contents
Introduction&history.
Dye chemistry
Theory of staining
H&E staining
Connective tissue stains
Histological Staining of teeth
Carbohydrate staining
Lipid staining
Microbial staining
Staining in exfoliative
cytology
DYE a coloured substance that has affinity for the substrate to which
it is employed.
Why a dye molecule appears in certain colour?
Colour is seen by eye as a result of effect of certain electromagnetic waves on rods and
cones of retina.
White light being composed of all colours of visible spectrum varies in wavelength from
4000-8000A .
If a light of specific wavelength is absorbed from white light the resultant light will then be
coloured depending upon particular wavelength to be removed.
Stained materials absorb certain components of white light illuminating the specimen and
therefore appear coloured.
For instance, if the red rays are removed from white light, the colour we detect is bluegreen. Blue-green is complementary to red, and red is complementary to blue-green.
Impregnation:
it is deposition of salts or heavy metals over certain selective cells, tissue
structures and processes. It has following main characteristics:
1.structures demonstrated are renderd opaque and black.
2.colouring matter is particulate.
3.the deposit is on or around but not in the element demonstrated.
STAINING:
1.vital staining- this involves staining of structures in living cells either in vivo or in
vitro.
2.vital staining by phagocytosis: particles of coloured matter are taken up by
phagocytic cells, and form colloid suspensions.
3.Histochemical reactions: in this reaction takes place between stain and tissue.
4.Fat stains:
the colouring agents are more soluble in element to be demonstrated
than vehicle.
5.Histologic stains: these stain killed or non living elements by methods that
CLASSIFICATION OF DYES
Depending upon chemical composition:
Nitroso group, nitro group, azo group, thiazol group etc.
Counterstain
Counter Stain: These are the substances, which are used to gain information supplementary to that given by
the Primary stain. This demonstrates the overall morphology or histology of the tissue concerned. It
may consist of single dye / dye mixtures. Depending upon the primary target, it may color the cell
nucleus, cytoplasm or specific tissue structures, e.g.-collagen.
Requirements of a counter stain: 1. It should be contrasting.
2. It should have subtle color.
Theories of staining
covalent bonding
Solvent solvent interactions:
hydrophobic bonding
Stain-stain interactions:
vanderwaals between dye molecules
RATE OF REACTION:
EFFECTS OF FIXATION:
Retention and reactivity of the tissues affect staining.
For ex: many lipids are well preserved after fixation in osmium tetroxides and
poorly preserved after formalin.
Steps in staining:
Removal of wax with xylol
Hydration with graded alcohols
Staining
Dehydration through graded alcohols
Clearing in xylol
Mounting under coverslip
Mordants
Some stains will not stain a tissue until third element is present this is called as mordant.
mordant forms a link between tissue and stain.
The combination of dye and mordant forms a compound which is called as lake which is
capable of attaching firmly to the tissues.
Mordants most commonly used are salts ,usually sulphates of chromium,aluminium,iron etc
Ex: Heidenhains iron hematoxylin.
ACCENUATORS
Name given to group of substances ,while not acting as mordants form lakes with dyes or
taking part in any obvious chemical union increase the selectivity or staining power of dyes
Ex: pottasium hydroxide in loefflers methylene blue
METACHROMASIA
Everson pearese defined metachromasia as the staining of a tissue component so that the
absorption spectrum of the tissue dye complexdiffers sufficiently from that of original dye.
Certain basic dyes will stain the tissue a colour other than the dye this is called
metachromatic staining.
Ex: mucin stains red with toludine blue .
methyl violet stains amyloid deposits red.
H&E STAINING
Introduction :
H&E stain, HE stain or hematoxylin and eosin stain, is a popular staining method used in
histology.
The staining method involves application of the basic dye hematoxylin, which colors basophillic
structures with blue-purple hue.
and alcohol-based acidic eosin Y, which colors eosinophilic structures bright pink.
ADVANTAGES:
most widely used stain.
simplicity and ability to demonstrate enormous number of different tissue structures.
disadvantages:
takes 2-3 months to ripen
HEMATOXYLIN
Alum hematoxylin
iron hematoxylin
Tungsten hematoxylin
Molybdenum hematoxylin
Haematoxyli
n
Alum hematoxylins
Routinely used
Mordant- potash alum, ammonium alum.
Bluing: all stain nuclei red colour which is converted to
blue black colour when section is washed with weak
alkali(tap water is usually alkaline or sometimes alkaline
solutions such as lithium carbonate are used.)
Alum hematoxylins can be used regressively i.e section is
over stained then differentiated in acid alcohol followed by
bluing or progressively.
EHRLICHS HEMATOXYLIN
Staining time30-40 min
Natural ripening ,retains its ability for months.
Ind:
nuclear stain, stains mucin, polysaccharides, cartilage, bone.
Comp:
hematoxylin-2 gm
absolute alcohol -100ml
glycerin-100 ml (slows down oxidation process)
distilled water-100 ml
glacial acetic acid-10 ml
Pottash alum-15gm
ADVANTAGES
It stains nuclei intensly.
Stained sections fade less slowly.
Particularly useful for sections exposed to acids and tissues stored in formalin for
long time.
DELAFIELDS
HEMATOXYLIN
Comp:
hematoxylin-4gm
95% alcohol
saturated aqueous ammonium alum
glycerin
Harris hematoxylin
MAYERS HEMATOXYLIN
Comp:
hematoxylin-1 gm
distilled water-1000ml
ammonium alum-50 gm
sodium iodate-0.2 gm
citric acid-1 gm
chloral hydrate-30 gm
GILLS HEMATOXYLIN
It is used as routine H&E staining.
Comp:
hematoxylin-2 gm
sodium iodate-0.2 gm
ammonium sulfate-17 gm
distilled water-750 ml
ethylene glycol-250 ml
glacial acetic acid-20 ml
CARAZZI HEMATOXYLIN
Chemically ripened using pottasium iodode.
Used as nuclear stain mainly used for frozen sections for
urgent biopsy.
Comp: hematoxylin 5 gm
glycerol
100 ml
potash alum 25 gm
distilled water 400 ml
potassium iodide 0.1 gm
RESULTS
Nuclei blue/black
Cytoplasm varying shades of
pink
Muscle fibres- deep pink or red.
Red blood cells-orange or red
Fibrin- deep pink
IRON HEMATOXYLINS
LOYEZ HEMATOXYLIN:
Ferric ammonium sulfate is mordant
Used to demonstrate myelin can be applied to paraffin and frozen sections
VERHOEFFS HEMATOXYLIN:
Used to demonstrate elastic fibers
Other hematoxylins
Tungsten hematoxylins
MOLYBDENUM HEMATOXYLIN
They are recommended for demonstration of collagen,reticulin
Ex: phosphomolybdic acid hematoxylin
LEAD HEMATOXYLIN:
Identification of endocrine cells in tumors, gastrin secreting cells in stomach.
Eosin
Eosin is a widely used counter stain. It was discovered in 1871.
In Greek, Eos means Dawn as it gives yellowish pink shades. The name came as
it imparts Fine rose red color to silk.
Eosin
The eosins are xanthenes dyes(acid aniline dyes) which are acidic in nature.
They are all halogenated derivatives of fluorocein.
Fluorecein---BR------------Eosin
Eosin is derived from fluorocein by the action of bromine. This eosin is used
as textile dye/ink preparation. The red sodium/potassium salt of this powder is used in
biology to stain cells.
FACTORS AFFECTING THE EOSIN-STAINING: 1. pH: 2. 2.Fixation: 3. 3.De-calcification: -
FACTORS AFFECTING THE EOSIN-STAINING: 4. Addition Of Glacial Acetic Acid: The amount added can vary from 1 drop/ litre to 0.5 ml/litre.
Glacial acetic acid
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Decrease in pH
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Increase in the ionization of tissue amino groups
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Increase in the attachment of dye
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Increase in depth of coloration (however differential effect is diminished)
Too much acid causes intense but flat and homogeneous staining. So it should be
avoided unless the effect is preferred.
Types Of Eosin
Types
Eosin y
45380
Acid red 87
Ethyl eosin
45386
Solvent red 45
Eosin B
45400
Acid red 91
Phloxine B
45410
Erythrocin B
45430
Acid red 51
Rose bengal
45440
Acid red94
Mercurochrome
EOSINY
EOSIN-Y
Common name: - Eosin y
Suggested names: - Eosin Y ws
Class: - Xanthene (Fluorone)
Empirical formula: - C 20 H 6 O5 Br 4 Na 2
Synonyms:-Bromofluorescein
-Disodium eosin
-Sodium eosin
-Tetra bromo fluorecein sodium salt.
-Eosine yellowish (ys)
-Bromoeosine
-Bromo fluoresceic acid
Chemical Structure: C20 H6 Br4 Na2 O5
EOSIN- Y
EOSIN-Y
1.
2.
3.
4.
5.
6.
7.
8.
Uses: Eosin y is the most common counterstain to alum haematoxylin in H & E method
It is one of the dyes in papanicolaus EA solutions for staining exfoliative cytology.
As a counterstain in the Gram-Weigert method for Gram positive bacteria and fibrin
In making Romanowskys stain.
It is one of the components of Wrights stain for blood corpuscles.
For staining bone marrow to study the cell morphology.
In modified Nochts stain.
In Eosin-Methylene blue medium.
Modified Eosin-Y
1. Edgar-degas eosin
2. Rubens-eosin-Phloxine
3. Treosin
4. Meter-Eosin
5. Eosinol
Modified Eosin-Y
Edgar-Degas Eosin-Y:
-Modified alcoholic Eosin-y
-It has been modified with the addition of stabilizers that have prolonged shelf life.
Rubens-Eosin-Phloxine: -Alcoholic eosin y-Phloxine B counter stain which gives vibrant staining result than other eosins.
-Moreover, staining intensity is more subtle than other eosin y-Phloxine B formulations.
-Pink shades are more Vivid.
Treosin: It is a slightly acidified combinations of Eosin y + orangeG + Acid fuschin. It provides greater
intensity and more vibrant colours than with traditional eosins. The shades of colour range from
pink, red orange to bright red.
Uses:-It is excellent for frozen sections / for intense histologic staining. This stain also
differentiates between collagen and smooth muscle. So it may reduce the need for
trichrome stains.
Modified Eosin-Y
Eosin- B
EOSIN-B:-
C.I. no:-45400.
C.I. name:-Acid red 91.
Synonyms:-Eosin bluish
-Imperial red
It gives bluish cast instead of yellow cast of eosin y.
PHLOXINE-B
ERYTHROCIN-B
ERYTHROCIN-B
C.I. no:-45430.
C.I no:-Acid red 51.
Synonym:-Food red 14
Uses:1.It is used with methylene blue as a plasma stain for
nerve
cells.
2.For staining bacteria in soil.
3. Phosphorescent triple probe for studying diffusion
of membrane proteins.
4.HPS-stain.
ROSE BENGAL
C.I. No:-45440.
C.I. No:-Acid red 94.
MERCUROCHROME:MERCUROCHROME:Synonyms:-Mercurochrome 220
-Merbromin.
Carbohydrate stains
Mucin staining:
Periodic acid schiff
Alcian dyes
Glycogen stains:
Periodic acid schiff
STAINING OF LIPIDS
Sudan III
Oil red in isopropyl alcohol
Sudan iv
Fetterot in propylene glycol
Micro organisms
Bacteria:
Simple stains: use only one dye that stains the cell wall. The cells are then visible against a light
background
Differential stains: use two or more stains and allow the cells to be categorized into various
groups or types.
The most common stain used in microbiology is the Gram stain.
Gram Staining Steps
1.
Crystal violet acts as the primary stain. Crystal violet may also be used as a simple
stain because it dyes the cell wall of any bacteria.
2.
Grams iodine acts as a mordant (Helps to fix the primary dye to the cell wall).
3.
Decolorizer is used next to remove the primary stain (crystal violet) from Gram
Negative bacteria (those with LPS imbedded in their cell walls). Decolorizer is
composed of an organic solvent, such as, acetone or ethanol or a combination of both.)
4.
Finally, a counter stain (Safranin), is applied to stain those cells (Gram Negative) that
have lost the primary stain as a result of decolorization.
Gram positive organisms- blue black
Gram negative organisms- red.
H&E STAINING
Staining
methods in cytolo
MYCOBACTERIA:
Zeihl-neelsens staining
SPIROCHATES:
Fontanas method and levaditis
method
FUNGI
Grocott methanamine silver for fungi
Gromoris silver methanamine
technique.
VIRUS
Phosphotungstic acid
Osmium tetroxide
Ruthenium tetroxide
Thank you
Thank you