Lessons from a proficiency testing event for acid-fast microscopy

Chest. 2001 Jul;120(1):250-7. doi: 10.1378/chest.120.1.250.


Objectives: To evaluate the routine performance and the technical parameters of different acid-fast staining methods: Kinyoun, Ziehl-Neelsen (ZN), auramine, and auramine-rhodamine.

Design and participants: The performance of 167 laboratories was analyzed using prestained and unstained slides.

Setting: Laboratories holding New York State permits.

Results: The results revealed that Kinyoun's cold carbol fuchsin method is inferior to both the ZN and fluorochrome (auramine and/or auramine-rhodamine) methods. Even though 91% of the participants used commercial staining kits, the study identified unexpected errors concerning the concentration of carbol fuchsin, time for staining and counterstaining, and the concentration of acid alcohol for decolorization, which may significantly influence the sensitivity. Besides these findings, the present study showed that the examination of < 300 view fields may also decrease the sensitivity of acid-fast microscopy. In addition, we found that the sensitivity and specificity of the ZN and fluorochrome methods are comparable if the procedural standards are followed.

Conclusions: The strict and ongoing quality control of the "simple to perform" acid-fast microscopy and the immediate review of commercially available staining kits are necessary. Because of the rapidity of the fluorochrome method, laboratories with large specimen numbers should use this technique. In all other cases, the ZN method should be used. Moreover, all clinicians should be aware of the method of acid-fast microscopy used and the proficiency of the laboratory in performing the assay.

Publication types

  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Benzophenoneidum / standards
  • Fluorescent Dyes / standards
  • Laboratories / standards*
  • Microscopy / standards
  • Mycobacterium tuberculosis / classification*
  • Quality Control
  • Reagent Kits, Diagnostic / standards*
  • Rhodamines / standards
  • Rosaniline Dyes / standards
  • Sensitivity and Specificity
  • Staining and Labeling / methods
  • Staining and Labeling / standards*


  • Fluorescent Dyes
  • Reagent Kits, Diagnostic
  • Rhodamines
  • Rosaniline Dyes
  • Benzophenoneidum
  • carbol-fuchsin solution