pH In Cell Culture – How Does pH Buffered Culture Media Work?

Changes in pH in the cell culture environment can alter virtually every cellular process like metabolism, cell growth, and membrane potential. Extracellular pH is slightly alkaline and typically 7.3-7.4, while intracellular pH is slightly lower at 7.2. Many organelles such as mitochondria maintain a slightly different pH from the cytoplasmic pH but if these differences are too great, there are dramatic functional consequences for the organelles.

Since pH is one of the most important parameters of cell culture, a deeper understanding of pH buffered growth media can help you get the best possible results.

pH Buffers & How pH Buffered Culture Media Works

In the simplest terms, a buffer is a mixture of a weak acid or weak base and its conjugate base or acid. They work by neutralizing any additional acid (H+ ions) or base (OH ions) to maintain the desired pH.

A pH buffer acts as either a weak acid or a weak base to ensure that the media will be somewhat resistant to change in pH. This works because the buffer is capable of donating or accepting hydrogen ions which are responsible for establishing pH.

Acid + Base  Conjugate Base + Conjugate Acid

The most common buffer used in mammalian cell culture is sodium bicarbonate. The sodium bicarbonate buffered media becomes sensitive to carbonic acid formed by the concentration of CO2. As long as the level of CO2 is controlled, the pH of the media can be maintained.

Biological processes are by and large only possible when the pH is within a certain range, and the correct buffer has to have a pKa close to the optimal pH of the desired cells. The pKa indicates the strength of an acid, with lower pKa indicating a stronger acid.

Does Temperature Play a Role in pH?

Temperature can play an important but non-linear role in pH. As temperatures increase, pH tends to decrease. This is best described by using water as an example.

Water has a lower pH when it is heated. At 100°C the pH is closer to 6.14 than it is to 7.00. This makes the new neutral pH at 100°C technically more acidic, but the components have not changed. No matter the temperature, water is always just hydrogen and oxygen.

This phenomenon has been measured for a variety of buffers and is particularly important when buffers are made at ambient room temperature (21-25°C) but used at a higher temperature like those inside an incubator (30-37°C). Taking this into consideration, a buffer that provides optimal capacity and range at one temperature may not be the right choice for experiments at different temperatures.

pH Buffer Capacity and Range

Each buffer has a specific effective capacity and range. The buffering capacity is the amount of acid or base that can be added before the pH begins to change. Alternatively, it’s the quantity of acid or base that must be added to change the pH of one liter of solution by one pH unit.

This means that the pH buffering capacity (pKa) is the negative log of the acid dissociation constant (Ka). It is the point where equilibration is reached, and equal portions of acid and conjugate base exist in solution. This is the point where the media has the highest buffering capacity and the greatest ability to resist changes in pH.

The buffer’s range is the pH range where a buffer effectively neutralizes added acids and bases while maintaining a relatively constant pH.

There are hundreds of pH buffered solutions but generally speaking there are three different types:


Acid buffers have a pH less than 7.0 and are made of a weak acid and one of its conjugates. The most commonly used acidic buffers have a pH of around 4.75. The pH of the buffer solution can be changed by adjusting the ratio of acid to salt.


Alkaline buffer solutions have a pH greater than 7.0. They are made of a weak base and one of its conjugates. The most commonly used alkaline buffer is ammonia-ammonium chloride and has a pH of 9.25 when mixed with equal molar proportions. The pH of this solution may be changed by adjusting the ratio of base to salt.


Neutral buffers are exactly what they sound like. They have a pH close to or at 7.0 and are used for a variety of reasons but generally to keep cells at the mammalian physiological range. These zwitterionic solutions have an equal number of positively and negatively charged ions.

The most commonly used buffer in cell culture is HEPES which is better at maintaining physiological pH than bicarbonate buffers and can be prepared between 6.8-8.2 pH.

If any of these buffers are pushed too far, they will break, and pH will have to be regulated with the addition of acid or base depending on environmental conditions inside the cell culture vessel.

What is Breaking the Buffer?

The buffer is considered broken when the entire base and its conjugate acid (or vice versa) are consumed in the process of neutralizing (adding acid or base.) The addition of any more acid or base at this point will rapidly, and often dramatically, alter the pH.

Once the buffer has broken all hope is not lost. It just means that the capacity has been filled and regulating pH is now a lot more hands-on.

No matter which buffer you choose for your cell culture and how you measure pH, nearly everyone agrees that maintaining consistent pH is crucial for cell growth. Adding a buffer to your media can increase your chances of a successful cell culture and make your life a little easier.

Guidelines for Controlling pH

An excellent open-access publication by Michl et al. provides a step-by-step flow chart with instructions on how to prepare media at a target pH and four specific recommendations on how to attain fine control over pH in cell culture and increase reproducibility.

To start a discussion on how you can use optical sensors to measure pericellular pH, reach out to our applications team.

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