C. elegans synchronization problems, Please help

Hello, i’m both new to this forum and to the field and have been having repeated problems trying to synchronize C.elegans for a longevity assay. I end up with no worms in my m9 the next day.

I’ve been using 2:1 house hold bleach (tesco’s own brand) to 5M NaOH.

After washing worms of the plate with sterile H2O and spinning down to give a volume of 3.5ml i add 1.5 ml of the above solution.

I keep shaking and checking the worms until there are neirly no whole worms left (about 8 minutes) and add about 3 ml of M9 to stop the reaction and quickly centrefuge (5000rpm 2 minutes) and remove supinatant and replace with M9 vortex and centrifuge ~3 times.

I then transphere whats left (which usualy looks mostly like chunks of worms) to M9 in a 100mm petridish and leave o/n agitated at about 88 rpm at 20’C.
The next day i have no L1s and no sign of life.

Does anyone have any idea where i’m going wrong?

As far as i can tell the only thing i could be doing wrong is either bleaching them too long, or too little. Or maby not centrifuging them long enough (can you over centrifuge?).

Any tips or advise would be much appritiated, repeated failure is getting quite frustrating.



there are a number of places where this could be failing, so let’s take these in turn.

  1. Are you using wildtype (N2) or a mutant strain of worms?

  2. Assuming you are using N2 worms, what stage of worms are you attempting to isolate eggs from?

The worms should be gravid adults…that is carrying eggs (typically, if you chunk L1 worms off a 5-10 day old plate onto a fresh OP50 plate, then 2+ days later @ 20 degrees you have gravid adults).

  1. Not sure what your bleaching mix is, are you using 2vol bleach/1vol 5N NaOH???

I use 1mL 10N NaOH, 2.5mL household bleach and 6.5mL sterile water per 10mL of bleaching solution, it works (5mL per worm pellet).

  1. Add bleach mix and vortex gently every minute, after 4 minutes start checking progress under the dissecting scope. I normally see a cloudy ‘soup’ of eggs and no worm debris after 6-7 minutes using my mix…but this varies so checking progress earlier than this is important.

  2. Add 5mL M9 to stop the reaction, vortex gently and then centrifuge @ 1100 x g for 2 minutes. wash pellet of eggs with a further 3 x 10mL of M9. I normally decant the supernatant, add 1mL of M9 and resuspend the clumped eggs thoroughly (but carefully) then add the remaining 9mL of M9. Finally, resuspend the eggs in a couple of Ml of M9 and pipette them into a couple of wells in a 24 well plate. This way I find no need to shake O/N, I just leave them on the bench to hatch. Others shake in falcon tubes and this is also fine.

The following morning you have worms that can be transferred to plates.

Try this protocol first and let us know whether you get L1 worms. I assume it’s your bleaching solution that’s causing problems as you shouldn’t see chunks of worms.

At the end of the egg isolation procedure, you should be able to see the eggs under your scope (total mag x50).



Thanks for your time i’ll try that protocol the next time i have worms.

I’m using both N2 and EAT-2 strains, i wait until 3 days after chunking (usualy chunk friday leave them over the weekend and syncronise on monday) when lots of eggs are already vissable on the plate.

per tube i use 0.5 ml 5M NaOH to 1ml of bleach.

How much do you put into each well?

OK. I think it’s worth checking whether you’re leaving your worms too long, as really you want the worms carrying most of the eggs rather than having laid most of them.

As for the bleach/NaOH…there’s too much (conc.) of both in your recipe…where did you get this from? The 1 in 4 dilution of bleach and 1N NaOH works fine.

I normally add 1mL of eggs/M9 per well.

In any case, you should really take some time and read through the wormbook methods as it’s an invaluable source of wisdom…

http://wormbook.org/ and go to WormMethods below left on the webpage.



Keep in mind the 0.5 ml 5M NaOH to 1ml of bleach then goes into a tube with 3.5ml of supernatant and was taken from the worm book methods originaly.

I’ll also try getting a plate of gravid adults just as eggs start to appear.


Ah, ok then, the method you’re using is the one in the wormbook for decontaminating strains? If so, then you’re right, it uses the concs and amounts you have tried. The only observation then would be, that the protocol suggests 10 minutes. Leaving them 8 minutes until no whole worms are present is probably why you’re getting so much debris and (probably eggs are there) but no L1s the following day.

The bleaching should go on until the traces of worms (and worm carcasses) have disappeared.

Good luck in your research and welcome to the C. elegans community!



You might take a look at this old thread, and at the thread it cites.

Different people use different recipes (I’ve never used commercial bleach, though not for any good reason; many people use it with great success), but the main points are covered already in this thread: vortex a bit gently (or just resuspend by swirling frequently - you aren’t trying to mechanically break up the worms so much as you’re trying to mix the slurry so their complete surfaces are exposed to the bleach solution), centrifuge gently (~ 1,000 rpm), and bleach until no worms are visible to the naked eye (typically 9-11 minutes using my protocol; you shouldn’t need to look under the dissecting scope, at least with a little practice). After rinsing away the bleach, you should see little other than eggs.


Sorry for the delay (I had to go for a training event)

I repeated the proticol, vortexing more gently and leaving the worms to dissolve a little longer (we also where having some minor issues with the centrifuge but not sure if these where relevent) and got worms, and my preliminary lifespan assay is underway highlighting many little problems to solve for my main experiment.

Thank you all for taking the time to help.


My experience suggest your blenching time is extended too much. Blenching hurts embryo if the treatment last too long. 5 mins or less is OK.