We have ordered new stocks of pax-2 from the CGC three times now and all have immediately developed contamination that I would describe as orange slime. It is in/on the op-50, not the agar itself. None of our other strains have ever had this type of contamination.
I have been picking rather than chunking for a few generations and the slime persists on every plate. It has not cleared by bleaching either.
Has anyone had this problem? Have you been able to clear the orange slime? Any suggestions?
Thank you!
Sounds like you have some kind of nasty biofilm-forming contaminant, likely Serratia marcescens or something similar, that is difficult to get rid of by bleaching. You should still be able to get rid of it by bleaching, but you may need to do this in tubes rather than just putting a drop of bleach on a plate. To do this, collect a bunch of gravid adults with a minimal amount of pink slime into a 1.5-ml eppendorf. Allow them to settle, aspirate most of the liquid. Optional: rinse once with water or M9 with light vortexing to remove more of the bacteria, and allow to settle again. Aspirate most of the liquid. Add ~0.5 ml of freshly made alkaline bleach solution, mix, spin worms down briefly, aspirate and replace bleach solution, spin down, and resuspend the embryos in a small volume of sterile water or M9 to transfer to a clean plate. The worms should spend a total of ~4 minutes in 2 changes of bleach solution; the carcasses should be efficiently disrupted and largely dissolved by this treatment, leaving mostly embryos.
Hello,
Thank you for the reply! This sounds similar to our bleaching protocol: we use 15ml conical tubes, worms suspended in 3.5ml sterile H2O, 1ml NaCL, and 0.5ml undiluted Clorox bleach, for 6-9 minutes. We spin down, aspirate, and wash with 5ml sterile H2O 3 times, leaving embryos.
I will try the extra pre-wash step to remove the bacteria. Should I just keep trying repeated bleaches until it clears?
Thank you!
Did you mean NaOH or NaCl? In any case, your bleach solution has about 3-fold less bleach than what I typically use, which is 3 parts bleach per 10 ml of solution. Specifically, I mix:
6.5 ml water
3 ml undiluted bleach
0.5 ml 10 N NaOH
10 ml total
If you are using NaCl instead of NaOH, that might account for the failure to completely eliminate the contaminant, and if you are using NaOH, I would suggest trying the more concentrated bleach recipe.
You can certainly do the bleaching in a 15 ml conical but I find it more convenient to do in a smaller tube and use a benchtop centrifuge to pellet the worms/embryos.
Some people also add a bit of nonionic detergent to their wash solution (e.g. 0.01% Triton or Tween-20) to reduce sticking of the embryos to plastic tubes/tips.
Good luck!
Abby
Oops! Yes, I meant NaOH, not NaCl. Thank you!
I usually clean C. elegans on plates, not in suspension; it’s moderately less work and it requires fewer gravid worms. My usual bleaching method, which has never failed to remove a contaminant if I try hard enough, is as follows (probably originally from a Michael Koelle protocol):
- Mix 7 parts 2.3M NaOH to 1 part sodium hypochlorite solution to make ~30 ul per strain
- Set a P20 pipetman to 5-6 ul, load it with the solution, set next to the scope
- Pick as many gravid animals as you can find onto your pick, certainly a couple of dozen
- Pipet the bleach solution outside the bacterial lawn of a NGM plate that has OP50 on it, ideally a plate that’s old enough that it will dry fairly quickly. Immediately swirl your pick and the couple of dozen worms it contains through this puddle over and over until the worms are off the pick and large clumps of bacteria have been broken up.
- Allow spot to dry. Apply another 5-6 ul of bleach solution to the spot.
- Continue waiting for the spot to dry and then applying more bleach until the worms have almost completely dissolved and only eggs and fragments of worms can be seen (usually 3-4 treatments; it is possible to overdo it, but harsh treatment can be necessary for robust contaminants)
- The next day, pick off animals that crawled from the spot into the lawn, put on the lawn of a new NGM plate with a lawn of OP50 (this step often isn’t necessary).