The identification of Mycobacteria with acridine orange is due to the affinity of the mycolic acid in the cell walls for the fluorochrome. The dye will bind to the Mycobacteria, which appear as bright orange, luminous rods against a dark background. Slides stained with acridine orange may be restained with Ziehl-Neelsen or Kinyoun stain directly, as long as the oil has been removed. This provides a convenient method of confirming and differentiating morphology of positive slides with the traditional stains.
Reagents

Specimen Collection
Organisms being stained by an acid fast method are usually taken from a solid or liquid medium on (in) which they have been cultured from their original source (e.g. wounds, throat, swabs, sputum, etc.). An aqueous suspension is made, in the case of the solid medium, by taking a small amount of the material and suspending it in a drop of distilled water on a microscope slide. Care should be taken not to make the smear to thick. In the case of a liquid medium, a drop is used directly from the culture container. However, due to the solids from the medium, this method is not always satisfactory. The suspension made by either method is air dried, then “fixed” by passing rapidly through a Bunsen burner flame two or three times. Allow the smear to cool before staining.
Procedure

1. Place the “fixed” smear on a staining rack and flood slide with Phenolic acridine orange for 15 minutes。
2. Wash with distilled water.
3. Flood slide with methylene blue decolorizer for 2 minutes.
4. Rinse thoroughly with distilled water.
5. Air dry. Do not blot.
6. Examine microscopically using a fluorescent microscope (i.e. a K530 excitation filter and a BG 12 barrier filter or G-365 excitation filter and an LP 420 barrier filter). Scan using X200 and verify using X600 without immersion oil. Examine at least 30 individual fields before deciding that the specimen is negative. Refrigerate smears that can not be read immediately.

Note: Staining times may vary to suit the individual.

Sources of Error

1. Overheating (burning) during fixation can be avoided by just touching the back of the slide to the back of the hand each time the slide has been passed though the flame.
2. Do not stain smears which have only been air dried. Smears must also be “fixed”.
3. Smears should not be to thick. After air drying, examine under a microscope. If there are no areas of bacteria separation, more water should be added to dilute the smear.
4. After staining it is essential that the back surface of the slide is wiped clean.
5. If If washing with distilled water is not done adequately, crystallization of the stain may appear on the slide.
6. Paper strips may inhibit AFB staining with acridine orange and should not be used.