Phenol red and pH buffering of cell culture media

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; RUclips: • 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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Another reason the media might turn purply is if it’s cold. Although I’ve been talking about the whole bicarb thing as the acid/base, water itself is also an acid/base we need to consider. Water can - and does - act as an acid, donating protons. It does this more at higher temperatures (its acid dissociation constant is higher at higher temps) and less at lower temperatures. So water contributes fewer protons to the party at lower temperature, and therefore the pH can rise. This is one reason why that cold media you take out of the fridge looks pinker than the media in your incubator, and one reason why you should pre-warm the media before sticking cells in it.
We’re typically more worried about the media turning orangey or, hopefully not, the dreaded yellow. This happens when the media becomes too acidic.
Even if you keep the growth conditions constant, working at the CO₂ concentration and temperature the media was made for, you still can (and typically do) get pH changes. Because there’s another source of acids you aren’t controlling, the cells! They produce acidic waste like lactic acid - which, by the way is not the cause of your muscle soreness (links at the end). Those waste products accumulate because there’s no waste disposal system or way to recycle them and use them for useful things like there is in the body. So the acid overwhelms the buffer, using up all the bicarbonate - we say the media becomes “spent.”
This will eventually happen even if the cells aren’t “overgrown” space-wise, but it will happen sooner if they are overgrown. And even sooner if the media is contaminated. So yellowish media could be a sign of time to split the cells (dilute them into a larger volume) or time for some decon and starting a fresh culture with better sterility.
more on splitting cells here: blog: bit.ly/passaging_cells ; RUclips: ruclips.net/video/qDLrNDV5754/видео.html  
The color changes happen because phenol red (aka phenolsulfonphthalein or PSP) has a couple of protonatable groups itself. There’s a ketone group (C=O with carbons on either side) which will protonate at really low pH (pKa ~1.2), when there’s tons of protons around. And a hydroxyl group (-OH), which will deprotonate ~pH 7.7 or higher, where protons are scarce. So you end up with a couple of different protonation states, and these affect what wavelength of light the molecule absorbs, and thus what color it appears.
much more on color and dyes here: bit.ly/lightleafcolor
This color change is useful for us to track pH, but it can be anti-useful (should be a word…) for some assays (experiments measuring things) with colorimetric (color-based) read-outs. So some media formulations leave it out.
Some people also leave it out because there are some reports it can cause problems by mimicking steroid hormones and stuff - but the evidence is mixed. Here are a couple conflicting results and I haven’t had time to dig deeper but if your cells are sensitive to estrogen, you may want to.
* Berthois, Y., Katzenellenbogen, J. A., & Katzenellenbogen, B. S. (1986). Phenol red in tissue culture media is a weak estrogen: implications concerning the study of estrogen-responsive cells in culture. Proceedings of the National Academy of Sciences of the United States of America, 83(8), 2496-2500. doi.org/10.1073/pnas.83.8.2496
* Jorge E. Moreno-Cuevas, & Sirbasku, D. A. (2000). Estrogen Mitogenic Action. III. Is Phenol Red a “Red Herring”? In Vitro Cellular & Developmental Biology. Animal, 36(7), 447-464. www.jstor.org/stable/4295107
more one the lactic acid muscle soreness myth:
* Lactic Acid…Let’s dispel the myth! by Peter J Vilasi, The Endurance Runner, March 31, 2015 theendurancerunner.wordpress.com/2015/03/31/lactic-acid-lets-dispel-the-myth/
* Lactic Acid Is Not What Causes Sore Muscles By: Ashley Hamer, Discovery, August 01, 2019 www.discovery.com/science/lactic-acid-is-not-what-causes-sore-muscles
Another helpful resource: Temperature, CO₂, and pH in Cell Culture Media, Alicia D Henn, BioSpherix biospherix.com/411-temperature-co2-and-ph-in-cell-culture-media/
more posts on cell culture: bit.ly/cell_culture  
 
more on osmosis and tonicity: blog form: bit.ly/osmolarityandmore ; RUclips: ruclips.net/video/RNu1vX8Mg18/видео.html  
 
more on cell culture media: blog: bit.ly/cell_culture_media ; RUclips: ruclips.net/video/8GKN60J9mC4/видео.html  
   
more about all sorts of things: #365DaysOfScience All (with topics listed) 👉 bit.ly/2OllAB0 or search blog: thebumblingbiochemist.com