Acid-fast Staining



Principle

The majority of the bacteria are stainable with simple or gram staining. While few cannot be stained with these techniques, e.g. Mycobacterium and Neisseria. For these specialized techniques is used called acid-fast staining. The characteristic difference between mycobacterium and other microorganisms in the presence of thick waxy (lipoidal substance mycolic acid) wall that makes the penetration of conventional stains very difficult. Once the stain is penetrated into the cell wall it cannot be easily removed by vigorous acid alcohol washing. Because of this property, the organisms are called acid-fast.  The acid-fast technique uses three different reagents.
Primary stain: Carbol fuchsin. Due to heavy, waxy contents, conventional aqueous stains such as crystal violet and methylene blue are able to penetrate the Mycobacterium cell wall. While Carbol fuchsin, a dark red stain mixed in 5% phenol is soluble in lipoidal materials that constitute the major cell wall component of the Mycobacterium spp. Stain penetration is enhanced by the application of heat, which drives carbol fuchsin through the cell wall into the cytoplasm (Ziehl-Nelson modification). Heat reduces the surface tension between the cell wall components and stain can easily penetrate.  
 Decolorizing Agent: Acid alcohol (3% HCl and 95% ethyl alcohol). Prior to decolorization smear is cooled which will harden the waxy cell wall substances. Then, upon application of acid-alcohol acid-fast microorganisms will resist decolorization. The bacterial cell will appear red. While the non-acid fast will be readily decolorized by the action of acid-alcohol.
Counterstain: Methylene blue. This is used as a final stain to stain on the previously decolorized cells. As only non-acid fast cells are decolorized they will absorb counterstain and will appear as blue. While the acid-fast cells are already stained with primary stain and appear as red.

Materials 

72-96 h tryptic soy broth culture of Mycobacterium smegmatis and 24 culture of S. aureus, carbol fuchsin, acid-alcohol, methylene blue, glass slides, staining rack, inoculation loop, hot plate, blotting paper, microscope.

Procedure

  1. Obtain a clean glass slide. Perform a bacterial smear by mixing both the cultures.
  2. Allow smear to air dry and heat fix the smear.
  3. Flood the smear with carbol fuchsin. Heat the smear with flame or placing over hot plate for 5 min. Do not allow the stain to completely evaporate. Also, prevent the stain to boil.
  4. Cool the slide and wash it with tap water.
  5. Decolorize with acid-alcohol.
  6. Wash the smear with tap water.
  7. Counterstain with methylene blue for 2 min.
  8. Wash the smear with tap water.
  9. Dry the smear with blot paper and examine under a microscope (oil immersion lens).

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