Synchronization and incubation


I would just like to know why is it important to put collected eggs on a shaker overnight to hatch. Without the shaker, the a lot of L1 were dead, and some eggs didn’t hatch.

They need oxygen, so if you don’t aerate them they will deplete the local source of oxygen and then undergo hypoxia followed by death. I presume you are hatching without food so that you can get synchronized L1s, based on your title. Two options: 1. conical tube and a nutator or a flask/shaker, depending on the volume. 2. allow eggs to hatch o/n on NGM plate (usually a 10cm size) without food.

Good luck!

So by moving the water, more air exchange occurs! (I guess I should not tighten the falcon tube lid very tight).
Thank you very much for the reply, makes a lot of sense and is very helpful.

just to say…and really only as an alternative that you and others might try…we regularly synchronise worms by incubating eggs O/N in M9 in 24 well plates. No shaking is necessary if egg preps are distributed across a number of wells. Mortality is low and subsequent development on plates is normal.

I imagine the shaking is only necessary where you have large numbers of eggs (and hence L1s) in a conical tube where they end up piled on top of each other.


And just to mention that M9 itself does affect worm physiology, as explained in a recent WBG article:


but are we talking about adult transgenic worms (wt;frIs7) worms O/N in M9 causing induction of this marker or eggs from transgenic worms left to develop to the L1 stage O/N in M9 which later manifest altered adult patterns of expression?

Also, have you tried using S buffer or egg buffer instead of M9?

Just for embryo work perhaps the use of egg buffer removes this issue?



We haven’t tried either S buffer or egg buffer and we’re in the process of quantifying the consequences of early incubation in M9 on adult gene expression.

BTW, Transient exposure of L2s to 200 mM sodium hypochlorite results in L4s more resistant to fast killing by Pseudomonas (Mahajan-Miklos et al. Cell. 1999 96:47-56). Admittedly a far harsher stimulus, but probably an example of persistent changes in gene expression.


That is good to know! Thank you for sharing.