and Thawing Rates on Cryonic
Treatment of Caenorhabditis
elegans
by Carmel L.
My display at the Mid-Columbia Regional
Science Fair.
The
purpose of my experiment is to reveal and state the survival rate of C.
elegans nematodes in different freezing and thawing procedures.
And I hope to learn which Experiment Group will determine the most effective
way of freezing and thawing C. elegans.
My
hypothesis is that the survival rate will be greater with experiment Group
1, the C. elegans that are frozen gradually into the liquid nitrogen
and slowly thawed rather than the other freezing and thawing methods used
in the other 3 experiment groups.
Some
constants in my experiment were:
- liquid nitrogen container
- dry ice container
- Styrofoam case
- freezing vials
- petri dishes
- centrifuge
- centrifuge tubes
- beaker
The manipulated variables in my experiment are the different methods
the C. elegans are frozen and thawed. There are 4 Experiment
Groups and 5 tests in each Experiment Group and also a Control Group.
The
responding variable in my experiment is how much the survival rate is,
compared to the each Experiment Group and Control Group. To measure
the responding variable I will count each individual C. elegans
nematode under a low powered microscope. Three counts for every test
and divide it by three to get the average for that one freezing vial.
The
limitations in my project were that I only had so much information on C.
elegans and how they worked. I also have no background in chemistry
so I had to make all this clear in my head so that I would be able to do
my experiments the way they should be.
Because of the results of my experiment I wonder if I had controlled each different environment with set temperatures if it would have any impact on causing more or less of the C. elegans to survive.
The
purpose of my experiment is to reveal and state the survival rate of C.
elegans nematodes in different freezing and thawing procedures.
This
year this project was chosen because I was very interested on making a
unique and different project. I wanted something eye-catching but
also, a project that dealt with chemistry and living specimens. And
when my teacher mentioned this project, I decided to take a risk with a
project that had very limited background information. It was first
supposed to be dealing with regenerating
planaria and seeing if
any survived the freezing process and that dealt with cryonics, too.
But I had to remake and do some changes since when I researched online
and got feed back planaria does not survive under those circumstances.
So I had to do more and more research and finally with help with my teacher
and a professor online from Canada. I found a site that had procedures
on freezing nematodes. So now my project has changed, not completely
but just different specimen. So my project has come to this, my final
product. The work has been worth it, and this is why I picked this
project.
Freezing
C. elegans nematodes would benefit society by helping cryonics researchers
and scientist understand and build a better way to freeze other specimens
or possibly humans. Scientist could also have a better understanding
of the thawing procedures I have done in my experiment. They can
build from my project and make it more realistic for humans to be frozen
in time. Also, the scientist and anyone else interested can take
the informations gained from my experiment and gain an idea of what the
survival rate and what thawing procedures are the best for their experiments.
My
hypothesis is that the survival rate will be greater with experiment Group
1, the C. elegans that are frozen gradually into the liquid nitrogen
and slowly thawed rather than the other freezing and thawing methods used
in the other 3 experiment groups.
I
base my hypothesis on my background research report stating that when water
cools becoming into ice, that it will expand while in the freezing process.
This means that the C. elegans will be slowly packed and maybe even
crushed. But while slowly cooling down the C. elegans I theorize,
will take longer for the ice to fully expand. Also, the worms while
being frozen slowly will adapt to the freezing temperature. Instead
of being frozen quickly. Also, the worms while being thawed slowly
will revive and slowly adapt back to the room temperature. Lastly,
this is a more realistic point of view when the worms are living near ponds.
The water does not freeze in an instant it is a gradual process.
The
constants in my experiment are:
- liquid nitrogen container
- dry ice container
- Styrofoam case
- freezing vials
- petri dishes
- centrifuge
- centrifuge tubes
- beaker
- balance scale
- bottle
- freezing solution
- S Basal
- glass pipettes
- freezer
- fridge
- freezing and thawing times (8 hours)
- amount of time in the liquid nitrogen (30 minutes)
The
manipulated variables in my experiment are the different methods the C.
elegans are frozen and thawed. There are 4 Experiment Groups
and 5 tests in each Experiment Group and also a Control Group.
The
responding variable in my experiment is how much the survival rate is,
compared to the each Experiment Group and Control Group. To measure
the responding variable I will count each individual C. elegans
nematode under a low powered microscope. Three counts for every test
and divide it by three to get the average for that one freezing vial.
Freezing
Solution
1.
Measure the exact amount of every ingredient needed for the freezing solution.
2. Mix 1 Molar of Sodium Chloride in 100 ml of distilled water
3. Then put 20 ml of that solution into a bottle
4. Next mix 1 Molar of Potassium Phosphate with a pH of 6.0 in 100
ml of distilled water.
5. Add that mixture with the 20 ml of Sodium Chloride.
6. Then pour into the mixed solution 60 ml of glycerol.
7. Make sure the bottle is marked and labeled Freezing Solution.
S
Basal
1.
Measure each ingredient needed for the S Basal solution.
2. Pour into a clean bottle, 95 ml of distilled water.
3. Next, put 2.3 grams of Potassium Phosphate dibasic and 11.8 grams
of Potassium Phosphate monobasic into 100 ml of distilled water.
4. Then put 0.58 grams of Sodium Chloride into the same bottle.
5. Finally, put 1 ml of cholesterol in 95% ethanol (5-10 mg/ml)
6. Mix all ingredients well and label the bottle S Basal.
Freezing
Method
1.
Don’t feed worms for 1 day because somehow food in the stomach prevents
the worms from surviving the freezing process. This has to have been
prior to freezing worms.
2. To wash worms off the petri dish pour S Basal into the plate.
3. Then use a sterilized 5-ml glass pipette to pour worms and S Basal
mixture into a sterilized centrifuge tube.
4. Spin then worms and S Basal mixture in a centrifuge for 30 seconds.
5. Then remove everything but 1.5 ml of the supernatant.
6. Next, add 1.5 ml of the freezing solution.
7. Mix together well and distribute solution with worms into freezing
vials.
8. Make sure each individual freezing vial has marked in permanent
ink, which experiment group it belongs to.
Slow
Freezing Method
1.
After putting in each individual freezing vial put all of Group 1 (slow/slow)
and Group 2 (slow/fast) into a Styrofoam case and seal tightly.
2. Put it into the fridge for 8 hours.
3. Then transfer the Styrofoam case with freezing vials in it into
a freezer for another 8 hours.
4. Before putting into the liquid nitrogen, put the Styrofoam case
and C. elegans into a cooler with dry ice for another 8 hours.
There are a total of 24 hours in steps of different cold temperatures.
Be careful with the dry ice because it is really cold.
5. Take the freezing vials out of the Styrofoam case and put into the
liquid nitrogen for 30 minutes.
6. After 30 minutes take out each freezing vial.
7. Take Group 1 (slow/slow) and put it back into the Styrofoam case
and into the dry ice.
8. Keep Group 1 in the dry ice for 8 hours and then into the freezer
for another 8 hours and lastly in the fridge for 8 hours.
9. Take Group 2 (slow/fast) and thaw in room temperature water bath.
Fast
Freezing Method
1.
After in the centrifuge and already into individual freezing vial.
Put Group 3 (fast/fast) and Group 4 (fast/slow) into the liquid nitrogen.
2. Put these 2 groups for the same time as the slow method which, is
30 minutes in the liquid nitrogen.
3. Take out all the freezing vials and take Group 3 (fast/fast) and
thaw out quickly with a water bath in room temperature.
4. Put Group 4 (fast/slow) into the Styrofoam case with Group 1 (slow/slow)
in the dry ice for 8 hours and then into the freezer for another 8 hours
and lastly in the fridge for 8 hours.
Counting
the C. elegans
1.
Put each individual freezing vial into different petri dishes with names
on them with permanent ink.
2. Take a dropper and put 3 drops of one petri dish (which has one
freezing vials worth of worms) and put it onto a depression slide that
has 3 depressions for each individual drop.
3. Count how many worms are living in each drop under a microscope.
4. Make sure to record this on a data table.
The
purpose of my experiment was to reveal and state the survival rate of C.
elegans nematodes frozen and thawed in different ways.
In
my data table it shows that Experiment Group 1 had the highest percentage
of C. elegans living after the experiment was over. Experiment
Group 1 was the experiment group that was frozen slowly into liquid nitrogen
and was thawed slowly too. Experiment Group 3 showed the least percentage
of C. elegans living during those tests. Group 3 was the experiment
group that was frozen fast and also thawed quickly. The pattern here
looks as if the C. elegans survived more when thawed and frozen
slowly instead of frozen and thawed quickly.
I
have included data tables and also graphs to show what my results were.
In my results you can identify which worms
survived in the different experiments and tests and in the different temperature
changes I put them through.
My
hypothesis was that the worms frozen and thawed slowly would have the most
worms living. So because of my results I think that my hypothesis
should be accepted.
Because
of the results of my experiment I wonder if I had controlled each different
environment with set temperatures if it would have any impact on causing
more or less of the C. elegans to survive.
If
I were to do my experiment over I would make sure to change procedures
like, having different cultured plates of C. elegans for each experiment
group instead of just having one cultured plate. Because in my experiment
in some of the freezing vials I did not find any C. elegans at all
and I think that having only one cultured dish was the main cause of this.
There weren’t enough worms to go all around for each freezing vial I guess.
Cryonics,
[Online] Available http://www.cryonics.org/prod/html
Harris,
Phillip Nematodes, [Online] Available http://nhm.ac.uk
Liquid
Nitrogen, [Online] Available
http://web.physics.twsu.edu/facsme/nitro/safte.htm,
http://lrc.3.monmouth.army.mil/cecom/lrc/pie/nitrogen.html,
http://capederm.com/info_liquid_nitrogen.htm
Making
Dry Ice, Dry Ice, [Online] Available
http://www.occc.com/abc/making.htm,
http://www.mcneilly-ice-co.com/dryice.html,
http://engr.uvic.ca/~sventure/project_book97/97_Project.3.book.html
Nematodes,
The World Book Encyclopedia, 1999.
Putting the worms through the freezing process
in liquid nitrogen
Freezing vials containing the worms being thawed after freezing process.
Me being judged at regional science fair.
