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ZN Staining Procedure: Principle, Method and Results.

Microorganisms in the genus Mycobacteria and Nocardia have complex cell walls with higher lipid content. The cell wall of these microbes contains mycolic acid, which is a complex hydrocarbon. Due to this waxy cell wall, bacteria don’t take up the stain easily. Therefore, microorganisms with a waxy cell wall are difficult to stain with routine staining methods such as gram staining or simple staining. To visualize such bacteria special staining method such as Ziehl Neelsen acid fast stain (ZN Stain) is very useful.

The Ziehl-Neelsen (ZN) staining technique is a differential staining technique. First, it was developed by Ziehl and later modified by Neelsen, therefore, this staining technique is named as Ziehl-Neelsen staining. Since we use heat in this procedure, the Ziehl-Neelsen Staining technique is also known as Hot Method of Acid-Fast Staining

The primary goal of this staining method is to distinguish bacteria into acid-fast and non-acid-fast groups.

Z N Stain Principle:

Mycolic acids in cell wall makes the bacterial cell surface waxy. Staining such waxy cell surfaces with aqueous-based stains like the Gram stain is difficult as mycolic acids don’t allow stain penetration.

In ZN staining the smear is first stained with primary stain Carbol Fuchsin. Moderate heat allows the stain to penetrate in the waxy cell wall surface.

Once the Carbol Fuchsin binds to mycolic acids in the cell wall it is not removed even with a strong decolorizer such as an acid alcohol solution.

This property of bacterial cell wall to retain primary stain Carbol fuchsin even after washing with a strong decolorizer (Acid in alcohol Solution) is known as acid fastness. Such bacteria are acid-fast and take up the red color of Crabol Fuschin.

In the next step, the smear is counterstained with secondary dye methylene blue. The bacteria which are decolorized with acid alcohol solution are non-acid fast in nature. only such non-acid fast bacteria take up counterstain methylene blue and appear blue in color.

Reagents for ZN Staining:

The main reagents in ZN staining are Carbol fuchsin, acid alcohol solution, and methylene blue.

Carbol fuchsin:

It is the primary stain. Most species of bacterial cells are stainable with standard aqueous stains like methylene blue and crystal violet. Similarly, mycobacteria are not stainable with common dyes like methylene blue and crystal violet. These bacteria are stainable with carbol fuchsin, a dark red stain prepared in 5% phenol.  Heat enhances the penetration of carbol fuchsin through the lipoidal waxy cell wall.
Carbol fuchsin preparation requires distilled water, basic fuchsin, ethyl alcohol, and phenol crystals.

Distilled water- 100ml

Basic fuschin- 1g

Ethyl alcohol (100% ethanol)- 10ml

Phenol crystals- 5ml

2. Acid alcohol solution:

Acid alcohol acts as a decolorizer, i.e., It removes the stain bound to cell surface.
Decolorizer Acid alcohol Solution preparation: (3 percent HCl in 95 percent ethyl alcohol)

Ethyl alcohol- 95 ml

Distilled water- 2 ml

Concentrated hydrochloric acid- 3 ml

3. Methylene blue:

It is the secondary stain which acts as counterstain.

Counterstain Methylene blue preparation:

0.25% methylene blue in 1% acetic acid

Methylene blue- 0.25g

Distilled water- 99ml

Acetic acid- 1ml

Materials required for ZN staining

1. Bacterial culture / sputum sample

2. Clean glass slide

3. Spirit lamp

4. ZN stain reagents

5. Microscope with 100x objective

6. Cedarwood oil or liquid paraffin oil

ZN Staining Procedure:

Image Source: Elizabeth Gray.

Smear Preparation:

1. Take a grease-free and clean glass slide.

2. Aseptically make a smear on the glass slide using the sputum sample or bacterial culture

3. Quickly pass the slide with the smear through the flame for 2-3 times and heat fix the smear or air dry.

Ziehl Neelsen Staining:

1. Flood the smear with primary stain carbon fuchsin. Keep the slide on steam for stain penetration. wait for 5 minutes

2. Allow the slide to cool and wash with tap water.

3. Now flood the smear with decolorizer (Acid alcohol) Solution until the alcohol runs almost clear.

4. Wash the smear on the slide with tap water.

5. Now counterstain the smear with methylene For about 2 minutes.

6. Wash the smear with tap water

7. Blot dry the slide and examine under oil immersion.

Results and Observation :

ZN Staining results

Figure: Mycobacterium tuberculosis visualization using the Ziehl–Neelsen stain. Image Source: CDC/Dr. George P. Kubica.

Acid-fast organisms, Mycobacterium spp., will appear pink-red colored

Nonacid-fast organisms will appear blue.

In addition, background material should stain blue because of methylene blue

Z N Stain Uses:

1. To examine, identify, and classify different Mycobacterium species.

2. To distinguish between acid-fast and non-acid-fast bacilli.

3. For identification of some fungal species, such as Cryptosporidium.

4. The main use of Z N stain is for the diagnosis of pulmonary tuberculosis from sputum samples of patient.

FAQ on ZN staining

Decoloring agent is nothing but Decolorizer. Acid alcohol solution is used as decoloring agent. 20% H2SO4 also can be used.
In ZN staining Moderate heat is applied after addition of Carbol fuchsine. This heat allows penetration of carbol fuchsine stain inside waxy lipoidal cell wall.
In ZN staining, bacterial smear is stained with carbol fuchsin and phenol in this procedure. The carbol fuchin stain binds to mycolic acid in the cell wall of mycobacteria. Following staining, an acid alcohol is added as a decolorizing agent to remove the stain from the cells. All organisms like mycobacteria retain the dye and are hence known as acid fast bacilli, or simply AFB. Following decolorization, the smear is counter stained with malachite green or methylene blue, which dyes the background and decolorised non acid fast cells in green or blue, allowing the red AFB to be seen.
Acid fast staining is a differential staining method used to distinguish acid fast bacteria from non-acid fast bacteria. Mycobacterium species have a high concentration of mycolic acid in their cell walls. Because of the high lipid content, most dyes cannot easily pass through the cell wall, hence Gram staining cannot be used to detect Mycobacterium species. To stain such organisms, an unique staining procedure known as acid-fast staining is created. It is often referred to as the Ziehl-Neelsen technique.
ZN staining is also known as Hot method Acid fast staining method
The property of bacterial cell wall to retain primary stain Carbol fuchsin even after washing with a strong decolorizer (Acid in alcohol Solution) is known as acid fastness
1. To examine, identify, and classify different Mycobacterium species. 2. To distinguish between acid-fast and non-acid-fast bacilli. 3. For identification of some fungal species, such as Cryptosporidium. 4. The main use of Z N stain is for the diagnosis of pulmonary tuberculosis from sputum samples of patient.
1. Acid-fast organisms, Mycobacterium spp., will appear pink-red colored 2. Nonacid-fast organisms will appear blue. In addition, background material should stain blue because of methylene blue
ZN staining is a differential staining method.
Carbol fuchsin (Primary Stain) Acid Alcohol Solution (Decolorizer) Methylene blue. (Secondary Stain)

Solve MCQ on ZN staining

Check your understanding on ZN staining procedure

https://rbrlifescience.com/qsm_quiz/zn-staining/

Which organisms are stained with ZN stain?

Why heat is used in ZN staining?

Which mordant is used in ZN staining?

Which organism is detected by the Ziehl-Neelsen method?

Which stain is used to diagnose tuberculosis?

Which type of bacteria appear red in Ziehl-Neelsen staining method?

Which is the primary stain in ZN staining?

Which reagents is used as counterstain in zn staining

Zn staining is also known as

In zn staining non acid fast bacteria get decolorised with acid alcohol and take up the counterstain. After successful zn staining non acid fast bacteria appear as

ZN staining is ______________ method of acid fast staining.

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