Acridine Orange
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Acridine orange is an
organic compound Some chemical authorities define an organic compound as a chemical compound that contains a carbon–hydrogen or carbon–carbon bond; others consider an organic compound to be any chemical compound that contains carbon. For example, carbon-co ...
that serves as a
nucleic acid Nucleic acids are large biomolecules that are crucial in all cells and viruses. They are composed of nucleotides, which are the monomer components: a pentose, 5-carbon sugar, a phosphate group and a nitrogenous base. The two main classes of nuclei ...
-selective
fluorescent Fluorescence is one of two kinds of photoluminescence, the emission of light by a substance that has absorbed light or other electromagnetic radiation. When exposed to ultraviolet radiation, many substances will glow (fluoresce) with color ...
dye Juan de Guillebon, better known by his stage name DyE, is a French musician. He is known for the music video of the single "Fantasy Fantasy is a genre of speculative fiction that involves supernatural or Magic (supernatural), magical ele ...
with cationic properties useful for cell cycle determination. Acridine orange is cell-permeable, which allows it to interact with
DNA Deoxyribonucleic acid (; DNA) is a polymer composed of two polynucleotide chains that coil around each other to form a double helix. The polymer carries genetic instructions for the development, functioning, growth and reproduction of al ...
by intercalation, or
RNA Ribonucleic acid (RNA) is a polymeric molecule that is essential for most biological functions, either by performing the function itself (non-coding RNA) or by forming a template for the production of proteins (messenger RNA). RNA and deoxyrib ...
via
electrostatic attraction Coulomb's inverse-square law, or simply Coulomb's law, is an experimental law of physics that calculates the amount of force between two electrically charged particles at rest. This electric force is conventionally called the ''electrostatic f ...
s. When bound to DNA, acridine orange is very similar spectrally to an organic compound known as fluorescein. Acridine orange is used in epifluorescence microscopy and
flow cytometry Flow cytometry (FC) is a technique used to detect and measure the physical and chemical characteristics of a population of cells or particles. In this process, a sample containing cells or particles is suspended in a fluid and injected into the ...
. The ability to penetrate the cell membranes of acidic organelles and cationic properties of acridine orange allows it to differentiate between various types of cells (i.e., bacterial cells and white blood cells). Acridine orange and
fluorescein Fluorescein is an organic compound and dye based on the xanthene tricyclic structural motif, formally belonging to Triarylmethane dye, triarylmethine dyes family. It is available as a dark orange/red powder slightly soluble in water and alcohol. ...
have a maximum excitation at 502 nm and 525 nm (green). When acridine orange associates with RNA, the fluorescent dye experiences a maximum excitation shift from 525 nm (green) to 460 nm (blue). The shift in maximum excitation also produces a maximum emission of 650 nm (red). Acridine orange is able to withstand low pH environments, allowing the fluorescent dye to penetrate acidic organelles such as
lysosomes A lysosome () is a membrane-bound organelle that is found in all mammalian cells, with the exception of red blood cells (erythrocytes). There are normally hundreds of lysosomes in the cytosol, where they function as the cell’s degradation cent ...
and phagolysosomes that are membrane-bound organelles essential for acid hydrolysis or for producing products of
phagocytosis Phagocytosis () is the process by which a cell (biology), cell uses its plasma membrane to engulf a large particle (≥ 0.5 μm), giving rise to an internal compartment called the phagosome. It is one type of endocytosis. A cell that performs ph ...
of apoptotic cells. The shift in maximum excitation and emission wavelengths provides a foundation to predict the wavelength at which the cells will stain.


Optical properties

When the pH of the environment is 3.5, acridine orange becomes excited by blue light (460 nm). When acridine orange is excited by blue light, the fluorescent dye can differentially stain human cells green and prokaryotic cells orange (600 nm), allowing for rapid detection with a fluorescent microscope. The differential staining capability of acridine orange provides quick scanning of specimen smears at lower magnifications of 400x compared to
Gram stain Gram stain (Gram staining or Gram's method), is a method of staining used to classify bacterial species into two large groups: gram-positive bacteria and gram-negative bacteria. It may also be used to diagnose a fungal infection. The name comes ...
s that operate at 1000x magnification. The differentiation of cells is aided by a dark background that allows colored organisms to be easily detected. The sharp contrast provides a mechanism for counting the number of organisms present in a sample. When acridine orange binds to DNA, the dye exhibits a maximum excitation at 502 nm producing a maximum emission of 525 nm. When bound to RNA, acridine orange displays a maximum emission value of 650 nm and a maximum excitation value of 460 nm. The maximum excitation and emission value that occur when acridine orange is bound to RNA are the result of electrostatic interactions and the intercalation between the acridine molecule and nucleic acid-base pairs present within RNA and DNA.


Preparation

Acridine dyes are prepared via the condensation of 1,3-diaminobenzene with suitable benzaldehydes. Acridine orange is derived from dimethylamino
benzaldehyde Benzaldehyde (C6H5CHO) is an organic compound consisting of a benzene ring with a formyl substituent. It is among the simplest aromatic aldehydes and one of the most industrially useful. It is a colorless liquid with a characteristic almond-li ...
and ''N'',''N''-dimethyl-1,3-diaminobenzene. It may also be prepared by the
Eschweiler–Clarke reaction The Eschweiler–Clarke reaction (also called the Eschweiler–Clarke methylation) is a chemical reaction whereby a primary (or secondary) amine is methylated using excess formic acid and formaldehyde. Reductive amination reactions such as this on ...
of 3,6-Acridinediamine.


History

Acridine orange is derived from the organic molecule acridine, which was first discovered by Carl Grabe and Heinrich Caro, who isolated acridine by boiling coal in Germany during the late nineteenth century. Acridine has antimicrobial factors useful in drug-resistant bacteria and isolating bacteria in various environments. Acridine orange in the mid-twentieth century was used to examine the microbial content found in soil and direct counts of aquatic bacteria. Additionally, the method of acridine orange direct count (AODC) proved useful in the enumeration of bacteria found within landfills. Direct epifluorescent filter technique (DEFT) using acridine orange is a method known for examining the microbial content within food and water. The use of acridine orange in clinical applications has become widely accepted, mainly focusing on highlighting bacteria in blood cultures. Past and present studies comparing acridine orange staining with blind subcultures for the detection of positive blood cultures showed that the acridine orange is a simple, inexpensive, rapid staining procedure that appears to be more sensitive than the Gram stain for detecting microorganisms in cerebrospinal fluid and other clinical and non-clinical materials.


Applications

Acridine orange has been widely accepted and used in many different areas, such as epifluorescence microscopy, and the assessment of
sperm Sperm (: sperm or sperms) is the male reproductive Cell (biology), cell, or gamete, in anisogamous forms of sexual reproduction (forms in which there is a larger, female reproductive cell and a smaller, male one). Animals produce motile sperm ...
chromatin Chromatin is a complex of DNA and protein found in eukaryote, eukaryotic cells. The primary function is to package long DNA molecules into more compact, denser structures. This prevents the strands from becoming tangled and also plays important r ...
quality. Acridine orange is useful in the rapid screening of ordinarily sterile specimens. When acridine orange is used with flow cytometry, the differential stain is used to measure
DNA denaturation In biochemistry, denaturation is a process in which proteins or nucleic acids lose folded structure present in their native state due to various factors, including application of some external stress or compound, such as a strong acid or base ...
and the cellular content of DNA versus RNA in individual cells, or detect
DNA damage DNA repair is a collection of processes by which a cell identifies and corrects damage to the DNA molecules that encode its genome. A weakened capacity for DNA repair is a risk factor for the development of cancer. DNA is constantly modified ...
in infertile sperm cells. Acridine orange is recommended for the use of fluorescent microscopic detection of microorganisms in smears prepared from clinical and non-clinical materials. Acridine orange staining has to be performed at an acidic pH to obtain the differential staining, which allows bacterial cells to stain orange and tissue components to stain yellow or green. Acridine orange is recorded as being used as a curing agent to cure
selectable marker A selectable marker is a gene introduced into cell (biology), cells, especially bacteria or cells in cell culture, culture, which confers one or more traits suitable for artificial selection. They are a type of reporter gene used in laboratory micr ...
in antibiotic resistant organisms present in a sample. When isolates are subjected to curing in the presence of acridine orange, a substantial number were recorded to have been cured of at least one resistant marker. Acridine orange is also used to stain acidic
vacuole A vacuole () is a membrane-bound organelle which is present in Plant cell, plant and Fungus, fungal Cell (biology), cells and some protist, animal, and bacterial cells. Vacuoles are essentially enclosed compartments which are filled with water ...
s (
lysosome A lysosome () is a membrane-bound organelle that is found in all mammalian cells, with the exception of red blood cells (erythrocytes). There are normally hundreds of lysosomes in the cytosol, where they function as the cell’s degradation cent ...
s,
endosome Endosomes are a collection of intracellular sorting organelles in eukaryotic cells. They are parts of the endocytic membrane transport pathway originating from the trans Golgi network. Molecules or ligands internalized from the plasma membra ...
s, and
autophagosome An autophagosome is a spherical structure with double layer membranes. It is the key structure in macroautophagy, the intracellular degradation system for cytoplasmic contents (e.g., abnormal intracellular proteins, excess or damaged organelles, i ...
s), RNA, and DNA in living cells. This method is a cheap and easy way to study lysosomal
vacuolation A vacuole () is a membrane-bound organelle which is present in plant and fungal cells and some protist, animal, and bacterial cells. Vacuoles are essentially enclosed compartments which are filled with water containing inorganic and organic mo ...
,
autophagy Autophagy (or autophagocytosis; from the Greek language, Greek , , meaning "self-devouring" and , , meaning "hollow") is the natural, conserved degradation of the cell that removes unnecessary or dysfunctional components through a lysosome-depe ...
, and
apoptosis Apoptosis (from ) is a form of programmed cell death that occurs in multicellular organisms and in some eukaryotic, single-celled microorganisms such as yeast. Biochemistry, Biochemical events lead to characteristic cell changes (Morphology (biol ...
. The emission color of acridine orange changes from yellow, to orange, to red as the pH drops in an acidic vacuole of the living cell. Under specific conditions of ionic strength and concentration, acridine orange emits red fluorescence when it binds to RNA by
stacking Stacking may refer to: Arts and media * ''Stacking'' (video game), a 2011 game from Double Fine * '' Stacking'', a 1987 TV movie directed and produced by Martin Rosen * Stacking, a technique in broadcast programming Language * Consonant stac ...
interactions, and green fluorescence when it binds to DNA by intercalation. Depending on acridine orange concentration, nuclei may emit yellowish-green fluorescence in untreated cells, and green fluorescence when
RNA synthesis Transcription is the process of copying a segment of DNA into RNA for the purpose of gene expression. Some segments of DNA are transcribed into RNA molecules that can encode proteins, called messenger RNA (mRNA). Other segments of DNA are transc ...
is inhibited by compounds such as
chloroquine Chloroquine is an antiparasitic medication that treats malaria. It works by increasing the levels of heme in the blood, a substance toxic to the malarial parasite. This kills the parasite and stops the infection from spreading. Certain types ...
. Acridine orange can be used in conjunction with
ethidium bromide Ethidium bromide (or homidium bromide, chloride salt homidium chloride) is an intercalating agent commonly used as a fluorescent tag (nucleic acid stain) in molecular biology laboratories for techniques such as agarose gel electrophoresis. It ...
or
propidium iodide Propidium iodide (or PI) is a Fluorescence#Biochemistry and medicine, fluorescent intercalating agent that can be used to Staining (biology), stain cell (biology), cells and nucleic acids. PI binds to DNA by intercalating between the bases with li ...
to differentiate between viable,
apoptotic Apoptosis (from ) is a form of programmed cell death that occurs in multicellular organisms and in some eukaryotic, single-celled microorganisms such as yeast. Biochemical events lead to characteristic cell changes ( morphology) and death. These ...
, and
necrotic Necrosis () is a form of cell injury which results in the premature death of cells in living tissue by autolysis. The term "necrosis" came about in the mid-19th century and is commonly attributed to German pathologist Rudolf Virchow, who is ...
cells. Additionally, acridine orange may be used on blood samples causing bacterial DNA to fluoresce, aiding in the clinical diagnosis of bacterial infections, such as meningitis.


References

{{reflist Acridine dyes Genetics techniques Vital stains Dimethylamino compounds