Hallo everybody 
I am a bachelor student working for the first time with C.elegans. I am trying to feed my worms with RNAi bacteria but my plates get directly contaminated with fungi. I was woundering whether I could use Nystatin. I read in the worm book that Nystatin is used for RNAi feeding in liquid culture, so I thought it should be fine.
I´ve seen that there is already a similar question,but no answer to it!
I would be glad if someone could answer me!
People do sometimes use Nystatin, and get away with it. Apparently it doesn’t kill E. coli nor C. elegans. But: it shouldn’t be necessary. and just because it’s possible to use it without utterly destroying your experiment doesn’t mean it’s a good idea. If you can keep your workspace sufficiently clean, you should be able to get rid of most fungal contamination; if you can’t do this, you have big reasons to worry. Aside from the issue of how to compare your results with Nystatin-treated worms to an extensive published literature that doesn’t use Nystatin, you have to worry that if your incidence of fungal contamination is so high it drove you to using Nystatin to keep it in check, what other contaminants are your worms being exposed to? Even if Nystatin were to get rid of the most obvious symptom, you’d still have an ongoing problem with frequent contamination of your plates. You’d be much better off solving that problem than using a questionable method of treating one of the symptoms.
Hi,
just out of interest, how do you know it is a fungal contamination and have you done the basic controls to identify the source of the contamination?
For example:
Do your (a) NGM plates (no OP50, no worms) become contaminated or (b) NGM/OP50 or © only when worms are added?
Is the contamination problem found in various parts (and/or also seen by others) of your lab?
Did the contamination start at a particular time and did anything change in your/the lab routine at that time?
In 1. above, if the answer is (a), then who makes your plates and with which reagents/equipment?
If the answer is (b), change your OP50 stock by going back to frozen or plated stocks.
If the answer is ©, then bleach your worms…or if the spores are resistant, chunk your worms from a uncontaminated portion of a newly colonised plate…or pick your worms onto intermediary plates (no OP50) and then onto clean plates (+OP50) and see if you are able to remove the contamination that way.
Steve
At the lab I’m currently at, part of our standard protocol is to use Nystatin in the plates we make. We use 10 mL of stock for each liter of NGM, where the stock is 10,000 U/mL. It really seems to help reduce mold contamination, and doesn’t hinder the growth of any of the E. coli that we use (currently, we use both HB101 and OP50) or the worms. We haven’t use Nystatin in RNAi feeding experiments, but if wormbook says that people do it for RNAi liquid cultures then it shouldn’t be a problem.
My lab uses nystatin in RNAi plates without any problems with growing the bacteria or seeing RNAi effects in the worms. Depending on where your lab is located the use of nystatin can be more or less necessary. When we were in Pittsburgh, we had to use nystatin in our plates or else we would see contamination that no amount of troubleshooting or optimizing would get rid of.
We buy a nystatin suspension from Sigma (N1638) and use 10 ml per liter of agar.
It’s pretty humid in the summer in our lab and fungus can get to be a problem. But, I agree with Hillel, its better to fight the source than add Nystatin. Nystatin acts by binding sterols and you just won’t know what effects it could have on your worms.
To keep fungus down, we wash the plastic boxes the plates are stored in after each use, seed plates in a bacterial hood, bag all used plates in ziploc bags before throwing out and do lots of wipes of benches/scopes with 70% ethanol.
With the exception of the hood, all of this should be possible in a undergrad-type lab. And it might be worth asking around if anyone has a hood you could seed in.
Good luck!
Amy