RPMI 1640 has phenol red as a pH indicator. Similar to other Culture medium it buffers ph on the basis of Co2- bicarbonate buffering system. When we prepare our medium and open it several times for use, it comes in contact with atmospheric Co2 repeatedly, which has a potential for changing pH. Its a rather small change but it does affect the pH of the media which in turn is indicated by change in phenol red indicator color. The attached image can be a good indicator if you want to make sure that your medium is still suitable for your cells and is in the range. Normally it doesnt really affect much if you use up your media quickly. 

The best way to tackle with this is to make your final medium (RPMI+ FBS) in smaller aliquots (say about 50 ml) so that you use up each aliquot in one or two culture days, in comparion to making a 500ml final volume which has to be reopened multiple times. and does to lead to a change in pH. Hope it helps :)

RPMI 1640 has phenol red as a pH indicator. Similar to other Culture medium it buffers ph on the basis of Co2- bicarbonate buffering system. When we prepare our medium and open it several times for use, it comes in contact with atmospheric Co2 repeatedly, which has a potential for changing pH. Its a rather small change but it does affect the pH of the media which in turn is indicated by change in phenol red indicator color. The attached image can be a good indicator if you want to make sure that your medium is still suitable for your cells and is in the range. Normally it doesnt really affect much if you use up your media quickly. 

The best way to tackle with this is to make your final medium (RPMI+ FBS) in smaller aliquots (say about 50 ml) so that you use up each aliquot in one or two culture days, in comparion to making a 500ml final volume which has to be reopened multiple times. and does to lead to a change in pH. Hope it helps :)

This really helps. I always aliquot 100mls and even with that, I noticed the change in color with time. Thank you Dharmat.

May be your medium is contaminated, causing the pH and color change. Just asking...

Hi Temitope

Bicarbonate buffering system is in equilibrium with the CO2 (which is a gas) of the atmosphere. In fact cells are cultivated in incubator with atmosphere containing 5% CO2. When you open your medium in the lab, the CO2 dissolved in the medium tends to evaporate, determining a pH change. The phenomenon is reversible, in fact the colour of your medium goes back to normal after you put it in the CO2 incubator. If you do not like this buffer system, or your cells are sensitive to pH change, use medium with Hepes.

Thank you Paola Di Bonito. I used 2g of Hepes while I prepared the medium.

Hi Adelusi!

Colour change in an indication of change in pH. Appropriately stored un-opened commercially purchase RPMI-1640 maintains the pick colour for a very long time. Colour change normally occurs in the presence of viable cells and the medium should thus be changed. Contamination of the medium results in colour change, usually from pink to yellow with or without visible sign of deterioration. The suggestions of Rachayate will help.

Hi Adelusi,

Put your bottle containing medium overnight at room temperature and next day check for contamination. Minimal change in ph is normal for Rpmi medium as suggested by others above. If you are preparing the medium by your own then check the quality of carbonate it should not be too old else it will not dissolve completely and alternatively change in ph occurs. You can also try using commercially available anti-bacterial anti- mycotic solutions that also helps sometime.