Nocardia in Acid Fast Stain: Introduction, Principle, Procedure and Result Interpretation

Nocardia in Acid fast stain

Nocardia in Acid-fast stain

Nocardia species in acid-fast stain showing acid-fast intertwining and branching threads like structure i.e. positive as shown above picture. Initially, the clinician advised the patient for the AFB test of sputum due to suspecting tuberculosis but during the  AFB stained smear examination, we found acid-fast structures resembling branching threads. From the same sputum, specimen smear prepared and performed modified Ziehl-Neelsen or Acid-fast bacilli (AFB) stain. After observation, we got this type of positive structure. Later organism confirmed as Nocardia,  growing on culture media and using biochemical tests.

Principle of AFB Staining for Nocardia

The presence of mycolic acid in the cell wall of organisms has been found responsible for keeping the acid-fast property. Nocardia has lower mycolic acid in the cell wall than M. tuberculosis and thus it needs a weaker decolorizer i.e. 1 % sulphuric acid. Therefore, AFB staining is useful to visualize acid-fast structures of various microorganisms especially Mycobacterium tuberculosis, and in modified form for Mycobacterium leprae, Nocardia species, Cryptosporidium parvum, Cyclospora cayetanensis , Isospora belli, and fungal spores.

Requirements for Ziehl-Neelsen or Acid-fast bacilli (AFB) stain

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 or 3% acid alcohol
  • 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 for modified Ziehl-Neelsen stain for Nocardia


  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. Wait for five minutes.
  5. Rinse with water.
  6. Decolorize by 1 % Sulphuric acid until the smear becomes pale pink in color. (wait for nearly five minutes)
  7. Rinse with water.
  8. Counterstain with methylene blue for one minute.
  9. Rinse with water.
  10. Drain and dry.
  11. Observe the smear first under the low power (10X) objective, and then under the oil immersion (100X) objective.

Result and Interpretation

Acid-fast organisms: pink or red bacillus

Background:  Blue ( as counterstain)

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 Handbook 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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