1. PURPOSE OF EXAMINATION: To lay down the procedure for ZN stain in Histo-pathology section at NCHLS Surat.
2. PRINCIPLE AND METHOD OF PROCEDURE USED FOR EXAMINATION: Acid fast bacilli cell wall contains waxy lipid, mycolic acid. Phenol and carbol fuscin helps dye to pass through lipid wall and attach with negatively charged ions present in bacteria. After staining with help of reducing substance cells are decolorized except acid fast bacilli.
3. STAFF RESPONSIBLE: lab. technician under supervision of resident doctor/tutor.
4. PERFORMANCE CHARACTERISTICS: NA
5. TYPE OF SAMPLE: Histopathology section slide
6. PATIENT PREAPARATION: NA
7. TYPE OF CONTAINER AND ADDITIVES:NA
8. POSITIVE CONTROL: Positive control for AFB
9. REQUIRED EQUIPMENT AND REAGENTS:
Equipments: Coplin jars, funnel, Whatman filter paper no. 4
Reagent: Basic fuchsin, Absolute alcohol, phenol, 20% sulfuric acid, Methylene blue, Distilled water, Harris hematoxylin, xylene, DPX
10. ENVIRONMENTAL AND SAFETY CONTROLS: Universal safety controls
11. CALIBRATION PROCEDURE (METROLOGICAL TRACEBILITY):NA
12. PROCEDURE(STEPS): * Carbol fuchsin preparation:
• Take Basic fuchsin 1 gm, 10 ml absolute alcohol and 5 gm phenol and dissolve in 95ml of distilled water put in water bath at 56C.
• Dissolve basic fuchsin in absolute alcohol and then add phenol in it.
• Mix well and filter it. Store in brown bottle.
20% sulfuric acid preparation:
• Take 20% of sulfuric acid and add in 80 ml of distilled water.
1%methelene blue preparation:
• Take 1 gm methylene blue powder and mix in 100 ml distilled water. Filter in glass bottle
Staining Procedure:
• Take test slide on which staining has to be performed along with positive control.
• Deparaffinize section by keeping in oven for 5 minute and clear it with 2 changes xylene each for 15 minute.
• Dehydration with graded alcohol 2 change for 2 min.
• Bring section to water.
• Put filter paper on the slide section and pour the ZNCF solution over the section.
• Heat section with spirit lamp till steam arises. Allow it to heat for 5 minute.
• Wash in tap water to remove excess stain.
• Differentiate with 20% sulfuric acid till section decolorize section till tissue is very pale pink
• Wash in water.
• Counter stain in 1%methylene blue 1 dip.
• Wash in water.
• clear in xylene, mount with DPX
Result
• AFB: Red
• Nuclei: blue
• Other tissue : Pale blue
13. QUALITY CONTROL PROCEDURE: The newer batch of stain is tested before being put in to use by checking their efficiency with the batch of stain already in use.
14. INTERFERENCE: Inadequate quality/improper dilution
15. PRINCIPLE OF PROCEDURE FOR CALCULATING RESULTS INCLUDING, WHERE RELEVENT, THE MEASUREMENT OF UNCERTAINITY OF MEASURED QUANTITY VALUES: NA
16. BIOLOGICAL REFERENCE INTERVALS OR CLINICAL DECISION VALUES:NA
17. REPORTABLE INTERVAL OF EXAMINATION RESULTS: NA
18. INSTRUCTION FOR DETERMINING QUANTITATIVE RESULTS WHEN RESULTS IS NOT WITHIN THE MEASUREMENT INTERVAL: NA 19. ALERT /CRITICAL VALUE, WHERE APPROPRIATE : NA
20. LABORATORY CLINICAL INTERPRETATION: Interpret test result after true positivity of control slide.
21. POTENTIAL SOURCE OF VARIATION: NA
22. REFERENCE: Histopathological laboratory techniques by Culling’s.