we are having some problems with large scale worm collections from 10 cm NGM plates for DNA/RNA.
Anyone have tips to reduce burrowing (except use of agarose, because we need a lot of plates for different strains ,
and it becomes to expensive to use agarose). Thanks!
I hadn’t heard of agarose to prevent burrowing - I know it’s in an old DNA prep protocol (for Southern blots), it’s claimed it gives cleaner DNA preps somehow.
The main thing I recommend is to make very sure you don’t have any bacterial contamination (streak out your bacterial food source and always grow from a single colony, dissolve mothers in bleach leaving just the eggs, etcetera). If that’s not enough then a higher concentration of agar may help to prevent burrowing. But, some strains really like to burrow.
Just increase a bit the agar on the plates or double checked if you made a mistake in the last batch and added less agar.
This is what happened to us a couple of times and correcting/increasing the agar amount solved the problem
Thanks for the suggestions, we do keep the food clean and bleach, and add 2% agar. Would you recommend going even higher with the agar?
2% agar sounds high already (without looking it up I think we use 1.5 or 1.7% agar). There are different sources of agar out there, and I have no idea how much of an effect that could have.
Low humidity can cause the surface of the agar to crack, especially with older plates, but I’d think you’d notice that. And, I assume you’re not damaging the surface when you add the bacteria or the worms.
I think the obvious things may be covered, I’m not sure what else to suggest.
Hi Sevinc. Is this N2? Some other strains are more likely to burrow as well.
Hi Dave, yes, it is N2, and we noticed some of our strains don’t do it as much, but it is not strictly strain specific.
I am wondering if we should be storing our plates after seeding and while growing worms in humid (wet paper towel lined?) plastic containers to prevent drying.
Is this something people even do?
I’ve never really worried that much about a “plate humidor.” I will leave them on the bench for a few days after spotting, and only put them into plastic boxes if they are going to be out a week or more. At that point they will start cracking, sooner if the plates are older to begin with. But I don’t think that’s it.
I checked our protocol (originally from the Meyer lab): 2% Bacto Agar.
Thank you, in that case, we will try a few things and see if we can mitigate the burrowing a bit more…
A thought: I have experienced a type of contamination that is very subtle, but results in burrowing. One can discern it by a lawn-shaped scum on the plate after starvation. I am fairly sure that this arises from over-grown OP50 bottles, which I think it pretty common. Whatever it is, it can be transmitted to new plates upon picking. Curious if otehrs have seen this.
So consider:
- First, look at starved plates. Do you see this scum? I also sometimes see bacterial papillae on a starved plate.
- If so, streak and grow fresh OP50, taking care not to overgrow. On plates spotted with this, hypochlorite decontaminate your strain, and grow on fresh OP50. Is burrowing still a problem?
Funny thing is, I was just freezing some strains yesterday that had this problem. I think it can arise with people who over-incubate their OP50 cultures. But I have never followed it systematically. Again, I am interested to hear if others have observed the same phenomenon anecdotally…