Special stains bmlt

special stains in histopathology

1. SPECIAL STAINS
IN
HISTOPATHOLOGY

2. ●H and E 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 for Lipids

5. ●LIPIDS
●Simple lipids
–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. ●Lipids with melting points below staining
temperature can be stained with fat stains.
●Lipids in solid or crystalline form at staining
temp. can not be stained.
●Melting point is inversely related to the length of
fatty acid.
●Best fixatives- Formal calcium (2% calcium
acetate and 10% formalin)

7. ●Staining techniques for 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
●60% isopropanol
●Alum hematoxylin
●Glycerin jelly mounting medium
●Red oil O working solution.

10. ●To make oil 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 slides in 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 in alum hematoxylin (5 dips) – stains
nucleus.
●Rinse with distilled water
●with aqueous mounting media, Glycerine Jelly.

13. ●Fat emboli in Oil red O stain.

14. Uses of Oil Red O stain
●Oil red O stain is used for staining neutral
triglycerides, lipids and lipoprotein.
● Tumors arising from fat cells (liposarcomas)
can be differentiated from other types of
tumors.
● 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 storage disorder 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
–Fix slides 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 in distilled water.
●Stain with nuclear fast red – 3 minutes.
●Wash in tap water
●Rinse in distilled water.
●Mount using aqueous media.

20. ●Sudan black B - myeloblasts

21. Uses of Sudan Black B
●Used for staining neutral triglycerides, lipids,
lipoproteins and phospholipids.
●In hematological disorders, it can be used to
differentiate blasts. Sudan black B stains
myeloblasts but does not stain lymphoblasts.

22. Thank You