I've been working on using an axenic media, CeHR, to culture large numbers of C. elegans, but even though through working in a sterile biosafety cabinet I constantly am experiencing bacterial contamination that is setting back my project. Has anyone had any experience working with CeHR and encountered this sort of issue?
No experience with CeHR, but plenty of experience trouble-shooting contamination. First question, are your reagents "cell tissue" grade? If not, then there is no guarantee that they are sterile to start with. Two, make sure your work area is clean - wipe everything down before you start. Looking at the protocol, two critical steps are filter-sterilizing the medium and then adding "ultra pasteurized milk". The milk seems a likely suspect for contamination. Same with the glucose solution. Can you autoclave the milk before adding it to the final medium? I would filter sterilize or autoclave all possible stock solutions, check to make sure the stocks solutions aren't cloudy (contaminated), wipe everything down before you start and make sure you are putting the medium into autoclaved & sterile containers. Good luck!
PS C. elegans will ALWAYS bring bacteria with them in their intestine if they have eaten. Are you adding bleached, newly hatched larvae? If your worms have been exposed to any bacteria, they will be covered with it and have it in their digestive tract.
Hi Andrew,
were you able to get rid of any bacterial contamination ?
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