Why Is Acid Fast Staining Used for Some Bacteria?

Acid-fast staining is a laboratory procedure used to identify bacteria that possess a waxy coat on their cell wall and stain poorly with Gram staining, principally members of the genus Mycobacterium and Nocardia. While acid-fast staining is effective for differentiating these bacteria from acid-fast negative bacteria, it is insufficient to differentiate between individual species of acid-fast bacteria.

Ziehl-Neelson Method

• In the Ziehl-Neelson method of acid-fast staining, a smear sample is heat fixed, stained with carbol fuchsin (a solution of phenol and fuchsin), heated, rinsed in water, decolorized with an acidic solution, again rinsed in water, counterstained with methylene blue followed by a final rinse and blotted dry. When the smear is then viewed under the microscope acid-fast bacteria appear red while other organisms appear blue.

Modified Kinyoun Method

• The modified Kinyoun method does not require heating after the initial staining since the concentrations of phenol and fuchsin in the carbol fuchsin are modified to increase the effectiveness of the stain. The remaining procedures and reagents remain the same.

Fluorochrome Methods

• In fluorochrome acid-fast staining, fluorochromes are used as the stain and potassium permanganate is used as the counterstain. These fluorochromes cause acid-fast bacteria to fluoresce while the potassium permanganate will create a dark background and prevent the other material and cells on the smear from fluorescing.

Why It Works

• The acid-fast bacteria possess mycolic acids on their cell wall which bind tightly with the dyes. Once bound these dyes resist removal by the acidic solution. The counterstain is provides contrast between the acid-fast bacteria and the background material.

Diseases from Acid-fast Bacteria

• Medically important acid-fast bacteria include Mycobacterium tuberculosis (tuberculosis), Mycobacterium leprae (leprosy), and Nocardia asteroides (nocardiosis).