Ziehl-Neelsen Staining (ZN Staining) – Principle, Procedure and Results

Ziehl-Neelsen Staining (ZN Staining) is a laboratory staining technique used in microbiology for the microscopic examination of specimen smears. In the general sequence, a smear is first prepared from the specimen and fixed on a glass slide. The fixed smear is stained with carbol fuchsin, which is heated until steam just begins to rise. The smear is then decolorized with an acid decolorizer and stained with a counterstain. The stained slide is finally examined under the light microscope.

The reagent concentrations, heating duration and staining times differ between laboratory protocols. The exact conditions depend upon the validated procedure followed in a particular laboratory.

In clinical laboratory practice, ZN staining is commonly carried out on sputum specimens and other clinical samples. Suitable laboratory biosafety procedures are required for the examination of suspected infectious specimens.

What is Ziehl-Neelsen Staining (ZN Staining)?

Ziehl-Neelsen staining (ZN staining) is a differential acid-fast staining technique used to detect acid-fast microorganisms, particularly mycobacteria. It is used to differentiate acid-fast organisms from non-acid-fast organisms. Mycobacteria have a cell envelope rich in mycolic acids, which is responsible for their acid-fast nature.

In Ziehl-Neelsen staining, carbol fuchsin is used as the primary stain along with heat. It is also known as the hot acid-fast staining method because heat is used during staining. The term ZN stain refers to Ziehl-Neelsen staining.

Ziehl-Neelsen staining is one of the acid-fast staining methods. Other methods include Kinyoun staining and fluorochrome staining.

Objectives of Ziehl-Neelsen Staining

The following are the objectives of Ziehl-Neelsen staining:

1. To demonstrate the presence of acid-fast microorganisms, particularly mycobacteria.
2. To differentiate acid-fast organisms from non-acid-fast organisms and other materials present in the smear.
3. To detect acid-fast bacilli (AFB) in stained specimen smears under a microscope.
4. To help in the microbiological examination of specimens from suspected mycobacterial infections.

Principle of Ziehl-Neelsen Staining

The principle of Ziehl-Neelsen staining is based on the ability of acid-fast bacteria to retain carbol fuchsin even after treatment with acid or acid-alcohol. Mycobacteria contain a high amount of mycolic acids in their cell envelope, which gives it a waxy nature. Due to this, these bacteria are difficult to stain with ordinary dyes. Once stained, they do not easily lose the primary stain during decolorization. This property is known as acid-fastness.

Carbol fuchsin contains basic fuchsin and phenol. Phenol acts as a mordant and helps in the penetration of the dye into the waxy cell envelope. In the conventional Ziehl-Neelsen method, heating or steaming also helps the carbol fuchsin enter the cells. Heat is used in the hot staining method, but cold acid-fast staining methods can be carried out without heating.

During decolorization, the acid-fast bacteria retain the red carbol fuchsin, whereas non-acid-fast cells lose the primary stain. Methylene blue is then used as a counterstain, which stains the decolorized cells and background material blue, providing a contrast with the acid-fast bacteria.

Reagents Used in Ziehl-Neelsen Staining

The following are the reagents used in Ziehl-Neelsen staining:

1. Carbol Fuchsin (Primary Stain)– Carbol fuchsin is the primary stain used in ZN staining. It contains basic fuchsin, phenol, alcohol and distilled water. Basic fuchsin gives the red colour, while phenol helps in the penetration of the dye into the waxy cell envelope of mycobacteria. It is used in 0.3% or 1% concentration, depending upon the staining method.
2. Decolorizer– It is used to remove the primary stain from non-acid-fast cells and other materials present in the smear. The commonly used decolorizers are 25% sulphuric acid and 3% acid-alcohol. Acid-alcohol is prepared using concentrated hydrochloric acid and ethanol.
3. Methylene Blue (Counterstain)– Methylene blue is used as the counterstain. It stains the decolorized cells and background materials blue. The acid-fast bacteria remain red. A 0.1% or 0.3% aqueous solution of methylene blue is used, depending upon the staining method.

Preparation of Ziehl-Neelsen Staining Reagents

The following method of reagent preparation is given in Annexure 7 of the Module for Laboratory Technicians (2005), published by the Central TB Division, Ministry of Health and Family Welfare, Government of India. It uses 1% carbol fuchsin, 25% sulphuric acid and 0.1% methylene blue.

1. Preparation of 1% Carbol Fuchsin Solution
   • Take 5 g of basic fuchsin, 25 g of phenol and 50 ml of methylated spirit.
   • Dissolve the basic fuchsin in methylated spirit by shaking the flask.
   • Melt the phenol and add it to the solution.
   • Heat the mixture gently in a water bath at about 60°C. Do not heat it directly over a flame.
   • Add distilled water to make the final volume 500 ml.
   • Filter the solution through Whatman No. 1 filter paper and store it in a labelled glass bottle.
   • If the basic fuchsin has 85% dye content, 5.88 g is required instead of 5 g.
2. Preparation of 25% Sulphuric Acid Solution
   • Take 375 ml of distilled water in a glass flask.
   • Slowly add 125 ml of concentrated sulphuric acid to the water.
   • Allow the solution to cool.
   • Store it in a labelled glass bottle. Water should never be added to concentrated acid.
3. Preparation of 0.1% Methylene Blue Solution
   • Take 0.5 g of methylene blue and 500 ml of distilled water.
   • Dissolve the methylene blue in distilled water by shaking the flask.
   • Store the solution in a labelled glass bottle.
   • For methylene blue having 82% dye content, 0.61 g is required instead of 0.5 g.

Phenol is toxic and corrosive. Concentrated sulphuric acid can cause severe burns. Suitable gloves, eye protection and a laboratory coat should be used during the preparation of these reagents. The solutions should be prepared with proper chemical ventilation. Methylated spirit is flammable and should not be heated directly over a flame.

The prepared reagents should be labelled with their names and dates of preparation. Carbol fuchsin should be filtered again if particles are formed in the solution. Each new batch of staining reagents should be checked using known positive and negative control smears.

The concentrations of staining reagents differ between laboratory methods. For example, the Médecins Sans Frontières ZN staining protocol uses 0.3% carbol fuchsin, 3% acid-alcohol and 0.3% methylene blue. The reagents should be prepared according to the validated method used in the laboratory, without mixing formulations from different protocols.

Procedure of Ziehl-Neelsen Staining

The following are the steps involved in Ziehl-Neelsen staining. The method is given in the Module for Laboratory Technicians (2005), published by the Central TB Division. It uses 1% carbol fuchsin, 25% sulphuric acid and 0.1% methylene blue.

Preparation and Fixation of Smear

1. Take a sputum specimen and select the thick, yellow purulent portion. Avoid the salivary portion of the specimen.
2. Take a new, clean, grease-free glass slide without scratches. Label the slide with the specimen identification number.
3. Transfer a small amount of sputum to the centre of the slide using a disposable wooden stick or loop. Spread it evenly over an area of about 2 × 2 cm.
4. The smear should not be too thick or too thin. The printed letters on a paper should be just visible through the smear.
5. Allow the smear to air-dry for 15–30 minutes. Do not dry it directly over a flame.
6. After drying, fix the smear by passing the slide over a flame 3–5 times, for 3–4 seconds each time. Avoid heating the slide for too long.
7. Use a separate stick or loop for each specimen to prevent contamination.

The suspected infectious specimens should be handled using suitable laboratory biosafety precautions. Gloves, a laboratory coat and proper ventilation are required. Avoid the formation of aerosols during smear preparation. Heat fixation does not completely kill all the mycobacteria present in the smear.

For other specimens, such as body fluids and tissues, the smear preparation and fixation methods may differ. Concentrated specimens may also require different preparation methods and additional biosafety precautions. These should be carried out according to the validated laboratory procedure.

Ziehl-Neelsen Staining Steps

1. Place the heat-fixed slide on a staining rack with the smear facing upwards.
2. Cover the entire smear with 1% filtered carbol fuchsin.
3. Heat the slide gently from underneath until vapours start rising. Do not allow the stain to boil or dry on the slide. The flame should not be kept at one place.
4. Allow the stain to remain on the slide for 5 minutes. Add more carbol fuchsin if the stain starts drying.
5. Wash the slide gently with tap water to remove the excess stain. Drain off the water.
6. Cover the smear with 25% sulphuric acid and allow it to stand for 2–4 minutes.
7. Wash the slide with tap water. If the smear is still red, repeat the decolorization with 25% sulphuric acid for another 1–3 minutes and wash again. The smear should appear light pink after decolorization.
8. Add 0.1% methylene blue to cover the smear and allow it to stand for 30 seconds.
9. Wash the slide gently with tap water to remove the excess methylene blue.
10. Drain off the water and allow the slide to air-dry completely. Do not wipe or blot the smear with tissue paper.

The staining time and concentration of reagents may differ in different laboratory protocols. The above method uses 1% carbol fuchsin and 25% sulphuric acid. Other validated methods may use different concentrations and staining times.

Microscopic Examination

1. Place the completely dried slide on the stage of a bright-field microscope.
2. At first, examine the slide under the 40× objective to select a suitable area of the smear.
3. Add a drop of immersion oil and examine the smear using the 100× oil-immersion objective. Use the fine adjustment knob to focus the smear.
4. Examine the microscopic fields systematically across the smear. At least 100 oil-immersion fields should be examined if no acid-fast bacilli are found initially.
5. Observe for red or pink, slender rod-shaped structures against the blue background. The acid-fast bacilli may be straight or slightly curved. Some may have a beaded appearance.

Results and Interpretation of Ziehl-Neelsen Staining

Positive Ziehl-Neelsen Staining Result

The acid-fast bacilli appear red or pink when the ZN-stained smear is examined under the oil-immersion objective of a light microscope. These bacilli are slender, rod-shaped and may be straight or slightly curved. Some bacilli have a beaded appearance. They may be present singly, in pairs or in small groups.

The presence of acid-fast bacilli in the smear is reported as an AFB-positive result. It indicates that acid-fast organisms are present in the specimen. However, the ZN staining result alone cannot confirm Mycobacterium tuberculosis infection.

Non-tuberculous mycobacteria can also give a positive ZN staining reaction. Therefore, other laboratory tests such as molecular testing or culture with species identification are required to identify Mycobacterium tuberculosis.

Negative Ziehl-Neelsen Staining Result

In a negative ZN stain, the non-acid-fast cells and other background materials appear blue due to methylene blue staining. No red or pink acid-fast bacilli are seen in the examined microscopic fields.

When no acid-fast bacilli are found in the smear, the result is reported as AFB negative. It does not mean that mycobacteria are absent from the specimen.

Sometimes the bacilli are present in very small numbers and cannot be detected under a microscope. Poor specimen quality or uneven distribution of bacilli in the smear may also give a negative result.

A negative ZN stain does not rule out tuberculosis infection. Further laboratory examination may be required when mycobacterial infection is suspected.

Grading of Ziehl-Neelsen Staining

The grading of Ziehl-Neelsen staining is based on the number of acid-fast bacilli (AFB) observed under the oil-immersion objective of a microscope. The number of bacilli and the microscopic fields examined are used to determine the grade of the smear.

The following grading system is given in the RNTCP Module for Laboratory Technicians and is used under the National Tuberculosis Elimination Programme (NTEP).

When no acid-fast bacilli are found after examining 100 oil-immersion fields, the smear is reported as negative or NIL.

If 1–9 AFB are found in 100 fields, the result is reported as scanty positive. The actual number of bacilli should be recorded instead of giving a numerical grade. For example, if 4 AFB are found in 100 fields, the result is reported as Scanty 4, not 1+.

The grades 1+, 2+ and 3+ indicate increasing numbers of acid-fast bacilli in the smear. When the bacilli are present in large numbers, fewer microscopic fields are required for grading.

This grading system is used for conventional ZN staining under bright-field microscopy. The reporting method may differ in other laboratory guidelines, particularly for fluorescence microscopy.

The AFB grade indicates the approximate number of acid-fast bacilli present in the examined smear. It cannot determine the species of mycobacteria or whether the bacilli are living or dead.

Modifications of Ziehl-Neelsen Staining

The Ziehl-Neelsen staining method is modified for certain organisms that are weakly acid-fast. These organisms may lose the primary stain when a strong acid is used for decolorization. Therefore, the concentration of staining reagents and the decolorization method may be changed. Some methods are also carried out without heating.

The following are the modifications of Ziehl-Neelsen staining:

Modified ZN Staining for Weakly Acid-Fast Bacteria

• Modified ZN Staining for Nocardia Species– Nocardia species are partially acid-fast bacteria. They may lose the primary stain when a strong acid is used for decolorization. In this method, a weaker solution of 0.5–1% sulphuric acid is used instead of 25% sulphuric acid. The bacteria appear as red or pink branching filaments against a blue background.
• Modified ZN Staining for Mycobacterium leprae– Mycobacterium leprae is less resistant to acid decolorization than Mycobacterium tuberculosis. The skin smears are stained with carbol fuchsin and decolorized using a weaker acid solution. A 5% sulphuric acid solution or 1% acid-alcohol may be used, depending upon the laboratory method. The bacilli appear red against a blue background.
• Fite-Faraco Staining– This is another modified acid-fast staining method used for the detection of Mycobacterium leprae in tissue sections. In this method, the paraffin-embedded tissue sections are treated with a mixture of xylene and peanut oil instead of xylene alone. It helps to prevent the removal of waxy materials from the bacilli. Carbol fuchsin is used as the primary stain, followed by mild decolorization. Some methods use 5% sulphuric acid in 25% alcohol. This method is mainly used for tissue sections and differs from the modified ZN staining of skin smears.

Modified ZN Staining for Intestinal Coccidian Oocysts

The modified ZN staining method is also used to detect intestinal coccidian oocysts in stool specimens. These are protozoan parasites. Their acid-fast staining is not due to a mycolic-acid-rich bacterial cell wall.

• Modified ZN Staining for Cryptosporidium– It is used for the detection of Cryptosporidium oocysts in stool specimens. The oocysts are small and rounded. They usually appear red or pink after staining, but some oocysts may remain unstained. In one heated modified ZN method, 1% sulphuric acid is used for decolorization, followed by methylene blue counterstaining.
• Modified ZN Staining for Cyclospora– The oocysts of Cyclospora cayetanensis show variable staining reactions. Some appear pink or deep red, while others remain unstained. A modified safranin staining method may also be used for these oocysts. However, it is different from the ZN staining method.
• Modified ZN Staining for Cystoisospora– The oocysts of Cystoisospora belli are large and ellipsoidal. They may appear red or pink after modified acid-fast staining. Their shape and size help in distinguishing them from other intestinal coccidian oocysts.
• Modification of Staining Reagents– Different concentrations of staining reagents are used in different laboratory methods. Some modified ZN methods use heated carbol fuchsin and a weaker acid solution. The CDC modified acid-fast method uses Kinyoun carbol fuchsin without heating, followed by decolorization using sulphuric acid in ethanol. The decolorizer is prepared using 10 ml of sulphuric acid and 90 ml of absolute ethanol. Malachite green is used as the counterstain in this method.

Kinyoun (Cold) Acid-Fast Staining Method

• Kinyoun Staining Method– Kinyoun staining is a cold acid-fast staining technique. Heating is not required in this method. It uses carbol fuchsin containing higher concentrations of basic fuchsin and phenol than the conventional heated ZN method. These help in the penetration of the stain without heating.
• Decolorization and Counterstaining– In the conventional Kinyoun method, 3% hydrochloric acid in alcohol may be used for decolorization. The smear is then counterstained with methylene blue. The acid-fast bacteria appear red, while the non-acid-fast materials take the colour of the counterstain.
• Difference from Modified ZN Staining– The Kinyoun method differs from the conventional ZN staining method mainly in the use of cold carbol fuchsin staining instead of heating. It is not the same as the modified ZN methods used for Nocardia, Mycobacterium leprae and intestinal coccidian oocysts. These methods may require different staining reagents and decolorizing solutions. A modified Kinyoun method is also used for intestinal coccidian oocysts, but its reagents differ from those used in conventional Kinyoun staining.

Applications of Ziehl-Neelsen Staining

The following are the applications of Ziehl-Neelsen staining:

• It is used for the examination of sputum smears for acid-fast bacilli (AFB) in patients suspected of pulmonary tuberculosis.
• Other respiratory specimens, such as bronchial washings and bronchoalveolar lavage fluid, can also be examined by ZN staining.
• The method is used to examine lymph node aspirates, cerebrospinal fluid, pleural fluid and tissue specimens in suspected extrapulmonary tuberculosis. These specimens may contain very few bacilli.
• Smears prepared from mycobacterial cultures are stained to examine the acid-fast properties of the bacteria.
• ZN staining is also used during the follow-up examination of patients receiving treatment for tuberculosis, according to the laboratory protocol. It cannot distinguish living bacilli from dead bacilli.
• The AFB smear result is used along with molecular tests and culture for the diagnosis of tuberculosis. A positive result does not confirm Mycobacterium tuberculosis infection. A negative smear also does not rule out tuberculosis.
• Modified ZN staining methods are used for the detection of Nocardia, Mycobacterium leprae and intestinal coccidian oocysts. These organisms require different staining and decolorization conditions.

Limitations of Ziehl-Neelsen Staining

The following are the limitations of Ziehl-Neelsen staining:

• ZN staining has low sensitivity when the number of acid-fast bacilli is very small. The bacilli may be present in the specimen but cannot be detected under a microscope.
• The staining result depends upon the quality of the specimen. Poorly collected sputum containing mostly saliva may give a false-negative result.
• The smear should not be too thick or too thin. Uneven spreading of the specimen may also affect the detection of acid-fast bacilli.
• Improper drying and fixation may damage the bacilli or cause the smear to detach during staining.
• The concentration and quality of staining reagents affect the staining result. Insufficient heating or staining with carbol fuchsin may prevent the bacilli from taking up the stain properly.
• Excessive decolorization may remove the primary stain from acid-fast bacilli, while insufficient decolorization may give a false-positive result.
• Stain precipitates, dust particles and other materials present on the slide may be mistaken for acid-fast bacilli.
• Contamination of slides, staining reagents or immersion oil may give incorrect results. The transfer of bacilli from one smear to another can also cause false-positive results.
• The result depends upon the experience of the person examining the smear. Some bacilli may be missed if an insufficient number of microscopic fields are examined.
• A properly maintained light microscope with a clean oil-immersion objective is required. Poor illumination or damaged lenses may affect the examination of the smear.
• ZN staining cannot differentiate Mycobacterium tuberculosis from other acid-fast mycobacteria on the basis of their microscopic appearance alone.
• The method cannot distinguish living bacilli from dead bacilli. It also cannot determine whether the bacteria are resistant or susceptible to anti-tuberculosis drugs.
• The specimens may contain infectious mycobacteria. Proper laboratory biosafety precautions are required during specimen handling and smear preparation to prevent the formation of aerosols. Heat fixation does not completely kill all the bacilli.

Advantages of Ziehl-Neelsen Staining

The following are the advantages of Ziehl-Neelsen staining:

• It is a simple and inexpensive staining method. The reagents required for ZN staining are commonly available in microbiology laboratories.
• The method does not require a special microscope. A light microscope with an oil-immersion objective is used to examine the stained smear.
• It is a rapid test and the stained smear can be examined on the same day.
• Acid-fast bacilli can be detected directly from sputum and other suitable specimens without growing the bacteria in culture.
• The method helps to differentiate acid-fast bacilli from non-acid-fast materials present in the smear.
• It is useful when a large number of acid-fast bacilli are present in sputum.
• The number of bacilli in the smear can be estimated using a standard grading system.
• ZN staining can be carried out in laboratories with limited facilities. However, proper biosafety precautions are required.
• The stained smears can be preserved and examined again for laboratory quality control.
• It is also used for follow-up smear examination during tuberculosis treatment, according to laboratory guidelines.

References

1. Central TB Division. (2005). Module for laboratory technicians. Directorate General of Health Services, Ministry of Health and Family Welfare, Government of India. https://tbcindia.mohfw.gov.in/wp-content/uploads/2023/05/6341531217Module-for-Laboratory-Technician.pdf
2. Paray, A. A., Chandra, M., Kaur, A., & Singh, M. (2026). Acid-fastness in microbiology: The versatility of Ziehl–Neelsen staining technique, its modifications, and integration of artificial intelligence – A brief review. Adesh University Journal of Medical Sciences & Research, 8(1), 4–9. https://doi.org/10.25259/AUJMSR_64_2025
3. Van Deun, A., Hossain, M. A., Gumusboga, M., & Rieder, H. L. (2008). Ziehl-Neelsen staining: Theory and practice. The International Journal of Tuberculosis and Lung Disease, 12(1), 108–110. https://pubmed.ncbi.nlm.nih.gov/18173887/