how to synchronize worms ? urgent~~~~~~~````

Hi,

does anyone know how to synchronize worms? I heard of two ways:

  1. after the adult worms laid eggs on the plates, wash them off and since the eggs are sticky, they can stay on the plates,so only eggs are left on the plates.
  2. pick several (maybe 3~4) gravid worms into the synchronization solution, after 6~7 minutes, the adult worms will be destroyed while the eggs can survive, and only eggs are left on the plates.

I tried the first way, it works, because after I washe the worms off, there were only eggs left on the plate. But one problem is that, since the eggs were laid by different adults, so they were laid at different time, the time difference can be 6~7 hrs, and even larger. so the so-called “synchronized worms” still have worms that are at different stage? Can anyone tell me how to solve this problem? and is there any other more convient and efficient way to synchronize the worms?

thanks a lot!

karena

Both of your methods are going to yield worms of a pretty wide range of ages. As you’ve found, the eggs on a plate can be several hours’ difference in age; the same is going to be true of the unlaid eggs you get with the one-step alkaline hypochlorite (“synchronization solution”) method.

You have two real options for precisely synchronizing worms. The first is to place a known number of adult hermaphrodites on a plate for a limited amount of time (say, an hour), then remove all the adults. This way you know all the eggs were laid within (say) an hour of each other. In my experience, this works best when the adults used to lay eggs are all of similar ages themselves.

The other is to start with the alkaline hypochlorite treatment, then let the eggs develop without food (either in M9 or on a plate without bacteria). Their development will arrest at the L1 stage until they get food, so they’ll all be developmentally synchronized even if their ages in hours differ.

Hi, Andy,

3Q a lot for your reply. It helps~~~~~~~~~~~!!

one more question, so as for the first method that you mentioned, I need to pick the worms that are going to lay eggs very soon, right? do you just observe this under the dissection microscope, and if you see the eggs inside their body , you just pick them? since I am kind of new to worms, I am not quite familiar with it. Is it very easy to observe the eggs inside them?

thanks again!

one more question, so as for the first method that you mentioned, I need to pick the worms that are going to lay eggs very soon, right? do you just observe this under the dissection microscope, and if you see the eggs inside their body , you just pick them? since I am kind of new to worms, I am not quite familiar with it. Is it very easy to observe the eggs inside them?

It is easy to see the eggs inside the animal, but it isn’t really necessary. I’d strongly endorse Andy’s advice about using similarly staged adults. For example, pick late L4 animals to a plate. After 24 hours, you will have a group of adults, each of which should have a fair number of eggs in them and be laying eggs, averaging one to two an hour per adult. Because you’ll have used similar adults, these newly laid eggs should be at a similar stage of development to each other (older adults tend to lay eggs that have developed somewhat further than those laid by younger adults). As Andy said, move these adults to a seeded plate, wait an hour, and move them on to yet another plate.

A lot of the answer here depends on what you are doing. If you need synchronized populations of larvae, especially if you need reasonably large numbers (say, more than a few dozen) or maximum convenience, I’d strongly encourage you to follow the second course Andy mentioned, of bleaching to collect eggs and letting the eggs hatch in S medium without food. If you wait about 16 hours for the animals to hatch and arrest, then when you start them growing on a food source the great majority of them will start growing and remain quite well synchronized, achieving adulthood within about an hour of each other (less than a 5% range in developmental time for the great majority of the animals). Note that (1) the tube containing the eggs in S medium should be kept moving on a wheel to maintain aeration and (2) for each additional day spent in L1 arrest it will take about an hour longer for the animals to reach adulthood. I didn’t readily find a good synchronizing protocol in Wormbook: mine is to take a small (6cm) plate with a lot of gravid animals (one day before the animals on the plate would use up the food is usually good), wash them off with water into a 15ml conical, spin down 1 minute 1k rpm, aspirate all but the pellet and a little water (maybe 0.2 ml), add a bleach solution (4.6 ml water, 0.8 ml 5M NaOH, 250 ul hypochlorite solution with 5% available chlorine), vortex very gently for about 10 seconds of every minute to keep the worms in suspension, and when I can no longer see worms with my naked eye (this takes about 10-12 minutes), spin 1 minute 1k rpm, aspirate, add 5ml water, spin again, aspirate, 5ml water again, spin, aspirate, add ~750 ul S medium and put on a wheel at 20 degrees C overnight. Dispense using a 20 ul glass micropipet.

If timepoints within embryonic development are critical to you, it may be best to monitor individual embryos from a precisely defined starting point. You may have to cut open the gravid animal to get, say, eggs at the two-cell stage.