User:Thomasmk1/Acridine orange

Unedited Version:

= Acridine orange = 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 acridine orange 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. The sharp contrast allows for the differentiation of cells and provides a mechanism for determining the number of cells present in a given sample.

When an acridine orange molecule 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 is capable of binding to the nucleic acid of multiple organisms, including live or dead bacterial cells.

Preparation[ edit]
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. It may also be prepared by the Eschweiler–Clarke reaction of 3,6-Acridinediamine

History[ edit]
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.

Uses[ edit]
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.

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.

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.

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

Edited Version:

Edited version contains only information I added to the particular section. Changes based on the peer review have been made.

= Acridine orange = The cell-permeable characteristic of both acridine orange and fluorescein, allows the stain to differentiate between various types of cells, especially bacterial cells and low pH. The shift in maximum excitation and emissions provides a foundation to predict the wavelength at which cells will stain.

Optical properties
Bright orange organisms are easily detected against a black to faint green background. The sharp contrast allows for the differentiation of cells and provides a mechanism for determining the number of cells present in a given sample.

.Acridine orange is capable of binding to the nucleic acid of multiple organisms, including live or dead bacterial cells. The sharp contrast serves for easy differentiation and detection of the type of cells present in a sample. The fluorescent stain can only be used with a fluorescent microscope and regarding bacterial cells, acridine orange cannot separate Gram-negative and Gram-positive cells.

History
The original history section did not contain any references.

A study involving McCarthy and Senne 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 appeared to be more sensitive than the Gram stain for detecting microorganisms.

Uses
Acridine orange stain is useful in the rapid screening of ordinarily sterile specimens. It is recommended for the use of fluorescent microscopic detection of microorganisms in smears prepared from clinical and non-clinical materials. 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.Acridine orange differentiates bacterial cells from white blood cells by staining bacterial cells green and white blood cells red (citation).