Ziehl Neelsen Stain Principle, Procedure, Result

Ziehl Neelsen stinging method is used to differentiate between the acid-fast and non-acid fast bacteria.

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Ziehl Neelsen Stain Principle, Procedure, Result.

Ziehl Neelsen Acid Fast Stain Overview

You must be wondering what is ziehl neelsen stain? Ziehl-Neelsen Staining is a type of acid-fast staining technique, and it is another important differential staining procedure used to differentiate between acid-fast and non-acid fast bacteria. You must be thinking what is acid-fast staining? It is a staining procedure that differentiates between bacteria based on their ability to retain a dye when washed with an acid alcohol solution. It mainly differentiates between acid-fast and non-acid-fast bacteria.

This acid fast staining method is used to identify Mycobacterium tuberculosis and M. leprae. These two pathogens are responsible for tuberculosis and leprosy, respectively. They posses a lipid-containing cell wall, made of mycolic acid, a group of branched-chain hydroxy fatty acids, this prevent the binding of dye with the cell wall.

In Ziehl-Neelsen Staining we use a high concentration of phenol and carbol fuchsin, as well as a wetting agent, to drive the stain carbol fuchsin into mycobacterial cells. Once this dye has entered, the cells are not readily decolorized by acidified alcohol (acid-alcohol) and thus are said to be acid-fast. Non-acid-fast bacteria are readily decolorized by acid alcohol and thus are stained another color by a second dye called a counterstain.

This staining method was first introduced by Paul Ehrlich and modified by two German doctors Franz Ziehl (bacteriologist) and  Friedrich Neelsen (pathologist).

Objective of Ziehl-Neelsen Staining

The main objective of this stinging method is to differentiate between the acid-fast and non-acid fast bacteria. It also used to stain Mycobacterium species.

Ziehl Beelsen Stain Principle

The cell wall acid-fast bacteria contain waxy mycolic acid, which is relatively impermeable to simple stains. But when the heat and Carbol fuchsin applied, the cell’s waxy mycolic acid layer become weaken and porous, thus the carbol fuchsin penetrates the cell wall.

When we applied heat, it softens the cell wall and permit the stain( carbol fuchsin) to penetrate it. Carbol fuchsin is more soluble in phenol as compared to alcohol and water, in turn, phenol is more soluble in lipid or wax, thus the phenol and fuchsin mixture easily enters the cell.

The cells are now decolorized by a decolorizer containing sulfuric acid or Hydrochloric acid. Thus, those bacteria are decolorized and appear colorless, are considered as Non Acid- fast bacteria, and those are do not get decolorized are known as acid-fast bacteria.

The non-acid fast bacteria easily resist the decolorization, this is because, phenol dye mixture is more soluble in waxy mycolic acid as compared to the acid or alcohol. In this way phenol act as a mordant.

After completion of decolorization, the acid-fast bacteria appear in red or pink color, while the non-acid fast bacteria appear colorless. Now, a counterstain or secondary stain (Methylene blue) is applied to visualize the non-acid fast bacteria, they retain the color of counterstain (Methylene blue) and appear in blue.

Requirement of Ziehl-Neelsen Staining

Culture and Instruments: Bacterial culture/ Sputum sample, Glass Slide, Microscope, Wash bottle, Distilled water, Inoculating loop, Spirit lamp / Bunsen burner.


  • Primary Stain: Carbol fuchsin stain (mixture of 100ml Distilled water, 1g Basic fuschin, 10ml of 100% Ethyl alcohol, and 5ml Phenol crystals)
  • Decolourizer: 20% sulphuric acid or acid-alcohol (Mixture of 95ml of 95% Ethyl alcohol, 2ml Distilled water, 3ml of 3% Concentrated hydrochloric acid)
  • Counterstain: methylene blue (mixture of 0.25g  Methylene blue, 99ml Distilled water, and 1ml Acetic acid).

Ziehl Neelsen Stain Steps/Procedure

Smear preparation

  1. Take a grease-free slide.
  2. Sterilize the inoculating loop by using a bunsen burner. Hold it on flame until it becomes hot red.
  3. Use the sterile loop to transfer a loop full culture on the slide, and then prepare the smear.
  4. Dry the smear in the air.
  5. Heat-fix the dried smear, by passing it through the flame of bunsen burner.

Acid-fast staining Procedure

  1. Now, flood the slide with the primary stain, carbol fuchsin, and apply heat gently until vapour just begins to rise. Don’t evaporate the stain, replenish the stain as required. Don’t boil the stain, Do not overheat. (For cold method, flood the smear with carbol fuchsin with Tergitol and wait for 5 to 10 minutes.)
  2. After that wait 5 minutes and then wash it off with flowing tap water.
  3. Now, decolorized it by applying 20% sulphuric acid and wait for 1-2 minutes. Repeat this step again and again until it turns pink in color.
  4. Wash off the decolorizer by tap water.
  5. Next, flood the smear with counterstain, the methylene blue dye, and wait for 2-3 minutes.
  6. Wash the counterstain by water.
  7. Dry the smear in air.
  8. Now smear in ready to observe under microscope.
ReagentAcid FastNon-Acid Fast
Carbol Fuchsin with heatRed (Hot Pink)Red (Hot Pink)
Acid AlcoholRedColorless
Methylene Blue/Malachite GreenRedBlue/Green
Ziehl Neelsen Stain Steps
Ziehl Neelsen Stain Steps | Created with

Result of Ziehl-Neelsen Staining or acid-fast staining

  • Acid-fast bacteria: These bacteria retain the color of primary stain, carbol-fuschin, and appear pink.
  • Non-acid fast: They take the color of methylene blue dye and appear blue.
Ziehl Neelsen Stain Result
Ziehl Neelsen Stain Result | This image modified from and modified by

Modified Ziehl Neelsen Staining Technique

  1. The smear can be treated with 95% alcohol as a secondary decolorizer after the treatment with primary decolorizer, sulfuric acid. Because there are few microorganism, which has both acid-fast and alcohol fast characteristics, such as Mycobacterium tuberculosis.  Whereas the saprophytic mycobacterium is only acid fast.
  2. 3% HCl and 95% alcohol can be used as a decolorizer instead of 20% sulfuric acid. This too distinguishes tubrcle bacilli from saprophytic mycobacteria. It is mainly used in the determination of renal tuberculosis.
  3. Modifications in the percentage of sulfuric acid; 5% H2SO4 for M.leprae, 1% H2SO4 for Actinomyces in tissue, 0.5% H2SO4 for cultures of Nocardia, 0.25-0.5% for spores and for oocysts of Cryptosporidium and Isospora,
  4. Where heating is not employed, we can use the cold method, by increasing the concentration of phenol and the duration of staining instead of heating (Kinyoun’s modification, Gabbett’s modification ).

Examples of Acid-fast

Nocardia spp (Partial Acid Fast), Rhodococcus spp (Partial Acid Fast), Legionella micdadei (Partial Acid Fast in tissue), Cyst of Cryptosporidium (Acid Fast), Cyst of Isospora (Acid Fast)

Importance of Ziehl-Neelsen Staining 

  • Used in identification of Mycobacterium species.
  • Helps in differentiate between acid-fast and non-acid fast bacilli.
  • To identify some fungal species i.e Cryptosporidium.
  • Can help differentiate and identify fungi ,i.e Histoplasma.



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