2. What is Stain?
A dye or chemical that has an affinity for the particular tissue
component that is to be demonstrated. They allow the presence/or
absence of certain cell types, structures and/or microorganisms to be
viewed microscopically
4. Types of staining techniques
Simple staining
(Use of of single stain)
Differential staining
(Use of two contrasting stains)
Direct
(Positive)
Indirect
(Negative)
Separation
into groups
1. Gram stain
2. Acid fast
stain
Visualization
of structures
1. Flagella stain
2. Capsule stain
3. Spore stain
6. Importance of fixing the smears
Fixation accomplishes three things:
• It kills the organisms;
• It causes the organisms to adhere to the slide; and
• It alters the organisms so that they more readily accept stains
(dyes).
7. Routine staining(H&E)
HAEMATOXYLIN
• Basic dye
• Stains acidic or basophilic
structure with a purplish blue
colour
• Cell nucleus (which contains
DNA and nucleoprotein)
• Organelles that contain RNA
such as ribosomes and the
rough endoplasmic reticulum.
EOSIN
• Acidic dye
• It stains basic, or acidophilic
structures with reddish or
pink colour
• Cytoplasm, cell walls, and
extracellular fibres.
8.
9. ADVANTAGES-
• Its merit is that it is not expensive
• Readily available in virtually all dermatopathology practice settings.
DISADVANTAGES-
• This stain, does not clearly demonstrate certain tissue components, such
as elastic fibres and does not allow differentiation between melanin,
haemosiderin and other skin pigments.
• Special stains are required for these purposes, and also for confirming the
nature of abnormal dermal deposits, such as calcium, mucin and amyloid
and for the demonstration of microorganisms.
10. Special Stains
• Used in addition to H & E staining to selectively
stain cells and cellular components
• Gives information on:
• Presence of certain class of molecules
• Their localization
• Number of molecules present
11. Special stains:
It covers a wide variety of methods that may be used to visualize
particular tissue structures, elements, or even microorganisms not
identified by H&E staining.
Can be grouped into:
1. Stains for detection of microorganisms
2. Connective tissues and lipids
3. Amyloid
4. Minerals .pigments and miscellaneous.
5. Carbohydrates(mucopolysaccharides)
13. GRAM’S STAINING
Dr Hans Christian Gram introduced it in 1884. This process is Carried out
by differential stain known as Gram’s stain.
It identifies and classifies two major groups of bacteria, i.e. Gram-positive
and Gram-negative.
14. Procedure:
Gram staining Protocol
Primary staining Heat fixed smear is flooded by crystal
violet and allowed to stand for 1min
Mordanting After washing, iodine is then flooded and
allowed to stand for 1min
Decolourization After washing, alcohol is added that is
washed immediately
Counter staining At last, safranin/Carbol fuchsin is flooded
over the smear and allowed to stand for
30sec, then washed by water.
Observation After air drying, place one drop of oil
immersion over the smear and adjust the
microscope to identify the specimen,
whether it is gram negative or gram
positive. Gram positive appears purple in
colour and Gram negative appears pink in
colour.
15. Basic classification of bacteria are based on this staining.
Gram positive-Bacteria with
large deposits of peptidoglycan in their cell
walls retain methyl violet
Gram negative-Bacteria with
large deposits of lipids and
lipopolysacharrides and less
peptidoglycan
16.
17. ACID FAST STAINING
Paul Ehrlich first developed it in 1882. And later, modified by Ziehl
Neelsen.
Used to stain Mycobacteria, oocysts of Cryptosporidium parvum,
Cyclospora, Isospora; also hooklets of cysticerci
Acid fast cells stain Red
non acid fast cells stain Blue
18. • Mycobacterial cell walls contain a waxy substance composed of mycolic acids. The
property of acid fastness (Property of the mycobacteria of being stained with
specific dyes and resisting de-colouring with acids and alcohol)is related to the
mycolic acid.
• The leprosy bacillus is much less acid and alcohol fast than the tubercle bacillus.
19. Procedure:
Acid fast staining Protocol
Primary staining Heat fixed smear is flooded with carbol
fuschin(heated) and allowed to stand for
10-15 min.
Decolourization After washing, acid alcohol is added.
Counter staining At last, methylene blue is flooded over the
smear and allowed to stand for 30 sec,
then wash it with water
Observation After air drying, place one drop of oil
immersion over the smear and adjust the
microscope to identify the specimen,
whether specimen is acid fast or not.
Acid fast appears red in colour and non
acid fast appears blue in colour.
22. Fig. Fite – Faraco stain for M. leprae where Lepra bacilli- Magenta Back ground- Pale blue
23. GIEMSA STAIN
it contains a mixture of Azure, Methylene blue, and Eosin dye. It is
specific for the phosphate groups of DNA and attaches itself to where
there are high amounts of adenine-thymine bonding.
Giemsa is a metachromatic stain.
Bacteria stains blue and cytoplasm stains pink and nuclei blue.
Giemsa is used to identify :
1. Mast cellls.
2. Several infectious organisms; malaria parasite, spirochetes, protozoans,
and cutaneous Leishmania in particular.
27. Gomori methenamine silver(GMS )Staining:
Stains fungi, and parasites
like Pneumocystis carnii,
histoplasma species
brown or black with
a green background .
28. Warthin starry stain:
-A silver nitrate stain Where Spirochetes
stain BLACK with a yellow background
-Tissue sections are incubated in a 1% silver
nitrate solution followed by a developer
hydroquinol.
-Used for diseases such as syphilis, Lyme
disease, H. pylori and Legionella.
30. • Periodic Acid Schiff (PAS) :
The periodic acid-Schiff (PAS) stain demonstrates the presence of certain
polysaccharides, particularly glycogen and mucoproteins containing
neutral mucopolysaccharides, by staining them red.
The PAS reaction is of value also in the study of basement membrane
thickening, such as in lupus erythematosus or porphyria cutanea tarda.
31. Because the cell walls of fungi are composed of a mixture of cellulose and
chitin and thus contain polysaccharides, fungi stain bright pink-red with the
PAS reaction.
For the distinction of neutral mucopolysaccharides and fungi from glycogen
deposits, it is necessary to compare two serial sections, one exposed to
diastase before staining and the other not.
Because glycogen is digested by the diastase and thus is no longer colored red
by the PAS reaction, it can be easily distinguished from neutral
mucopolysaccharides and fungi that are diastase resistant.
Because glycogen is present in outer root sheath cells and eccrine gland cells,
demonstration of glycogen may be of diagnostic value in adnexal tumors with
outer root sheath or eccrine differentiation. Demonstration of neutral
mucopolysaccharides is of value in Paget’s disease of the breast and in
extramammary Paget’s disease.
32. • Fig. Periodic acid–Schiff (PAS)
stain, showing thickening of the
basement membrane zone in
cutaneous lupus erythematosus.
• Fig. Periodic acid–Schiff (PAS) stain,
showing numerous hyphae within the
hairshaft in an endothrix infection.
33. • Alcian blue:
Demonstrates the presence of acid mucopolysaccharides by staining them
blue.
Acid mucopolysaccharides are present in the dermal ground substance but in
amounts too small to be demonstrable in normal skin.
However, in the dermal mucinoses, there is a great increase in nonsulfated
acid mucopolysaccharides, mainly hyaluronic acid, so that the mucin stains
with alcian blue.
In extramammary Paget’s disease of the anus with rectal carcinoma and in
cutaneous metastases of carcinoma of the gastrointestinal tract containing
goblet cells, tumor cells in the skin, like their parent cells, secrete sialomucin.
Sialomucin contains nonsulfated acid mucopolysaccharides staining with
alcian blue, as well as PAS-positive neutral mucopolysaccharides.
34.
35. Whereas non-sulfated acid mucopolysaccharides stain with alcian blue at
pH 2.5 but not at pH 0.5, strongly acidic sulfated acid
mucopolysaccharides, such as heparin in mast cell granules and
chondroitin sulfate in cartilage, stain with alcian blue both at pH 2.5 and at
pH 0.5.
36. fig: in papular mucinosis ;epidermis shows no alterations. There are focal
area in the upper and mid reticular dermis due to a separation of collagen
fibers. The area is positively stained with alcian blue, indicating deposit of
mucin (Alcian blue, x40)
38. • Fontana-Masson(black in colour):
-Stains melanin and argentaffin
granules black (nuclei will be
red) .
-Useful for quantifying
melanocytes (e.g. in vitiligo)
and in cases of minocycline
pigmentary alteration.
-Also stains Cryptococcus
42. • Prussian blue stain:
-Stains iron blue.
-The Prussian blue reaction-
tissue treated with dilute
hydrochloric acid and
potassium ferrocyanide .
This stain is used to detect and identify ferric (Fe3+) iron in tissue
preparations, blood smears,or bone marrow smears. Minute amounts of
ferric iron (haemosiderin) are commonly found in bone marrow and in the
spleen. Abnormal amounts of iron can indicate hemochromatosis and
hemosiderosis
44. • Massons
Trichrome
Stain:
1. Used to differentiate
between collagen and
smooth muscle in tumor.
2.Stains cytoplasm, muscles,
keratin –red
,collagen- Blue, elastic
fibres- black.
45. Van Gieson
Stain:
• Used to differentiate collagen and
smooth muscle
• Can be used to demonstrate the
presence of collagen in pathological
conditions
• Stains nuclei blue, Collagen bright
red, Cytoplasm, muscle, fibrin and red
blood cells yellow, elastic fibers black
46. Stains for amyloid :
• Congo red
• Thioflavin T and S
• Crystal violet and methyl violet
• RIT NO. 5 SCARLET
• PAGODA RED
47. Congo red:
Brick red color of amyloid as viewed
on normal microscopy
Apple green birefringence imparted
by amyloid under polarized light
51. • Oil red O :
-Stains fat red; needs
frozen/fresh tissue
(once tissue is fixed and
processed into paraffin blocks,
this method does not work).
-This may be very helpful
in seeing the fat globules
in sebaceous carcinoma
53. • Sudan black B
• Paraffin-embedded
tissue
• stains fat black
54. Stains for mast cells:
• Giemsa and toluidine blue are metachromatic stains for mast cells
• chloroacetate esterase (Leder stain)
55. • Toluidine Blue :
-Used to stain mast cells
-Mast cells stain red- purple
(Metachromatic staining)
and the background
stain blue (orthochromatic
staining) .
56. • Chloroacetate esterase :
(Leder stain)
-The lineage-specific
cytoplasmic granules of
myeloid cells are readily
Identified.
-Give an intense bright red hue.
-It can help distinguish
acute lymphocytic leukemia
from acute myeloid leukemia.
-Mast cells also stain positive.
57. Tissue material to be demonstrated
Tissue material to be demonstrated
• PAS (periodic acid Schiff) for neutral mucin
• Alcian Blue for acid mucin
• Mucicarmine
Mucin (mucopolysaccharides)
• Fontana-Masson
Melanin
• Perl's Prussian Blue
Iron (haemosiderin)
• Von Kossa
• Alizarin red
Calcium
• MSB (Martius Scarlet Blue)
Fibrin
• EVG (Elastic Van Geisen) for reticular dermis
• Orcein for papillary dermis
Elastic fibres
•Oil Red-O (Fat is dissolved in tissue
processing, frozen section required)
Fat
• Toluidine Blue
• Giemsa
Mast cells
58. Collagen Massons trichrome stain
Van Geisons stain
Bacteria •Gram (gram negative very difficult to
demonstrate)
• Ziehl-Neelsen for most mycobacteria (ZN for AFB)
• Wade-Fite for M. Leprae
Fungi • PAS
• Grocott / Gomori methenamine silver (GMS)
59. Immunohistochemistry
stains
• Immunohistochemistry (IHC) is considered to be an advanced form
of histopathology. Immunohistochemistry is not usually used initially but is
added when routine/regular histological testing is insufficient to form a
diagnosis.
• The IHC pattern is considered diagnostic, demonstrating nuclear,
membranous or cytoplasmic patterns.
• IHC is often used in situations where a presence or absence of certain
proteins can form a basis for a diagnosis. It can also be used to distinguish
between two different disease processes that may otherwise appear
similar to the pathologist.
60. Common Antigens distribution in Normal Skin
Antigen Location
HMB45 Melanoma cells
Cytokeratins, including AE1, AE3, CAM 5.2,
CK20 .
Epidermis and its appendages and their
tumors.
Vimentin Mesenchymal cells, melanocytes,
lymphomas, sarcomas, melanomas.
CD1a ,CD207 Langerhans cells.
MART 1/Melan A Melanocytes
Loricrin Granular cells
K20, Neuron specific enolase,
Synaptophysin, Chromogranin
Merkel cells
CD3 ,CD4,CD45RO Lymphocytes
BPAG1 & BPAG2 D-E Junction
c-kit, CD10, Tryptase Mast cells
