I have been doing cell culture for 15 years with no issues and all of a sudden have a type of contamination that I have not seen described. I am growing mouse melanoma cell lines, and their growth rates have slowed dramatically, yet the media turns yellow long before cells are confluent. One cell line is also exhibiting cell death. The media is not turbid, and I cannot see budding yeast cells in the media. I threw out all cultures and thawed freezes that have grown well before, but the same thing happened within a week of growth. It does appear that there are small "blobs" on the surface of some cells. Has anyone experienced this type of contamination? Any insights into what it might be and how it can be eliminated? Thank you!
Thank you both for the replies. I feel like an idiot, but I actually think I figured things out. A few weeks ago I moved all of my RPMI-1640 to a glass-door refrigerator and have had the light on in the fridge. I just read that this media should be stored dark and that light will break down L-glutamine and other compounds, depleting nutrients and creating toxic by-products. The change in cell behavior began about 1 week after moving the media. This would explain the poor cell growth/death in media where contamination is not visible. The one culture that grew well for me this week was grown in media taken from a bottle in the back of the fridge (where it is darker). I don't know for sure yet, but I am hopeful that this is what was wrong and will be a quick fix. Let me know if this all sounds plausible/likely.
Hi, melanoma cells may produce melanin, which firstly appears yellow and may eventually turn the medium dark brown to black. I don't think this is your issue as it is not really toxic to cells. It may be a simple case of a very high metabolic rate, depleting the medium quickly and acidifying it. You may wish to try a medium containing a higher buffering capacity (e.g. 25mM HEPES) which will maintain a neutral pH for much longer. It is about the same price as medium without the extra buffer. Hope this helps,
Jon
Thank you, Jon. I am looking into mycoplasma. I've been growing the melanoma cells in the same media for years and have never had this problem. Regarding mycoplasma, I have read that it is often slow growing. But is it also possible that it could spread to all cultures within a week and have this effect? Does mycoplasma contamination typically turn the media yellow (even when melanoma cells are only ~25% confluent) but not cloudy? Thanks again for your quick response.
Not only can it spread quickly, but can contaminate all other cultures in the lab as well. If the cells are readily available I would personally discard all old stocks and replace them with new ones. Treatment is costly and not guaranteed to work 100% effectively as they may be both inside and outside of the cells. And yes, if they are capable of utilising the medium they will acidify it in much the same way as your cells do. The fact that your cells previously grew OK gets me thinking though. Have you tried making fresh medium with a completely different batch of serum and supplements, as well as replacing every other solution? (including PBS/HBSS, trypsin etc). The fact that all your newly thawed cells show the same yellowing, in previously untainted cells, suggests that there is a common underlying cause, and this could be as simple as just one of your components being contaminated with whatever it is causing the problem. This includes pipette tips as well. I've seen a similar effect with contaminated PBS due to an inefficient autoclave.
It might be so that the media from which you are making the stock is contaminated. Why don't you create a new stock from a different source and check the result on a small scale? Also please ensure that you clean the incubator / freezer before you try out the fresh experiment. The cleaning procedure might also be a possible source of contamination. So, please do take care in the process.
Thank you both for the replies. I feel like an idiot, but I actually think I figured things out. A few weeks ago I moved all of my RPMI-1640 to a glass-door refrigerator and have had the light on in the fridge. I just read that this media should be stored dark and that light will break down L-glutamine and other compounds, depleting nutrients and creating toxic by-products. The change in cell behavior began about 1 week after moving the media. This would explain the poor cell growth/death in media where contamination is not visible. The one culture that grew well for me this week was grown in media taken from a bottle in the back of the fridge (where it is darker). I don't know for sure yet, but I am hopeful that this is what was wrong and will be a quick fix. Let me know if this all sounds plausible/likely.
Hi, I think you do not have a biological problem, Is better use the Standard Tissue Culture Treated Surface and DMEM, high glucose.
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For me, it turned out that my media was light sensitive and the glutamine had broken down. If you are not storing your media in the dark, this may be causing your problem as well.
Just adding onto what Jon has stated in his last answer. Try check for contamination of pipettes or tips. We had a batch of contaminated tips that gave us the problem. Also he has mentioned about PBS. When u r done with autoclaving, then if u can always spread the PBS on the bacteriological and/or fungal media/broth. You know it doesn't take much time to spread a plate or a two of the NAM/PDA media. Its a quick troubleshoot and is always helpful. Anyways if u r reading this then what have u finally concluded about the problem that u had faced. I mean was it just the light sensitivity of the L-Glutamine or something else?
Good Luck !!
Thank you, Vikrant - it did indeed just turn out to be the light sensitivity of the media! Cells have been growing happily ever since correcting this!
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