Hi,
I am just starting to work with C. elegans,. The goal of the project is to test the affect of drugs on the lifespan of C. elegans. The first question is how do you count them? I am thinking of using 60 mm plates with a grid on them and a hand counter (assuming the adults will not move out of the “square grid” they are in during the counting). Does that seem reasonable?
The second issue is assaying just adults. Have seen the postings about FUDR, have also read about using a temperature sensitive nasophangeal mutant in which all the embryonic forms die at 25oC leaving just infertile adults. I do not want to have to transfer adults every 2 days to new plates for the life time of the study. Have thought about generating a large population of males, getting them onto their own plates and then not having to transfer them again.
Any and all suggestions/comments are welcome.
Thanks,
Gary
After a long time spent in the absence of hermaphrodites, males are inclined to leave the plate in search of a mate (and desiccate on the plate wall); see Lipton et al, and papers citing it. So you probably don’t want to circumvent the problem of self-progeny by using males. There are methods to discourage animals from leaving plates, but I’m not sure you want to introduce such complications. In any case, if you want to compare your results to previously published lifespan work, the vast majority will have been done in hermaphrodites, so you will probably make things easier for yourself by using similar conditions - unless you are hoping to find previously overlooked lifespan regulators that affect the longevity of males more greatly than that of hermaphrodites.
If you put so many animals on the plate that you really need a hand counter (instead of just muttering a running total to yourself, leaving your hands free to move the plate and adjust the focus and prod worms with a pick), I suspect the plate might be too crowded - or at least, crowded enough for the crowding to have possible effects on physiology. Normally my recommendation would be to use multiple 6cm plates, and a modest number of animals (under 40, certainly) on each. With only a couple of dozen animals on the plate, you don’t even need a grid; you can mark the lawn with your pick and make it around the plate rapidly and, with a little practice, without fear of double-counting an ambiguously placed worm.
On the other hand, I note that you are proposing to test the effects of drugs. Drugs are often expensive and limiting, which might be problematic using even a single 6cm plate, let alone multiple plates. And I don’t know how many animals you intend to use for each experiment, which would rather depend on how modest an effect you might need to detect.
You might also look into lifespan methods that have been done using liquid (which might be the easiest way to examine a large panel of drugs available in limited quantities) and into automated methods (in particular, there was a report at the last international meeting where plates were photographed at each time point, in order to determine whether animals had moved and were thus still alive).
As Hillel points out, the majority of work has been done in hermaphrodites and it would be much easier if you followed similar procedures/conditions. For lifespan experiments, I normally put 25 worms per plate and try to make a lawn directly in the center of the plate to avoid worms crawling on the side and off the plate. I normally have 2 experimental plates and a couple backup plates for each condition.
Lifespan experiments do require a lot of manual labour, but are rewarding in the end. Changing 50 or so worms every couple of days isn’t as bad as you think it is. I personally do not use the fem-1 background or fudr in my experiments. It can be a lot of work for the first few days, but afterwards it is smooth sailing.
Testing the effects of drugs is indeed challenging. You will need to test multiple things before you begin, including determining the concentration of the solution that you are adding to your plates. You’ll need to make sure your drugs aren’t crystallizing and you will need to decide if you’re going to be adding the drug to the media or spreading it onto the plates. Also, the frequency at which you transfer your worms will also ultimately affect the ‘dosage’ that the worms receive. Indeed, as Hillel mentions, it may be worth while to investigate lifespans performed in liquid culture.
Thank you Hillel and Snug for the insightful advice. So how does one go about doing lifespan studies in liquid culture, I thought that would be impossible due to the new progeny mixing with the adults.
Best,
Gary