ZIEHL-NEELSEN STAIN: RATIONALE, REAGENTS, AND TECHNIQUE - BIOLOGY - 2025

The Ziehl-Neelsen a coloring technique to identify microorganisms resistant alcohol acid (ARA). The name of this microbiology procedure refers to its authors: bacteriologist Franz Ziehl and pathologist Friedrich Neelsen.

This technique is a type of differential staining, which implies the use of different dyes in order to create contrast between the structures that you want to observe, differentiate and later identify. The Ziehl-Neelsen stain is used to identify certain types of microorganisms.

Ziehl-Neelsen stain

Some of these organisms are mycobacteria (eg, Mycobacterium tuberculosis), nocardia (eg, Nocardia sp.), And some unicellular parasites (eg, Cryptosporidium parvum). Many of the bacteria can be classified through a common technique called a Gram stain.

However, some bacterial groups require other methods to be able to identify them. Techniques such as the Ziehl-Neelsen stain require combinations of dyes with heat to fix the former to the cell wall.

Then comes a bleaching process that allows for two results: resistance or sensitivity to discoloration by acids and alcohols.

Basis

The rationale for this staining technique is based on the properties of the cell wall of these microorganisms. The wall is made up of a type of fatty acids called mycolic acids; These are characterized by having very long chains.

When fatty acids have very long structures, they can retain dyes more easily. Some bacterial genera are very difficult to stain by Gram stain, due to the high content of mycolic acids in the cell wall.

The Ziehl-Neelsen stain uses the phenolic compound carbol fuchsin, a basic stain. This has the ability to interact with the fatty acids of the cell wall, which is waxy in texture at room temperature.

Carbol fuchsin staining is enhanced in the presence of heat, as the wax melts and the dye molecules move more rapidly into the cell wall.

The acid that is used later serves to discolor cells that were not stained because their wall was not sufficiently related to the dye; therefore, the strength of the acid bleach is able to remove the acid dye. Cells that resist this discoloration are called acid-fast.

Secondary colorant

After decolorization of the sample, it is contrasted with another dye called secondary dye. Generally, methylene blue or malachite green is used.

The secondary dye stains the background material and consequently creates contrast to the structures that were stained in the first step. Only discolored cells absorb the second dye (counterstain) and take on their color, while acid-fast cells retain their red color.

This procedure is frequently used for the identification of Mycobacterium tuberculosis and Mycobacterium leprae, which are called acid-fast bacilli.

Reagents

Primary colorant

0.3% carbol fuchsin (filtered) is used. This dye is prepared from a mixture of alcohols: phenol in ethanol (90%) or methanol (95%), and in this mixture 3 grams of basic fuchsin are dissolved.

Bleaching solution

In this step, solutions of 3% alcoholic acid or 25% sulfuric acid can be used.

Secondary dye (counter-dye)

The dye most used to contrast the samples is usually 0.3% methylene blue. However, others can also be used, such as 0.5% malachite green.

Technique

Acid-fast staining procedure

Prepare a bacterial smear

This preparation is done on a clean, dry slide, following sterility precautions.

Smear drying

Allow the smear to dry at room temperature.

Heat the sample

The sample should be heated by applying fire to the slide below. An alcohol fixation can be done when the smear has not been prepared with sputum (treated with sodium hypochlorite to whiten it) and if it is not going to stain immediately.

M. tuberculosis is removed with bleach and during the staining process. Heat fixation of untreated sputum will not kill M. tuberculosis, while alcohol fixation is bactericidal.

Cover the stain

The stain is covered with the carbol fuchsin solution (primary basic stain).

Heat the stain

This is done for 5 minutes. You should notice an evolution of steam (approximately 60 ° C). It is important not to overheat and avoid burning the sample.

With regard to heating the stain, great care must be taken when heating the carbol fuchsin, especially if the staining is carried out on a tray or other container in which highly flammable chemicals from the previous staining have been collected.

Only a small flame should be applied under the slides using a previously lit swab moistened with a few drops of acidic alcohol, methanol, or 70% ethanol. Avoid using a large swab soaked in ethanol as this is a fire hazard.

Wash the stain

This wash must be done with clean water. If the tap water is not clean, wash the smear with filtered or distilled water, preferably.

Cover the smear with acid alcohol

This acid alcohol should be at 3%. Coverage is carried out for 5 minutes or until the smear is sufficiently discolored, i.e. pale pink in color.

It must be taken into account that acidic alcohol is flammable; therefore, it must be used with great care. Avoid being near sources of ignition.

Wash the stain

Washing should be with clean, distilled water.

Cover the smear with stain

It can be malachite green (0.5%) or methylene blue (0.3%) stain for 1 to 2 minutes, using the longer time if the smear is thin.

Wash the stain

Again clean (distilled) water should be used.

To drain

The back of the slide should be cleaned and the stain placed on a drainage rack to allow it to air dry (do not use absorbent paper for drying).

Examine the smear under the microscope

The 100X objective and immersion oil must be used. Scan the smear systematically and record the pertinent observations.

Interpret the results

Theoretically, microorganisms that stain a reddish color are considered acid-fast positive (AAR +).

On the contrary, if the microorganisms stain blue or green, depending on the dye used as a counter-dye, they are considered acid-fast negative (AAR-).

References

1. Apurba, S. & Sandhya, B. (2016). Essentials of Practical Microbiology (1st ed.). Jaypee Brothers Medical Publishers.
2. Bauman, R. (2014). Microbiology with Diseases by Body System (4th ed.). Pearson Education, Inc.
3. Heritage, J., Evans, E. & Killington, A. (1996). Introductory Microbiology (1st ed.). Cambridge University Press.
4. Morello, J., Granato, P. Wilson, M. & Morton, V. (2006). Laboratory Manual and Workbook in Microbiology: Applications to Patient Care (11th ed.). McGraw-Hill Education.
5. Vasanthakumari, R. (2007). Textbook of Microbiology (1st ed.). BI Publications PVT.