lytechinus variegatus) were available for each group to work with. These can be a bit tricky to find online, but
companies like http://www.marinusscientific.com/
should have them available as long as they have a week or two notice. These sea urchins arrived in a large
insulated container with each sea urchin sequestered to a plastic bag filled
with water. We kept them at room
temperature in the bags they shipped with.
Most survived several days in those bags will no ill effects. We could tell that they were alive because
they’d wiggle when we held them.
Several sea
urchins used in this experiment
The full lab protocol is included below, but I’ll describe
how it works with pictures here as well.
Sea urchins are spiny balls, but half two distinct sides. One side as a mouth and the other has an
anus. All lab groups would grab a single
sea urchin and inject 0.5 M KCl around the edges of the mouth with a syringe.
Injecting a
sea urchin.
The syringe had to be sharp enough to pierce the soft tissue
around the mouth of the sea urchin, but students had to be careful to not
damage the organism too much or inject too much liquid. The 0.5 M KCl would cause the sea urchins to
release gametes out their back sides to the sea urchins then had to be placed
ass down over a beaker.
A Sea urchin
releasing eggs
Within minutes the sea urchin would start to release either
sperm (white) or eggs (yellow). Students
would do this for multiple sea urchins until they had both a sperm and egg,
then they’d mix the sperm and egg together in a petri dish to fertilize
them. We’d analyze the gametes under a
microscope to see if fertilization occurred.
The presence of an envelope around an egg usually indicated successful
fertilization.
A
fertilized egg with an envelope
Students had to then come in the next day to calculate the
percentage of eggs that had been fertilized.
Students were also instructed to design an additional experiment
to push their protocol beyond a simple fertilization. For example, they could adjust temperature
and see if that affected fertilization efficiency.
Protocol
Materials
0.5 M KCl
Artificial
Sea Water:
28.32 g NaCl
0.77 g KCl
5.41 g MgCl2·6H2O
7.13 g MgSO4·7H2O
1.18 g CaCl2
H2O to 1
Liter
Protocol
for collecting sea urchin gametes
1. Induce spawning – inject 1 ml of
0.5 M KCL into the soft membrane around the mouth
2. Gametes should appear within
minutes
Sperm are off-white
Eggs are tan-orange
3. Collect the sperm by inverting
the male onto a small beaker à
cover the sperm and place on ice
4. Collects the eggs by inverting
the female onto a beaker filled with artificial sea water à allow eggs to settle.
Pour out ASW and replace with fresh ASW.
Standard
Fertilization protocol
1. Wash eggs with 10 mL artificial
sea water (3 times)
2. Dilute 50 uL sperm in 5 mL ASL
3. Add 100 uL diluted sperm to 10
mL artificial sea water containing eggs in petri dish
4. Check for signs of fertilization
under the dissecting scope
5. Cover petri dishes and wait 24
hours. Calculate percentage of
fertilized eggs
Turning
this into an experiment
- You will perform both a
standard fertilization and an experimental fertilization
The experimental fertilization
will change one variable to the standard protocol to see if it affects
fertilization efficiency - Adjusting temperature, ion
concentration, pH, light conditions, exposure to UV light, etc. - In your lab notebooks, you will
include an extra paragraph in your conclusion explaining your results
Final Thoughts
This lab went very well. My only real complaint is that it
was my first time working with these creatures and I wasn’t as effective an
instructor due to my own inexperience.
Fortunately, my lab assistant had worked with them before and saved my
bacon on a number of occasions.
I’m not sure if I’ll continue this series. I’ve highlighted
my favorite labs and I may want to go back to covering research papers for a
bit. We’ll see what happens. Thanks for reading!