Ziehl-Neelsen (AFB) Staining SOP

Purpose

To outline the procedure for performing Ziehl-Neelsen staining to identify acid-fast bacilli in clinical specimens.

Scope

This SOP applies to all laboratory personnel involved in staining procedures.

Responsibilities

• Technician: Perform staining and ensure accuracy of results.
• Quality Manager: Review and approve SOP and training records.
• Laboratory Supervisor: Ensure compliance with SOP and provide training.

Materials & equipment

• Ziehl-Neelsen staining kit
• Microscope
• Glass slides
• Bunsen burner
• Staining rack
• Distilled water

Procedure

1. Prepare the smear by spreading a thin layer of the specimen on a glass slide.
2. Air dry the slide completely before heat fixing.
3. Pass the slide through the flame of a Bunsen burner three times to fix the smear.
4. Flood the slide with carbol fuchsin stain and incubate for 5 minutes at room temperature.
5. Rinse the slide gently with distilled water to remove excess stain.
6. Decolorize the slide with acid-alcohol for 2-3 minutes until the color stops running.
7. Rinse the slide again with distilled water.
8. Counterstain with methylene blue for 1 minute.
9. Rinse with distilled water and air dry the slide.
10. Examine the slide under the microscope using oil immersion.

Records

• Staining log
• Quality control records
• Training records

Safety

• Wear appropriate personal protective equipment (PPE) including gloves and lab coat.
• Handle all specimens as potentially infectious.
• Dispose of all waste according to biohazard regulations.