AFB Staining: Introduction, Principle, Procedure, Result and Interpretation

AFB staining

Introduction of AFB Staining

This acid-fast bacilli in brief AFB staining or Ziehl-Neelsen staining method is a modification of Ehrlich’s (1882) method. Its name is from the surnames of German doctors, bacteriologist Franz Ziehl(1859-1926), and pathologist Friedrich Neelsen (1854-1898).

Principle of AFB Staining

The presence of higher alcohol, glycerol, fatty acid, and especially mycolic acid in the cell wall has been found responsible for keeping the acid-fast property of bacteria. Therefore, AFB staining is useful for Mycobacterium tuberculosis, an etiological agent of tuberculosis.

Requirements for AFB Staining

a) Compound light microscope

b) Reagents and glasswares

  • Bunsen flame
  • Wire loop
  • Clean grease-free slides
  • Marker pen
  • Sprit lamp
  • Carbol fuchsin
  • 20% Sulphuric acid
  • Methylene blue

c) Specimens

In the case of primary tuberculosis

  • sputum
  • bronchial or laryngeal washing
  • Gastric lavage when sputum is swallowed as in children

 In miliary tuberculosis

  • bone marrow
  • Liver biopsy

Tuberculous meningitis

  • Cerebrospinal fluid (CSF)

Renal tuberculosis

  • urine

d) Quality control strains

  • Positive control (PC): Mycobacterium tuberculosis
  • Negative Control: Escherichia coli

Procedure of AFB Staining

  1. Make smear on a clean glass slide.
  2. Dry and fix the smear.
  3. Cover the smear with a strong carbol fuchsin solution.
  4. The heat from underneath the slide until just steam comes from the stain. Do not boil.
  5. Wait for five minutes.
  6. Rinse with water.
  7. Decolorize by 20% Sulphuric acid or 3% acid alcohol until the smear becomes pale pink in color. (wait for nearly five minutes)
  8. Rinse with water.
  9. Counterstain with methylene blue for one minute.
  10. Rinse with water.
  11. Drain and dry.
  12. Observe the smear first under the low power (10X) objective, and then under the oil immersion (100X) objective.

Result and Interpretation of AFB Staining

AFB: pink or red bacillus

Background:  Blue ( as counterstain used )


There are various ways of a reporting system for AFB stainings such as the Center for Disease Control and Prevention (CDC), the World Health Organization (WHO), and the International Union Against Tuberculosis and Lung Disease (IUATLD). The most common and widely accepted classification is IUATLD and according to it as follows.

  • No organism seen: Negative
  • 1-9/100 OIF ( oil immersion field): Exact number
  • 10-99/100 OIF: +
  • 1-10/OIF : ++
  • 10/OIF: +++

Further Readings

  • Bailey & Scott’s Diagnostic Microbiology. Editors: Bettey A. Forbes, Daniel F. Sahm & Alice S. Weissfeld, 12th ed 2007, Publisher Elsevier.
  • Clinical Microbiology Procedure Hand book Chief in editor H.D. Isenberg, Albert Einstein College of Medicine, New York, Publisher ASM (American Society for Microbiology), Washington DC.
  • Mackie and Mc Cartney Practical Medical Microbiology. Editors: J.G. Colle, A.G. Fraser, B.P. Marmion, A. Simmous, 4th ed, Publisher Churchill Living Stone, New York, Melborne, Sans Franscisco 1996.
  •  Manual of Clinical Microbiology. Editors: P.R. Murray, E. J. Baron, M. A. Pfaller, F. C. Tenover and R. H. Yolken, 7th ed 2005, Publisher ASM, USA
  •  Textbook of Diagnostic Microbiology. Editors: Connie R. Mahon, Donald G. Lehman & George Manuselis, 3rd edition2007, Publisher Elsevier.


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