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Saturday, April 5, 2014

Fluorescent Acid Fast Staining Protocol - Mycobacterium tuberculosis

Acid-fast mycobacteria resist decolorization by acid-alcohol after primary staining owing to the high lipid (mycolic acid) content in their cell walls. The identification of mycobacteria with auramine O is due to the affinity of the mycolic acid in the cell walls for the fluorochromes. The dye will bind to the mycobacteria, which appear as bright yellow, luminous rods against a dark background. The potassium permanganate helps prevent non-specific fluorescence. All acid-fast organisms will be stained by auramine O, including some parasites. Slides tained with auramine O 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. The fluorochromes stains are recommended for specimen examination because of their increased sensitivity and speed.

Sample / Specimen

Clinical specimens or pure cultures suspected of harboring mycobacteria are stained for acid fastness. A direct smear (not concentrated) from a clinical specimen is discouraged because it lacks the sensitivity of a concentrated(centrifuged) smear. A negative result from a direct smear must be followed by a concentrated smear. Preparation of smear for staining is as follows:

  • In the case of a solid medium, an aqueous suspension is made.

Take a small amount of material and suspend it in a drop of distilled water on a microscope slide. Do not make the smear too thick.

  •  In the case of a liquid medium, A drop is used directly from the culture container. 

Air dry the smear, then fix by passing the slide through a Bunsen burner flame two or three times. A better fixation method is to allow the slide to remain on an electric slide warmer at 65 to 75ÂșC for at least 2 h. Allow the slide to cool prior to staining.


Reagents: Reagents may be purchased commercially or prepared in the laboratory.

Fluorochrome acid-fast stain

  • Auramine O
  • 0.5% Acid-alcohol
  • Counterstain (potassium permanganate or acridine orange)
  • Glass slides, Coverslips 

Procedure / Protocol for Fluorescent Acid Fast Staining

  • Flood the slide with fluorochromes stain.
  • Stain for 15 min.
  • Rinse the slide with water; drain excess water from the slide.
  • Flood with 0.5% acid-alcohol
  • Decolorize for 30-60 s. (some protocols call for 2 min.)
  • Rinse the slide with water; drain excess water from the slide.
  • Flood the slide with potassium permanganate or acridine orange
  • Counterstain for 2 min; do not allow the slide to dry. NOTE: Timing is critical during the counterstaining step with potassium permanganate.
  • Counterstaining for a longer time may quench the fluorescence of acid fast organisms.
  • Rinse the slide with water; drain excess water from the slide.Air dry; do not blot.
  • Examine the smear with a fluorescent microscope (K530 excitation filter and a BG 12 barrier filter or G365 excitation filter and an LP 420 barrier filter).
  • Examine smears using the high power objective (40X, total magnification, X400); verify using the oil immersion objective (100X, total magnification, X1,000). Some recommend screening with a 25X objective.

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