Acridine orange stain: Introduction, Principle, procedure and result

Acridine Orange Stain: Introduction, Principle, Procedure and Result Interpretation

Introduction of Acridine orange

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.

Principle of Acridine orange

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

Requirement for Acridine orange

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

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

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 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.

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