Ziehl-Neelsen (acid-fast) stain

Principle

Mycobacteria have mycolic acid in their walls. Once heated carbol fuchsin enters, acid-alcohol cannot remove it, so they stay red ("acid-fast"). Other cells lose the dye and take the blue counterstain.

Reagents

• Strong carbol fuchsin
• Decolouriser: 3% acid-alcohol (or 20–25% sulphuric acid)
• Counterstain: methylene blue (or malachite green)

Procedure

• Fix the smear by heat.
• Flood with carbol fuchsin and heat gently until steam rises (do not boil); keep hot for about 5 minutes.
• Rinse, then decolourise until only a faint pink remains; rinse.
• Counterstain for about 1 minute, rinse, dry and examine at least 100 oil-immersion fields before reporting negative.

Results

• Acid-fast bacilli: red, straight or slightly curved, sometimes beaded
• Background and other cells: blue

Notes

• M. leprae and Nocardia are only weakly acid-fast; a weaker decolouriser (e.g. 1% sulphuric acid for Nocardia) is used in modified methods.
• Kinyoun is a cold modification; auramine fluorescence staining is faster to screen.
• Grade results using the WHO/IUATLD scale (scanty, 1+, 2+, 3+).