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The purpose of this staining method is to differentiate between acid-fast and non–acid-fast bacteria. This method is used for organisms not stained by the Gram stain method, particularly organisms of the genus Mycobacterium. This is called the Ziehl-Neelsen staining technique.


Glass slide, carbol fuchsin stain, 3% acid alcohol, and malachite green stain.


  1. Prepare bacterial smear on a clean slide using sterile technique.

  2. Allow smear to air dry and then heat fix.

  3. Cover the smear with carbol fuchsin stain.

  4. Heat the stain until vapor just begins to rise (ie, about 60°C). Do not overheat. Allow the heated stain to remain on the slide for 5 minutes.

  5. Wash off the stain with water.

  6. Cover the smear with 3% acid alcohol for 5 minutes or until the smear is sufficiently decolorized (ie, pale pink).

  7. Wash well with water.

  8. Cover the smear with malachite green stain for 1 to 2 minutes, using the longer time when the smear is thin.

  9. Wash off the stain with clean water.

  10. Wipe the back of the slide clean, and place it in a draining rack for the smear to air dry (do not blot dry).

  11. Examine the smear microscopically, using the ×100 oil immersion objective.

FIGURE 25.35

Acid-Fast Bacilli. Mycobacterium tuberculosis seen on Ziehl-Neelsen staining technique for acid-fast bacilli. (Photo contributor: George P. Kubica, PhD. CDC Public Health Library. PHIL ID# 5789.)

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