Acid Fast Bacteria and Acid Fast Staining

Several special stains can be used to screen for the presence of bacteria. Other special stains can help categorize these bacteria into groups of bacteria. One of the more common special stains used to classify bacteria is Acid Fast staining. This stain is used to sort bacteria into acid-fast or non-acid-fast groups.¹

The Acid Fast stain determines if the blood, tissue, or other body fluid is infected with Mycobacteria, which can cause diseases such as leprosy or tuberculosis (TB) or Nocardia (bacteria found in soil and water that may affect the lungs, brain, and skin).² According to the World Health Organization, tuberculosis alone is the leading cause of death from an infectious agent, and worldwide, an estimated 9.9 million people became ill with TB in 2020. Tuberculosis is curable.³ Diagnosis and treatment are critical in the treatment of these diseases.

The acid-fast bacteria (AFB) cell consists of a gram-positive cell envelope with high lipid content and includes the bacterias - Mycobacteria (M. leprae, M. tuberculosis, M. smegmatis, M. Avium complex, M. kansasii) and Nocardia ( N. brasiliensis, N. cyriacigeorgica, N. farcinica, and N. nova.)^{4, 5} These cell walls are waxy like and difficult to penetrate; therefore the laboratory must use special stains to identify the organisms.

In 1882, the discovery of the tubercle bacillus by Robert Koch with a complex staining method prompted other researchers to attempt to improve on Koch’s process. This finding by Koch led to the development of various approaches to staining acid-fast bacteria – four are listed below.

Named for two German physicians, one of the earliest and standard staining techniques is Ziehl-Neelson (ZN) staining. The staining is performed by placing cells in suspension on a slide, air-drying the liquid then fixing the cells with heat. The primary reagent used is Carbol Fuchsin, followed by a decolorizer and acid alcohol, then counterstained with methylene blue/malachite green. If the cell is acid-fast, it will remain red from the Carbol Fuchsin. The non-acid fast cell will pick up the methylene blue.⁶ Slides viewed using the ZN method are with a standard bright-field microscope.

Developed by Joseph Kinyoun, the Kinyoun stain is similar to the Ziehl-Neelson stain without heating. This staining is referred to as the cold method. The primary reagent used is Kinyoun Carbol Fuchsin Stain (a mixture of basic fuchsin, phenol, ethanol, and deionized water), followed by Carbol Fuchsin Decolorizer (a combination of hydrochloric acid with ethanol), then counterstained with methylene blue/malachite green. With the Kinyoun staining method, high concentrations of Carbol Fuschin are used instead of heat.⁷ Again, the acid-fast cells will remain red, while the non-acid fast cells will retain the blue from the methylene blue used. These slides can also be viewed under a standard microscope.

Auramine-Rhodamine Fluorochrome uses fluorescence microscopy to visualize acid-fast bacilli. The primary reagent used is a fluorescent dye, Auramine-Rhodamine, followed by the decolorizer, acid-alcohol, then counterstained using potassium permanganate. Acid-fast bacilli will fluoresce red-orange, yellow, or reddish-yellow. Non-acid-bacilli do not fluoresce and appear pale yellow.⁸

The Fite stain is used to diagnose leprosy and other bacterial infections such as Nocardiosis. This staining method was worked on by others and was also known as the Fite-Faraco method and the Wade-Fite method. Using peanut oil mixed with xylene in the deparaffinization, the oil coats the cell walls and protects the acid fastness. Mineral oil is sometimes substituted in this staining protocol.⁹ The primary reagent in this protocol is Carbol-fuchsin, followed by a rinse in water, acid alcohol, and counterstained with Methylene blue. The acid-fast bacilli will be red and the background blue.¹⁰

To learn more about acid-fast and special stains, visit Knowledge Pathway – Special Stains in Microbiology - Bacteria & Fungi, GMS & AFB Stains. www.leicabiosystems.com. Accessed April 19, 2022.