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To dye or not to dye? About phenol red in culture media

One of the respected clients of VitaVitro suggested us to omit phenol red from our culture medium. He argued that the phenol red is just a pH indicator, has no nutrition and support function, and may have some adverse effects. For the colour, most commercial culture media have a very narrow pH range (say 7.2-7.6); in this range, you can't judge the pH by colour.
He also said many commercially available media and are free of phenol red. When they introduced their product, they called it a New Generation medium.

I even wrote a book (now in press) to the new generation of embryologists - admitting that old men won't listen, anyway. However, in some issues, we have to be careful. We have seen the rise and fall of new generation two-phase embryo culture media; also the spectacular fiasco of new generation dry incubators (see discussed in our Opinion Paper ①) Or the "non-toxic" new generation vitrification solutions, where propylen glycol replaced the (in fact much less toxic) DMSO...
Let me express my doubts regarding this innovative attempt, too.

We always used phenol red for the most efficient bovine in vitro embryo production system in the world. I also used it for all vitrification media in various species. There is zero confirmed evidence of harmfulness in any animal or human embryos. Out of the 32 million citations analysed by PubMed, not a single one focuses on it (do a simple search just with "phenol red": 36 items, none of them is relevant). Some abstracts and reviews express doubts, mostly because of the potential contaminants that may be present in some phenol red samples, but we are very far from any conclusive evidence. I am 100% sure that the hysteria around is similar to that of the ethanol in the lab. Fashion, marketing, saying "our medium is better because"; then other companies start to follow them, too.

I may have made more mammalian embryos than most of you, a total close or above a 7 digit number, so may I respectfully suggest considering the following points:

1.   I disagree that phenol red does not show differences between pH 7.2-7.6. Maybe 0.1 difference is hard to detect, but 0.2 - yes, and 0.4 can be invisible for only blind or colour-blind people. We had a reference phenol red solution (with Hepes buffered media) showing the scale between 7 and 7.8. It was prepared every year and held on the shelves of the lab for easy evaluation.
2.  Also, in contrast to the common belief, 0.2 alterations in pH (if the starting point is well between the physiological range) are NOT harmful to the embryos. However, you can immediately see any drastic, harmful changes in the pH without opening the second (glass) window of your box-type incubator or with a single glance at the dishes in a top loader benchtop. Machines and sensors may break down; a second (or third) level of control in mammalian embryology is never useless.
3.  Additionally, there are some situations when phenol red may be extremely helpful. One example: your gas supply is stopped overnight. You come to the lab, and you see zero carbon dioxide influx. Most incubators alarm you, but - except for a few - they do not tell you when did the problem occur. Maybe at 7 am, maybe at 10 pm the previous day. Moreover, dishes at a different position, different shelves, different compartments may react differently to lack of gas. A single glance at the given culture dish will tell you: pink colour - no worries, we can still proceed; blue colour - hmmm...
4.  (I also tell you a secret. When we cultured 50 bovine embryos in 400 µl medium covered with 400 µl oil in four-well dishes, without somatic cells or medium change from Day 1 to 7, I could tell you the outcome by the naked eye. If the medium was a bit yellowish, that resulted from the high metabolic activity of viable embryos. With a bit of practice, the colour allowed a ±5% estimation of blastocyst/oocyte rates. A marked change toward yellow indicated 50%+, i.e. satisfactory results, and healthy, happy embryos.)

You may say that a pH meter may also help, but it is complicated. For example, in a breakdown situation described in point 3, the pH measurement itself may kill embryos, even if they survived the gas problem.

So, again, it is an option. Milk? Sugar? in your morning coffee. Some people do not want sugar because it may make them fat (will not, if they ONLY put a single cube of sugar in). Some people are sensitive to lactose (but the 5ml milk will not burn up their stomach). The real determinant is their own taste (or superstition), not the outcome, because the result, i.e. embryo development, will be just the same.

In summary, phenol red does not harm embryos, but may support embryologists. That is why VitaVitro embryo culture medium - in contrast to some 'New Generation' products - contains phenol red.



①Vajta, Parmegiani, Chen, Yakovenko: Back to the future: optimised micro-well culture of individual human preimplantation stage embryos. J. Assist. Reprod. Genet. ( in pres)