Liquid culture help: worms are retaining embryos

Hello, I’m a master’s student starting my first solo assay and I have run into some problems. I’m working with a drug that requires I use liquid cultures, but no one in my lab had done them before. So, my PI suggested I post here for some help troubleshooting and general tips, since I’m kind of on my own.

I have been following the procedures on worm base for liquid culture (S basal complete and spun down OP50, in a shaker at 20C and 200 RPM), a modification being that I’m using a small culture size (50mL). They look healthy for the most part, now that I have figured out how to maintain the right amount of food (I had some trouble keeping them well fed at first). However, I’m still having problems with my adults retaining embryos, instead of the usual egg laying. This wasn’t a problem in my first few generations, but as the culture goes on, it seems to be getting worse. Now, very few if any are exhibiting normal egg laying. My PI says this is a stress response, but we are unsure of the cause. She has also suggested I switch to liquid NGM instead of S basal and maybe change the peptone ratio.

Anyone who does long term liquid cultures have any suggestions or tips?

Also, if it helps to know, my drug is a polycyclic aromatic hydrocarbon and I will be looking at toxicity, stress and fat accumulation and will be doing RNAi assays later on.

Thanks!

If I recall correctly, people often use HB101 bacteria instead of OP50 for liquid culture and other unconventional strain prep conditions. It’s basically baby food for worms, and they love it (on plates, too). That may help with your egg-laying issues.

i think that unless you use an Egl-c (egg-laying constitutive) mutant background you will continue to find that your worms are Egl when grown in liquid. this is normal for wild type worms.
i love HB101 (or the related strain, AMA1004), but in my experience worms grown in liquid with this as food source are still fairly Egl.

see:

Genetic and cellular basis for acetylcholine inhibition of Caenorhabditis elegans egg-laying behavior.
Bany IA, Dong MQ, Koelle MR.
J Neurosci. 2003 Sep 3;23(22):8060-9.

People often grow in flasks with shaking because they plan to do for biochemistry purposes, but if your drug suspensions are meant for other experimental purposes I would suggest a no-shake condition.

Shaking is good for oxygenation in large volumes of liquid but a small amount of liquid like 500µL in the center of a small culture dish doesn’t need to be shaken. Worms swim to the periphery if they want more oxygenated conditions and aren’t constantly bombarded by the stimulation of moving liquid.

I cultured this way for 10 generations in NGM salts with only with trace cholesterol and concentrated OP50 added and they are pretty happy.