Egg laying assay

Model Organism: Worms Scientific Discussion
1

Hello,

Can someone please share a protocol or a description for an egg laying assay to be done…
(Not counting the eggs inside the worm)

Thanks.

2

That’s a pretty vague request. There are several kinds of egg assays, including the unlaid egg assay that you aren’t interested in. What question are you trying to address?

3

I want to compare the eggs laid by my mutant of interest to that of N2 and some other positive controls like egl mutants.

4

You could count the number of eggs laid by one or more staged (24-40 hours post L4) animals on a plate in one to two hours. Alternatively, we have picked 5 late-late L4’s to a single plate (3 plates per genotype), and counted the number of eggs laid after 18 hours. N2 typically lays 100 eggs, really Egl guys, like egl-1(gf), lay zero. Your guy may lie between. It can be sensitive to brood size, so it might be useful to count the number of eggs left in the Mom at the end of the assay to make sure the total adds up to around the same as N2.

But, we usually just do the unlaid egg assay:

Stage worms about 30-40 hours post-L4, 40 worms per genotype. Eggs will start to hatch by 40 hours from truly Egl animals (and also crush the gonad), though, so we often just go with 30 hours. Spot 5-7uL of 20% bleach sol’n (2 mls neat bleach + 8 mls ddH20) in the circles of the LID of a 96 well plate. Pick a single worm into each bleach drop, and after about 5 min, the worm will be dissolved, leaving the eggs which are resistant to the bleach and can be easily counted. I usually do them in groups of 10 per genotype till I get to an n=30 animals. To assay animals with transgenes, we obtain five lines and assay 10 animals per line for an n=50.

It’s really easy, and the data look good.

-Kevin.

5

Some people recommend tabulating the stages at which eggs are laid rather than the number of eggs that are laid (or the stages of eggs still within the mother rather than the number of eggs in the mother). The argument is that these data may be less affected by brood size and the rate at which fertilized embryos are generated - though they still won’t be unaffected. You can find descriptions of such assays in the published literature or in the relevant section of WormMethods at WormBook, which you should check out if you haven’t already.

6

I used 6-cm seeded NGM plates and left females (my species is C. remanei) on each plate for 2-3 hours. After females were removed I added in a small drop of NaOH to dissolve OP50 - which made it much easier to spot the eggs. And then just count number of eggs present on the plate.

The crucial thing is the amount of OP50 seeded on the plates and incubation time, something between 50-60 microliter, seeded in the center, and 3-6 hours in room temperature worked fine in our lab, the bacteria was not too thin to hold the female, and not too thick. Number of females is important too. I did some trials and 5 females per plate laid ‘countable’ number of eggs.

I also tried 24-well plates with seeded NGM in each well, it didn’t work at all. It took me ages to make the plates and the escaping rate was much much too high.

Hope this helps!

7

Thanks a lot … :slight_smile:

8

Remember, it matters whether you are looking at the behavior or brood size. Very different assays.