Embedding processes-pptx

By Geoffrey Mutale (MMLS-MUST)
1
H ISTOPATH OLOGY LEC TU R E SLID E
EMBEDDING PROCESSES
MUTALE
GEOFFREY
Masters in -Advanced Histopathology & Diagnostic Cytology

OBJECTIVES
At the end of this lesson, you will be
able to:
• Describe the process of embedding
• Different embedding media
• Tissue orientation
By Geoffrey Mutale (MMLS-MUST) 2

INTRODUCTION
Embedding is the process in which the tissues
or the specimens are enclosed in a mass of the
embedding medium using a mould.
Since the tissue blocks are very thin in
thickness they need a supporting medium in
which the tissue blocks are embedded.
This supporting medium is called embedding
medium.

CONT’
•Various embedding substances are
paraffin wax, celloidin, synthetic
resins, gelatine, etc.
•The choice of embedding media
depends upon Type of microscope
,Type of microtome & Type of tissue
eg. hard tissue like bone or soft
tissue like liver biopsy

EXAMPLES OF IMPREGNATING MEDIA
• Paraffin wax
• Agar
• Gelatin
• Low viscosity nitrocellulose( L.V.N)
• Water soluble wax (carbo wax)
• Celloidin
• Epoxy Resin

CONT’
• Paraffin wax – Paraffin is solid at room
temperature.
• The melting point of paraffin ranges from
40-60°C.
• For tropical countries hard wax having a
melting point of 58-60°C is suitable.
• This is the most commonly used tissue
embedding medium.

CONT’
Carbowax:
• It is a water soluble wax.
• Therefore tissues are directly transferred
to water soluble wax after fixation and
washing.

CONT’
Paraplast
• This is a mixture of purified paraffin and
plastic.
• It is easy to make the serial sections of 4
microns with this embedding medium due
to its elasticity.

CONT’
Ester wax
• It is harder than paraffin but has a low
melting point of 46-48°C.
• It is soluble in alcohol so there is no need
for the clearing step when using this tissue
embedding medium

CONT’
Epoxy Resin
• Epoxy resins are used for electron microscopy.
• Epoxy polymers of araldite differ from
methcrylate in that they are crosslinked causing
the cured solid block of araldite to be insoluble in
any solvent.
• Longer filtration is required because the
viscosity of resin is greater than methacrylate.

CONT’
Agar embedding:
• It is mainly used in double embedding.
• Multiple fragments and friable tissue may
be impregnated in one block when
sectioning on the cryostat.
• Another use of agar embedding is for
FNAC specimens.

CONT’
Celloidin media:
Celloidin (colloidin) is a purified form of
nitrocellulose soluble in many solvents,
suitable for specimens with large hollow
cavities which tend to collapse, for hard
and dense tissues such as bones and
teeth and for large tissue sections of the
whole embryo

CONT’
• Advantages:
• Its rubbery consistency allows tissue blocks that
are either very hard or of varying consistency, to
be cut without undue distortion.
• Dense tissues which are hard to infiltrate (e.g.
bones and brains) and specimen which tend to
collapse easily due to air spaces (e.g. eyes) are
supported better, thereby avoiding the crumbling
of tissues during sectioning.

CONT’
• It permits cutting of tissue sections which are
thicker than paraffin wax, and is therefore
recommended for processing of neurological
tissues.
• It does not require heat during processing;
hence, producing minimum shrinkage and tissue
distortion especially for cutting large bone
sections.

CONT’
It is, therefore, recommended in cases
when minimum shrinkage is required and
frozen section technique cannot be done.

DISADVANTAGE
• Celloidin impregnation is very slow (lasting for
several days or weeks)
• Very thin sections (less than 10u) are difficult to
cut.
• Photomicrographs are difficult to obtain.
• It is very volatile and therefore must be kept in
bottles with ground – glass stoppers to prevent
evaporation.

CONT’
Gelatin:
• Gelatin impregnation is rarely used except
when dehydration is to be avoided and when
tissues are to be subjected to histochemical
and enzyme studies.
• It is used as an embedding medium for
delicate specimens and frozen tissue
sections because it prevents fragmentation of
tough and friable tissues when frozen
sections are cut.

PROPERTIES OF AN IDEAL EMBEDDING MEDIUM
The embedding medium is considered as
ideal if it bears the following qualities:
• The melting point of the embedding
medium in the pure state should be below
65°C to avoid damage to the tissues by
heating during impregnation.
• The embedding medium must be
solidifying at room temperature.

CONT’
• The embedding medium must penetrate
the tissues replacing the fluid in which it is
saturated, usually a clearing agent.

DOUBLE EMBEDDING OF TISSUES
• As the name suggests, in this type of embedding
two types of embedding medium are used.
• This term particularly refers to the embedding in
which the tissues are first penetrated with
nitrocellulose and then embedded in the Paraffin
wax.
• The goal of this is to produce a block that can be
sectioned with a matrix of nitrocellulose in
addition to paraffin.

DOUBLE EMBEDDING

CONT’
• Tissue embedding machine
All the blocking steps can be performed with the
help of tissue embedding machine.
The embedding machine contains the following
parts;
Mould warmer, cassette bath, working surface
warmer with a nozzle for pouring the wax, forceps
well and cold plate.

TISSUE EMBEDDING MACHINE

CONT’
• The cold plate is of high efficiency refrigeration
system having temperature control ranging from
different freezing points to 4 or 5 degree C.
• It can occupy about 50-60 blocks.
• Large 3-5 litre capacity paraffin reservoir with
adjustable temperature
• Optional vacuum lids, which allows for vacuum
infiltration of tissues.

METHOD OF EMBEDDING
• Open the tissue cassette, check
requisition form entry to ensure the correct
number of tissue pieces is present.
• Select the mould; there should be
sufficient room for the tissue with
allowance for at least a 2 mm surrounding
margin of wax.

CONT’
• Simple glossed wall or floor tiles may also
be used in place of glass plate.
• Fill the mould with paraffin wax.
• Using warm forceps select the tissue,
taking care that it does not cool in the air;
at the same time.

CONT’
• Glycerine may be applied to the L pieces
and also to the metal or glass plate on
which the moulds are placed for
embedding.

CONT’
• Place the tissue in the mould according to the
side to be sectioned.
• This side should be facing down against the
mould.
• A small amount of pressure may be used in
order to have more even embedding.
• Chill the mould on the cold plate, orienting the
tissue and firming it into the wax with warmed
forceps.

CONT’
• This ensures that the correct orientation is
maintained and the tissue surface to be
sectioned is kept flat.
• Insert the identifying label or place the
labelled embedding ring or cassette base
onto the mould
• Add more paraffin into the mould to fill the
cassette and mould.

CONT’
• Cool the block on the cold plate.
• Remove the block from the mould.
• Cross check block, label and requisition
form

TYPES OF MOULDS
This is the traditional embedding method.
The “L moulds (Leuckhart) are adjusted according to the shape and
size of the tissue

CONT’
Reusable metallic moulds
Most used moulds PVC disposable moulds

ORIENTATION OF DIFFERENT TISSUE
• During embedding the orientation of tissue is
important.
• Correct orientation of tissue in a mould is the
most important step in embedding.
• Incorrect placement of tissues may result in
diagnostically important tissue elements being
missed or damaged during microtomy.

TISSUE ORIENTATION

CONT’
• During embedding it is important to orient
the tissue in a way that will provide the
best information to the pathologist.
• Embedding should be done according to
the type of tissue.
• The requisition form should always be
read during embedding for proper
orientation.

GENERAL CONSIDERATIONS
• Elongate tissues are placed diagonally
across the block.
• Tubular and walled specimens such as vas
deferens, cysts and gastrointestinal tissues
are embedded so as to provide transverse
sections showing all tissue layers.

CONT’
• Tissues with an epithelial surface such as
skin, are embedded to provide sections in
a plane at right angles to the surface (hairy
or keratinized epithelia are oriented to face
the knife diagonally).
• Usually tissues are embedded with the
surface to be cut facing down in the
mould.

CONT’
• Multiple tissue pieces are aligned across the
long axis of the mould, and not placed at
random. Incorrect placement of tissues may
result in diagnostically important tissue elements
being missed or damaged during microtomy.
• In circumstances where precise orientation is
essential, tissue should be marked or agar
double embedded.

POINTS TO NOTE
• Embedding is the process in which tissues or
specimens are enclosed in a mass of the
embedding medium using a mould.
• Embedding medium are supporting medium into
which the tissue block are embedded.
• Various embedding substances such as paraffin
wax, celloidin, synthetic resins, gellatine are
used depending the type of microscope, type of
microtome, type of tissue.

POINTS TO NOTE…..
• Epoxy resin is used for electron microscopy
• Agar embedding is used in double embedding
and FNAC specimens
• Celloidin media is used for cutting hard tissues
Gelatin is used when frozen sections are
required on friable tissues
• A variety of moulds are used for embedding.
• There may be L moulds or plastic moulds

CONT
• Various substances can be added to paraffin
wax in order to modify its consistency and
melting point to improve efficiency during
microtomy
• Additives increase the hardness of block
• Correct orientation of tissue in a mould is the
most important steps in embedding

THE END
Thank you

REFERENCES
• Bancroft, J.D. and Stevens, A.: theory and practice of
histological
• techniques ed.3, Churchill livingstone inc. 1990.
Edinburgh. London,
• Melbourne and New York.
• 2. Lillie, R.D.: Histopathologic technique and practice
histochemistry ed.
• 3, New York, 1965 McGraw Hill Book co.
• 3. Manual of histologic and special staining techniques
ed. 2, New York,
• 1960, The Blakiston Division McGraw Hill Book Co.

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