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, clinician advised the patient for AFB test of sputum due to  suspecting tuberculosis but  during the  AFB stained smear examination, we found   acid fast structures resembling to 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 structures. Later organism conformed  as Nocardia,  growing on culture media and using biochemical tests.

Principle of AFB Staining for Nocardia

Presence of mycolic acid in the cell wall of organisms  have 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 weaker decolorizer i.e. 1 % sulphuric acid. Therefore, AFB staining is useful to visualize acid fast structures of various microorganisms specially 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 glass wares

  • 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 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 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. Counter  stain  with methylene blue for one  minute.
  9. Rinse with water.
  10. Drain and dry.
  11. Observe the smear first under low power (10X) objective, and then under oil immersion (100X) objective.

Result and Interpretation

Acid fast organisms: pink or red bacillus

Back ground:  Blue ( as counter stain)


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  • 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
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