1. Capt Rishi Pokhrel
Dr Swati Patil
Dr Kirti Solanke
Dr Anil Dwivedi

3.  Introduction STAINING
 Dyes
 Types
 Equipments
 Steps
 H&E
 Special stain

4.  Term 1st used by LEEUWENHOEK in1719.used
saffron for muscle fibre
 GOPPERT& COHN in 1849 used carmine
 GERLACH in1858 –selective nuclear staining
for nerve cells,regressive stain-weak acetic
acid
 WALDEYER in 1863-used hematoxylin

5.  Stains are colored substances which
dye tissue
 Dyes-staining agent whose chemical
formula is known,mixture of very
closely related compounds with alike
properties
 Stain-dyes which are metallic salts of
animal and vegetable origin

6.  NATURAL
 SYNTHETIC

7. DYES
BASIC ACIDIC NEUTRAL

8.  Acidic dyes-color acid is combined with non
coloring metallic base(sodium & potassium).
Eosin-Y,light green.Acid fuchsin is sodium
salt of acidic sulphonated derivative of
rosaniline
 Basic dyes-color base is combined with non
coloring metallic acid
(acetate,chloride,sulphate radicle)
Basic Fuchsin(colored rosaniline base and
colorless acidic CI radicle &,haematoxylin
 Neutral dyes- compounds of color base with
color acid.
neutral red.

9. auxochrome
Benzene Quinone Aniline
chromophore chromogen

10.  Dye must ionize in solution to produce
colored cations or anions –unite with proteins
and other tissues to form colored compounds

11. Haematoxylin oxidation Haematein

12.  Stored for long time ;-ve charge possesed by
haematein loses its affinity,mordanting is an
essential
 A mordant ammonium alum, potash alum
,iron alum(aluminium compound) forms lake
with strong basic dye.
 Water soluble lakes from aluminium
compounds are blue.progressive staining
 Lakes from ferric compounds-regressive
staining

13. ALUM IRON
HAEMATOXYLINS HAEMATOXYLINS
DELAFIELD‟S HEIDENHAIN‟S
EHRLICH‟S
WEIGERT
ACID
MAYER
HARRIS

14.  ROUTINE –with hematoxylin &eosin, provides
only little differentiation
 SPECIAL-in selective instances,eg fuelgen.
 VITAL-to stain living tissue eg tyrptan
blue,lithium carmine for histiocytes.

15.  Vital staining
 elective solubility
 Metallic impregnation- Ag(NH3)2OH
 Staining with dyes

16.  SUPRAVITAL-when stain is applied to a tissue
which has already been removed before it is
stained.(dissociation)e.g.-R.E. cells by trypan
blue,lithium carmine stains
histiocytes,alizarine stains bones red
 INTRAVITAL-by injecting dye into the living
org e.g. Janus Green stain mitochondria.
 NON-VITAL-for fixed cells

17.  Substances that dissolve in tissue –
lysochrome
 Fat droplets electively stained in alcoholic
solution if stain is more soluble in fat than in
alcohol

18.  Metallic compounds can be reduced by
tissues to stable metallic state
 Ammoniacal silver –deposited silver is stable
after reduction.
 Argentaffin cell-tyrosin derivative melanin,
phenolic compound-
kultschitzky cells
 Argyrophil cells

19.  Certain basic dyes react with tissue
components such that their normal color
changes from blue to red or purple
 Reason-b‟cos of presence of polyanions
within the tissue
 Metachromatic Dyes –thiazines-toludine blue
 Eg metachr staining in cartilage,epithelial
mucins,mast cell granules

20.  Assist interaction of tissues & dyes
 Mercuric chloride,formaldehyde,ethyl alcohol
split chromatin-DNA & protein
 Trichloroacetic acid,picric acid,chromium
compounds facilitate-acidic dyes
 After fixation ethyle alcohol or acetic acid –
both acidic & basic dyes
 Blockage of carboxyl group with preserved
amino group-basic dyes

21. • Tissue sections placed in
Progressive ascending soln of dye
• Selective affinity of dye for
staining different tissue
• Less sharper
• Tissue over stained
Regressive • Differentiated
• Routinely used
staining

22.  Methyline blue, eosin
 Indirect-dye+mordant=colored lake;combine
with tissue-mordant-dye complex
 Insoluble in ordinary acqeous or alcoholic
solvent,allowing counterstaining &
dehydration

23.  De-staining basic dyes with- weakly acidic
medium mordant
oxidizing agent
dyes
 Aqueous haematoxylin differentiated in
acidified alcohol(1% HCl in 95% alcohol)
 Eosin differentiated in alcohol 0.5%conc
ammonium hydroxide

24.  Ripening of stain
 Well at room temperature
 some require refrigeration –schiff‟s
reagent,aldehyde fuschin,methyl-
green,azocarmine,silver nitrate.

25. EQIPMENT AND MATERIALS
 15cm deep sink,two taps,white
background
 A slide washing tray
 Staining rack-two stout glass
rods,4cm apart
 Bunsen burner

26.  Bright daylight
 Microscope
 Glass lidded jars-for stains,grooved to hold 6
slides-coplin jars
 Stainless steel racks-10-20 slides
COPLIN JAR

27. UniMailer
Slide Storage System
Slide Folder Rack

28.  Removal of paraffin wax-two changes
 Removal of xylene with absolute alcohol
 Treatment with descending grades of alcohol
 Water
 Staining
 Dehydration
 Clearing
 mounting

29. Hydration
water
Differentiation
+Blueing Mounting
+ Dehydration
eosin

30. • Xylene, decreasing concentration of
I. Hydration
alcohol-water
II. Staining with
• over stained 2-20 minutes
haematoxylin
III. Differentiation • By acidified alcohol
IV. Blueing • Water or lithium carbonate
V. Dehydration • Increasing con. Of alcohol up to 95%
VI. Staining with eosin • 0.5-1 % eosin in 90% alcohol 3 sec. to 1 min.
VII. Clearing • Xylene
VIII. Mounting • With Canada balsam

31.  MOUNTING-used between section and coverslip-
1.resinous media(xylene preparation)
2.aqueous media(water preparations)-
KAISER‟S GLYSERINE-JELLY
APATHY‟S MOUNTANT
 1.RESINOUS MEDIA-xylene balsam
colophonium –terpentine
euparal
xam
D.P.X.(distrene-polystyrene
plasticizer-tricresyl phosphate,xylene
B.P.S.(butyl,phthalate,styrene

32.  RINGING MEDIA-mount which fail to set
completely hard sealed at margin
 Solid media-paraffin wax,kronig‟s cement
 Commercially available-cellulose adhesive
Durofix

33.  Labelling of slides-
 Fading of stained section-

35.  1- PERIODIC ACID SCHIFF'S (PAS )-
 Principle: periodic acid oxidizes the carbon to
carbon bond forming aldehydes which react to the
fuchsin-sulfurous acid which form the magenta color.
(Periodic Acid cleaves sugars into aldehyde groups. Aldehydes react
with Schiff Reagent- RED)
 Amyloid ,BM,cartilage,cellulose,cerebrosides,epithelial
mucins,fungi,glycogen,hyaline membrane fetal
lung,lipochrome pigment,mucoid cells of ant lobe of
pituitary,pancreatic zymogen granules,starch,thyroid
colloid
 Results:
Glycogen: magenta (red)

36. H&E PAS

37. Feulgen Reaction:
 Active aldehyde group by breaking purine-
deoxyribose bond
 - DNA (not RNA) is cleaved by HCl, reacts
w/Schiff.
 Acidic phosphate radicle is reason for
basophilia-methyl green pyronin technique

38. Feulgen stain

39. Methyl Green Pyronin
Stain
DNA: blue-green to green
RNA: pink to red

40. PURPOSE: Alcian blue stains acid mucus substances and acidic mucins.
 PRINCIPLE: 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.
- Alcian blue stains both sulfated and carboxylated acid
mucopolysaccharides and sulfated and carboxylated glycoproteins.
- It is believed to form salt linkages with the acid groups of acid
mucopolysaccharides.
 RESULTS:
Acid mucins/mucus substances: blue
cell nuclei:red
background:yellow

42.  Purpose: To differentiate between neutral and acidic mucus
substances.
 Routine stain for G.I. biopsies.
 Results:
Acid mucus substances: blue
Neutral polysaccharides: magenta

43. Acid mucus substances: blue
Neutral polysaccharides:
magenta

44. PURPOSE: acid mucopolysaccharides (mucin), which is a secretion produced
by a variety of epithelial cells and connective tissue cells.
 The mucicarmine technique is also useful in determining the site of a primary
tumor in that finding mucin positive tumor cells.
 Principle: aluminum is believed to form a chelation complex with the carmine,
changing the molecule to a positive charge allowing it to bind with the acid
substrates of low density such as mucins.
 Results:
Mucin: deep rose
Nuclei: black
Other tissue elements: yellow

45. Mucin: deep rose
Nuclei: black
Other tissue elements: yellow

46.  LIPIDS-SUDANlll,SUDAN4,SUDAN BLACK,OIL
RED O
 PROTEINS
 NUCLEOPROTEINS(DNA)-FUELGIN REACTION
 HEMOGLOBIN-BENZIDINE
 COPPER ASSOCIATED PROTEIN-ORCEIN
 FIBRIN-PTAH STAIN

47. OSMIUM TETRAOXIDE

48.  VAN GIESON‟S STAINING-1% acid fuchsin 10ml
aqueous picric acid -100ml
collagen fibre-red,deep red
nuclei-blue to black
other-bright lemon yellow-epidermis
olive –muscle & nerve
 TAEZER-UNNA ORCEIN –for elastic fibre
orcein-1g
alcohol-100ml
HCl-1ml
elastic fibre-dark brown
nuclei-blue

49.  GOMORI‟S STAINING-silver nitrate(10%sol) -20ml
potassium hydroxide(10%sol)-4ml
nuclei-grey
reticulin fibre-black
collagen fibre-greyish purple

50. Stains Reticular Fibers and
Basement Membrane Black.

51.  MASSON‟S TRICHROME-for
collagen,hypophysis cerebri,thyroid gland
mordant-phosphomolybdic acid 5g
phosphotungstic acid 5g
distilled water 200 ml
stain-weigert‟s iron haematoxylin
biebrich scarlet in 1% acetic acid
2.5%fast green in 2.5% acetic acid
result-nuclei –black
cytoplasm-pink to brown
muscle-red
RBC-brilliant scarlet
mylinated nerve -red

52. Figure 2. Muscle and collagen demonstrated
by Masson Trichrome in gastrointestinal
tract. 20X

53.  THIONIN STAINING-nissl substance,decalcified
bone ,mucin ,mucopolysaccharide,mast cell,sex
chromatin
staning sol A-lithium caronate 5.5g
distilled water-1000ml
sol B-thionine -0.25 g
lithium carbonate-10ml
nissle sub-bright blue

54.  CAJAL‟S GOLD SUBLIMATE METHOD-Astrocyte
staining sol-distilled water 100ml
1%aqueous gold chloride 20ml
mercuric chloride 1g
results-astrocytes-reddish purple to black
nerve cells-pale rose to violet
nerve fibres-unstained or stained pale
 DEL RIO HORTEGA „S METHOD-
Oligodendrrocyte,microglia
fixation –FAB(formaline ammonium bromide)
preparation of ammoniacal siver carbonate-
silver nitrate 5ml
sodium carbonate 15ml

58. cresyl violet Golgi's gold method.

59. HAPPY DASERA

60.  The Azan-Mallory stain is one of several commonly
used techniques in which three or more dyes are
combined. These multiple-dye stains have the
advantage of showing a large number of tissue
structures. The Azan-Mallory's stain combines aniline
blue, orange G (stains proteins) and acid fuchsin
(stains DNA and RNA). Collagen-containing
connective tissue is shown as blue, erythrocytes as
orange, and chromatin, nucleoli, basophilic
cytoplasm, and muscle cell cytoplasm as red. With
azocarmine and aniline blue (Azan) stain, a
combination of the basophilic dye (azocarmine) with
aniline blue stains nuclei and basic structures are
stained red and collagen, mucus, and cartilage matrix
are stained blue

61. Figure 1. Weigert‟s Iron Hematoxylin
demonstrating nuclear detail prior to
muscle and collagen staining. 20X

62.  Used to differentiate between collagen and smooth muscle in
 tumors, and the increase of collagen in diseases such as cirrhosis.
 Routine stain for liver and kidney biopsies. the name implies, three dyes
are employed selectively
 staining 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.
 Others suggest that the tissue is stained first with the acid dye, Biebrich
 Scarlet, which binds with the acidophilic tissue components. Then when
 treated with the phospho acids, the less permeable components retain
the
 red, while the red is pulled out of the collagen. At the same time causing
a
 link with the collagen to bind with the aniline blue.

63.  The trichrome stain is utilized as the stain of choice
of distinguishing
 histologic changes in tumors, connective tissue
diseases, muscle
 and fibroblast tumors, renal diseases and
dermatology cases. Even
 the disciplines of forensics, archaeology and
hematopathology
 incorporate the trichrome stain for specific tissue
entities and
 structures. With the utilization of
immunohistochemistry expressions,
 the trichrome techniques still offer a great deal of
diagnostic results

64. Cresyl Violet & Luxol Fast Blue

65.  There are hundreds of other staining
routines, most of which involve the use of
gold or silver salts. Among the most
elegant of these stains are the ones
developed by Camillo Golgi (1843-1926)
or Santiago Ramon y Cajal (1852-1934),
who shared a Nobel prize for their work in
1906. These methods are especially
useful for visualizing glial elements. Both
these men are great figures of the history
of the life sciences and the study of the
nervous system in particular. Golgi
developed several stains that are still used
today, was the discoverer of several
important nervous system structures, and
won the Nobel Prize for his work. Golgi's
stains comprise a set of methods for
nerve cells and fibers; they're
characterized by fixation in an aldehyde-
osmium-dichromate solution, followed by
impregnation with silver salts. As you can
see here, the process renders the subject
as several shades of golds, browns and
blacks. Neuron somata are golden and
their processes black. This stain permits
the definition of much detailed
information about the structure of the
nervous system.

69. unmylinated

70. Mallory's connective
L S OF NERVE
tissue stain

71. PURKINJE CELLS H&E

72. Wilder‟s retinaculin method

75.  Suprarenal capsule
 Masson‟s fontana method for argentaffin
granules-direct reduction after non alcoholic
fixation
 Argyrophil cells-silver impregnation
techniqes demonstrate argentaffin cells after
alcoholic fixation
 Diazo reaction for argentaffin granules-red
 Gibbs‟ method

76.  Romanowsky mixed methylene blue and
eosin

77.  Heidenhain‟s method and masson‟s trichrome
stain

78.  Commence fixation with tissue intact ,bisect
later on same day.
 Acetic formaline-penetrate rapidly
 Acid fixation prevent “pink disease”
 Reticulin stains

79.  Posterior pituitary-neurosecretory substance
& hypothalamus is rich in cystine-acid alcian
blue technique is better than gomori‟s
aldehyde fuchsin

80.  BASEMENT MEM- P.A.S
 CONNECTIVE TS FIBRE
a- COLLAGEN FIBRE- VAN GIESON‟S
b- ELASTIC FIBRE- TAENZER UNNA ORCEIN
METHOD
c- RETICULIN FIBRE- GOMORI‟S
TRICHROME,GOLGI SILVER/SILVER STAIN

81.  Basic stain-base contains coloring substance
combined with acidic radicle-Basic fuchsin
 Acidic stain-
 Romanowsky-combination of polychrome
methylene blue and eosin
 Colorless leucobases-dyes can be reduced
easily

82. Masson's Trichrome Stain Muscles (red) Masson's Trichrome Stain Collagen
(green or blue) Masson's Trichrome Stain Mucus (green or blue) Masson's
Trichrome Stain Cytoplasm of most cells (pink) Masson's Trichrome Stain
Glycogen (deep red or magenta) Periodic Acid Schiff (PAS) Reaction
Contents of goblet cells (red or magenta) Periodic Acid Schiff (PAS)
Reaction Basement membrane (positive or pink) Periodic Acid Schiff (PAS)
Reaction Brush borders in kidney tubules (positive or pink) Periodic Acid
Schiff (PAS) Reaction Elastic fibers (jet black) Verhoeff's Stain for Elastic
Tissue Nuclei (gray) Verhoeff's Stain for Elastic Tissue Remaining
structures (pink) Verhoeff's Stain for Elastic Tissue Fibrous c.t. (deep
blue) Mallory-Azan Stain Mucus (deep blue) Mallory-Azan Stain
Erythrocytes (red-orange) Mallory-Azan Stain Cytoplasm of liver (pink)
Mallory-Azan Stain Cytoplasm of kidney (pink) Mallory-Azan Stain Nuclei
(red) Mallory-Azan Stain Erythrocyte cytoplasm (pink) Mallory-Azan Stain
Lymphocyte nuclei (dark purple-blue) Mallory-Azan Stain Lymphocyte
cytoplasm (pale blue) Mallory-Azan Stain Monocyte nuclei (medium blue)
Mallory-Azan Stain Monocyte cytoplasm (pale blue) Mallory-Azan Stain
Neutrophil nuclei (dark blue) Mallory-Azan Stain Eosinophil nuclei (dark
blue) Mallory-Azan Stain Eosinophil granules (bright pink) Mallory-Azan
Stain Basophil granules (deep purple) Mallory-Azan Stain Platelets (light
blue) Mallory-Azan Stain Myelinated fibers (blue-black) Cajal's and Del
Rio Hortega's Methods (silver and gold) Unmyelinated fibers (blue-black)
Cajal's and Del Rio Hortega's Methods (silver and gold) Neurofibrils
(blue-black) Cajal's and Del Rio Hortega's Methods (silver and gold)
General background (nearly colorless) Cajal's and Del Rio Hortega's
Methods (silver and gold) Astrocytes (black) Cajal's and Del Rio Hortega's
Methods (silver and gold) End product of stain (can be black, brown or
gold) Cajal's and Del Rio Hortega's Methods (silver and gold) Lipids in
general (black) Osmic Acid (Osmium Tetroxide) Stain Lipids in the myelin
sheath of nerves (black) Osmic Acid (Osmium Tetroxide) Stain Elastic
fibers (brown-reddish) Orcein Stain

83.  Widely utilized
 techniques are the Masson, Gomori One Step, Martius
Scarlet Blue
 and Mallory. ionized acid dyes react with the ionized basic
tissues.
 fibrils of collagen stained blue, fibroglia, neuroglia and
muscle
 fibers stained red and fibrils of elastin stained pink or
yellow.
 The trichrome stain is also used to
 distinguish tumors that have arisen from muscle cells and
fibroblasts.
 Gomori‟s trichrome is the trichrome stain of choice for
distinguishing
 histological changes that occur in neuromuscular diseases.