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Malay - Aim of special stain:
- Demonstrate Acid Fast Bacilli in tissue sections
- Diagnostic applications:
- Tuberculosis (TB) and Leprosy
- Material supplied for surveys:
- Gastric tissue or skin lesion containing AFB cut at 3-4µm
- Recommended control:
- Tissue/s containing AFB – Veterinary samples can also be used
1. Classic Ziehl Neelsen Method
Reagents – commercial source unknown, unless specified:
| 1. Concentrated Carbol Fuchsin Solution (commercial preparation – Australian Biostain) | |
| a) Basic fuchsin | 0.5 g |
| b) Absolute alcohol | 5 mL |
| c) 5% aqueous phenol | 100 mL |
| 2. 1% Acid Alcohol | |
| a) 70% Ethanol | 400 mL |
| b) Concentrated Hydrochloric Acid | 4 mL |
| 3. Methylene blue stock solution | |
| a) Methylene blue | 1.4 g |
| b) 95% alcohol | 100 mL |
| 4. Working Methylene solution | |
| a) Methylene blue stock solution | 10 mL |
| b) Distilled or tap water | 90 mL |
Method:
- Dewax sections and take to tap water.
- Carbol Fuchsin at room temperature for 20 minutes.
- Rinse in 70% ethanol.
- Differentiate in 1% Acid alcohol – several changes over 1-2 minutes until no more colour comes away from the section.
- Wash in tap water.
- Counterstain in working Methylene Blue Solution for 30 seconds.
- Rinse in tap water.
- Rapidly dehydrate, clear and mount.
Results:
Acid fast bacilli – magenta/deep red
Nuclei – blue
Other tissue components – pale blue
2. Ziehl-Neelsen (ZN) PUTT Modification:
Reagents – commercial source unknown, unless specified:
| 1. New fuchsin | |
| 2. Saturated lithium carbonate | |
| 3. 3% hydrochloric acid in 60% alcohol (ZN differentiator) | |
| 4. 1 % Methylene Blue in absolute alcohol |
Method:
- Take sections to 60% alcohol and rinse in pure distilled water.
- Stain in new fuchsin at room temperature for 5 minutes.
- Treat with saturated aqueous lithium carbonate for 1 minute.
- Differentiate until pale pink in 3% HCI in 60% alcohol.
- Rinse in absolute alcohol and dehydrate thoroughly.
- Counterstain in 1 % Methylene blue in absolute alcohol for 1 minute.
- Rinse very quickly in absolute alcohol.
- Clear in xylene and mount.
Results:
Acid fast bacilli – red
Red blood corpuscles – pink
Mast cell granules – blue
Other bacteria – blue
Nuclei – blue
Disclaimer:
These methods are intended as a guide only. Laboratories that wish to implement these methods should perform internal validation prior to use. The RCPAQAP does not make any claim or warranty for the accuracy or performance of these methods.
References
2022 RCPAQAP Technical General Survey 1.
2013 RCPAQAP Technical General Survey 2.
Ellis, RC; LA Zabrowarny. Safer staining method for acid fast bacilli. Journal of Clinical Pathology 1993;46: 559–560. doi:10.1136/jcp.46.6.559. PMC 501296. PMID 7687254.
Jeanine H Bartlett. Theory and practice of Histological Techniques. John D Bancroft; Marilyn Gamble. 6Th edition. New York, NY: Churchill Livingstone; 2008:314-315.
Putt Fa. A modified Ziehl-Neelsen method for demonstration of leprosy bacilli and other acid-fast organisms. American journal of clinical pathology. 1951 Jan; 21(1):92-5. PMID: 14799470.