1.0 Purpose
To establish a uniform procedure for Ziehl-Neelsen (ZN) staining of tissue sections.
2.0 Scope
This SOP applies to all tissue sections and smears received in the Surgical Pathology section for ZN staining.
3.0 Introduction
ZN staining is used to demonstrate acid-fast bacteria, specifically Mycobacterium species, including the causative agent of tuberculosis.
4.0 Principle
The lipoid capsule of acid-fast organisms absorbs carbol-fuchsin and resists decolorization with a dilute acid rinse.
5.0 Analytical Measuring Range and Limit of Detection
Not applicable.
6.0 Responsibility
- Technical Staff
- Pathologist
7.0 Abbreviations
- ZN Stain: Ziehl-Neelsen Stain
8.0 Requirements
Kit Reagents
a) Carbol Fuchsin: Dissolve 1 g Basic Fuchsin in 10 ml absolute alcohol, then add 100 ml of 5% Phenol.
b) 0.1% Methylene Blue
c) 1% Acid Alcohol for Lepra: 1 ml HCl in 70% alcohol.
d) 3% Acid Alcohol for Mycobacterium tuberculosis: 3 ml HCl in 70% alcohol.
Instrumentation and Software
- None specified.
Disposables
- None specified.
Other Materials Required but Not Provided with the Kit
- None specified.
Specimen Collection and Handling
- Specimen Collection: Room Temperature.
- Specimen Transport: Room Temperature.
- Specimen Storage: Room Temperature.
- Specimen and Control Preparation: Room Temperature.
9.0 Precautions
- Handle all specimens with gloves, mask, and goggles.
- Handle all instruments with care to prevent injury.
10.0 Limitations & Interferences
Not applicable.
11.0 Documentation
Maintain all documents in the following:
- Special Stains Log/Register
- Daily Routine Register
12.0 Instructions
12.1 Pre-Analytical Steps
- Sample preparation and reagent preparation.
12.2 Analytical Steps
12.2.1 Sample Preparation
1. Deparaffinize sections by dipping in two changes of xylene, then in two changes of alcohol to remove xylene.
12.2.2 Staining
2. Pour carbol fuchsin on the slide, heat until the stain steams, and leave for 10 minutes.
12.2.3 Rinse
3. Wash in running tap water.
12.2.4 Differentiation
4. Differentiate with acid alcohol until a pale pink color appears.
5. Wash in running tap water.
12.2.6 Counterstaining
6. Counterstain with 0.1% methylene blue for 30 seconds.
12.2.7 Rinse
7. Wash in running tap water.
8. Dehydrate, clear, mount with DPX, and label.
12.2.9 Reporting
9. Submit the slides to the pathologist for reporting.
12.3 Quality Control and Assurance
1. Run a known positive control slide with each batch of samples.
12.4 Calibration and Calibration Verification
Not applicable.
12.5 Storage of Samples
- Smear-stained slides: Store for 10 years at room temperature.
13.0 Reference Range
Not applicable.
14.0 Critical Value
Not applicable.
15.0 Reporting of Results and Interpretation
Acid-fast bacilli are evaluated under an oil immersion lens. Report positive if slender rods or beaded rods matching the morphological characteristics of Mycobacteria are found.
16.0 Contingency Plan
- In case of reagent shortage, manpower shortage, or instrument breakdown, multiple staff are trained, and backups are available.
17.0 References
- Bancroft’s Theory and Practice of Histological Techniques, by John D. Bancroft and Marilyn Gamble, 2007 (6th Edition).
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