Auramine–rhodamine fluorescent stain (AFB)

Principle

• Fluorochrome dyes bind the mycolic acid-rich cell wall of acid-fast bacilli (AFB) and resist decolorization with acid-alcohol.
• A counterstain such as potassium permanganate quenches background fluorescence.
• AFB glow yellow to orange against a dark background under a fluorescence or LED microscope.

Specimen

• Sputum (ideally early morning, 2–3 specimens), BAL, tissue, body fluids, often after digestion–decontamination (NALC–NaOH) and concentration.

Reagents and equipment

• Auramine O with or without rhodamine B, acid-alcohol (about 0.5% HCl in 70% ethanol), potassium permanganate.
• Fluorescence or LED microscope; biosafety cabinet for smear preparation.

Procedure

• Prepare an even smear, air-dry and heat-fix.
• Flood with fluorochrome for about 15 minutes, rinse with water.
• Decolorize with acid-alcohol for about 2 minutes, rinse.
• Counterstain with permanganate briefly (about 2 minutes); longer exposure can quench the bacilli.
• Rinse, air-dry (do not blot); examine with a 20–25× objective and confirm morphology at 40×.

Results

• Positive: slender, slightly curved yellow-orange rods; report with a standard grading scale for fluorescence microscopy.
• Scanty or doubtful positives are confirmed by re-examination or Ziehl–Neelsen staining.

Quality control and pitfalls

• Include positive and negative control smears with each batch.
• Fluorescence fades; store stained smears in the dark and read within 24 h.
• Debris and some artifacts fluoresce; check shape carefully.
• Nonviable bacilli stain too, so smears do not show treatment success reliably.
• A negative smear does not exclude TB; culture and NAAT are more sensitive.

Clinical use

• Faster and more sensitive screening than Ziehl–Neelsen because more fields are examined at lower magnification; WHO recommends LED fluorescence microscopy.