Fixation on Histology

Acid Fast Bacteria Stains


Acid_Fast_Bacteria.jpgThere are several histology stains, such as Ziehl-Neelsen, that demonstrate acid fast bacteria. Acid fastness is a physical property of the cell wall of this group of bacteria, that makes them resistant to decolorization by acids. This is important because during staining, the bacteria resists decolorization with the acid-alcohol solution used and retains the carbol fuchsin dye to appear a red/magenta color. It uses a methylene blue dye counterstain, contrasting the bacteria which retained the carbol dye and the cells that were decolorized with the acid-alcohol solution and appear blue.

Mycobacterium tuberculosis, the bacteria causing Tuberculosis, is one of these acid-fast bacteria, which has mycolic acid in its cell wall, making it resistant to gram staining. The TB bacteria was first identified in the 1880s by Robert Koch. Several scientists then contribute improvements to his methods of identifying this bacteria, resulting in the Ziehl-Neelsen staining method. Ziehl introduces the use of carbolic acid (phenol) as the mordent, which Neelsen combines with basic fuchsin to make what we see today as carbol fuchsin solution. The Ziehl-Neelsen staining method is known as the “hot-staining” method because it uses heat to penetrate the waxy cell walls. This is in comparison to other acid-fast staining methods, including Kinyoun and Auramine-Rhodamine. In the Kinyoun acid fast stain, instead of heat, you increase the concentration of the carbol fuschin.

Auramine-Rhodamine, sometimes also known as Truant Auramine-Rhodamine, is a method of fluorescent staining for acid-fast bacteria. Its use in acid-fast bacteria dates back to the early 1960s in experiments by Truant, Brett, and Thomas, which used auramine and rhodamine dyes with a potassium permanganate counterstain. The auramine and rhodamine binds to the mycolic acid in the cell wall, which allows the stain to penetrate it. The bacteria will show up as a yellow with a green background. This fluorescent method of bacteria detection works well, but it is not as specific as the Ziehl-Neelsen stain.

There are different types of acid-fast bacteria; Mycobacterium tuberculosis is just one of them. Mycobacterium leprae is another type of acid-fast bacteria that causes leprosy. Mycobacterium leprae is less acid-fast and more easily decolorized by the Ziehl-Neelsen stain. Therefore, a modification known as the Fite stain is often used for diagnosis of leprosy. In this stain, peanut oil is used with the xylene to protect the cell wall from too much contact with the organic solvents, protecting its acid-fastness. Why peanut oil? We actually did a post about that before! Click here to read more about peanut oil in the Fite stain.

Learn more about acid-fast bacteria stains in NSH’s HT Prep Course, which covers histology basics, such as staining, microtomy, processing, and fixation, to prepare you for the HT exam.