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 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