I use DMEM to culture MA104. normally it's of pink color because of DMEM, but it becomes orange overnight. Also cell cultures are not grown very well and there is no contamination. We suspect that inefficiency of incubator, so we put one cell culture in another incubator and put one into our incubator. Two day later we compare them. Medium of cell culture that grown in our incubator became yellow-orange and medium of other culture was still pink, so we understand that our incubator is not good enough. We measure temperature of incubator, it is 32...
I want to ask, what is the reason of color changes? Why incubator leads to make medium acidic and how effect the growth of cell?
DMEM contains phenol red, which is solely a pH indicator. At physiological pH, the media is a pink-red color. When the media is acidic it turns a yellow-orange color as you describe, and when basic it turns a deep purple (such as if you add bleach). Most of the time when the media turns yellow it is because you have too many cells on the plate and they are consuming all of the substrates from the media and producing acidic byproducts into the media, and thus the media needs to be changed and the cells split. In your case, it appears there in fact is an incubator issue, as you noted the media does not change color in another incubator.
First, why is your incubator temperature 32 degrees Celsius? Unless you have a special cell line, most incubators should be set at 37 degrees Celsius, which is human body temperature.
Second, most incubators are set at 5% CO2 (unless you have a specific experiment testing different gas concentrations). Additionally, DMEM is a bicarbonate buffered system, which relies heavily on this CO2 concentration. If the gas in your incubator is off, this will dramatically change the pH in your media.
I would recommend getting the temperature AND CO2 levels checked/equilibrated to the appropriate levels before continuing with the experiment. In the mean time you can perform a quick/cheap experiment by adding HEPES (10mM) to your media before adding it to the cell. HEPES is a non-bicarbonate dependent buffering system, so even if the CO2 is off in your incubator, the pH should remain in the physiological range.
Last, when the media is changing to a yellow color... are the cells dying? Just curious how the cells look after being incubated in this faulty incubator.
Good luck,
~Adam
DMEM contains phenol red, which is solely a pH indicator. At physiological pH, the media is a pink-red color. When the media is acidic it turns a yellow-orange color as you describe, and when basic it turns a deep purple (such as if you add bleach). Most of the time when the media turns yellow it is because you have too many cells on the plate and they are consuming all of the substrates from the media and producing acidic byproducts into the media, and thus the media needs to be changed and the cells split. In your case, it appears there in fact is an incubator issue, as you noted the media does not change color in another incubator.
First, why is your incubator temperature 32 degrees Celsius? Unless you have a special cell line, most incubators should be set at 37 degrees Celsius, which is human body temperature.
Second, most incubators are set at 5% CO2 (unless you have a specific experiment testing different gas concentrations). Additionally, DMEM is a bicarbonate buffered system, which relies heavily on this CO2 concentration. If the gas in your incubator is off, this will dramatically change the pH in your media.
I would recommend getting the temperature AND CO2 levels checked/equilibrated to the appropriate levels before continuing with the experiment. In the mean time you can perform a quick/cheap experiment by adding HEPES (10mM) to your media before adding it to the cell. HEPES is a non-bicarbonate dependent buffering system, so even if the CO2 is off in your incubator, the pH should remain in the physiological range.
Last, when the media is changing to a yellow color... are the cells dying? Just curious how the cells look after being incubated in this faulty incubator.
Good luck,
~Adam
thanks for this quick answer . Cells are not dying but dont grow well ... They grow slowly Also trypsinization is not effective (I wait 20 or 30 min for trpysinization)
We call technical service of incubator because of inappropiate temperature. They try to fix it. But, they meausure 5% CO'2 so there isnt any problem associated with CO2... I wonder that can temperature affect production of metabolic compounds of cell culture that make medium acidic..
Hi Efe,
Recently we are also having same problem in our lab, could you diagnose your problem. We are having trouble with HEK-293 grown in DMEM also with our primary neuronal culture but we do not face this issue if we place them in other incubator. Technicians calibrated incubator but problem doesn't get solved. they say CO2 percentage is fine. Please let me now if you could find reasons
PS: we cleaned everything and fumigated to cut the possibility of contamination.
Hi Disha,
You might have to check the water tray in your CO2 incubator.. Believe me I had the same problem then it turned out that there wasn't enough water in the tray.
Thank you Ahmed , but we already checked and changed water in first instance when this problem started.
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