Phenol red
Encyclopedia
Phenol red is a pH indicator
that is frequently used in cell biology
laboratories.
. It is a weak acid
with pKa
= 8.00 at 20°C.
A solution of phenol red is used as a pH indicator
: its color exhibits a gradual transition from yellow to red over the pH range 6.8 to 8.2. Above pH 8.2, phenol red turns a bright pink (fuchsia
) color.
This solution contains a pH indicator which helps in monitoring of the pH changes in the cell culture. It will change from red to yellow color when the pH value decreases.
This observed color change is caused by phenol red losing protons (and changing color orange or yellow) as the pH increases. In crystalline form, and in solution under very acidic conditions (low pH), the compound exists as a zwitterion
as in the structure shown above, with the sulfate
group negatively charged, and the ketone
group carrying an additional proton. This form is sometimes symbolically written as H2+PS− and is orange-red. If the pH is increased (pKa = 1.2), the proton from the ketone group is lost, resulting in the yellow negatively charged ion denoted as HPS−. At still higher pH (pKa = 7.7), the phenol
's hydroxide group loses its proton, resulting in the red ion denoted as PS2−.
In several sources, the structure of phenol red is shown with the sulfur atom being part of a cyclic group, similar to the structure of phenolphthalein
. However, this cyclic structure could not be confirmed by X-ray crystallography
.
Several indicators share a similar structure to phenol red, including bromothymol blue
, thymol blue
, bromocresol purple, thymolphthalein
, and phenolphthalein
. (A table of other common chemical indicators is available in the article on pH indicator
s.)
and is now obsolete.
The test is based on the fact that phenol red is excreted almost entirely in the urine. By measuring the amount of phenol red excreted colorimetrically
, kidney function can be determined. Phenol red solution is administered intravenously, all of the urine
produced is collected and the phenol red present determined.
prosper at a near-neutral pH
; that is, a pH close to 7. The pH of blood
ranges from 7.35 to 7.45, for instance. When cells
are grown in tissue culture
, the medium in which they grow is held close to this physiological pH. A small amount of phenol red added to this growth medium will have a pink-red color under normal conditions. Typically 15 mg / 1 L is used for cell culture.
In the event of problems, waste products produced by dying cells or overgrowth of contaminants will cause a change in pH, leading to a change in indicator color. For example, a culture of relatively slowly-dividing mammal
ian cells can be quickly overgrown by bacteria
l contamination. This usually results in an acid
ification of the medium, turning it yellow. Many biologist
s find this a convenient way to rapidly check on the health of tissue cultures. In addition, the waste products produced by the mammalian cells themselves will slowly decrease the pH, gradually turning the solution orange and then yellow. This color change is an indication that even in the absence of contamination, the medium needs to be replaced (generally, this should be done before the medium has turned completely orange).
Since the color of phenol red can interfere with some spectrophotometric
and fluorescent assays, many types of tissue culture media are also available without phenol red.
mimic, and in cell cultures can enhance the growth of cells that express the estrogen receptor.
An important breakthrough in biotechnology was reported in the scientific literature on May 5th, 2005. Using phenol red as a differentiation factor, scientists at the University of Tennessee produced human oocyte
s (eggs) from cells scraped from the surface of adult ovaries. These cells on the outer ovarian surface are known as ovarian surface epithelial cells. Such cells had been taken from five women aged 39 to 52 and were cultured in the presence of phenol red, inducing oogenesis
.
Previously, human eggs had only been produced in vitro
from totipotent, blastomeric embryonic stem cells. One of the significant aspects of this experiment is that it demonstrated viable human egg cells can easily be produced from an adult cell that already has some degree of specialization. Furthermore, it lessens the implications associated with the fact that women are born with all of the egg cells they will ever have throughout their lives. While this breakthrough was not without controversy, it provides hope for infertile women wishing to undergo in vitro fertilization, and hints at the possibility of new options for post-menopausal women as well. It also suggests that the future of stem cell research may not have to rely as heavily on human embryos as a source of unspecialized, totipotent cells for research.
PH indicator
A pH indicator is a halochromic chemical compound that is added in small amounts to a solution so that the pH of the solution can be determined visually. Hence a pH indicator is a chemical detector for hydronium ions or hydrogen ions in the Arrhenius model. Normally, the indicator causes the...
that is frequently used in cell biology
Cell biology
Cell biology is a scientific discipline that studies cells – their physiological properties, their structure, the organelles they contain, interactions with their environment, their life cycle, division and death. This is done both on a microscopic and molecular level...
laboratories.
Chemical structure and properties
Phenol red exists as a red crystal that is stable in air. Its solubility is 0.77 grams per liter (g/L) in water and 2.9 g/L in ethanolEthanol
Ethanol, also called ethyl alcohol, pure alcohol, grain alcohol, or drinking alcohol, is a volatile, flammable, colorless liquid. It is a psychoactive drug and one of the oldest recreational drugs. Best known as the type of alcohol found in alcoholic beverages, it is also used in thermometers, as a...
. It is a weak acid
Weak acid
A weak acid is an acid that dissociates incompletely. It does not release all of its hydrogens in a solution, donating only a partial amount of its protons to the solution...
with pKa
PKA
PKA, pKa, or other similar variations may stand for:* pKa, the symbol for the acid dissociation constant at logarithmic scale* Protein kinase A, a class of cAMP-dependent enzymes* Pi Kappa Alpha, the North-American social fraternity...
= 8.00 at 20°C.
A solution of phenol red is used as a pH indicator
PH indicator
A pH indicator is a halochromic chemical compound that is added in small amounts to a solution so that the pH of the solution can be determined visually. Hence a pH indicator is a chemical detector for hydronium ions or hydrogen ions in the Arrhenius model. Normally, the indicator causes the...
: its color exhibits a gradual transition from yellow to red over the pH range 6.8 to 8.2. Above pH 8.2, phenol red turns a bright pink (fuchsia
Fuchsia (color)
Fuchsia is a vivid reddish or pinkish purple color named after the flower of the fuchsia plant, itself named after the German scientist Leonhart Fuchs...
) color.
This solution contains a pH indicator which helps in monitoring of the pH changes in the cell culture. It will change from red to yellow color when the pH value decreases.
This observed color change is caused by phenol red losing protons (and changing color orange or yellow) as the pH increases. In crystalline form, and in solution under very acidic conditions (low pH), the compound exists as a zwitterion
Zwitterion
In chemistry, a zwitterion is a neutral molecule with a positive and a negative electrical charge at different locations within that molecule. Zwitterions are sometimes also called inner salts.-Examples:...
as in the structure shown above, with the sulfate
Sulfate
In inorganic chemistry, a sulfate is a salt of sulfuric acid.-Chemical properties:...
group negatively charged, and the ketone
Ketone
In organic chemistry, a ketone is an organic compound with the structure RCR', where R and R' can be a variety of atoms and groups of atoms. It features a carbonyl group bonded to two other carbon atoms. Many ketones are known and many are of great importance in industry and in biology...
group carrying an additional proton. This form is sometimes symbolically written as H2+PS− and is orange-red. If the pH is increased (pKa = 1.2), the proton from the ketone group is lost, resulting in the yellow negatively charged ion denoted as HPS−. At still higher pH (pKa = 7.7), the phenol
Phenol
Phenol, also known as carbolic acid, phenic acid, is an organic compound with the chemical formula C6H5OH. It is a white crystalline solid. The molecule consists of a phenyl , bonded to a hydroxyl group. It is produced on a large scale as a precursor to many materials and useful compounds...
's hydroxide group loses its proton, resulting in the red ion denoted as PS2−.
In several sources, the structure of phenol red is shown with the sulfur atom being part of a cyclic group, similar to the structure of phenolphthalein
Phenolphthalein
Phenolphthalein is a chemical compound with the formula C20H14O4 and is often written as "HIn" or "phph" in shorthand notation. Often used in titrations, it turns colorless in acidic solutions and pink in basic solutions...
. However, this cyclic structure could not be confirmed by X-ray crystallography
X-ray crystallography
X-ray crystallography is a method of determining the arrangement of atoms within a crystal, in which a beam of X-rays strikes a crystal and causes the beam of light to spread into many specific directions. From the angles and intensities of these diffracted beams, a crystallographer can produce a...
.
Several indicators share a similar structure to phenol red, including bromothymol blue
Bromothymol blue
Bromothymol blue is a chemical indicator for weak acids and bases. The chemical is also used for observing photosynthetic activities or respiratory indicators .Bromothymol blue acts as a weak acid in solution...
, thymol blue
Thymol blue
Thymol blue is a brownish-green or reddish-brown crystalline powder that is used as a pH indicator. It is insoluble in water but soluble in alcohol and dilute alkali solutions. It transitions from red to yellow at pH 1.2–2.8 and from yellow to blue at pH 8.0–9.6.-Bibliography:* Merck. "Thymol...
, bromocresol purple, thymolphthalein
Thymolphthalein
Thymolphthalein is an acid-base indicator. Its transition range is at approximately pH 9.3-10.5. Below this pH, it is colorless; above this pH, it is blue. The molar extinction coefficient for the blue thymolphthalein dianion is 38000 M-1cm-1 at 595 nm....
, and phenolphthalein
Phenolphthalein
Phenolphthalein is a chemical compound with the formula C20H14O4 and is often written as "HIn" or "phph" in shorthand notation. Often used in titrations, it turns colorless in acidic solutions and pink in basic solutions...
. (A table of other common chemical indicators is available in the article on pH indicator
PH indicator
A pH indicator is a halochromic chemical compound that is added in small amounts to a solution so that the pH of the solution can be determined visually. Hence a pH indicator is a chemical detector for hydronium ions or hydrogen ions in the Arrhenius model. Normally, the indicator causes the...
s.)
Phenolsulfonphthalein test
Phenol red was used in the phenolsulfonphthalein test, also known as the PSP test. This test was used to estimate the overall blood flow through the kidneyKidney
The kidneys, organs with several functions, serve essential regulatory roles in most animals, including vertebrates and some invertebrates. They are essential in the urinary system and also serve homeostatic functions such as the regulation of electrolytes, maintenance of acid–base balance, and...
and is now obsolete.
The test is based on the fact that phenol red is excreted almost entirely in the urine. By measuring the amount of phenol red excreted colorimetrically
Colorimetry
Colorimetry is "the science and technology used to quantify and describe physically the human color perception."It is similar to spectrophotometry, but is distinguished by its interest in reducing spectra to the physical correlates of color perception, most often the CIE 1931 XYZ color space...
, kidney function can be determined. Phenol red solution is administered intravenously, all of the urine
Urine
Urine is a typically sterile liquid by-product of the body that is secreted by the kidneys through a process called urination and excreted through the urethra. Cellular metabolism generates numerous by-products, many rich in nitrogen, that require elimination from the bloodstream...
produced is collected and the phenol red present determined.
Indicator for cell cultures
Most living tissuesBiological tissue
Tissue is a cellular organizational level intermediate between cells and a complete organism. A tissue is an ensemble of cells, not necessarily identical, but from the same origin, that together carry out a specific function. These are called tissues because of their identical functioning...
prosper at a near-neutral pH
PH
In chemistry, pH is a measure of the acidity or basicity of an aqueous solution. Pure water is said to be neutral, with a pH close to 7.0 at . Solutions with a pH less than 7 are said to be acidic and solutions with a pH greater than 7 are basic or alkaline...
; that is, a pH close to 7. The pH of blood
Blood
Blood is a specialized bodily fluid in animals that delivers necessary substances such as nutrients and oxygen to the cells and transports metabolic waste products away from those same cells....
ranges from 7.35 to 7.45, for instance. When cells
Cell (biology)
The cell is the basic structural and functional unit of all known living organisms. It is the smallest unit of life that is classified as a living thing, and is often called the building block of life. The Alberts text discusses how the "cellular building blocks" move to shape developing embryos....
are grown in tissue culture
Tissue culture
Tissue culture is the growth of tissues or cells separate from the organism. This is typically facilitated via use of a liquid, semi-solid, or solid growth medium, such as broth or agar...
, the medium in which they grow is held close to this physiological pH. A small amount of phenol red added to this growth medium will have a pink-red color under normal conditions. Typically 15 mg / 1 L is used for cell culture.
In the event of problems, waste products produced by dying cells or overgrowth of contaminants will cause a change in pH, leading to a change in indicator color. For example, a culture of relatively slowly-dividing mammal
Mammal
Mammals are members of a class of air-breathing vertebrate animals characterised by the possession of endothermy, hair, three middle ear bones, and mammary glands functional in mothers with young...
ian cells can be quickly overgrown by bacteria
Bacteria
Bacteria are a large domain of prokaryotic microorganisms. Typically a few micrometres in length, bacteria have a wide range of shapes, ranging from spheres to rods and spirals...
l contamination. This usually results in an acid
Acid
An acid is a substance which reacts with a base. Commonly, acids can be identified as tasting sour, reacting with metals such as calcium, and bases like sodium carbonate. Aqueous acids have a pH of less than 7, where an acid of lower pH is typically stronger, and turn blue litmus paper red...
ification of the medium, turning it yellow. Many biologist
Biologist
A biologist is a scientist devoted to and producing results in biology through the study of life. Typically biologists study organisms and their relationship to their environment. Biologists involved in basic research attempt to discover underlying mechanisms that govern how organisms work...
s find this a convenient way to rapidly check on the health of tissue cultures. In addition, the waste products produced by the mammalian cells themselves will slowly decrease the pH, gradually turning the solution orange and then yellow. This color change is an indication that even in the absence of contamination, the medium needs to be replaced (generally, this should be done before the medium has turned completely orange).
Since the color of phenol red can interfere with some spectrophotometric
Spectrophotometry
In chemistry, spectrophotometry is the quantitative measurement of the reflection or transmission properties of a material as a function of wavelength...
and fluorescent assays, many types of tissue culture media are also available without phenol red.
Estrogen mimic
Phenol red is a weak estrogenEstrogen
Estrogens , oestrogens , or œstrogens, are a group of compounds named for their importance in the estrous cycle of humans and other animals. They are the primary female sex hormones. Natural estrogens are steroid hormones, while some synthetic ones are non-steroidal...
mimic, and in cell cultures can enhance the growth of cells that express the estrogen receptor.
An important breakthrough in biotechnology was reported in the scientific literature on May 5th, 2005. Using phenol red as a differentiation factor, scientists at the University of Tennessee produced human oocyte
Oocyte
An oocyte, ovocyte, or rarely ocyte, is a female gametocyte or germ cell involved in reproduction. In other words, it is an immature ovum, or egg cell. An oocyte is produced in the ovary during female gametogenesis. The female germ cells produce a primordial germ cell which undergoes a mitotic...
s (eggs) from cells scraped from the surface of adult ovaries. These cells on the outer ovarian surface are known as ovarian surface epithelial cells. Such cells had been taken from five women aged 39 to 52 and were cultured in the presence of phenol red, inducing oogenesis
Oogenesis
Oogenesis, ovogenesis or oögenesis is the creation of an ovum . It is the female form of gametogenesis. The male equivalent is spermatogenesis...
.
Previously, human eggs had only been produced in vitro
In vitro
In vitro refers to studies in experimental biology that are conducted using components of an organism that have been isolated from their usual biological context in order to permit a more detailed or more convenient analysis than can be done with whole organisms. Colloquially, these experiments...
from totipotent, blastomeric embryonic stem cells. One of the significant aspects of this experiment is that it demonstrated viable human egg cells can easily be produced from an adult cell that already has some degree of specialization. Furthermore, it lessens the implications associated with the fact that women are born with all of the egg cells they will ever have throughout their lives. While this breakthrough was not without controversy, it provides hope for infertile women wishing to undergo in vitro fertilization, and hints at the possibility of new options for post-menopausal women as well. It also suggests that the future of stem cell research may not have to rely as heavily on human embryos as a source of unspecialized, totipotent cells for research.