Acridine orange is an

organic compound In chemistry, organic compounds are generally any chemical compounds that contain carbon-hydrogen or carbon-carbon bonds. Due to carbon's ability to catenate (form chains with other carbon atoms), millions of organic compounds are known. The ...

that serves as a

nucleic acid Nucleic acids are biopolymers, macromolecules, essential to all known forms of life. They are composed of nucleotides, which are the monomers made of three components: a 5-carbon sugar, a phosphate group and a nitrogenous base. The two main cl ...

-selective

fluorescent Fluorescence is the emission of light by a substance that has absorbed light or other electromagnetic radiation. It is a form of luminescence. In most cases, the emitted light has a longer wavelength, and therefore a lower photon energy, tha ...

dye A dye is a colored substance that chemically bonds to the substrate to which it is being applied. This distinguishes dyes from pigments which do not chemically bind to the material they color. Dye is generally applied in an aqueous solution an ...

with cationic properties useful for cell cycle determination. Acridine orange is cell-permeable, which allows the dye to interact with DNA by intercalation, or

RNA Ribonucleic acid (RNA) is a polymeric molecule essential in various biological roles in coding, decoding, regulation and expression of genes. RNA and deoxyribonucleic acid ( DNA) are nucleic acids. Along with lipids, proteins, and carbohydra ...

via

electrostatic attraction Coulomb's inverse-square law, or simply Coulomb's law, is an experimental law of physics that quantifies the amount of force between two stationary, electrically charged particles. The electric force between charged bodies at rest is convention ...

s. When bound to DNA, acridine orange is very similar spectrally to an organic compound known as fluorescein. Acridine orange and

fluorescein Fluorescein is an organic compound and dye based on the xanthene tricyclic structural motif, formally belonging to triarylmethine dyes family. It is available as a dark orange/red powder slightly soluble in water and alcohol. It is widely used ...

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 found in many animal cells. They are spherical vesicles that contain hydrolytic enzymes that can break down many kinds of biomolecules. A lysosome has a specific composition, of both its membrane prote ...

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 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 phagocytosis is ...

of apoptotic cells. Acridine orange is used in

epifluorescence microscopy A fluorescence microscope is an optical microscope that uses fluorescence instead of, or in addition to, scattering, reflection, and attenuation or absorption, to study the properties of organic or inorganic substances. "Fluorescence microsc ...

and

flow cytometry Flow cytometry (FC) is a technique used to detect and measure 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 flo ...

. The ability to penetrate the cell membranes of acidic organelles and cationic properties of acridine orange allows the dye to differentiate between various types of cells (i.e., bacterial cells and white blood 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 In microbiology and bacteriology, 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. The name comes from the Danish ...

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 the simplest aromatic aldehyde and one of the most industrially useful. It is a colorless liquid with a characteristic almond-like odor. ...

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 is the male reproductive 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 with a tail known as a flagellum, whi ...

chromatin Chromatin is a complex of DNA and protein found in 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 roles in 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 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. In human cells, both normal metabolic activities and environmental factors such as radiation can cause DNA da ...

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 also used to stain acidic

vacuole 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 ...

s (

lysosome A lysosome () is a membrane-bound organelle found in many animal cells. They are spherical vesicles that contain hydrolytic enzymes that can break down many kinds of biomolecules. A lysosome has a specific composition, of both its membrane prot ...

endosome Endosomes are a collection of intracellular sorting organelles in eukaryotic cells. They are parts of endocytic membrane transport pathway originating from the trans Golgi network. Molecules or ligands internalized from the plasma membrane can ...

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 Ancient Greek , , meaning "self-devouring" and , , meaning "hollow") is the natural, conserved degradation of the cell that removes unnecessary or dysfunctional components through a lysosome-dependent re ...

, and

apoptosis Apoptosis (from grc, ἀπόπτωσις, apóptōsis, 'falling off') is a form of programmed cell death that occurs in multicellular organisms. Biochemical events lead to characteristic cell changes (morphology) and death. These changes incl ...

. 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 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. The segments of DNA transcribed into RNA molecules that can encode proteins are said to produce messenger RNA (mRNA). Other segments of DNA are copied into RNA molecules called ...

is inhibited by compounds such as

chloroquine Chloroquine is a medication primarily used to prevent and treat malaria in areas where malaria remains sensitive to its effects. Certain types of malaria, resistant strains, and complicated cases typically require different or additional medi ...

. 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 i ...

propidium iodide Propidium iodide (or PI) is a fluorescent intercalating agent that can be used to stain cells and nucleic acids. PI binds to DNA by intercalating between the bases with little or no sequence preference. When in an aqueous solution, PI has a fluo ...

to differentiate between viable,

apoptotic Apoptosis (from grc, ἀπόπτωσις, apóptōsis, 'falling off') is a form of programmed cell death that occurs in multicellular organisms. Biochemical events lead to characteristic cell changes (morphology) and death. These changes includ ...

, and

necrotic Necrosis () is a form of cell injury which results in the premature death of cells in living tissue by autolysis. Necrosis is caused by factors external to the cell or tissue, such as infection, or trauma which result in the unregulated dige ...

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