Comparison of Ziehl Neelsen & Auramine O staining methods on direct and concentrated smears in clinical specimens

Indian J Tuberc. 2011 Apr;58(2):72-6.


Background: In developing countries like ours with a large number of tuberculosis (TB) cases and limited resources, the diagnosis of TB relies primarily on smear microscopy for Acid Fast Bacilli (AFB) but its sensitivity is limited in paucibacillary cases.

Aim: To evaluate the increase in efficacy of smear microscopy when smears are prepared from clinical samples after concentration by Petroff's method and stained by Auramine O (AO) fluorescent dye as against Ziehl Neelsen (ZN) staining of similar taking culture as the gold standard.

Methods: Smears were prepared from 393 clinical samples both by direct and after Petroff's concentration and examined by fluorescent microscopy and Ziehl Neelsen method .The concentrated material was also cultured on Lowenstein Jensen media and the results of the two microscopy methods were compared with the culture results taken as the gold standard.

Results: Mycobacterial growth was detected in 137 (35.77%) specimens, out of which three were non-tubercular mycobacteria. Using culture as the reference method, the sensitivity of direct staining was 55.55% for ZN and 71.85% for AO. Direct fluorescent microscopy detected 9.29% paucibacillary sputum samples that were missed on ZN staining. On concentration, the sensitivity increased by 6.67% for ZN and 11.11% for AO. The sensitivity of AFB smear microscopy increased by 27.41% and was statistically significant (p = < .001) when both methods were combined. The specificity was 99.19% for both ZN and AO.

Conclusion: Fluorescent microscopy has higher sensitivity and comparable specificity which is further enhanced by concentration. Now with the advent of newer inexpensive Light Emitting Diode (LED) based fluorescent microscopes (FM), which are easier to use, fluorescent microscopy can be widely used even in peripheral laboratories where culture facilities are not available.

Publication types

  • Comparative Study

MeSH terms

  • Bacteriological Techniques
  • Benzophenoneidum*
  • Coloring Agents*
  • Female
  • Humans
  • Male
  • Predictive Value of Tests
  • Sensitivity and Specificity
  • Sputum / microbiology*
  • Staining and Labeling / methods
  • Tuberculosis, Pulmonary / diagnosis*


  • Coloring Agents
  • Benzophenoneidum