Acridine Orange Stain- Principle, Procedure and Result Interpretation

Objective

Differential staining of nucleic acids including DNA and RNA.

Principle

Acridine orange, a vital stain, will intercalate with nucleic acid, changing the dye’s optical characteristics so that it will fluoresce bright orange under ultraviolet light. All nucleic acid–containing cells will fluoresce orange. Acridine orange is a metachromatic stain and under appropriate conditions, RNA will stain orange and DNA will stain green.

Methods

1. Properly prepare and fix the smear prior to staining.
2. Flood slide with acridine orange stain (available from various commercial suppliers). Allow stain to remain on surface of slide for 2 minutes without drying.
3. Rinse with tap water and allow moisture to drain from slide and air-dry.
4. Examine the slide using fluorescent microscopy.

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Result Interpretation

Bacteria and yeasts will fluoresce bright orange against a green-fluorescing or dark background. The nuclei of host cells may also fluoresce.

Note: RNA is more abundant during cellular growth and may mask the green fluorescence of the DNA within the cell.

Limitations

1. Cellular debris within a sample such as white blood cells, epithelial cells, and dead bacteria may distort the microscopic image.
2. Acridine orange is a very sensitive stain, and caution should be used when interpreting results.