Many bacteria of the genera Mycobacterium and Nocardia are medically significant causes of infectious diseases, such as tuberculosis, leprosy and other lung and skin infections. It is therefore obviously important to be able to accurately identify members of these genera.
Cell Wall of Mycobacteria and Nocardia
The Gram Stain is a differential stain reaction that is used to categorize most bacteria as either Gram+ or Gram-. This distinction is due to differences in the cell wall structure of these two categories of bacteria and has significance with respect to which antibiotics can be used to kill or control bacteria.
Bacteria of the genera Mycobacterium and Nocardia have unusual cell walls that are waxy and nearly impermeable due to the presence of mycolic acid, and large amounts of fatty acids, waxes, and complex lipids. These organisms are highly resistant to disinfectants, desiccation and are difficult to stain with water-based stains such as the Gram.
Because the cell wall is so resistant to most compounds, acid-fast organisms require a special staining technique involving heat to drive stain into their waxy cell wall.
Acid Fast Differential Stain
Heat Fixing a Bacteria Smear
Prior to staining bacteria, a bacterial smear must be heat fixed onto a microscope slide. A smear is a sample of bacteria suspended in a small amount of water on a slide. That sample is then dried using heat. The heat kills the bacteria and attaches the sample to the slide so that it does not easily wash away.
Acid Fast Staining Procedure
The protocol for staining acid-fast organisms is as follows:
- Place a strip of blotting paper over the slide.
- Place the covered slide over a screened water bath and then saturate blotting paper with primary stain Ziehl’s carbol fuschion.
- Allow the slide to sit over water bath for 3 – 5 minutes, reapplying stain if it begins to dry out.
- Remove blotting paper and rinse slide until water runs clear.
- Flood slide with decolorizer, Acid Alcohol, for 10 – 15 seconds and then rinse.
- Flood slide with counterstain, Crystal Violet, for one minute and then rinse.
- Gently blot the slide dry. It is now ready to be viewed under oil immersion (1000x TM) with a bright-field compound microscope.
After this staining procedure, the Acid-fast cells will appear pink, because the primary stain, Ziel’s carbol fuschion, has been driven into the bacteria’s waxy cell wall with the heat from the water bath. Acid-fast cells also typically clump together, due to the wax in their cell wall. The Nonacid fast cells (bacteria that do not have a waxy cell wall) will appear purple, having retained the counterstain, crystal violet, after the primary stain was removed by the decolorizer.
Additional Microbiology Information
For more information on microbiology, see the Virtual Microbiology Classroom of the science education website Science Prof Online or Todar's Textbook of Bacteriology.
Sources
Bauman, R. (2005) Microbiology. Pearson Banjamin Cummings.
Park Talaro, K. (2008) Foundations in Microbiology. McGraw-Hill.
Tami Port - Tami Port is a college professor of cell and microbiology and creator of ScienceProfOnline.com, a free science education website.
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