Synchronisation & M9

Model Organism: Worms Methods & Reagents
1

Hi,

Apologies for this naive question. I’m new to this field!

I’ve been trying to synchronise N2 worms as part of my UG project, with no luck whatsoever.

The general protocol I’ve been using is to get a large plate of almost starved N2s, use M9 buffer to collect the eggs into a fulcan tube,
I then add bleach (500µL 10M NaOH, 2mL sodium hypochlorite and 7.5mL de-ionsed water). Shake for 2-3 minutes, centrifuge (2500rpm for 1minute),
remover supernatant and proceed to ‘wash’ the eggs with M9 using this procedure roughly 5 times. I leave the eggs on a rotator overnight and so far, have
never managed to produce L1s the following day. I was wondering if anyone could see where I’m going wrong?

Also, I was wondering if the M9 makes a difference? The ‘M9 salts’ available in my lab and online differ slightly to the m9 recipe found on this website - was wondering why there’s a difference and if it has an effect?!

Thanks in advance for any help!

J

2

A few things:
The bleach solution you report using is a bit different from any I’ve seen before (though this probably is OK), the bleach incubation is shorter than I’ve seen before, and I’m nervous about what you might mean by “shake for 2-3 minutes”; all you need is enough agitation to stop animals from falling out of suspension and forming a clump with surface areas protected from bleach, and I’ve been told excessive vortexing can cause physical damage.
You don’t need to use M9 to rinse the animals off the plate nor to rinse the bleach off the animals; water is fine.
I’ve not tried to hatch and arrest animals in M9; every protocol I’m aware of says to use S medium.
This topic has been covered many times on the forums (I’d link, but you can’t link search function output). In one of these threads, six years ago almost to the day, I posted a protocol that should work for you.

3

Hi,

a few basic questions/observations…some of which might seem blindingly obvious (so bear with me) and some of which echo what Hillel mentioned.

So…basic questions/observations/suggestions.

  1. ‘…a large plate of nearly starved N2 worms…’ Do you check the plates visually to ensure that the worms are at the correct stage of development? That is, can you see eggs in the worms and a few on the plates?

  2. ‘…use M9 buffer to collect the eggs into a fulcan tube…’ Spare the M9, water is fine and so is a 15mL Falcon tube.

  3. ‘…2mL sodium hypochlorite …’ Are we talking household bleach here or actually sodium hypochlorite? If the latter, what concentration are you using?

  4. ‘…Shake for 2-3 minutes, centrifuge (2500rpm for 1minute), …’ As Hillel mentioned, shaking (continuously?) for 2-3 minutes, if that’s what you meant is not necessary. A brief vortexing every minute is sufficient and a visual check to see whether the worms have ‘dissolved’ (in the latter stages of the alkaline bleach procedure) is also necessary. For the centrifugation step, we regularly use 1100 x g for 2 minutes and that works fine.

  5. Have you checked that you have eggs at the end of your protocol and if so, roughly how many have you got?

  6. Have you tried putting the eggs onto an NGM/OP50 plate to see if they develop?

Regarding using M9 rather than S medium to hatch and arrest, we do this routinely and the L1s are fine. We have also used 24 wells with M9 and no shaking or mixing O/N and the L1s were fine.

Steve

4

I’ve hatched overnight in M9 many times without issue. I’ve had better rates of survival if the embryos are left in a petri dish with just enough M9 to cover the bottom, and left on an orbital shaker or rocking platform set low enough to just keep the buffer moving.

As for hypochlorite treatment, this is the protocol currently used by the CGC:

Protocol 4. Egg prep: Removing bacterial or yeast contaminants from C. elegans stock plates
Reagents
• 5 N NaOH
• Household bleach (5% solution of sodium hypochlorite)
• Sterile water

Methods

  1. Use contaminated C. elegans stock plates that have many gravid hermaphrodites.† Wash the plates with
    sterile H2O. Pipet the H2O across the plate several times to loosen worms and eggs that are stuck in the bacteria.

  2. Collect the liquid in a sterile 15 ml conical centrifuge tube with cap. Add H2O to total 3.5 ml.

  3. Mix 0.5 ml 5 N NaOH with 1 ml bleach. Make this solution fresh just before use! Add to the conical tube with the worms.

  4. Shake well or vortex the tube for a few seconds. Repeat shaking/vortexing every 2 minutes for a total of 10 minutes.
    [NOTE: I prefer not to vortex; simply inverting the tube a few times is almost always enough.]

  5. Spin the tube in a table top centrifuge for 30 seconds at 1300 x g to pellet released eggs.

  6. Aspirate to 0.1 ml.

  7. Add sterile H2O to 5 ml. Mix and allow to rest for a few seconds.

  8. Repeat steps 5 and 6. The solution containing the embryos should not smell strongly of bleach.

5

The method that I have always used is described in detail in this paper: http://www.biomedcentral.com/1741-7007/4/1

You can hatch L1s overnight in M9 with this method but it requires 24 well tissue culture plates and small volumes so that there is enough oxygen. They won’t survive in an eppendorf tube.

6

If you are to synchronize worms at starved L1 stage, how about repeating the washing step more?

First, bleach the gravid adults with 3 mL of bleaching solution just until their bodies are broken in the middle, and immediately add 9 mL of M9.
Then centrifugate at 1000 rpm for 1 min.
Remove the supernatant just leaving 3 mL. Then add 9 mL of M9 again and repeat the same washing step at least 8 times.

If you are to synchronize the worms on agar plates, it is okay to repeat the washing step just 5 times.

7

I think 5…let alone 8 times is a little excessive, but if it works it works.

8

Why are you leaving so much supernatant when washing? Embryos will pellet well even at low speeds, so it should be easy to remove much more liquid without disturbing the pellet. A few washes like this should remove nearly all the NaOCl and reduce the number of spins – and the abuse of the embryos that goes with it.

9

Come in Jaanki, come in Jaanki…where are you? We (other forum members) seem to be spending more time discussing this than you do… ??? It’s a bit like that scene in Trainspotting where the guy throws a beer glass over the balcony in a bar (walks out unnoticed) and a fight starts downstairs!

In the end, we can keep suggesting variants of the general method that all, in their own way, work. BUT, that amounts to nothing when we don’t know what you did, how you did it or whether you were aware of the potential stumbling blocks in the protocol you used…

But anyway, it’s interesting how questions like these exemplify how much ritual, myth and magic is interwoven in the basic facts.

My pick of the week is as follows;

Dauer (sorry) at #6: Household bleach is the work of the devil: Basic bleach (5%) is fine, no fragrances, no thickening agents etc.

Steady at #5: M9 bad for hatching, S medium good. Probably not that simple, both work and the L1s that pop out are apparently normal in both. Whether over the longer term L1 worms at arrest sitting in M9 do less well than those in S medium is an interesting question that needs some empirical input.

Climbing to #4: bleach the worms until they break in the middle: Sure the eggs come out but there’s also a hell of a lot of junk there too. Bleaching longer such that the eggs float in a worm soup has a purpose.

Solid at #3: You need to leave a large supernatant layer above the egg pellet because…: a combination of ritual & myth here. Spin the eggs @ 1100xg and they pellet such that you can DECANT the supernatant. The eggs are fine (stop egg abuse NOW!).

Poised at #2: shaking the eggs O/N is essential: No, it’s not. Sure, under certain circumstances I would imagine it is better (for example high density of eggs in solution), but leaving the eggs in a 24 well plate O/N without shaking works.

Straight in at #1: Washing the eggs 8 times with M9 is necessary or the eggs DIE. Use your nose, three time tops. Any more and you are wasting time, energy and M9.

Have a good weekend everybody…

Steve

10

To steve, daul and snug

Maybe I have been too timid when washing worms… I learned a lot this time. Actually I was tired of washing worms that many times. Thanks for the correction.
And I usually spend longer time to bleach worms so that the eggs come out and float around but here I just suggested to spend shorter time…

Baek

11

Firstly I apologise for my absence / late reply - been too busy in the lab trying out variations & recommendations!
Thank you all for you overwhelming responses!

To answer the questions earlier mentioned…

  1. I visually check the worms on the ‘nearly starved’ plates to ensure there’s plenty of worms.
    Unfortunately, I am unable to see eggs under my microscope, even at the highest magnification, so I just assume there are, judging by the rest of the plate.

  2. Noted - tried water instead of M9…

  3. The sodium hypochlorite is actually 5% sodium hypochlorite.

  4. I had been shaking continuously, but have tried gentle vortexing since…

5/6. As previously mentioned, I can’t actually check if there are eggs present. I leave the ‘eggs’ in M9 overnight, and transfer them to plates the next day…
So far I’ve had very very small yields of L1s, and am still struggling to synchronise properly!

To clarify, is it best to bleach until the worms are totally disintegrated? I always have the fear of damaging the eggs too!
I have yet to try s-basal but will probably give that a go from Monday…
I have been trying to take the best from each of the methods proposed and am getting some progress, but still haven’t perfected this technique!
Thought it would be much more simple & have a clarified method :’(

Thank you all once again for your helpful comments!

Jaanki

12

Hi, welcome back and no problem…research is engrossing…

  1. there should be no problem seeing the eggs under a dissecting microscope at x50 (total) mag…what kind of microscope are you using?

  2. Welcome back from the dark side Luke…

The eggs are robust, that’s the idea of the protocol. You can wait until the worms have just ‘dissolved’.

Steve

13

If you are having trouble seeing anything with your microscope, with a little experience you can follow the progress of your eggs with the naked eye.
In a 15ml falcon tube you should be able to clearly see adult worms swimming in the bleach, and you should be able to see when they start to dissolve. I use a bleach solution of half 5% sodium hypochlorite and half 1M NaOH, and the worms take 6 or 7 minutes to dissolve depending on strain. Best to wait until they’re completely dissolved.
After you centrifuge them, you should see a decent sized white pellet in the very bottom of your falcon tube. If you don’t see that, you can immediately tell you don’t have many eggs, so the problem would be with your worms - not enough of them or not at the right stage of development.
If you do have the large pellet, make sure you remove almost all bleach liquid from it before your first wash. Then when you re suspend the pellet, you can actually see the eggs with the naked eye if you get the light right. It looks like a fine dust swirling in the water. This should come back into a clean white pellet again each time you wash. If the pellet starts getting smaller and disappearing you’re losing eggs in the washes. Centrifuge longer or do fewer washes - three is usually fine for me.

14

Regarding the washing of the pellet of eggs after bleach: Shaking them loose between steps is essential in my experience, and much more important than the number of washes. I use a sharp flick of the wrist to break them up.

15

to follow up on the ‘washing the eggs’ story, I decant of the wash buffer (M9) after pelleting the eggs, add 1mL of M9 to the pellet and resuspend it with a Gilson.

Not as energetic as the flick of the wrist but just as effective.

16

I too personally prefer the finesse of pipetting. I feel like the eggs have gone through enough trauma as it is…(thank goodness they don’t suffer from shaken baby syndrome!)

17

If you prepare small numbers of eggs in an eppendorf tube, pipetting will not necessarily break up the pellet and you risk losing eggs that stick to the side of the pipette tip.
That’s why I flick the tube :slight_smile:

18

Hello I was wondering if you can help answer my question. I am new to c elegans. I am trying to help my daughter with a science showcase and I was wondering is it absolutely always necessary to use a centrifuge? What else would she need other than the solution? She doesn’t have a lab.

19

Hi,

For the protocol discussed in this thread, yes, you will need a centrifuge. There are other ways of synchronizing smaller populations of worms, such as picking several gravid adults to plates, letting lay embryos for a given time (~1-2 hrs), and then removing the adults so that all the embryos left on the plate will be approx. the same age. However, I expect that your daughter is going to encounter more significant problems if trying to do much without common lab equipment. How will you be preparing sterile media and solutions? How will you be culturing, and handling the worms and bacteria, and safely disposing of waste materials?