STAINING & H&E

INTRODUCTIONa histological dye.

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

Staining is an auxiliary technique

used to enhance contrast in the
microscopic image.
The objective of staining tissue
sections is to impart colour and
therefore contrast to specific tissue
constituents.
HISTORY:
1714 Leewenhock was first person
to use dye as biologic stain.(saffron to
muscles)
1849 Goppert &cohn introduced
caramine.
1863 waldeyer introduced
hematoxylin.
1856 major advance was introduction
of aniline dyes.
1891 Heidenhain devised iron
hematoxylin still used today.

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.

Methods of use of dyes

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.

Depending upon their source:

Natural dyes carmine,orcein,hematoxylin etc
Synthetic dyes aniline dyes

Other classification acidic dyes(acid fuschin)

basic dyes(basic fuschin)
neutral dyes(azure eosin)

chemistry of aniline dyes

Benzene :it is formed by six carbon atoms and 6 hydrogen atoms .

Benzene absorbs colour in the ultraviolet region so colourless .
When two hydrogen atoms are replaced by oxygen a readjustment of double bond
takes place and a new compound is formed which is coloured.
CHROMOPHORES
Chemical structures absorb certain wavelengths of light and therefore confer colour
are known as chromophores.
Chromophoric groups are usually linear sequence of atom in which there is linear
sequence of double and single bonds.
Ex: quinonoid arrangement is a chromophore.
AUXOCHROMES
To be a dye, a compound must contain in addition to chromophoric group another
group which gives new compound power of electrolytic dissociation.such groups are
called auxochromes.Ex: if chromogen trinitrobenzene is added to hydroxyl group
which is an auxochrome the chromogen will be converted to dye picric acid.

STAIN & STAINING

STAIN A marker or a reagent used to generate the marker.

A permanent discolouration that can clearly be distinguished from the surface or
medium it is found upon.
STAINING an element is defined as stained ,when following treatment with reagent
or series of reagents it aquires colour.

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

Physical theories: these depend upon

simple solubility
EX : fat stains which are more soluble in fat than solvent in which it is dissolved.
adsorption
Large body attracts to itself minute particles in sorrounding medium.

Chemical theory of staining:

According to this theory actual chemical combination of dye and tissue takes place.
Ex: acidic dyes stains basic elements(cytoplasm).
The stain theory is based on the attraction of oppositely charged tissues and dye molecules.
Most histologic dyes are classified either as acid/as basic dyes. An acid dye exists as anionic
(Negatively charged) in solution where as basic dye exists as a cation (positively charged)
tissue groups.
Acidic dye + Basic tissue group-----------Stained tissue
In H&E, haematoxylin-metal complex are cationic (positively charged) eosin is anionic (negatively
charged)
Basophilic: Any substance that is stained by the basic dye such as haematoxylin is considered to be basophilic.
Acidophilic: A substance that is stained by the acidic dye such as eosin is considered to be Acidophillic.

Why are stains taken into tissues?

Stain uptake is due to dye-tissue or reagent tissue affinities.
Affinity: attractive forces binding dye to tissue.
Various contributions to stain tissue affinity are:
Stain tissue interactions:
coloumbic attractions
vanderwalls forces
hydrogen bonding

covalent bonding
Solvent solvent interactions:
hydrophobic bonding
Stain-stain interactions:
vanderwaals between dye molecules

Why are stains not taken up in to every part

of tissue?

NUMBER AND AFFINITY OF BINDING SITES:

For ex: negatively charged dyes have affinty for tissue structures carrying
cationic charge.
Sudan dyes have high affinty for fat but low affinity for sorrounding
hydrated proteins.

RATE OF REAGENT UPTAKE:

For ex: mucin staining using alcian blue requires short period

RATE OF REACTION:

RATE OF REAGENT LOSS:

regressive stain involves selective loss of stains from tissues dye is lost
from permeable structure first and impermeable structures retain stain

What are the effects of specimen

geometry on staining?

1. THE THREE DIMENSIONAL FEATURES OF SPECIMENS:

Specimens are objects with not only breadth and width but also depth.
Dipersed cells stain differently ,than tissue blocks and thin sections stain
differently from thick.

SIMPLE GEOMETRICAL INFLUENCE ON STAINING:

Thin specimens stain faster than thick,specimens with irregular surfaces stain
faster than smooth, dispersed specimens stain faster than uniform slabs.

What are effects of tissue modifications prior

to staining?

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.

stain is applied until the desired intensity of tissue

coloration is attained.

REGRESSIVE STAINING: type of staining in which

ning
tissues are overstained and excess dye then removed
selectively until the desired intensity is obtained.

Equipment for staining

staining bench slide washing tray
staining rack
sink
gas point for bunsen burner
shelf space to carry stocks of stains
&drop bottles
Wide mouthed screw capped bottles
Couplin jars
Wide mouthed glass stoppered bottles
Drop bottles
Coverslips and ancilliary instruments

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

it is an extract derived from heartwood of tree hematoxylin campechianum.

It is extracted from logwood with hot water and then precipitated with aqueous solution
of urea.
Hematoxylin itself is not a stain ,its oxidation product hematin is responsible for colour
properties.
Hematoxylin is anionic and has poor affinity for tissues and is inadequate as nuclear
stain without mordant
Mordants commonly used aluminium,iron,tungsten.
HEMATIN IS PRODUCED BY 2 WAYS:
1.natural oxidation also called ripening
HEMATIN
this takes place by exposure to air,light
Slow process ,takes 2-3 months, retain stability for long time.
2.chemical oxidation:
Uses sodium iodide /mercuric oxide
Advantage: they can be used immediately.
Disadvantage: Shorter useful lif

Classified according to mordant used:

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

Staining time-15-20 min

Naturally ripened.

Ind: nuclear stain along with eosin.

Comp:

hematoxylin-4gm
95% alcohol
saturated aqueous ammonium alum
glycerin

Harris hematoxylin

Staining timeprogressive in cytology-4-30 sec

Regressive 5-15 min.
Chemically ripened with mercuric oxide.

INDICATIONS: nuclear staining in exfoliative cytology.

Used regressively and progressively.
Composition:
hematoxylin-2.5 gm
absolute alcohol-50 ml
potash alum-50 gm
ditilled water- 500 ml
mercuric oxide-1.5 gm
glacial acetic acid-20 ml
Hematoxylin is dissolved in absolute alcohol and then alum is added which has been
previously dissolved in warm water. mixture is brought to boil then mercuric oxide is
added.
The quality of stain detoriates after a few months .this detoriation is marked by the
formation of precipitate.
It is better to prepare fresh stain for every month.

MAYERS HEMATOXYLIN

Staining time- as progressive stain-15 min, regressive stain-40-60 min.

Chemically ripened with sodium iodate.

Ind: used as nuclear counter stain in the demonstration of glycogen& in

various histochemical techniques.

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

Disadv-staining of gelatin adhesive &glass itself.

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

2.remove fixation pigments if neccesary.

3.Stain in alum hematoxylins
4.Wash well in running tap water until section
blue for 5 min or less.
5.Differentiate in 1% acid alcohol for 5-10 sec.
6.Wash well in tap water until sections again
blue(10-15 min)
7.Blue by dipping it in alkaline solution followed
by 5 min tap water wash.
8.Stain 1% eosin y for 10 min.
9.Wash in running tap water for 1-5 min
10.Dehydrate through alcohol ,clear &mount.

Disadvantages of alum hematoxylins

Sensitivity to any susequent acid staining

solutions
Ex:van geison stain application of picric acid
fuschin mixture in von geison stain removes
hematoxylin so nuclei are barely discernable.

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

Iron salts are used as mordants & oxidising agents.

Ferric chloride &ferric ammonium sulfate are most common salts .
Over oxidation is problem with this stains.
WEIGERTS HEMATOXYLIN
Ferric chloride is used as mordant/oxidant.
Iron and hematoxylin solutions are prepared seperately and mixed before
use.

Ind: along with eosin it is used for staining CNS tissues.

Comp: a. Hematoxylin solution:
hematoxylin-1 gm
absolute alcohol 100 ml
b.iron solution:
30%aqueous ferric chloride -4 ml
Hcl-1 ml

EIDENHAINS IRON HEMATOXYLIN

STAINING TIME-30-40 MIN

This technique differs from other hematoxylins in following respect:
Mordant is employed seperately from hematoxylin
Mordant is employed as differentiating agent.
INDICATIONS:- mitochondria,muscle striations and nuclear stains can be demonstrated.
Composition:
Iron alum solution:
Ferric ammonium sulfate-5gm
Distilled water-100 ml
Hematoxylin solution:
Hematoxylin-0.5 gm
Absolute alcohol- 10 ml
Distilled water-90 ml

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

Method of staining with iron hematoxylins

Dewax and hydrate to graded alcohols of water.

Mordant in iron solution for 1 hr
Rinse in distilled water
Stain in iron hematoxylin for 1 hr
Wash in tap water
Differentiate in iron solution
Wash in running tap water ,
Dehydrate,clear and mount
RESULTS
Mitochondria, muscle striations myelin stains grey black

Other hematoxylins

Tungsten hematoxylins

Mallory phoshotungstic acid hematoxylin

(P.T.A.H):
staining time: 12-24 hrs
comp: hematin-1 gm
phophotungsic acid-20 gm
Distilled water-1000 ml
It is good stain for tissues of the nervous system.

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.

HEMATOXYLIN WITHOUT MORDANT:

Used to demonstrate various materials in tissue sections like iron,copper.

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 is one of the two Corner stone dyes for all

techniques, the other being hematoxylin.

hematoxylin and Eosin

It is mainly a cytoplasmic stain and it has an ability, with proper differentiation, to

distinguish between the cytoplasm of different types of cells, and between different
types of connective tissue fibers and matrices,
by staining them differing
shades of red and pink.
Eosin is most suitable stain to combine with alum hematoxylin to demonstrate the
general histologic archetecture of tissue.
It has ability to distinguish between cytoplasm of different types of cells and different
types of connective tissue fibres by staining them different shades of pink.

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
|
|
Decrease in pH
|
|
Increase in the ionization of tissue amino groups
|
|
Increase in the attachment of dye
|
|
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.

FACTORS AFFECTING THE EOSIN-STAINING:

5. Staining Colour: When used in conjunction with haematoxylin, the nuclei
come out dark blue and the cytoplasm and nucleoli-red.
Eosin y can demonstrate different structures by
differences in the intensity with which they are stained.

Other Eosins are not as good as Eosin y, at producing

these differences. It will provide at least 3 shades of
pink showing different intensities on erythrocytes,
collagen and the cytoplasm of muscle and epithelial
cells. The single dye can stain different shades because
the dye molecules can aggregate in a concentrated

Types Of Eosin
Types

Colour index no.

Colour index name

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

Eosin Y :Is widely used as cytoplasmic stain.

Addition of little acetic acid is said to sharpen
staining.

EOSINY

Eosin y is most commonly used as it exhibits the comparative

differences between the various tissue elements. Ethyl eosin and Eosin B
come close. Other types tend to be more homogeneous and some what
redder in shade.

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

Composition: 1. Aqueous-0.5% aqueous solution

2. Alcoholic-0.5% acidic ethanol
3. Eosin-y solution(alcoholic) and Phloxine :- 0.5% eosin y +
0.1% Phloxine B
-Addition of
Phloxine can further

improve the result

Thymol is added to inhibit the growth of fungi

Commercial preparations may also contain fluorocein and

Physical Properties: EOSIN-Y

1. Physical state: - Dry powder.
2.Colour: -Red to Brown
3.Odour: -None (Solution has odour of alcohol).
4.Solubility: - In water - 40%
In alcohol 2%
In cellosolve -25%
In Glycol- 27.5%
In Xylene-0 %
5. Molecular weight: - 691.88
6. Stability: -Stable under ordinary conditions
7.Dye content: - 90%
8.Boiling point: -181F
9.Specific gravity: - 0.81(at 68F)
10.Autoignition: -At 363C
11.The molecule carries one negative charge between pH 3 & 5 and two negative charges
above pH 5.
12.Fluorescence:-It is strongly Fluorescent (But this property is hardly ever used).
13.Combustion:-On combustion, the material emits toxic fumes.
14.Reaction with air and water:-Does not react.

EOSIN-Y

Types Of Eosin-Y: 1. Eosin y Alcoholic: - It produces 3 distinct colour variations enhancing

differentiation between muscle, RBCs and connective tissue.
2. Eosin yw-phloxine:-It yields a brighter reddish hue and a more
differentiation of cytoplasmic colour than eosin y.
3.Eosin y saturated: -Very intense and rapid counterstain.

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

Meter-Eosin: It is a mixture of Bjiebrich scarlet + Eosin y + Phloxine B. Tissue is

stained intense red-Displaying various shades.
Eosinol: It is the colored free acid radical of eosin y. It is made from eosin-y
by treatment with hydrochloric acid Eosinol-Y
If it is prepared from eosin BIt is called as Eosinol-B.
It is soluble in ethanol and slightly soluble in xylene, but not in water.
USES:-It is sometimes useful for staining Difficult tissues. To use, small quantity of
eosinol is dissolved into one of the dehydrating alcohol or into the first of xylenes used
for clearing prior coverslipping.

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.

Uses:1.Counterstain for collagen following Mayers haemalum.

2.Used with Azure A as a tissue stain for cell granules, nuclei, and
microorganisms.
3.For differential staining of cells of anterior pituitary.
4.Used in Ramanowskys stain.
5.In preparation of eosinol B.
6.Trichrome staining.

PHLOXINE-B

PHLOXINE-B :C.I. no:-45410

Synonyms:-Cyanosine WS.
-Magdala red.
-Eosin 10B.

Uses:1.In Mallorys Phloxine methylene blue stain.

2.In modified eosin-y preparation.
3.HPS stain.
4.Demonstrates Paneth cell granules in phloxine tartrazine
method.
5.To demonstrate Alcoholic hyaline

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.

Uses:1.Fluorochrome for the study of fats by a uv-technique.

2.Bacteriostatic agent as it represses bacterial growth but permits
growth of soil fungi.
3.For plate counts of soil fungi.
ETHYL EOSIN:USE:-Instead of eosin y, it can be used as a
counterstain for Alum haematoxylin.

C.I. no:- 45386

C.I. name:-Solvent red.
Synonyms:-Eosin S.
-Eosin alcohol.

MERCUROCHROME:MERCUROCHROME:Synonyms:-Mercurochrome 220
-Merbromin.

Uses:1.In the past it was use to mark tissues for orientation

before paraffin processing.
But it contains significant amount of mercury and is
poisonous. So it should be avoided.
2.Used as an antiseptic.
3.To mark the tissue margins.

Health Hazards Of Eosin:

Health Hazards Of Eosin: The product usually does not present a significant hazard in normal laboratory
use as a light microscope stain. But prolonged contact/ with large quantity may cause
health problems.
Eosin powder: 1.Eye contact: -Mechanical irritation and redness.
2.Ingestion: -Nausea, Stomachache and vomiting
3.Inhalation: -The dust is irritating to respiratory tract.
4.Carcinogenicity:-It is suspended carcinogen.
Alcohol: 1. Cumulative poison: -CNS depression , Headache, visual
disturbances and blindness.
2.Damage to liver and kidney.
3.Methanol has shown genetic toxicity in some animals.

Standard hematoxylin and eosin stain for

paraffin sections.
Method :

1. Dewax sections, hydrated through graded alcohols to water.

2. Fixation pigments are removed if necessary.
3. Stained in an alum hematoxylin of choice for a suitable time.
4. Washed well in running tap water until sections blue for 5 minutes or
less.
5. Differentiated in 1% acid alcohol for 5-10 sec.
6. Washed well in tap water until sections are again blue (10-15 minutes).
7. Or blue by dipping in an alkaline solution (e.g. ammonium water),
followed by a 5- min tap water wash.
8. Stained in 1% eosin Y for 10 min.
9. Washed in running tap water for 1-5 min.
10.Dehydrated through alcohols , clear and mount.

Some connective tissue stains

Collagen von geison, Massons trichome.

Reticular fibres: silver impregnation techniques, periodic acid schiff.
Elastic fibres: periodic acid schiff method,weigert resorcin fuschin
method.
Basement membrane: periodic acid shiff technique, methenamine
siver ,bauer feulgen etc
CARTILAGE: alcian blue, periodic acid schiff, toludine blue.
DECALCIFIED SECTIONS OF BONE: H&E ,schmorls picrothionin method,
vonkossa method, solochrome cyanine method,goldners trichome method
etc.

Carbohydrate stains

Mucin staining:
Periodic acid schiff
Alcian dyes
Glycogen stains:
Periodic acid schiff

The Romanowsky stains are all

STAINING
OF BLOOD
based on a combination
of
eosinate (chemically reduced
SMEARS
Eosin) and Methylene blue.

Common variants include Wright

stain, Jenners stain Leishman
stain and Giemsa stain.
All are used to examine blood or
bone marrowsamples. They are
preferred over H&E for inspection
of blood cells because different
types of white blood cells can be
readily distinguished. All are also
suited to examination of blood to
detect blood-borne parasites like
malaria.

TRICHOME- collagen fibres

STAINING
OF TEETH
VAN
GEISON- periodontal
ligament
PICROTHIONIN-dentinal tubules
SILVER IMPREGNATION- nerve
fibers
SUDAN BLACK-lipid&myelin.
GROUND SECTIONS:
Hematoxylin
Picrothionin

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.

STAINING METHODS OF MICRO

ORGANISMS

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

STAINS FOR ELECTRON

MICROSCOPY

As in light microscopy, stains can

be used to enhance contrast in
transmission electron microscopy
..

Phosphotungstic acid
Osmium tetroxide
Ruthenium tetroxide

Thank you

Thank you