Ziehl-Neelsen stain: Foundation, Reagents and Technique

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

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

Ziehl-Neelsen stain

Some of these microorganisms are mycobacteria (for example, Mycobacterium tuberculosis ), nocardias (for example, Nocardia sp.) and some unicellular parasites (for example, Cryptosporidium parvum ). Many of the bacteria can be classified through a common technique called Gram stain.

However, some bacterial groups require other methods to identify them. Techniques such as Ziehl-Neelsen staining require combinations of dyes with heat to fix the first to the cell wall. Then comes a decolorization process that allows two results: resistance or sensitivity to discoloration by acids and alcohols.

Basis

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

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

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

The carbol fuchsin stain is improved in the presence of heat, because the wax melts and the dye molecules move more rapidly into the cell wall.

The acid that is used later serves to discolor the cells that were not stained because their wall was not sufficiently related to the colorant; therefore, the strength of the acid decolorizer is capable of removing the acid dye. The cells that resist this discoloration are called acid-resistant.

Secondary coloring

After the discoloration of the sample, this is contrasted with another dye called secondary dye. Methylene blue or malachite green is generally used.

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

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

Reagents

Primary coloring

Carboxin 0.3% 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.

Decolorizing solution

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

Secondary coloring (anti-colorant)

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

Technique

Acid-fast staining procedure

Prepare a bacterial smear

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

Drying the smear

Allow the smear to dry at room temperature.

Heat the sample

The sample must be heated by applying fire to the slide below. A fixation with alcohol 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 be stained immediately.

M. tuberculosis It is eliminated with bleach and during the staining process. Heat-setting of untreated sputum will not kill M. tuberculosis , whereas fixation with alcohol 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 a vapor release (approximately 60 ° C). It is important not to overheat and avoid burning the sample.

With regard to the heating of 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 lighted swab previously moistened with a few drops of acid alcohol, methanol or 70% ethanol. Avoid using a large swab soaked in ethanol because this is a fire hazard.

Wash the stain

This washing should 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, that is, pale pink.

It must be taken into account that acid alcohol is flammable; therefore, it should be used very carefully. Avoid being near sources of ignition.

Wash the stain

The washing should be with clean, distilled water.

Cover the smear with dye

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

Wash the stain

Again clean water (distilled) must be used.

To drain

The back of the slide should be cleaned and the stain should be placed on a drainage shelf, so that it is air-dried (do not use absorbent paper for drying).

Examine the smear under the microscope

The 100X objective and the immersion oil should be used. Scan the smear systematically and write down the relevant observations.

Interpret the results

Theoretically, microorganisms that are dyed a reddish color are considered acid-fast positive (AAR +). On the contrary, if the microorganisms are stained blue or green, depending on the dye used as a counter-dye, they are considered negative alcohol-resistant acid (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.