Getting crazy because of cell contamination - (Sep/29/2005 )

Yes, that's what I thougt too: I find it very strange that the colour of the medium doesn't change that much. In the past, I already had a clear bacterial contamination once and that time, the medium really turned yellow. In contrast, I now see cloudy strings floating around. They have a bit a "milky" character (if you see what I mean, sorry for my English..)

So, maybe it could be mycoplasma you think?
And maybe after the weekend I can try to plate things on blood agar. I haven't tried that one yet.

@ Aimikins: your bubble-story was quite funny and made me laugh! (it was a funny note, very welcome amongs all my cell culture troubles) But I am quite sure the contamination I am dealing with aren't air bubbles.

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Well Anneke I propose that you wait to see after the weekend if your test flask is turning also to Gloubi Boulga ! What is strange is the milky stuff cause mycoplasma doesn't do that they usually slow down the growth of the celles but don't show such a behaviour ! Might be slow growing fungi.

pesji

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i suppose the pH change would be different for different types of bacteria; depending on the waste products they produce, you would not necessarily get yellow media.

the one time I had an issue with bacterial contamination, it looked very much like what you are describing...once the bacteria went nuts reproducing, the stuff in the wells looked a little milky and icky. there were clumps of tiny black dots floating around. the monolayers continued to stay attached, but grew a little more slowly and looked a bit sickly (they entered senescence early, that sort of thing). it only happened in one set of plates, I am assuming staph from my skin because of the clumpy appearance. but the media did not really change color.

good luck!

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Hellow again!

Unfortunately the contamination hasn't been eliminated yet...

As I told you, I had made some new flasks of medium on Friday and without using them for cell culture, I had put an little flask with medium alone in the incubator. On Monday, all of these medium flasks were contaminated...
Because I doubted if I maybe had made some mistake on Friday (e.g. with the pipetting or so), I made new little medium flasks, but indeed: today they were all contaminated again.

This lets me suppose it must be some component of the medium itselfs (and not the cells) that causes my contamination. However, on Monday I made some new little medium-flasks of RPMI, adding sequencially one more component:
1) medium alone
2) medium + FBS
3) medium + FBS + glutamine
4) medium + FBS + glutamine + sodiumpyruvate.
To my surprise, all these flasks still look uncontaminated... and consequently I still have no idea of the causing factor...

I asked my colleagues if it would be possible to ask the microbiology-lab of our university to plate out some contaminated material, but they were not very enthousiastic about it... They said it would probably be a bacterial contamination and plating it out wouldn't give us more information about the cause. Are they right or do I have to insist?

Ciao, An

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Are you preparing the medium yourself from powder ? If yes it might well be coming from the water itself cause that's the only component which is common in each of your contrôls

Of course it might just come as I told you before from the incubator ! If it's the case you would have to use fumigation to kill all possible sources of contamination.

Hope it helps

Pesji

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why are you not using antibiotics?

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First of all some cells just don't grow well with antibiotics like myeloma cell line PAI for example they don't grow well with Pen strep. Second a lot of lab prefers to see right away the contamination and it's also a good (bu tough) way to learn how to work under sterile conditions !

But yes it can be pretty painful

pesji

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@ pesji: We buy the medium in flasks and we only have to add the serum, glutamine and sometimes sodiumpyruvate. So, the water can't be the cause I guess.

On Monday, I also cleaned the warmwatherbad and the incubator. But now I am wondering if it maybe could be caused by the little filter in the incubator? ... At the end, you begin to doubt about everything...

Why we don't use antibiotics? Well, we indeed prefer to detect contaminations as early as possible. Because we believe the results you obtain from a contaminated culture that is controlled by antibiotics can't be 100% reliable. Up till now, I had only a few minor contaminations. But yes... I now experience that working without antibiotics can be pretty hard!

Still hoping to find a solution as soon as possible...

An

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Here are some possibilities to decontaminate an co2 incubator I guess this is now your last chance to get rid of this annoying story
http://www.shellab.com/whitepapers/Elimina...ntamination.pdf

It looks quite complete !

hope it will solve your problem

pesji

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One additional measure that has been mentioned on this site before (in reference to mycoplasma contamination), is to raise the temperature of the incubator to it's highest operating temperature (sometimes above 80 C) for several hours. The thought is that an elevated temperature in such a humid environment may kill off any contaminating organisms.

As an aside, you did a good job with the media controls...you have some flasks that aren't growing your contamination, now try the last step and add some cells! You may have identified the incubator as a possible source of contamination, but is there another incubator close by that could serve as a (hopefully) negative control?

Good luck!

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