AFB Stain Assistant Professor

Dr. Issam Jumaa

Acid Fast (Ziehl-Neelsen or Hot Method)
‫قسم تقنيات المختبرات الطبية‬
Purpose ‫المرحلة الثانية‬
Practical
Identification of acid-fast Mycobacterium spp.
Principle
Acid-fast mycobacteria contain mycolic acid in their outer membrane, making the cells waxy and resistant to
staining with aqueous based stains such as the Gram stain. The primary stain, carbolfuchsin is applied to the cells,
and heat and phenol are used to allow the stain to penetrate into the waxy surface of acid-fast microorganisms.
The excess stain is removed with treatment by acid alcohol (ethanol and hydrochloric acid). A secondary stain,
methylene blue, is then applied to the cells.
Method
1. Prepare and fix the specimen smear prior to staining.
2. Place a small strip of blotting or filter paper over the top of the specimen, and place the slide over a boiling hot
water bath on a mesh surface.
3. Cover the filter paper with the primary stain, carbolfuchsin. Leave the slide on the
water bath for 3 to 5 minutes. Continue to apply stain if the filter paper begins to dry.
4. Remove the filter paper and rinse the slide with water until the solution runs clear.
5. Run acid-alcohol decolorizer over the slide for approximately 10 to 15 seconds.
6. Rinse the slide with water.
7. Cover the smear with the secondary or counterstain, methylene blue, for 1 minute.
8. Gently rinse the slide with water.
9. Blot the slide dry with bibulous paper.
Expected Results
Acid-fast organisms, Mycobacterium spp., will appear pink. Nonacid-fast organisms will appear dark blue. In
addition, background material should stain blue.

Application of Reagent
Primary dye Carbol fuchsin
Decolorizer Acid alcohol
Counter stain Methylene blue