PHENOL RED: CHARACTERISTICS, PREPARATION, APPLICATIONS - BIOLOGY - 2025

The phenol red is a substance of organic nature having the property of changing color when it passes from alkaline to acid and vice versa. For this characteristic it is used as an acid-base indicator. It is also known by the name of phenolsulfonphthalein, and its chemical formula is C ₁₉ H ₁₄ O ₅ S.

This pH indicator is yellow below 6.8, and red above 8.4. This substance is not carcinogenic, but it is capable of irritating the skin and mucous membranes. Phenol red is one of the most widely used pH indicators in laboratories.

Chemical structure of phenol red. Colors according to pH. Source: File: Phenol-red-zwitterionic-form-2D-skeletal.png. Wikipedia.com/Pxhere. Edited image.

It is used in the preparation of important biochemical tests in the diagnosis and identification of bacterial microorganisms. Biochemical tests and culture media that use phenol red as a pH indicator include: Triple Sugar Iron (TSI) agar, Kligler, urea, salty mannitol agar, XLD agar, bright green agar and Vogel-Johnson agar.

Phenol red has also been used in the diagnosis of Helicobacter pylori infections through the diagnostic methodology known as chromoendoscopy.

Recently, phenol red has been found to have estrogenic activity, due to its similar structure. Therefore, it binds to the estrogen receptors present in some cells.

This finding causes the use of phenol red in the preparation of cell culture media to be reconsidered when hormone sensitive cells are to be used.

characteristics

The phenol red indicator is characterized by being a powder formed by tiny crystalline particles of a bright dark red color. In solution the liquid is translucent and light red in color. It is soluble in alkali hydroxides and carbonates, but is moderately soluble in water, ethyl alcohol, and acetone, and insoluble in chloroform.

Its molecular mass is 354.59 g / mol, and its melting point is 285 ° C. Its density is 0.972.

Preparation

To prepare the solution, it is necessary to weigh 0.10 g of phenol red and dissolve in 14.2 ml of NaOH (sodium hydroxide) 0.020 M. Then, make up with distilled water to a final volume of 250 ml.

It should be stored at room temperature, in a dry and well-ventilated place.

Applications

As a pH indicator

Its main use is limited to being added to culture media for the diagnosis and identification of bacterial microorganisms. The phenol red pH indicator is included in those media in which it is desired to show whether the microorganism has been able to ferment certain carbohydrates.

The fermentation of carbohydrates generates the formation of acids. Therefore, the colonies and the culture medium will turn yellow. If, on the other hand, there is no carbohydrate fermentation, then the microorganism will use the peptones present. This alkalizes the medium, which will turn red.

Biochemical tests using phenol red include the following: triple sugar iron (TSI) medium, Kligler, and phenol red broth. Whereas the selective and differential culture media using phenol red are salty mannitol agar, XLD agar, bright green agar and Vogel-Johnson agar.

On the other hand, the urea test also uses phenol red as a pH indicator, but in this case what we want to show is whether the microorganism is capable of splitting urea, which generates a product that slightly alkalizes the medium. (ammonia). In this case there is a color shift to fuchsia.

In the event that urease is not present, the medium will remain the same color.

Chromoendoscopy with phenol red

Hernández et al. Used the 0.1% phenol red indicator plus 5% urea to evaluate the gastric mucosa and diagnose the presence of Helicobacter pylori. This technique is called chromoendoscopy, and it is part of the so-called reactive tests.

This technique has the advantage of efficiently locating the site of lesions, avoiding contamination, and being immediately readable. The yellow coloration is interpreted as negative, and the red-fuchsia coloration is interpreted as a positive reaction.

Research Involving Phenol Red pH Indicator

Various investigations have shown that phenol red has a similar chemical structure to certain non-steroidal estrogens; and in tissue culture media it has estrogenic activity, since it has the property of binding to the estrogen receptor when it is found at concentrations between 15-45 µg.

Especially, it has been found to bind efficiently to the estrogen receptor of MCF-7 human breast cancer cells, with an affinity of 0.001%.

This represents a very important finding, as researchers have tried to obtain estrogen-free cellular media so that cells are not stimulated. For this they had tried to eliminate estrogens from the serum, but they never imagined that a substance that was part of the culture medium could simulate the hormonal action.

Therefore, in this regard, the use of cell culture media containing phenol red should be reconsidered when using estrogen sensitive cells.

Toxicity

For the handling of phenol red, the use of instruments and biosafety measures is recommended.

In case of accidental spillage, it is necessary to collect the substance mechanically with absorbent materials. Dispose of in an appropriate container. It should not be poured down the drain.

Risks

The NFPA (National Fire Protection Association) classifies phenol red as a health risk 2. This means a moderate risk. On the other hand, in terms of flammability and reactivity it is classified as 1 and 0 respectively; that is, there is a slight risk of flammability and there is no risk of reactivity.

Regarding the damage caused by phenol red on direct contact, we can mention irritation of the skin and the ocular mucosa. Likewise, it is harmful if ingested only in large quantities. It is not dangerous to inhale and it is not carcinogenic.

First aid

In the case of contact with skin and mucous membranes, it is recommended to wash the affected area with plenty of water for 15 minutes, lifting the eyelids.

At the skin level, an emollient cream can be placed on the affected area to relieve irritation. If irritation persists, it is necessary to seek medical advice.

In case of mild ingestion, rinse your mouth. If the ingestion was in considerable volume, it is essential to seek immediate medical assistance. Do not induce vomiting, or give milk.

Incompatibility with other substances

Care must be taken that phenol red does not come into contact with the following substances with which it is incompatible: perchlorates, peroxides, permanganates, phosphides, tin II, metal chloride and hydrides. With all of them it can react violently (explosively) and cause fire.

References

Berthois Y, Katzenellenbogen JA, Katzenellenbogen BS. Phenol red in tissue culture media is a weak estrogen: implications concerning the study of estrogen-responsive cells in culture. Proc Natl Acad Sci USA. 1986; 83 (8): 2496–2500.

Phenol Red MSDS Safety Sheet. National University of Heredia. Costa Rica. School of Chemistry. Available at: Users / Team / Downloads / red% 20fenol.pdf

ROTH. Safety data sheet for phenol red. 2015. Spain. Available at: carlroth.com

Chemical Engineers and Associates. Phenol red pH waters. Safety sheet. Colombia. Available at: enclosuredelpensamiento.com

Neogen. Red phenol broth base. Available at: foodsafety.neogen.com

Hernández H, Castellanos V, González L, Infante M, Peña K, Andrain Y. Chromoendoscopy with phenol red in the diagnosis of Helicobacter pylori infection. Spanish Journal of Digestive Diseases. 2012; 104 (1). Available at: scielo.org

Marín J, Díaz J, Solís J. Chromoendoscopy in Helicobacter pylori infection: is it reaction time? Spanish journal of digestive diseases: official organ of the Spanish Society of Digestive Pathology. 2011; 104 (1): 01-03. Available at: researchgate.net