Special stains lecture 1 (1)

Special stains

1. Special StainsSpecial Stains
Dr.Sara Seif el Din

2. Stains for collagen
Stains for muscle
Stains for elastic tissue
Stains for reticulin fibres
Stains for carbohydrates
Stains for amyloid
Stains for lipid
Stains for pigments & minerals
Stains for nerve tissue
Stains for microorganisms
Stains for decalcified bone
Broadly classifying the special stains under
following category…..

3. Demonstration of
Collagen
oMassons trichrome stain
oVan Geisons stain

4. MASSON'S TRICHROME
STAIN
PRINCIPLE:
The term ‘trichrome stain’ is a general name for a number
of techniques for selectively demonstration of muscle,
collagen fibers, fibrin, and erythrocytes.
The general rule in trichrome staining is that the less
porous tissues are colored by the smallest dye molecule;
whenever a dye of large molecular size is able to
penetrate, it will always do so at the expense of the
smaller molecule.

5. Human skin
Results
Nuclei Blue- Black
Cytoplasm, keratin, Red
muscle fibers,
intercellular fibers
RBCs, Fibrin

Collagen, mucous, Blue
Cartilage, Amyloid,
adult or mature bone

6. Van Gieson’s Stain (1889)
oVan Gieson’s mixture of picric acid and acid
fuchsin

oThe simplest method for the differential staining
of collagen.
Principle
Picric acid is employed. It provides acidic pH and
acts as a counterstain for muscle and cell
cytoplasm. It forms with dyes a complex which
has affinity for collagen.

7. Results:

 Collagen – deep red

 Muscle, – yellow
cornified epithelium

 Nuclei – blue to black

8. Demonstration of muscle
striations
-Haematoxylin and eosin and trichrome methods can
demonstrate muscle striations.
- They may also be stained by using Heidenhain iron
haematoxylin and Mallory’s phosphotungstic acid
haematoxylin. Both these methods will give better
definition of muscle striations than the trichromes.

9. Mallory's PTAH stain
Purple
skeletal muscle striations in tumor with rhabdomyoblasts.

10. Demonstration of elastic tissue fibres
- Numerous techniques have been evolved for the demonstration of
elastic tissue fibres, although few are in current use.

- Of these, the most popular are
. Verhoeff ,
. Orcein,
. Weigert’s resorcin-Fuchsin,
. Aldehyde fuchsin.

11. Verhoff’s Elastic
Stain
Verhoeff's Elastic stain is used to demonstrate
pathologic conditions such as atrophy, breaks,
thinning, loss etc. in elastic fibers.

12. •Principle
•The tissue is overstained with a soluble lake of hematoxylin-
ferric chloride-iodine. Both ferric chloride and iodine serve as
mordants, but they also have an oxidizing function that assists
in converting hematoxylin to hematein.
• The mechanism of dye binding is probably by formation of
hydrogen bonds, but the exact chemical groups reacting with the
hematoxylin have not been identified.
• This method requires that the sections be overstained and then
differentiated, so it is regressive.

13. Results :

 Elastic Fibres – Bluish Black
to Black
Nuclei - Blue to Black
Collagen - Red
Other tissue elements – Yellow

14. This section shows elastic cartilage stained with hematoxylin
and potassium iodine to reveal elastic fibers (arrow),
which are dark-stained linear structures embedded
in the cartilage matrix.

15. Elastic fibres
•Elastic fibres are thinner than collagen fibres
and are arranged in a branching pattern to
form a three dimensional network. They give
tissue the ability to cope with stretch and
distension. Elastic fibres are interwoven with
collagen fibres in order to limit distensibility
and to prevent tearing.

16. •Elastic material is found in certain ligaments
(elastic ligaments), some cartilage (called
elastic cartilage) and in large arteries (elastic
arteries). Instead, the elastin is laid down in
fenestrated (having gaps or openings) sheets
or lamellae arranged in concentric layers
between layers of smooth muscle.

17. Methods of demonstartion
•aldehyde Fuchsin Elastic Stain

18. Movat’s Pentachrome Stain

19. Verhoeff’s Elastic Stain

20. Demonstration of reticulin
fibres:
Reticulin fibres are demonstrated either by using dyes dyes as means of
coloring agent or by metal impregnation methods.
techniques :1.Gordon and sweet’s method,
2. Gomori’s method,

Silver impregnation is the best method because it provides good
contrast enabling even the finest fibers to be resolved.

21. Wilder’s Reticulin Stain
oUsed to demonstrate reticular fibers in tissue
sections
oDifferential diagnosis of carcinomas, sarcomas, and
lymphosarcomas.
oCertain liver diseases where a change from normal
reticular fiber pattern is seen.

22. Result
oReticulin fiber – Black
oCollagen – Rose color
oOther tissue elements –
Depending on counter
stain used

24. •Gomori Stain for Reticular Fibers

25. Gordon & Sweets Stain for Reticular Fibers

26. Diagnostic applications
•Liver cirrhosis
•Liver fibrosis

27. •Stains for carbohydrates ?
•Mechanism of PAS technique ?
•Differentiation of the mucin by
different stains ?
•Clinical application of PAS stain?

28. STAINS FOR CARBOHYDRATE
oPeriodic Acid Schiff Stain
oAlcian Blue
oMucicarmine Stain – Southgate’s

29.  PERIODIC ACID SCHIFF (PAS )FOR THE
DEMONSTRATION OF GLYCOGEN 
•McManus, 1946)
•Glycogen is a simple intracytoplasmic polysaccharide found
in greatest amount in liver, cardiac and skeletal muscle, with
significant quantities also present in hair follicles,
endometrial glands, vaginal and cervical epithelium,
umbilical cord, neutrophil leucocytes and megakaryocytes.

30. •PRINCIPLE 
•Tissue containing glycol groups or their amino or
alkylamino derivatives are oxidized by periodic acid to form
dialdehydes, which combine with Schiff's reagent to form
an insoluble magenta compound.
•The chromphoric groups of basic fuchsin in Schiff's reagent
are broken by sulphuration to form a colorless solution. In
the presence of free aldehyde groups an insoluble colored
compound similar to the original dye is formed.

31. Results:
PAS positive substances
- magenta

Nuclei - blue

32. PERIODIC ACID SCHIFF (PAS) WITH DIASTASE FOR THE ENZYME
EXTRACTION OF GLYCOGEN
PRINCIPLE 
Glycogen is digested with certain forms of amylase.
Commercially available diastase, which á amylase or
salivary amylase from saliva can be used to digest glycogen
in tissue sections.
Purpose :
- Glycogen is present in mucosa, skin, liver, parathyroid
gland and skeletal & cardiac muscle.
- The PAS stain is used for demonstration of basement
membrane, mucosubstances secreted by the epithelia of
various organs, fungi, etc.

33. •RESULTS 
Presence of glycogen will be evidenced by loss of
staining after enzyme treatment when compared to the
untreated sections.
PAS Stain
PAS with Diastase

34. Glycogen in Ewing Sarcoma

35. Note
oThe most important PAS positive carbohydrates in
tissues are polysaccharides (glycogen),Neutral
mucopolysaccharides, mucoproteins, glyco proteins, and
glycolipids.
oAcid mucopolyssacharides are only weakly positive or
negative.
oThe PAS reaction can be used to demonstrate many
other normal and pathological tissue constituents, the
most important of which are amyloid, basement
membrane, cartilage, cerebrosides, epithelial mucins,
fungi, hyaline membrane of neonatal lung, lipochrome
pigments, mucoid cells, mucoid granules, zymogen
granules, starch, thyroid colloid etc.

36. PAS stain for fungi
oThe fungal cell wall contains polysaccharides that are
oxidized by the periodic acid to aldehydes.
oThe aldehydes react with Schiff's reagent to stain the
fungi rose pink.

37. Candida Hyphae in chronic hyperplastic Candidosis
PAS-positive
materials can
simply be recognized
by their shape
(morphology) ,
eg.Fungal hyphae

38. Clinical Application of PAS Stain
oSalivary Gland Tumors
oSoft tissue tumors
Alveolar Soft-Part Sarcoma (Diastase resistant
crystals)
oFungal Infection
Candidiasis
Actinomycosis

39. ALCIAN BLUE Ph2.5 –
ACID MUCOPOLYSACCHARIDES
Alcian blue is a group of polyvalent basic dyes that are
water soluble. The blue color is due to the presence of
copper in the molecule.
Acid mucin - strongly sulphated connective tissue mucins
react at low pH values with suitable cationic dyes and
are usually PAS-negative.

40. Submandibular salivary gland mucous cell showing
sulfomucin Brown black, sialomucin blue, -Iron
alcian blue technique
RESULTS:

Acid mucins
/mucosubstances - blue
Nuclei
(using Nuclear fast red)
-reddish pink
Background - Very pale red
or colourless

41. Colonic mucosa showing sialomucin that have acid and neutral mucopolysaccharide
stain purple

42. The goblet cells of the gastrointestinal tract are filled with
abundant acid mucin and stain pale blue with this Alcian blue stain.

43. MUCICARMINE STAIN –
SOUTHGATE’S – MUCIN
PRINCIPLE :-
Mucicarmine has large molecular size which allows the dye to
penetrate and bind to acidic substrates of low density, i.e.
mucins. Neutral mucins and some strongly acidic sulphated
mucins do not show appreciable staining.

44. Colonic Mucosa showing sialomsucin
stain --- magenta
RESULTS:
Mucin - deep rose
Nuclei - black
Other tissue elements- yellow

45. Mucoepidermoid carcinoma, mucin positive

46. Demonstration of
amyloid
o Congo red
o Thioflavin T
o Crystal Violet

47. Congo-Red - Putchler’s
Modification – Amyloid
Principle :
Amyloid is homogeneous and eosinophilic, the deposits are
extracellular and may become sufficiently large enough to
cause damage to surrounding tissues. When stained with the
congo red stain the amyloid, with aid of polarizing lenses,
will birefringe an apple green color, under the microscope.
Purpose :
To demonstrate amyloid deposits in tissue sections

48. RESULTS:
Amyloid -red to pink
Nuclei -blue

49. Puchtler and
Sweat
Congo Red
Method
Polarised Light
showing
“apple
green”birefringence
of amyloid deposits

50. Positive red staining is present around the large central artery and
a smaller vessel to its upper right. The right panel shows the green
birefringence that is diagnostic of amyloid when the Congo red
stain is viewed with polarized light.
All amyloids have a fibrillar ultrastructure that gives this reaction.

51. StainS for
demonStration
of lipid and
principle?

52. Stains for Lipid

- Oil Red O
- Sudan Black B

53. PRINCIPLE :
Staining with oil-soluble dyes is based on the greater
solubility of the dye in the lipid substances than in
the usual hydroalcoholic dye solvents.

54. sebaceous gland stained
with oil red O stain
Cytoplasm – red
Nuclei - blue

55. Oil red O stain of fat emboli in lung.

56. RESULTS :
Fat blue -black
Nuclei -red

57. VON KOSSA METHOD FOR CALCIUM
PRINCIPLE:
Tissue sections are treated with silver nitrate solution,
the calcium is reduced by the strong light and replaced
with silver deposits, visualized as metallic silver.
PURPOSE:
Abnormal deposits of calcium may be found in any
area of the body. With the H&E stain, calcium appear
deep blue-purple.

58. Coronary artery showing
calcified atheromatous plaque
RESULTS :

Calcium salts -black
Nuclei -red

Cytoplasm -pink

59. PERL’S- PRUSSIAN BLUE REACTION
PRINCIPLE :

The reaction occurs with the treatment of sections in acid
solutions of ferrocyanides. Any ferric ion in the tissue
combines with the ferrocyanide and results in the formation
of a bright blue pigment called 'Prussian blue" or ferric
ferrocyanide.

60. Hemosiderin, liver, iron stain.
RESULTS:

Iron (hemosiderin) -blue

Nuclei -red

Background - pink

61. Staining for
Microorganism
-Gram staining
-Ziehl- Neelsen (ZN) stain
-Warthin-Starry for spirochetes
-Gomori Methenamine (hexamine) silver for fungi
-Giemsa stain for parasite

62. gram stain of the abscess shows thin, gram
positive rods in chains.
Result
Gram positive organism
- blue-black
Gram negative organism
- red

64. Mycobacterium tuberculosis in lung, Ziehl-Neelsen acid fast stain.

66. Giemsa stain
A stain for hemopoietic tissue and hemoprotozoa consisting
of a stock glycerol methanol solution of eosinates of
Azure B and methylene blue with some excess of the basic
dyes.

67. oGiemsa stain is used for the histopathological diagnosis of
Malaria and other parasites. It is named after Gustav Giemsa,
an early malariologist.
oGiemsa stain is also a differential stain. It can be used to
study the adherence of pathogenic bacteria to human cells.
oIt differentially stains human and bacterial cells pink and
purple respectively. It can be used for histopathological
diagnosis of malaria and some other spirochete and
protozoan blood parasites.

68. Designed to differentiate blood cells and to stain intracellular
parasites in red blood cells and plasma, e.g. Plasmodium falciparum
(malaria parasite(.

69. Toxoplasma gondii

70. Stains for smears
•Blood smears
•Mucosal scrapes : buccal ,tongue
•Mucinous sample : sputum
•Fine needle aspiration

71. Hematologic stains
•Romanowsky stains
Giemsa stain
Leishman stains
Wright’s stains

72. Romanowsky stains
It will stain both nucleus and cytoplasm.
These histology stains are used for blood smear and bone
marrow.
Examples of Romanowsky histology stains include Wright's stain,
Giemsa stain and Jenner's stain.
These histology stains are based on a combination of eosin and
methylene blue. .

73. PAPANICOLAOU STAIN
•Papnicolaou formula
•Harris’s hematoxylin
Hematoxylin - 5g
Ethanol - 50ml
Potassium alum - 100g
Mercuric oxide – 2-5g
Glacial acetic acid - 40ml
•Orange G 6
Orange G (10% aqueous) – 50ml
Alcohol – 950ml
Phosphotungstic acid – 0-15g
EA 50
0.04 M light green SF -10ml
0.3 M eosin Y – 20ml
Phosphotungstic acid - 2g
Alcohol -750ml
Methanol -250ml
Glacial aceticmacid -20ml
Filter all stains before use.

74. •Results:
Nuclei are stained blue while cytoplasm displays varying shades
of pink, orange, yellow and green

75. • Cytologic image of a scraping
of the buccal mucosa. Intermediate
squamous cell with sex chromatin
body (Barr body) (arrow) lying
against the inner nuclear membrane
(Papanicolaou stain).
- Cytology smear showing clusters
of keratinizing squamous carcinoma
indicating metastasis in the lymph
node.