egg laying assay to determine egg laying rate

Model Organism: Worms Methods & Reagents
1

I’m an UG doing my final year project on the effect of clozapine and other drugs on the egg laying rate of C. elegans so am required to determine the number of eggs laid in x hours. I did an egg preparation and put individual, synchronised egg laying adult C. elegans into wells of a 96 well-plate filled with 100ul M9 buffer. However, I’m finding it difficult to see the eggs under our stereoscope even under full magnification. Is there another way I can do an ‘egg laying’ assay? I was thinking I could pre-expose worm(s) to drug in, say, 100ul M9 buffer and then, after an hour, retrieve worms to small seeded plates, let them lay eggs over 4-5 hours, remove adult and then count hatched worms 2 days later.

2

We use flat-bottom, but round bottom might allow the eggs to accumulate in one spot (conical might make things hard to see).

  • Stage 40-50 L4 stage animals to a fresh plate with food. Assay at 24-30 hours post-L4.
  • Pipet 50µl of either M9 buffer, M9 with serotonin (5 mg/ml; 12.5 mM final concentration; 176.215 g/mol creatine sulfate complex), or M9 plus drug into wells of a 96-well plate. No more than 1% DMSO / solvent final concentration for your drugs.
  • Pick single worms gently into each well, trying not to carry over any food.
  • Incubate at room temperature for 90 minutes, and then count the eggs that are laid.
  • Assay 30 animals per genotype. I recommend staggering it into 3 groups of 10 animals per genotype, starting 30 minutes apart.

N2 should lay very few or zero in M9, many more w/ serotonin (about 10-20).
ser-1 mutants or ser-1, ser-7 double mutants should be resistant to exogenous serotonin (about 0-5).

I wouldn’t move treated animals to plates because the drug will wash out and you’ll see nothing. If you do it on plates (which should work fine), you’ll want to make up NGM plates with the drug, and you’ll probably need to do a (new) dose-response curve.

I’d stick to the 96-well plate format, particularly if you’re looking for egg-laying activators.

-Kevin.

3

Hello,

I did egg laying assay on separate plates containing the drugs, Levamisole and Serotonin.

So when I make the medium size NGM plates I added these drugs on the agar itself.

Then , when they are ready to use, I transferred the 1-Day old adults on them, and started the Assay.

In this way, I feel its much more easy to control everything, including the counting of exact number of eggs on the plates. :slight_smile: