We have recently encountered a problem with our OP50. The colonies develop a “crust”, which crumbles when it is touched with a pick. The worms will eat it, but they definitely grow more slowly. N2 has trouble moving through the colony and sort of surfs along top. Wild isolates just burrow through it. I have encountered this phenomenon occasionally in the past, but it always seemed to resolve itself over a short time, which this time it hasn’t. The problem apparently isn’t the OP50 itself (we made a new stock plate from a frozen stock of OP50, problem not solved) or the culture media for the bacteria (fresh media from a different lab’s stock, same problem). Problem also apparently not the ancillary reagents in the plates (we made new stock solutions, same problem). Not the incubator (grew plates in a different incubator, same problem). I have asked several experienced worm hands, they all seem to recognize the phenomenon, but no one knows a solution.
For us, it was always streaking out fresh OP50 that did the trick. Odd that wasn’t the case for you.
Off the top of my head, oddball things that can cause issues:
residual soap in the flask the bacteria are grown in or the bottle that media/reagents are stored in
change in water quality. Even had a problem with some experiments repeating when the university altered their system for generating DI water.
Swings in relative humidity
No idea if any of those could be the root cause of your problem, just brainstorming things that sometimes are taken for granted that have caused me weird problems in the past.
Too much LB when spreading the plates with a bacterial culture can cause this. In our lab, we use low salt LB medium to grow the OP50-1 (half the NaCl as standard LB medium). This can make a difference. But as pointed out there can be other contributors.
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We have seen this problem a few times. Each time we were just happy to solve it and didn’t go at it in a very scientific one change at a time way, so I still don’t know exactly what the problem was. We believe once that it was a change in peptone source. We now only use Difco Bacto Peptone. The other 2 times we believe it was a problem with SOLUTION D, likely that the monobasic and dibasic phosphates were mixed up or one was measured incorrectly. Let us know what fixes your problem as we would like to add it to our possiblities should we see it again.
We have had this issue too, and from conversations with other labs + own tests we concluded it was the peptone content: switching from 7.5 g/L to 5 g/L solve the issue. However, I think there can be different reasons, and a lot of them have been discussed on the C. elegans researchers Facebook group: Facebook Groups. If you search for “crusty” you can find the post.