This test is used to distinguish acid-fast bacteria based on their appearance after staining with acid-fast stain containing 20% sulphuric acid (Ziehl-Neelsen stain) as a decolorizer.
Principle of Ziehl-Neelsen stain
- Because of the high lipid content of the cell wall, bacteria such as mycobacteria are extremely difficult to stain using conventional methods.
- The first staining is done with Carbol fuchsin, phenol, and heat. The stain binds to the cell wall’s mycolic acid.
- An acid decolorizing solution is applied after staining. This removes the red dye from the background cell tissues and other organisms, with the exception of Mycobacteria and other acid fast organisms such as Nocardia, which retain the dye.
- After decolorization, the smear is counter stained with malachite green or methylene blue, which stains the background material and provides a contrast color against which the red acid fast bacilli can be seen.
- Nocardia, Cryptosporidium, and Microsporidium are also partially acid fast, with staining resistance of 0.5–1 percent.
Type of Sample used for Ziehl-Neelsen stain
Sputum: Instruct the patients to collect expectorated sputum or sputum obtained by deep cough, in a sterile wide-mouthed container after rinsing the mouth with water.
- Instruct patients to collect sputum from a deep cough for three days in a row and to avoid contaminating it with saliva.
- Sputum samples collected on a single day can be investigated at the request of the clinician.
- Label the container with the date and the patient’s name.
Urine: Instruct patients to collect a first morning urine sample on three consecutive days in a clean, dry, and sterile container provided by the laboratory.
- Instruct patients to clean the genitals and surrounding area with moist cotton and copious amounts of water while collecting urine to avoid contamination by normal flora.
- Instruct patients to collect a midstream urine sample for acid fast bacilli (AFB) culture.
Consumables and Reagents
- Clean, dry glass slides
- Carbol fuchsin stain
- Acid – 20% H2SO4 , 5% or 1% or 0.5% H2SO4 (for modified Ziehl-Neelsen stain)
- Methylene blue
Procedure for Ziehl Neelson Stain
- A thin smear of the specimen should be prepared. Dry in the air, then gently heat for 2 minutes over a Bunsen burner flame to fix the smear.
- Flood the heat-fixed smear with carbol fuchsin slide, heat from below the slide until fumes appear, and leave for 5-7 minutes. Do not allow the stain to boil.
- Wash off the stain with distilled water
- Cover the smear with 20 % sulfuric acid for 3 minutes
- Wash well with distilled water.
- Decolorization is repeated until the smear appears pale pink.
- Cover the slide with methylene blue for 2 minutes.
- Wash with distilled water, dry and examine the slide under oil immersion microscope.
Ziehl-Neelsen stain Results Interpretation
If any definite bacilli (pink/red rods) are observed, the smear should be reported as acid fast bacilli (AFB) positive, with an indication of the number of bacteria present as follows:
|More than 10 AFB/field||Report as 3 + (plenty)|
|1–10 AFB/ field||Report as 2+ (many)|
|10–100 AFB/100 fields||Report + (moderate)|
|1–9 AFB/100 fields||(few)|
Limitation of Ziehl-Neelsen stain
Ziehl-Neelsen stain cannot determine the exact species of Mycobacteria. It can only differentiate whether the bacteria is acid fast or not. Further tests must be performed to identify the species of Mycobacteria.
- Follow the usual precautions as for handling any biological fluid
- Wear disposable gloves and face mask while doing the procedure
- Temperature: 25 to 30°C.
Laboratory strategy in the diagnosis of infective syndromes. Mackie & McCartney. Practical Medical Microbiology. Edited by Collee, Dugid, Fraser, Marmion. 1989;14. 600-50.