Dr Piyush Tailor

1. PURPOSE OF EXAMINATION: To lay down the procedure for GMS staining in Histo-pathology section at NCHLS Surat.

2. PRINCIPLE AND METHOD OF PROCEDURE USED FOR EXAMINATION-Fungal cell wall is rich in polysaccharide which is converted by oxidation with chromic acid to dialdehyde. This is demonstrated by reduction of an alkaline hexamine silver complex. Upon reduction, the solution precipitates nascent silver ions thus blackening of site. This is known as Argentaffin reaction.

3. STAFF RESPONSIBLE: lab. technician under supervision of resident doctor/tutor.

4. PERFORMANCE CHARACTERISTICS: NA

5. TYPE OF SAMPLE: Histopathology section slide

6. PATIENTS PREPARATIONS: NA

7. TYPE OF CONTAINER AND ADDITIVES:NA

8. POSITIVE CONTROL: Known Positive Control.

9. REQUIRED EQUIPMENT AND REAGENTS:

EQUIPMENTS: Acid cleaned glasswares, Coplin jars, Oven

REAGENTS: 5% Aqueous sodium tetraborate, 5% silver nitrate, 3% Sodium thiosulfate, 1% aqueous sodium metabisulphite, 3%methenamine(hexamine),borax solution,0.2% aqueous gold chloride solution, 0.2% light green in 1% acetic acid, Xylene, DPX.

10. ENVIRONMENTAL AND SAFETY CONTROLS: Universal safety precautions

11. CALIBRATION PROCEDURE (METROLOGICAL TRACEBILITY):NA

12. PROCEDURE(STEPS):

A) REAGENT PREPARATION-

• STOCK HEXAMINE SOLUTION-Take 5ml of 5% silver nitrate solution and add to 100 ml 0f 3% aqueous hexamine to prepare stock solution. The white precipitates which are formed will dissolve on shaking. The solution can be kept for 1-2 months at 40C.

• WORKING SOLUTION- Dilute 2ml of freshly prepared 5% aqueous sodium tetraborate solution with 25ml of distilled water. Mix well and add 25ml of stock hexamine silver solution. Mix well.

• 5% aqueous chromium trioxide( 5gm chromic acid in 100ml distilled water)

• 1%aqueous sodium metabisulphite( 1gm sodium thiosulphate in 100ml distilled water)

• 5% aqueous thiosulphate ( 5gm sodium thiosulphate in 100ml distilled water)

• 0.2% light green( prepare 1% acetic acid by dissolving 1ml of acetic acid in 99ml distilled water. Add 0.2gm light green in 100ml of 1% acetic acid)

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 with2 changes of xylene each for 15 minutes.

• Bring section to water.

• Oxidize in 5% chromic acid for 1 hour.

• Wash in distilled water for 1 minute.

• Bleach with 2% sodium metabisulphite for 1 minute.

• Wash in running tap water for 2 to 5 minute.

• Wash in several changes of distilled water for 2 minutes.

• Place sections in preheated hexamine silver solutions in a coupling jar at 560C in a water bath.

• Examine the sections after 10minute and then after 5min interval until the fungi are blackened and the background is clear.

• Wash in several changes of distilled water for 2 minute.

• Treat section with hypo solution for 3minute

• Wash in running water for 5 minute.

• Counter stain with light green solution for 30 seconds

• Wash in running tap water for 1 minute

• Dehydrate in alcohol, clear in xylene, mount with DPX

Result

• Fungi, some mucin, glycogen: Black

• Background: Green

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.