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Ziehl-Neelsen Staining for Acid-Fast Bacilli

SOP for Ziehl-Neelsen staining to detect acid-fast bacilli (Mycobacterium tuberculosis and other mycobacteria).

Last verified 1 month ago 2 min read Microbiology Staining Bacteriology Mycobacteriology #Ziehl-Neelsen #ZN Stain #Acid-Fast Bacilli #AFB #tuberculosis #mycobacterium #staining
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Document Control
Version
1.0
Effective
Aug 28, 2026
Reviewed
Aug 29, 2026
Department
Microbiology
Principle

Mycobacteria have cell walls rich in mycolic acids (waxy lipids), making them resistant to decolorization by acid-alcohol after staining with carbol fuchsin. The phenol in the stain helps penetrate the waxy cell wall. Heating enhances penetration. Once stained, the organisms resist decolorization with 20% sulfuric acid (hence "acid-fast") and retain the red/pink color. Non-acid-fast organisms and background material are decolorized and take up the blue counterstain.

Standard Operating Procedure: Ziehl-Neelsen Staining

1. Purpose and Scope

To perform Ziehl-Neelsen (ZN) staining for detection of acid-fast bacilli (AFB), primarily Mycobacterium tuberculosis, in clinical specimens.

2. Specimen Requirements

  • Type: Sputum (preferred early morning), BAL, CSF, tissue, body fluids, urine.
  • Volume: Sputum: 3-5 mL; Other specimens: as much as available.
  • Storage: Process fresh or refrigerate at 2-8°C.
  • Rejection Criteria: Improperly labeled or contaminated specimens.

3. Safety and Precautions

  • BIOSAFETY LEVEL 2 or 3 - Perform in biological safety cabinet.
  • Wear appropriate PPE (gloves, gown, N95 mask, eye protection).
  • Mycobacteria are airborne pathogens.
  • Use dedicated AFB staining area.
  • Dispose of waste in biohazard bags; decontaminate with 5% phenol or bleach.

4. Equipment and Reagents

  • Clean glass slides, Bunsen burner or hot plate.
  • Reagents:
  • Primary stain: Ziehl-Neelsen carbol fuchsin (0.3% basic fuchsin in 5% phenol + ethanol)
  • Decolorizer: 20% sulfuric acid (H₂SO₄) or 3% acid-alcohol
  • Counterstain: Methylene blue (0.3%) or malachite green
  • Microscope with 100x oil immersion objective.

5. Step-by-Step Procedure

  1. Smear Preparation:
  • For sputum: Select purulent portions, prepare thin smear (2x3 cm).
  • Air dry completely.
  • Heat fix by passing through flame 2-3 times.
  1. Staining:
  • Flood smear with carbol fuchsin.
  • Heat gently until steam rises (do not boil). Maintain steaming for 3-5 minutes. Add more stain if it dries.
  • Cool, rinse gently with water.
  • Decolorize: Flood with 20% H₂SO₄ for 2-3 minutes or until pink color stops running. Rinse with water. Repeat if necessary.
  • Rinse well with water.
  • Flood with methylene blue counterstain for 1-2 minutes.
  • Rinse with water, air dry.
  1. Microscopy: Examine under 100x oil immersion objective.

6. Interpretation

  • AFB positive: Bright red/pink rods against blue background.
  • AFB negative: Only blue background, no red rods.
  • Grading (WHO/IUATLD):
  • Scanty: 1-9 AFB/100 fields (report exact number)
  • 1+: 10-99 AFB/100 fields
  • 2+: 1-10 AFB/10 fields
  • 3+: >10 AFB/field

7. Quality Control

  • Include known AFB-positive slide (M. tuberculosis control) with each new batch of reagents.
  • Check reagent quality monthly.

8. Reporting Results

  • Report as "AFB seen" with grading, or "No AFB seen."
  • Report immediately for positive results (critical value).

Frequently Asked Questions

Heating helps the carbol fuchsin penetrate the thick, waxy mycolic acid layer of the mycobacterial cell wall.
Kinyoun (cold) method uses higher phenol concentration and does not require heating; ZN (hot) method uses heat for penetration.