ACID-FAST BACTERIA STAIN (AFB)

 

 

Uploaded: 2008-04-07, Updated: 2008-04-07
Ziehl-Neelsen Stain (hot stain)
  1. Primary Staining: Basic Ziehl-Neelsen carbolfuchsin, 60 ēC, one hour;
  2. Gentle wash with water to cool the slide;
  3. Decolorizing: acid ethanol (1% HCL);
  4. Wash the slide in water;
  5. Counterstaining: with methylene blue;
  6. The acid-fast bacteria retain the red color. Non acid-fast bacteria will not be red.
Kinyoun Stain (cold stain)

  1. Primary staining: Basic Kinyoun carbolfuchsin (prepared in phenol to help the dye penetrate), 2 minutes;

  2. Rinse with water;

  3. Decolorizing: acid ethanol (1% HCL);

  4. Rinse with water;

  5. Counterstaining: methylene blue;

  6. Rinse with water.
Auramine O Stain (increased sensitivity and speed)
  1. Primary staining: fluorochromes stain with phenolic auramine,15 min;
  2. Rinse the slide with water; drain excess water from the slide;
  3. Decolorizing: acid ethanol (1% HCL);
  4. Rinse the slide with water; drain excess water from the slide;
  5. Counterstaining: potassium permanganate or acridine orange, 2 min;
  6. Rinse the slide with water;
  7. Examine the smear with a fluorescent microscope: mycobacteria are bright yellow, luminous rods against a dark background. The potassium permanganate helps prevent non-specific fluorescence.
Sensitivity of Smear
  • ~ 50%  in newly diagnosed patients;
  • ~ 30% in HIV co-infected patients;
  • Requires at least 5 ×103 to 1×104 AFB/ml to be detected;
  • Ziehl-Neelsen stain is more sensitive than Kinyoun stain in detecting lightly staining organisms, especially rapid growing mycobacteria.
  • Fluorochrome stain is more sensitive, less time-consuming, but less specific than fuchsin stains. Fluorochrome is preferred for screening and fuchsin is used for confirming.