Microtomy, or the preparation of tissue slides, is the foremost technique used in histological studies. This presentation is a brief overview of the technique and the steps involved.

1. MICROTOMY
PREPARATION OF HISTOLOGICAL SLIDES
SYED MUHAMMAD KHAN
BS HONS. ZOOLOGY

2. HISTOLOGY
 Histology is the study of tissues and how these tissues are
arranged into organs (“Histo” in Greek means tissue or web).
 Tissues consist of cells and extra cellular matrix.
 The function of the tissue depends on the interaction
between the cells and the extracellular matrix.
 The study of tissues is dependent on microscopy and other
advances in biological techniques.

3. MICROTOMY
 Microtomy/Section Cutting is the technique of making
very thin slices of tissue specimens for the microscopic
examination.
 It is used to study various components of the cells or
tissues like lipids, enzymes, antigens or antibodies
(Immunohistochemistry), organelles, etc.
 It can also be used to identify abnormalities or atypical
appearance in the tissue (if present).

4. MICROTOMY
 The main stages involved in the preparation of histology
slides via microtomy are:
1. Fixation: to prevent cell decay and to preserve it in a
life-like appearance.
2. Processing: dehydration, clearing and embedding.
3. Sectioning: cutting very thin sections of the wax
embedded block.
4. Staining: to create visible contrast.
5. Mounting: to create a permanent slide.

5. FIXATION
 Fixation is the preservation of biological tissues from
decay due to autolysis or putrefaction.
 Samples of biological tissue are fixed to preserve the
cells/tissues in as natural a state as possible.
 Chemical fixatives are very carefully selected
substances whose properties must meet many criteria.
 Even the most careful fixation alters the sample to a
certain extent and may potentially introduce artifacts.

6. FIXATION
 Artifacts are structures or features in tissue that interfere
with normal histological examination, i.e. pigments
formed by fixatives.
 The choice of fixation method and specific fixative may
depend on the subsequent processing steps.
 Fixation is a reaction between the fixative and proteins
in the specimen which form a gel, keeping everything as
their in vivo relation to each other.

7. FIXATION
 The aims of fixation are as follows:
1. To prevent autolysis and bacterial attack.
2. To fix the tissues so they will not change their volume
and shape during processing.
3. To prepare tissues and leave them in a condition which
will allow clear staining of sections.
4. To leave the tissues in a life-like state.

8. FIXATION
 Chemical Fixation: biological structures are preserved in a
state as close to that of the living tissue as possible.
 This requires a chemical fixative that can stabilize proteins,
nucleic acids and muco-substances of tissues by making
them insoluble.
 Some chemical fixatives include: (1) Acetic acid, (2)
Formaldehyde – 10%, (3) Ethanol, (4) Glutaraldehyde, (5)
Methanol, (6) Picric acid and (7) Osmic acid (Osmium
tetroxide).
 The tissue samples must be cut into small cubes, around 1 cm
x 1 cm x 1 cm in size, and be put in the fixative (i.e. 10%
formaldehyde / formalin) for 24 – 28 hours.

9. FIXATION
 Frozen Sections
1. Small pieces of tissue (typically 5 mm x 5 mm x 3 mm) are placed in
a cryoprotective embedding medium.
2. Then they are snap frozen (rapid cooling for preservation) in
isopentane (an alkane) – cooled by liquid nitrogen.
3. The tissue is then sectioned in a freezing microtome
(cryomicrotome – discussed later).
4. Sections are then fixed by immersion in a specific fixative or series
of fixatives for a carefully controlled period of time.

10. PROCESSING
 Aim: to embed tissues in a solid medium firm enough to support
them and give them sufficient rigidity to enable thin sections to be
cut, and yet soft enough not to damage the knife or the tissues.
 The stages of processing are:
1. Dehydration (removal of water)
2. Clearing (removal of alcohol)
3. Infiltration & Embedding (preparation of a paraffin wax block)

11. STEPS INVOLVED IN TISSUE PROCESSING

12. PROCESSING
 Dehydration
1. Dehydration removes the fixative and water from the tissues
and to replace them with a dehydrating fluid.
2. Specimens are dehydrated in an ascending ethanol series: 10%,
20%, 50%, 70%, 95% and 100% – absolute (around 30 minutes in
each).
3. Some common dehydrating agents include: (1) Ethanol, (2)
Methanol and (3) Acetone.
4. Tissues may be held and stored indefinitely in 70% ethanol
without harm.

13. PROCESSING
 Clearing
1. In this process, an organic solvent such as xylene is used
to remove the alcohol and allow infiltration with paraffin
wax.
2. Some clearing agents include: (1) Xylene, (2) Toluene,
(3) Chloroform, (4) Benzene and (5) Propylene oxide.

14. PROCESSING
 Infiltration & Embedding
1. The tissue is first put in 58oC hot
paraffin wax for one hour.
2. The wax infiltrates it and
replaces xylene.
3. Tissues are surrounded by a
medium such as paraffin wax
(to make a block).
4. When the wax solidifies, it will
provide support and firmness to
the tissue during sectioning.
Wax embedded tissue

15. PROCESSING
 The overall aims of embedding are:
1. To improve ribboning
2. To increase hardness
3. To decrease melting point
4. To improve adhesion between specimen and wax

16. PROCESSING
 Embedding is done in molds / cases, for example: (1) paper boat
mold, (2) metallic boat mold, (3) peel-away disposable mold, and
(4) base mold.
Peel-away
Disposable Mold
Embedding Rings
Metallic
Base Mold
Cassette Bases

17. SECTIONING
 Sectioning is the production of
thin slices of wax-embedded
tissues via a microtome.
 Sections are 5 μm thick for light
microscopy and 80-100 nm for
electron microscopy.
 Microtome is a mechanical
instrument used to cut biological
specimens into very thin sections
for microscopic examination.
Thin sections of paraffin embedded
tissue being cut by a microtome

18. SECTIONING
 Most microtomes use a steel blade (ultramicrotomes use a
diamond knife).
 They are used to prepare sections of animal or plant tissues for
histology.
 Once the sections have been extracted, they are put on
warm water (to flatten them).
 Then they are picked from underneath by a glass slide.
 The slide with the section on it, is allowed to dry at 37oC, so
that the section adheres to it.

19. TYPES OF MICROTOMES
 Rotary Microtome
• It is the most commonly used
microtome.
• It is used for sectioning of paraffin
embedded blocks.
• It can also be used for frozen
sections in cryostat and also for
resin embedded cases.
• Sectioning occurs by rotational
movement of the microtome
head containing the block across
the blade.
Rotary Microtome

20. TYPES OF MICROTOMES
 Rocking Microtome
• It is a small microtome that has
two rocking arms.
• One cuts the sections.
• The other feeds through the
tissue block.
• It is limited to sectioning small
soft blocks as it uses spring
action to cut.
Rocking Microtome

21. TYPES OF MICROTOMES
 Base Sledge Microtome
• In this microtome, the sample is
placed into a fixed holder
(shuttle), which then moves
backwards and forwards across a
knife.
• The pressure applied to the
sample during the cut can be
reduced.
• Typical cut thickness achievable
on a sledge microtome is
between 1 and 60 µm.
Base Sledge Microtome

22. TYPES OF MICROTOMES
 Sliding Microtome
• It is an unusually designed
microtome with a blade
moving over the block, rather
than the block moving.
• It is good for celloidin
sectioning, although it can
produce good paraffin
sections too.
Sliding Microtome

23. TYPES OF MICROTOMES
 Ultramicrotome
• It is a microtome used mainly for
electron microscopy.
• It allows the preparation of extremely
thin sections.
• Diamond knives (preferably) and glass
knives are used in this microtome.
• To collect the sections, they are
floated on top of a liquid as they are
cut and are carefully picked up onto
grids suitable for TEM specimen
viewing. Ultramicrotome

24. TYPES OF MICROTOMES
 Cryomicrotome
• It is used for cutting frozen
samples.
• The reduced temperature allows
the hardness of the sample to be
increased which allows the
preparation of semi-thin samples.
• However the sample temperature
and the knife temperature must
be controlled in order to optimize
the resultant sample thickness.
Cryomicrotome

25. TISSUE SECTIONS
Transverse Section (T.S)
 Horizontal section cut made in a plane
at right angle to the longitudinal axis of
the body of a subject.
 T.S goes between lateral ends.
 It is usually comparatively shorter.
 The number of possible transverse
sections through a specimen is
comparatively more.
Longitudinal Section (L.S)
 Vertical section that is cut along the
longest axis of a subject.
 L.S runs through the anterior posterior
axis.
 It is usually comparatively longer.
 The number of possible longitudinal
sections through a specimen is
comparatively lesser.

26. STAINING
 Staining refers to the use of stains to make cells and/or cellular
structures visible and to enhance the contrast of a microscopic
image.
 The mounted sections are treated with an appropriate histology
stain.
 Biological tissues have very little variation in colors/shades when
viewed using either under a microscope.
 Staining biological tissues is done to both increase the contrast of
the tissues and also to highlight some specific features of interest.

27. STAINING
 The staining of paraffin embedded sections is done in the following
way:
 Deparaffinization: The wax of the section is removed by applying
xylene.
 Re-hydration: The specimen is rehydrated via a descending ethanol
series: 90%, 70%, 50%, 20%, 10% and distilled water (around 30
minutes in each).

28. STAINING
 Hematoxylin & Eosin Staining
1. The tissue is first stained with hematoxylin for 10 minutes (this stain gives
a blue color to the nuclei).
2. Then the tissue is rinsed in distilled water and then dipped in acid
alcohol until only the nuclei are stained blue.
3. The slide is again washed with distilled water and the neutralized with
alkaline tap water (Scott’s tap water).
4. It is washed again in distilled water and then stained with eosin for just
10 seconds to stain the cytoplasm, extracellular matrix, collagen and
erythrocytes red.

29. STAINING
 Dehydration: Specimen are dehydrated once again, in an
ascending ethanol series: 10%, 20%, 50%, 70%, 95% and 100%
ethanol (around 30 minutes in each).
 Clearing: The specimen is cleared in a clearing agent such as
xylene for 15 minutes to remove all traces of alcohol and raise
the refractive index of the specimen to make it more
transparent.

30. MOUNTING
 Mounting is done to preserve and
support a stained section for
microscopic examination.
 To mount a slide:
A. Apply a single drop of DPX
Mountant upon tissue section.
B. Hold coverslip at 45o allowing the
drop to spread along the edge of
the slip.
C. Let go of slip and allow medium
to spread slowly
Mounting Procedure

31. HISTOLOGICAL SLIDES
H&E stained sections (top left to bottom right) – lung tissue (of an emphysema patient),
retina, cartilage, muscle tissue, kidney and liver.