contamination of DH5alpha cell line - (Aug/05/2008 )
Hello,
I've experienced some problems with contamination of my e.coli cells. It did a ligation, plated and did a miniprep to a few colonies to to sequence their plasmids, picked out one that was correct and grew it for maxiprep (500 ml) with ampicillin. The next day, the medium was somewhat more cloudy than usual and had a strange, unpleasant smell. Anyways, tried to get some plasmid from it, and it didn't work. So, I grew a few ml of the colony over night and the next morning the smell was there again! I looked under the microscope to check it and saw something strange, alive and ampicillin resistant. I am not a microbiologist so I don't know what it is, but it is about the size of e.coli, perhaps somewhat more cocci-shaped and grew in clumps. I'll attach a picture of it as well as a picture of what my cells normally looks like.
Does anybody have a idea what this might be?
(couldn't attach the photos, so here are some links:)
http://akira.ruc.dk/~fha/contamination.jpg
http://akira.ruc.dk/~fha/contamination2.jpg
http://akira.ruc.dk/~fha/normal.jpg
I've experienced some problems with contamination of my e.coli cells. It did a ligation, plated and did a miniprep to a few colonies to to sequence their plasmids, picked out one that was correct and grew it for maxiprep (500 ml) with ampicillin. The next day, the medium was somewhat more cloudy than usual and had a strange, unpleasant smell. Anyways, tried to get some plasmid from it, and it didn't work. So, I grew a few ml of the colony over night and the next morning the smell was there again! I looked under the microscope to check it and saw something strange, alive and ampicillin resistant. I am not a microbiologist so I don't know what it is, but it is about the size of e.coli, perhaps somewhat more cocci-shaped and grew in clumps. I'll attach a picture of it as well as a picture of what my cells normally looks like.
Does anybody have a idea what this might be?
(couldn't attach the photos, so here are some links:)
http://akira.ruc.dk/~fha/contamination.jpg
http://akira.ruc.dk/~fha/contamination2.jpg
http://akira.ruc.dk/~fha/normal.jpg
Hi,
Your Pics are not really clear but some looks like diplococci. Why you dont gram stain theim to make sure that you still have your Gram negative E.coli?
yeah I know, sorry for the picture quality but our camera is crap. Diploccoci sounds resonable, I looked at some photos and it resembles what I saw. I'll try some other things before I gram stain - I may have an older freeze culture before the infection, I'll try and look into that before I start considering killing the diploccoci. If I were to kill them though, something like vancomycin or another gram-positive antiobiotic should do the trick yes?
why not start over and redo the transformation - after you check that your competent cells aren't contaminated, of course. Just seems like it would be much cleaner than trying to get rid of your contamination with an antibiotic.
What else I think you can do instead of antibiotic is taking your samples to the agar plate and try to get separate colony. I think you know the method. Then try to take your ecoli colony. Or if you are working in the Microb lab try to separate your gram neg bactery in EMB. Hope that your problem be whit gram Pos.
Any way I hope that your problem be solved whit out need to this solutions.
Stay in touch.
As you're not a microbiologist, I'm not sure why you care what the bug might be and I doubt you'll be able to identify it. It's not even clear you have a single contamiant.
The problem is in your aseptic tecncique and I suggest you work on it.
The problem is in your aseptic tecncique and I suggest you work on it.
Agree with jorge, work on aseptic technique. start with new cell line and check your primary frozen stock for any contamination.
i would recommend, In addition , DH10b or top10 cell by invitrogen. They have gene to develop resistance to streptomycin in their chromosomal dna. That might help you a lil bit with your cloning.
However this should be the last resort as aseptic technique is the most crucial thing whether u're doing microbiology, molecular or whatever related. it really sucks to hvae it on the plate, ruins ur day..