PREPARATION OF THE

SPECIMEN FOR
MICROSCOPY
• Histochemistry – defined by Pearse as “the identification,
localization & quantification, in cells & tissues by chemical or
physical tests, of specific substances, reactive groups &
enzyme catalyzed substances”.

• Any chemical procedure that localizes a substance within cells

or tissues for subsequent microscopy is a histochemical
technique

• Thus histochemistry encompasses immunologic & molecular

biologic techniques when they are combined with histology
 Methods
1. Paraffin embeded section of soft tissues
2. Decalcified sections for hard tissues
3. Ground sections for calcified tissues
4. Frozen sections for soft tissues
 Paraffin embeded section of
soft tissues
Use : Soft tissue of gingiva, cheek, tongue, lip,
salivary glands
Steps
1. Fixation
2. Processing
a. Dehydration
b. Clearing
c. Infiltration
3. Embedding
4. Sectioning
5. Mounting
6. Staining
Step 1. Specimen Orientation

The Requisition Form

Step 2. Dissecting the Specimen

Fixing the Specimen

Storing the Specimen

Step 3. The Gross Description

Fixation

Def: Complex series of chemical events and

differs for the different groups of chemical
substances found in tissues
Aims
• Coagulate the proteins
• Prevention of autolysis & bacterial attack
• Preserve the tissue structure
• Prevent alteration during subsequent
procedure
• To make the tissues permeable
to the reagents used for staining of
sections
Types of fixatives
1. Aldehydes
Formaldehyde, Glutaraldehyde
2. Oxidising agents
Osmium tetroxide, potassium permagnate
3. Protein denaturing agents
Acetic acid, methyl alcohol, ethyl alcohol
4. Physical
Heat, microwave
5. Others
Mercuric chloride, picric acid
• Commonly used fixative for oral tissues –
10% neutral formalin
• Fixation period – Hours – days depending on
the size & density of the specimen & on type
of fixing solution used
• Rossman’s fluid ( Formaldehyde, alcohol,
picric acid & acetic acid) – visualization of
glycogen, glycoproteins, prteoglycans.
• Carnoy’s mixture ( ethyl alcohol, acetic acid,
Chloroform) – nucleic acid
• Feulgen’s reaction – visualization of DNA
Common fixatives
10% Neutral buffered
formalin (4% Formalde-
hyde, 7.2 pH methanol)
2% Glutaraldehyde
(50% Glutaralde- hyde, 0.2
M -Routine fixative
cacodylate buffer)
Zenker’s (Mercuric chloride,
potassium di-chromate,
sodium sulfate, glacial acetic
acid)
Ethyl alcohol
Tissue Advantages and disadvantages
All Routine fixative
Preservation, general staining
Immunohistochemistry
-Molecular analyses
-Long-term storage
All Electron microscopy
Preservation of collagen
-Slow penetration
-Must be refrigerated
All Routine fixative
Preserves mitochondria
-Must be washed overnight to remove excess
chromate
-Must be prepared fresh
-Molecular analyses
-No metal instruments
-Immunohistochemistry
All Enzyme histochemistry, Molecular analyses,
Impression smears, Blood smears, Preserves
glycogen,
-Routine fixative, Causes excessive hardening,
Tissue processing
Aim : to embed the tissue in a solid
medium firm enough to support the tissue
& give it sufficient rigidity to enable thin
sections to be cut, & yet soft enough not
to damage the knife or tissue
Dehydration : to remove fixative & water from
the tissue & replacing them with
dehydrating fluid
Dehydrating agents
1.Ethanol
2. Methanol
3. Propan -2-ol, isopropylalcohol
4. Acetone
• used in increasing percentages of alcohol (
40%, 60%, 80%, 95% & absolute alcohol)
• Time for each step – Size & density of the
specimen
Clearing : Replacing the dehydrating fluid
that is totally miscible with both the
dehydrating fluid & the embedding
medium
Clearing agents
1. Xylene
2. Toluene
3. Chloroform
4. Cedarwood oil & clove oil
• Impregnation : Replacing the clearing agent
with the embedding medium
• Infiltration of the specimen with
molten paraffin in temperature oven ( 2-3
successive dishes of paraffin)
• Time : size & density of the specimen
Embedding
Types
1. Paraffin wax
2. Resins ( acrylic, epoxy)
3. Polyethylene wax,Ester wax
microcrystalline wax
4. Agar, gelatin, Celloidin
 Procedure
• Specimen is removed from the molten wax
• Placed in the center of a paper box or a small
metallic tray or L- molds
• Cut surface is placed facing the bottom of
the box
• Filled with molten wax & is allowed to solidify
• Immerse in cool water or refrigerator to
harden the wax.
• Separate the paraffin wax block from the
mold & mount on a wooden block or metallic
block holder.
Mounting
• Paraffin ribbon are mounted on
prepared microscopic slides.
• Clean slides are coated with thin film of
Meyer’s albumin adhesive
• Warm water bath , paraffin sections is
floated & the prepared slides are slipped
under it & lifeted from water with ribbon
• slide is placed on drying table or slide
warming table ( 42c )
Haematoxylin & Eosin stain:

• The
haematoxylin component stains the cell
nuclei blue/ black, with good intranuclear
detail.

• The
eosin stains the cell cytoplasm & most
C/T fibers in varying shades of pink, orange
& red.
Haematoxylin:
• Is extracted from the heartwood of the tree
Haematoxylon campechiam.
• It is extracted from logwood with hot water.
• Then precipitated out from the aqueous
solution using urea.
• The major oxidation product of
haematoxylin, hematein is a natural dye that
is responsible for the color properties.
Production of hematein:

a. Natural oxidation:
• By exposure to light & air.
• This is a slow process.
• But the resultant solution retains its staining ability
for a long time.
Eg: Ehrlich’s & Delafield’s Haematoxylin solution.
b. Chemical oxidation:

• These oxidizing agents converts the

haematoxylin to hematein instantaneously.
• They have a shorter lifetime.
Eg: Sodium iodate – Mayer’s
Mercuric chloride – Harri’s
Mordants:
• Hematein is anionic, having a poor affinity
for tissue.
• Is inadequate as a nuclear stain without the
presence of a mordant.
• Eg: Aluminium, iron & tungsten.
• Mordant ( Metal cation) confers a net positive
charge to the dye mordant complex.
• And enables it to bind to anionic tissue sites
such as nuclear chromatin.
• Haematoxylinsolutions can be classified
according to which mordant is used:

a. Alum Haematoxylin
b. Iron haematoxylin
c. Tungsten haematoxylin
d. Molybdenum haematoxylin
e. Lead haematoxylin
f. Haematoxylin without mordant.
Alum haematoxylin:

• The mordant is aluminium, in the form of

potash alum or ammonium alum.
• All stain the nuclei a red color, which is
converted into the blue black when the
section is washed in a weak alkali solution.
Different alum haematoxylins are:
• Ehrlich’s
• Mayer’s
• Harri’s
• Gill’s
• Cole’s
• Delafield’s
Mayer’s haematoxylin:
This is an alum hematoxylin that is
chemically ripened with sodium iodate.
Uses:
• As a regressive stain .
• As a progressive stain where a nuclear
counter stain is needed to emphasize a
plasmic component which has been
demonstrated by a special stain.
• And where the acid alcohol differentiation
might destroy or de color the stained
cytoplasmic component.
• Itis used as a nuclear counterstain in the
demonstration of glycogen.
• In various enzyme histochemical
techniques.
• The stain is applied for a short time (usually
5-10 minutes), until the nuclei are stained .
• And is then ‘blued’ without any
differentiation.
Harris’s hematoxylin (Harris 1900) :
• Was traditionally chemically ripened with
mercuric oxide.
• Mercuric oxide is highly toxic and can have
detrimental corrosive long term effects on
some automated staining machines.
• So sodium or potassium iodate is frequently
used as a substitute for oxidation.
• It gives particularly clear nuclear staining &
for this reason has been used as a progressive
stain in diagnostic exfolitative cytology.
Uses:
• It is generally used regressively but can be
very useful when used progressively.
• When using as a progressive stain, an acetic
acid/alcohol rinse provides a more
controllable method in removing excess stain.
• The hydrochloric acid/alcohol acts quickly &
indiscriminately is more difficult to control &
can result in a light nuclear stain.
• A 5-10% solution of acetic acid, in 70-95%
alcohol, detaches dye molecules from the
cytoplasm nucleoplasm while keeping nucleic
acid complexes intact (Feldman and Dapson
1985).
Staining times with alum hematoxylins:
The time will vary according to the following
factors:
• Type of hematoxylin used
Eg: Ehrlich’s 20-45 min. Mayer’s 10-20 min.
• Age of stain :
• Intensity of use of stain:
A heavily used hematoxylin will lose its
staining powers more rapidly.
• Whether the stain is used progressively or
regressively.
Eg: Mayer’s hematoxylin used progressively
5-10 min, used regressively 10-20 min.
Eosin :
• To demonstrate the general historical architecture
of a tissue.
• It distinguishes between the cytoplasm of different
types of cells.
• And between the different types of C/T fibers and
matrices, by staining them differing shades of red &
pink.
 The serous cells are stained purple with the basic dye hematoxylin
due to their high content of cytoplasmic RNA - cytoplasmic
basophilia. The mucous cells are unstained, because neither dye
stains mucus. The cytoplasm of cells forming the ducts is stained
red-pink with eosin due to the presence of cytoplasmic proteins -
acidophilic or eosinophilic staining. A nucleus in the duct cell is
basophilic, due to its containing DNA and RNA.
Eosin Y is the most widely used eosin.
• As a cytoplasmic stain it is used as a 0.5 to
1.0 per cent solution in distilled water, with
a crystal of thymol added to inhibit the
growth of fungi.
• The addition of a little acetic acid is said to
sharpen the staining.
• Differentiation of the eosin staining occurs
in the subsequent tap water wash.
 Procedure for H&E sections
1. Xylene – 2min 11. Sodium bicarbonate – 1-
2 min
2. Xylene – 2min
3. Absolute alcohol – 2 12. Distilled water – 1min
min 13. 80% alcohol – 1min
4. 95% alcohol – 1min 14. 95% alcohol – 1min
5. 80% alcohol – 1min 15. Eosin – 1-2 min
6. 60% alcohol – 1min 16. 95% alcohol – rinse
7. Distilled water – 1min 17. 95% alcohol – rinse
8. Hematoxylin ( Harris’s 18. Absolute alcohol – 1min
) – 3 -10 min
19. Absolute alcohol – 2 min
9. Ditilled water – rinse
20. Xylene – 2 min
10. Amonium alum – 2 -10
min 21. Xylene – 2min
 PAS technique
• Periodic acid schiff technique.
• Detection of carbohydrates &
prteoglcans,glycoproteins, studying the
ground substance of teeth & bones
• Developing or resorbing bone & dentin stain
intensily with PAS
• Interglobular dentin, less calcified dentin,
dentinogenesis imperfecta & odontoma
exhibits distinct PAS reaction
``
• Epithelial
mucopolysaccharides, BM, mucins of salivary
glands, fungal hyphae.
• Periodic acid oxidises the glycol groups to
aldehydes
• Aldehydes are revealed as a reddish purple
dye product on treatment with leucofuchsin
( Schiff’s reagent)
 • 8.
Wash in tap water, 2
Periodic Acid-Schiff (PAS) minutes
Approximate time: 35 minutes
Technique: • 9. 50% Alcohol, five dips
Fixative: Absolute alcohol • 10. 70% Alcohol, five dips
Procedure 1. Absolute alcohol, 60 • 11.
95% Alcohol, five dips (two
seconds changes)
2. Rinse in distilled water (one
• 12.Absolute alcohol, five dips
change)
(two changes)
3. 0.5% Periodic acid solution, 5
minutes • 13.Xylene (two changes); dip
4. Rinse in distilled water (three until clear
changes) • 14.Mount and coverslip with
5. Schiff reagent, 15 minutes resinous media
6. Wash in running tap water, 2 to • Results:
Fungi: red to rose
5 minutes or until pink color Glycoproteins,
develops mucopolysaccharides: red to
7. Hematoxylin (Harris), 60 rose
seconds • Nuclei: blue
• Cytoplasm: light pink
Decalcification
• Calcified tissues – bone & teeth
• Removal of calcium from the tissue before
processing – Decalcification.
• Fixed specimen decalcified - 5%
nitric acid or 10% formic acid
• 8 – 10 days while changing the solution
daily.
• Washing the specimen -24hr to remove
the acid
Testing completion of
decalcification
1. Piercing the hard tissue with a sharp
& fine needle.
2. Keeping the specimen in a test
tube containing 5-6ml of nitric acid
&adding 1 ml of concentrated
ammonium hydroxide & few drops of
saturarted aqueous solution of
ammonium oxalate ( precipitation of
calcium – traces of calcium present)
Ground section
• Teeth preserved in 10% formalin to prevent
them from becoming brittle.
Use
1. Essential for the study of enamel, dentin &
cementum
Procedure

• Tooth is cut longitudinally in a mesiodistal

plane
• Buccal surface is applied firmly to the
rapidly rotating carborundum disk.
• Fine abrasive lathe wheel is used to ground
the tooth
• 13 mm adhesive tape is wrapped round the
wooden block & ground surface of the
tooth is wiped dry & pressed onto the
adhesive tape .
• Lingual surface of tooth is applied to
coarse abrasive lathe wheel & ground to
0.5mm.
• Fine abrasive lathe wheel – ground to
thickness of 50 -70 microns
• The section is further ground on fine &
smooth flat carborundum stone slab –
thickness of 30 -50 microns
• Section is dried for several minutes
• Sectionis lifted with a camel,s brush & placed
on a drop of mounting medium placed on glass
slide
• another drop of mounting media is put on the
section & covered with a cover glass.
`
MICROTOMES
AND
MICROTOMY

53
 CONTENTS
• Introduction
• History
• Different
types of Microtomes
• Microtome Knives
• Description of Rotary Microtome
• Paraffin block Section Cutting
• Non-paraffin blocks Section Cutting
• Faults and Remedies in Paraffin Embedded Microtomy
• References

54
 Greek : Mikros – Small
Tome – Incision

Microtome is a mechanical device used for cutting

thin uniform slices of tissues to examine under
microscope.
Syn – Histotome

55
History of Microtomes
• Herman Welker of Halle - 1856 introduced apparatus
for cutting tissues.

• Wilhem His – in 1866 improved this apparatus.

• His and Ranvier –Introduced Mechanical movement of

object .

• Rivert – Introduced Movement of knife.

• Charles & Minol of Boston – Introduced first rotary,

sliding and sledge type Microtomes.

• Lang and Mogensen designed 1st cryostat is 1938.56

Further improvements
……….

57
Types
• Rocking Microtome
• Rotary Microtome
• Base sledge Microtome
• Sliding Microtome
• Vibrating Microtome
• Freezing Microtome
• Cryostat
• Ultra Microtome
• Saw Microtome
• Histo-saw Microtome
58
Rocking Microtome
• Oldestin design, cheap and
simple to use.

• The knife is fixed and block

of tissue moves through an
arc to strike the knife.

• Between 2 strokes block is

moved towards knife for
required thickness of
sections by the help of
rachet operated micrometer
thread.

59
• Movement of cutting arm depend on the type of tissue
to be cut.

• Steady forward and backward movement of handle

will give ribbons of good sections.

Apart from this other techniques.

• Puttinghandle forward and releasing it from this

position i.e. allowing the spring to pull it back
sharply.

• Pulling handle forward and leaving back very slowly.

60
 Disadvantages

• Size of block that can cut is limited.

• Sectionsare cut in a curved plane so the sections are

not evenly thick.

• Light in weight.

61
Rotary / Minot Rotary Microtome

• It
is named so because
rotator action of hand-
wheel activates the
cutting movement.

• Contains a block-holder
mounted on a steel
carriage which moves up
and down in grooves

• Blockis advanced by a
micrometer screw
towards blade.
62
Advantages
• Cutting of large number of sections from each block.

• Larger blocks of tissue may be cut on this machine.

• Cutting angle of knife is adjustable.

• Less vibrations when cutting hard tissue.

• It
can be used to cut celluloid – embedded sections
with the help of special holder to set the knife
obliquely.

• Heavier machine so stable. 63

Base Sledge Microtome
• Block holder is mounted on a
steel carriage which slides
backwards and forwards on .
guides against a fixed
horizontal knife.

• Knife used is 24 cm in length

and wedge shaped.

• Knife holding clamps are

adjustable and allow the tilt
and angle of knife to the block
for easy setting.

64
• This is an all- purpose microtome.

• Various sizes of stages are available for use with

blocks of various sizes.

• Designed originally for cutting sections of large

blocks of tissue.

• Can be used for sectioning tissues of all types, sizes

and degree of hardness.

• Additional equipment allows the production of

frozen sections by using either carbon di oxide gas or
thermo electric modules. 65
• Advantages

• Celluloidin
sections can also be cut due to
adjustable knife holder.

• Freezing stage is also available in this type.

• Disadvantages

• Slower in use .
66
Sliding Microtome
• In this knife is moved
horizontally against a
fixed block.

• Knife is moved up or
advanced against block
in an inclined plane.

• Best to cut celluloid in

embedded sections.

• It can be used for

paraffin wax embedded
sections also.

67
Vibrating Microtome
• This instrument is designed to
cut tissue which has not been
fixed, processed or frozen.

• During sectioning tissue is

immersed in either water, saline or
fixative and cut by a vibrating
razor blade.
• Tissues are cut at a very slow
speed to avoid disintegration.

• Used in Enzyme histochemistry

and ultrastructural
histochemisry

68
Freezing Microtome

• In this type microtome is

usually clamped to the
bench.

• This is connected to a
cylinder of CO2 by means of
flexible metal tube such that
liquid carbon-di-oxide must
reach the microtome chuck.

• Along with this knife freezing

attachment is supplied with
most machines.
69
• Thermoelectric module
Enables tissue to be
frozen without necessity
of solid CO2 or liquid
nitrogen.

• This is based on
“peltier effect” i.e. when 2
dissimilar metals are
placed in opposition and
a direct electric current
passed through them
heat is generated on one
surface and lost from the
other.
70
• In freezing microtome thermo-
modules used consist of Bismuth
telluride and copper laid side by
side and connected in series.

• When current is passed it

generates heat on one surface and
it is lost from other.

71
• This modules are clamped
into microtome block and knife
holder.

• As the temperature achieved

is directly related to current
applied it is possible to
regulate it accurately down to
at least
- 250C and lower.

• Uses : Urgent surgical

biopsies if cryostat is not
available.
72
Cryostat
• Consist of rust proof
microtome of any type.

• Enclosed and operated with

in a deep freeze cabinet where
temperature is regulated at
-10 to -400 C.

• Facilities exist in many for

controlling knife and block
temperature individually.

• The cabinet is fitted with a

double glass window and a
door through which material
may be passed in and out. 73
74
• Sections formed are thin 3-5 of frozen fresh tissue
free of ice crystal defects.

• This is due to quick freezing at a very low

temperature and sections are cut without allowing
tissue to thaw.

• Uses :
• Urgent surgical biopsies
• Enzyme histochemistry
• Autoradiography
• Diagnosis of muscle biopsies
• Fluorescent antibody studies.
75
Ultra Microtome

• Ultra microtomes are

used for cutting ultrathin
sections for viewing in
the T.E.M

• They cut semithin and

ultrathin sections of
resin embedded
biological and materials
specimens.

• Sections are cut by glass

or diamond knives at a
thickness of around
70nm. 76
77
 Cryo Ultra Microtome
• Has capablities of cutting
semithin and ultrathin sections of
biological or materials specimens at
sub zero temperatures.

• Uses high pressure and liquid

nitrogen to rapidly freeze
specimens. It can freeze up to 200
microns.

• The specimens are subjected to

very high pressure immediately
before contact with cryogen to
minimize or eliminate ice crystal
formation.

• Semi thin or ultra thin sections are

78
collected for immuno-labelling.
Saw Microtome

• Specifically used for cutting

extremely hard and brittle
materials such as teeth, bone,
ceramics and reinforced
plastics.

• Heart of this microtome is

the diamond coated
innerhole-saw The thickness
is only 300 m.

• The section thickness is set

manually with a knurled
screw on the object arm.
79
 Important parts
• Objectarm with object
holder and object mount.

• Nozzlewith valve for

regulation of water.

• Knurled screw for setting

section thickness.

• Scale ring for zero

setting.
• Clamp for section
thickness setting.

• Saw blade. 80
OPERATION

• Mounting the object

• Setting the height of the object

• Trimming the object surface

• Making the section

• Removing the section

• Changing the object

81
 Faults And Remedies In Paraffin
Embedded Microtomy
A. Alternate sections thick and thin

1. Wax too soft for tissue • Cool block with ice or

or conditions. re-embed in higher
melting point wax.

2. Block or knife loose

• Tighten block and knife.

3. Insufficient clearance
angle. • Slightly increase
clearance angle.

4. Mechanism of
microtome faulty. • Cheek for any obvious
faults, e.g. Pawls may82 be
worn
B. Thick and thin zones parallel to knife edge
(Chatters)
1. Knife or block loose in • Tighten
holder

2. Excessively steep knife • Reduce angle to

angle. minimum but still
leaving clearance
3. Calcified areas in • Rehydrate and
tissue decalcify, or surface
decalcify.
4. Tissue or wax too hard • Use sharp heavy duty
for sectioning knife. Reduce slant
condition. angle of knife. If
available, use heavy
duty microtome. Use
83
softening fluid on
C. Sections will not join to form a ribbon

• Breathe on block to
1. Wax too hard for warm or re-embed in
sectioning lower melting point
conditions. wax.

• Clean with xylene

moistened cloth.
2. Debris on knife
edge
• Adjust to optimal angle

3. Knife angle too

steep or too 84

shallow
 D. Areas of tissue in block not present in
sections
1. Incomplete • Return tissue to
impregnation of tissue. vacuum impregnating
bath for a few hours or
reprocess if fault is
excessive.

2. Wax block becoming • Re-attach with hot

detached from wood. spatula

85
 E. Sections roll into a tight coil instead of
remaining flat on knife
• Resharpen to produce
1. Too little rake angle.
shallower cutting angle
or reduce knife tilt if
clearance angle is
excessive
• Sharpen Knife
2. Knife blunt

• Reduce section
3. Section thickness too thickness or use
great for wax in use. slightly higher melting
point wax. Breathe on
block as sections are
being cut.
86