I am helping an undergraduate student set up an experiment and would appreciate help with optimizing conditions. Design: expose worms to a drug through
day 2 of adulthood, then zap with a stressor e.g. oxidative, heat, cold and see how many survive vs. control. Questions:
We want to avoid transferring or using FUDR - how do we decide on a strain that is as close to N2 as possible, but sterile at a restrictive temperature
(e.g. fem-1, spe-9 etc.)? Or is it not a good idea to use such a strain when doing temperature stress assays?
Liquid or solid culture? So far, we have been growing on solid through L4, then transfer to liquid (M9) and add OP50 for 48 hours then stress. I noticed
that the bacteria forms clumps in the media, and am concerned that some worms are getting more food than others. Also, worms are more stressed in liquid
culture, so is liquid a good setup? Original thinking was that worms take up more of the drug if they are swimming in it vs. crawling.
To kill or not to kill bacteria? Whether in liquid or solid culture, should we kill the bacteria? If yes, what is the best way? Otherwise, how do we know that the
drug is acting on the worms directly and not through the bacteria?
For cold or heat shock, I have heard that people transfer animals to plates that are first subjected to cold or heat rather than just sticking a plate to hot or
cold temperature incubator? Can anyone share their technique for this?
Others might have better…or more right on suggestions, but here’s mine under the influence of travel lag and a healthy dose of flu…
So your student is doing the old one-two experiment, hit with drugs and count the survivors?
no transfer…if you go down the heat stress route (my personal favourite) then there are a number of advantages and considerations for transferless expts, some of which are also applicable to the other stress methods;
a. number of eggs laid drops off dramatically anyway at higher temperatures so the background is lower
b. you could mark the experimental worms with fitc or a vital stain and use this to distinguish expt worms from offspring (at least over a short period of time).
c. there will be intra-strain variation in response anyway that needs to be taken into consideration, hence control and treated worms should be subject to the same stress conditions (incubators are notoriously variable, within (shelf to shelf) and repeated measure wise.
swim or crawl? If the drug is easily dispersed/dissolved in agar then I would go the agar route…you can handle the plates easily enough and count the survivors. I would a) treat, b) stress and then c) (as a kind of reward) feed. This avoids messy setups and clumping bacteria. The worms are then on a plate as usual and the killing curve or survival curve can be calculated.
Kill? no…and if you follow 2 above then this is not a problem as the bacteria don’t get to the drug beforehand.
Plates take time (about 40 minutes for a 10cm plate to heat up…so this should be in the calculations…popping them in for 2 hours means that almost half (ok less but i wanted to be dramatic) would be the plates heating up to the required temp
i hope others will jump in and add to my answers (with constructive critique) or offer additional nuggets…but come back if you want further ideas, suggestions etc.