Mammalian cell culture media (liquid cell food) is often red. This doesn’t have anything to do with blood* - instead it’s phenol red dye, a pH indicator. If the pH gets too low (solution becomes too acidic), which can happen if the cells overgrow or the culture gets contaminated, it turns orange and then yellow. If the pH gets too high (solution becomes too basic/alkaline), the dye, and thus the media, turn pink-purple. This can happen if the cells get too much CO₂, like if they’re out of the incubator too long and/or the media is too cold. So we can look at our cell culture vessel (dish, plate, flask, etc.) and get a quick readout as to what the environment’s like and whether the cells are hopefully comfortable.
blog form: bit.ly/phenol_red_pH
Where’d that CO₂ thing come in? This comes back to the way in which there is one kinda-connection between the dye and blood - blood, and most tissue culture media, relies on CO₂ and the bicarbonate buffer system it feeds into, to keep its pH steady. How? Much more here: bit.ly/bloodgasesandbuffering
but here’s the gist…
pH is a measure of free proton (H⁺ ) availability. And it’s an inverse log scale, so the more protons there are, the lower the pH and the more “acidic” we say the solution is. Conversely, the fewer protons there are, the higher the pH and the more “basic” or “alkaline” we say the solution is. How many protons there are (and thus the pH) depends on the relative amounts of proton-givers (acids) and proton-takers (bases). If you have roughly equal amounts of both, you get a “pH buffer” - it’s able to buffer the pH, meaning keep it constant, by counteracting any added acids or bases.
for more: bit.ly/phbuffers; KZitem: • Choosing and preparing...
What pH it keeps steady depends on the initial relative amounts of acid and base - and in the case of our cell culture this depends on CO₂ (from the incubator) and bicarbonate (provided in the media).
The bicarbonate buffer system depends on carbonic acid as the acid and bicarbonate as the base. Bicarbonate is what we call the conjugate base of carbonic acid, meaning that you get bicarbonate when carbonic acid deprotonates (once something gives up a proton it can take it back).
When CO₂ dissolves in water, you get carbonic acid (H₂CO₃), which can then deprotonate to form bicarbonate (HCO₃⁻ ).
CO₂ + H₂O ⇌ H₂CO₃
So overall you have
CO₂ + H₂O ⇌ H₂CO₃ ⇌ H⁺ + HCO₃⁻
And we can provide CO₂ using CO₂-controlled incubators, which pump in CO₂, typically to around 5% (compared to room air which is less than 1% or so CO₂). So we can get a steady stream of CO₂. But that’s not enough to keep the pH at the pH we want. Instead, the balance would weigh in favor of the carbonic acid and the solution would be too acidic. If we want the solution to be at a physiological (bodily) pH, ~7.4, we need to tip the starting scales by using media that has bicarbonate in it already. Typically, media has it in the form of sodium bicarbonate salt, where sodium is the cationic (positively-charged) “counter-ion” to neutralize the negative charge of bicarbonate. For example, typically DMEM formulations include 44mM sodium bicarbonate.
Those concentrations are precisely calculated and optimized to keep pH where you want it under the temperature and CO₂ concentrations you grow the cells at. But if you take the cells out of the incubator - such as to feed or experiment with them, the pH can change. And thus the color of the media can change. As you decreases CO₂ concentrations, the media becomes more basic and thus purply. And the cells are likely starting to act funny. So you don’t want to take them out of the incubator for too long. And you want to make sure you use vented caps so CO₂ can get into the growth vessels (it doesn’t do you - or your cells - any good to pump CO₂ at the cells if it can’t reach them!)
If you’re really concerned about pH fluctuations, you can add a buffer like HEPES to help stabilize things since it doesn’t rely on a constant supply of gas. But then you potentially introduce other problems, such as increasing osmolarity (increasing the amount of stuff outside the cell which can lead water to exit the cell) and potentially chelating (binding tightly to and thus “hiding”) metals like calcium which the cells need. So you typically only add it if needed. And, if you add it, it’s not as a full replacement for the bicarb system because the cells still need CO₂ and bicarb to function properly (it’s important for more than just pH balance)
Here’s a really great article I found about this: Michl, J., Park, K. C., & Swietach, P. (2019). Evidence-based guidelines for controlling pH in mammalian live-cell culture systems. Communications biology, 2, 144. doi.org/10.1038/s42003-019-03...
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