Protocol Online logo
Top : Forum Archives: : Cell Biology

why does DMEM pH increase - Tissue culture (Aug/23/2005 )

When I make flesh DMEM solution, it is red in color. But when I almost use up the solution, it appears light purple. I think this is because of the pH increase (DMEM contain phenol red).

Could anyone tell me the reason....



The reason is that when you have not your medium in CO2 buffering atmosphere, it becomes alcaline. For the reason of buffering CO2 chamber are used. And for this reason it s not goot to provide the manipulations on the cell culture for too long time out of CO2 chamber.

If you are interested in the very stable pH, you can commande the special DMEM, which is additionally buffered ( but it costs 3 times more, than standard DMEM)


Sigma cell culture handbook rulez!

Buffering Systems

Most cells require pH conditions in the range 7.2 - 7.4 and close control of pH is essential for optimum culture conditions. There are major variations to this optimum. Fibroblasts prefer a higher pH (7.4 - 7.7) whereas, continuous transformed cell lines require more acid conditions pH (7.0 - 7.4). Regulation of pH is particularly important immediately following cell seeding when a new culture is establishing and is usually achieved by one of two buffering systems; (i) a "natural" buffering system where gaseous CO2 balances with the CO3 / HCO3 content of the culture medium and (ii) chemical buffering using a zwitterion called HEPES (Prod. No. H4034).

Cultures using natural bicarbonate/CO2 buffering systems need to be maintained in an atmosphere of 5-10% CO2 in air usually supplied in a CO2 incubator. bicarbonate/CO2 is low cost, non-toxic and also provides other chemical benefits to the cells.

HEPES (Prod. No. H4034) has superior buffering capacity in the pH range 7.2 - 7.4 but is relatively expensive and can be toxic to some cell types at higher concentrations. HEPES (Prod. No. H4034) buffered cultures do not require a controlled gaseous atmosphere.

Most commercial culture media include phenol red (Prod. No. P3532 / P0290) as a pH indicator so that the pH status of the medium is constantly indicated by the color. Usually the culture medium should be changed / replenished if the color turns yellow (acid) or purple (alkali).