Experiment With C. Elegans

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Experiment With C. Elegans

Postby KingPtolemy » Sat Sep 29, 2012 6:49 am

My topic is testing the effects of different chemicals found in red wine on the longevity of C. Elegans. This topic has been approved by my science fair coordinator, but I have no idea where to go from here. My specific question is how to expose the roundworms to these chemicals for them to take effect. My school has a lab, but I am not completely sure of it's limits. How do I go about conducting this experiment? What materials and methods would this involve? Also, if anyone could share some links or resources on how to write a detailed project proposal on these experiments, that would be helpful.
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Re: Experiment With C. Elegans

Postby KingPtolemy » Sat Sep 29, 2012 12:05 pm

Can anyone help? I need to submit a proposal by tomorrow, I found this http://www.docstoc.com/docs/7602175/SCI ... R-PROPOSAL document which outlines what I need to put in into a proposal, but I really don't have any clue. I would greatly appreciate it if someone who have done this ebfore or with experience could help me.
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Re: Experiment With C. Elegans

Postby Rooz » Sun Sep 30, 2012 5:25 am

Hey KingPtolemy,

I would start with defining what will be the outcome(s) of your study/experiment. For example, you would look into the survival of the worms exposed to reagent X compared to not-exposed to the reagent X. Let's say the exposed worms live 2-3 days more. I am not vary familiar with the life span of C. elegans so make sure that it matches with your study time frame.

I would limit the outcome and the reagents (stuffs in wine) to 2-3 factors.

You will have an outline after determining these factors to write your proposal.

Hope that this helps. Feel free to write me back if you have any specific question.

Good luck,
Rooz
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Re: Experiment With C. Elegans

Postby KingPtolemy » Tue Oct 02, 2012 8:04 am

Thanks for your reply, I'm working on my proposal right now.
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Re: Experiment With C. Elegans

Postby KingPtolemy » Wed Oct 03, 2012 8:49 am

I have a question regarding the amount of chemical/chemicals to add in each culture. Should it be the same or different? for example if I use 50mM of a single chemical in one culture, should I use 25mM each of 2 chemicals in another culture (for another experimental group) or should it be 50 each. How is this done generally and what is the justification to use that specific amount of chemical.
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Re: Experiment With C. Elegans

Postby sarahlaugtug » Thu Oct 04, 2012 3:00 pm

Hello,
Thanks for your questions. I can't wait to hear about your results and follow the progress of your project. I worked with Caenorhabditis elegans during my undergraduate work (cell biology), so I am pleased to see them resurface, so to speak.

So on with your question. You are wondering if you should keep the chemical components of wine the same or different quantities? First, I would like to direct you to the "Scientific Method" page on science buddies to find out more about setting up an experiment. You are right to ask these questions now. You will need to decide what your variables and controls are, read this article first,
Check out this article about "Controls":
http://www.sciencebuddies.org/science-f ... bles.shtml

Your control will be non-exposed worms. You need to have a fairly large sample size (maybe about 25, but that's up to you) to ensure that worms aren't dying from natural causes before you begin your experiment. For example since they only live 2-3 weeks, you wouldn't want to run an experiment that would last 3 weeks.

After reading that, there are a couple options you can choose to try in your experiment:
1) You can look at how the same amount of 3 or 4 chemicals affects the animal, or
2) You can look at how different amounts of 1 chemical affect the animal

Option #2 might be easier to test for since you are only looking at one chemical component of the wine. Also, true scientific experiments only look at 1 variable, for the purposes of eliminating other factors. It depends what you are looking for, so you should decide that first. You mentioned adding 50 units of 1 chemical, with 25 units each of 2 chemicals...this creates a lot of variables. Think long term about your results: you may have results that don't make sense; were your results based on one chemical or the other? Were they a result of too much of 1 chemical and not enough of the other? It will be difficult to tell.

Here is what I would do, sequentially:
1. Decide what you are testing for. Why do you want to do this experiment? Do you want to see how different chemicals affect C. elegans, OR do you want to see how one particular chemical affects C. elegans? What would be the significance of doing either?

2. Do some research on the life of C. elegans, and how to control their environment. Since they don't live very long, and you have a lot of time, it might be a good idea to grow some and that will help you determine how to keep them alive and how long they live, for your experimental phase.
A great resource for scientific research is to use Google Scholar-lots of different papers and experiments on this species. This will also give great examples on what the control and variables are. Here is one example of an article you might find useful; I found it on: http://scholar.google.com/
http://onlinelibrary.wiley.com/doi/10.1 ... ated=false


3. Above, you mentioned that you were thinking of adding 50mM? Your measurements will actually be in liquid form, since they live in agar, aka gelatinous goo, lol. So the units of measurement will be, microliters (uL). Your teacher should be able to help you set this up. Here's an article about agar:
http://www.sciencebuddies.org/science-f ... Agar.shtml

4. Figure out your variables:
A. Controlled variable--animals exposed to nothing (normal)..the food, agar, temperature, etc. should remain the same in all animals, including the experimentals
B. Independent variable--your experimental change
C. Dependent variable--how the animals change (if any) due to the independent variable.

:D
So, that was a lot of information. I hope I didn't overwhelm you, but please ask me more questions if any of that confuses you or you need help on the next steps. I can't wait to see how this experiment unfolds.
Always remain curious,
Sarah
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Re: Experiment With C. Elegans

Postby heatherL » Fri Oct 05, 2012 7:27 am

Hi KingPtolemy,

You have a really interesting project, and have gotten some excellent advice from the other experts. I just wanted to provide some additional resources for you regarding C. elegans.

WormBook (http://www.wormbook.org/) is an online resource about the biology of C. elegans, which could help you with your background research.
Wormatlas (http://www.wormatlas.org/) is another online resource about C. elegans biology.
WormBase (http://www.wormbase.org/) is mostly about C. elegans genetics, but may have some useful information.

I hope this helps!

Heather
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Re: Experiment With C. Elegans

Postby KingPtolemy » Tue Oct 16, 2012 8:19 am

Thank you Sarah and Heather for your replies. I need to know how to calculate nanomolars, I'm experimenting with a few chemicals like resveratrol and melatonin and would like to use 500 nanomolars of each. How do I do it? This is urgent and any response will be appreciated. Thanks.
KingPtolemy
 
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Project Question: Please suggest some science fair experiments for high school using Python programming
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Re: Experiment With C. Elegans

Postby heatherL » Tue Oct 16, 2012 9:20 am

Hi KingPtolemy,

Nanomolars is a measure of concentration, which is one million times smaller than 1 Molar (1 mole of solute per liter of solution).

Here is the Wikipedia page about Molar concentration: http://en.wikipedia.org/wiki/Molar_concentration

If your chemicals come in solid form, you would use the molecular weight of the compound to determine how much to dissolve in water to get your desired concentration.
Here is the Wikipedia page about concentration in general: http://en.wikipedia.org/wiki/Concentration

If your chemicals are already in solution form, you may need to perform a dilution to get them to 500 nanomolars.
Here is the Wikipedia page about serial dilution: http://en.wikipedia.org/wiki/Serial_dilution

I hope that helps you get started with your calculations. If you are still unsure, try posting again with more specifics so we can help ensure your calculations are correct.

Heather
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Re: Experiment With C. Elegans

Postby KingPtolemy » Thu Oct 18, 2012 7:40 am

Thank you once again for the prompt reply.

I have a few more questions.

I will be starting the experiment with 9 petridishes of worms, 1 control group and 8 experimental groups (with different combination of chemicals). I would like to complete the entire experiment with the adult worms I started the experiments with.

1. How many worms should I start with in each petridish?
2. How do I separate the progeny or what is the best way to prevent mixing of adult and progeny? Do I have to check on them everyday and manually remove them? How do I identify the adults from the progeny?
3. What proportion of males and hermophrodites should there be in each petridish?


thank a ton
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Re: Experiment With C. Elegans

Postby heatherL » Fri Oct 19, 2012 7:12 am

Hi KingPtolemy,

It sounds like Sarah has a lot more experience working with C. elegans than I do, so I hope she chimes in on this one.

I think that you would likely get primarily hermaphrodites and should not have to worry about males. They are self-fertilizing, so the best way to keep the adults separate from the progeny is to remove the eggs when you find them. I would suggest that you check every day for new eggs. You'll have to work under a microscope for this, and it will be a delicate process. It might be interesting to see how the chemicals affect development, though, so you might consider transferring some eggs to a petri dish with no chemicals and some to a petri dish with the same chemical on which it was laid. Just a thought.

Hopefully Sarah has some more specific advice for you!

Heather
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Re: Experiment With C. Elegans

Postby sarahlaugtug » Fri Oct 19, 2012 5:04 pm

Hello again,
I'm glad to see you are pursuing this project. Heather has some valuable information, so I think between the both of us we should be able to assist you when needed.

I highly recommend you do more research on the care of the worms, their environment, their life cycle; many of your questions can be answered through research. Check out some of the resources I listed toward to the bottom of this message. :D

Yes the eggs and small worms (should they hatch) will need to be removed and put into their own petri dishes or discarded. I would suggest saving offspring and using them as an additional control group--just remember that if you choose to do that, label the petri dishes (i.e. eggs collected on day 1 from generation L1 could be labeled, "L2, day 1"). I highly suggest having more than 1 control group, as well as experimentals. I would say use at least 2 controls and experimentals (ideally 3). Many of your worms will die (usually not by the experimenter's fault)...so you want to make sure your sample size is abundant. I have had to restart colonies because the worms died...good thing they are always producing!

Here are some answers to your questions:
1. Since you will not know the ages of the first generation (L1), unless you receive them as eggs, I would wait until several hatch (L2) and begin your experiment with those. Use about 5-10 worms per plate, in case some die.

2. How do I separate the progeny or what is the best way to prevent mixing of adult and progeny? Do I have to check on them everyday and manually remove them? How do I identify the adults from the progeny?
You don't really need to separate males from hermaphrodites, it shouldn't affect your results (hermaphrodites have thin, whip-like tails; while males have blunt tails...see pictures in the link below). I'm not sure if you will need to or want to separate the sexes out or not, but if you do, let me know and I can explain that. Check your dishes daily for eggs and hatched worms (they will be smaller and thinner than adults, and will move very quickly) and remove them. On that note, if your worms are getting sluggish/ not moving, they are going to die soon (they live only a couple of weeks). Also, since you will be transferring a known quantity of worms into each plate, you will know how many are new by the number of worms.

Question 2. 2. How do I separate the progeny or what is the best way to prevent mixing of adult and progeny? Do I have to check on them everyday and manually remove them? How do I identify the adults from the progeny?
How to remove L2 worms (and subsequent generations). You will be working under a dissecting microscope and using aseptic technique, you will need a Bunsen burner to heat your "inoculating loop" before scraping into your agar to retrieve new worms. Careful to let loop cool, so as to not singe the worms.

Question 3: You don't need any particular proportion; hermaphrodites self-fertilizing, and all offspring are clones (identical genetics). There will be very few males, as Heather stated.

Please check out these resources for information on worm care and anatomy.

Here is a site about how to grow them:
http://www.wormclassroom.org/c-elegans-cultivation

Here is an anatomical picture of worms:
http://www.wormatlas.org/ver1/handbook/anatomyintro/anatomyintro.htm

One important thing to mention is to label your dishes, or else your results will be very confusing later. Your labeling can include:
1. the generation (if you are keeping more than one, L1, L2, etc.)
2. Date of addition of worms onto dish.
3. Temperature worms are kept at.
4. your initials (if you aren't the only one working in the lab)

Let me know if this information is too confusing or you need further explanation.
Always remain curious,
Sarah
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Re: Experiment With C. Elegans

Postby KingPtolemy » Mon Oct 29, 2012 5:26 pm

I apologize for not replying earlier. It's been a hectic few weeks for me. The strains will be arriving tomorrow and I will be starting my experiment soon. I'll be back if I need your help, and thank you for all the valuable information you have given me. I really appreciate it :D
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Re: Experiment With C. Elegans

Postby sarahlaugtug » Fri Nov 02, 2012 2:11 pm

Hello and welcome back KingPtolemy,
No problem! We are here to help you when you need it. Glad to hear your worms will be arriving soon. I look forward to hearing how your project is coming along.
Have fun!
Always remain curious,
Sarah
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Re: Experiment With C. Elegans

Postby sarahlaugtug » Wed Nov 21, 2012 6:07 pm

Hello again!

Just curious to see how your project is going and if you received the worms yet?

Happy Holidays.
Always remain curious,
Sarah
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