Help with Bacterial Contamination

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effervimus
effervimus's picture
Help with Bacterial Contamination

My lab has quite a puzzle on our hands.  We seem to have a bacterial contamination in our cell lines and need to figure out where it is coming from and how to get rid of it.  its a lot of information so:

  • multiple cell lines infected: PC3, 293, U2OS, etc.
  • seems to grows in the presence of pen/strep, zeocin, and gentamicin
  • tried treatment with Normocure and if anything they're growing faster
  • cleaned out hood, water bath, and all parts of the incubator with ethanol; didn't help
  • got new medium, trypsin, and PBS; no luck
  • examined the medium (new and old); no sign of bacteria
  • cells taken from stock in the liquid nitrogen tank show contamination after 1-2 days when grown in new medium even when grown in a different incubator
  • it seemed to begin a few days after adding new (autoclaved) water to the humidifier unit in the incubator
  • autoclaved pipette tips sometimes show condensation after being removed from the autoclave
  • lab, which only shares our microscopes and autoclave, say they are beginning to show signs of contamination

We are trying to figure out where the contamination is coming from and how to save the cells.  The cell lines are mutants and took us quite some time to get them so we really, really don't want to toss them out unless we absolutely must.  Any ideas?

Mobi_Dot
Mobi_Dot's picture
Hi effervimus,

Hi effervimus,

Please go to you tube and type cell culture contamination the first video and there is a link for a preprint paper might help?

EricP
EricP's picture

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Hi,
The fact that this is resistant to Normocure is very worrying… We really worked hard on making this product very efficient against a lot of difficult strains. This was definitely your best chance on the market !
The last time we had occurrence of such a contamination, it was due to bacterial spores (bacillus / Gram+). You may notice some bulges on your bacteria if this is the case, like little matches. The source was the environment (water bath probably or sink). We could not rescue the cells as the bank was contaminated…. Spores are extremely hard to kill. They are resistant to UV, high temperature, extreme freezing and chemical disinfectants….
 We are trying to develop a special protocol or product for this, but this work is still in progress...

Kristina Holmberg
Kristina Holmberg's picture
 It is always good to do a

 It is always good to do a good cleaning of the culture hood by formaldehyde fumigation, perhaps once a year. Ethanol and similar solutions are good for daily cleaning. Also try to place other equipments in the hood to fumigate them too.

EricP
EricP's picture
 Hi,

 Hi,

Could we get a sample to try decontamination ? We are always keen on getting very resistant strains to improve our knowledge or products !

Thanks