Mycobacterium tuberculosis: Introduction, Auramine-Phenol Stain and Its Significance

Mycobacterium under Fluorescence microscope

Introduction of Mycobacterium tuberculosis

Mycobacterium tuberculosis is a species of pathogenic bacteria in the family Mycobacteriaceae, It is the causative agent of tuberculosis and it may be pulmonary tuberculosis, miliary tuberculosis,  tuberculous meningitis, and renal tuberculosis. First discovered in 1882 by a German physician and microbiologist, Robert Koch. M. tuberculosis has an unusual lipid content, mycolic acid, and this coating makes  Gram reaction variation and thus acid-fast stains such as Ziehl-Neelsen is a useful solution for staining Mycobacterium tuberculosis. Fluorescent stain such as auramine-phenol is more sensitive than the Ziehl-Neelsen to identify this acid-fast bacillus.

Specimens may be for tuberculosis according to the location of involvement e.g. pulmonary tuberculosis-sputum, bronchial or laryngeal washings, gastric lavage ( in children when swallowed). Miliary tuberculosis – bone marrow, liver biopsy. Tuberculous meningitis-Cerebrospinal fluid (CSF). Renal tuberculosis-urine.

Mycobacterium under Fluorescence Microscope

The fluorochrome dye, auramine-phenol, forms a complex with mycolic acids found in the acid-fast cell wall of test organisms which resist decolorization by acid-alcohol. Potassium permanganate ( counterstain), renders tissue and its debris non-fluorescent, therefore reducing the possibility of artifacts. The cells visualized under ultraviolet light appear bright yellow against a dark background ( but the above image missing may be due to improper use of counterstain).

Mycobacterium tuberculosis pictures of auramine-phenol stained slide under a fluorescence microscope having the following features- Bright yellow rods, variable in length, often curved or also fragmented, glowing against a background.

Reporting sputum smear

According to IUATLD (International Union against Tuberculosis and Lung Diseases) and WHO guidelines-

Report using 40 x objective and 10 x eyepiece

  • Scanty  AFB  1-19 AFB in 40 fields
  • +                         20-199 AFB in 40 fields
  • ++                       5-50 AFB per filed (examine 20 fields)
  • +++                   >50 AFB per field ( examine 8 fields)

To say No AFB seen: Examine 40 fields that should be free from AFB


AFB seen (++)

From the field as shown above and below pictures.

Advantage of fluorochrome stain over Ziehl-Neelsen stain

It is highly sensitive than Ziehl-Neelsen stain because of observation at 40X objective whereas in Ziehl-Neelsen stain at 100X i.e. oil immersion field.


  • Fluorescent stain such as auramine-phenol is more sensitive than the Ziehl-Neelsen to identify this acid-fast bacillus because of fields observation at 40 X objective whereas in Ziehl-Neelsen at 100 X objective.
  • Reagents like Auramine-phenol are possible carcinogens, acid –alcohol and potassium permanganate is also a strong irritant to skin, eyes, and respiratory system. Precaution is required while handling and staining using such reagents.
  • Excessive exposure to the counterstain may result in a loss of the brilliance of the fluorescing organism.
  • Stained smears should be observed within a day of staining because of the possibility of the fluorescence fading.
  • AFB stands for acid-fast bacilli.

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.
  • Jawetz, Melnick and Adelberg’s Medical Microbiology. Editors: Geo. F. Brook, Janet S. Butel & Stephen A. Morse, 21st ed 1998, Publisher Appleton & Lance, Co Stamford Connecticut.
  • 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.
  • District Laboratory Practice in  Tropical Countries  –  Part-2-   Monica Cheesebrough-   2nd Edn Update


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