The wet plate protocol: an efficient way to obtain dauer larvae

Developmentally arrested dauer stages in nematodes for laboratory assays have so far been obtained either by simply starving culture plates or by liquid culture in large Erlenmeyer flasks. Harvesting dauers from starved agar plates is straightforward but the yield is usually very low. Liquid cultures on the other hand produce high yields, but their maintenance is labor-intensive. We have combined the simple setup of starved plates with the increased yield of liquid cultures in the "wet plate protocol":

  1. Wash 3 well-populated culture plates of worms into a centrifuge tube using sterile dH2O. Pellet down and remove supernatant.
  2. Add 20ml of fresh E. coli OP50 grown in L-Broth medium.
  3. To start the wet plates, use 9 freshly poured 10cm NGM plates. The plates should not have been dried after pouring.
  4. Seed each plate with 2ml of worm/OP50 mixture.
  5. Place the plates in a Tupperware® box. Add moistened tissue to keep the interior of the boxes humid and incubate at 25° C.
  6. Check the plates every second day to ensure conditions are still humid.
  7. Depending on species, the plates are ready to harvest with lots of dauer larvae present after about 10 days.

It is important to maintain a layer of liquid on the plates at all times. If the worms are able to crawl instead of swim, the plates are already too dry. Just like liquid cultures in an Erlenmeyer flask, liquid plates may become contaminated. It is therefore important to prepare the plates to be as sterile as possible (i.e. under a flowhood). If problems persist, the culture may either be started with freshly bleached eggs instead of a mixed culture. Additionally, antibiotics and fungicides may be added to the worm/OP50 mixture.

For large-scale experiments that require huge numbers of dauers (e.g. microarrays), the use of liquid cultures is preferable. For smaller experiments however, the wet plate protocol has proven to be simpler, quicker and less prone to contamination in many assays in our lab.

Published: December 6, 2010 in


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