INFILTRATION AND EMBEDDING

• 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. Various embedding substances are paraffin wax,
celloidin, synthetic resins, gelatine, etc.
 PARAFFIN WAX
• Paraffin wax is a polycrystalline mixture of solid hydrocarbons produced during the refining of coal and mineral oils.
Wax hardness (viscosity) depends upon the molecular weight of the components and the ambient temperature.
• High molecular weight mixtures melt at higher temperatures than waxes comprised of lower molecular weight fractions.
• Paraffin wax is traditionally marketed by its melting points which range from 39°C to 68°C. Tissue-wax adhesion
depends upon crystal morphology of the embedding medium.
• Small, uniform sized crystals provide better physical support for specimens through close packing.
• Early histological wax formulations have largely been replaced by uniform, high quality proprietary blends of
histological paraffin waxes.
• Additives recently incorporated in proprietary waxes include plastic polymers such as polyethylene wax (which improves
adhesion, hardness and plasticity) and dimethyl sulphoxide (DMSO) which has the ability to reduce infiltration times and
facilitate thin sectioning.
• Carbowax: It is a water soluble wax. Therefore tissues are directly transferred to water soluble wax after fixation
and washing.
•  Methacrylate: It is easily miscible with alcohol and gives a clear and hard block when polymerised Polymerization
takes place in the presence of a catalyst. Any trace of water causes uneven polymerization and formation of bubbles
in the block around the tissue.
• 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.
•  Celloidin media: Celloidin is a purified form of nitrocellulose. It is used for cutting hard tissues.
•  Gelatin:Its melting point is less than the melting point of agar. Gelatin may be used when frozen sections are
required on friable and necrotic tissues.
• Epoxy Resin (Araldite): Epoxy polymers of araldite is used in higher resolution work and to
see greater details. 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.
• For electron microscopy araldite is obtained as casting resin CY212, a hardner DDSA and an
amine accelerator, DMP (ditrimethylamino methyl phenol) Blocks are suitably cured before
sectioning for 48 to 60 hours at 60oC.
•  
TYPES OF MOULDS

• A variety of moulds are used for embedding. These may be LEUCKHARD embedding
moulds (L mould) paper blocks, plastic moulds. Most of the laboratories use L moulds. L
moulds are made up of metal, easy to procure, reusable and may be adjusted to make
different size of blocks. One limb of the.”L” is longer than the other. The two “Ls” are
joint
• Plastic moulds: Most of the laboratories use plastic embedding rings now. These are
relatively inexpensive, convenient and support the block during sectioning and are designed to
fit it on the microtome. This eliminates the step of mounting or attaching the block on a holder
(metal or wooden holder).
• Tissue-Tek System1 or Mark1 system: In this system plastic embedding rings with stainless
steel moulds allow rapid embedding and cutting of tissues. In this system the blocks are stored
with the plastic rings; the angle does not change for further requirement of sections.
• The disadvantage of this method is that the space required for storing is more.
PLASTIC EMBEDDING RINGS
• Tissue-Tek system 2or Mark 2 system: The Mark 2 system has provided a cassette to hold tissue during
processing and has a stainless steel lid on the plastic cassette. The cassette has a rough surface on one
side of it with a slope where the accession number or the marking is done using a permanent marker.
• Advantages
o Since the cassette is processed with the tissues and afterwards used for embedding, the writing has to be done
once.
o Cassettes are thin so less wax is required.
The space required for filing the blocks is less.
• Disadvantages
▪ A special clamp has to be used in the microtome for this technique.
Thecassettesareshallowhencethinsectionsshouldbetakenforprocessing.
TISSUE EMBEDDING MACHINE:

• The embedding machine contains the following parts -

• 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.
• Leuckhart mould method-This is the traditional embedding method. The “L moulds are adjusted
according to the shape and size of the tissue. Glycerine may be applied to the L pieces and also to
the metal or glass plate on which the moulds are placed for embedding. 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.
• 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.
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.
• Cool the block on the cold plate.
• Remove the block from the mould.
• Cross check block, label and requisition form.
 FACTORS AFFECTING TISSUE PROCESSING

TEMPERATURE

Lower temperatures protect tissues from the destructive effects of processing reagents. At these temperatures, structural
elements of tissues are stabilized against the destructive effects of solvent changes. Unfortunately at low temperatures
reagent viscosities increase and diffusion rates decrease which result in prolonged processing times. Although mild heat can
reduce processing times considerably, it may increase shrinkage. Tissue shrinkage during infiltration in paraffin wax results
mainly from the effect of heat on collagen.

High infiltration temperatures cause marked tissue shrinkage and hardening which can be avoided by maintaining embedding
waxes 2-3°C above their melting points. Prolonged immersion in paraffin wax at the correct temperature results in only slight
tissue shrinkage though tissues such as blood may harden and become brittle. The extent to which tissues are affected during
paraffin wax infiltration depends upon the combination of fixative, dehydrant and solvent used as well as the tissue type.
Microwave processing involves internal heating with reduction in duration.
• PRESSURE AND VACUUM

High pressure facilitates infiltration of dense specimens with the more viscous embedding
media. Positive pressure for fluid transfer is probably too low to have a significant
influence on infiltration. Vacuum applied during dehydration, clearing and infiltration
improves the quality of processing in tissues such as lung which become de-aerated
during the process. However, duration of wax infiltration is dependent upon viscosity and
is not generally reduced by applying a vacuum.
• AGITATION

Agitation of tissues during processing ensures an adequate fluid exchange and in automatic
tissue processors, continual motion of tissue containers and flow of processing fluids is
maintained. Fluid interchange between processing reagents and tissues is promoted by exposure
of the maximum tissue surface area. Therefore, tissues should be loosely packed in baskets to
facilitate exchange of reagents and increase diffusion. Ideally, the cassette perforations should be
perpendicular to the fluid flow. If tissues are allowed to settle on the bottom of a container or are
too tightly packed, tissue surface area available for fluid exchange will be severely restricted.