Bacteria with an acid-fast cell wall (see Fig. 1) when stained by the acid-fast procedure, resist decolorization with acid-alcohol and stain red, the color of the initial stain, carbol fuchsin. The genus Mycobacterium and the genus Nocardia are acid-fast. All other bacteria will be decolorized and stain blue, the color of the counterstain methylene blue.

The acid-fast stain is an especially important test for the genus Mycobacterium. Besides the many saprophytic forms of mycobacteria, there are two distinct pathogens in this group: M. tuberculosis, the causative organism of tuberculosis, and M. leprae, the causative agent of leprosy. Mycobacterium tuberculosis (the tubercle bacillus) causes tuberculosis, although atypical species of Mycobacterium may occasionally cause tuberculosis-like infections, especially in the debilitated or immunosuppressed host. Mycobacterium avium-intracellulare complex (MAC), for example, frequently causes systemic infections in people with HIV/AIDS.

PROCEDURE (Ziehl-Neelsen Method)

1. Heat-fix a smear of a bacterium as follows:

a. Using the dropper bottle of distilled water found in your staining rack, place 1/2 a drop of water on a clean slide by touching the dropper to the slide.

b. Aseptically remove a small amount of the bacterium from the agar surface and mix it with the water. Flame the loop and let it cool.

c. Using the loop, spread the mixture over the entire slide to form a thin film.

d. Allow this thin suspension to completely air dry.

e. Pass the slide (film-side up) through the flame of the bunsen burner 3 or 4 times to heat-fix.

2. Cover the smear with a piece of blotting paper and flood with carbol fuchsin.

3. Steam for 5 minutes by passing the slide through the flame of a gas burner.

4. Allow the slide to cool and wash with water.

5. Add the acid-alcohol decolorizing slowly dropwise until the dye no longer runs off from the smear.

6. Rinse with water.

7. Counterstain with methylene blue for 1 minute.

8. Wash with water, blot dry, and observe using oil immersion microscopy.

 

Acid-fast bacteria will appear red; non-acid-fast will appear blue.