| Acridine orange |
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Preferred IUPAC name
N,N,N',N'-Tetramethylacridine-3,6-diamine
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Systematic name
3-N,3-N,6-N,6-N-Tetramethylacridine-3,6-diamine
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Other names
3,6-Acridinediamine
Acridine Orange Base
Acridine Orange NO
Basic Orange 14
Euchrysine
Rhoduline Orange
Rhoduline Orange N
Rhoduline Orange NO
Solvent Orange 15
Waxoline Orange A
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| Identifiers |
| CAS number |
494-38-2 N |
| PubChem |
62344 Y |
| ChemSpider |
56136 Y |
| EC number |
200-614-0 |
| KEGG |
C19315 N |
| MeSH |
Acridine+orange |
| ChEBI |
CHEBI:234241 N |
| ChEMBL |
CHEMBL81880 Y |
| RTECS number |
AR7601000 |
| Jmol-3D images |
Image 1 |
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n1c3c(cc2c1cc(N(C)C)cc2)ccc(c3)N(C)C
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-
InChI=1S/C17H19N3/c1-19(2)14-7-5-12-9-13-6-8-15(20(3)4)11-17(13)18-16(12)10-14/h5-11H,1-4H3 Y
Key: DPKHZNPWBDQZCN-UHFFFAOYSA-N Y
InChI=1/C17H19N3/c1-19(2)14-7-5-12-9-13-6-8-15(20(3)4)11-17(13)18-16(12)10-14/h5-11H,1-4H3
Key: DPKHZNPWBDQZCN-UHFFFAOYAJ
|
| Properties |
| Molecular formula |
C17H19N3 |
| Molar mass |
265.35 g mol−1 |
| Appearance |
Orange powder |
| Hazards |
| EU classification |
Xi N |
| S-phrases |
S26 S28 S37 S45 |
| NFPA 704 |
|
N (verify) (what is: Y/N?)
Except where noted otherwise, data are given for materials in their standard state (at 25 °C, 100 kPa) |
| Infobox references |
Acridine orange is a nucleic acid selective fluorescent cationic dye useful for cell cycle determination. It is cell-permeable, and 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 and become protonated and sequestered. In these low pH conditions, the dye will emit orange light when excited by blue light. Thus, acridine orange can be used to identify engulfed apoptotic cells, because it will fluoresce upon engulfment. The dye is often used in epifluorescence microscopy.
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Contents
- 1 Introduction
- 2 Properties
- 3 History
- 4 Reactions
- 5 Uses
- 6 References
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Introduction[edit]
Acridine orange can be used to differentiate between deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) very easily. When acridine orange bonds with DNA and forms a complex, the emitted radiation is green. When it bonds with RNA and forms a complex, the emitted light is orange.
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 microorganisms bright orange for detection with a fluorescence microscope. This differential staining capability allows more rapid scanning of smears at a lower magnification (400x), than by Gram stain (1000x). Bright orange organisms are easily detected against a black to faint green background6.
The ring structure of the acridine orange can absorb the incoming radiation. It is referring to the hydrophobic nature of the compound; acridine orange tends to diffuse spontaneously into the membrane surrounding the microorganisms.
When an acridine orange bonds with DNA, it has an excitation maximum at 502 nm (cyan) and an emission maximum at 525 nm (green); when it bonds with RNA, the excitation maximum shifts to 460 nm (blue) and the emission maximum shifts to 650 nm (red). This is all due to the intercalation or electrostatic attractions4.
Acridine orange binding with the nucleic acid occurs in both living and dead bacteria, also other microorganisms. This does not mean that the acridine orange can be used to distinguishing living from dead microbes; however, it has been proved that the acridine orange is a very useful tool to enumerating the total number of microbes in a sample3.
History[edit]
In 1942, Hilbrich and Strugger were first described using acridine orange to detect the fluorchromatic 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 orange1.
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 epifluoresent filter technique (DEFT) using acridine orange is specified in methods for the microbial examination of food and water3.
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 materials1. 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 Senne1.
Reactions[edit]
The crystal structure of the biological stain, acridine orange, when crystallized from ethanol, is shown to be a zinc chloride double salt of acridine orange, containing, in addition, acetic acid of crystallization2.
Absorbance
Figure 2: Molar extinction coefficient of acridine orange dissolved in basic ethanol
Fluorescence
Figure 3: fluorescence emission spectrum of Acridine orange dissolved in basic ethanol
Infrared spectrum of a pronated fluorescence dye
The infrared spectrum (IR) of protonated acridine orange (AOH+) has been measured in the fingerprint range (600–1740 cm-1) by means of IR multiple photon dissociation (IRMPD) spectroscopy. The IRMPD spectrum of mass-selected AOH+ ions was recorded in a Fourier Transform Ion Cyclotron Resonance Mass Spectrometer equipped with an electrospray ionization source using an IR free electron laser. The fragmentation process of AOH+ upon IR activation in the ground electronic state is analyzed in some detail, revealing that elimination of CH4 is thermodynamically favored over loss of CH3NCH2. The effects of protonation on the geometric and electronic structure are revealed by comparison with neutral acridine orange5.
Uses[edit]
Acridine orange has been widely accepted and used in many different areas, such as epifluorescence microscopy, the assessment of sperm chromatin quality, and preparation for the coal tar and creosote oil.
Acridine orange stain is particularly useful in the rapid screening of normally sterile specimens, and it’s recommended for the use of fluorescent microscopic detection of microorganisms in direct smears prepared from clinical and non-clinical materials6.
Acridine orange is a versatile fluorescence dye to stain acidic vacuoles (lysosomes, endosomes, and autophagosomes), RNA, DNA in living cells.[1] This method is a cheap and easy way to study lysosomal vacuolation, autophagy, and apoptosis.[2] Acridine orange emits from yellow, orange, to red fluorescence when the pH get lower in the acidic vacuole of the living cell. Acridine orange emits yellow fluorescence when it binds RNA and green fluorescence when it binds DNA. Nucleus emit yellowish-green fluorescence in normal condition, and deep green fluorescence when the RNA synthesis is inhibited by inhibitors such as chloroquine.[3]
Acridine orange is prepared from coal tar and creosote oil.
Acridine orange can be used in conjunction with ethidium bromide to differentiate between viable, apoptotic and necrotic cells. Additionally, Acridine orange may be used on blood samples to fluoresce bacterial DNA, aiding in clinical diagnosis of bacterial infection once serum and debris have been filtered.
Acridine orange can be used in the assessment of sperm chromatin quality.
References[edit]
[4]
- ^ Chloroquine inhibits cell growth and induces cell death in A549 lung cancer cells. Bioorgnic & Medicinal Chemistry. 2006 May 1; 14(9):3218-3222.
- ^ Chloroquine inhibits cell growth and induces cell death in A549 lung cancer cells. Bioorgnic & Medicinal Chemistry. 2006 May 1; 14(9):3218-3222.
- ^ Chloroquine inhibits cell growth and induces cell death in A549 lung cancer cells. Bioorgnic & Medicinal Chemistry. 2006 May 1; 14(9):3218-3222.
- ^ Acridine Orange Stain. Infection Control.
