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Malay - Aim of special stain:
- Identification of Helicobacter pylori
- Diagnostic applications:
- Chronic gastritis and duodenal ulcer
- Material supplied for surveys:
- Gastric mucosa positive for Helicobactor pylori organisms, cut at 3µm
- Recommended control:
- Gastric tissue containing Helicobacter pylori
1. Diff-Quik staining protocol for Helicobacter pylori
Reagents – commercial source unknown, unless specified:
| 1.Commercial Diff-Quik Solution 1 (Red) (commercial preparation – Australian Biostain) |
| 2. Commercial Diff-Quik Solution 2 (Blue) (commercial preparation – Australian Biostain) |
| 3. 0.5% aqueous acetic acid | |
| a) Glacial acetic acid | 5 mL |
| b) Distilled water | 995 mL |
Method:
- Deparaffinise sections and hydrate to water.
- Stain slides in Diff-Quik Solution 1 (Red) for 10 seconds.
Allow excess stain to drain off the slide. - Stain slides in Diff-Quik Solution 2 (blue) for 40 seconds.
Agitate slides while staining.
Allow excess stain to drain off the slide. - Rinse rapidly in running tap water.
- Differentiate in 0.5% acetic acid -1 dip.
- Rinse rapidly in tap water.
- Allow sections to air dry.
- Air dry, clear and mount.
Results:
H Pylori and background – blue
2. Warthin-Starry (Modified) for Spirochetes
Reagents – commercial source unknown, unless specified:
| 1. Stock Acetate buffer (pH 3.6) | |
| a) Solution 1: | 0.2 M Sodium acetate |
| i) Sodium acetate | 16.4 g |
| ii) Distilled water | 1 L |
| b) Solution 2: | 0.2 M Acetic acid |
| i) Glacial acetic acid | 11.8 mL |
| ii) Distilled water | 1 L |
| 2. Working Acetate buffer | |
| a) Solution 1 (from 1 a) | 1.5 mL |
| b) Solution 1 (from 1 b) | 18.5 mL |
| Distilled water | 480 mL |
| 3. 0.2% Silver solution | |
| a) AgNO3 | 0.1 g |
| b) Working acetate buffer | 50 mL |
| 4. Developer* | |
| a) Gelatine Solution: | |
| i) Gelatine | 7.5 g |
| ii) Working acetate buffer | 150 mL |
| Dissolve by heating at 60°C. | |
| b) Hydroquinone Solution: | |
| i) Hydroquinone | 0.3 g |
| ii) Working acetate buffer | 10 mL |
| c) Silver Solution: | |
| i) AgNO3 | 0.6 g |
| ii) Working acetate buffer | 30 mL |
| Filter before use. | |
| 5. 5% Sodium thiosulphate | |
| a) Sodium thiosulphate | 5 g |
| b) Distilled water | 100 mL |
*Reagent preparation for reagent 4 – developer:
Note: All glassware should be clean, rinsed in distilled water and dried.
- Immediately before use, add 2 mL Hydroquinone Solution (4b) to 30 mL Gelatine in acetate buffer (4a), and mix rapidly.
- Then add 6 mL AgNO3 acetate buffer (4c) and mix rapidly.
Method:
(Use plastic forceps)
- Bring sections down to water.
- Rinse section thoroughly with distilled water and place into a slide mailer (or a Coplin jar) with filtered 0.2% silver solution. Put the slide mailer (or Coplin jar) on the centre of the turntable inside the microwave oven.
- Microwave the section for 20 seconds using “HIGH” power (output 800 watt).
- Pre-warm 15 ml developer solution in mailer by microwave for 15-20 seconds using “HIGH” power.
- Transfer directly the section to developer solution and dip for 2 to 6 seconds.
- Transfer section to working acetate buffer.
- Fix section with 5% Sodium thiosulphate (hypo) for 1 minute.
- Rinse with distilled water.
- Dehydrate, clear and mount.
Results:
Spirochaetes and helicobacter pylori– black
Background – yellow/brown
3. Giemsa-Modified for Demonstration of Helicobacter pylori
Reagents – commercial source unknown, unless specified:
| 1. Stock Giemsa Solution | |
| a) Giemsa stain | 2.0 g |
| b) Glycerol | 102 mL |
| c) Methanol | 102 mL |
| Reagent preparation: | |
| Dissolve Giemsa stain in Methanol (heat to 45 °C with regular stirring). | |
| Warm glycerol to 45°C (or pouring temperature). | |
| Mix together for 30 minutes and filter with filter paper (commercially sourced from Whatman No 4) immediately while warm. | |
| Ripen 1 week before use. |
| 2. Working Giemsa Solution | |
| a) Stock Giemsa Solution | 1 mL |
| b) Distilled water (buffered to 6.0 – 6.8) | 10 mL |
Method:
- Rehydrate section to water.
- Stain in fresh working Giemsa for 15 minutes.
- Rinse in distilled water.
- Differentiate on 100% alcohol as you dehydrate.
- Clear and mount.
Results:
H pylori – blue
Note: Modified from Lillie and Fulmer (1976), Original by Giemsa (1904)
4. Cresyl Fast Violet method for Staining H pylori
Stained on Sakura Prisma Automated stainer.
Reagents – commercial source unknown, unless specified:
| 1. Cresyl fast violet (commercial preparation – Australian Biostain) |
| 2. 0.2% Acetate | |
| a) Sodium acetate | 0.2 g |
| b) Distilled water | 100 mL |
| 3. Cresyl fast violet differentiator (commercial preparation – Australian Biostain) |
Method:
- Xylene 3 minutes.
- Xylene 2 minutes.
- 100% Ethanol 1 minute.
- 95% Ethanol 1 minute.
- 70% Ethanol 1 minute.
- Wash 1 minute.
- Cresyl Fast Violet 0.2% Acetate 6 minutes.
- Wash 1 minute.
- 95% Ethanol 5 seconds.
- Cresyl Violet Differentiator 1 second.
- 100% Ethanol 30 seconds.
- 100% Ethanol 30 seconds.
- 50/50 Ethanol Xylene 1 minute.
- Xylene 1 minute.
- Xylene 2 minute.
Results:
H pylori – 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
2017 RCPAQAP Technical General Survey 1.
2018 RCPAQAP Technical General Survey 3.
Lott. Robert Richard. Helicobacter pylori in W. Brown. Histologic preparations Common Problems and Their Solutions. The College of American Pathologists press 2009:65-73.
Ahmed N. 23 years of the discovery of Helicobacter pylori: is the debate over? Annals of Clinical Microbiology and Antimicrobials. 2005 Oct 31;4:17. doi: 10.1186/1476-0711-4-17. PMID: 16262889; PMCID: PMC1283743.
Wilson J.D. Harrison’s Principles of Internal Medicine 12 Edition Vol. 2. McGraw-Hill; 1992:1245-1246.
Jeanine H Bartlett. Theory and practice of Histological Techniques. John D Bancroft; Marilyn Gamble. 6th edition. New York, NY: Churchill Livingstone; 2008:314-318.
Clark G. Staining Procedures Used by the Biological Stain Commission. 4th Edition, Williams & Wilkins, Baltimore, London, 412. 1981:173 – 174.
K.S. Suvarna, C. Layton and J.D. Bancroft. “Microorganisms”, in Theory and Practice of Histological Techniques, 3rd Edition; New York, NY: Churchill Livingstone; 1990:307.