Hi all,
I have a problem of growing worms. Worms have broken, not smooth surface on their body. They look not healthy and contain less number of eggs in their body. Brood size is also low.
We tried two or more strains(N2 and other mutants) but they looked all the same. So I guess this is not due to worms but NGM media. Anyone has idea how to fix ?
It affect everyone in the lab and we can’t figure it out. :-[
Best,
H
It sounds to me like the obvious culprits would be infection or bad media.
Regarding the media, I’m curious about cholesterol. From what I’ve read - I’ve never tried it - leaving cholesterol out of the media can cause progressive effects, especially in the F2, including small body size and small brood size. I couldn’t quickly find a report that low cholesterol causes visible cuticle defects, but it would make some sense. Are you sure you’re adding the cholesterol? And that you’re doing it at the right point in making the media (it should not be in the media when it’s autoclaved, should be added after the media has cooled enough that you can safely leave your hand or forearm on the flask, reportedly if the cholesterol is heated too much it becomes insoluble). That’s about the only part of the NGM recipe that might easily be gotten wrong, although of course if your ingredients are somehow bad despite having the right labels on them it could be a pain to figure out.
One other possibility regarding the media: I haven’t seen this with NGM and C. elegans, at least not very strongly, but automatic plate pourers can make bad or even toxic media, possibly because of the tubing they use. Hand-pouring some plates can help.
On the possibility of infection, all the usual advice applies: grow your bacteria from a single streaked colony, and bleach your strains to get (largely) infection-free eggs.
Hi Hillel,
Thank you for your comments, we do add cholesterol when media is cool down. but we have some issue of bacterial contamination recently and I might need to check the contamination of OP50 as you mentioned.
We had some issue of PH of the K buffer and I was thinking that might be an issue. Thanks again for point out multiple potential issues.