Miramar College
Biology 205 Microbiology
Background
Differential Staining: The Acid Fast Stain
The acid fast stain is one of the most medically important stains, second only to Gram
staining. This is due to the highly pathogenic nature of certain members of the genus
Mycobacterium-M. tuberculosis. Because of a high concentration of mycolic acid in their
cell wall, all members of this genus will not completely destain during the Gram stain
process, producing that second example of Gram positive irregular. Additionally,
this waxy material allows for these bacteria to be identified easily in the acid fast stain.
During the acid fast stain, heat is used as a mordant to allow the primary stain to
penetrate the waxy mycolic acid layer. The heat will prevent the cells from being
destained using acid-alcohol. Because these cells hold fast to the primary stain with acid
alcohol treatment, they are termed acid fast positive. All other cells will easily be
destained by the addition of acid alcohol and are termed non-acid fast. These non-acid
fast cells are counterstained with methylene blue.
Introduction
The method you will be using for your acid fast stain, the Ziehl-Neelsen
method, uses carbolfuchsin mixed with phenol as a mordant. The waxy mycolic acid of
acid fast bacteria is quite sticky and makes preparing a thin smear difficult. Keep this in
mind as you are preparing your acid fast stain.
Objectives
1. Perform a successful acid fast stain.
2. Be able to distinguish acid fast from non-acid fast cells under oil immersion.
3. Be able to explain the steps in the AF stain.
4. Explain why AF bacteria hold the stain when decolorized with acid-alcohol.
5. Give examples of AF bacteria.
Acid Fast Staining Protocol
Table supplies
Individual supplies
Culture of Mycobacterium smegmatis
Acid Fast staining kit (carbolfuchsin, acid
alcohol, methylene blue)
Culture of Staphylococcus aureus
Microscope slides
Staining tray
Water bottle
Bibulous paper
