Increased embryonic lethality in worm stocks

Hello worm community,

Over the past several weeks, our lab has noticed a dramatic increase in embryonic lethality in all strains that we carry (10-30% as opposed to ~0.7%). The phenotype of the dead eggs is unusual, as they appear on the plate as brown nucleated circles as opposed to clear, oval unhatched embryos. Animals maintained over several weeks from healthy frozen stocks exhibit ~50% decrease in brood size as well, whereas thawed animals maintained for less than three passages appear to have normal broods (270-300).

We have replaced all of our bacterial stocks, remade our NGM plate reagents, used different sources of water (from the same building), and we have also used worms, NGM plates, and food from another lab on the same floor and observed the same problem.

Our best guesses as to what the problem is are either the plastic petri dishes (they are 60mm from Fisher), unclean glassware (we have in-house dishwashers with a history of leaving detergent residue behind), or a water contaminant that can fit through a 0.2 um filter (the water system in our building was updated in late July). All three of these items are common between the two labs whose worm stocks we tested.

Does anyone have experience with one of these issues affecting embryonic viability?
Has anyone seen an instance of either bacterial stocks or NGM plates causing embryonic lethality like this before, or does anyone have a guess as to what we could do to identify and resolve this issue?

For two years we have not had an issue with maintaining our worm stocks in our current location, and we have been struggling with this issue since late August. Any insight into resolving this issue would be greatly appreciated.

Thank you,

Allen Andrews

I don’t have any helpful suggestions as to the source of your problem, and I hope that you are able to successfully solve it and that when you do you will let people know what it was. One note I do have, regarding the phenomenon you describe:

The phenotype of the dead eggs is unusual, as they appear on the plate as brown nucleated circles as opposed to clear, oval unhatched embryos
These sound like unfertilized, likely endomitotic, oocytes, not dead embryos.

Could the temperature of your incubator be off? Higher temperature can give the “phenotype” that you describe. If so, I guess you should also see more males among the surviving worms.


Thank you for your comments thus far.

The number of endomitotic nuclei has decreased since receiving a fresh stock of OP50 from the CGC and seeding a 1:10 dilution of an overnight culture in LB. They now only appear at the very end of egg-laying and in modest numbers (~5-15 in 1 of every 5 animals).

I have now done embryonic lethality experiments in several incubators and a 20 C room in several types of containers (fly boxes, tupperware boxes, on top of bench, etc) and this does not seem to change the drop in brood size. Also, close monitoring of temperature with both digital and mercury thermometers suggests that the temperature in our incubators is not fluctuating significantly.

I have since dropped out each component of the NGM plate mix out, and I have found that dropping out peptone from the NGM mix seems to restore wild-type brood counts (266+/-14; n=9; 2 independent experiments). With peptone, the brood counts are significantly lower (205+/-9; n=10; 2 independent experiments). In both cases, embryonic lethality is wild-type (0.8% +/- 0.23). We are receiving our peptone from BD cat# 211667 (500g-Lot 9247363) or 211820(2 kg-Lot 8339901). I have done this drop-out experiment with both reagents and the numbers are virtually identical.

Has anyone ever observed this dramatic effect on brood size based on NGM composition? Did we win the lottery and get two bad lots of peptone? Does anyone get peptone from another manufacturer?

Any thoughts would be most appreciated.

Thank you,

Allen Andrews

Hi Allen,

We haven’t observed any peptone-dependent lethality with NGM plates, but have seen a similar effect with enriched peptone plates and bacterial strain NA22. The cause seems to be related to some volatile product of bacterial growth b/c it was worse when the plates were partially sealed in trays or plastic bags to minimize evaporation. We also observed the problem when we filled our incubator with the same type of plates for a large-scale mutant screen; plates from the same experiment incubated in the open at room temperature were not as affected.

It’s probably worth making plates with peptone borrowed from other labs at your institution, just to confirm that the problem is batch/source-specific.

Good luck,