Acid-Fast Bacillus (AFB) Smear


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Subject: Acid-Fast Bacillus (AFB) Smear

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Definition and Use

  • Smears of patient specimens are stained and examined for the presence of mycobacteria. They may provide early evidence of TB or other mycobacterial diseases. Certain dyes bind to the thick, mycolic acid–rich cell walls of mycobacteria. The lipids of the cell wall make the cells resistant to decolorization with acid–alcohol solutions. AFB staining should be undertaken for most specimens that are submitted for mycobacterial culture.

  • Methods:

    • There are two types of AFB stains: chromogenic (carbol-fuchsin stains [hot: Ziehl-Neelsen; cold: Kinyoun]) and fluorogenic (auramine O + rhodamine). After staining, the smear is destained with an acid–alcohol solution, typically HCl in ethanol. Mycobacteria retain the stain.

      • In chromogenic methods, slides are examined using a 100× oil immersion objective with light microscopy. Nonmycobacterial cells are counterstained with methylene blue. Mycobacteria appear red, whereas other bacteria and background are stained blue.

    • In fluorogenic methods, auramine-stained slides are examined by fluorescence microscopy using a 25× or 40× objective. Mycobacteria are yellow-orange against a dark background. The improved signal-to-noise of auramine fluorochrome staining, allowing scanning with lower power objective, results in examination of a greater area of the slide at a given time, and therefore greater sensitivity. Any detected organisms should be confirmed by examination for typical morphology using the 100× objective. Some laboratories confirm positive fluorochrome smears with a carbol-fuchsin–based stain.

  • Specimens should be collected and transported to the laboratory according to recommendations for mycobacterial cultures.

  • Turnaround time: <24 hours


  • Expected results: Negative. Detection of mycobacteria requires 10,000 or more organisms per milliliter or gram of sample for consistent detection. Sensitivity may be improved by concentration of specimen, such as by centrifugation, and by examination of multiple specimens. Rapidly growing mycobacteria, such as Mycobacterium fortuitum, have relatively thin layers of cell wall mycolic acid and may decolorize with acid–alcohol decolorizing solutions. These organisms may be stained using a weaker acid in aqueous solution.

  • Positive results: Positive specimens are very likely (>90%) to yield growth of mycobacteria in culture. In a minority of patients, usually with cavitary or extensive tuberculosis, sputum AFB stains may remain persistently positive for weeks after patients have converted to negative cultures. Nonviable organisms may be detected by AFB stains. Nocardia and related species are weakly acid fast and may give false-positive results if staining protocols are not followed closely.


  • Standardized protocols, such as those published by the American Thoracic Society, should be followed carefully to ensure sensitive detection and accurate interpretation of smears.

  • Common pitfalls: Care must be taken to avoid contaminating slides with acid-fast organisms. Common causes of slide contamination are use of tap water for solution preparation, carryover between slides with immersion oil, and use of common staining chambers.