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my cells love infection, any thoughts on antibiotics?


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#1 SStamis

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Posted 20 September 2010 - 05:06 AM

Dear Cell Culture experts,
I have started cell culture, and neither I or my boss have much experience with it. We ordered a slow growing human epithelial cancer cell line (MDA PCa-2b) and it got infected. I took the cell line down and washed my incubator with 70% EtOH. This is the second time I've gotten infection with this cell line, but my 2 other cell lines are infection free. I'm a little concerned that my stock is contaminated. We have penn-strep from sigma in the freezer, does that work against mycoplasma? any thoughts on concentration?
thanks!
-Sarah

#2 Piersgb

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Posted 20 September 2010 - 05:50 AM

Penn-step is used quite commonly in cell culture media. I use it in my culture with primary mouse bone marrow cells and have had very few obvious infections. I must say that I have never tested for mycoplasma infections, but if you're worried about this then perhaps you should run some tests on this?

So I guess I would recommend you try Penn-Strep for a bit and see how that goes. I use it at 100 IU/mL penicillin and 10 g/mL streptomycin. I don't think this would work against mycoplasma infections as they don't have cell walls. They are also very small and so you probably wouldn't be able to filter these out and you wouldn't see them under the microscope. There are commercial treatments available that can rid infections.

#3 leelee

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Posted 20 September 2010 - 07:26 PM

What you really need to do is improve your aseptic technique. Find somebody in your institute who has experience with cell culture and have them first show you how to do it, and then watch you to see where you are going wrong.

You should always aim to culture your cells without antibiotics, as these can not only affect the nature/behaviour of your cells, but also hide low level contaminations.

Also, if you do have contamination, throw your cells out. You should never treat or cure your cells of any infection as the nature/behaviour of your cells has now been compromised.

No, pen/strep won't work against mycoplasma- but you shouldn't try to treat it anyway, just throw away contaminated cells. Mycoplasma should be routinely tested for by every lab doing cell culture. Mycoplasma will affect cells and hence your results. Experimental results generated with cells infected with mycoplasma are invalid.

#4 labrat612

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Posted 21 September 2010 - 06:34 AM

If you're seeing contamination within a short period of culturing time, chances are that mycoplasma is not to blame. However, routine checking for it can't hurt at all.

Primary cells love love love to get infected. I've cultured primary neuronal cells and had the same issue. Add a little pen/strep to your mix. Just keep in mind that adding antibiotics can affect the behaviour of your cells.
Also, using antibiotics can cover up sloppy technique. So don't use that as a crutch. I don't know if you routinely culture your other cells without antibiotics, if you do and don't suffer any repercussions, then your technique is probably fine.

Do be weary if your primary cells have contamination and your other cells do not. It might be worthwhile to clean out the incubator completely, and then restart your culturing. And make sure to clean well with 70% EtOH (at least) in between working with different cell lines.


Hope that helps!




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