Acridine Orange Staining: Introduction, Principle, Procedure, Result Interp

Acridine Orange Staining: Introduction, Principle, Procedure, Result Interpretation and Uses

Acridine Orange Staining: Acridine orange is a fluorochrome stain and uses the rapid identification of Trichomonas vaginalis, yeast cells, and clue cells in vaginal smears. It can also use to detect intracellular gonococci, meningococci, and other bacteria particularly in blood cultures. It intercalates with the nucleic acid i.e. either DNA or RNA present in organisms and fluoresce to emit various colors that help in differentiation. This binding is the result the electrostatic interactions of acridine molecule between the nucleic acid base pairs. It shows metachromatic properties, and thus is commonly used in fluorescence microscopy and flow cytometry analysis of cellular physiology and cell cycle status, even including the fluorescent microscopic examination of microorganisms.

Introduction of Acridine Orange Staining

Acridine orange is a fluorochrome stain. Acridine orange staining uses the rapid identification of Trichomonas vaginalis, yeast cells, and clue cells in vaginal smears. It can also use to detect intracellular gonococci, meningococci, and other bacteria, particularly in blood cultures. It intercalates with the nucleic acid i.e. either DNA or RNA present in organisms and fluoresces to emit various colors that help in differentiation. This binding is the result of the electrostatic interactions of acridine molecules between the nucleic acid-base pairs. It shows metachromatic properties, and thus is commonly used in fluorescence microscopy and flow cytometry analysis of cellular physiology and cell cycle status, even including the fluorescent microscopic examination of microorganisms.

Principle of Acridine orange Staining

Acridine orange is a fluorochrome that causes DNA to fluoresce green and
RNA to fluoresce orange-red.

Requirement for Acridine orange Staining

Acridine orange acid stain
Alcohol saline solution
physiological saline
Test specimen
Glass Slide
Inoculating loop or sterile bamboo stick
Fluorescence microscope having a BG 12 exciter filter and No. 44 and No. 53 barrier filters

Procedure of Acridine orange staining

Cover the unfixed dried smear with the acridine orange acid stain for 5–10 seconds and smear fixes due to being fixative is contained in the stain.
Now, wash off the stain, and decolorize the smear with alcohol saline solution for 5–10 seconds.
Rinse the smear with physiological saline, and place the slide in a draining rack.
Add a drop of saline or distilled water to the smear, and cover with a cover glass.

Observation of Acridine orange Stained Smears

Examine first with the 10X objective to see the distribution of fluorescing material, and then with the 40X objective to identify Trichomonas vaginalis and to detect yeast cells, bacteria, and parasites( chromatoid bars of Entamoeba histolytica/ dispar).

Results interpretation of Acridine orange

Trichomonas vaginalis : Orange-red with the yellow-green nucleus

Yeast cells: Orange

Bacteria: Orange

Pus cells ( WBCs): Yellow-green
Epithelial cells: Yellow-green

Keynotes on Acridine Orange Staining

The appearance of chromatoid bars of Entamoeba histolytica/ dispar in an acridine-stained smear is shown in this video clip as well as the above image.
In bacterial vaginosis, the orange staining bacteria adhering to the green epithelial cells (clue cells) can be clearly seen.
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 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.
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.
RNA is more abundant during cellular growth which may mask the green fluorescence of the DNA within the cell.

Acridine orange acid stain preparation

To make 100 ml
Acridine orange: 26 mg
Acetic acid, glacial (concentrated): 2 ml

Distilled water: 98 ml

Distilled water (D/W), acridine orange, and transfer to a
brown bottle of 100 ml capacity.
Fill a cylinder to the 98 ml mark with distilled water.
Add 2 ml of glacial acetic acid, i.e. to the 100 ml mark.
Add about half of the acid solution to the acridine orange, and mix until the dye is completely dissolved.
Add the remaining acid solution, and mix well.
Label the bottle, and store it at room temperature.
The stain is stable for several weeks.
Caution: Glacial acetic acid is a corrosive chemical with an irritating vapor, therefore handle it with care in a well-ventilated room.

Alcohol saline solution

To make 250 ml

Ethanol or methanol, absolute: 5 ml
Physiological saline or Sodium chloride,

0.85 g/dl i.e 0.85% w/v : 245 ml

Fill a cylinder (250 ml capacity) to the 245 ml mark with the saline solution.
Add 5 ml of absolute ethanol or methanol to the 250 ml mark.
Transfer to a screw-cap bottle, and mix well.
Label the bottle, and store it in a cool place.
The reagent is stable for several months.

Caution: Ethanol and methanol are highly flammable, therefore use these chemicals well away from an open flame.

Physiological saline, 8.5 g/l (0.85% w/v)

To make 1 liter
Sodium chloride: 8.5 g
Distilled water : upto 1 litre

Weigh the sodium chloride, and transfer it to a leak-proof bottle premarked to hold 1 liter.
Add distilled water to the 1-liter mark, and mix until the salt is fully dissolved.
Label the bottle, and store it at room temperature.
The reagent is stable for several months. Discard if it becomes contaminated.

Uses of Acridine Orange Staining

Following are the applications of this stain-

It uses the rapid identification of Trichomonas vaginalis, yeast cells, and clue cells in vaginal smears which can also use to detect intracellular gonococci, meningococci, and other bacteria, particularly in blood cultures.
It uses for analyzing mitochondria and lysosomal content by flow cytometer.
It is also applicable for the visual detection of DNA or RNA ( nucleic acids) on agarose and polyacrylamide gels.
Acridine Orange Staining may enumerate the microbial load in a sample because acridine orange binds with the nucleic acid of both living and dead organisms.
It also uses for identifying engulfed apoptotic cells, because they will fluoresce upon engulfment.
It acts as differential staining for human cells and prokaryotic cell with a fluorescence microscope.
To differentiate cells in the live and dead tumor cell groups.
Acridine Orange Test for Assessment of Human Sperm DNA Integrity

Limitations of Acridine Orange Staining

Despite various useful applications, it has some limitations and they are-

Cellular debris within a specimen such as WBCs, epithelial cells, and dead bacteria may distort the microscopic image.
Due to being a very sensitive stain, caution should be used when interpreting tests results.

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