Special staining for Elastic fibers,Parasites,Pigments and Minerals

1. Special Stain For Elastic Fibers
Prepared By :
Yasir Bashier Bhat
byasir385@gmail.com

2. Verhoeff- van Gieson Stain :
Ira Van Gieson first described the Verhoeff van Gieson
staining in 1889 as a method of evalvating collagen
fibers in neural tissue.
Frederick Herman Verhoeff, then modified the stain in
1903 as a method to differentiate collagen and other
connective tissues and highlight elastic fibers in
particular.

3. What does it stain ?
Elastic fibers are connective tissue fibers that allow tissues
to stretch and are abundant in the aorta ,for example,
where they provide flexibility to this large blood vessel.
They are also present in other tissues that need flexibility,
such as urinary bladder, skin and lung.
These fine elastic fibers cannot typically be seen on
routine H & E – stained tissue sections, therefore special
stains are required to highlight them. Although there are
numerous special stains for identification of elastic fibers,
VVG is most commonly used because it is quick, and
produce intense staining of elastic fibers.

4. Preparation of Reagent/ Solution :
(1) Weigerts Iron Hematoxylin Solution
Solution A Solution B
Hematoxylin= 5gm 29% ferric chloride = 20ml
95 % alcohol = 500ml Distilled water = 475ml
HCL = 5ml
Mix well ,its stable now for 1 year
(2) Van Gieson Solution
Acid Fuschin = 10ml
Picric Acid = 100ml

5. Principle : VVG is a two part combination stain
that enables differentiation of some conective
tissue components in a tissue which are not easily
distinguished by H & E staining :
An iron hematoxylin stain that is specific for elastic
fibers.It forms strong bonds with elastin, the main
component of elastic connective tissue.
And a counterstain comprises two acid dyes ; Picric
acid & acid fuchsin that is specific for collagen.

6. Procedure :
1. Deparraffinize the section and hydrate to distilled
water.
2. Stain with weigerts hematoxylin for 5 minutes
3. Rinse well with running tap water
4. Place in Van Gieson stain for 3 minutes
5. Complete dehydration in three changes of alcohol
(absolute)
6. Clear in Xylene
7. Mount with synthetic resin

7. RESULTS :
Elastic Fibers – Black
Collagen – Red
Other tissue elements - Yellow

8. Special Stain For Parasites

9. Giemsa Stain :
Giemsa stain was a name from a Germany Chemist
scientist, for his application of a combination of
reagents in demonstrating the presence of parasite in
Malaria.
It belongs to a group of stains known as Romanowsky
stains.

10. Principle :
Giemsa stain is used for both thin and thick smears to
examine blood for malaria parasite, a routine check up
for other blood parasites and to morphologically
differentiate the nuclear and cytoplasm of Erythrocytes,
leukocytes and platelets and parasites.
It composed both the Acidic & Basic dyes;
Azure and methylene blue a basic dye binds to the acid
nucleus and Eosin an acidic dye that is attracted to the
cytoplasm.

11. Reagents Used :
Methanol
Giemsa powder
Glycerin
Water

12. Procedure :
Preparation of the Giemsa stain stock solution
(1) Into 200ml of methanol. Add 3.8 g of Giemsa powder and
dissolve.
(2) Heat the solution up to 600C
(3) Then, add 250ml of glycerin to the solution slowly
(4) Filter the solution and leave it to stand for about 1-2 months
before use.
Preparation of Working solution
Add 10 ml of stock solution to 80ml of distilled water and 10 ml of
methanol.

13. Staining Procedure :
Thin Film Staining
1. On a clean glass slide make a thin film of the
specimen (blood) and leave to air dry.
2. Dip the smear into pure methanol for fixation of
the smear and leave to air dry for 30 seconds.
3. Stain with diluted Giemsa stain solution (1:20
vol/vol) for 20-30 minutes. (For 1:20 dilution,
add 2ml of stock Giemsa to 40 ml of buffered
water)
4. Flush with tap water and leave to dry

14. Thick Film Staining
(1) Add a thick smear of blood and air dry for 1 hour
on a staining rack.
(2) Dip the thick blood smear into diluted Giemsa
stain (1:50,vol/vol) (for a 1:50 dilution add 1 ml of
stock Giemsa to 50ml of buffered water)
(3)Wash the smear by dipping in in buffered water of
distilled water for 3-5 minutes
(4) Leave it to dry

15. Results :
Malaria parasite
Nucleus – red or pink
Cytoplasm - blue

16. Staining For Pigments

17. Fontana Masson Silver Staining :
It is used for the demonstration of melanin and
argentaffin granules in tissues.
(Argentaffin granules are found in carcinoid tumors)
Melanin is a non lipid, brown-black pigment present
normally in the hair,skin,retina, iris and certain parts of
CNS.

18. Principle :
Melanin has the ability to reduce solutions of
ammonical silver nitrate to metallic silver without the
use of an external reducing agent.
Results :
Melanin- Black

19. Special Stain For Minerals

20. Von Kossa Stain For Calcium :
The technique is for demonstration deposits of calcium or
calcium salt so it is not specific for the calcium ion itself.
In this method tissue sections are treated with a silver
nitrate solution and the silver is deposited by replacing
the calcium reduced by the strong light, thereby
visualized as metallic silver.
Reagents :
1% Silver nitrate solution
5% Sodium Thiosulfate
0.1 % Nuclear Fast Red Solution

21. Procedure :
1. Deparrafinize the section and hydrate to water
2. Place the section in 1% silver nitrate solution and expose to
bright sunlight or under the light of 100 watt bulb for 30
minutes
3. Wash in distilled water
4. Place in 5% sodium thiosulfate for 3 minutes
5. Wash throughly in tap water
6. Stain in nuclear fast red
7. Dehydrate and mount
Results :
Calcium deposits – Black
Cell nuclei – red
Cytoplasm - pink

22. Staining Of The Section For Hemosiderin

23. Principle :
Hemosiderin – a protein compound that stores iron in
your tissue- can accumulate under your skin. Most often
appear on the lower leg, sometimes covering the space
between your knee and ankle.
This happens because of hemoglobin , a protein molecule
that contains iron. When sometimes Rbc’s breaks down
,the haemoglobin releases iron. The trapped iron is then
stored as hemosiderin in tissues beneath your skin.
Hemosiderin reacts with potassium ferrocyanide in acid
medium and yields a Prusian blue color.

24. Procedure :
1. Deparrafinize the section
2. Take the section to water (hydration)
3. Put the slide in working acidified ferrocyanide solution
for 5 – 20 minutes
4. Wash throughly with distilled water
5. Stain with nuclear fast red for 5 minutes
6. Rinse with distilled water
7. Dehydrate and mount
Results :
Hemosiderin : Prussian blue
Nuclei : Red
Cytoplasm : Pink