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Chapter 2 - Microscopy and Staining -- Virtual

2 - 1 The acid-fast stain

Because of the waxy substance (mycolic acids) present on the cell walls, cells of species of Mycobacterium do not stain readily with ordinary dyes. However, treatment with cold carbol fuchsin for several hours or at high temperatures for five minutes will dye the cells. Once the cells have been stained, subsequent treatment with a dilute hydrochloric acid solution or ethyl alcohol containing 3% HCl (acid-alcohol) will not decolorize them. Such cells are thus termed acid-fast in that the cell will hold the stain fast in the presence of the acidic decolorizing agent. This property is possessed by few bacteria other than Mycobacterium.

Microscopic examination of tissues or of sputum stained by the acid-fast staining procedure is an aid in the diagnosis of tuberculosis. If an individual has pulmonary tuberculosis, and if the tubercles in the lungs are open, the bacteria (Mycobacterium tuberculosis) will be present in the sputum. The bacteria which cause leprosy (Hansen's disease; caused by M. leprae) can also be detected with this staining procedure. The finding of acid-fast cells in milk, on the skin, or in feces is of no great signifi-cance, because these bacteria may be commonly-found saprophytic species of Mycobacterium.

  1. After preparation of the heat-fixed smear, place the slide over a steaming water bath.
  2. Place a piece of paper towel or blotting paper over the smear. The paper should be about as wide as the slide and cover an area just slightly greater than the smear itself. Saturate the paper with carbol fuchsin and let the slide remain above the steaming water bath for five minutes. Add more carbol fuchsin to the paper if it appears the stain is drying out.
  3. Allow the slide to cool to room temperature. Remove the paper and wash off the excess stain with water.
  4. Decolorize the smear with acid-alcohol for 10-15 seconds. Wash gently with tap water.
  5. Counterstain with methylene blue for 3 minutes. Rinse the slide gently and dry.
  6. Examine the smear first with the 10X and then the 100X (oil-immersion) objective. Those cells which retained the primary stain (carbol fuchsin) through the acid-alcohol treatment are stained red; these are the acid-fast organisms. Mycobacterium cells characteristically appear as clusters of long, red rods. All other cells are blue.

The above video demonstrates the acid-fast stain protocol.

Below is pictured an example of an acid-fast stain

The acid fast stain

Figure 3.17. The acid fast stain. A photomicrograph of Mycobacterium smegmatis (pink) and Micrococcus luteus (blue) at 1000x magnification. M. smegmatis is acid-fast, retaining the carbol fuchsin dye, thus appearing pink. M. luteus is not acid-fast, loses the carbol fuchsin during decolorizaiton, and is counter-stained with methylene blue.