Ziehl -Neelsen Stain: Introduction, Principle, Test Requirements, Staining Procedure, Result Interpretation, Keynotes, and Z-N Stained Smear Footages Collections

Ziehl -Neelsen Stain

Ziehl–Neelsen stain is a type of acid-fast stain and it is named after the surnames of two German doctors who modified the stain. One is the bacteriologist Franz Ziehl (1859–1926) and other is the pathologist Friedrich Neelsen (1854–1898). It was initially introduced by Paul Ehrlich but his stain was not able to penetrate the acid-fast bacilli. It is most commonly used to stain Mycobacterium tuberculosis in a variety of specimens like sputum, urine, pus, CSF, etc. Its various modification is available according to the nature of testing microbes. Original acid-fast stain for M. tuberculosis is also called hot stain (due to applying heat) while for Mycobacterium leprae is called cold stain since it needs heating. Similarly for staining Nocardia, oocysts of coccidian parasites another type of modification is needed.

Principle of Ziehl -Neelsen Stain

The presence of higher alcohol, glycerol, fatty acid, and especially mycolic acid in the cell wall has been found the conduct to keeping the acid-fast property of bacteria. This is the reason, the Ziehl-Neelsen stain (AFB stain) is useful for Mycobacterium tuberculosis, an etiological agent of tuberculosis. Acid-fast structures take primary stain pink or red while non-acid fast takes counter stain blue ( in case of methylene blue) or green ( in case of malachite green).

Requirements

a) Compound light microscope

b) Reagents and glassware

• Bunsen flame/Sprit lamp
• Wire loop
• Clean grease-free slides
• Marker pen/ diamond pencil
• Carbol fuchsin (primary stain)
• 20% Sulphuric acid or 3 % acid alcohol
• Methylene blue (counter stain or secondary stain)

c) Specimens

In the case of primary tuberculosis

• sputum ( We used this specimen for staining as shown above image.)
• 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 (NC): Escherichia coli

Procedure of Ziehl -Neelsen Stain

1. Prepare smear on a clean glass slide.
2. Dry and fix the smear.
3. Flood the smear with a primary stain, strong carbol fuchsin solution.
4. Heat the preparation from underneath the slide until just steam comes from the stain. Remember-Do not boil.
5. Wait for five minutes.
6. Rinse with water.
7. Decolorize by 20% sulphuric acid or 3% acid alcohol until the smear becomes pale pink in color. (wait for nearly five minutes)
8. Rinse with water.
9. Cover the smear with counterstain, methylene blue for one minute.
10. Rinse with water.
11. Drain and dry.
12. Focus the smear first under the low power (10X) objective, and then observe under the oil immersion (100X) objective of compound microscope.

Result and Interpretation

Acid Fast Bacilli (AFB): pink or red bacillus

Background:  Blue (as counterstain used )

Reporting

There are various ways of reporting systems for Ziehl-Neelsen staining findings 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+)
• 1-10/OIF : ++ (2+)
• 10/OIF: +++ (3+)

Keynotes

1. 5% sulphuric acid is used as a decolorizing agent for staining Mycobacterium lepare while 1% sulphuric acid uses as a decolorizing agent for staining Nocardia, Cryptosporidium, Isospora oocyst.
2. Kinyoun’s modification of Ziehl-Neelsen is preferred for staining coccidian parasites like Cryptosporidium, Cyclospora, Isospora oocysts.
3. 0.25% sulphuric acid is applied as a decolorizing agent for staining spores.
4. Acid-fast bacterial list contains Mycobacterium tuberculosis, Mycobacterium lepare, Mycobacterium smegmatis, Mycobacterium fortitum, Nocardia asteroids, Nocardia brasiliensis, Nocardia caviae.
5. Acid-fast positive parasites are Cryptosporidium parvum, Cyclospora cayetanensis and Isospora belli.
6. Fungal spores are also acid-fast positive.
7. Spermatozoa head is acid-fast positive.
8. The application 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.
9. Composition of Acid-Fast Stain and Reagent are as follows-

Carbol Fuchsin

• Basic Fuchsin: 0.3 gm
• Ethyl alcohol,95%: 10.0ml
• Phenol: 5.0 ml
• Distilled Water: 95.0ml

Acid Fast Decolorizer

• Hydrochloric acid concentrated: 3.0 ml
• Ethyl alcohol,95%: 97 ml

Methylene Blue (Loeffler’s)

• Methylene Blue:0.3 gm
• Ethyl alcohol,95%:30.0 ml
• Distilled Water:100.0ml

Ziehl -Neelsen Stained Smear Footage Collection

Mycobacterium leprae bacilli in modified Ziehl-Neelsen (Cold stain) slit skin smear Microscopy

Sputum in Ziehl-Neelsen stained smear exhibiting Mycobacterium leprae

Acid Fast Bacilli (AFB) of Mycobacterium tuberculosis in Ziehl-Neelsen stained sputum smear showing pink beaded, thin slender rod with sometime curved

Numerous Acid Fast Bacilli (3+) of Mycobacterium tuberculosis in Clinical Specimen

Plenty of Mycobacterium tuberculosis acid fast bacilli in Ziehl-Neelsen stained smear of sputum Microscopy

Nocardia asteroides in modified Ziehl-Neelsen stained smear of clinical specimen Microscopy

Actinomyces in Ziehl-Neelsen stained smear showing acid fast negative

Fungus in Acid Fast Stained Smear showing Yeast cells and pseudohyphae

Cyclospora cayetanensis in modified Ziehl-Neelsen stained smear of feces

Contd…

Further Reading

1. Bailey & Scott’s Diagnostic Microbiology. Editors: Bettey A. Forbes, Daniel F. Sahm & Alice S. Weissfeld, 12th ed 2007, Publisher Elsevier.
2. Textbook of Diagnostic Microbiology. Editors: Connie R. Mahon, Donald G. Lehman & George Manuselis, 3rd edition2007, Publisher Elsevier.
3. Clinical Microbiology Procedure Handbook Chief in editor H.D. Isenberg, Albert Einstein College of Medicine, New York, Publisher ASM (American Society for Microbiology), Washington DC.
4. Manual of Clinical Microbiology. Editors: P.R. Murray, E. J. Baron, M. A. Pfaller, F. C. Tenover and R. H. Yolken, 7th ed 2005, Publisher ASM, USA
5. Mackie and Mc Cartney Practical Medical Microbiology. Editors: J.G. Colle, A.G. Fraser, B.P. Marmion, A. Simmous, 4th ed, Publisher Churchill Living Stone, New York, Melborne, Sans Franscisco 1996.