EMBEDDING AND

MICROTOMY

CECILIA LEKPOR
LEARNING OBJECTIVES
❖At the end of the Lecture student should be able to
❖Explain the techniques involved in embedding
❖Know the different media used in embedding-
❖Advantages and Disadvantages Embedding media
❖Processes involved in embedding and microtomy
❖Faults and remedy in sectioning
EMBEDDING-OVERVIEW
❖After infiltration by molten paraffin wax, tissue specimens are ready to be
embedded into moulds

❖Specimens unloaded from the processor are removed from their cassettes
and placed into moulds, into which molten paraffin is poured

❖ During the embedding step, the tissue should be oriented in the mould to
provide optimal sectioning

❖ Tissue can be oriented on edge, or on face, depending on the tissue type

OVERVIEW CONT…
❖When tissue orientation is critical to histologic assessment, it is important to
convey clear embedding directions for the desired orientation

❖Specimen are carefully oriented to determine the plane through which the
section will be cut

❖ Proper orientation also determines the area of interest to view under the
microscope

❖Cassettes carries the processed tissue with the identification details, which
is place on top of a moulds and is attached by adding further wax to form a
specimen block
OVERVIEW–CONT…
❖Specimen block is allowed to solidify on a cold surface and when
removed from the moulds, a tissue block is made

❖The cassette filled with wax provides a stable base for clamping in the
microtome

❖The block containing the specimen, is now ready for Microtomy

❖Paraffin wax for tissue embedding should be clean, filtered and held at 2–
4℃ above its melting point
EMBEDDING MEDIA
❖There are various types of embedding media used in histopathology
such as:

➢Paraffin wax
➢Resins
➢Agar
➢ Gelatine
➢Celloidin
WAXES
❖PARAFFIN WAXES
➢Paraffin wax is a polycrystalline mixture of solid hydrocarbons produced
during the refining of coal and mineral oils

➢Its melting point ranges between 40-70oC

➢For routine work, wax of 56-58oC melting point is satisfactory

➢However, the melting point has influence on the histological properties

➢High molecular weight mixtures melt at higher temperatures than waxes

comprised of lower molecular weight fractions
WAXES CONT…
➢It is most widely used embedding media for histological specimen is
paraffin wax

➢Due to its ease in processing large number of tissues within a short

period

➢Sectioning and staining present less difficulties than other media

➢Paraffin is comparatively cheaper

➢Tissue-wax adhesion depends upon crystal morphology of the embedding

medium
WAXES CONT…
➢Small, uniform sized crystals provide better physical support for
specimens through close packing
➢To improve consistency of wax additives are added to:
▪ Alter the crystalline morphology of paraffin wax
▪ This result in a less brittle, more homogeneous wax with good cutting
characteristics
▪ Gives better support to harder tissue

➢Heating for prolonged periods at 10-20oC above melting point hardens

the wax
OTHER TPYES OF WAXES
❖Microcrystalline Waxes
➢These waxes differ from paraffin wax in having much finer
microcrystalline structures and higher melting points 62 to 88oC

❖Paraplast:
➢This is a mixture of greatly purified paraffin wax and several synthetic
plastic polymers with melting point 56 to 57oC
➢ Sections can be cut without cooling the block surface on ice
➢ Larger and bone block may be cut with ease as it is more resilient than
paraffin wax
OTHER TPYES OF WAXES CONT…
❖Water soluble wax (Carbowax):
➢Have melting point of 35-50 oC
➢Soluble in water and other liquids except mercury
➢Useful in demonstration of lipids and some enzymes

❖Resins:
➢Are embedding media ideal for electron microscopy
➢May work for semi thin sections for light microscopy
➢Used for embedding undelcalcified bone, e.g., Epon, acrylic
OTHER TPYES OF WAXES CONT…
❖Agar:
➢When employed alone, does not give sufficient support to tissues
➢Used in combination with ester wax and paraffin wax in the application of
double embedding

❖Celloidin (Low viscosity nitrocellulose-LVN):

➢ Insoluble in water but soluble in diethyl ether, methanol and methyl benzoate
➢Very useful in the study of the central nervous system
EMBEDDING

❖The process by which tissues are surrounded by a medium such as

agar, gelatin, or wax which when solidified provides sufficient
external support during sectioning/microtomy
EMBEDDING CENTRE
TISSUE EMBEDDING STEPS
1. Turn the heat block on to melt the paraffin one hour before adding the
tissue cassettes

1. Place the entire cassettes containing the tissue in the moulds compartment
(65°C) for 15 minutes to melt the wax

1. Open cassette to view tissue sample and choose a moulds that best
corresponds to the size of the tissue
 EMBEDDING STEPS-CONT…
4. Put a small amount of molten paraffin in the mould, dispensing from the
paraffin reservoir

5. Using warm forceps, transfer tissue into mould, placing cut side down,
as it was placed in the cassette

6.Transfer mould to cold plate, and gently press tissue flat. Paraffin will
solidify in a thin layer which holds the tissue in position

7. Top up with more wax and transfer mould to cold plate to solidify
ORIENTATION OF TISSUE
➢For tubular structures such as arteries, veins, fallopian tube- the cross
section of the wall and lumen should be visible

➢Skin biopsies such as punch or excision biopsy- cross section of the

epidermis, dermis and subcutaneous layers must be visible-cut in a plane at
right angle to the surface

➢For most tissue blocks, sections are cut from the largest area of the tissue.

➢Muscle biopsies are cut in both longitudinal and transverse planes

➢When a particular tissue feature is present on one surface only, mark the
tissue on the opposite aspect with Indian ink
TISSUE ORIENTATION
MICROTOMY
❖Microtomy
➢A technique or procedure of cutting thin slices/section from paraffin-
wax embedded tissue blocks for microscopic examination
➢Helps in the study of different types of tissue components

❖ This process utilizes a microtome in conjunction with a microtome

knife or disposable microtome blade and blade holder
MICROTOMES
❖Microtomes are mechanical device designed for the accurate cutting of thin
uniform slices of tissues sections
❖Normally modern microtomes allow cuts of a thickness of 0.1 to 100
microns (μm)
❖ The history of the microtomes began with the beginning of light
microscopes
❖In order to analyze objects, they had to be thin enough for light to penetrate
them

❖For paraffin wax embedded tissue block, the range of sections is 1–5µm in
thickness
MICROTOMES CONT…
❖Variety of microtomes have common features as:

➢Knife holder
➢Device for firm clamping of tissue blocks and knife
➢A mechanism to advance the tissue block at a predetermined thickness
➢ A mechanism to advance knife
TYPES OF MICROTOMES
❖There are five 5 basic types of microtomes named according to their
mechanism

➢Rocking
➢Rotary
➢Base sledge
➢Sliding
➢Freezing
ROCKING MICROTOME
➢Name derived from the rocking
action of the cross arm

➢Oldest in design, cheap, simple

to use

➢Extremely reliable

➢Minimal maintenance
ROTARY MICROTOME
❖A general-purpose machine to produce semi-thin tissue sections

❖MECHANISM:
➢The handle wheel rotates through 360℃ moving the tissue block vertically
past the cutting surface and returning it to the starting position

➢Block holder mounted on a steel carriage which moves up and down and is
advanced by the micrometer screw-cutting flat sections
ROTARY MICROTOME CONT…
❖Manual ones are completely manipulated by the operator

❖Semi-automated (one motor to advance either the fine or the coarse hand-
wheel)

❖Mechanism of block advancement-retracting or non-retracting

❖Retracting action moves the tissue block away from the knife on upstroke,
producing a flat surface to the tissue
LOCK KEY

HAND-WHEEL

BLOCK HOLDER
 HistoCore AUTOCUT

❖It allows users to select

among automated, semi
auto-automated, or
manual sectioning modes
based on their personal
preferences.
HistoCore AUTOCUT

A
B
C
• A. Feed Wheel or
user-motorized with
the push of a button

• B. Enable fast and

effortless trimming

• C. Shorten cleaning
time from minutes to
seconds with the
Antistatic Waste Tray.
ROTARY MICROTOME CONT…
❖Ability to cut thin 2-3mm sections

❖Easy adaption to all types of tissues (hard, fragile, fatty) sectioning

❖Ideal for cutting serial sections-large number of sections from each block

❖Cutting angle of block is adjustable

❖Heavier and more stable

ROTARY MICROTOME CONT…
❖Only small length of knife is available so larger tissue block cannot be
sectioned
❖Celloidin embedded tissue can not be cut
BASE SLEDGE MICROTOME

KNIFE

BLOCK

BLOCK HOLDER

SLEDGE
BASE SLEDGE MICROTOME CONT…
❖Originally designed for cutting sections of very large block of tissue (e.g.
whole brain)
❖Used primarily for large blocks, hard tissue, whole mount
❖Useful in neuropathology and Ophthalmic pathology
❖MECHANISM:
➢The block holder is mounted on a steel carriage which slides backwards
and forwards on guide against a fixed horizontal knife
BASE SLEDGE MICROTOME CONT…
❖ADVANTAGES

➢Heavy, very stable, on subject to vibration

➢Large knife (24cm in length) and usually wedge shaped-less vibration

➢Adjustable knife holding clamps allow tilt and angle of the knife to
touch the block to cut sections more easily

❖DISADVANTAGE
➢Slower in use than rocking or rotary
SLIDING MICROTOME
KNIFE

BLOCK HOLDER
SLIDING MICROTOME
❑ADVANTAGES
❖Designed for cutting celloidin embedded tissue block
❖The knife or blade is stationary, specimen slides under it during sectioning
❖Also use for paraffin wax embedded sections
❖Due to its design, it causes few breakdowns
❑DISADVANTAGES
❖Does not allow serial cuts, which slows down the process
❖Exposure of the blade can cause accidents
❖It is almost impossible to obtain sections with a thickness of less than 8
microns
MICROTOME KNIVES
❖Developed to fit specific types of microtomes and coped with different
degrees of hardness of tissues and embedding media
❖There are disposable steel blade, diamond knife, heavy steel blade and
glass

❖Paraffin-wax embedding tissues knives are made of steel

❖Glass and diamond knives used in electron microscopy and with plastic
resin-embedded tissue block

❖Knives are classified according to their shape when viewed in profile as:
Profile A-Biconcave Profile B-Planoconcave Profile C-Wedge shaped Profile D-Tool-edge
❖Profile A is biconcave
➢ Sectioning celloidin embedded tissues
➢ It is rarely used in routine histopathology
➢Less rigid, prone to more vibration

❖Profile B is Plano-concave
➢Can be used to section wax-embedded soft tissues, celloidin-embedded
tissues and botanical specimens
➢Profiles A and B produce the sharpest edges
❖Profile C is wedge-shaped
➢ Most utilized steel knife for routine histopathology

➢ It is a more rigid knife than profiles A and B

➢ Used for paraffin wax sectioning of all tissues, as well as being employed in
cryostats for sectioning frozen material
➢This profile cannot be ground as sharp as profiles A and B

❖Profile D is tool-edge or plane-shaped

➢Sectioning hard materials, such as resins and extremely hard paraffin wax tissue
blocks
➢Decalcified dense cortical bone

➢This knife produces the least sharp edge when ground

DISPOSABLE STEEL BLADE
❖Cheap knives used for routine microtomy or cryotomy

❖Blades are sold in dispensers of 50 with which one or two out of the lot can
produce scores in section

❖The thin blade is held rigidly in its own special holder to minimize vibration
during microtomy

❖ The blades may be coated with platinum or chromium for a longer cutting
life
❖Provide a flawless 2-5 micrometer sections
❖ADVANTAGES
➢A damaged or dull cutting edge can be replaced by a new perfect edge within
seconds without the time consuming and costly process of sharpening
➢There is little, if any, variation in the cutting quality of the new blade unlike
sharpened conventional knives
➢The operating costs of disposable knife system are lower than the
conventional knife system

❖DISADVANTAGE
➢Due to the thin dimensions of the blades, there is the tendency for some
minor vibrations to occur when cutting very dense fibrous tissues or heavily
keratinized tissues.
PARAFFIN WAX SECTIONING
❖All operations concerning the placement or removal of the block into and out
of the block holder should be carried out with the safety of the user in mind

❖The expertise needed to become a competent microtomist requires practical

experience under the guidance of a skilled tutor

❖Blades are extremely dangerous and should be treated with respect

❖The hand wheel of rotary microtomes should be clamped before the block
carrier on the microtome should be positioned
❖ Clamped where available, at the end of the microtome furthest from the
blade during these procedures
EQUIPMENT
➢Microtome
➢Floatation (water bath)-thermostatically controlled
➢Thermostatically controlled slide drying oven or hot plate
➢Fine pointed or curved forceps
➢Sable or Camel haired brush
➢Slide rack
➢Clean microscope slide
➢Teasing needle
➢Ice tray
➢Chemical resistant pencil
CLAMPING THE BLADE IN THE KNIFE HOLDER
SETTING OF THE CUTTING ANGLE OF MICROTOME KNIFE
❖Setting of microtome knives to cut paraffin sections is important

❖Knives have two facets which are set at different angles

❖The upper and lower edge facets must produce a narrow bevel which
ideally should not be greater in width than 1 micrometer
SETTING OF THE CUTTING ANGLE OF MICROTOME KNIFE
CONT…
❖These are:
➢Cutting angle/Bevel Angle-Angle sustained by two facets

➢Clearance angle-Angle between the lower facet of the knife and the surface
of the tissue block. This angle prevent compression of sections of the tissue
block

➢Rake angle- angle between the upper facet of the knife and the surface of the
tissue block-It also prevent compression of sections

➢Tilt angle/knife angle-angle at which the knife is clamped in the microtome

(i.e. Vertically)
SETTING CUTTING ANGLE
TECHNIQUES INVOLVED
❖Insert appropriate knife in the knife holder and screw it tightly in
position

❖Correctly set the adjustable angles

❖Fix the block in the block holder of the microtome

❖Move the block holder forward or upward until the paraffin wax is
almost touching the knife edge
TECHNIQUE INVOLVED CONT…
❖Trim excess wax from the block surface until a representative cross-
sectional area of the tissue is obtained

➢Thickness adjuster is set at 10–15μm intervals and sections are removed

until the full face of the tissue is available (Coarse feed mechanism can
also be used)

➢An older blade could be used for that purpose

TECHNIQUES INVOLVED CONT…
❖Blocks for diagnostic sectioning are pre-cooled on an ice tray or chiller
plate prior to cutting

❖Chilling causes compression of the block and offers a more rigid block
face to the knife for ease of sectioning

❖Reset the thickness gauge to 3–5 micrometer and replace the blade with a
sharp one

➢Bring the block face up until its nearly touches knife edge

➢As the block hits the knife edge, the friction produced causes ‘melting front’
in the wax
ADJUSTING
THE
MICROMETER
SCREW
SECTIONS CUTTING
 TECHNIQUES INVOLVED CONT…
➢Thus, subsequent sections adhere to each other, to create a ribbon

➢The first tissue ribbon is held up with a pointed fine forceps and floated out on a
water bath

❖The sections are teased apart at their points of adhesion and attached to glass
microscope slides by part submersion of the slide in the water bath-FLOATING
OUT

❖Floating out is done in a thermostatically controlled bath

➢ The temperature of the water should be about 10oC below the melting point of
the wax
TECHNIQUES INVOLVED CONT…
➢Section must be laid with the shining side down
➢Folds should be removed and flattened before sections are attached to slides- This is
usually done by Floating out sections on water bath

➢Single or multiple sections can be separated with fine forceps and picked onto a slide

➢Distilled H2O may be used to prevent water bubbles from being trapped under the sections

➢Also, sections can be placed on a slide and run 20% alcohol under them. Any fold or
bubbles are removed

➢Alcohol or small drop of detergent may be added to the water to reduce surface tension to
flatten out the sections with ease
FLOATING OUT AND PICKING OF SECTIONS
TECHNIQUES INVOLVED CONT…
➢Label slides immediately

➢Excess water is drained from the slide, which is left to dry face up on a 60oC on
hotplate or drying oven

➢The slide is then placed in contact with the hotplate for 5–10 minutes to facilitate
adhesion of the paraffin wax section to the glass
PROBLEMS AND SOLUTIONS IN PARAFFIN WAX SECTIONING
PROBLEM AND CAUSE
❖A. When ribbon and consecutive
sections are curved
• Leading and trailing edges of block not
parallel
• Knife blunt in one area
• Surplus area of wax at one side
• Tissue varying in consistency
❖B. Alternate sections thick and thin
1. Wax too soft for tissue or conditions
2. Block or knife is loose
3. Insufficient clearance angle
4. Mechanism of microtome faulty
SOLUTION
• Trim with sharp scalpel until parallel
• Sharpen or use different part of knife
• Trim away excess wax
• Re-orientate block, cool block
• Cool block with ice or re-embed tissue
in higher melting-point wax.
• Tighten block and knife
• Slightly increase clearance angle
• Check for obvious faults on microtome
PROBLEMS AND SOLUTIONS IN PARAFFIN WAX SECTIONING
PROBLEM AND CAUSE
❖C.Thick and thin zones parallel to knife
edge (chatters)
1. Knife or block loose in holder
2. Excessively steep knife angle
3. Tissue or wax too hard for sectioning
conditions
4. Calcified areas in tissue
❖D. Scoring or splitting of sections at
right angles to knife edge
1. Nick in knife edge
2. Hard particles in tissue
3. Hard particles in wax
SOLUTION
1. Tighten
2. Reduce angle to minimum but still leaving
clearance
3. Use sharp heavy-duty knife or microtome,
reduce slant angle, use softening fluid on
tissue.
4. Rehydrate and decal. Or surface decal.
1. Use different part of knife or change
2. If calcium, decalcify. If other, remove with
fine sharp pointed scalpel
3. Re-embed in fresh filtered wax
PROBLEMS AND SOLUTIONS IN PARAFFIN WAX SECTIONING
PROBLEM AND CAUSE
❖E. Sections will not join to form ribbon.
1. Wax too hard for sectioning conditions
2. Debris on knife edge
3. Knife angle too steep or too shallow.
❖F. Sections attached to block on return
stroke
1. Insufficient clearance angle
2. Wax debris on knife edge
3. Debris on block edge
4. Static electrical charge on ribbon
SOLUTION
1. Breath on wax to warm or re-embed in
lower melting point wax
2. Clean with xylene moistened tissue
3. Adjust to optimal angle
1. Increase clearance angle.
2. Clean knife with xylene moistened tissue
3. Trim edge with sharp scalpel
4. Place a Bunsen burner near knife to ionise
air
PROBLEMS AND SOLUTIONS IN PARAFFIN WAX SECTIONING
PROBLEM AND CAUSE SOLUTION
G. Areas of tissue not present in sections 1. Return the tissue to impregnation and
1. Incomplete impregnation of tissue. re-embedding

2. Wax block becoming detached from 2. Re-attach by re-embedding

holding cassette
❖H. Excessive compression of sections
1. Blunt knife 1. Sharpen or change knife or blade

2. Bevel of knife too wide 2. Reground the steel knife

3. Wax too soft for tissue or sectioning 3. Cool block with ice or use higher
conditions melting point ice
PROBLEMS AND SOLUTIONS IN PARAFFIN WAX SECTIONING
PROBLEM AND CAUSE SOLUTION
❖ I. Sections expand and disintegrate
on water surface are curved 1. Return tissue to impregnation for a few hours
1. Poor impregnation of tissue
2. Cool by adding cold water and re-set the
temperature of the water bath
2. Water temperature too high.

❖J. Sections roll into a tight coil

1. Sharpen or change knife or blade
1. Knife blunt
2. Reduce knife tilt if clearance angle is
2. Too little rake angle excessive
3. Reduce section thickness or change wax to
slightly higher melting point
3. Section thickness too great for wax
REFERENCES
❖Sue E. Knoblaugh, Julie Randolph-Habecker, in Comparative Anatomy and
Histology (Second Edition), 2018

❖Cellular pathology techniques, C.F. A CULLING

❖Carson, Freida L; Christa Hladik ( 2009 .) Histotechnology: A Self-Instructional

Text (3ed.). Hong Kong :American Society of Clinical Pathology Press.

❖Histological Techniques- J D Bancroft