I just started to work with C. elegans and should establish some methods, as I am the only one working with nematodes so far in my lab… Unfortunately, problems already start with the plates Sad
I am using the standard-protocol (Brenner 1974), but after 3 - 4 days the agar surface becomes uneven and the plates become somehow soft, so that the worms crawl into the agar and I lose a lot of them. Also, when I washed some off the plates with water, about 80% those worms, who still were on the surface, adhered there and could not be washed of. Therefor I had a lot of agar in my tube afterwards… It’s really weird… And annoying.
So, does anyone have some advise or a trick or anything that could help me???
Would be great, because I’m just starting and it’s already stuck…
Worms digging in is normal but if that happens more than often, you can try increasing the concentration of AGAR so that the plates are HARDER and little problematic for worms to dig in.
Try using 0.1% Tween+S basal solution…to wash of worms so they don’t stick to the plates.
And to me the agar gets washed too if I use the pipette and use a little more force, so try to just pour in S basal+tween, wait for 30 sec and just shake it a bit, don’t use any other thrust or force.
Hi,
I have another suggestion, make sure the OP50 and your worms are clean of any bacterial or mold/yeast contamination. In my experience, some contaminations cause softening of agar allowing worms to burrow more. Usually the agar surface starts to form depressions around where the worms were put down with the pick or where OP50 is seeded and once the surface becomes irregular, worms burrow even more. Some worm strains can be prone to infections, their guts or cuticles may become colonized with the contaminant and cleaning them up may be difficult, but with some effort (bleaching, serial transfer from plate to plate, etc) you can keep your worms and plates clean. Good luck,
wonder if anyone could tell what’s the purpose of adding cholestrol in NGM … b’cos i was following the protocol, and tried to look for the explaination behind this.
What I’ve always been told (and what I see Todd Harris said in this Wormbase Forum post) the reason is that worms need cholesterol, and cannot synthesize it themselves.
I don’t think I’ve ever looked into how this is known. In Chapter I of Worm II, it says:
C. elegans does not biosynthesize ecdysteroids, although cholesterol is required in the diet (Chitwood and Feldlaufer 1990).
if you are interested in “outsourcing” your plate-making job, please check my business out: www.lab-express.com.
I used to be a worm scientist and started the business a year ago. I provide worm plates to researchers.
Thanks.