Help on Egg Picking Methodology

Model Organism: Worms Methods & Reagents
1

Hello everyone,

I am working on C. elegans, more specifically on monitoring some endpoints from egg to egg-laying individuals. To achieve this, I need to individualize the eggs. Our current protocol involves a bleaching to synchronize organsims and pool the eggs in M9 solution. After that, the goal is to pick about 50 (more for future experiments) with platinum wire.

However, we’re not quite able to individualize the organisms starting from the egg for various reasons, the main one being the confusion between the eggs and residue from the bleaching, the agar plate, … As of now, we can start the individual monitoring as soon as they hatched, because L1-stage nematodes are much easier to pick + identify.

If you’ve ever encountered such problem, or if you’re working with this organism (or similar) please share some advice on how to pick the eggs and with certainty !

Thanks a lot for your answers !

2

With a good bleaching protocol there should be nothing left but eggs, certainly nothing that can be confused with an egg.

My standard protocol is:

  1. make fresh bleach solution (proportions: 4 ml water, 850 ul 5M NaOH, 250 ul sodium hypochlorite solution).

  2. Wash gravid worms or eggs off of plate in water. Pellet (1k rcf for 1 minute) and remove supernatant. Rinse pellet in water if desired.

  3. Add bleach solution to pellet and start a stopwatch. Volume depends on size of pellet, at least 10x the volume of the pellet. Resuspend pellet by swirling and keep in suspension by occasional swirling (once a minute or so). Do not vortex vigorously.

  4. At the 5 or more minute point, pellet the dissolving worms (1k rcf, 30 seconds), remove and discard supernatant. The pellet will be faintly yellow and nearly as large as when you started.. Add more bleach solution, the same volume as before, so you’ve replaced the bleach solution. You may want to pipet the worms up and down to physically break some cuticles.

  5. You’re done when you can no longer see worms with the naked eye, holding the tube up to the light. For C. elegans this is probably about 10-14 minutes total time. Other species take longer and might benefit from replacing the bleach solution again. When this is so, pellet the eggs as before (1k rcf, 30 seconds), remove and discard supernatant. Pellet should be smaller than before and white. Add sterile water (at least the same volume as during the bleach treatment).

  6. Swirl to resuspend, pellet as before, remove and discard the supernatant. Add more water for a second rinse. Swirl to resuspend.

  7. Pellet the eggs, remove supernatant. Resuspend eggs in a small volume of water or in your buffer or medium of choice. Examine an aliquot on the dissecting scope, it should consist entirely of eggs. You should be able to see embryonic morphogenesis in some of the eggs, indicating their shells were not bleached and the embryos were not destroyed during the bleaching process.