Dr Piyush Tailor

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

2. PRINCIPLE:

It is a type of Romanowsky stain which has two components - azure B (trimethylthionine) and eosinY (tetrabromofluorescein).

Azure B binds to anionic molecules &eosinY binds to cationic site on proteins.

Acidic grouping of nucleic acids, proteins of cell nuclei and primitive cytoplasm determine the uptake of basic dye azure B.

Basic grouping of cytoplasm results in its affinity for acidic dye and its staining by eosin.

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

4. REAGENTS: Giemsa powder (5gm), glycerin (350ml), methanol (1650ml), May - Grünwald powder (5gm), NaH2PO4.2H2O (6gm), K2HPO4 (4.5 gm), Distilled water (1000 ml)

5. PERFORMANCE SPECIFICATION: NA

6. SAMPLE: Histopathology section slide

7. EQUIPMENTS: Clean labeled containers, Coplin jars, Oven, Measuring cylinder,

pH meter/strip

8. CALIBRATION PROCEDURE:NA

9. POSITIVE CONTROL:

10. PROCEDURE (STEPS):

REAGENT PREPARATION-

GIEMSA POWDER:

Take 5 gm of Giemsa powder and grinding it in 350 ml of glycerin for one hour. Allow it at least for two days for ripening in the dark place. After two days add 600 ml of methanol to it. Shake well and use it after filtering by doing dilution 1:4

MAY - GRÜNWALD POWDER:

Take May - Grünwald powder 5gm in 1000 ml methanol dissolve it. Then filter the stain with filter paper. Store it as stock solution. At the time of staining 1:10 dilution of the stain is prepared for use.

SORENSON PHOSPHATE BUFFER SOLUTION:

Take 1000 ml of distilled water in glass/plastic container. Add 6gm of NaH2PO4.2H2O & 4.5 gm of K2HPO4 in it. Mix properly with glass rod. Measure pH of prepared solution with pH meter/pH strip. Store at room temp.

STAINING PROCEDURE:

• Take test slide on which staining has to be performed along with positive control.
• Deparaffinize section by keeping in oven for 5min and clear it with 2 changes of xylene each for 15 minute.
• Bring section to water.
• Dilute the MGG stain with buffer (1Apply the stain over the tissue for 5 minute.)
• Remove the stain from tissue.
• Dilute the Giemsa stain with tap water (1:4).
• Apply the stain over the tissue for 15- 20 minutes.
• Wash with tap water.
• Dry the tissue and mount with D.P.X. Label it properly.

11. 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.

12. INTERFERENCE: Inadequate quality/improper dilution

13. CALCULATION OF RESULTS AND UNCERTAINITY:NA

14. REFERENCE INTERVALS:NA

15. LABORATORY INTERPRETATION: interpret test result after true positivity of control slide.

16. REPORTEBLE INTERVAL FOR EXAMINATION RESULTS: NA

17. ALERT CRITICAL VALUE: NA

18. POTENTIAL SOURCE OF VARIABILITY: NA

19. REFERENCE: Theory & Practice of Histological Techniques, 4th Edition, 1996, John D. Bancroft