Contaminations in tissue culture

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jojo90
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Contaminations in tissue culture

Post by jojo90 » Sun Apr 20, 2008 5:13 am

What are possible sources of contamination
in tissue culture work?

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MrMistery
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Post by MrMistery » Sun Apr 20, 2008 8:52 am

bacteria, if you don't work sterile.
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canalon
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Post by canalon » Tue Apr 22, 2008 1:03 pm

bacteria and fungi. Good sterile technique is necessary (and a laminar flow 'hood helps keep the bad guys away... sorry for the lame joke).
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Post by MrMistery » Tue Apr 22, 2008 8:29 pm

and an broad spectrum antibiotic in the growth medium is not bad in addition to your sterile technique. Just in case you're clumsy one day :D
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biohazard
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Post by biohazard » Thu Apr 24, 2008 8:35 am

Some people advise to avoid antibiotics, since they render cultures susceptible to so called cryptic contaminants (e.g. mycoplasmas), that have some degree of resistance to these antibiotics and thus are able to slowly grow in tissue/cell culture without causing any visible effects. So basically your culture can be badly contaminated without you having any clue, save from some dodgy research results you may get.

This being said, our lab uses antibiotics to keep our cultures safe, but we mostly use short-term cultures that don't allow cryptic contaminants to develop (so easily), and the cultures are usually discarded after a period of time, thus discarding the possible contaminants as well. For any long-lasting cultures, you may need to consider the pros and cons of using antibiotics.

Oh, and to add some (rare) contaminants: viruses can infect certain tissue cultures. Also, some labs have even reported insects - they get to surprisingly many places, even though finding a fly from your culture dish must be both very rare and extremely embarrassing! Finally, there can be chemical contaminants or endotoxins e.g. in the growth medium or some other reagents. Most well-known suppliers have very strict quality controls, but if you, say, buy your medium in powder form and dissolve it in your lab, the water from the lab can be a source of contamination.

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biohazard
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Post by biohazard » Thu Apr 24, 2008 8:54 am

I came up with one more contaminant that mostly applies to cell cultures, but could in theory affect tissue cultures as well: other cell/tissue cultures. At least with long-lasting, immortalised cell cultures it is not uncommon, that the cultures contaminate each other - you basically need to transfer just one living cell to another culture's container and that's it. It could happen via aerosoles, droplers, contaminated equipment etc.

Like canalon said, good sterile technique is essential!

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Post by MrMistery » Thu Apr 24, 2008 10:27 am

Yeah, i heard HeLa cells are the baddest of all, they contaminate everything :lol:

I would ask something. I know Mycoplasma can dodge the antibiotics, but what would the disadvantages of using them be? I mean, your culture is vulnerable to them either with or without the antibiotics, so why would be using antibiotics a disadvantage?
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Post by biohazard » Thu Apr 24, 2008 11:45 am

As far as I've understood, it is their "cryptic" nature: they can spoil your cells without you knowing it, and thus alter the results which you then may publish. According to Sigma's cell culture manual, the culture almost always gets contaminated with something else along with mycoplasmas, if it gets contaminated at all. If you do not use antibiotics, you notice the contamination within 24-48 h and can discard the culture. But if the antibiotics inhibit the other contaminants, you may falsely believe that your cells are ok. So that's what Sigma's experts tell us.

Personally I feel much safer with antibiotics, though, as long as I regularly switch to new cultures from our N2 stock and get rid of the old ones :)

For longer, immortalised cells - well I do use antibiotics there too, but I'm little worried about whether I actually should.

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