Hi! I have just started to work with C. elegans and I need your help.
I am planning to run a killing assay on liquid medium and infect around 20 hermaphrodites (C. elegans N2) with different pathogens. Here is my question: the killing assay will extend over the generation time of the worm so that new progeny may interfere with the counting of surviving nematodes…how can I overcome this problem? I also concern that some nematods killing is due to internal hatching and this may affect my results.
Does anyone have any advice or trick that can help me?
Thanks a lot
The simplest solution is to just transfer your animals every day or two as long as they’re laying eggs (depending on the rate of development under your assay conditions). However that can be very labor intensive, and if you’re not careful you can injure animals as you pick transfer them repeatedly.
Alternatively, FUDR is a chemical commonly used in the aging field to accomplish what you want. It blocks development and embryo formation in adults. Usually the way I use it I’ll egg prep my genotype of interest to plates without FUDR, and transfer them as late L4s or young adults to plates with FUDR. An earlier transfer can result in arrested larvae or adults with morphological defects. In my hands, putting gravid adults on FUDR increases bagging, and you get lots of arrested L1s on the plate (which may or may not be ignorable…)
However, there are increasing anecdotal reports that FUDR has some other effects; in particular I’ve heard that FUDR causes burst vulva in certain genotypes, and some assays can have different results with or without FUDR. FUDR might also inhibit the growth of your pathogens. It’s also fairly expensive – $700/gram if I recall correctly.
If I were you, I’d use FUDR, but only after running a few experiments to confirm that FUDR isn’t doing anything weird in your assay. Then, after you’ve tested all of your pathogens, repeat the most interesting experiments without FUDR.
In aging experiments, I censor animals that die by some unusual cause like bagging or a ruptured vulva (anything that doesn’t look like a natural death from old age, or in your case infection). That does NOT mean that you can simply ignore those animals – when you censor an animal, record it since they still give some information about population survival. Most standard statistical packages have a tool to handle this automatically for you, and if you really want you can figure out how to calculate Kaplan-Meier product-limit survival functions in Excel.
Also, you could consider using a mutant strain that’s sterile or doesn’t develop at 20°. Again, you’d have to validate that your chosen strain doesn’t alter immunity to the sorts of pathogens that you’re interested in.