I am looking for advice in using levamisole/tetramisole to reduce worms pharynx pumping.
I am interested in feeding worms with a pathogenic bacteria and then shifting them to OP50 E. coli after certain time and continue to grow them in the new food (similar to the methods used by Portal-Celhay & Blaser, Inf. and Imm., 2011).
Published methods suggest washing 1 day-old adult worms using 25 mM of levamisole (dissolved in M9) to reduce movement, then washing worms with M9 and 100 ug/ml gentamicin and finally washing only using M9. Each of this washes are performed twice.
I have been having some issues.
First, worms are not surviving the levamisole washing. I am losing around 90% of my sample because worms don’t recover after the anaesthetising or worms fail to develop to adulthood.
Second, after the washing and moving the worm (or worms) to a new plate, I have seen a couple suspicious colonies growing where I transferred the drops of recently washed individuals (this colonies appear obvious after one day at 25 deg C). This suggests to me that some bacteria are surviving the washing perhaps.
why do you need to add levamisole to wash the worms? they will pellet fine even if they aren’t paralyzed. and as elegantlife says, you don’t need much levamisole to slow them way down. even 1 mM will greatly reduce movement without killing them. if you want to get rid of the previous bacteria you could use a drug resistant e. coli strain (like DA837 or OP50-1 available via the CGC), or let the worms eat op50 for a few hours, then wash them again to purge the bad bugs.
Yes, my first thought was to reduce the concentration, I did this by reducing the levamisole concentration by 100 fold. I re-run the washing with 0.25 and 0.0025 mM. The worms didn’t stop moving 100%, so I got a bit frustrated…
I will tray the 1mM, thanks for the advice. The reason why I want to paralyse them is to reduce pharynx pumping, so theoretically there is not ingestion of antibiotic, but at the same time the antibiotic kills any external pathogen. I am interested in recovering the previously ingested pathogenic bacteria later on. Perhaps like you mentioned, 1 mM is enough. I certainly will try.
oh, i see… what is the antibiotic that you are using? some colicin or ampicillin type thing that will kill the bacteria you are trying to get rid of? and then you do the Lev wash in medium with bacterial food? seesm like you would need some kind of incubation to kill off the bugs.
that sounds encouraging. i just learned that gentamicin is bactericidal since it binds irreversibly to the 30S ribosomal subunit (unlike tetracyline, which is only bacteriostatic since it binds reversibly). so, i guess protracted incubation probably isn’t necessary once the drug has been taken up…