N2 Lethality on Agar Pads

Hello; I am currently trying to assay embryo lethality when embryos are left to incubate in an agar pad setup. I have been using the following protocol to microscopically study how mutants behave, but noticed significant embryo lethality in the wild type N2 strain when mounting them overnight on agar pads at 20C. This lethality can vary between 0 to 40%, even between mounts set up at the same time with the same equipment/protocol. I would like to find mounting conditions that reliably permit all N2 embryos to develop, and am not sure if I am introducing some error or if this is a common or unavoidable phenomenon when incubating embryos for long times on agar pads. Any advice or insight is appreciated!

  1. Melt 2% agarose and Vaseline in the microwave and maintain in a heat block on my bench set to 95C.
  2. Prepare bench and dissection scope by wiping down with ethanol and assembling coverslip, slides, needle, buffer, and brushes.
  3. Drop hot agar onto slide, drop other slide perpendicular on top and allow to solidify.
  4. Take worm plate out of the low precision incubator, then prepare a coverslip with 4uL of freshly made M9 media in the middle.
  5. Isolate three gravid (36 Hr post-L4) adult N2 worms cultured on OP50 overnight at 20C into pH- and osmotically-balanced M9 with as little bacteria as possible.
  6. With a needle freshly cleaned before use, dissect the worms. Wipe off needle with Kimwipe. Restart dissection if too few embryos are obtained.
  7. Clean out the carcasses using an eyelash brush. Clean the brush on sterile paper/plastic between putting it in M9.
  8. Remove one slide from the agar pad setup. Place the coverslip gently on top of the agar pad and allow buffer to spread out.
  9. Quickly and carefully use a paintbrush to spread molten Vaseline around the edge of the coverslip, allowing some Vaseline to get under the slip to create an airtight seal.
  10. Take slide for incubation in the high-precision incubator at 20C. Slides may be stored next to each other in sealed containers.

One possible cause of lethality is sealing the slide with vaseline that is too hot. 70C is warm enough to keep agarose and vaseline molten; you don’t need to keep them at 95. When sealing the slide, leave it sitting on the bench to dissipate heat (don’t hold it in your hand). If the problem persists, try waiting a few seconds after dipping the paintbrush in the vaseline, before spreading it on the slide.

One possibility: do eggs need oxygen? When setting up lineage slides with larvae rather than eggs according to a similar protocol you are supposed to supply a small amount of bacteria as food and a small air bubble to supply oxygen; something similar might be the case for eggs (not the food, of course).

The fact that the original poster observed such inter-slide variability suggests to me something else… that human fallibility is at play here.

Embryo age will play a role in lethality, e.g. variation in the age you mount.

Mechanical stress is also a factor that increases lethality.

Grouping too many together on the slide will cause anoxia and increase the lethality.

Of course, too hot agar or seeping vaseline onto the mat might also do the trick.

Practice is always useful…some things take time before one gets consistent results.

Finally, are you the only one mounting embryos? If not, it might be worth looking at what your colleagues do and also whether they have the same issues.

Zhirong Bao and John Murray published a paper on keeping embryos alive for time lapse. It may have useful suggestions.

Cold Spring Harb Protoc. 2011 Sep 1;2011(9). pii: pdb.prot065599. doi: 10.1101/pdb.prot065599.
Mounting Caenorhabditis elegans embryos for live imaging of embryogenesis.
Bao Z, Murray JI.


  • MM

From the paper Morris links:

Problem (Step 14): Embryos are not hatching overnight.
Solution: There are several different scenarios.

  1. The buffer evaporated and the embryos dried out. Check your Vaseline seal.
  2. Embryos developed normally for a while and then stopped/arrested. This is most likely caused
    by anoxia. If five or more embryos cluster tightly together, some may suffocate. Dirty mounts
    (see Discussion) are particularly prone to arrest over time (e.g., after 2 h). Bacterial growth may
    also deplete the oxygen supply. Clean the worms well in Step 6. Adding antibiotics to the
    buffer will also help by inhibiting bacterial growth.
  3. Embryos from starved or old mothers may die with gross defects (early cytokinesis defects, gastrulation
    failure, exploding embryo during morphogenesis). Use young, well-fed adults.
  4. The one-cell embryo is more vulnerable to osmotic damage than the later stages, because the
    eggshell is not yet mature. Boyd buffer is presumed to be a better osmotic match with the early
    embryo than M9 buffer. However, use embryos that are two cells or later, unless you study
    processes earlier than that.
  5. The imaging process may kill embryos either through heat or phototoxicity.

Great! Thank you all for your suggestions and the link, I will start testing some of these variables very soon.