Mycobacterium avium, Ziehl-Neelsen staining
Ziehl-Neelsen Staining
DESCRIPTION
Acid fast organisms have a lipoid capsule that can resist decolorization with a dilute acid rinse (previously stained with Ziehl-Neelsen
fuchsin solution). Method is used for genus Mycobacterium.
REAGENTS
Ziehl-Neelsen fuchsin solution: basic fuchsin 2.5 g, distilled water 250 ml, 100%
alcohol 25 ml, phenol crystals melted 12.5 ml.
Acid solution: H
2SO4 in H2O  1:4.
Loffler methylene blue solution: methylene blue crystals 1 g, phenol crystals melted,
96% alcohol 10 ml, distilled water 100 ml.

PROCEDURE
Dry and heat-fix the slide then cover it with Ziehl-Neelsen fuchsin solution. Heat the
slide for 10 minutes; avoid boiling. Wash in running tap water.
Add H
2SO4 solution and wait few seconds. Remove acid solution and wash in running
tap water.
Cover the slide with Loffler methylene blue solution and wait 1 minute. Remove
solution, wash and dry the slide.
RESULTS
Acid-fast bacteria are red colored, others are blue. After discoloring only acid fast cells remain red.

NOTES
Solutions are flammable and corrosive.
(c) Costin Stoica
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