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Three Types of Acid-Fast Stains

Microscopy is using technology to make things that are very small visible to the human eye. Scientists who view microorganisms and bacteria through microscopes must sometimes use a procedure called staining to better see or differentiate specific parts of these microscopic creatures. The stains themselves are substances that adhere to the cells, giving them color. There are several kinds of stains that suit certain microscopic organisms better than others. There are simple stains, differential stains, gram stains, negative stains and acid-fast stains.

Acid-Fast Organisms and Acid-Fast Stains
- "Acid-fastness" refers to a specific physical property of certain bacteria, namely their ability to resist acid decolorization during staining procedures. These resistant organisms are called "acid-fast organisms," and can only be colored for viewing purposes using acid-fast stains. There are three primary stains used for successfully staining acid-fast organisms: the Ziehl-Neelsen stain; the Kinyoun method; and certain fluorescent dyes.
Ziehl-Neelsen Staining
- The Ziehl--Neelsen stain is a special bacteriological stain that is used to view acid-fast organisms, primarily Mycobacteria. Acid-fast organisms that are dyed using the Ziehl-Neelsen stain turn bright red. The procedure for Ziehl-Neelsen staining is as follows:
  
  1. Cover with tissue paper (optional).
  
  2. Cover microscope slide with carbolfuchsin, the primary stain, for 3 to 5 minutes while heating with steam or heating on a Bunsen burner.
  
  3. Remove tissue paper if used, decolorize slide using a mixture of ethanol and hydrochloric acid."
  
  4. Counterstain using malachite green or methylene blue.
Kinyoun Staining
- The Kinyoun stain is another method used in staining acid-fast microorganisms. It is similar to the Ziehl-Neelsen stain, with the primary difference being that you do not heat up the slide when applying Kinyoun stains. Underneath a microscope, stained organisms will appear blue. The procedure for applying Kinyoun stains is as follows:
  
  1. Cover slides with Kinyoun carbolfuchsin for 5 minutes and rinse with water until it comes off clear.
  
  2. Cover slides with acid-alcohol -- 3 percent HCl in ethanol -- for 3 minutes and rinse again with water until it comes off clear.
  
  3. Cover slides with methylene blue dye for 3 minutes and rinse gently with water until it comes off clear.
  
  4. Let slides air-dry before viewing.
Auramine-Rhodamine Staining
- The auramine-rhodamine stain, sometimes called the Truant auramine-rhodamine stain, is a technique used to view acid-fast microorganisms using fluorescence microscopy. When specific compounds are illuminated using high-energy light, they tend to emit a different, lower frequency light as a result of exposure. This is called fluorescence. Acid-fast microorganisms give off a reddish-yellow fluorescence with this method. The auramine-rhodamine stain might not be as specific for acid-fast organisms as the Ziehl-Neelsen or Kinyoun stains, but it is more affordable and is often used as a screening tool.

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