Acridine Orange Staining: Principle, Procedure, Results

Acridine orange is a fluorescent dye that intercalates or binds with the nucleic acid (either DNA or RNA) present in organisms and fluoresces to emit various colors that help differentiate cellular organelles. This binding results from the electrostatic interactions of acridine molecules between the nucleic acid-base pairs. Due to its metachromatic properties, acridine orange (AO) is commonly used in fluorescence microscopy and flow cytometry analysis of cellular physiology and cell cycle status, including the fluorescent microscopic examination of microorganisms.

Fluorescent acridine orange stain coryneforms bacteria
Fluorescent acridine orange stain coryneforms bacteria (Image source)


Acridine orange is a cell-permeable, nucleic acid selective fluorescent dye that emits green fluorescence when bound to dsDNA (at 520 ) and red fluorescence when bound to ssDNA or RNA (at 650 nm). Since it is a cationic dye, it also enters acidic compartments such as lysosomes which, in low pH conditions, will emit orange light.

Acridine orange is a carcinogen when absorbed through the skin. Wear gloves when working with this stain.

Staining procedure:

Staining procedures vary according to their use

  1. For staining clinical specimens with acridine orange at low pH (Acridine orange acid stain)
  • Requirements: acridine orange, glacial acetic acid, distilled water
  • Preparation of reagent: 50 mg acridine orange is dissolved in 10 ml of distilled water to prepare a stock solution and stored in the refrigerator.1 ml of acridine orange stock solution and 0.5 ml of glacial acetic acid is added to 50 ml of distilled water to prepare a working solution.
  • Staining procedure:
  1. Prepare a smear in a clean grease-free slide and allow it to air dry.
  2. The slide is then fixed with methanol and dried again.
  3. It is then put in a trough with an acridine orange staining working solution (i.e 0.01 percent).
  4. After 2 minutes of staining, the slides are washed gently with water, dried, and examined in a fluorescent microscope.

Observance: Bacteria stain orange against a green to a yellow background of human cells and debris.

  1. For staining cells for analysis by flow cytometry.
    Requirements: 0.1M Citric Acid (dissolve 1.921g per 100ml distilled water), 0.2M Dibasic Sodium Phosphate  (dissolve 2.839g per 100ml distilled water) ,Triton X-100 (Baker), 0.5M EDTA, Sodium chloride(NaCl), Acridine Orange (Powder) and Sucrose.
  2. Preparation of reagents:
    • Stock Buffer I:20mM Citrate-Phosphate, pH 3.0, 0.1mM EDTA, 0.2M Sucrose, 0.1% Triton X-100
      (To 125ml distilled water add 40µl 0.5M EDTA, 26.48ml 0.1M Citric Acid, 6.85ml 0.2M Dibasic Sodium Phosphate, 13.69g Sucrose, 0.2ml Triton X-100 .QS to 200ml and 0.2µ filter. Store at 4°C)
    • Stock Buffer II:10mM Citrate-Phosphate, pH 3.8, 0.1M NaCl (To 150 ml distilled water, add 9.92ml 0.1M Citric Acid, 5.46ml 0.2M Dibasic Sodium Phosphate, 1.7g NaCl. QS to 200ml and 0.2m filter. Store at 4°C)

Staining Procedure

  1. Make a 2mg/ml solution of acridine orange in distilled water and dilute to 1:100 in Buffer II
  2. Aliquot cells: 105- 106 in 100µl PBS or media.
  3. Add Buffer I (0.5ml) at room temp, agitate to suspend.
  4. Add Buffer II + AO (0.5ml) at room temp, agitate to suspend.
  5. Run on flow cytometer. Excitation 488 nm; dot plot of green fluorescence at 530nm versus red fluorescence >600 nm).


  • Green fluorescence when bound to dsDNA and
  • Red fluorescence when bound to ssDNA or RNA.


Acridine orange stain is used to confirm the presence of bacteria in positive blood cultures when Gram stain results are difficult to interpret or when the presence of bacteria is highly suspected, but none are detected using light microscopy. Other applications of acridine orange stains are listed here;

  • For enumerating the microbial load in a sample since acridine orange binds with the nucleic acid of both living and dead bacteria.
  • Detection of cell wall-deficient bacteria (e.g., mycoplasmas) grown in cultures. Cell wall deficient bacteria are hard to visualize in Gram stain as they cannot retain Gram stain dyes.
  • For differential staining of human cells and prokaryotic cell with a fluorescence microscope. Human cells are stained black to faint green in which bright orange organisms are easily detected.
  • Acridine orange is also used in a method referred to as the quantitative buffy coat (QBC), a rapid screening tool for the detection of malaria.
  • For analyzing mitochondria and lysosomal content by flow cytometry.
  • For visual detection of nucleic acids on agarose and polyacrylamide gels.
  • For identifying engulfed apoptotic cells because they will fluoresce upon engulfment.

 Research showed that acridine orange staining is a sensitive, rapid, and reliable method for detecting bacteria in blood cultures early during incubation and can be substituted for blind subcultures. Acridine orange is better than Gram stain in cases with low amounts of organisms.


  • Acridine orange stain is non-specific. It stains all nucleic acid-containing cells, so it does not discriminate between gram-negative and gram-positive bacteria.

Quality Control

  1. Examine the acridine orange staining solution for color and clarity. The solution should be clear, orange, and without evidence of precipitate.
  2. Each use time, stain a prepared slide of known bacteria, such as Escherichia coli mixed with staphylococci, and examine for the desired results. Record results and refer out-of-control results to the supervisor.
    1. Gram-negative rods and Gram-positive cocci are fluorescent (orange).
    2. The background is nonfluorescent (green-yellow).


Nisha Rijal

I am working as Microbiologist in National Public Health Laboratory (NPHL), government national reference laboratory under the Department of health services (DoHS), Nepal. Key areas of my work lies in Bacteriology, especially in Antimicrobial resistance.

2 thoughts on “Acridine Orange Staining: Principle, Procedure, Results

  1. Does anyone know how long acridine orange stain lasts in C. Elegans? Protocols call for staining an hour before observing, but can the fluorescence last longer? Could they be stained 24 hours before and still fluoresce under the fluorescent microscope?

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