Ziehl-Neelsen Stain: Introduction, Principle, Procedure, Result Interpretatio

Ziehl-Neelsen Stain: Introduction, Principle, Procedure, Result Interpretation and Keynotes

Introduction of Ziehl-Neelsen Stain

This acid-fast bacilli in brief AFB staining or Ziehl-Neelsen staining method is a modification of Ehrlich’s (1882) method. Ziehl-Neelsen Stain name is from the surnames of German doctors, bacteriologist Franz Ziehl(1859-1926), and pathologist Friedrich Neelsen (1854-1898).

Principle of Ziehl-Neelsen Stain

The presence of higher alcohol, glycerol, fatty acid, and especially mycolic acid in the cell wall has been found responsible for keeping the acid-fast property of bacteria. Therefore, AFB staining is useful for Mycobacterium tuberculosis, an etiological agent of tuberculosis.

Requirements for Ziehl-Neelsen Stain

a) Compound light microscope

b) Reagents and glasswares

Bunsen flame
Wire loop
Clean grease-free slides
Marker pen
Sprit lamp
Carbol fuchsin
20% Sulphuric acid
Methylene blue

c) Specimens

In the case of primary tuberculosis

Sputum
Bronchial or laryngeal washing
Gastric lavage when sputum is swallowed as in children

In miliary tuberculosis

Bone marrow
Liver biopsy

Tuberculous meningitis

Cerebrospinal fluid (CSF)

Renal tuberculosis

urine

d) Quality control strains

Positive control (PC): Mycobacterium tuberculosis
Negative Control: Escherichia coli

Procedure of Ziehl-Neelsen Stain

Make smear on a clean glass slide.
Dry and fix the smear.
Cover the smear with a strong carbol fuchsin solution.
The heat from underneath the slide until just steam comes from the stain. Do not boil.
Wait for five minutes.
Rinse with water.
Decolorize by 20% sulphuric acid or 3% acid alcohol until the smear becomes pale pink in color. (wait for nearly five minutes)
Rinse with water.
Counterstain with methylene blue for one minute.
Rinse with water.
Drain and dry.
Observe the smear first under the low power (10X) objective, and then under the oil immersion (100X) objective.

Result and Interpretation of Ziehl-Neelsen Stain

AFB: pink or red bacillus

Background: Blue ( as counterstain used )

Reporting of Ziehl-Neelsen Stain

There are various ways of a reporting system for AFB stainings such as the Center for Disease Control and Prevention (CDC), the World Health Organization (WHO), and the International Union Against Tuberculosis and Lung Disease (IUATLD). The most common and widely accepted classification is IUATLD and according to it as follows.

No organism seen: Negative
1-9/100 OIF ( oil immersion field): Exact number
10-99/100 OIF: +
1-10/OIF : ++
10/OIF: +++

Keynotes on Reporting of Ziehl-Neelsen Stain

5% sulphuric acid uses as a decolorizing agent for staining Mycobacterium lepare.
1% sulphuric acid uses as a decolorizing agent for staining Nocardia, Cryptosporidium, Isospora oocyst.
Kinyoun’s modification of Ziehl-Neelsen is preferred for staining coccidian parasites like Cryptosporidium, Cyclospora, Isospora oocysts.
0.25% sulphuric acid uses as a decolorizing agent for staining spores.
The list of acid-fast bacteria are Mycobacterium tuberculosis, Mycobacterium lepare, Mycobacterium smegmatis, Mycobacterium fortitum, Nocardia asteroids, Nocardia brasiliensis, Nocardia caviae.
Acid-fast positive parasites are Cryptosporidium parvum, Cyclospora cayetanensis and Isospora belli.
Fungal spores are also acid-fast positive.
Spermatozoa head is acid-fast positive.
The use of a 3% acid alcohol makes Mycobacterium smegmatis acid-fast negative while 20% sulphuric acid can’t do this. This is the reason, in a 24 hours urine sample a 3% acid alcohol decolorizer is preferred.

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