Keeping to the wormbook I want to get started with liquid culture of c.elegans. We are a new c.elegans lab, and I am trying to avoid as many mistakes as possible from the getgo. Hence a couple questions:
what are common mistakes with liquid culture, that could easily be avoided if known beforehand?
S Medium: Is everyone making its own trace metal solution? I have been looking to buy it somewhere, but couldn’t find a supplier.
Thank you for taking the time to read, and possibly answer this.
dealing with your last question first, as the trace metals soln. in protocol 2 contains just 4 trace metal compounds; it’s easily made up and can be stored in a dark cupboard…
For the S-Basal, I add the cholesterol in a sterile hood after the autoclaving when the solution is at 50 degrees or so…like I do for NGM plates.
Step #5 of the protocol is important…too little or too much food (OP50) can cause problems either because you get a too concentrated bacterial soup or because they may enter dauer where food is short. Also worm density (too high) may induce the latter.
You can use OP50 that carries antibiotic resistance to make things easier as contamination is always an issue to consider See Solis & Petrascheck;
Last, (well from me at least) is that liquid cultured worms do look a little different from plate-grown worms…(see protocol in Wormbook).
I think the main thing to remember is that after synchronising your worms in the initial stages of preparing a starting population for liquid culture, you should be careful to keep everything sterile and to work accordingly…when a liquid culture fails, you lose a lot of worms, time and work.
Hi Stefan,
I usually omit the trace metals and never ran into problems !
It is the best to start with a synchronized population, this way you can cultivate roughly 2500-3000 worms per ml.
As steve mentioned, liquid culture easily gets contaminated, I usually add a mixture of antibiotics (Pen.-Strep.) and antimycotics (nystatin or amphotericin B) into the liquid culture.
let me know if you need a detailed protocol.
best,
Marlon