Acridine Orange Staining: Principle, Procedure, Results

Last updated on June 2nd, 2021

Acridine orange is a dye that intercalates or binds with the nucleic acid ( either DNA or RNA) present in organisms and fluoresce to emit various colors that help in the differentiation of cellular organelles. This binding is the result of the electrostatic interactions of acridine molecules between the nucleic acid-base pairs. Acridine orange (AO), due to its metachromatic properties, 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 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 its use

  1. For staining clinical specimen 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 and dried, and then 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.


  • For analyzing mitochondria and lysosomal content by flow cytometry.
  • For visual detection of nucleic acids on agarose and polyacrylamide gels.
  • For enumerating the microbial load in a sample since acridine orange binds with the nucleic acid of both living and dead bacteria.
  • For identifying engulfed apoptotic cells, because it will fluoresce upon engulfment.
  • 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.

 Researches 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.

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 time of use, 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).
About Nisha Rijal 48 Articles
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.