How to stain Acid Fast Bacilli (AFB) in the microbiology lab (Ziehl-Neelsen vs Kinyoun stain) -- Staining methodology:

Carbolfuchsin: 4 minutes
Acid alcohol decolourizer: 3-5 seconds
Methylene Blue: 30 seconds
- Remember to always use a control slide for AFB staining


Staining principle: Mycolic acid in cell wall of Acid Fast Bacilli (AFB) bacteria resists the acid decolourizer; therefore maintains the primary stain while other genera do not.

AFB positive genera: Mycobacterium and Mycolicibacterium (differentiated in 2018).

Mycobacterium encompasses TB and non-TB organisms
Mycolicibacterium (e.g. M. smegmatis) was formerly known as Mycobacterium until a 2018 phylogenetic reshuffling.
Other genera can be ‘partially acid fast’ (e.g. Rhodococcus, Tsukamurella, Dietzia, Nocardia) but are not Kinyoun positive nor AFB organisms. Partially acid fast means they will stain positive with a Modified Kinyoun stain, not a regular Kinyoun stain.


Staining interpretation:
- Kinyoun positive = pink/red
- Kinyoun negative = blue
- Gram positive = purple
- Gram negative = pink
Note that AFB organisms will stain gram positive and Kinyoun positive


When would you do it?
- Suspected AFB in a gram stain
- Performed directly from culture work-up suspected of being AFB
- Performed after positive fluorescent AFB stain (Auramine O stain) as a confirmatory stain
o Fluorescent AFB stain is performed on almost all specimens with an AFB culture order (e.g. sputum) and is quicker to read and interpret than a Kinyoun stain.


Corrections: Kinyoun stain @3:32


End credit microbe: Pseudomonas aeruginosa

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