When should I suspect there is a Mycoplasma contamination? - (Sep/13/2007 )

Since you can't see it in light microscopy- when should I suspect that there is mycoplasma contamination?
If I suspect mycoplasma contamination in my culture, and I threw all of them out. cleaned the incubator and made new medium-
is this enough to try culturing again? thanks!

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You should routinely test all of your cell lines for mycoplasma contamination, as you are right, you can't determine this through light microscopy. In our lab, when a new cell line is received, it is considered mycoplasma positive (until tested) and is dealt with accordingly, ie. kept in a seperate incubator, seperate media, seperate laminar flow hood etc

That said, you did the right thing about your suspected contamination, too many researchers seem not to care at all about mycoplasma contamination!!!

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You throw away your cells just because you assume that they are infected?
Didnt you run any assay at all to prove it?

You could run a DAPI test next time. You will need a fluorescence microscope for that.
You could also ask labs near you if they could perform a PCR assay for you.
Both ways are not that expensie (compared to discarding cells because of mere guesswork).

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Hi,

it is correct. You should do the test regularly. We use a luminescence-kit (MycoProbe® Mycoplasma Detection Kit). It`s very easy and quick. You could also do DAPI-staining but most of the time one is not really accurate.

Many people think that they don`t have any contamination but they have never tested it.

Cheers

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Yea I did throw all of them away- their morphology changed rather drastically... I was seeing the "cells breaking up into thousand little pieces" picture as posted in the thread that was pinned. Not only did I see a flower, but the cells also had a lot more debris than usual. Not being a biologist, I'm not competent/confident enough to try to de-contaminate anything that might be remotely contaminated. And I wasn't even sure how to go about cleaning my incubator, or that I should. I'm the only one in my lab( and most of my department) that does cell culture. So- given my cells are not too expensive (1.5 weeks of my time, which I could work on other things) I chose to be safe and behind by 2 weeks. But that's a good suggestion- at least I can run the DAPI test fairly quickly. Thanks for the tip!

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we just cleaned our incubator with "mycoplasma off" - its toxic but does the job

rhombus will probably start shouting at me (30 years in cell culture...) but theres always the pcr test if you are lacking in any other method

dom

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Hi again,

what kind of a incubator do you have in the cell culture? I know that some incubators have a autoclaving program.

In my lab we autoclave the shelves and clean inside of the incubator with 70%EtOH. Of course, we try to open/close the incubator as less as possible.

Cheers

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we do have some shiny high tech ones which as we speak (type) are running a high temp cleansing program (dont know about autoclaving - boom?)
- our quaranteen incubator on the other hand looks like an old tank and has a thing for toxic sprays and elbow grease (bit of the old alcohol too)

they are heraeus (old) and hera-cell (new)

dom

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Dear Dom,

You did not mention the 2 Nobel prizes given to the labs I was trained in..... anyway there are specialist contracting companies in the UK which will screen your cells. This costs £80/cell line and is done by EXPERTS in the field of mycoplasma detection and identification. As Dom states in his reply, PCR kits are available but give too many false negatives. That is why the ATCC no longer use PCR as a detection assay.

Lauralee has the correct advice and that should be standard in all cell culture labs.

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dominic, what kind of stuff is "mycoplasma off"?

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