Description

Acridine Orange (chemical formula: C₁₇H₁₉N₃) is a fluorescent cationic dye widely used in molecular and cellular biology for staining nucleic acids. It is a cell-permeable dye that intercalates into DNA and RNA, exhibiting different fluorescence colors depending on the nucleic acid type and environment. DNA-bound Acridine Orange fluoresces green, while RNA-bound dye fluoresces red/orange under blue or ultraviolet excitation.

Acridine Orange is a planar aromatic molecule that can cross cell membranes, making it useful for live-cell imaging and viability assays.

Common Uses in a Biology Lab

Nucleic Acid Staining:

• Used to stain both DNA and RNA in cells and tissues for fluorescence microscopy and flow cytometry.
• Enables differentiation between DNA and RNA based on distinct emission spectra.

Cell Viability and Apoptosis Assays:

• Can distinguish live, apoptotic, and necrotic cells by differential uptake and fluorescence patterns.
• Often combined with other dyes (e.g., ethidium bromide) for dual staining.

Micronucleus and Chromosome Studies:

• Used in genotoxicity assays to detect micronuclei formation.
• Enables visualization of chromosomes in metaphase spreads.

Microbial Identification:

• Used for staining bacteria and protozoa in environmental samples or cultures.
• Allows rapid visualization of microorganisms under fluorescence microscopy.

Safety Hazards

Moderate Toxicity and Mutagenicity:

• Acridine Orange is considered a potential mutagen and carcinogen due to its intercalating properties.
• Handle with care to avoid ingestion, inhalation, or skin contact.

Irritation:

• Can cause irritation to the skin, eyes, and respiratory tract.

Proper PPE and Handling:

• Use gloves, lab coat, and eye protection.
• Work in a fume hood or well-ventilated area.

Handling

Personal Protective Equipment (PPE):
Wear gloves, lab coat, and goggles. Use a fume hood or biological safety cabinet for solution preparation.

Ventilation:
Prepare and handle solutions in a well-ventilated area.

Storage:
Store in a cool, dry, dark place, protected from light to prevent photodegradation.

Disposal:
Dispose of waste solutions as hazardous chemical waste. Avoid release into the environment.

Additional Notes

• Fluorescence Excitation/Emission:
  • Excitation: ~502 nm (blue light)
  • Emission: ~525 nm (green) when bound to DNA, ~650 nm (red) when bound to RNA.
• Concentration for Use:
  Typically used at micromolar concentrations in staining protocols.
• Photostability:
  Acridine Orange is sensitive to light and can photobleach; samples should be protected from prolonged illumination.
• Applications in Research:
  Extensively used in cell cycle analysis, DNA quantification, apoptosis studies, and microbial detection.

Conclusion

Acridine Orange is a versatile fluorescent dye critical for nucleic acid visualization and live-cell imaging. Its unique ability to differentiate DNA and RNA via fluorescence makes it invaluable in cytology, microbiology, and molecular biology research. However, its mutagenic potential requires careful handling, appropriate PPE, and disposal measures.