Acridine orange is an organic compound that serves as a nucleic acid-selective fluorescent dye 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 via electrostatic attractions. When bound to DNA, acridine orange is very similar spectrally to an organic compound known as fluorescein. Acridine orange and fluorescein have a maximum excitation at 502nm 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 and phagolysosomes that are membrane-bound organelles essential for acid hydrolysis or for producing products of phagocytosis of apoptotic cells. Acridine orange is used in epifluorescence microscopy and flow cytometry. 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 stains 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 dimethylaminobenzaldehyde and N,N-dimethyl-1,3-diaminobenzene. It may also be prepared by the Eschweiler–Clarke reaction 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.