Acid Fast Staining

Acid-fast staining was first introduced by a scientist named Paul Ehrlich in the year 1882. Later, this method was modified by Ziehl and Neelson in the year 1883. Thus, acid-fast staining sometimes refers to Ziehl and Neelson staining. It is a type of differential staining method, which is used to distinguish between the acid-fast and non-acid fast bacteria. Mycobacterium is an acid-fast bacteria which retains the colour of Carbol fuschin even by the addition of acid alcohol. The mycobacterium species retain the colour of the primary stain because they contain mycolic acid in their cell wall.

Mycolic acid is a waxy substance that does not allow the decolourizer to enter into the cell wall due to its waxy nature. Therefore, acid-fast staining is used to differentiate the mycobacterium species from the other groups of bacteria. Non-acid fast bacteria quickly lose the colour of the primary stain due to the absence of mycolic acid and appears blue.

Content: Acid Fast Staining

  1. Definition of Acid-Fast Staining
  2. Components of Acid-Fast Stain
  3. Principle
  4. Process
  5. Observation
  6. Interpretation of Result

Definition of Acid-Fast Staining

Acid-fast staining can define as one of the staining methods which differentiate the Mycobacteria species from the other bacterial groups based on the staining and cell wall differences. The cell wall of Mycobacterium species contains mycolic acid, which makes it resistant towards the effect of acid decolourizer and thus refers as “Acid-fast bacteria”. In contrast to this, non-acid fast bacteria lacks mycolic acid in their cell wall and loose up the colour of the primary stain.

The positive result of acid-fast staining indicates the presence of acid-fast bacteria, which appears red. The negative result indicates the presence of non-acid fast bacteria which appears blue.

Examples of acid-fast bacteria: Mycobacterium tuberculosis, M. leprae, M. smegmatis, M. phlei etc.

Examples of non-acid fast bacteria: Escherichia coli, Staphylococcus aureus etc.

Components of Acid-Fast Stain

Acid-fast staining is a differential staining procedure, which uses the combination of three reagents like:

components of acid fast stain

  1. Ziehl Neelson Carbol fuschin
  2. Acid alcohol
  3. Loeffler’s Methylene blue

Ziehl Neelson Carbol Fuschin (ZNCF)

It uses as a Primary stain. To stain a mycobacterium, one needs a special stain like ZNCF. An ordinary stain cannot stain the mycobacterial cell. ZNCF is composed of a phenolic base and a Carbol fuschin dye. A phenolic base of ZNCF shows high affinity towards the lipid content.

Thus, ZNCF dye can solubilize the lipoidal material in the cell wall and stain the cell red. Due to high lipid content, a mycobacterial cell wall is less permeable. So, a phenol base increases the cell permeability by which a cell allows the stain to penetrate.

Acid Alcohol

It uses as a decolourizing agent, which contains 3% of HCL along with 95% ethanol. It plays an essential role in the identification of acid-fast and non-acid fast organisms. Acid-fast bacteria contains high lipid content, which prevents the cell from binding with stains and decolourizers.

Thus, acid-fast bacteria will retain the colour of the primary stain and appears red. In contrast to this, a non-acid fast bacteria lacks such a large amount of lipid content, as a result of which a cell loses the colour of primary stain and decolourizes.

Methylene Blue

It uses as a counterstain, which consists of 3% of methylene blue. Methylene blue stains the decolourized cells of non-acid fast bacteria and make it appear blue. Unlike non-acid fast, an acid-fast bacteria will not take up the colour of methylene blue and will appear red.


Acid-fast staining is based upon the principle of staining of the bacterial cell relative to their cell wall differences. The cells of Mycobacteria species appears red whereas non-acid fast bacteria appear blue. A smear is subjected to heat after staining with Zeihl Neelson Carbol fuschin. During steam, Carbol fuschin penetrates the cytoplasm into the bacterial cell. After 2-3minutes, all the cells appear red, because a primary stain (Carbol fuschin) is added at several internal during the steaming process, to prevent cell drying.

After this, a decolourizer (3% HCl) is added to the smear. Now, this is the essential step which helps us to distinguish between the mycobacteria and the other bacteria. The mycobacteria will resist the effect of decolourizer, because of the large amount of lipoidal material or mycolic acid in their cell wall. Thus, a mycobacterium refers as “Acid-fast bacteria” as they don’t allow the penetration of the acid decolourizer into the cell and remains red.

On the contrary, other bacteria will not resist the effect of decolourizer because of these lack lipoidal content. Therefore, the other bacteria will refer as “Non-acid fast bacteria”, as a decolourizer enter into the cell and create pores in the cell wall. thus, a primary stain leaks out of the cell wall, leaving a non-acid fast cell colourless.

cell wall of acid fast bacteria

At last, counterstain a smear with methylene blue. During this stage, only the decolourized or non-acid fast cells will take the blue colour of methylene blue stain. In opposite to this, the acid-fast cells will remain red in colour.


To carry out the process of acid-fast staining, we require the following:
Requirements: Clean glass slide, slide holder, bacterial suspension, inoculating loop, Bunsen burner, distilled water, water bath, ZNCF, acid alcohol, Loeffler’s methylene blue, oil immersion and microscope.

The process includes the following steps:

process of acid fast staining

  • Take a clean, sterilized slide.
  • Then, add a drop of distilled water on the centre of the glass slide. After doing this, thoroughly mix the inoculum by a sterilized inoculating loop. Then, heat fixes the smear and air dry the slide.
  • Flood the smear with the primary stain (Carbol fuschin) and allow it to stand for 1minute.
  • Then flood the smear with the decolourizer (3% HCl) and then wash the glass slide with distilled water.
  • Finally, flood the smear with the counterstain (Loeffler’s methylene blue) and allow it to stand of 1minute. Subject the glass slide to the water wash treatment.
  • Air-dry the glass slide.
  • Observe the glass slide under oil immersion at 100X objective.


An observation table is given below:
observation of acid fast stainsWithout staining, the bacteria appear colourless. So, it becomes necessary to stain the bacteria to identify and classify them based on their physical and chemical differences. By the addition of primary stain, i.e. ZNCF, both the acid-fast bacteria and non-acid fast bacteria appear red.

Then, by the addition of acid decolourizer, non-acid fast bacteria lose the colour of primary stain and appears colourless whereas acid-fast bacteria appear red. At last, the colourless non-acid fast cells take up the colour of methylene blue and appears blue. The acid-fast bacteria will remain red by the addition of methylene blue.

Interpretation of Result

introduction image
Acid-fast cells: These appear red coloured, rod shaped and can occur singly or in small groups.
Non-acid fast cells: These appear blue coloured.

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