What are the procedure for acid-fast staining?
Acid-Fast Staining Instructions
- Air dry and heat fix a thin film of microorganisms.
- Flood the slide with Carbolfuchsin.
- Flood slide with Acid Alcohol for 30 seconds.
- Counterstain by flooding the slide with Methylene Blue for 30 seconds.
- Dry the slide by putting it between the pages of a book of Bibulous paper.
Why is methylene blue used in acid-fast staining?
After the auramine dye has fully stained the smear, a drop of acid alcohol is applied for one to two minutes to decolorize the smear. Methylene blue or potassium permanganate is used as a counterstain to provide background color.
How is a smear for an acid fast stain fixed?
To perform the acid-fast stain, a heat-fixed smear is flooded with the primary stain carbol fuchsin, while the slide is heated over a steaming water bath. The heat “melts” the waxy cell wall and permits the absorption of the dye by the cells.
What are the reagents used in the acid fast stain?
The acid-fast bacilli will stain bright red, and the background will stain blue. Reagents used in the procedure include Ziehl–Neelsen carbol-fuchsin solution, 1% acid alcohol, and methylene blue solution [15].
What dyes are used in acid-fast staining?
The dyes used in the acid-fast staining technique include carbol fuchsin (primary dye), acid alcohol (decolourizer), and methylene blue as a counterstain.
What are the three components of an acid-fast stain?
Acid-fast stain is a differential stain used to identify acid-fast organisms such as members of the genus Mycobacterium. Acid-fast organisms are characterized by wax-like, nearly impermeable cell walls; they contain mycolic acid and large amounts of fatty acids, waxes, and complex lipids.
Why is carbol fuchsin used in acid-fast staining?
Carbol fuchsin is used as the primary stain dye to detect acid-fast bacteria because it is more soluble in the cells’ wall lipids than in the acid alcohol.
Which is Decolorizer used in acid-fast staining?
Remel TB Decolorizer is a reagent recommended for use with the Kinyoun or Ziehl-Neelson carbolfuchsin staining procedure to differentiate acid-fast bacteria from nonacid-fast bacteria. The microscopic acid-fast staining technique is one of the earliest methods used for detection of the tubercle bacillus.
What is the mordant in acid-fast staining?
During the acid fast stain, heat is used as a mordant to allow the primary stain to penetrate the waxy mycolic acid layer. The heat will prevent the cells from being destained using acid-alcohol. Because these cells hold fast to the primary stain with acid alcohol treatment, they are termed acid fast positive.
What color is acid-fast positive?
pink/red color
Acid Fast positive cells are stained the pink/red color of carbolfuchsin. Acid Fast negative cells are stained the light blue color of methylene blue.
What is the role of phenol in acid-fast staining?
In acid fast stains, the phenol allows the stain to pen- etrate, even after exposure to decolourisors. If an organism is to be termed Acid Fast, it must resist decolourisation by acid-alcohol. A counterstain is then used to emphasise the stained organism.
What is the Decolorizer in acid-fast stain?
Principle of Acid-Fast Stain Then the smear is decolorized with decolorizing agent (3% HCL in 95% alcohol) but the acid fast cells are resistant due to the presence of large amount of lipoidal material in their cell wall which prevents the penetration of decolorizing solution.