Hi everybody,
I have a problem I hope someone might be able to help me with. We started with compound screenings in liquid medium in 96-well plates (similarly as described in this protocol http://www.ncbi.nlm.nih.gov/pubmed/21445049) and everything worked fine for more than a year.
Several months ago we started having troubles - basically every time I put the worms on 96 well plate they die overnight. I don´t recall us doing any major change around that time. And the weirdest thing is that it happens on plates only - when I take part of the worm solution I am putting on plates (same worm population, same bacteria, same S complete) and leave it on Petri dish the worms are OK. Our maintenance is also OK.
I tried:
- preparing new media, buffers, stock solutions (3 times)
thawing new stocks of worms and bacteria (twice), when it didn´t help we also ordered new worms and bacteria (OP50 and OP50-1) from CGC
lowering the concentration of bacteria
heat-killing the bacteria
using 4 different seals and getting rid of a seal altogether using parafilm instead
changing the plastics (5 different manufacturers)
lowering the volume in wells
Nothing really helped. The only way the worms survive in plates is when they are in clean S complete without any bacteria at all (which is unusable for lifespan experiments).
I also noticed that the effect in less severe (meaning “only” part of the population die overnight and the rest die within the next day) when I cultivate the worms in lower temperature + I lower the concentration of bacteria. So I thought the bacteria could be the problem here, but neither new stocks nor heat killing helped. I also thought that gas exchange could be the deal and that the worms could be suffocating, but lower volume in wells didn´t really help either. I also checked the pH and didn´t see any changes.
I have to admit I am out of ideas what could be wrong here.
Thanks in advance for any advice.