Acridine Orange Stain- Principle, Procedure and Result Interpretation
The objective of acridine orange stain is to differentiate between nucleic acids, such as DNA and RNA, in biological samples. Nucleic acids are essential molecules that store and transmit genetic information in living organisms. They are composed of nucleotides, which are units of a nitrogenous base, a sugar, and a phosphate group. DNA (deoxyribonucleic acid) and RNA (ribonucleic acid) are the two main types of nucleic acids found in cells. DNA is usually double-stranded and forms a helical structure, while RNA is usually single-stranded and can fold into various shapes. DNA contains the bases adenine (A), thymine (T), cytosine (C), and guanine (G), while RNA contains the bases adenine (A), uracil (U), cytosine (C), and guanine (G).
Acridine orange is a synthetic dye that belongs to the group of acridines, which are organic compounds with a flat structure and three fused rings. Acridine orange can bind to both DNA and RNA molecules by inserting itself between the base pairs, a process known as intercalation. This alters the optical properties of the dye and makes it emit fluorescence when exposed to ultraviolet light.
The fluorescence color of acridine orange depends on the type and amount of nucleic acid present in the cell. When acridine orange binds to double-stranded DNA, it emits green fluorescence. When it binds to single-stranded RNA, it emits orange or red fluorescence. Therefore, acridine orange can be used as a differential stain to distinguish between DNA and RNA in cells.
Acridine orange is also a metachromatic stain, which means that it can change its color depending on the pH and concentration of the dye solution. At low pH and high concentration, acridine orange stains all nucleic acids orange or red. At high pH and low concentration, acridine orange stains DNA green and RNA orange or red.
Acridine orange stain is useful for detecting microorganisms such as bacteria and yeasts in clinical specimens, as well as for studying the nucleic acid content and distribution in cells. Acridine orange stain can also be used to detect apoptotic cells, which are cells that undergo programmed cell death and have fragmented DNA. Apoptotic cells show bright orange fluorescence due to the accumulation of RNA in the cytoplasm.
Acridine orange stain is a simple, rapid and sensitive technique that requires only a fluorescent microscope and a suitable filter set. However, some limitations of acridine orange stain are that it is not very specific for nucleic acids and can also bind to proteins and lipids, causing background fluorescence. Moreover, acridine orange stain is not very stable and can fade over time or under light exposure. Therefore, slides stained with acridine orange should be examined as soon as possible after staining and stored in the dark.
To perform the acridine orange stain, you will need the following materials:
- A bacterial or yeast smear on a glass slide
- Acridine orange stain solution
- Tap water
- A fluorescent microscope
Follow these steps to stain and observe the nucleic acids:
- Prepare and fix the smear according to the standard protocol. Make sure the smear is thin and evenly spread on the slide.
- Flood the slide with acridine orange stain solution and let it sit for 2 minutes. Do not let the stain dry on the slide.
- Rinse the slide gently with tap water and drain off the excess water. Air-dry the slide completely before examining it under the microscope.
- Use a fluorescent microscope with an appropriate filter to view the stained cells. Adjust the focus and brightness to obtain a clear image.
You have now completed the acridine orange stain procedure. You can proceed to interpret the results in the next section. 😊
The acridine orange stain allows the visualization of nucleic acids in cells under fluorescent microscopy. The stain intercalates with the nucleic acid molecules and changes its fluorescence depending on the type of nucleic acid. RNA stains orange and DNA stains green. This can help differentiate between different types of cells and organelles based on their nucleic acid content.
The acridine orange stain is especially useful for detecting microorganisms such as bacteria and yeasts, which have a high nucleic acid to cytoplasm ratio and fluoresce bright orange against a dark or green-fluorescing background. The stain can also reveal the presence of host cells, such as epithelial cells or leukocytes, which may have orange-fluorescing nuclei and green-fluorescing cytoplasm.
The acridine orange stain can also be used to identify the metabolic state of cells, as RNA is more abundant during cellular growth and may mask the green fluorescence of the DNA within the cell. Therefore, cells with a high RNA content may indicate active protein synthesis and cell division, while cells with a low RNA content may indicate quiescence or senescence.
The acridine orange stain is a simple and rapid technique that can provide valuable information about the nucleic acid composition and activity of cells. However, it also has some limitations and sources of error that should be considered. For example, the stain may fade over time or be affected by pH, temperature, or light exposure. The stain may also bind to other molecules besides nucleic acids, such as proteins or lipids, and cause false-positive or false-negative results. Therefore, it is important to use proper controls and validation methods when using the acridine orange stain for nucleic acid detection.
Acridine orange stain is useful for detecting microorganisms in clinical specimens, especially when the number of organisms is low or when they are difficult to culture. It can also be used to differentiate between gram-positive and gram-negative bacteria, as well as to identify acid-fast bacilli and fungi. Acridine orange stain has some limitations, such as the need for a fluorescent microscope, the interference of background fluorescence from some materials, and the instability of the stain over time. Additionally, acridine orange stain cannot distinguish between viable and non-viable cells, nor between different types of nucleic acids within the same cell. Therefore, it should be used with caution and in conjunction with other methods for accurate identification and characterization of microorganisms.
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