I have a question concerning FUdR and I hope that those of you who have more experience using this chemical might be able to help me with that.
I read that FUdR inhibits DNA and RNA synthesis which causes mitotic cells to die etc. I also read that bacteria should get concentrated before being added on the FUdR plates because they won’t grow there.
I want to do infection experiments over a few days with a large amount of worms and I don’t want progeny. The FUdR seems to be an option, but since bacteria don’t grow on the plates because of FUdR, isn’t this also interfering with my infection then or are the pathogens replicating normal again in the worms intestine? :-\
I’m afraid I don’t know much about the effects of FUdR on bacteria. Just thought I’d advise caution using the FUdR as it interferes with the physiology of the adult worm too, including lifespan (Aitlhadj and Sturzenbaum, 2010; Van Raamsdonk and Hekimi, 2011), morphology and motility (Bolanowski et al., 1981; Glenn et al., 2004). It’s lifespan effects can also vary according to genotype.
When I prevent contamination by progeny in ageing experiments, I pick the worms on each day during their reproductive period, although this can be quite time-consuming (and morale-draining!).
I’m sure FUdR can be of use in some situations. I guess whatever strategy you use to prevent progeny is a trade-off.
I don’t think using FUdR would be a good idea. It will severely limit the growth of bacteria. There are many other ways to avoid progeny contamination, including frequent transfers as Ben has mentioned. Perhaps you should explore using genotypes that lack a germline or are unable to reproduce at certain temperatures?
Either picking nor mutant strains seem to be an option for me. I have large amounts of worms, so I cannot pick them, and the mutant strains develop too slow at 15 degrees, the temperature where they still reproduce. Therefore both is too time consuming and that is why I went back to thinking about the FUdR method, since the nematodes are only the host and I’m more interested in the bacteria. Maybe I should just try it and compare the growth with/without FUdR. I might report my results here if I don’t forget it
I have a comment on supplementing FudR: we do not concentrate the bacteria prior to the addition of FudR, what we do is actually seeding our NGM plates with bacteria (day 0), let it dry for one day and then add a drop of FudR directly on top of the bacteria, let it dry for another day (day 1) and then transfer the worms onto those plates (day 2). The optimum final concentration of FudR varies according to the genotype (a final concentration of 10uM works fine for me to prevent progeny).
As for the effect of FudR on the bacteria, I think it is worth trying the addition of FudR on bacteria that is known to be pathogenic to the worm and see if the effect is diminished with the addition of FudR. I am saying this because the effect of FudR on the bacteria pathogenesis depends on whether your bacteria needs to be alive to kill/affect the worm. May be adding FuDR on top of the bacteria as described above would work. Also, it is worth optimizing the final concentration of FudR in a way that allows you to inhibit the production of progeny with minimal effect on bacterial proliferation.
thanks for your detailed answer. I will try it like you described! I was anyways unsure concerning the FUdR concentrations, as everyone seems to use differen amounts…
In this publication for example (Aitlhadj and Stürzenbaum, 2010), they use 0,1 mg/ml which is something around 0,4 µM only, if I calculated correctly… I tried this in a small plate with 10 worms and it worked. Then I tried 10 µM in large square plates directly in the plates (10x10) and it didn’t work! When I have those large plates, putting a “drop” of FUdR on top is not possible, I need some milliliters to soak the whole bacterial lawn with FUdR, I guess I would have to adjust the concentration to the whole volume of what I put on the plate… or do you adjust the 10 µM with the amount of agar that is in the plate?
Do you think I’d need a higher concentration of FUdR if I have large plates with so many worms since it obviously didn’t work? Or may I have made something wrong during production of the plates, but I wouldn’t know what, I put FUdR into the agar at around 60 °C…