Ziehl-Neelsen Staining Technique Virtual Lab | PraxiLabs

Ziehl-Neelsen Staining Technique Virtual Lab Simulation

Biology | Genetics | Microbiology | Microscopy



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General Aim

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 can not 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.

 

Method

1.Place slide with heat fixed smear on staining tray. 2.Gently flood smear with concentrated carbol fushin. 3.Heat the stain until vapour just begins to rise. N.B. Do not overheat. Heating the stain: Great care must be taken when heating the carbol fuchsin. Only a small flame should be applied under the slides using an ignited swab previously dampened with a few drops of acid alcohol or 70% ethanol or methanol. Do not use a large ethanol-soaked swab because this is a fire risk. 4.Allow the heated stain to remain on the slide for 5 minutes 5.Tilt the slide slightly and gently rinse with tap water (or distilled water using a wash bottle if the tap water is not clean). N.B. While washing the slide after staining, do not let the water stream fall directly on the smear. This may disrupt the smear. Let the stream of water flow slowly along the surface, such that only the stain is flooded, and the smear is intact. 6.The smear will appear as a red circle on the slide. 7.Decolorize using 20% H2SO4 (or 3% HCL). Add the acid and leave it for 1-2 minutes. 8.Repeat this step until the smear appears pink in color. Caution: Acid is flammable, therefore use it with care well away from an open flame. 9.Immediately rinse with water. 10.Flood the smear with methylene blue dye and leave it for 1-2 minutes. 11.Tilt the slide slightly and gently rinse with tap water or distilled water using a wash bottle. 12.The smear will appear as a blue circle on the slide. 13.Blot dry the slide with bibulous paper. 14. View the smear using a light-microscope under oil-immersion lens (100x).

Learning Objectives (ILO’s)

  • Become proficient at performing the Ziehl-Neelsen stain consistently and accurately.

  • To differentiate between acid-fast bacilli and non-acid-fast bacilli.

  • To stain Mycobacterium species.

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 technique 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 Staining 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 can not 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 Ziehl-Neelsen stain 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-fuschin and phenol allows penetration through the bacterial cell wall for visualization.


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-fuschin which is basic strongly binds to the negative components of the bacteria which include the mycolic acid and the lipid cell wall. addition of acid alcohol along with the application of heat forms a strong complex that can not be easily washed off with solvents.


The acid-fast bacilli take up the red color of the primary dye, carbol-fuschin.


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
This technique has been used in the identification of Mycobacterium tuberculosis and Mycobacterium leprae.

 

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