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Special stains bmlt
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- 2. ● H andE stain – Preliminary stain ( first stain) applied to the tissue sections. – Provides diagnostic information in most of the cases.
- 3. ● Special stain –Staining technique that is used to highlight various individual tissue components which can not be identified in routine H and E stain or are better appreciated on special stains. – Eg. special stains for carbohydrates, amyloids, nucleic acids, lipids, microorganisms etc.
- 4. Special Stains forLipids
- 5. ● LIPIDS ● Simplelipids – Fat – Oil – waxes ● Compound lipid – C/o fatty acids, alcohol and another group eg. phosphorus or nitrogen ● Derived lipids – Derived from simple or compound lipids by hydrolysis – Cholesterol – Bile acids
- 6. â—Ź Lipids withmelting points below staining temperature can be stained with fat stains. â—Ź Lipids in solid or crystalline form at staining temp. can not be stained. â—Ź Melting point is inversely related to the length of fatty acid. â—Ź Best fixatives- Formal calcium (2% calcium acetate and 10% formalin)
- 7. ● Staining techniquesfor lipids – Oil red O – Sudan black B
- 8. Oil Red O ●Principle – Staining with oil soluble dyes is based on greater solubility of the dye in lipid substances than in usual hydroalcoholic dye solvents. ● Result Lipid – RED Nucleus – blue
- 9. Reagents â—Ź 60% isopropanol â—ŹAlum hematoxylin â—Ź Glycerin jelly mounting medium â—Ź Red oil O working solution.
- 10. ● To makeoil red O stock solution – Oil Red O 0.5g – Isopropanol 100 ml dissolve the dye in isopropanol, using very gentle heat of water bath. This is the stock solution. ● To make working oil red O solution – Dilute 30ml of stock solution in 20ml of distilled water and allow it to stand for 10 min. – Filter into coplin jar and cover immediately. – Working solution should be prepared fresh each time.
- 11. Procedure ● Fix slidesin 10% formalin if fresh. ● Wash well in tap water for 1 – 10 min to drain of excess water. ● Rinse with 60% isopropanol. ● Stain with freshly prepared Red oil O working solution for 15 min.
- 12. ● Dip inalum hematoxylin (5 dips) – stains nucleus. ● Rinse with distilled water ● with aqueous mounting media, Glycerine Jelly.
- 13. â—Ź Fat emboliin Oil red O stain.
- 14. Uses of OilRed O stain â—Ź Oil red O stain is used for staining neutral triglycerides, lipids and lipoprotein. â—Ź Tumors arising from fat cells (liposarcomas) can be differentiated from other types of tumors. â—Ź Fat occurring in an abnormal place, such as fatty emboli that may develop after either a bone fracture or an injury that crushes a fatty body area.
- 15. ● Lipid storagedisorder like nieman-pick disease ● To demonstrate fat or lipids in condition like fatty liver. ● In hematological condition like burkitt’s lymphoma and sea-blue histiocytic syndrome.
- 16. Sudan Black B ●Principle – Sudan Black B is a dye insoluble in water but dissolves in fat. Therefore the dye accumulates in fat globules within the cells. – It is a slightly basic dye and combines with acidic groups in compound lipids, thus staining phospholipids too. ● Results – Fats : blue/black – Nucleus : red
- 17. ● Reagents – 85%propylene glycol Propylene glycol : 85 ml Distilled water : 15 ml – Hematoxylin – Sudan black B/ Propylene Sudan Black B : 0.7 g Propylene glycol : 100ml
- 18. ● Procedure – Fixslides in 10% formalin. – Wash well in tap water, rinse in distilled water, and drain off excess water. – Dip in propylene glycol – 2 changes – five minutes each (dehydration) – Stain with sudan black B – 7 minutes – Dip in 85% propylene glycol – 3 minutes. (differentiation)
- 19. ● Rinse indistilled water. ● Stain with nuclear fast red – 3 minutes. ● Wash in tap water ● Rinse in distilled water. ● Mount using aqueous media.
- 20. â—Ź Sudan blackB - myeloblasts
- 21. Uses of SudanBlack B â—Ź Used for staining neutral triglycerides, lipids, lipoproteins and phospholipids. â—Ź In hematological disorders, it can be used to differentiate blasts. Sudan black B stains myeloblasts but does not stain lymphoblasts.
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