N2 'growth arrest'

Hello worm community,
Our lab is new to C. elegans research and we are having a problem with worm growth. We want to treat N2 worms with a drugs that slows growth. However, we are finding that the worms will not mature past the L3 stage after synchronization by bleaching and treatment with DMSO (0.1%) lacking drugs. Previous experiments performed with the same protocol had no issue of treated worms growing to adulthood and laying viable eggs. We have not been able to find an explanation for this ‘growth arrest’ and would greatly appreciate any feedback.

Experiment Parameters:

  • Drug is dissolved in DMSO and then serially diluted using heat-killed OP50 suspended in M9 until the final DMSO concentration is 0.1% and the drug is in the low Nano molar range.
    -Worms are bleached and then eggs are allowed to hatch overnight on NGM plates without food.
    -The following day the L1 larva are collected with M9 and about 150 L1 larva are placed on a 10cm NGM plate with 300ul of the OP50 + drug + DMSO (all in M9) treatment.
    -Everything is incubated at 20°C.
    -Worms grow as expected for about 2 days then ‘arrest’ in approximately the L3 stage. They seem to stay at this stage for about 2 weeks while becoming more and more lethargic before dying.
    -Extra treatment food is added as worms eat through what is on the plate – this is only at the beginning of the experiment. As they become more lethargic, their eating slows.
    -Additionally, there is a control treatment of No Drug using the heat-killed OP50 in M9 with 0.1% DMSO and these worms do also have the ‘growth arrest’ problem.

This experiment has been attempted about 3 times with the same results, but previously, this was working and worms that were bleached and treated reached adulthood in about 3-4 days and started laying viable eggs that hatch and grow to adulthood.

-Initially we thought over-bleaching might be the problem, but generally there is a good hatch rate of about 90% of the bleached eggs. Since having trouble with this experiment, we have taken special care to ensure the bleach time is at an absolute minimum by ensuring not all worms are dissolved as there is usually a light scattering of adult worm bodies left after bleaching.
-A few months ago there was an issue of the cholesterol (dissolved in ethanol) that is added to the NGM plate media not being properly dissolved as it was being added to the media – it made the media visibly cloudy – but that should now be remedied by making a fresh solution of cholesterol every time NGM plate media is made.
-There is no visible contamination on the plates.
-The OD of the OP50 suspended in M9 is adjusted so the worms are getting the same amount of food in every trial of this experiment.
-Also, all components of the plate media (KPO4 buffer, etc) and M9 have been remade and sterilized before this most recent experiment was performed.
-The N2 worms that are kept on a live bacterial lawn that are used to keep the line going are passaged regularly and are not allowed to enter daur stage or even a state of mild starvation (as in, care is taken to ensure they are passaged before the old plate runs out of food). They do not appear to have this inability to mature that is affecting the experimentally treated worms.

We cannot think of any other condition that would be affecting the worms so strongly as to prevent them from growing to adulthood. Is it possible for N2 passaging lines to get ‘old’ and less hearty (in a similar way that mammalian cell lines age)? We also frequently re-streak the OP50 on NZY plates that are then used to make saturated cultures in L broth, but potentially the original line is contaminated?

Thank you for any advice/feedback that you have to offer.

You shouldn’t have to make the cholesterol fresh each time. If it was crashing out of solution when added the likely explanation is that you didn’t let the media cool enough before adding cholesterol.

You don’t mention a couple of obvious controls: heat-killed OP50 without DMSO, or living OP50 without heat-killing (with or without DMSO). Either of these might indicate the problem. I haven’t treated worms with DMSO and haven’t done much feeding worms heat-killed OP50, so I don’t really know whether either is usually a problem. I know at least one old story with people seeing developmental arrest because they fed their worms with killed bacteria, but that was in liquid culture rather than on plates (in that instance they later determined the problem could be bypassed with additional magnesium, which living but not dead bacteria concentrate from the media - but I’d not really expect that’s the problem in this case).

Offhand I’d say that your cholesterol problems have not really been solved. Either way it would seem to be your plates. Get some worm plates from a nearby worm lab.

  • MM

I think the problem is the heat-killing of the bacteria.
How about adding some kanamycin, which will prevent the E. coli from growing but doesn’t have the potential to ruin the food?
Other people have mentioned UV killing as an option, so that would also be worth a try.

It’s actually a known phenomenon, indeed caused by heat-killed bacteria: see this old thread…