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Microbiology

Acridine Orange

Acridine orange is a valuable fluorescent stain used in clinical microbiology. It’s particularly useful for rapidly detecting bacteria and fungi in clinical specimens. Unlike traditional stains that rely on binding to specific cell wall components, acridine orange stains nucleic acids (DNA and RNA), making it a broad-spectrum stain

Principle of Acridine Orange Staining

  • Nucleic Acid Binding: Acridine orange is a fluorescent dye that binds to nucleic acids (DNA and RNA) in cells. When bound to nucleic acids, acridine orange emits green fluorescence when excited by blue light
  • Differential Staining: While acridine orange binds to both DNA and RNA, the fluorescence intensity and color can vary depending on the amount and type of nucleic acid present. Generally, DNA-rich structures (like bacterial chromosomes) fluoresce brighter than RNA-rich structures (like ribosomes)
  • Mechanism
    1. Intercalation: Acridine orange intercalates (inserts itself) between the base pairs of DNA and RNA molecules
    2. Fluorescence: When excited by blue light (typically in the range of 450-490 nm), the acridine orange emits green fluorescence. The intensity of the fluorescence is proportional to the amount of nucleic acid present

Procedure

  1. Smear Preparation: Prepare a thin smear of the specimen (e.g., blood, CSF, urine, wound exudate) on a clean microscope slide. Allow the smear to air dry completely
  2. Fixation (Optional): Fixation is not always necessary for acridine orange staining, but it can improve cell adherence and reduce background fluorescence. If desired, fix the smear by immersing it in absolute methanol for 1-2 minutes, or by heat-fixing
  3. Staining: Flood the smear with acridine orange stain (typically a 0.01% solution in phosphate-buffered saline) and incubate for 2-5 minutes
  4. Rinsing: Rinse the stained smear gently with phosphate-buffered saline to remove excess stain. Avoid prolonged rinsing, as this can remove too much stain and reduce the fluorescence intensity
  5. Mounting (Optional): Mount the smear with a coverslip using a non-fluorescent mounting medium. This can help preserve the fluorescence and improve image quality
  6. Microscopy: Examine the slide under a fluorescence microscope using a blue light excitation filter (typically in the range of 450-490 nm) and a barrier filter to block the excitation light and allow the green fluorescence to be observed. Use appropriate magnification (typically 40x or 100x oil immersion) to identify cells and organisms

Interpretation

  • Bacteria: Bacteria appear as bright green fluorescent cocci or bacilli against a dark background. The fluorescence makes it easier to detect even small numbers of bacteria
  • Fungi: Fungi appear as bright green fluorescent yeast cells or hyphae against a dark background. The fluorescence makes it easier to visualize fungal structures, even in specimens with a lot of debris
  • Host Cells: Host cells (e.g., white blood cells, epithelial cells) also stain with acridine orange, but they typically exhibit weaker fluorescence than bacteria or fungi. The nuclei of host cells may appear brighter than the cytoplasm
  • Artifacts: Be aware of potential artifacts, such as stain precipitates and fluorescent debris, that can resemble cells or organisms. Use careful focusing and higher magnification to confirm the presence of true biological structures
  • Reporting: Report the presence or absence of bacteria or fungi, and describe their morphology. Estimate the quantity of organisms observed (e.g., rare, few, moderate, many). Note the presence and appearance of host cells
    • Example: “Acridine orange stain positive for bacteria, gram-positive cocci present, moderate quantity”

Common Problems and Troubleshooting

  • Weak Fluorescence: Weak fluorescence can result from old or contaminated reagents, improper pH of the staining solution, insufficient staining time, or excessive rinsing. Ensure that all reagents are fresh, the pH of the staining solution is correct, and the staining procedure is followed carefully
  • High Background Fluorescence: High background fluorescence can make it difficult to distinguish cells and organisms from the background. This can be caused by autofluorescence of the specimen, contamination of the reagents, or improper cleaning of the microscope optics. Use fresh reagents, clean the microscope optics regularly, and consider using a quenching agent to reduce autofluorescence
  • Fading Fluorescence: The fluorescence of acridine orange can fade over time, especially with prolonged exposure to UV light. Examine the slides promptly after staining, and use a non-fluorescent mounting medium to help preserve the fluorescence

Advantages

  • Rapid and sensitive method for detecting bacteria and fungi
  • Broad-spectrum stain that can be used to detect a wide range of organisms
  • Relatively simple and inexpensive to perform
  • Can be used to stain a variety of clinical specimens

Disadvantages

  • Does not differentiate between different types of bacteria or fungi (only detects their presence)
  • Requires a fluorescence microscope, which can be expensive
  • Can be difficult to distinguish between true organisms and artifacts

Key Terms

  • Acridine Orange: A fluorescent dye that binds to nucleic acids (DNA and RNA)
  • Fluorescence Microscope: A microscope that uses UV light to excite fluorescent dyes, causing them to emit visible light
  • Intercalation: The insertion of a molecule between the base pairs of DNA or RNA
  • Autofluorescence: The natural emission of light by some substances when exposed to UV light, which can interfere with the interpretation of fluorescent stains
  • Artifact: A structure or substance that is not part of the original specimen but may resemble a cell or organism under the microscope
  • Phosphate-Buffered Saline (PBS): A balanced salt solution used to dilute acridine orange stain and rinse specimens
  • Excitation Filter: A filter in a fluorescence microscope that allows only light of a specific wavelength to pass through, used to excite the fluorescent dye
  • Barrier Filter: A filter in a fluorescence microscope that blocks the excitation light and allows only the emitted fluorescent light to pass through, used to observe the fluorescence signal