Thank you in advance for the answers to this question (I’m new to C. elegans research)!
In synchronizing worms by putting the eggs on a clean plate until L1 stage, but also in general: do the worms eat debris that may have been leftover after bleach prep, or eggs that may be unhatched, or debris left (e.g. cuticle remnants…)? Sometimes my worms are out of synch the night after I put bleached eggs to a clean plate and my guess is that perhaps I had some bleached, but dead OP50 that was left on the plate (which I’m guessing worms can eat), but I have also been told that worms may eat the remnants of dead worms, eggs, etc… Is this true?
Yes they do, and if you have too much debris then they won’t be synchronized, which appears to be the problem you are having. In my grad lab we filtered bleached embryos through a 53um nylon mesh (Fisher #08-670-201) that we placed into an embroidery hoop. In my post-doc lab the preferred filter is a 40um ‘cell strainer’ nylon filter (bdbiosciences #352340).
Both help to separate the carcasses from the embryos and should allow for better synchronization. If you have a lot of embryos that burst and don’t make it, those can be used as food as well, so my advice would be to bleach until 75% of the moms have burst, which should allow for good embryonic viability. Good luck!
did you filter the eggs out of the suspension or did you wait until L1-larvae hatched before you filtered away the debris? in the last case, there is still the problem that dead worms etc. are still in my suspesion, isn’t it? I’m also looking for a good way for synchronizing, without loosing too many eggs/L1s.
Take a pellet of worms (less than 3 mL per 50mL conical) and add up to 20 mL of M9, then add 2.5 mL of bleach (Clorox from store, but NOT the splashless formula!) and 2.5 mL of 10N NaOH.
Then bleach for 3-5 minutes. You can monitor the bleaching if you’d like, but in my experience, you always over-bleach when monitoring the bleach. You are better off just going 3-5 minutes.
Then add M9 up to 50 mL and spin down pellet. (this should be done relatively slowly).
Take off supernatant and wash with M9 and spin. Repeat washing at least 2x times. This makes sure all bleach is gone from tube.
Then filter the pellet through a 40 micro mesh onto a plate (or into liquid culture). This should be 98% embryos, which will lead to excellent synchronization the next day.
ah… the rich tapestry of C elegans research… we use an alternative, but seemingly as effective approach to synchronising our worms.
Chunk a day 5 plate and grow worms for 2 days. Wash the worms off the plate using sterile water (fairly ubiquitous step), pellet (300 x g, 2’, RT) and wash with sterile water. Pellet again and then add 5 mL of fresh bleach/NaOH solution (per 10mL: 2.5 mL household bleach, 1 mL 10N NaOH, 6.5 mL H2O). Incubate for about 5-7 minutes at RT until the worms break open.
The time scale reflects that it is important to judge by eye when the worms ‘just disappear’ as Hillel said in an earlier forum post. Also important, vortex gently every minute. One can also monitor the progress of the reaction under the dissecting microscope by tipping the Falcon on its side…but by eye is just as good.
Neutralise the reaction with 5mL of M9 and then pellet the eggs at 1100 x g, 2’, RT, repeat washing with 5mL of M9 twice and then resuspend your eggs in M9 overnight…Hey presto, sync’d L1 worms, no debris…
As you can read from the medawg’s post, one filters immediately after bleach. But as steveh points out, if you dissolve all the carcasses first, then there are no worries. Either approach is valid. There are some folks in my post-doc lab that filter and others that dissolve. I’ve always found that if one does this in a small scale, then the dissolving trick works. If one needs to work on a larger scale (say, bleaching a 5ml pellet of adults), then the filtering trick works much better as if you wait for all the carcasses to dissolve you will rapidly diminish the amount of embryos that are unaffected by bleach.
