Acid Fast Stain (Kinyoun-Cold Method)- Principle, Procedure and Result Interpretation

The Kinyoun-Cold Method is a staining technique that is used to identify acid-fast microorganisms, such as Mycobacterium spp. and some parasites, such as Cryptosporidium and Isopora spp. Acid-fast microorganisms are those that retain the primary stain, carbolfuchsin, even after being treated with a decolorizing agent, such as sulfuric acid. This property is due to the presence of mycolic acid in their cell walls, which makes them resistant to staining with water-based dyes.

Acid-fast staining is important for the diagnosis and treatment of various diseases caused by acid-fast microorganisms, such as tuberculosis, leprosy, nocardiosis, and cryptosporidiosis. The Kinyoun-Cold Method is a modification of the Ziehl-Neelsen Method, which requires heating the specimen during staining. The Kinyoun-Cold Method does not require heating, but uses a more concentrated solution of carbolfuchsin and phenol to penetrate the cell wall of acid-fast microorganisms. The Kinyoun-Cold Method is also faster and simpler than the Ziehl-Neelsen Method, making it suitable for routine laboratory use.

The Kinyoun-Cold Method is based on the differential staining of acid-fast and nonacid-fast microorganisms. Acid-fast microorganisms are those that retain the primary stain, carbolfuchsin, even after treatment with a decolorizing agent, such as sulfuric acid. Nonacid-fast microorganisms are those that lose the primary stain and take up the secondary stain, methylene blue.

The reason why some microorganisms are acid-fast and others are not lies in the composition of their cell walls. Acid-fast microorganisms, such as Mycobacterium spp. and some parasites, have a high content of mycolic acid in their cell walls. Mycolic acid is a long-chain fatty acid that makes the cell wall waxy and impermeable to most stains. To stain these microorganisms, a special technique is required to penetrate their cell walls and bind the dye to their cytoplasm.

The Kinyoun-Cold Method uses carbolfuchsin as the primary stain and phenol as a mordant. Phenol is a chemical that enhances the staining ability of carbolfuchsin by increasing its solubility and affinity for the cell wall. Carbolfuchsin is a red dye that contains basic fuchsin and phenol. When applied to the specimen smear, carbolfuchsin and phenol enter the cell wall of both acid-fast and nonacid-fast microorganisms. However, only acid-fast microorganisms can retain the carbolfuchsin-phenol complex after decolorization with sulfuric acid. This is because the sulfuric acid forms an insoluble salt with the mycolic acid in the cell wall of acid-fast microorganisms, preventing the carbolfuchsin-phenol complex from being washed out. Nonacid-fast microorganisms, on the other hand, do not have mycolic acid in their cell walls and thus lose the carbolfuchsin-phenol complex during decolorization.

To differentiate between acid-fast and nonacid-fast microorganisms, a secondary stain or counterstain is applied after decolorization. The Kinyoun-Cold Method uses methylene blue as the secondary stain. Methylene blue is a blue dye that stains the cytoplasm of nonacid-fast microorganisms that have lost the primary stain. Acid-fast microorganisms do not take up the secondary stain because they are already saturated with the primary stain.

Therefore, by using the Kinyoun-Cold Method, acid-fast microorganisms can be identified by their pink color while nonacid-fast microorganisms can be identified by their blue color.

The Kinyoun-Cold Method is a simple and rapid staining technique that does not require heating or prolonged staining time. The following steps describe how to perform the Kinyoun-Cold Method on a specimen smear:

1. Prepare and fix the specimen smear prior to staining. The specimen can be sputum, urine, feces, tissue, or any other material that may contain acid-fast microorganisms. Spread a thin layer of the specimen on a clean glass slide and let it air-dry. Then, pass the slide through a flame several times to fix the smear and kill any pathogens.
2. Cover the smear with carbolfuchsin for 3 to 5 minutes at room temperature. Carbolfuchsin is a red dye that contains phenol, which helps the stain penetrate the waxy surface of acid-fast microorganisms. You can use a dropper or a staining rack to apply the stain evenly over the smear. Do not let the stain dry on the slide.
3. Gently rinse the slide with water to remove any excess stain. Hold the slide at an angle and run tap water over it until the water runs clear. Do not rub or wipe the slide as this may remove some of the stained microorganisms.
4. Run 1% sulfuric acid decolorizer over the slide for approximately 3 minutes. Sulfuric acid is used to remove the carbolfuchsin from nonacid-fast microorganisms and background material, leaving only acid-fast microorganisms stained red. You can use a dropper or a staining rack to apply the decolorizer over the slide. Check the slide under a microscope every 30 seconds to monitor the decolorization process. Stop when you see faint blue color on the slide.
5. Rinse the slide with water and decolorize again for 1 to 2 minutes until the solution runs clear. This step ensures that all nonacid-fast microorganisms and background material are completely decolorized and do not interfere with the result interpretation.
6. Cover the smear with the secondary or counterstain, methylene blue, for 1 minute. Methylene blue is a blue dye that stains nonacid-fast microorganisms and background material, making them visible under a microscope. You can use a dropper or a staining rack to apply the counterstain over the slide.
7. Gently rinse the slide with water to remove any excess counterstain. Hold the slide at an angle and run tap water over it until the water runs clear.
8. Blot the slide dry with bibulous paper or air-dry it in a horizontal position. Do not rub or wipe the slide as this may damage or remove some of the stained microorganisms.
9. Examine the slide under a microscope using an oil immersion objective (100x magnification). Look for pink rods (acid-fast bacilli) and blue rods (nonacid-fast bacilli) on a blue background.

After performing the Kinyoun-Cold Method, the stained specimen smear can be examined under a light microscope using an oil immersion objective (100x magnification). The acid-fast organisms will appear pink while the nonacid-fast organisms and the background material will appear blue. This is because the acid-fast organisms retain the primary stain, carbolfuchsin, even after decolorization with sulfuric acid, while the nonacid-fast organisms lose the primary stain and take up the secondary stain, methylene blue.

The most common acid-fast organisms that can be identified by this method are Mycobacterium spp., which are rod-shaped bacteria that cause diseases such as tuberculosis and leprosy. The presence of a single acid-fast bacillus in a sputum sample is considered diagnostic for tuberculosis. Other acid-fast organisms that can be detected by this method include parasites such as Cryptosporidium and Isopora spp., which are protozoans that cause diarrheal infections in humans and animals.

The interpretation of results should be done carefully and with caution, as some factors may affect the accuracy and reliability of the staining method. Some of these factors are:

• The quality and thickness of the specimen smear: A thin and evenly spread smear is preferred to avoid clumping and overlapping of cells that may obscure the staining results.
• The duration and intensity of decolorization: Over-decolorization may result in false-negative results, while under-decolorization may result in false-positive results. The decolorization should be stopped when the solution runs clear from the slide.
• The presence of other substances or microorganisms that may interfere with the staining: Some substances such as lipids, waxes, or resins may also resist decolorization and appear pink. Some microorganisms such as Nocardia spp. or Rhodococcus spp. may also show partial or weak acid-fastness and appear pinkish-red.

Therefore, it is recommended to use the Kinyoun-Cold Method in conjunction with other methods such as culture, biochemical tests, molecular tests, or serological tests to confirm the identification of acid-fast microorganisms.

The Kinyoun-Cold Method is a simple and effective staining technique for identifying acid-fast microorganisms such as Mycobacterium spp. and some parasites. It is based on the principle that acid-fast organisms have a waxy surface that prevents them from being stained by aqueous based stains, but allows them to retain the primary stain, carbolfuchsin, when treated with phenol and sulfuric acid. The nonacid-fast organisms and background material are stained by the secondary stain, methylene blue, to provide contrast. The result is that acid-fast organisms appear pink while nonacid-fast organisms appear blue under the microscope. This method is useful for diagnosing diseases such as tuberculosis, leprosy, cryptosporidiosis and isosporiasis. It can also be used for quality control of cultures and specimens.

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