Ziehl Neelsen Stain Technique

Theoretical Background / Context

• Staining is an auxiliary technique used in microscopic techniques to enhance the clarity of the microscopic image.
• Stains and dyes are widely used in the scientific field to highlight the structure of the biological specimens, cells, tissues, etc.
• The Ziehl-Neelsen staining technique is a differential staining techniques in microbiology that was initially developed by Ziehl and modified later by Neelsen, hence the name Ziehl Neelsen stain.
• Neelsen used carbol-fuschin from Ziehl’s experiment, with heat and added a decolorizing agent using acid-alcohol and a counterstain using methylene blue dye, thus developing the Ziehl-Neelsen Technique of staining.
• The use of acid-alcohol in the technique earned it the name Acid-Fast Stain and the application of heat in the technique gives it the name the hot method of Acid-Fast staining which is a synonymous name for the Ziehl Neelsen stain technique.
• This technique is used on microorganisms that are not easily stained by basic stains such as Negative staining or Gram staining.
• One of the most complex micro-organisms that require harsh treatment of the Ziehl-Neelsen compounds is the Mycobacterium spp.
• Mycobacterium, Actinomycetes, Norcadia, Isospora, Cryptosporidium, and some fungi contain a thick cell wall made up of lipoidal complexes known as mycolic acid.
• Mycolic acid is difficult to stain and therefore simple stains like gram staining cannot penetrate the thick cell wall of these organisms.
• They require harsher treatments to allow stain penetration for identification and examination and hence the use of the Ziehl-Neelsen or the hot method of Acid-fast stain.

Principle of Work

• The principle of ziehl neelsen staining uses basic fuchsin and phenol compounds to stain the cell wall of Mycobacterium species.
• Mycobacterium does not bind readily to simple stains and therefore the use of heat along with carbol-fuchsin and phenol allows penetration through the bacterial cell wall for visualization in the staining of microorganisms.
• Mycobacterium cell wall contains high lipid content made up of mycolic acid on its cell wall making it waxy, hydrophobic, and impermeable. These are ß-hydroxycarboxylic acids made up of 90 carbon atoms that define the acid-fastness of the bacteria.
• Use of carbol-fuchsin which is basic strongly binds to the negative components of the bacteria which include the mycolic acid and the lipid cell wall. The addition of acid alcohol along with the application of heat forms a strong complex that cannot be easily washed off with solvents.
• The acid-fast bacilli take up the red color of the primary dye, carbol-fuchsin, while non-acid-fast bacteria easily decolorize on the addition of the acid-alcohol and take up the counterstain dye of methylene blue and appear blue following the steps of ZN staining.
• The Ziel Neelsen Stain technique has been used in the identification of Mycobacterium tuberculosis and Mycobacterium leprae.