Exercise 6-C
STAINING OF MICROORGANISMS
ACID-FAST STAIN
Introduction
The acid-fast stain is a differential stain that separates bacteria on the basis of the lipid content of their
cell walls. Bacteria categorized as acid-fast (members of the genus Mycobacterium and some other
Actinobacteria) have Gram-positive walls with thick peptidoglycan containing large quantities of a wax-
like lipid called mycolic acid. These cells are resistant to staining with normal procedures, and require
severe treatment, e.g., heat and potent dyes, in order to be stained. Once stained, their walls do not
readily decolorize, and so can be differentiated from non-acid-fast cells. The acid-fast stain is used
primarily in the diagnosis and study of diseases caused by acid-fast bacteria (Tuberculosis and Leprosy),
however it can also be used on a variety of non-pathogenic organisms. The basic steps in this technique
are as follows:
1. Bacteria cells are heat-fixed and stained with a potent primary staining reagent called carbol fuchsin
while being subjected to steam heat. An alternative procedure uses a different formulation of carbol
fuchsin (“Kinyoun’s Acid-Fast Reagent”) which does not require the application of steam heat.
2. The stained cells are subjected to decolorizing with an acid-alcohol solution; 95% ethyl alcohol
containing 2.5% concentrated nitric or hydrochloric acid (HNO3 or HCl). This step will remove the
primary stain from cells that are not acid-fast, rendering them colorless.
3. Non-acid-fast cells are counterstained with a basic dye of a contrasting color (e.g., methylene blue).
When a mixed culture containing both acid-fast and non-acid-fast cells is subjected to this procedure, the
acid-fast cells will be stained red and the non-acid-fast cells will be stained blue. The presence of
purple-colored cells usually indicates the procedure has been improperly completed; however, some
cells are only partially acid-fast and others may contain acid-fast structures. The endospores formed by
bacteria in the genus Bacillus will often stain red when subjected to the acid-fast stain.
Standard Procedure Using Steam Heat
1. Place about 200 ml of water in a 400 ml beaker on a wire gauze platform over a Bunsen burner
and heat the water to generate steam.
2. At one end of a clean glass slide, prepare a small, mixed smear containing a Mycobacterium
species and Staphylococcus aureus. For best results, thoroughly mix a small quantity of the
Mycobacterium with a loopful of water and break up the cell clusters before adding the
Staphylococcus. At the other end of the slide, prepare a small smear of your morphological
unknown.
3. Allow the smears to air dry and then heat-fix them. It is important to insure that the cells used
are dead, but do not apply excess heat.
4. Place a wire mesh platform over the beaker and place the slide on the platform smear side up.
