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Identify cell culture contamination - (Feb/03/2013 )

Hi there,

I have been having severe contamination issues (since Nov). My cell lines are cultured in RPMI +FCS+Pen/Strep and quite often, but randomly flasks are contaminated (after subculturing one clean and one contaminated flask originating from the same flask).
Media does not turn yellowish but remains pinkish. With 40x, I could see some rod-like shapes.
Weird thing is that it does not occur within a day or two, this pic is from a PC-3 flask that has not been touched for 3days.
Other people in the lab using different hoods and different incubators experience the same issue. However, the other incubators are for primary cells and the throughput is much less than in the cell line incubator I am using. All hoods and incubators are in the same room. The neighbouring group with the same gas supply does not even use P/S and never experiences any issues with contamination. They do far less cleaning than we do. I personally have worked in cell culture for >2yrs w/o antibiotics (in other labs) and never had problems.
We all use our own media. We switch on UV after one person has finished (4-5times a day) so I guess we are really cautious about aseptic techniques.
Would you have any clue about what kind of contamination it might be and what might be improved?
Attached Image


Rod shapes could be fungal hyphae potentially, but these are normally quite large and easily visible with a 10x lens. Lactobacilli (any cheese or yoghurt makers in your group?) are rod shaped and large bacteria, but they acidify things by production of lactic acid; E. coli are rod shaped too and very common gut flora. Pen/strep should be effective against both gram+ and gram- bacteria, unless you have a resistant strain.

Have you checked the medium and FCS for contamination?

A photo of the rods would be helpful.


Thanks for the suggestions. With the medium not becoming acidic, I thought of anaerobic bacteria; however, the lag time is really long for bacterial contamination (or it crawls through filter caps into the flasks inside the incubator). I ll get a pic tomorrow.

If it was the medium or FCS, wouldn't there be a systemic contamination of all flaks?
I was thinking of putting AB-free agar plates into the hood while working or into the incubator for some time and then wait for colonies to grow to nail down hood or incubator.

Is anyone experienced with Novocin in culture media? It seems to be the magical supplement to cope with contamination.