I have a few research students in my undergraduate lab that want to treat C. elegans with various chemicals and compounds (ex. estrogen, caffeine, PCBs, environmental toxins, etc.)
I am familiar with C. elegans but not how to expose them to different chemicals. Is the common practice to add the chemical to the media immediately before pouring? Or, would I add the chemical to the bacterial broth either before or after the bacteria are grown overnight and seed the plates with the chemical/bacterial broth?
Any input, experiences, and references would be great. Thanks!
Generally I dissolve the chemicals in distilled water or in appropriate medium and then add to autoclaved and cooled to 60C NG agar medium.
And then pour in the plates and leave them overnight.
Following day, I seed the plates with the chemical broth.
Adding to the media, adding to the dried plate, and adding to the bacterial lawn are all fairly common methods. Some notes:
Unless you add to the media while it’s liquid, you don’t really know the concentration.
Even if you know the concentration in the media, this doesn’t mean you know the concentration experienced by the worms; for one thing, the bacteria may concentrate the drug.
More importantly, the bacteria might metabolize the drug. For this reason, you should consider doing your drug studies feeding your worms killed bacteria.
Different media recipes can alter the effectiveness of a drug.
You could also consider doing the assays in liquid culture in 96 well plates. Adding DMSO or detergent is an option with this approach; it helps some chemicals get into the worm. Liquid culture would let you assay a range of doses pretty easily and reduces the amount of chemical that one needs to use (unless you’re doing the assay on NGM agar in 96-well plates). The worms might be a bit unhappy in liquid growth, but one can use daf-22 mutants, which grow nicely in liquid culture. Also, Hillel’s point about the bacteria metabolizing the drug is a very good one. If you don’t kill the bacteria in the first pass, you should confirm any effects using dead bacteria.
Great points by Hillel and Jordan. I think it might actually boil down to the experimental design. Depending on your selection of drugs, there may be different tolerances to heat. The best thing to do would be to check out to see if any of that is an issue, and then perhaps figure out your delivery method (ie. adding it to hot molten agar or by spreading the compounds on top later, or even addition to liquid media as Jordan suggests).
Is there a particular phenotype the students are interested in? How will they assay the phenotype (will they use a microscope and look at the plates, or will they mount the animals on slides?)?
Thanks everybody for your thorough and informative responses. All of these considerations will help us in our design and controls.
At first, students will mostly observe the worms on the plates and collect data related to fecundity, changes in developmental timing, behavior, lifespan, etc. Later studies might include mounting and imaging worms depending on our initial results.