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 ; RU-vid: ru-vid.com/video/%D0%B2%D0%B8%D0%B4%D0%B5%D0%BE-qDLrNDV5754.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 ; RU-vid: ru-vid.com/video/%D0%B2%D0%B8%D0%B4%D0%B5%D0%BE-RNu1vX8Mg18.html more on cell culture media: blog: bit.ly/cell_culture_media ; RU-vid: ru-vid.com/video/%D0%B2%D0%B8%D0%B4%D0%B5%D0%BE-8GKN60J9mC4.html more about all sorts of things: #365DaysOfScience All (with topics listed) 👉 bit.ly/2OllAB0 or search blog: thebumblingbiochemist.com
