Infection assay, liquid culture and starving adults

Model Organism: Worms Methods & Reagents
1

Hallo,

since you guys seem to be really helpful in this forum, I decided to ask you some more questions while trying to set up an assay for infecting my nematodes. I went to all past articles in “Methods, Protocols,…” so far to not ask unnecessary questions :slight_smile: To summarize it shortly:

I want to do infection assays, using bacterial pathogens that cause permanent infection, and re-isolate the bacteria in the end!

1.) I have to make some decisions, starting with the question: liquid culture or plates?
I’ve heard that liquid cultures are critical with dauer formation, contamination etc. It also seems to be much effort to grow enough E. coli. I need to grow/bleach the worms for a about 4 generations before infection starts (with a temperature sterile strain, growing them at 15°C and then switching to 25°C for infection after enough generations/big enough nematode count). The number of worms I need depends on how good my infection works. I guess it will be at least 1 x 10^5, or even higher potencies that get infected! This would also be a consequence of my next question, what will I do after the infection, so

2.) what happens if I stop feeding adult nematodes? (I do not want to feed more E. coli nor more pathogenic bacteria)
How long can they survive without food and could there be any unwanted consequences for my assay?

3.) In case I’d do liquid culture, some of the protocols I went through do not use OP50 but other strains, and add antibiotics, wormbook doesn’t, not even nystatin. Do you think this should be done? I would not detect bacterial contamination I guess because I do have the feeding bacteria in the medium, but then again, I don’t know how well OP50/other bacteria can grow in liquid NGM or S-Medium in general?

Thank you in advance for any hint or advice which I do really appreciate, unfortunately I have no one to ask who has experience, so you are my only helpful input :slight_smile:

Greetings,
Tanja

2

hi, hope to just share some of my experience as i’m doing the infection stuff. :slight_smile:

  1. I prefer solid assays over liquid assays, more reproducible and less conditions you cannot control. that is if you can deal with smaller numbers of worms and/or more plates and be patiently transferring worm by worm and counting them. all of them will just eat the pathogen and be infected, they die at different rates and hence the survival curves.

  2. if you don’t feed adults, they might bag and that will interfere with your infection assays.

  3. OP50 grows rather well in S Medium. No matter how much you wash the worms, there is still bound to be some E. coli/bacteria stuck to them (did some lysing and actual CFU counts to warn me that).

  4. if you want to recover bacteria from infected worms, be careful some bacteria lysates might not be as easily dissociated to give you reproducible CFU counts every time.

  5. hope the pathogen you have worked with, is one of the more common ones where reviews and publications are available. check out their protocols. also, the types of plates you use will influence the way / rate at which the pathogen kills.

3

Thank you very much for your answer. Meanwhile I also think I will choose plates, because some of the problems just don’t arise when taking plates, although the handling might be more complex.

The problem with restricting food is, as you also pointed out for S-Medium, that E. coli that is stuck to the nematodes starts growing on even empty plates, so it seems to be difficult to let them starve… I will use sterile mutants, so bagging shouldn’t be a problem. I just wonder how I can handle it. Maybe I should use antibiotics for washing/plates after the infection.

Do you have experince with recovering bacteria from the nematodes? I do FastPrep with silica beads, but I wonder how much is lost because worms are not ground well enough.