The identification of Mycobacteria with rhodamine-auramine is due to the affinity of the mycolic acid in the cell walls for the fluorchromes. These dyes will bind to the Mycobacteria, which appear bright yellow or orange against a greenish background. The potassium permanganate helps prevent non-specific fluorescence. All acid-fast organisms will be stained by rhodamine-auramine, including the sporozoan parasites. Slides stained with rhodamine-auramine 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.
|AAD-8oz||Fluorescent Decolorizer||8 oz.|
|AAD-1gl||Fluorescent Decolorizer||1 gallon|
|PPC-1-8oz||Potassium Permanganate||8 oz.|
|PPC-1-1gl||Potassium Permanganate||1 gallon|
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 too 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.
- Place the “fixed” smear on a staining rack and flood slide with rhodamine-auramine for 15 minutes. Do not let surface dry.
- Wash off the stain with distilled water.
- Flood slide with fluorescent decolorizer for 2-3 minutes.
- Rinse thoroughly with distilled water.
- Flood slide with potassium permanganate for 3-4 minutes. Do not allow slide to dry.
- Rinse thoroughly with distilled water and air dry.
- Examine microscopically under the same light source as used for fluorescent microscopy (i.e. a K530 excitation filter and a BG 12 barrier or G-365 excitation filter and an LP 420 barrier filter). Slides can be screened on high power (400X) and verified under oil immersion.
Note: Staining times may vary to suit the individual.
Sources of Error
- 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.
- Do not stain smears which have only been air dried. Smears must also be “fixed”.
- Smears should not be too 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.
- After staining it is essential that the back surface of the slide is wiped clean.
- If washing with distilled water is not done adequately, crystallization of the stain may appear on the slide.