Liofilchem Ziehl-Neelsen staining kit, 3x250mL

The Ziehl-Neelsen Kit is a diagnostic kit designed for staining acid-alcohol resistant bacteria in clinical samples or culture media. This kit is primarily used for detecting mycobacteria, such as those in the Mycobacterium genus, using microscopic examination. The test exploits the acid-alcohol resistance of mycobacteria, providing a differential staining method to distinguish them from other bacterial species.

Kit Contents

Each kit includes:

1. Fuchsin Solution (250 mL): Contains Basic Fuchsin (0.3%), Ethyl Alcohol (10%), and Phenol (5%).
2. Acid-Alcohol Solution (250 mL): Contains Ethyl Alcohol (97%) and Concentrated HCl (3%).
3. Methylene Blue Solution (250 mL): Contains Methylene Blue (0.3%).
4. Instruction Sheet.

The solutions are housed in plastic bottles with dropper lids and sealed for stability.

Principle of the Method

• The Ziehl-Neelsen staining method leverages the ability of phenol to facilitate the penetration of Basic Fuchsin into bacterial cells.
• Acid-alcohol resistant bacteria retain the red Basic Fuchsin stain even after treatment with the Acid-Alcohol Solution due to their unique cell wall composition rich in mycolic acids.
• Non-resistant bacteria lose the red color and take up Methylene Blue during counterstaining, appearing blue.

Procedure

Sample Preparation and Fixing

1. Smear pathological material or bacterial culture onto a clean slide.
2. Allow the smear to air dry.
3. Fix the smear by passing the slide quickly over a flame or using other heat-fixing methods. Avoid overheating.

Staining Steps

1. Primary Stain (Fuchsin Solution):
   • Cover the slide with Fuchsin Solution.
   • Heat gently until steam forms (3-5 minutes). Do not boil.
   • Allow the slide to cool and rinse with distilled water.
2. Decolorization (Acid-Alcohol Solution):
   • Apply Acid-Alcohol Solution until no further red color is released (about 1-2 minutes).
   • Rinse with distilled water.
3. Counterstaining (Methylene Blue Solution):
   • Cover the slide with Methylene Blue Solution.
   • Leave for 30-60 seconds, rinse with distilled water, and allow to air dry.
4. Microscopic Examination:
   • Observe the slide under an oil immersion microscope.

Interpretation of Results

• Acid-alcohol resistant bacteria (e.g., Mycobacterium): Appear red.
• Non-acid-alcohol resistant bacteria and background material: Appear blue.

Quality Control

Each lot undergoes quality control with:

• Mycobacterium smegmatis (ATCC 14468): Positive control for acid-alcohol resistance (red staining).
• Staphylococcus aureus (ATCC 25923): Negative control for acid-alcohol resistance (blue staining).

Limitations

1. The Ziehl-Neelsen stain provides presumptive evidence of acid-alcohol resistant microorganisms. Further confirmation with additional diagnostic methods is necessary.
2. Rapidly growing mycobacteria may retain the stain to varying degrees.
3. Care must be taken to ensure proper decolorization with the Acid-Alcohol Solution to avoid insufficient staining results.

Storage Instructions

• Store at 10-25°C in the original packaging.
• Avoid heat and excessive temperature fluctuations.
• Shelf life: Valid until the expiry date indicated on the label. Discard if signs of deterioration (e.g., color changes, precipitation) are present.

Precautions

• The kit contains hazardous substances as per current legislation. Consult the Safety Data Sheet (SDS) before use.
• For in vitro diagnostic use only in professional laboratory settings.
• Use approved aseptic and safety methods to handle potentially infectious materials.

Waste Disposal

After use, slides and materials that come into contact with samples must be decontaminated and disposed of according to laboratory protocols for handling potentially infectious waste.

Applications

• Clinical Diagnostics: Detecting Mycobacterium spp. in respiratory samples (e.g., sputum for tuberculosis diagnosis).
• Research: Studying acid-alcohol resistant bacteria in microbial cultures.
• Environmental Monitoring: Identifying mycobacteria in water and soil samples.

Specifications

• Product Code: 80276
• Packaging:
  • Fuchsin Solution: 250 mL
  • Acid-Alcohol Solution: 250 mL
  • Methylene Blue Solution: 250 mL

Compliance and Standards

• Based on protocols outlined in:
  • Manual of Clinical Microbiology, 7th edition.
  • American Society for Microbiology guidelines.

This comprehensive kit simplifies the detection of acid-alcohol resistant bacteria, offering reliable and reproducible results for laboratory professionals.