HISTOPATHOLOGY LEC

LECTURE 11: MICROTOMY

PROF. KYLE SOLANO, RMT, MD
JULY 1, 2021
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MICROTOMY
● The word “microtomy” originated from the Greek language.
○ Mikros = small
○ Temnein = cut
● The word “microtomy” means to cut the tissue in thin
sections.
● For successful microscopic examination, it is necessary to
have thin sections of the tissue by microtomy. ● Before putting in the microtome, it is placed in ice to even
● Types of Microtomes: out the temperature
○ Rotary microtome
■ most common ROTARY MICROTOME
■ There is a rotating flywheel that actuates ● This is the most commonly used microtome in routine
the movement of the block laboratory.
○ Rocking microtome ● The cutting blade is kept in horizontal position, and the
○ Base sledge microtome block containing tissue moves up and down with the help of
○ Sliding microtome rotatory handle attached with the microtome.
○ Cryomicrotome ● In each 360° rotation of the wheel handle, the block moves
■ Used for frozen section biopsy down followed by up, and the tissue is cut as thin ribbon.
■ Similar to rotary microtome but the
environment in which the tissue is
exposed to the blade to make thin
sections is sub zero - 35
○ Ultramicrotome
■ Used to make thin slices for electron
microscopy
■ Focuses on organelles and more of a
subcellular study
○ Laser microtome
● Cutting it continuously creates tissue ribbon
○ Happens when subsequent sections attach
together at the ends so that the edges will fuse
together

Advantages
● Good-quality 2–3-μm-thin section is possible.
● Heavy and stable automated rotary microtome reduces
health hazard and gives the best-quality section.
● Good tissue ribbon production.
● Trimming = to even the surface of the block ● Easy-to-cut various types of tissue: firm, fragile, small
● Rotating flywheel causes the block to move up and down biopsy, etc.
forcing the specimen impregnated with the wax and the ● It is consistent
block to encounter the blade
● When it encounters the blade, small part of it is sectioned Disadvantages
● Expensive.
● Unsuitable to cut large blocks.
○ Block attachment is quite small
● Knife faces up and so may be dangerous to the technical
staff
○ Blade is very sharp
○ If u change the knives often, it will be less blunt

ROCKING MICROTOME
● Floatation bath
● The rocking microtome is also known as Cambridge
○ important because it reduces tension within the
rocking microtome.
tissue
● The word “rocking” is used as there is a rocking action of
○ It stretch the tissue so that it becomes flat
the microtome like arm movement. In this type of
○ The flatter the tissue, the better it adheres to the
microtome, the knife is static, and the block of tissue moves
slide
in a rocking motion (arc-like movement of the block).
○ If there are no wrinkles within the section, the
● This is one of the oldest designs of the microtome. The
viewing is better in the microscope
microtome can cut thin section with ribbons and is ideal for
○ If the blade is dull, there will be plenty of artifacts
serial section. The sections are slightly curved in this
and spaces in which the morphology cannot be
microtome
clearly seen
● One of the oldest and rarely used microtome

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 CRYOMICROTOME
● This type of microtome is used for the cutting tissue for
frozen sample. The sample is made hard in liquid nitrogen
and then cut by the microtome in the chamber that contains
liquid nitrogen.
● In frozen sections, temperature is used as fixatives (rapid
fixative) in a sense that it fixes a tissue in a certain time to
inhibit all progression of metabolic processes
○ Makes it hard to produce ribbons
● With frozen section, you can have a diagnosis of a
● The block moves in an arc because there is a pivot area
processed slide in as early as 15 minutes during the
operation (intraoperative procedure)
Advantages ○ Surgeon takes a mass in the body and sends it to
1. Thin section histopath to do frozen section (know if its
2. Easy to operate malignant or benign)
3. Low-cost instrument and reliable ○ If malignant, it will take the whole organ
○ If benign, close and leave it be (can also be
Disadvantages removed depends on the surgeon)
1. Tissue is curved and the microtome does not provide a flat ● Frozen section dictates what's the next move in managing
section. the patient
2. As the microtome is of light weight so vibration may occur ○ Final diagnosis is reserved for the H&E because
even if frozen section is rapid it is still lacking
NOTE: Every time you rotate a rotary microtome, apart from going up because the morphology of the cells and the
and down, the block is also moving forward (to shave a deeper tissues isn't as clear and as helpful compared to
section) H&E processing
- Pathologist and technologist should be wary of consuming ● Freezing media also facilitates the even freezing of the
all of the specimen (don't want to consume all the specimen tissue
because it may be needed for second reading) ● Ideal slides for frozen sanction are charged slides
- Dapat tipid in making ribbons After partial freezing of the media, place the tissue
↓
● Heat transfer apparatus or tool
BASE SLEDGE MICROTOME
○ Transfers the residual heat of your specimen into
● In sledge microtome the block is fixed in a static position
the tool
within a steel carriage. The knife slides to and fro over the
○ Evens the interface between the balde and the
top of the block. This microtome is the best for large tissue
specimen
sample or the hard tissue. The tissue sections are usually
↓
thick (more than 10 μm) in base sledge microtome.
Trim to expose the specimen; cut the excess frozen freezing media
○ Best for large or hard tissue because:
↓
■ The block is static (adequate control)
● Freezing media acts as a structural support so that the
tissues are intact
↓
Section after trimming and put it on a slide
● Not for rapid diagnosis but rather its purpose is to change
the course of management
↓
Submerge in ethyl alcohol
↓
Rinse
↓
Immerse in hematoxylin - a basic stain in a sense that
↓
Advantages
Stain
1. Hard tissue can be cut. Advantages
2. Large tissue sample can be cut.
1. To get rapid section for rapid diagnosis
3. The best microtome for ophthalmology and large
2. To study nerve biopsy
neuropathology section.
3. To study enzyme histochemistry
● Machine is turned on 24/7
Disadvantages
● Fresh tissue is required
1. Difficult to get thin section. ○ Needs to be rapidly transported to the
2. Large slides are required. histopathology section because of the on going
metabolism
SLIDING MICROTOME ○ If it contains formalin, it is hard to stick to the
● In this microtome the knife is static, and the block moves freezing media
horizontally over the knife.
Disadvantages
Advantages 1. It needs continuous supervision to maintain the
1. Large sections can be cut temperature.
2. Mainly used for celloidin-embedded tissue 2. Freezing artefacts are often seen.
3. Simpler design and easy maintenance 3. Very expensive instrument.
4. Brain sections can be cut better by this type of microtome 4. Fixed tissue is very difficult to cut.

Disadvantages MICROTOME KNIVES

1. The knife may glide in case of hard tissue and may jump ● Most of the knives that we use are disposable
2. Long knives are difficult to sharpen ● It is time consuming to use knives that require to be
sharpened first
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 ● It is the angle between the lower bevel of the knife and the
surface of the block. It is usually around 5°. The angle of
clearance is related with the friction between the block and
knife. Lower the angle of clearance, the less will be the
compression on the block.
Bevel angle:
● This is the angle in between the two planes of the knife.
Cutting edge:
● This is the straight line formed by the intersecting of the two
planes of the bevel.

Box 5.1 Microtome Knives.

- Most of the knives that we used are disposable type.
Types based on shape:
● Plano concave (Profile A): Nowadays disposable blade is used in many laboratories to save
○ Very sharp knife and is used for cutting soft time to sharpen.
tissues
● Biconcave (Profile B): Two types of disposable blades are available:
○ Used for rocking microtome ● Low-profile blade: These blades are used to cut small
○ Vibrates during cutting biopsy or soft large tissue.
● Wedge (Profile C): ○ No need to sharpen
○ Most commonly used ○ They are not very rigid compared to your ordinary
● Tool edge (Profile D) knife.
○ Used for hard tissue ○ Some vibration effect may be seen.
○ chisel ○ Vibration effect- there are shears; ma shear konti
Types based on disposability: ang tissue but for some, it is quite negligible
● Permanent because the newer rotary microtomes are very
● Disposable stable.
○ Low-profile blade: To cut small biopsy ● High-profile blade: These are used to cut firm to relatively
■ Usually used hard tissue such as the uterus, heart, etc.
○ High-profile blade: To cut firm to hard tissue Advantages:
Materials used in knife: ● Easy to replace within seconds.
● Conventional knife: Steel ● No need to sharpen.
● Diamond knife: Made of diamond and used to cut epoxy ● The overall cost of disposable blade system is low as there
resin blocks is no need of any knife sharpener or abrasive powder to
○ For electron microscopy sharpen the knife.
○ You need very hard embedding media so that for Disadvantages:
electron microscopy because the sections are so ● They are not very rigid like ordinary knife, and therefore
thin, you need to support the thin sections. vibration effect may be seen.
DIFFERENT ANGLE IN MICROTOMY
How do you sharpen your conventional knife?
● These angles dictate the quality of the sections you have Honing :
and how smooth your microtome…. Ribbons
● If the angle is too high, you might not make ribbons or it
doesn't cut deep enough.
○ Your sections might be short and uneven.

● Abrasive grindings are used to sharpen the knife. Stone

can be used to sharpen the knife. Light oil can be used to
lubricate the stone before use.Iron oxide, aluminium oxide
and silicon carbide are usually used as abrasive. The
handle of the knife is hold by one hand, and the other hand
holds the front end of the knife. The knife is pushed
forward and diagonally over the slab Perpendicular linerom
surfaceof block several times. Same procedure is followed
in other surface of the knife also.
● Heel to toe movement
Stropping:
Rake angle:
● It is the angle between the upper bevel of the knife and the
perpendicular line drawn from the surface of the block.
Increased rake angle makes the section cutting easier.
Angle of clearance:

3

● It helps to polish the cutting edge of the knife that is
already sharped by honing and also to remove any “burr”
formed during honing. Strop is made of leather and it
should be free from any dust or grit.
● Polishing
● It is to to remove the BURR
● Usually use leather “strop”
● Toe to heel movement
Factors Involved in Cutting
● Temperature: Lowering the temperature facilitates section
cutting.
● Angle of rake: Higher rake angle helps in smooth flow of
ribbons. Lower rake angle is used for hard tissue.
● Consistency of tissue: Soft tissue is cut at a slow rate
than the hard tissue.
The following instruments are essential for section cutting:
● Microtome with blade
● Water bath
● Paraffin block with embedded tissue to cut
● Ice tray
● A blunt forceps or camel brush
● Slide rack with slides
Water Bath (Floatation Chamber) Water bath is used to float the
tissue after cutting. The temperature of the water bath is usually
controlled automatically by a thermostat. The temperature of water in
the water bath should be 10 °C below the melting point of the
embedded paraffin wax and is usually kept in 40–50 °C.
It is necessary to prevent formation of any air bubbles within the
water bath. For adequate floating ofthe tissue, one can add a few
drops of alcohol or little amount of detergent. This reduces the
surface tension of the water and tissue floats smoothly.Blunt
Forceps and Camel Hair Brush Blunt forceps helps to manipulate the
floating tissue section.
Camel hair brush is used to clean the blade. Slide Rack with Clean
Glass Slides The clean slides are kept in the slide rack. The slides
can be already labelled by diamond pencil or on the frosted side by
lead pencil. Alternatively this can be marked after lifting the tissue
section.Adhesive In case of routine section and staining, no adhesive
is required. However, in certain situations we use cell adhesive
material such as:
● We usually label slides with a pencil, the writing of the
pencil can survive decolorization, alcoholization and
staining.
● Staining process is quite abrasive to pen marks, especially
on the xylene part.
1. Brain sections
2. Decalcified tissue
3. Using strong alkali at the time of staining
The most commonly used adhesives include:
● Charge slides: it attaches negatively charged tissues.
○ It pilits coz there is an attraction because of
positively charge slide and there is an
electrostatic forces that binds them together.
○ Not that strong but it is enough
● Mayer’s egg albumin and glycerol: This is prepared by
mixing
○ White part of egg: 100 ml
○ Glycerol: 100 ml.
○ Homogenize the mixture thoroughly, and filter it
by gauze piece. Add a few crystals of thymol to
prevent bacterial growth.
○ Most commonly used.
● Poly-l-lysine: This is a good adhesive and does not
produce any background staining. The slides are coated
with poly-l-lysine diluted by distilled water (1:10) before
using.
● 3-Aminopropyltriethoxysilane (ACEP): The slides are
dipped in the dilute ACEP solution in acetone (2%) and
then dried before using.
● Permanent positively charged slides: Here the slide is
tempered in such a way that the slide surface is always
positively charged. It is easier to lift the tissue section with
these slides. The positively charged slides are also
excellent for lifting frozen section tissues.
Adhesive- you put it in a slide before you fish out the floating section.
STEPS OF TISSUE SECTIONING
1. Trimming the tissue: Trimming of the tissue is needed to
expose the tissue piece within the paraffin wax for cutting.
The block is fixed in the chuck of the microtome, and the
paraffin is cut till the tissue is fully exposed
.
Adequate caution should be taken not to overcut tissue as
this may produce various artifactual changes.
● We really need to trim so that the tissue is
exposed because there is still a layer of paraffin
on top of the tissue and we need to cut that off so
that the tissue will be exposed.
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2. Cooling the block: After the initial trimming, the blocks are
kept for cooling for 15–20 min.

This will help to maintain the same consistency of the

paraffin and tissue. This helps in easy cutting.

3. Cutting proper: The block is fixed in the chuck of the

microtome. The cutting surface of the block should be
parallel to the knife. The angle of clearance should be
only 2–5° to have good section. The tissue in the block is
cut by gentle, smooth and slow stroke. The ribbon-like
tissue sections are produced. The tip of the ribbon is held
by forceps, and the end part of the ribbon is removed from
the knife edge by brush. In case of any difficulty to get the
flat section, the cutting surface should be gently warmed by
warm water.

5. Picking up the tissue: The slide is placed vertically within

the water bath in front of the tissue, and when the tissue is
touched, the slide is withdrawn vertically from the water.
The tissue pickup process must be gentle and smooth. To
prevent any mix-up, the water bath should be cleaned
immediately after cutting each block.
● Those slides already have their adhesives applied
before you fish out
● For frozen section, we usually used charged
slides

6. Drying the section: The slide containing the picked-up

section is kept in slide rack. The slides are now kept in hot
oven to get dry. The temperature of the oven should be
slightly more than the melting point of the paraffin.
● We put it in the oven to melt the paraffin otherwise
it will affect the staining process.