This staining technique has been recommended for the rapid identification of Trichomonas vaginalis ,clue cells and yeast cells in genital tract specimens.It is also used to detect intracellular meningococci,gonococci in cervical and urethral smears and other bacteria such as Helicobacter pylori in gastric biopsies mostly culture or isolated in blood and the enumeration of mycoplasmas in broth culture
Principles of Acridine Orange stain
- Acridine Orange is a fluorochrome dye and binds to nucleic acids of bacteria and other cells, either in the native or the denatured state.
- DNA will fluoresce green while RNA fluoresce Orange red
- Due to the low pH of the buffer solution,this results in an orange staining of bacteria and fungi, and a green to yellow staining of human epithelial and inflammatory cells and background debris.
Material Required for Acridine Orange stain
- Alcohol saline solution
- Acridine orange acid stain
- 8.5 g/l Sodium chloride or normal saline
- Clean slide
- Inoculum or specimen
Procedure for Acridine Orange stain
- Make a smear of the specimen on a clean slide
- Allow the smear to air dry
- Don’t fix the smear.Cover the dried smear with acridine orange acid stain for 5-10 seconds (It contains an acid fixative)
- Wash off the stain with physiological saline or normal saline then decolorize the smear with the alcohol saline solution for 5-10 seconds
- Rinse the smear with normal saline and place it in a draining rack for air drying
- Now add a drop of normal saline or distilled water to the smear and cover it with a cover glass
- Examine the smear using a fluorescent microscope starting with 10X objectives to see the distribution of fluorescing material then switch to 40X objectives to identify Trichomonas vaginalis,yeast cells or bacteria.
Observation and interpretation
|Trichomonas vaginalis||Orange red with yellow green nucleus|
In cases of Bacterial vaginosis,orange staining bacteria are seen adhering to the green epithelial cells term clue cells
- Monica cheesbrough Part 2,p45