I’m a high school student looking to do a science fair project dealing with Parkinson’s Disease and C. elegans. Since I am unfamiliar with working with worms, I was hoping that you guys could answer some of my questions:
What is the purpose of washing worms?
What is the purpose of adding CaCl2, MgSO4, KH2PO4, cholesterol, and ethanol to Nematode Growth Agar (NGA)? What about Ampicillin in LB broth?
Can Lewy-bodies form in wild-type C. elegans or only in mutant types?
How should I transport worms from one location to another (ie from school to a university lab) without harming them?
Would it be possible to standardize the amount of bacteria on a plate?
If I were to expose the worms to nicotine, how would I do that and what amount would be appropriate?
What are some good websites to visit for general info?
Others can give you advice on how to administer nicotine to the worms. I’m thinking it could be included in the agar pad in a fixed percentage, and/or in the bacterial food that you seed on top. You might vary the percentage to see what effects you can get in terms of behavior, growth, or pathology. A good place to start.
Lewy bodies are well defined for vertebrate brain, but to my knowledge they have not been described in any invertebrate tissue. They were first described by transmission electron microscopy (TEM) as inclusions in dying brain cells. You may be able to find a stain for Lewy bodies that could be tried for light microscopy? I’m not sure. Try a literature search with Google to find such a stain? Some of these stains may be toxic, and require careful handling of the solutions. It may be easier and safer to stain the live worms, or you can learn to “fix” them (kill them) first before staining. Here again, the fixative solutions also require careful handling, as many are toxic to humans too.
Light microscopy seems easier, cheaper and faster than trying to learn TEM. Methods for TEM are available at WormAtlas. General methods for culturing worms and for light microscopy are available at WormBook. (see clickable links at the top of this page)
You wash worms to remove bacteria that may be stuck to them, possibly interfering with additional treatment / microscopy.
2) What is the purpose of adding CaCl2, MgSO4, KH2PO4, cholesterol, and ethanol to Nematode Growth Agar (NGA)? What about Ampicillin in LB broth?
You probably don’t want to grow worms in liquid culture (although I’m not familiar with nicotine assays). You can add antibiotics to plates but it’s generally not necessary with good technique. The other ingredients are metabolites and things worms can’t synthesize. NGA/NGM is probably (hopefully!) described in more detail in WormBook.
4) How should I transport worms from one location to another (ie from school to a university lab) without harming them?
You can transport worms with ease. Use parafilm to attach the lid. If the plates aren’t vented (vented plates have a little ridges on the lid that keep it from resting flat on the bottom half of the plate), poke some holes in the parafilm. You can carry them just like that. You might not want to leave them in the car on a cold and blustery day.
5) Would it be possible to standardize the amount of bacteria on a plate?
You could create serial dilutions of your bacterial liquid culture, seed plates with a specific amount of bacteria, and incubate for a controlled period of time. Unless your assay depends on this, I don’t recommend this. It’s not really necessary.
The D.A.R.T. (Drug Abuse Research Team) at Abraham Lincoln HS in San Francisco has a good website describing alcohol/drug research in C. elegans. URL: http://www.edserv.sjcoe.net/dart/julie/index.htm - follow the links to “Prep It” and “Do It” for recipes, instructions, and good suggestions on setting up to do worm experiments.
Bill Schafer’s lab (at the MRC in Cambridge, England) studies worm responses to nicotine and would have advice on how to expose worms to nicotine. It is my understanding that you can either “seed” plates with a 30 mM nicotine solution (obtained from Sigma) - let the nicotine solution soak into the agar before adding worms. Alternatively, you can let the worms swim around in a liquid solution (such as water) containing nicotine (you’d probably have to test which concentrations give you a phenotype) [the cap of a 1.5ml microcentrifuge tube makes an ideal well] - after a certain amount of time, remove the worms from the liquid with a pipet, transfer to a bacteria-seeded plate, then observe the worms for any phenotypes. The Schafer Lab’s website is: http://www2.mrc-lmb.cam.ac.uk/groups/wschafer/