2. 2
Microtomy. Technique by
which tissues can be sliced into
thin sections and then
attached to a surface for
microscopy
Microtomes. These are
instruments used to cut
uniformly thin sections of the
tissues for further study.
Microtomes use sharp glass or
steel blades/diamond knives to
cut the tissues into thin
sections of uniform thickness
Introduction
3. Thickness of the sections is
predetermined at regular
distance on the microtome
The sections are attached to a
surface like glass slides for
staining
Tissues like muscles, bones, hair
are cut of thickness from
50 nm and 100 µm
Thickness of the sections may be
fixed according to the need of
the study and material involved
The stained sections can be
studied by light or electron
microscopy
Introduction
5. Types of microtomy
Main types include
◦ Rotary microtome
◦ Cryotome
◦ Ultra microtome
◦ Laser microtome
Use depends on the material
under study and the nature of
the work
Simple studies involve rotary
microtome while detailed
studies may involve ultra
microtome, etc.
Similarly cryomicrotome
involves sectioning of the
frozen tissues.
6. The most common type of the
microtome
Rotary action involves the
sectioning process at
predetermined thickness on
every rotation of the flywheel
Blade is fixed at horizontal
position
The sample holder moves the
sample ahead by the fixed
distance for cutting
Flywheel of the instrument may
be automatic or manual
Section thickness may vary from
0.5 µm to 60 µm
Types of microtomy
7. Types of microtomy
Used for making extremely thin
sections (40 nm to 500 nm)
Used mostly for biological
samples but samples may also
be processed
The sections are studied in
transmission or scanning
electron microscopy
The cut sections are floated on
the top of a liquid
These are then mounted on
a copper, nickel, gold, or other
metal grid
9. It consists of following
main steps
Fixation
Processing
Dehydration
Clearing
Embedding
Section cutting
Staining
Deparaffinization
Method (fixation and processing)
10. 10
It is the most important
step in histological studies
The histological details will
only be demonstrated if
the tissue is promptly and
adequately fixed
It is the process of
preserving the tissues in
the natural condition
Poorly fixed specimens are
more difficult to section
than the well fixed ones
Method (fixation and processing)
11. Commonly used fixatives
are alcohol, formalin,
glutaraldehyde, etc.
Factor affecting fixation
are temperature, change in
pH, penetration of the
fixative, volume, time, etc.
The lowest concentration
of the fixative is preferred
than the higher one
10% formalin or 2.5%
glutaraldehyde is used
Method (fixation and processing)
12. 12
It is a process in which tissues
are treated to make the thin
sections
A specimen is generally
processed as follows
Dehydration. It is the removal
of water from the tissues by
immersing serially in 50%, 70%,
85% and 100% alcohol for some
time
Clearing. It is removal of the
dehydrant from the tissues by
immersing in paraffin miscible
medium like xylene
Method (fixation and processing)
14. 14
It is the process of placing the
processed samples into molds
like paraffin blocks, etc.
The blocks are then used on
the microtome to make thin
sections
There are a number of
techniques that can be used to
make sections from difficult
blocks.
If the block is hard or friable,
the exposed surface can be
soaked in cold water or a
softening agent
Method (embedding)
15. 15
Embedding is an important
step that always requires a
care
Any excess wax on the
outside of a cassette
should be removed to
ensure the block is firmly
held during the sectioning
Specimen orientation is
very important
Paraffin embedded
specimens may be stored
indefinitely for future use
Method (embedding)
17. 17
It is process of cutting
the embedded samples
into thin sections
It requires great care as
tissues of the diagnostic
importance can easily be
lost or the block surface
may be damaged.
We should be sure that
all of the clamping
mechanisms are
tightened securely
Method (sectioning and staining)
18. 18
The thickness of the
sections is normally
between 10 μm to 30 μm.
The cuts sections are then
floated on warm water (at
40 °C) bath that helps to
remove wrinkles
These are then placed on
the glass microscopic
slides or some other
support for staining, etc.
Method (sectioning and staining)
19. 19
The slides must always
be grease and dust free
and stored and handled
correctly.
It involves reversal of the
process of embedding to
remove paraffin and
then staining by water
soluble stains
The slides are treated
with xylene and alcohol
to remove the paraffin
Method (sectioning and staining)
20. 20
Some of the stains need
mordants (Iodine acts as a
mordant in Gram staining
of bacteria)
The stains used depend on
their differential affinity
for various cellular
organelles
Some of the special stains
are used in special cases
Slides must always be
properly labeled
Method (sectioning and staining)