WEEK 6- HISTOPATH LAB PARAFFIN WAX IMPREGNATION

❖ simplest, most common and best embedding

IMPREGNATION AND EMBEDDING
Impregnation
❖ Impregnation (Infiltration) is the process whereby
the clearing agent is completely removed from the
tissue and replaced by a medium that will
completely fill all the tissue cavities and give a firm
consistency to the specimen.
❖ allows easier handling and cutting of suitably thin
sections without any damage or distortion to the
tissue and its cellular components.
Embedding
❖ Embedding (Casting or Blocking) is the process by
which the impregnated tissue is placed into a
precisely arranged position in a mold containing a
medium which is then allowed to solidify.
An infiltrating and embedding medium should be:
medium used for routine tissue processing.
❖ Paraffin wax- polycrystalline mixture of solid
hydrocarbons produced during the refining of coal
and mineral oils.
❖ solid at room temperature but melts at
temperatures up to about 65°C or 70°C
❖ most common for histological use being about 56°C
to 58°C
❖ traditional advice with paraffin wax is to use this
about 2°C above its melting point.
❖ Wax hardness (viscosity) depends upon the
molecular weight of the components and the
ambient temperature.
➢ To decrease viscosity and improve infiltration of
the tissue, technologists often increase the
temperature to above 60°C or 65°C.
❖ Paraffin wax is traditionally marketed by its melting
points which range from 39°C to 68°C.

❖ soluble in processing fluids

❖ suitable for sectioning
and ribboning
❖ molten between 30°C
and 60°C
❖ translucent or
transparent; colorless
❖ stable
❖ homogeneous
❖ capable of flattening
after ribboning
❖ non-toxic
❖ odorless
❖ easy to handle
❖ inexpensive

Embedding Medium

❖ medium used to infiltrate the tissue is usually the

same medium utilized for impregnation ❖ Tissue is submerged in two or more changes of
Four types of impregnation and embedding medium melted paraffin wax --- either in a paraffin oven or
an incubator which has been regulated at 55 – 60°C.
1. Paraffin wax ❖ Used for routine work – 56°C
2. Celloidin (collodion) ❖ If laboratory temperature is from 20 – 24°C ---
3. Gelatin paraffin wax with a melting point of 54 – 58°C is
4. Plastic indicated.
❖ If the laboratory temperature is between 15-18°C, ➢ To avoid spillage, fluid and wax containers must
the melting point of wax to be used should be be filled to the appropriate level and correctly
between 50 and 54°C. located in the machine.
➢ Hard tissues require wax with a higher melting ➢ Wax bath thermostats should be set at least 3
point than soft tissues. degrees above the melting point of the wax,
❖ There are three ways by which paraffin wax and timing should be checked when loading the
impregnation and embedding of tissues may be machine, especially if the machine is equipped
performed: with a delay mechanism.
➢ 1. By manual processing ➢ If the processor is to be run overnight it should
➢ 2. By automatic processing be programmed to hold on the first ethanol
➢ 3. By vacuum embedding bath and not finish until the next morning so
the specimens do not sit in hot paraffin longer
Manual Processing
than the time indicated.
❖ At least four changes of wax are required at 15
➢ Fresh specimens- they may incubate in formalin
minutes intervals in order to insure complete
in the first stage on the machine.
removal of the clearing agent from the tissue.
➢ important to not keep the tissues in hot paraffin
❖ immersed in another fresh solution of melted
too long or else they become hard and brittle
paraffin for approximately 3 hours to ensure
complete embedding or casting of tissue. Vacuum Embedding
❖ involves wax impregnation under negative
Automatic Processing
atmospheric pressure inside an embedding oven
❖ makes use of an automatic tissue processing
❖ It reduces the time when tissues are subjected to
machine (i.e., Autotechnicon) which fixes,
high temperatures thus minimizing heat-induced
dehydrates, clears and infiltrates tissues, thereby
tissue hardening
decreasing the time and labor needed during the
❖ Vacuum hastens the removal of air bubbles and
processing of tissues.
clearing agent from the tissue block, thereby
❖ only 2- 3 changes of wax are required to remove the
promoting a more rapid wax penetration of the
clearing agent and properly impregnate the
tissue.
specimen
❖ Recommended for urgent biopsies and for delicate
❖ made possible due to constant tissue agitation
tissues:
which accelerates and improves tissue penetration
➢ Lungs
giving rise to more consistent results.
➢ Brain
❖ Precautions with Automatic Processing
➢ Connective tissue
➢ The presence of any odor in the clearing agent
➢ Eyes
during final paraffin wax bath indicates that the
➢ Decalcified bones
paraffin wax needs to be changed.
➢ Spleen
➢ Dehydrating fluids should be changed
➢ Central nervous system
frequently since dehydration is the most critical
❖ the time required for complete impregnation is
stage of tissue processing and inadequate
reduced by 25% -75% of the normal time required
dehydration is difficult to correct once the
for tissue processing
tissue is in paraffin.
❖ vacuum embedding oven- consists of a flat-
▪ the first 100% ethanol bath should be
bottomed heavy brass chamber covered with a
discarded, and the others moved down, so
heavy glass lid resting on a wide and thick rubber
that the final bath has fresh 100% ethanol
valve which produces an airtight seal when the
after two complete processing runs of loads
chamber is being used.
of at least three-quarters capacity.
➢ vacuum chamber is enclosed in a
➢ The clearing agent and the dilute ethanols
thermostatically controlled water-jacket, usually
should be changed at least once a week.
 maintained at a temperature of 2-4°C above the
melting point of the wax.
➢ The degree of the vacuum should not exceed
500 mm. Hg
➢ Stopcock- prevent water from being sucked
back into the trap bottle and vacuum chamber
when the water or suction pump is closed.
Factors Affecting Paraffin Wax Impregnation
❖ Vacuum impregnation gives the fastest result.
❖ Total impregnation time generally depends upon
the nature and size of the tissues to be processed
and the type of clearing agents to be used.
❖ Larger and denser tissue blocks usually require
longer periods and more frequent changes of wax.
❖ Easily removed from the tissues --- BENZENE and
XYLENE
❖ More difficult to remove --- CHOLOROFORM and
CEDARWOOD OIL
➢ Addition of benzene may hasten displacement
of cedarwood oil with less tissue shrinkage
Practical Considerations
❖ The tissue should not be left in the paraffin oven for
more than 4 hours
❖ Infiltration in overheated paraffin (above 60°C) will
also produce shrinkage and hardening of tissues and
destroy lymphoid tissues completely.
➢ To avoid this, paraffin oven must be maintained
at a temperature 2 to 5°C above the melting point
of paraffin to be used for impregnation.
❖ Fresh wax should be filtered before use in a wax oven
at a temperature 2°C higher than its melting point.
❖ Wax that has been trimmed away from the
impregnated tissue may be melted and filtered for
future use, with a coarse filter paper, e.g. Green's No.
Substitutes
904. for Paraffin Wax
❖❖ Paraplast
Water must therefore be removed by heating the wax
to 100 -105°C, thereby
➢ a mixture of highlyraising its melting
purified paraffinpoint
and
❖ Paraffin wax may be used only twice, after which,
synthetic plastic polymers, with a melting point
fresh wax must be utilized.
of 56-57°C.
➢ more elastic and resilient--thereby permitting
large dense tissue blocks such as bones and
brain to be cut easily with the same result as in
double embedding.
➢ No deposit is left on the slide after staining, and
no special processing schedule is required.
➢ Paraplast with a melting point of 56 to 58 oC is
recommended.
❖ Embeddol
➢ synthetic wax substitute similar to Paraplast
with a melting point of 56-58°C.
➢ less brittle and less compressible than
Paraplast.
➢ Bio/aid-semisynthetic wax recommended for
embedding eyes.
➢ Tissue Mat is a product of paraffin, containing
rubber, with the same property as Paraplast.
❖ Ester Wax
➢ a lower melting point (46-48°C), but it is harder
than paraffin
➢ not soluble in water, but is soluble in 95% Ethyl
Alcohol and other clearing agents; hence, it can
be used for impregnation without prior clearing
of the tissue.
➢ Clearing agents: Cellosolve (ethylene glycol
monoethyl ether) or xylene
➢ Tissue must be placed in a solution containing
equal proportion of clearing agent and ester
wax for 3 – 6 hours before finally transferring it
to pure wax.
➢ Three to four changes of wax are required to
ensure complete tissue impregnation
➢ Sectioning of ester wax-impregnated tissues
should be done on a heavy duty microtome
❖ Water Soluble Waxes
➢ plastic polymers, mostly polyethylene glycols
with melting points of 38-42°C or 45-56°C.
➢ Carbowax- most commonly used, a
polyethylene glycol containing 18 or more
carbon atoms, which appears solid at room
temperature.
▪ soluble in and miscible with water; hence
does not require dehydration and clearing
of the tissue
▪ Processing time is reduced with the special
advantage that harmful effects produced by
ordinary dehydrating agents are
consequently avoided.
▪ It does not remove neutral fats and lipids
which are soluble in reagents used for
routine processing with paraffin
▪ suitable for many enzyme histochemical
studies
▪ Routine processing- four changes of
Carbowax, one each in 70% and 90% and 2
times in I 00% concentration, at a
Of the three methods of paraffin wax impregnation,
vacuum impregnation gives the fastest result
 temperature of 56°C are used, at 30
minutes, 45 minutes and 1 hour (with
agitation), respectively.
▪ Tissues are very difficult to float out and
mount --- add soap to water or use 10%
polyethylene glycol 900 in water
❖ Dimethyl sulphoxide (DMSO)
➢ reduces infiltration times and facilitates thin
sectioning
➢ Some individuals who handle DMSO-paraffin
wax may experience an unpleasant and
annoying oyster or garlic taste probably caused
by DMSO metabolites.
CELLOIDIN IMPREGNATION
❖ 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.
❖ Supplied in thin (2%), medium (4%) or thick (8%)
solutions of cellulose dissolved in equal parts of
ether and alcohol.
❖ used mainly for preparing soft tissue sections of
mixed consistency such as eyes and brain.
❖ No heat is required, and the resultant block has a
rubbery consistency which gives good support to
the tissues
❖ Very volatile and inflammable
❖ Disadvantages
➢ inability to cut thin sections,
➢ storage of blocks in alcohol
➢ speed of technique (which can take several
weeks or months).
Two methods are used for celloidin impregnation of
tissue:
❖ Wet Celloidin Method
❖ Dry Celloidin Method
Wet Celloidin Method
❖ recommended for bones, teeth, large brain sections
and whole organs.
❖ After the usual fixation and dehydration of the
tissue, it is placed in equal parts of ether and
alcohol for 12-24 hours.
➢ then placed in thin celloidin (2-4%) for 5-7 days,
transferred to medium celloidin (4-6%) for
another 5-7 days, drained off and poured with
thick celloidin (8-12%) until the specimen has
become impregnated, usually between 3-5
days.
➢ embedding medium containing freshly poured
thick celloidin and kept in a tightly covered jar
or dessicator in order to evaporate the alcohol-
ether solvent
➢ When the ball of the finger leaves no mark on
the surface of the tissue block, evaporation and
consequently, embedding, is considered to be
complete.
❖ The tissue block is then stored in 70-80% alcohol
until ready for cutting. This is done to avoid
dehydration and shrinkage of tissues.
Dry Celloidin Method
❖ preferred for processing of whole eye sections
❖ principle and procedure of this method is similar to
wet celloidin method, except that 70% alcohol is not
used for storage before cutting.
➢ Gilson's mixture, made up of equal parts of
chloroform and cedarwood oil, is added to the
celloidin block before hardening, to make the
tissue transparent
Low Viscosity Nitrocellulose (L.V.N.)
❖ another form of celloidin soluble in equal
concentration of ether and alcohol, with a lower
viscosity, allowing it to be used in higher
concentrations and still penetrate tissues rapidly
❖ forms a harder tissue block and makes cutting of
thinner sections possible
➢ The tendency of tissues to crack may be
prevented by adding plasticizers
❖ more explosive than celloidin and should therefore
be handled with care
➢ dry, striking or dropping the container will
cause the substance to explode.
GELATIN IMPREGNATION
❖ rarely used except when dehydration is to be
avoided and when tissues are to be subjected to
histochemical and enzyme studies
❖ 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.
❖ water-soluble, and does not require dehydration
and clearing
➢ fixatives (such as 10% formalin) should still be
washed out by running water whenever
indicated
❖ has a low melting point and does not cause over-
hardening of tissues by heating
❖ Process:
➢ After the fixative has been completely washed
out, the tissue is placed in 10% gelatin with 1%
phenol for 24 hours, transferred to 20% gelatin
with 1% phenol for the next 12 hours, and
finally to another fresh solution of 20% gelatin
with 1% phenol which is then allowed to cool in
a refrigerator until impregnation and
embedding are completed. Gelatin-embedded
tissues are then transferred to l 0% formalin for
12-24 hours in order to harden the tissue.
EMBEDDING
❖ the tissue is placed into a mold containing the
embedding medium and this medium is allowed to
solidify.
❖ embedding medium should match the tissue type in
strength and hardness
➢ too soft- the tissue will not be supported and
sections will be torn or shredded.
➢ too hard-sections will be brittle and will shatter
❖ To infiltrate the tissues with supporting embedding
medium, tissues must be free of all water
❖ ORIENTATION – process by which a tissue is
arranged in precise positions in the mold during
embedding, on the microtome before cutting, and
on the slide before staining.
Several types of Blocking-out Molds may be used:
❖ recommended for routine use, although, too slow
and cumbersome for use in a busy laboratory.
Compound Embedding Unit
❖ made up of a series of interlocking plates resting on
a flat metal base, forming several compartments
❖ advantage: embedding more specimens at a time,
thereby reducing the time needed for blocking
Plastic Embedding Rings and Base Mold
❖ consist of a special stainless steel base mold fitted
with a plastic embedding ring, which later serves as
the block holder during cutting.
❖ Tissue Tek- is equipped with a warm plate to
manage the impregnated specimen, and a cold
plate at -5°C for rapid solidification of the block.
➢ consists of a white plastic cassette mold with
detachable, perforated stainless steel hinge and
Snap-On lid, used to hold the tissue specimen
through-out fixation, dehydration, clearing and
wax impregnation.
Disposable Embedding Molds
❖ Peel-Away- disposable thin plastic embedding
molds, available in 3 different sizes, are simply
peeled off one at a time, as soon as the wax has
solidified, giving perfect even block without
trimming. It may be placed directly in the chuck or
block holder of the microtome.
❖ Plastic Ice Trays - may be recommended for busy
routine laboratories. Each compartment may be
utilized for embedding one tissue block, which may
then be removed by bending the plastic tray once
the wax has solidified or by smearing the inner mold
with glycerin or liquid paraffin before embedding.

Leuckhart’s Embedding Mold

❖ consists of two L-shaped strips of heavy brass or

metal arranged on a flat metal plate and which can
be moved to adjust the size of the mold to the size
of the specimen.
❖ Blocks produced are even, with parallel sides, and
with a fairly shaped initial setting of the wax.
❖ Paper Boats- are normally utilized for embedding ❖ Acrylic plastics- ade up of esters of acrylic or
celloidin blocks but are equally useful for paraffin methacrylic acid, and are used extensively for light
wax blocks. microscopy
➢ Advantage: cheap and easy to make; provide ➢ Low viscosity
easy and accurate identification of specimen,
thereby avoiding confusion and interchange of
tissue blocks. Rapid embedding of small or large
volume of individual specimen is possible, since
paper molds can be made to suit any size of
tissue.

Celloidin or Nitrocellulose Embedding Method

❖ recommended for embedding hard tissues such as

bones and teeth, and for large sections of whole
organs like the eye,
❖ Tissues are embedded in shallow tins of enamel
pans which are covered by sheets of weighted glass
❖ Not anymore used—special requirements needed
for processing

Double-Embedding

❖ process by which tissues are first embedded or fully

infiltrated with a supporting medium such as agar or
nitrocellulose, then infiltrated a second time with
paraffin wax in which they are subsequently
embedded
❖ used to facilitate cutting of large blocks of dense
firm tissues like the brain.
❖ Availability of paraffin waxes containing different
types of resins has made this technique obsolete.

PLASTIC (RESIN) EMBEDDING

❖ has provided superior results for light microscopic

studies, particularly in hard tissues such as
undecalcified bone and for high resolution light
microscopy of tissue sections thinner than the usual
4-6 µm
❖ Plastics are classified into epoxy, polyester, or
acrylic, based on their chemical composition.
❖ Epoxy- are made up of a carefully balanced mixture
of epoxy plastic, catalysts and accelerators.
➢ Three types of epoxy plastics are used in
microscopy
▪ bisphenol A (Araldite), or glycerol (Epon), or
cyclohexene dioxide (Spurr)
❖ Polyester plastics- originally introduced for electron
microscopy