BASIC

HISTOTECHNIQUES
• Presented by: Dr. Madhu Dethariya
• Under guidance of :
Dr. P. M. Santwani
madam
Dr. Nayan Koitiya sir

• ;
OBJECTIVES
• INTRODUCTION
• SOURCES OF SPECIMEN
• PROCESSING
INTRODUCTION :
▪ Histopathology is the branch of pathology which
concerns with the demonstration of minute
structural alterations in tissues as a result of
disease.
▪ For the demonstration of minute histological
changes, the tissue must be processed in such a
manner that it will provide maximum information.
▪ Most convenient way for study of morbid tissue is
to use permanent section.
▪ The histopathological procedures produce
Permanent slides, which can be stored for a longer
period and one can’t manipulate the findings;
therefore, it is considered best reliable technique.
 specimens:
1. Surgical biopsy 2.Autopsy Specimens
o Needle biopsy 3. Resected Specimens
o Endoscopy biopsy 4. Slides and Blocks
o Incision biopsy
o Excision biopsy
o Punch biopsy
o Cone biopsy
Specimen rejection criteria:
• Timing
• Patient’s details on requisition form and specimen
label are different.
• Details of site/nature of specimen not
mentioned.
• Specimen not received in specified container.
• Improperly packed container
• In case of slide or blocks: Broken slides,
improperly processed blocks or insufficient
contents in paraffin blocks.
Instruments used in grossing:
Processing:
1. Fixation
2. Dehydration
3. Clearing
4. Embedding
5. Sectioning/Cutting
6. Staining
7. Mounting
8. Microscopy
PROCESSING
FIXATION
Tissue Fixatives:
❑ A fixative may be defined as a substance that
preserves the morphological and chemical
characteristics of cells and tissues and prevents
autolytic and putrefactive changes.

❑ Fixation is a very important step and is the

foundation of all good histological preparations
INTRODUCTION
• It is necessary to fix the tissue in such a manner that
subsequent examination may be made to determine
its microanatomy.
• It is essential to fix tissue as soon as possible
• Amount of fluid in jar should be 15-20 times
• AIMS & EFFECT:
.Inhibits autolysis & putrefaction
.Preservation of tissue in a life like manner
.Hardening-allow easy manipulation of soft tissue
.Alters optical differentiation thus making tissues
to appear clearer
IDEAL FIXATIVE
AN IDEAL FIXATIVE HAS THE FOLLOWING
PROPERTIES
• Cheap & easily available
• Stable & safe to handle
• Causes quick fixation
• Minimal loss of tissue
• Even penetration
• Retain normal colour of tissue
• Should not bind to reactive groups
•

Reagents employed as fixatives:

1 . Formaldehyde :
-is a water soluble gas
-40% formaldehyde is formalin---polymeric form
-10% formalin( 40% hcho 10 ml+ water 90 ml)---monomeric form
-on long storage, ppt of paraformaldehyde is formed,due to poly merization of formaldehyde. Formation of this ppt can be retarded by addition of methanol.
-Side effects include -dermatitis
- damage to nasal mucosa
- sinusitis
• For fixation,best thickness is 3-5 mm
• Speed of fixation depends upon speed of
reaction between fixative & tissue & rate
of diffusion
• 10% formalin penetrates:
4 hrs - 2.7 mm
8 hrs - 4.7 mm
12 hrs - 5 mm
• Effect of formalin fixative on proteins:
Formation of cross links between
molecules giving rise to insoluble
product.It reacts virtually with all amino
acids
• Effect of formalin on lipids:
Reacts with phosphatidyl ethanolamine
causing degradation
Cholesterol & cerebroside remain
unaffected
• Commercial formalin is acid,either due to
formic acid as impurity or to oxidation of
formaldehyde
• It should be made neutral or basic by use
of phosphate buffer or calcium acetate(use
of neutral formalin results in absence of
formalin pigments & demonstration of
iron)
2. GLUTARALDEHYDE
• -Used for electron microscopy with osmium tetroxide.
-efficient cross linking agent for collagen.
-gives better preservation of structures.
-rapid fixing action but poor penetration.
3. TRICHLOROACTEIC ACID
Never used alone due to swelling effect on many tissues used
as compound fixative.
4. ACETIC ACID
never used alone, because of swelling effect.
• used as compound fixative.
5. ACETONE
• Used as cold acetone for histochemical demonstration
of tissue enzymes.

6. ETHYL ALCOHOL
Slow penetration, harden the tissue after long
exposure.
• When combined with other reagent , fixation is rapid.
• Denatures protein by precipitation and precipitate
glycogen .
• Used in histochemical method for enzyme.
 7.PICRIC ACID
• Explosive nature
• Precipitate protein and combine with them to
form picrate.

8. CHROMIC ACID
• Precipitate protein and preserve –CHO(aldehyde
group)
• Powerful oxidising agent.
• Hydrolyse DNA with conversion of pentose sugar
to aldehyde.
9.POTASSIUM DICHROMATE
• Has binding effect on protein,
gives fixation of cytoplasm
without precipitation.
• Used for fixation of mitochondria.
10.OSMIUM TETROXIDE
• Very expensive
• Demonstrates lipids-gives excellent
preservation and details of single cell.
• Used in electron microscopy.
• Gives uneven fixation
• Side effects-conjunctivitis
11.MERCURIC CHLORIDE
• Protein precipitant which rapidly
penetrate
• Rate of penetration is decreased after 1st
few mm.
• Intolerant fixative-exposure of tissue to
their action in excess of time will produce
excessive hardness
• Staining of cytoplasm tend to be brilliant.
COMPOUND FIXATIVE
• Choice of fixative will be governed by type
of investigation:

• Divided into 3 main groups:

• 1.microanatomical
• 2.cytological
• 3.histochemical
 MICROANTOMICAL
used when it is desired to preserve the anatomy

Routine formalin fixative Formalin fixative for carbohydrate

1.Formol saline • 1.buffered
2.Formol calcium gluteraldehyde
3.Buffered formalin • 2.haidenhains susa
4.Buffered formol sucrose • 3.zenker’ fluid
5.Alcoholic formalin • 4.zenker’ formol
6.Acetic alcoholic formalin • 5.bouin’s fluid
• 6.gendre’s fluid
• 7.rossman fluid
FORMOL SALINE
• 10% Formalin in 0.9% sodium chloride
• Neutral ph
• Adv: FORMALIN PIGMENT IS NOT FORMED since
its occurrence is due to interaction of formalin
solution at acid ph with hemoglobin(encounterd
in section of bone,spleen &liver)
• TISSUE CAN BE LEFT FOR LONGER PERIODS
without excessive hardening
• Fixation is completed in 6-12 hrs.
BUFFERED FORMOL SUCROSE
• Gives excellent PRESERVATION OF FINE
STRUCTURES,PHOSPHOLIPIDS and some
enzymes
• Used for combined CYTOCHEMISTRY &
ELECTRON MICROSCOPICAL STUDIES

• Formalin-10ml
Sucrose-7.5gm
Phosphate buffer-to 100ml
ACETIC ALCOHOL
FORMALIN
• Excellent for GLYCOGEN
• Addition of acetic acid ensures FIXATION
OF NUCLEAR PROTEIN
• Tissue upto 5 mm are fixed in 4 hr.
• Formalin-5ml
Glacial acetic acid-5ml
70%alcohol-90ml
HEIDENHAIN’S SUSA
FORMULA:
Mercuric cholride-4.5gm
Sodium cholride-0.5gm
Trichloroacetic acid-2gm
Acetic acid-4ml
Formalin-20ml
DW-to 100ml
• Advantage: Excellent fixative allows brilliant
staining
Good cytological detail
Gives rapid penetration with min shrinkage
• Disadvantage: Excessive hardening of tissue
Transfer it to 96% alcohol,or it causes
excessive hardening
Contains mercury pigment, so treat with
iodine to remove it
• Tissue of 7-8 mm thickness fixed in 12-24 hrs
ZENKER’S FLUID
FORMULA-
Mercuric chloride-5gm
Potassium dichromate-2.5gm
Sodium sulphate-1gm
DW to 100ml
Add 5ml of glacial acetic acid
immediately before use
ZENKER’S FLUID
• ADVANTAGE:
for SKIN,LIVER,& SPLEEN BIOPSY
excellent nuclear & connective tissue staining
• DISADVANTAGE:
Tissue must be washed in running water to
remove excess dichromate
Mercuric chloride pigment shoud be removed
with iodine
tissue becomes brittle due to longer time of
fixation
• Fixation completes in 12 hrs.
ZENKER’S FORMOL(HELLY’S FLUID)
FORMULA-
Mercuric chloride-5gm
Potassium dichromate-2.5gm
Sodium sulphate-1gm
DW- to 100ml
Add 5ml of formalin immediately before
use
ZENKER’S FORMOL (HELLY’S FLUID)

• Irrational combination as it contains both

oxidising agent(pot dichromate) & reducing
agent(formaldehyde),but still excellent fixative
• ADVANTAGE:
Good fixative for bone marrow,spleen & blood
containing organs
• DISADVANTAGE:
Poor penetration,brittleness of tissue &
formalin pigment formation
• Fixation is complete in 6-24 hrs
BOUIN’S FLUID
FORMULA-

Picric acid-75ml
Formalin-25ml
Glacial acetic acid-5ml
BOUIN’S FLUID
• Advantage:
For KIDNEY,TESTIS & EMBRYO
Brilliant staining with trichrome stains
Causes little shrinkage
Demonstrates glycogen
• DISADVANTAGE:
Yellow color of picric acid need to be removed
by treating with alcohol or by washing.
• Fixation complete in 24 hrs
GENDRE’S FLUID
• FORMULA-
picric acid-80ml
formalin-15ml
glacial acetic acid-5ml
• Good fixative for glycogen, fixes in 3-4 hrs.
ROSSMAN’S FLUID
• FORMULA-
formalin-10ml
absolute ethyl alcohol-90ml

• For CARBOHYDRATE FIXATION.

CYTOLOGICAL FIXATIVE
• NUCLEAR FIXATIVE
• CYTOPLASMIC FIXATIVE
NUCLEAR FIXATIVE
1.Carnoy’s fluid:
Abs alcohol - 60 ml
Chloroform - 30 ml
Glacial acetic acid - 10 ml
• Advantage:
-Gives excellent nuclear fixation(with
dissolution of cytoplasmic contents)
-can be used when diagnosis is required in
emergency, due to rapidity of fixation. Small
pieces 2-3 mm are fixed in 15 min
 2.CLARK’S FLUID:
Abs alcohol - 75 ml
glacial acetic acid - 25 ml
• Excellent for smears or coverslip
preparation of cell cultures for
general fixation or chromosome
analysis.
3.NEWCOMER’S FLUID:
isopropanol - 60ml
propionic acid - 30ml
petroleum ether – 10ml
acetone - 10ml
dioxane - 10ml
• Advantage:
- Preserves chromatin better than
carnoy’s fluid
- Penetrates rapidly
• Fixation occurs in 12-18 hrs
4.FLEMMING’S FLUID:
1% aq. Chromic acid -15 ml
2% aq osmium tetroxide -4 ml
glacial acetic acid -1 ml
• DISADVANTAGE:
Poor & uneven penetration
Following fixation tissue should
be washed overnight.
CYTOPLASMIC FIXATIVES
• 1.CHAMPY’S FLUID:
3% potassium dichromate – 7 ml
1% chromic acid - 7 ml
2% osmium tetroxide -4 ml
• ADVANTAGE : Preserves
mitochondria,fat,yolk,lipids
• Disadvatange:
- Penetrate poorly &unevenly
- Requires washing overnight
- Tissue thickness not be more than 2mm
- Deterioates rapidly,so requires fresh solution
2.Regaud’s fluid:
3% pot dichromate - 80ml
Formalin - 20ml
• Advantage:
-Penetrates evenly & rapidly
-Good for mitochondria & chromaffin tissues
• Disadvantage:
-Overhardens tissue
-Solutions need be fresh
3.MULLER’S FLUID:
pot dichromate -2.5 gm
sodium sulphate – 1 gm
Dist water to 100 ml
• rarely used as fixative except for
bone specimen
• 4.FORMOL SALINE & FORMOL
CALCIUM

• 5.ZENKER’ FORMOL:
Used as cytoplasmic fixative &
microanatomical fixative for bone
marrow , blood containing organs &
spleen
 6.SCHAUDINN’S FLUID:
mercuric chloride - 2 parts
abs alcohol - 1 parts
• ADVANTAGE:
for exfoliative cytology
rapid penetration
• DISADVANTAGE:
longer time for fixation
excessive shrinkage
HISTOCHEMIAL FIXATIVE
A good fixative should
A)preserve the constituent to be
demonstrated
B)preserve the specific tissue constituent
FOR HISTOCHEMICAL REACTION,IT IS BEST TO
USE CRYOSTAT CUT SECTIONS.

1.FORMAL SALINE
2.COLD ACETONE
3.ABSOLUTE ALCOHOL
 1.FORMOL SALINE : It is buffered to
prevent the formation of formalin
precipitate.
Tissue is well washed to remove the
excess fixative.
• MOST COMMON HISTOCHEMICAL
FIXATIVE USED
• 2.COLD ACETONE : Immersion in
acetone(0-4 ⁰C) for tissue in which it
is intended TO STUDY
ENZYMES(phosphatases)

• 3.ABSOLUTE ALCOHOL : fixation of

section cut from fridge dried material
may be affected by immersion in abs
alcohol for 24 hrs.
VAPOUR FIXATIVES
Used TO FIX CRYOSTAT CUT SECTIONS of fresh
tissue & section of frozen dried tissue.This is
used to demonstrate amines,the amines
being converted into flourescent isoquinoline

1.FORMALDEHYDE:
• vapour is obtained by heating
paraformaldedyde at temp 50-80oC.
• section require ½ to 1 hr
• Block of tissue require 3-5 hr at 50-60 oC
 2.ACETALDEHYDE:heat meta-
aldehyde at 80 oC for 1-4 hr. gives
good general flourescence
3.GLUTARALDEHYDE:50% aq sol at
80oC for 2 min to 4 hr.
4.ACROLEIN /CHROMYL CHLORIDE :
liq reagent for 1-2 hr at 30 oC

Urgent paraffin section
Thin slices are fixed in alcohol
containing fixatives Carnoy’s ,
formol alcohol
 Enzyme Histochemistry-
Fix in cold formol calcium

 Transmission Electron Microscopy

Osmium tetroxide, glutaraldehyde.

 Electron microscopy - 2%
glutaraldehyde


Immunoflourescence – unfixed
tissu Time fixativ
e interval e
Adrenal 1 or 2 Formaldehy
gland hr de
ey Immediately Formol
e after death or saline
within 2 hrs
Alimentary Immediately Susa
tract after death fixative
Blood forming - Zenker’s
organ do- fluid
Testis/ - Susa
ovary do- fixative
Lung/kidney/ Fairly resistant Susa
bone to postmortem fixative
changes
Renal Neutral
biopsy buffer
formalin
▪Tissue fixed with 2 fixatives in
succession
▪Improved preservation & staining
▪Tissues fixed with gluteraldehyde is
post fixed with osmium tetroxide for
electron microscopy
•Treatment of tissues with 3% potassium
dichromate following normal fixation.
•Before processing – tissue in dichromate solution
for 6- 8days
•After processing – for 12- 24 hrs
•Both followed by washing in running water
•Mitochondria & myelin demonstrated.
•Improved preservation and staining of elements.
LABELLING OF SPECIMENS
• Correct labelling & identification is must
• Printed labels should be used
• When fixing in osmium tetroxide, these
labels should not be used as they turn the
paper black
• Transfer of tissues best done by use of
tissue processing baskets, which are
perforated so can be transferred from
reagent to reagent.
DEHYDRATION

• Water has to be removed from the

tissue in order to embed the tissue in
paraffin wax.
• This is achieved by immersion in
increasing strengh of ethyl alcohol,
KNOWN AS DEHYDRATION.
• Conc. Of alcohol in 1st bath depends on
fixative, size and type of tissue
• Delicate tissue dehydrated slowly starting
at 50%
• Most other tissue be dehydrated at 70%(as
too high conc. Of alcohol causes high
degree of shrinkage)
• Exception is haidenhain’susa, where it is
done directly at 96% alcohol.
• Minimum duration of treatment in graded
alcohol depends on size & type of tissue.
• Giant section done in 50%, 70%, 96%,
100% for 24-48 hrs each
• Delicate section in same solution for 2-4
hrs each.
• For routine biopsy specimen (<7 mm
thickness) 70%, 90% & absolute alcohol for
2-4 hrs each.
• Agitation of tissue mechanically or in
automated tissue processor may
speed up the process.
• Heating has the same effect.
• In our laboratory, by manual method,
three changes of ACETONE was used
as dehydrating agent, each change
being of 25 min.
USE OF CuSO4 IN FINAL
ALCOHOL
• Layer of anhydrous CuSO4 placed at the bottom
of dehydrating bottel & is covered with 2-3 filter
papers(to avoid staining of tissue)
• Anhydrous Cuso4 is obtained by heating hydrous
form in porcelain jar
• advantage: CuSO4 removes water from alchol as
it in turn removes it from tissue
• Changes of colour of CuSO4 from white to blue
indicates that alcohol & Cuso4 be changed.
SUBSTITUTES FOR
ABSOLUTE ALCOHOL
• 1. ACETONE: used previously for manual
method.
• 2. METHANOL
• 3. DIOXANE: Mixes freely with water,
alcohol & paraffin so less shrinkage &
hardening.
• 4.TETRAHYDROFURAN.
• 5.METHYLATED SPIRIT
• 6.ISOPROPYL ALCOHOL
CLEARING
• Alcohol & wax are not miscible so
alcohol must be replaced by wax
solvent. Since majority of wax
solvents have effect of raising the
refractive index of tissue, it makes
them appear clearer, this is KNOWN
AS CLEARING.
1. XYLENE, BENZENE&
TOLUENE
• ADVANTAGE: tissue becomes
clear as alcohol is replaced
owing to diff. in refractive
index.
• DISADVANTAGE: causes
hardening of tissue & makes
tissue brittle.
2. CHLOROFORM
• ADVANTAGE: minimal brittleness
to tissue
• DISADVANTAGE: does not affect
refractive index, so that end point
of clearing can not be made out
• Most expensive clearing agent.
3. CEDAR WOOD OIL

• ADVANTAGE: best reagent for research

& treatment of delicate tissue, since it
has least hardening effect
• Used as clearing agent for hard tissue
as dense fibrous tissue & bone as
sections are then easier to cut.
• DISADVANTAGE: cost & slow clearing
action.
4.CARBON
TETRACHLORIDE
5. HISTOCLEANERS
6.AMYL ACETATE

In our laboratory by manual

method we used two changes of
xylene as clearing agent, with
each change of 25 min.
IMPREGNATION WITH WAX
• This is the process in which empty spaces in the
tissue & cells are taken up by paraffin wax.
• OVEN: Heated to 54-60oC, routinely at 58oC

• PARAFFIN WAX Must be free from water as it

may cause wax to crystallise.

• Melting point of wax depends on type of tissue

but is usually 54oC,which gives support for hard
tissue & easy ribboning.
• For research purpose:
Hard wax(M.P. 600C):for hard
fibrous tissue
Soft wax(M.P 450C):for fetal &
areolar tisue
PARAFFIN WAX ADDITIVES
These modify the consistency of the wax &
its melting point:
• BEES WAX
• RESINS
• STEARIC ACID
• PLASTIC WAXES
• AGAR
• BAY-BELLY WAX
TECHNIQUE OF IMPREGNATION
• Tissue is transferred from clearing agent to
molten paraffin wax
• Vol. of wax should be 25-30 times
• Length of time & no of changes will
depend on
size & type of tissue
clearing agent employed
SUBSTITUTES FOR PARAFFIN
WAX
• 1.PARAPLAST
• 2.PARAPLAST PLUS
• 3.HISTOWAX
• 4.EMBEDOLOL
• 5.BILWID
PARAPLAST
• Mixture of purified paraffin & several
plastic polymers
• Causes greater degree of elasticity
• Section of bone are cut easily
• Ribbons well allowing wrinkle free
serial sections.
PARAPLAST PLUS

• Contains DMSO( dimetyl sulfoxide ),which

gives rapid penetration reducing the time
of tissue processing
TIME OF IMPREGNATION DEPENDS ON

• 1.Size &type of tissue: hard tissues require

more time than soft ones.

• 2.Clearing agent employed:

xylene - easiest to remove
CCL4,chloroform - two changes reqrd

• 3.use of vacuum embedding oven: time is

halved
• In our laboratory, for manual method
we used to impregnate for 7 hrs in
paraffin wax
CASTING OR BLOCKING
• Done by transferring tissue from final wax
bath to a mould filled with molten wax.

Types of mould:
1.leuckart’s L-pieces
made of brass
available in various sizes
 2.Glass petridish:
• Should be previously smeared with
glycerine.
• Used to embed several tissue at one time.

3.Metal petridish:
• Made of aluminium.
• Final block is easily removed due to its
contraction during setting
• 4.PAPER BOAT
• 5.WATCH GLASS
• 6.TEST TUBE: may be used for small
fragments as bone marrow
• 7.PLASTIC EMBEDDING RINGS: adv of
rapid embedding & cutting of tissue &
simple to use
TECHNIQUE OF CASTING
• Fresh molten wax is poured over the mould to
form a solid thin layer.
• With forceps previously warmed to prevent wax
setting on them, tissue is lifted from final wax
&placed at the bottom of mould. gently press
the tissue against the solid layer that will hold it
in position.
• As block cools to form a skin on its surface,
immerse in cold water
• Once mould sets hard, blocks may be removed,
trimming done& tissue cut.
WATER SOLUBLE WAXES
• Are made of solid polyethylene glycols
• ADVANTAGE : due to bypassing of stages of
dehydration & clearing
A. shrinkage effect is reduced
considerably
B. time consumed is decreased.
TECHNIQUE
• Transfer tissue to 50% polyethylene glycol
in DW.
• Transfer to 4 successive changes , allowing
45 min in each.
• Transfer to equal parts of polyethylene
glycol & Nonex 63B for 30-40 min.
• Embed in a paraffin wax.
• CUTTING
• FIXING TO SLIDES: most difficult task as, if
they are floated on water violent diffusion
currents are set up.
• Overcome by adding trace of soap or few
drops of teepol to water.
GELATIN EMBEDDING
• Used primarily for
embryo
endometrium
eye
brain
• USED WHEN FROZEN SECTION OF FRIABLE
OR PARTIALLY NECROTIC TISSUE ARE
REQUIRED.
• Following embeding, block is immersed in
10% formalin to convert gelatin into
irreversible gel.
• DISADVANTAGE : during staining gelatin
tends to hold the stain , giving an
indifferent background.
CELLOIDIN EMBEDDING
• Used primarily for
bone
Sclera

• It is purified form of nitrocellulose

ADVANTAGE: does not require heating at any
stage
. Has rubbery consistency which supports
hard tissue, so cut section of mixed hard &
soft tissue are of even thickness eg eye
• Disadvantage:
. Difficult to cut thin section
. Serial section difficult to prepare
. Processing is slow, taking several weeks
. Block need to be stored at 70% alcohol
DOUBLE EMBEDDING
• celloidin impregnated can not give serial
sections.
• This can be overcome by 1st impregnating
the tissue in celloidin & then blocking in
paraffin wax.
OTHER EMBEDDING MEDIA
• ETHER/POLYESTER WAX
• MICROCRYSTALLINE WAX
• ACRYLIC/EPOXY RESINS
• AGAR
AUTOMATED TISSUE PROCESSOR
• IT IS A FULLY
AUTOMATED
MACHINE THAT
DOES ALL OF
TISSUE
PROSESSING BY
ITSELF
-PROCEDURE:

• BUCKET NO 1 - 10% FORMALIN - 0 HOUR

• BUCKET NO 2 - 50% ALCOHOL - 1 HOUR
• BUCKET NO 3 - 60% ALCOHOL - 1 HOUR
• BUCKET NO 4 - 70% ALCOHOL - 1 HOUR
• BUCKET NO 5 - 80% ALCOHOL - 1 HOUR
• BUCKET NO 6 - 90% ALCOHOL - 1 .15 HOUR
• BUCKET NO 7 & 8- 100% ALCOHOL-2.45 HOURS

• BUCKET NO 9 & 10– XYLENE – 4 HOURS

• BUCKET NO 11 - WAX - 2 HOURS

• BUCKET NO 12 - WAX - 2 HOURS

• ADVANTAGE:

• Transfers tissue mechanically from

reagent to reagent, so error of
forgetting to transfer is avoided.
• reduces time required in each fluid by
the action of continual agitation.
MICROWAVE
ROLE OF MICROWAVE IN HISTOPATHOLOGY
• Tissue is fixed more effectively than routine
methods, by heat which coagulates protein, as
heat is evenly & consistently applied throughout.
fixation may be performed in normal saline or
tris buffer, but pretreatment fixation with
formalin is preferred. cryostat sections are better
fixed by microwave
• Dehydration & impregation are accelerated .thick
tissue require 3 hr, while small biopsy can be
done in 15 min.
• Reduces the time exposure to various solutions
in staining.
• Tissue antigens are well preserved following
microwave irradiation.
• Have advantages in electron microscopy also
USING THIS MODALITY,TISSUE BIOPSY CAN BE
FIXED,PROCESSED & STAINED IN 30-45 MIN.
• DISADVANTAGE:
Limited no of slides can be kept in the
microwave at a time
OBJECTIVES
• Decalcification
• Frozen section
• Section cutting
• Staining
• Microtomes
• Honing
• Stropping
• Mounting
• Microscopy
DECALCIFICATION
DECALCIFICATION
• Presence of calcium salts in tissue
prevent preparation of good
section
1. Result in torn and ragged
section
2. Damage to cutting edge of
microtome knife
TECHNIQUE
• Divided into following stages:
1.Selection of tissue
2.Fixation
3.Decalcification
4.Neutralization of acid
5.Thorough washing
SELECTION OF TISSUE
• a. Bone- thin slices of bone are obtained.
slices should not exceed 4-5mm in
thickness
• b. Calcified tissue-type of section
depends on degree of calcification
OSTEOTOME
FIXATION
• Aqueous Nitric acid is preferred
• Bone marrow is best fixed in zenker
formol
DECALCIFICATION
• Methods of decalcification are:

-By an acid reagent ,usually dilute mineral acid

-Ion exchange resins
-Chelating agents
-Electrophoretic removal of calcium
• Criteria for good decalcifying agents
are:
. Complete removal of calcium
. Absence of damage to tissue
. Non impairment of staining
. Speed of decalcification
END POINT DETERMINATION
CHEMICAL TEST
• 5ml of decalcifying fluid neutralized with
dinitrogen sodium hydroxide,& 1ml of 5%
sodium or ammonium oxalate is added
• Interpretation-presence of turbidity indicate
presence of calcium in the fluid.Absence of
turbidity after 5 min indicate fluid is free from
calcium.
• It is obvious that fluid be changed every time it is
tested.
NEUTRALIZATION OF ACID
• Following treatment with acids, tissues need be
neutralised TO PREVENT SWELLING OF TISSUES.
• Done by treatment with alkali--5%lithium or
sodium sulphate
WASHING
• Thorough washing is essential to remove acid
which would interfere with staining reaction
• Washing should be carried out for 3-4 hrs in
alcohol
1. DECALCIFING FLUIDS
• Gooding and stewart
• Citrate citric acid buffer
• Jenkins’ fluid
• vonebner’s fluid
1.Gooding and Stewart fluid:
Formic acid- 5 to25 ml
Formalin- 5ml
Distilled water-10ml
Decalcification complete in 2 to 4 days
ADVANTAGE:
Rapid decalcification
Minimal damage to tissue
2.Citrate citric acid buffer
7% citric acid- 5ml
7.54% ammonium citrate-95ml
1% zinc sulphate
Chloroform –few drops

DISADVANTAGE:
Takes longer time. Human rib decalcify in 4 to
6 days
3.Jenkins fluid:

Absolute alcohol 73ml

DW - 10ml
Chloroform- 10ml
Glacial acetic acid- 3ml
Hcl-4ml
Not only decalcify BUT ALSO DEHYDRATES
4.Vonebner’s fluid:

Hcl- 15ml
Sodium chloride -175gm
DW- to 1000ml
commonly used agent in Great Britain
Human rib is decalcified in 36 to 72 hours
FLUIDS CONTAINING NITRIC
ACID
• Disadvantage of using these fluids is YELLOW
COLORATION due to formation of nitrous acid.
• This can be avoided by adding 0.1% urea to pure
nitric acid
1. Formol nitric acid
2.Phloroglucin- nitric acid
3.Aqueous nitric acid
4.Perenys fluid
FORMOL NITRIC ACID
Formalin- 5ml
Nitric acid- 7.5 to 15ml
DW – to 1000ml
• HCHO partially inhibit the tendency of maceration

AQUEOUS NITRIC ACID

Nitric acid- 5ml
DW – to 95mL
• Most commonly used decalcifying agent including OUR
LABORATORY.
• ADVANTAGE:
Is rapid, causes little damage to tissue and allows most staining
techniques can be applied
PERENY’S FLUID
10% nitric acid- 40ml
Absolute alcohol – 30ml
0.5% chromic acid -30ml
• ADVANTAGE:
Excellent reagent for small deposit of
calcium
Good for cytological preparation
little hardening effect
2. ION EXCHANGE RESINS
• Used TO REMOVE CALCIUM IONS FROM
DECALCIFYING FLUID
• Is an ammonium form of a sulphonated
polystyrene resin
• It is layered on bottom of a container to a depth
of half inch
• Specimen is allowed to rest on it
• Volume of fluid is 20-30 times bulk of tissue
• X-ray used for end point determination
CHELATING AGENTS
• These are organic compound whichs have the
power of binding certain metals
• Ethylene diamine tetraacetic acid; disodium salt
– binds with calcium
• ADVANTAGE:
Tissue decalcified by this method show minimum
artefact and stain by most techniques
ELECTROPHORETIC DECALCIFICATION

• Based on attraction of calcium ions to a

negative electrode
• ADVANTAGE-
Decrease the length of time required for
decalcification
• Abandoned in most institutes.
FROZEN SECTION
INTRODUDUCTION
• When a fresh tissue is rapidly frozen, the
matter within the tissue turns into ice & in
this state the tissue is firm, as the ice acts
as embedding medium. So the sections
are produced without the use of
dehydrating solution, clearing agents or
wax embedding
• Frozen section cutting is a quick
diagnostic procedure for tissues
before proceeding to a major radical
surgery. this procedure can be carried
out in OT near operating table.
MERITS:
• Quick diagnostic procedure. Time taken
from receipt of specimen to the study of
slides is approx 10 min. while for routine
paraffin section, at least 2 days are
required.
• Every type of staining can be done
• Minimal shrinkage of tissues as compared
to paraffin sections.
• Lipids & enzymes that are lost in routine
paraffin sections can be demonstrated.
DEMERITS:
• Difficult to cut serial sections.
• Not possible to maintain tissue blocks for
future use
• Sections cut are thicker
• Structural details tend to be distorted due
to lack of embedding medium.
METHODS FOR FROZEN
SECTIONS
• 1.FREEZING MICROTOME USING CO2 GAS.
• 2.REFRIGERATED MICROTOME(CRYOSTAT)
• Best results are obtained from:

Fresh unfixed tissues

Freezing the tissues as fast as
possible
FREEZING MICROTOME USING CO2
GAS
• Is a sliding microtome.
• SETTING OF MICROTOME & SECTION CUTTING:
• Microtome screwed firmly to the edge of
table by means of a stout screw.CO2 gas
cylinder placed near the microtome.
Cylinder connected to microtome by a
special tubing without any bends &
creases.
• Adjust the gauge of microtome to a
required thickness of sections. knife is
inserted in its place.
• Few drops of water placed over freezing
stage & selected piece of tissue placed
over it. short bursts of CO2 applied till the
surface of tissues is completely covered
with ice. alternatively, solid CO2(dry ice/
cardice) can be used to freeze.
• Then sections are cut by swinging
movement of knife forward & backward
with a regular rhythm. cut sections come
over knife.
• From the knife, sections are picked with a
camel brush & transferred to a petridish
containing water. Then put over slide with
the help of a dropper. Remove the folds in
sections by tilting the slides.
• Then slides are passed over flame for a
few seconds for fixing the sections ,then
stain the slides.

ADVANTAGE:
• Cheap
• Requires less space
• Equipment is portable
• DISADVANTAGE:
• Sections cut are
thick
• CO2 may run out
between
procedure
• Connecting tube
may block due to
solidified CO2
REFRIGERATED MICROTOME
(CRYOSTAT)
• Microtome is fitted in a thermostatically
controlled refrigerated cabinet. temp upto
-30 oC can be obtained.
• Type of microtome---Rotary type with an
antiroll plate
SETTING OF MICROTOME & SECTION
CUTTING
• Switch on the cryostat along with the knife
inserted in position several hrs before the
procedure for attaining the operating
temperature
• A small piece of fresh unfixed tissue is placed on
object disc of deep freeze shelf of the cryostat
for 1-2 min.
• The tissue is rapidly frozen.Now the object disc
with tissue is inserted into microtome object
clamp. place antiroll plate in its position.
• By manual movement sections are cut at
desired thickness. Antiroll plate prevents
folding of sections
• The section is picked from the knife &
taking the section directly on albuminised
slides.
• Slide is lowered on to the knife 1mm from
section. the section come automatically on
the slide because of difference of temp
between section & slide.
• Now stain the section
• Defrost the cryostat &Clean it Weekly

❑ ADVANTAGE:
• Thin section
• Better control of temperature
• Portable equipment

❑ BUT – it is expensive.
SECTION CUTTING
• MICROTOME KNIVES:
knife is greatest factor in producing good
sections.

• Types of microtome knives:

a)heiffor knife with fixed handle(used)
on rocking microtome
b)larger knives ranging from 8-24 cm.
These have detachable handles.
 HEEL of knife: is the
angle made by cutting
edge & end of knife
nearest to handle.

TOE of knife: is angle

formed by cutting edge
& the end of knife
farthest from handle.
⮚TYPES OF KNIFE:

• Planoconcave: used on sledge type & rotary

microtome
• Planoconcave(very concave): used for celloidin
work.
• Planewedge: used in cutting frozen sections & for
giant & ordinary paraffin sections.
• Bi-concave:for paraffin sections cutting on rocking
& sledge type microtome.

✔IN OUR LAB,WE ARE ROUTINELY USING

BICONCAVE KNIVES.
⮚SHARPENING OF MICROTOME KNIFE:

Cutting edge of ideal microtome

knife should be straight line
formed by intersection of two
planes.
Technique of cutting:
• Fix the block on the block holder.
• Insert knife in knife holder & screw it
tightly.
• Move the block holder until it just touches
the knife
• Ensure that whole surface of block moves
parallel to the edge of knife to ensure a
straight ribbon of sections.
• To trim block, set the section thickness to
15 micron with rough knife.
• Replace the rough knife with a sharp one.
• For routine work, 6 micron will give
moderately thin section.
• Microtomes operated till whole sections
are cut.
• Formation of ribbon is said to be due to
the heat generated during section cutting
which causes the edges of sections to
adhere.
• Routine sections
may be directly
floated onto
slides.
During cutting, wax embedded sections may
become compressed & creased. these
must be removed before attaching them
to the slides, achieved by floating them
onto warm water by one of these
methods:
• WATER BATH
• HOT STAGE METHOD
• WARM SLIDE METHOD
⮚ WATER BATH METHOD:
• Baths are controlled at a
temp 5-60C below M.P. of
wax.
• Baths are from inside
black as the creases are
made visible
• Sections are divided into
lengths convenient to go
on a slide with a scapel.
• Water bath showing
inner black surface.

• WE IN OUR
LAB,ROUTINELY ADD
ABS ALCOHOL TO
WATER BATH TO AVOID
FORMATION OF
CREASES.
⮚ USE OF ADHESIVES(used sections do not adhere
to the slides properly):
1. albuminised slides(this we routinely use in our
lab)
DISADVANTAGE: Albumin retains some of the
stain & gives a dirty background.
2. starch paste(is superior to albumin)
3. chrome gelatin
MICROTOMES
• Are mechanical devices for cutting
thin uniform slice of tissue sections.
• Tissue is supported in hard paraffin
wax & is moved automatically toward
knife.
TYPES OF MICROTOME
• 1. ROCKING
• 2. ROTARY
• 3. SLEDGE
• 4. SLIDING
• 5. FREEZING
ROCKING MICROTOME
• Knife is fixed.
• Block of tissue moves
through an arc &
strikes against the
knife
• Disadvantage:
size of block that can
be cut is limited.
block moves through
an arc, so sections are
cut in a curved plane.
ROTARY MICROTOME
Is so called due to rotary
action of handwheel, that
actuates the cutting
movement.
Block holder is mounted on
steel carriage which moves
up & down ;it cuts perfectly
flat sections.
Advantage: being heavier, is
more stable for cutting
serial sections.
Larger blocks of tissue may be
cut on this machine
SLEDGE MICROTOME
• Designed for cutting sections of very large
blocks.
• Block holder is mounted on a steel
carriage.
• Fixed horizontal knife.
• Microtome is heavy& stable, so not
subject to vibration.
• Knife used is large 24 cm & usually wedge
shaped.
SLIDING MICROTOME
• Knife is moved horizontally against a
fixed block which is advanced against
it on inclined plane
• Designed for cutting celloidin
embedded sections.
FREEZING MICROTOMES
For cutting frozen sections.
HONING
• After prolonged use, the cutting edge
of knife becomes so damaged that it
causes lines or even tears in sections
during cutting.
• A straight cutting edge & a correct
bevel must be restored by grinding
the knife on a hone.
TYPES OF HONES
• BELGIAN BLACK
hone:(best hone)
• Yellow stone about
½ inch thick & is
backed with a black
stone.
• Fast hone & is used
for coarse grinding &
finishing.
• Arkansas:
. Very hard
. Not very fast
. Used to finish a knife after
honing on coarse hone
• Alaxite:
. Fairly fast but coarse
. Not good for finishing a knife

• Carborundum:
. Used for removing large nicks in badly
damaged knife
• PLATE GLASS:
Used as hone after applying abrasive such as
aluminium oxide to surface.
• ALOXITE
• Tam O Shanter Scotch hone
GENERAL PRECAUTIONS
• Size of hone will depend on the type of
knife to be sharpened:
. For smaller knife-stone of 8 x 2 inch is
used
. For large knife- stone of 12 x 3 inch is
used
• Hones should be lubricated with
light oil or soap & water before
use.
• After use it should be thoroughly
cleaned
METHOD OF USING
• Hone should be placed on a non-skid
surface.
• Small quantity of lubricating oil is poured
• Knife is laid on hone with cutting edge
facing away from operator, hold the knife
between thumb & forefingers.
• Tip of finger & thumb of other hand rest
on other end of knife ensuring even
pressure along the whole edge of knife.
• Knife is pushed forward diagonally from
heel to toe tuned over its back & move
across hone untill the heel is in the centre.
it is turned over its back & moved across
the hone to its original position,thus
completing a ‘figure of 8’ movement.
• Process is continued untill all ragged edges
have been trimmed.
STROPPING
• Is the process of
polishing an already
sharp edge.

• TYPES OF STROPE:
• best stropes are
made from hide from
rump of horse.
PRECAUTIONS
• Strope should be kept soft by pouring veg
oil
• It should be kept free from grit & dust
TECHNIQUE
• Knife is laid on strope
with cutting edge
toward operator,
action is exact
opposite of that used
in honing.
AUTOMATED KNIFE
SHARPNER
• Precise, efficient & safe method of honing
• PRINCIPLE:
knife is locked in a knife holder. high
frequency vibrating honing plate is
lubricated. Electrical timing device is set &
switch is turned on. after four exactly
equivalent strokes on one side, knife is
automatically flipped over for four strokes
on other side.
this stroking cycle is repeated
continuously for the duration of electrical
setting.
• THESE AUTOMATED SHARPNERS HAVE
LIMITED UTILITY IN PRESENT DAY WORK
BECAUSE OF THE AVAILABILITY OF
DISPOSABLE KNIVES.
FAULTS IN SECTION
CUTTING
1.Section cut vertically -Knife edge is dragged &
needs honing
-Knife is dirty & needs
cleaning

2.Section curl or roll up

-Knife is blunt
-Tilt of knife is too great

3.Sections crumble on -Knife is blunt

cutting
-Wax is too soft
 -Wax is crystallised due
to slow cooling
4.Sections are
alternately thick & -Microtome adjusting
thin screws need
tightening
-Tissue very hard &
needs to be softened
-Tilt of knife is too
great
5.Ribbons of sections -Block edges are not
are curved parallel to each other
-Block edges are not
parallel to knife edge
6.Breadth of section is
less than that of -Due to compression of
breadth of block block ;occurs due to
loss of bevel on the
knife which needs to
be honed to restore it
STAINING
MOUNTANTS
• Section that need to be examined for any length
of time must be covered under coverslip. media
in which sections are mounted, are KNOWN AS
MOUNTANTS.

• TYPES OF MOUNTANTS:
• AQUEOUS
• RESINOUS
AQUEOUS MEDIA
• Used for material which is
unstained
stained for fat
metachromatically stained

• Have Low refractive index

• OF THREE MAIN TYPES:
• Syrup
• Gelatin media
• Gum arabic media

• Last two incorporate glycerin to prevent cracking

& splitting on drying.
• All should contain bacteriostatic agent as crystal
of thymol to prevent growth of moulds.
• 1.GLYCERINE JELLY: Used as standard mountant
for fat stains.
• 2.APATHY’S MEDIUM: Used for flourescent
microscopy.
• 3.HIGHMAN’S MODIFICATION OF APATHY’
MEDIUM: Used with metachromatic stains.
• 4.FARRANT’S MEDIUM
• 5.FRUCTOSE SYRUP
RESINOUS MEDIA
• Used for routine staining
• Mountant used should not fade the
particular stain used eg. basic aniline dyes
be mounted in non acid containing
mountants.
• Mountant should have correct refractive
index
• Best refractive index is 1.54.
TYPES:

1.NATURAL
2.SYNTHETIC
NATURAL MOUNTANTS
1.CANADA BALSAM:
. From canadian fir tree
. H& E stained slides are very well
preserved, but basic aniline dyes tend to
fade.

• 2.DAMMAR BALSAM
• 3.COLOPHONIUM RESIN
 SYNTHETIC RESINS
1)NEW UNIMOUNT (refractive index 1.50):
. Universal mountant
. Has tendency to trap air bubbles
. Recommended as routine mountant
. Best mountant for flourescent
antibody stained sections
2)KIRKPATRACK & LENDRUM’S(D.P.X.)
refractive index 1.52
distrene 80 10 gm
dibutylpthalate 5ml
xylol 35ml

• Most commonly used mountant

• 3)EUPARAL(R.I. 1.48)
• 4)NEUTRAL MOUNTING MEDIUM(R.I.1.52)

• RINGING MEDIA
• Mounting media that develop air bubbles
due to evaporation can be coated with
substance KNOWN AS RINGING MEDIA.eg
paraffin wax, cement
MICROSCOPY
REFERENCES
1.Handbook of histopathological &
histochemical techniques.by C.F.A. Culling
2.Manual of histological technique & their
diagnostic application.by John D Bancroft
3.Pathology practical book.by Harsh mohan
4.Recent advances In histopathology