Acridine Orange stain is a fluorescent dye that differentiates between DNA and RNA. This versatile stain can bind to both kinds of nucleic acids, making it suitable for a wide range of scientific and medical applications. This binding is the consequence of acridine molecules’ electrostatic interactions with nucleic acid-base pairs. Because of its metachromatic qualities, acridine orange (AO) is widely employed in fluorescence microscopy and flow cytometry analyses of cellular physiology and cell cycle state, as well as fluorescent microscopic study of microorganisms.
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Acridine Orange Stain Definition
A chemical substance called acridine orange has cationic qualities that make it a good fluorescent dye for determining the cell cycle and is selective for nucleic acids.
Principle of Acridine orange stain
Acridine orange is a cell-permeable, nucleic acid selective fluorescent dye that emits green fluorescence when bound to DNA and red fluorescence when bound to DNA or RNA. Since it is a cationic dye, it also enters acidic compartments such as lysosomes which, in low pH conditions, will emit orange light. Also, when absorbed via the skin, acridine orange can cause cancer. When working with this stain, you should wear gloves
The procedure required for Acridine orange stain
Required
- Acridine orange acid stain (stored at 15-30°C in the dark).
- Alcohol saline solution (absolute methanol).
- Microscopic glass slides
- Heat block at 45-60°C (optional).
- Fluorescent Microscope
- Immersion Oil
Procedure
1. Prepare a smear of the specimen on a clean, grease-free glass slide
3. Allow to dry naturally, or use a heating block to speed up the process.
2. Spread the samples thinly using a sterile stick.
4. Alcohol fixing: Fix the smear with methanol by soaking the slide and draining the excess before allowing to air dry.
5. Flood the slide with acridine orange stain for 2 minutes.
6. Drain any excess stains and carefully rinse with tap water.
7. Allow to air-dry.
8. To speed up the drying process, gently wipe the slide with a clean piece of filter paper or paper towels.
9. Examine the smear with the fluorescent microscope. Look for the distinctive shape of bacteria or fungus. No coverslip is required.
Result interpretation after the procedure
- Bacteria and fungus show in brilliant orange.
- Trichomonas vaginalis trichomonads resemble orange or red, with a yellow-green nucleus.
- The background seems black to yellow-green.
- Human epithelium, inflammatory, and tissue debris look pale green to yellow.
- Activated leukocytes will look yellow, orange, or red depending on their level of activation and the amount of RNA generated, whereas erythrocytes will either not fluoresce or glow pale green.
- Ureaplasma and Mycoplasma can be seen using an acridine stain.
Applications of Acridine Orange Stain
- Living/Dead Staining: Acridine Orange can be used in conjunction with other dyes (such as Ethidium Bromide) to distinguish between living and dead cells depending on membrane integrity.
- Chromatin Condensation: It helps detect apoptotic cells by showing chromatin condensation and nuclear fragmentation.
- Bacterial Staining: It is used to stain bacterial cells in samples to help identify and differentiate bacterial species.
- Tumor Analysis: Acridine Orange can reveal changes in nucleic acid composition between normal and malignant cells.
Acridine Orange Stain’s limitations
1. Photobleaching: This shortens the time available for monitoring samples and can hinder long-term imaging investigations.
2. Non-Specific Binding: Non-specific binding might cause background fluorescence, making it harder to evaluate data.
3. Toxicity: This restricts its application in live-cell imaging and long-term research.
4. Differential Staining Sensitivity: This might make it difficult to quantify and compare samples or sections within the same sample.
5. pH Sensitivity: Changes in pH might affect the staining pattern and fluorescence intensity, potentially resulting in conflicting findings.
6. Overlapping Fluorescence Emissions: This overlap can make it difficult to discriminate between DNA and RNA, particularly in samples with high nucleic acid concentrations.
7. Need for Fluorescence Microscopy: This requires access to specialist equipment, which may not be accessible in all facilities.
8. Quantification Challenges: Acridine Orange’s utility in accurate quantitative experiments is limited without further validation and controls.
9. DNA Damage Risk: This can be troublesome in investigations where the integrity of genetic material is critical.
What is acridine orange?
Acridine orange is an organic molecule that functions as a cationic fluorescent dye that selectively detects nucleic acids.
What is acridine orange staining?
Acridine orange staining is a scientific method for seeing nucleic acids (DNA or RNA) in cells and microorganisms.
What is acridine orange stain used for?
Acridine orange stain is a fluorescent dye commonly used in cell biology and microbiology.
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