Acridine orange

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Acridine orange
Acridine orange
Ball-and-stick model of the acridine orange molecule
Preferred IUPAC name
Systematic IUPAC name
Other names

Acridine Orange Base
Acridine Orange NO
Basic Orange 14
Rhoduline Orange
Rhoduline Orange N
Rhoduline Orange NO
Solvent Orange 15

Waxoline Orange A
3D model (JSmol)
ECHA InfoCard 100.122.153
EC Number 200-614-0
MeSH Acridine+orange
RTECS number AR7601000
Molar mass 265.360 g·mol−1
Appearance Orange powder
Irritant Xi Dangerous for the Environment (Nature) N
S-phrases (outdated) S26 S28 S37 S45
NFPA 704
Flammability code 0: Will not burn. E.g., waterHealth code 2: Intense or continued but not chronic exposure could cause temporary incapacitation or possible residual injury. E.g., chloroformReactivity code 0: Normally stable, even under fire exposure conditions, and is not reactive with water. E.g., liquid nitrogenSpecial hazards (white): no codeNFPA 704 four-colored diamond
Except where otherwise noted, data are given for materials in their standard state (at 25 °C [77 °F], 100 kPa).
☒N verify (what is ☑Y☒N ?)
Infobox references

Acridine orange is an organic compound. It is used as a nucleic acid-selective fluorescent cationic dye useful for cell cycle determination. Being cell-permeable, it interacts with DNA and RNA by intercalation or electrostatic attractions respectively. When bound to DNA, it is very similar spectrally to fluorescein, with an excitation maximum at 502 nm and an emission maximum at 525 nm (green). When it associates with RNA, the excitation maximum shifts to 460 nm (blue) and the emission maximum shifts to 650 nm (red). Acridine orange will also enter acidic compartments such as lysosomes where it becomes protonated and sequestered. Within these low pH vesicles the dye emits red fluorescence when excited by blue light. Thus, acridine orange can be used to visualize primary lysosomes and phagolysosomes that may include products of phagocytosis of apoptotic cells. The dye is often used in epifluorescence microscopy and flow cytometry.

Optical properties[edit]

At a low pH (3.5), when acridine orange is excited by blue light, it can differentially stain human cells green while staining prokaryotes bright orange for detection with a fluorescence microscope. This differential staining capability allows more rapid scanning of smears at a lower magnification (400×), than by Gram stain (1000×). Bright orange organisms are easily detected against a black to faint green background.

When an acridine orange binds with DNA, it exhibits an excitation maximum at 502 nm (cyan) and an emission maximum at 525 nm (green). When it binds with RNA, the excitation maximum is located at 460 nm (blue) and the emission maximum is located at 650 nm (red). This is all due to the electrostatic interactions occurring when the acridine molecule intercalates between the nucleic acid base pairs.

Acridine orange binding with the nucleic acid occurs in both living and dead bacteria, also other microorganisms. Acridine orange is useful for enumerating the microbes in a sample.


Acridine dyes are prepared via the condensation of 1,3-diaminobenzene with suitable benzaldehydes. Acridine orange is derived from dimethylaminobenzaldehyde and N,N-dimethyl-1,3-diaminobenzene.[1]


In 1942, Hilbrich and Strugger were first described using acridine orange to detect the fluorochromatic staining of microorganisms. Since then the use of acridine orange has been performed frequently in the examination of soil and water for microbial content. Direct counts of aquatic bacteria by using epifluorescent methods were evaluated by Jones and Simon in 1975. They also determined that the best estimation of the bacterial population in lake, river, and seawater samples can be achieved using acridine orange.

Acridine orange direct count (AODC) methodology has been used in the enumeration of landfill bacteria. A study shows that the use of AODC in marine bacterial populations can be compared favorably to fluorescent oligonucleotide direct counting (FODC) procedures. Direct epifluorescent filter technique (DEFT) using acridine orange is specified in methods for the microbial examination of food and water.

The use of acridine orange in clinical applications has become widely accepted; mainly focusing on the use in highlighting bacteria in blood cultures. In 1980, a study involved the comparing acridine orange staining with blind subcultures for the detection of positive blood cultures was done by McCarthy and Senne. The results showed that the acridine orange is a simple, inexpensive, rapid staining procedure that appeared to be more sensitive than the Gram stain for detecting microorganism in clinical materials. Later on, Lauer, Reller and Mirret performed a similar study, compared acridine orange with the Gram stain for detecting the microorganisms in cerebrospinal fluid and other clinical materials. As a result, they reached the same conclusion that was reported by McCarthy and Senne.


Acridine orange has been widely accepted and used in many different areas, such as epifluorescence microscopy, the assessment of sperm chromatin quality. Acridine orange stain is particularly useful in the rapid screening of normally sterile specimens, and its recommended for the use of fluorescent microscopic detection of microorganisms in direct smears prepared from clinical and non-clinical materials. The staining has to be performed at an acid pH in order to obtain this differential staining effect with bacteria showing orange stain and tissue components yellow to green.[2]

Acridine orange is a versatile fluorescence dye used to stain acidic vacuoles (lysosomes, endosomes, and autophagosomes), RNA, and DNA in living cells. This method is a cheap and easy way to study lysosomal vacuolation, autophagy, and apoptosis. Acridine orange emission changes from yellow, to orange, to red fluorescence as the pH drops in an acidic vacuole of the living cell. Under specific conditions of ionic strength and at specific 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 is inhibited by compounds such as chloroquine.[3]

Acridine orange can be used in conjunction with ethidium bromide or propidium iodide to differentiate between viable, apoptotic and necrotic cells. Additionally, acridine orange may be used on blood samples to cause bacterial DNA to fluoresce, aiding in clinical diagnosis of bacterial infection once serum and debris have been filtered.[4]

Acridine orange has been widely used in flow cytometry to differentially stain and measure content of cellular DNA versus RNA,[5] to measure DNA denaturation in individual cells, in situ,[6] and to detect DNA damage in infertile sperm cells.[7]


  1. ^ Gessner, Thomas; Mayer, Udo, "Triarylmethane and Diarylmethane Dyes", Ullmann's Encyclopedia of Industrial Chemistry, Weinheim: Wiley-VCH, doi:10.1002/14356007.a27_179
  2. ^ "Review" (PDF).
  3. ^ "Chloroquine inhibits cell growth and induces cell death in A549 lung cancer cells". Bioorganic & Medicinal Chemistry. 14 (9): 3218–3222. 2006-05-01.
  4. ^ Mirrett, Stanley (1982). "Acridine Orange Stain". Infection Control. 3 (3): 250–252. JSTOR 30142217.
  5. ^ Darzynkiewicz, Z.; Juan, G.; Srour, E.F. (2004). "Differential staining of DNA and RNA". Curr. Protoc. Cytom. 7: 7.3. doi:10.1002/0471142956.cy0703s30. PMID 18770805.
  6. ^ Darzynkiewicz, Z.; Juan, G. (2001). "Analysis of DNA denaturation". Curr. Protoc. Cytom. 7: 7.8. doi:10.1002/0471142956.cy0708s03. PMID 18770735.
  7. ^ Evenson, D.P.; Darzynkiewicz, Z.; Melamed, M.R. (1980-12-05). "Relation of mammalian sperm chromatin heterogeneity to fertility". Science. 210 (4474): 1131–1133. PMID 7444440.