Acid-fast Bacilli (AFB) Smear Microscopy

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The page below is a sample from the LabCE course Tracking Antibiotic-Resistant Tuberculosis. Access the complete course and earn ASCLS P.A.C.E.-approved continuing education credits by subscribing online.

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Acid-fast Bacilli (AFB) Smear Microscopy

Globally recognized as a transmission-risk indicator, the AFB smear exists as a rapid, less expensive answer to tuberculosis (TB) control strategy when culture and antimicrobial susceptibility testing (AST) are not available. Ideally, the smear is preliminary to a complete work-up that includes culture and AST, procedures that are mandated when the likelihood of resistance occurs. Treated specimens, those which have been decontaminated and concentrated, increase the sensitivity of the process by 30%.
The mycobacterial cell wall contains mycolic acids, which are fatty acids that contribute to the characteristic of "acid-fastness." The principle of the AFB smear is based on the fact that mycolic acid in the cell wall of AFB render them resistant to decolorization with acid alcohol. Three different staining procedures are used for the screening and/or confirmation of AFB:
  • Fluorochrome - Screening procedure that is more sensitive than conventional carbol fuchsin stains (Ziehl-Neelsen or Kinyoun).
  • Ziehl-Neelsen - Classic procedure that allows stain to persist after heating (required for better penetration of the stain into the cell wall)
  • Kinyoun - Carbol fuchsin has a higher concentration of phenol and basic fuchsin, which allows the stain to penetrate without the use of heat.
Each of these procedures uses acid alcohol as the decolorizer. When counterstained, the organism color is accentuated with either carbol fuchsin (Ziehl-Neelsen or Kinyoun stains) or auramine O/rhodamine B (fluorescent acid-fast stain), depending on the method used.
Fluorescent microscopy (shown in the upper image), though more costly, has the advantage of decreasing time to detection or chance of missed opportunity to observe AFB morphology. Use of a lower power (250X rather than 1000X) allows for greater slide coverage, 30 fields examined in 90 seconds, for example. Confirmation should follow with a Ziehl-Neelsen (as shown in the lower image) or Kinyoun stain, which is retained for reference. In a sputum specimen with a volume more than 5 mL, the sensitivity of the fluorescent method may be as high as 80% for pretreated specimens or as low as 20% for less adequate, or untreated specimens.

Fluorescent auramine stain w/acridine orange counterstain
Ziehl-Neelsen stain of M. tuberculosis in sputum