CHAPTER 11

IMPREGNATION AND EMBEDDING

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.
This allows easier handling and cutting of suitably thin sections without any
damage or distortion to the tissue and its cellular components.
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. Ideally, an infiltrating and
embedding medium should be:
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
The medium used to infiltrate the tissue is usually the same medium utilized for
impregnation, and for general purposes is known as an Embedding Medium.
There are generally four types of impregnation and embedding medium,
namely: 1. Paraffin wax
2. Celloidin (collodion)
3. Gelatin
4. Plastic

PARAFFIN WAX IMPREGNATION

Paraffin is the simplest, most common and best embedding medium used
for routine tissue processing. Paraffin wax is a polycrystalline mixture of solid
hydrocarbons produced during the refining of coal and mineral oils. It is solid
at room temperature but melts at temperatures up to about 65°C or 70°C.
Paraffin wax can be purchased with melting points at different temperatures,

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the most common for histological use being about 56°C to 58°C. At its melting
point, it tends to be slightly viscous, but this decreases as the temperature is
increased. The 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.
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 the crystal morphology of the
embedding medium. Small, uniform sized crystals provide better physical
support for specimens through close packing. Crystalline morphology of
paraffin wax can be altered by incorporating additives which result in a less
brittle, more homogeneous wax with good cutting characteristics. There is
consequently less deformation during thin sectioning. Setting temperature does
not appreciably affect crystal size.
Advantages:
1. Thin individual serial sections may be cut with ease from the
majority of tissues without distortion.
2. The process is very rapid, allowing sections to be prepared within
24 hours.
3. Tissue blocks and unstained mounted sections may be stored in
paraffin for an indefinite period of time after impregnation without
considerable tissue destruction.
4. Because formalin-fixed, paraffin-embedded tissues may be stored
indefinitely at room temperature, and nucleic acids (both DNA and
RNA) may be recovered from them decades after fixation, they are an
important resource for historical studies in medicine.
5. Many staining procedures are permitted with good results.
Disadvantages:
1. Overheated paraffin makes the specimen brittle.
2. Prolonged impregnation will cause excessive tissue shrinkage and
hardening, making the cutting of sections difficult.
3. Inadequate impregnation will promote retention of the clearing
agent. Tissues become soft and shrunken, and tissue blocks crumble
when sectioned and break up when floated out in a water bath.

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 4. Tissues that are difficult to infiltrate, e.g. bones, teeth, brains and
eyes, need long immersion for proper support; otherwise, they will
crumble on sectioning. Prolonged immersion in paraffin, on the other
hand, is not advisable.
5. Paraffin processing is not recommended for fatty tissues. The
dehydrating and clearing agents used in the process dissolve and remove
fat from the tissues.

After being completely cleared, the tissue is submerged in two or more

changes of melted paraffin wax, either in a paraffin oven or in an incubator
which has been regulated at 55-60°C. The duration and number of changes
required for thorough impregnation of tissue depends on: –
Size and type of tissues: Longer time is required for thicker
tissues.
Use of vacuum imbedding: Vacuum reduces the time required
for complete impregnation.
Clearing agent employed
Common waxes have melting points of 45°C, 52°C, 56°C and 58°C. The
56°C wax is normally used for routine work. In a laboratory with temperature
ranging from 20-24°C, paraffin wax with a melting point of 54-58°C is
indicated. If the laboratory temperature is between 15-18°C, the melting point
of wax to be used should be between 50 and 54°C. Hard tissues require wax
with a higher melting point than soft tissues.
There are three ways by which paraffin wax impregnation and embedding
of tissues may be performed:
1. By manual processing
2. By automatic processing
3. By vacuum embedding

1. Manual Processing
At least four changes of wax are required at 15 minutes intervals in order
to insure complete removal of the clearing agent from the tissue. The specimen
is then immersed in another fresh solution of melted paraffin for approximately
3 hours to insure complete embedding or casting of tissue. The following is an
example of a time schedule for manual processing of tissues about 3 mm. thick:
Fixation:
10% Buffered Formalin 24 hours
Dehydration:

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 70% Alcohol 6 hours
95% Alcohol 12 hours
100%Alcohol 2 hours
100% Alcohol 1 hour
100%Alcohol 1 hour
Clearing:
Xylene or Toluene 1 hour Xylene or Toluene 1 hour
Impregnation:
Paraffin wax 15 minutes
Paraffin wax 15 minutes
Paraffin wax 15 minutes
Paraffin wax 15 minutes
Embedding:
Paraffin wax 3 hours

2. Automatic Processing
This method makes use of an automatic tissue processing machine (i.e.,
Autotechnicon) which fixes, dehydrates, clears and infiltrates tissues, thereby
decreasing the time and labor needed during the processing of tissues. This
results in a more rapid diagnosis with less technicality. Usually, only 2- 3
changes of wax are required to remove the clearing agent and properly
impregnate the specimen. This is made possible due to constant tissue agitation
which accelerates and improves tissue penetration giving rise to more
consistent results. One example of an automatic tissue processing machine is
the Elliott Bench-Type Processor. The machine is mounted on rollers to permit
the turning of platforms and easy access to beakers and wax baths. It makes use
of 12 individual processing steps, with ten 1-liter capacity glass beakers and
two thermostatically controlled wax baths with a safety device cut-out switch to
protect the wax against over-heating. A transfer arm controlled by electrical
current moves the tissues from one processing reagent to another (by clock
schedules). It can be removed by raising a spring-loaded plunger in the center
of the cover plate, thereby allowing the tissue to be arranged manually anytime
during the processing. Agitation of fluid is accompanied by a continuous
vertical movement or rotation of the specimen carrier by a mechanism
connected to the transfer arm. An electrical clock connected to a metal disc
notched in positions of 15 minutes or more, serves to control the time needed
for each processing step. The clock rotates and sets the transfer arm and
mechanism into motion, moving the tissue to the next position. A delay
mechanism is provided in instances where processing time may exceed 24

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hours.

REAGENT PROCESSING TIME

Schedule 1 Schedule 2 Schedule 3

10% Buffered 2 hrs. 2 hrs. 2 hrs.
Formalin

10% Buffered 2 hrs . 2 hrs. 2 hrs.

Formalin
70% Alcohol 2 hrs . 2 hrs. 2 hrs.
Absolute Alcohol (1) 1 hr. 3 hrs. 2 hrs.
Absolute Alcohol (2) 1 hr. 3 hrs. 2 hrs.
Absolute Alcohol (3) 1 hr. 3 hrs. 2 hrs.
Xylene or Toluene (1) 1 hr.
Xylene or Toluene (2) 1 hr.
Benzene (1) 30 mins.
Benzene (2) 1 hr.
Chloroform ( I ) 2 hrs.
Chloroform (2) 2 hrs.
Chloroform (3) 2 hrs.
Wax (1) 2 hrs. 3 hrs. 2 hrs.
Wax (2) 3 hrs. 3 hrs. 2 hrs.
TABLE 11-1. Sample Time Table for Automatic Processing of tissues 3-5 mm.
thick
Precautions with Automatic Processing
The frequency with which fluids are changed depends on the number and
sizes of the tissues processed. The presence of any odor in the clearing agent
during final paraffin wax bath indicates that the paraffin wax needs to be
changed. Dehydrating fluids should be changed frequently since dehydration is
the most critical stage of tissue processing and inadequate dehydration is
difficult to correct once the tissue is in paraffin. The first 100% ethanol bath
should be discarded, and the others moved down, so that the final bath has
fresh 100% ethanol after two complete processing runs of loads of at least
three-quarters capacity. The clearing agent and the dilute ethanols should be
changed at least once a week.
To avoid spillage, fluid and wax containers must be filled to the
appropriate level and correctly located in the machine. Wax accumulating on

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any surface or beaker leads must be removed and any spillage should be wiped
away. Wax bath thermostats should be set at least 3 degrees above the melting
point of the wax, and timing should be checked when loading the machine,
especially if the machine is equipped with a delay mechanism.
Due to the viscosity of molten paraffin wax, some form of gentle agitation
is highly desirable. If the processor is to be run overnight it should be
programmed to hold on the first ethanol bath and not finish until the next
morning so the specimens do not sit in hot paraffin longer than the time
indicated. If specimens are fresh they may incubate in formalin in the first stage
on the machine. It is important to not keep the tissues in hot paraffin too long or
else they become hard and brittle. Processed tissues can be stored in the
cassettes at room temperature indefinitely.

3. Vacuum Embedding
Vacuum embedding involves wax impregnation under negative atmospheric
pressure inside an embedding oven. It reduces the time

Fig 11 -1. Tissue processor with vacuum and fume control

when tissues are subjected to high temperatures thus minimizing heat-induced

tissue hardening. It facilitates complete removal of transition solvents, and
prolongs the life of wax by reducing solvent contamination. Vacuum hastens the
removal of air bubbles and clearing agent from the tissue block, thereby
promoting a more rapid wax penetration of the tissue. This technique is
particularly recommended for urgent biopsies, for delicate tissues such as lung,
brain, connective tissues, decalcified bones, eyes, spleen and central nervous
system.
Vacuum infiltration requires a vacuum infiltrator or embedding oven,
consisting of wax baths, fluid trap and vacuum gauge, to which a vacuum of up
to 760 mm Hg is applied using a water or mechanical pump. Modern tissue

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processors are equipped to deliver vacuum, or vacuum and pressure, to all or
some reagent stations during the processing cycle.

Fig. 11-2. Vacuum embedding oven

With vacuum embedding, the time required for complete impregnation is

reduced by 25% -75% of the normal time required for tissue processing. The
tissue is not over-exposed to heat; brittleness, shrinkage and hardening of
tissues consequent to overheating is therefore prevented. The tissue can also be
transferred after clearing to a heated bath of paraffin wax from which air can be
evacuated.
The vacuum embedding oven consists of a flat-bottomed heavy brass
chamber covered with a heavy glass lid resting on a wide and thick rubber valve
which produces an airtight seal when the chamber is being used. The vacuum
chamber is enclosed in a thermostatically controlled water-jacket, usually
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. A stopcock is provided to
prevent water from being sucked back into the trap bottle and vacuum chamber
when the water or suction pump is closed.

Embedding Procedure: After fixation and dehydration, proceed as follows:

1. Clear in two changes of xylene, for 1 hour each.
2. Place the tissue in molten wax, in vacuum chamber and make the oven
airtight. Exhaust the air slowly by means of a vacuum pump or
Venture suction pump until there is a negative pressure of 400 to 500
mm. mercury.
3. Leave for 15 minutes, then slowly readmit air until normal atmospheric
pressure is reached.

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 4. Place the tissue in fresh wax.
5. Repeat steps 3 and 4.
6. Place the tissue into fresh wax.
7. Repeat step 3 and leave for 30 to 45 minutes.
8. Bring to normal atmospheric pressure and embed the tissue.
NOTE: The exhaustion and readmission of air must be gradual or
the specimen may be ruined.

Of the three methods of paraffin wax impregnation, vacuum impregnation

gives the fastest result. Total impregnation time, however, generally depends
upon the nature and size of the tissues to be processed, and the type of cleari ng
agents to be used. Larger and denser tissue blocks (e.g. bones, fibroids, brains)
usually require longer periods and more frequent changes of wax. Benzene and
xylene are easily removed from the tissues while chloroform and cedarwood oil
are more difficult to remove and require more frequent wax changes. Addition
of benzene may hasten displacement of cedarwood oil with less tissue
shrinkage.

Practical Considerations
Since prolonged treatment in melted paraffin causes shrinkage and
hardening of tissues, making cutting difficult, the tissue should not be left in the
paraffin oven for more than 4 hours. The shorter the time in the hot oven with
adequate paraffin impregnation and evaporation of clearing agent, the better it
is for the tissue. Tissues become increasingly harder and more brittle as they are
heated. Infiltration in overheated paraffin (above 60°C) will also produce
shrinkage and hardening of tissues and destroy lymphoid tissues completely. To
avoid this, the paraffin oven must be maintained at a temperature 2 to 5°C
above the melting point of paraffin to be used for impregnation.
Paraffin wax must be pure, i.e. free from dust, water droplets and other
foreign matter. 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. 904. When wax has been reused, some
amount of water inevitably is mixed with it. If excessive, this may impair the
impregnating capacity of the medium and prevent formation of a good tissue
block. Water must therefore be removed by heating the wax to 100 -105°C,
thereby raising its melting point. Paraffin wax may be used only twice, after
which, fresh wax must be utilized.

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 When using an automatic tissue processing machine, wax usually becomes
admixed with the clearing agent, especially in the first beaker; hence, water
must be discarded.
For fixed knife microtomes, a relatively hard wax with a higher melting
point is recommended. Heavier microtome knives require harder paraffin wax
than lighter ones.

SUBSTITUTES FOR PARAFFIN WAX

Paraplast is a mixture of highly purified paraffin and synthetic plastic
polymers, with a melting point of 56-57°C. It is more elastic and resilient than
paraffin wax thereby permitting large dense tissue blocks such as bones and
brain to be cut easily with the same result as in double embedding. Blocks
obtained are more uniform than with any other medium, with better ribboning of
sections. Serial sections may be cut with ease, without cooling the tissue block,
thereby preventing the formation of ice crystal artefacts. No deposit is left on the
slide after staining, and no special processing schedule is required. It is soluble
in common clearing agents and follows the same time schedule for paraffin
impregnation, and does not tend to crack like other paraffin wax substitutes.
Generally, Paraplast with a melting point of 56 to 58oC is recommended. During
the winter, 54 to 56oC Paraplast may be used if the tissue is cut in a cool room.
During the summer it may be necessary to use 60 to 63oC, although this is to be
avoided if possible in order to not to "cook" the tissue. "Cooked" tissue does not
section well or, if it does, it does not stain well and most details are destroyed.
Embeddol is synthetic wax substitute similar to Paraplast with a melting
point of 56-58°C. It is less brittle and less compressible than Paraplast. Bio/aid
is a semisynthetic wax recommended for embedding eyes. Tissue Mat is a
product of paraffin, containing rubber, with the same property as Paraplast.
Ester Wax has a lower melting point (46-48°C), but it is harder than
paraffin. It is 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. Cellosolve (ethylene glycol monoethyl ether) or xylene may be used
as clearing agents, if indicated. In such instances, removal of the clearing agent
must be gradual; that is, the 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 (e.g. sliding or sledge type
microtome) due to the relative hardness of the wax.
Water Soluble Waxes are plastic polymers, mostly polyethylene glycols

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with melting points of 38-42°C or 45-56°C. Polymer waxes are incorporated in
the majority of proprietary histological paraffin wax blends presently available
to improve adhesion, hardness and plasticity. The most commonly used is
Carbowax, a polyethylene glycol containing 18 or more carbon atoms, which
appears solid at room temperature. It is soluble in and miscible with water; hence
does not require dehydration and clearing of the tissue. The tissues are fixed,
washed out and transferred directly into the melted Carbowax. 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,
hence, allowing these substances to be demonstrated in thin sections. Tissues are
not exposed to too much heat so that excessive hardening, shrinkage and
brittleness of tissue is avoided; hence, making Carbowax technique suitable for
many enzyme histochemical studies. Cytologic details are excellently preserved.
For routine processing, four changes of Carbowax, one each in 70% and
90% and 2 times in I 00% concentration, at a temperature of 56°C are used, at
30 minutes, 45 minutes and 1 hour (with agitation), respectively. Specimens are
then embedded in fresh Carbowax at 50°C and rapidly cooled in a refrigerator.
Due to its hygroscopic nature, Carbowax is very easily dissolved in water.
Hence care must be taken to avoid contact of the block with water or ice.
Tissue sections are very difficult to float out and mount due to its extreme
solubility in water, dehydrating and clearing agents. Adding soap to water or
using 10% Polyethylene Glycol 900 in water will reduce tissue distortion and
promote flattening and "floating out" of sections.
Dimethyl sulphoxide (DMSO) added to proprietary blends of plastic
polymer paraffin waxes reduces infiltration times and facilitates thin sectioning.
DMSO scavenges residual transition solvent and probably alters tissue
permeability by substituting for or removing bound water thus improving
infiltration. Some individuals who handle DMSO-paraffin wax may experience
an unpleasant and annoying oyster or garlic taste probably caused by DMSO
metabolites. Possible health risks associated with the use of DMSO-paraffin
wax are minimal if correct laboratory hygiene is observed.

CELLOIDIN IMPREGNATION
Celloidin (Collodion) 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. It is supplied in thin (2%), medium (4%) or thick
(8%) solutions of cellulose dissolved in equal parts of ether and alcohol. This is

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 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. Disadvantages include
inability to cut thin sections, storage of blocks in alcohol and speed of
technique (which can take several weeks or months).
Advantages:
1. It permits cutting of tissue sections which are thicker than in paraffin
wax, and is recommended for processing of neurological tissues.
2. Its rubbery consistency allows tissue blocks that are either very hard or of
varying consistency, to be cut without undue distortion.
3. Dense tissues which are hard to infiltrate (e.g. bones and brain) and
specimens which tend to collapse easily due to air spaces (e.g. eyes) are
supported better, thereby avoiding the crumbling of tissues during
sectioning. When eye sections are embedded by the paraffin method, the
retina may be detached from the harder tissues (e.g. sclera and choroid)
that encircle it. The cedarwood oil used in the dry celloidin technique
helps to soften the brittle layers.
4. I t does not require heat during processing; hence, producing minimum
shrinkage and tissue distortion especially for cutting large bone sections.
It is, therefore, recommended in cases when minimum shrinkage is
required and wh en frozen section technique cannot be done.
Disadvantages:
1. Celloidin impregnation is very slow (lasting for several days or weeks).
2. Very thin sections (less than I 0 µ) are difficult to cut.
3. Serial sections are difficult to prepare.
4. Vapor of the ether solvent is very flammable; hence, it should never
be used near an open flame.
5. Photomicrographs are difficult to obtain.
6. It is very volatile and therefore must be kept in bottles with ground​-
glass stoppers to prevent evaporation.

Two methods are used for celloidin impregnation of tissue:

Wet Celloidin Method - is recommended for bones, teeth, large brain

sections and whole organs. After the usual fixation and dehydration of the

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tissue, it is placed in equal parts of ether and alcohol for 12-24 hours. The
tissue is 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. The specimen is removed from the celloidin, transferred to
an embedding medium containing freshly poured thick celloidin and kept in a
tightly covered jar or dessicator in order to evaporate the alcohol-ether solvent.
The dessicator top is removed for a few seconds, time and again, to admit fresh
air and harden the tissue block. Evaporation must be gradual to achieve a
consistent, uniform degree of hardness throughout the block and prevent the
formation of air bubbles.
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 is preferred for processing of whole eye sections.
The principle and procedure of this method is similar to wet celloidin method,
except that 70% alcohol is not used for storage before cutting. Instead, 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. The dry
method does not make use of alcohol due to the presence of cedarwood oil in
the block.

Nitrocellulose
Low Viscosity Nitrocellulose (L.V.N.) is 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. Because of
this, many workers prefer L.V.N. to the ordinary celloidin for impregnation and
embedding. It forms a harder tissue block and makes cutting of thinner sections
possible. The tendency of tissues to crack may be prevented by adding
plasticizers (e.g. oleum ricini or castor oil) when embedding chrome-mordanted
tissues.
Low viscosity nitrocellulose is more explosive than celloidin and should
therefore be handled with care. When dry, striking or dropping the container
will cause the substance to explode. It is usually marketed while wet with
alcohol. The container must be kept tightly covered and protected from sunlight
to avoid evaporation of alcohol. When no longer needed for future use, the
nitrocellulose should be carefully destroyed, since the material becomes

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increasingly dangerous as the alcohol continues to evaporate .

GELATIN IMPREGNATION
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. It is water-soluble, and does not require
dehydration and clearing, although fixatives (such as 10% formalin) should still
be washed out by running water whenever indicated. It has a low melting point
and does not cause over-hardening of tissues by heating.
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.
Tissues should not be more than 2-3 mm. thick since gelatin-embedded
specimens are harder to freeze than non-impregnated tissues. The 1 % phenol
serves to prevent the growth of molds. Excess gelatin may be removed by
floating the sections oh to paper and trimming them with scissors. The volume
of the impregnating medium should be at least 25 times the volume of the
tissue.

EMBEDDING
After impregnation, the tissue is placed into a mold containing the
embedding medium and this medium is allowed to solidify. Ideally the
embedding medium should match the tissue type in strength and hardness. If
the embedding medium is too soft for the material, the tissue will not be
supported and sections will be torn or shredded. If the medium is too hard for
the tissue, sections will be brittle and will shatter. To infiltrate the tissues with
supporting embedding medium, tissues must be free of all water (since usually
embedding medium is not miscible with water). Paraffin embedded tissues are
arranged at the bottom of the mold together with their proper labels and
immersed in melted paraffin at a temperature between 5-10°C above its melting
point and then cooled rapidly in a refrigerator at -5°C or immersed in cold
water to solidify. This will allow hardening of tissues, giving them a firmer
consistency and better support, thereby facilitating the cutting of sections.
The process by which a tissue is arranged in precise positions in the mold

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during embedding, on the microtome before cutting, and on the slide before
staining, is known as Orientation. Generally speaking, the surface of the
section to be cut should be placed parallel to the bottom of the mold in which it
is oriented. Several types of Blocking-out Molds may be used: 1. 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. The mold is adjustable to
give a wide variety of sizes to fit the size of the tissue block for casting. It is
recommended for routine use, although, too slow and cumbersome for use in a
busy laboratory.
2. Compound Embedding Unit is made up of a series of interlocking
plates resting on a flat metal base, forming several compartments. It has
the advantage of embedding more specimens at a time, thereby reducing
the time needed for blocking.
3. 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.
One model, the so-called 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. It 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.
With the Tissue Tek system, the specimen is placed on the base
mold, the plastic embedding ring is placed in position, filled up with
wax, and then placed on a small cool area to allow the wax in the base of
the mold to semi-harden. This will allow easy orientation of the block.
Once the tissue has been properly oriented, the base of the cassette is
placed on top and together, they are placed on the cold plate so that the
paraffin wax can cool and harden quickly. After the paraffin wax has
solidified (usually 5 minutes), the block is taken out together with the
embedding ring and is immediately ready for cutting without having to
undergo trimming or mounting, thereby saving time and effort.
The advantages of Tissue Tek include ease of use, less paraffin
wax needed, faster embedding, firmly attached tissue and holder, and
permanent identification. It produces easier orientation when re-
sectioning of tissue is required, and blocks can be filed immediately after

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 sectioning.
4. Disposable Embedding Molds a. 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.
b. Plastic Ice Trays -such as those used in ordinary refrigerators
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.
c. Paper Boats are normally utilized for embedding celloidin
blocks but are equally useful for paraffin wax blocks. They have
the advantage of being cheap and easy to make. They provide 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.
To mark the position of small tissues in the paraffin block, a mark such as
an "X" is drawn with soft lead pencil on the inner surface of the bottom of the
boat. This will attach and be visible on the wax block when solidified and
removed from the paper boat. Embedding molds should bear the case number,
and other identification data of the tissue block within. Once tissues have been
embedded, they may be stored in a cool place indefinitely until they are cut.
The choice of embedding mold will depend on the type of chuck in the
microtome you will use to section the tissue. Stainless steel, ceramic, paper,
plastic, and aluminum foil molds can be used. The basic method is the same for
each.
1. Open cassette to view tissue sample and choose a mold that best
corresponds to the size of the tissue. A margin of at least 2 mm of paraffin
surrounding all sides of the tissue gives best cutting support. Discard
cassette lid.
2. Put small amount of molten paraffin in mold, dispensing from paraffin
reservoir.
3. Using warm forceps, transfer tissue into mold, placing cut side down,
as it was placed in the cassette.
4. When the tissue is in the desired orientation, add the labeled tissue

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 cassette on top of the mold as a backing. Press firmly.
5. Hot paraffin is added to the mold from the paraffin dispenser. Be sure
there is enough paraffin to cover the face of the plastic cassette.
6. Cool the top surface of the Paraplast by blowing gently on it. Tissues at
this stage are very brittle and should be handled with care. If necessary,
fill cassette with paraffin while cooling, keeping the mold full until solid.
7. Cool thoroughly in cold running tap water. If you use ice water for the
final cooling, you may split the block owing to too rapid shrinkage.
Paraplast naturally splits in the line of least resistance-right through the
tissue 8. Paraffin should solidify in 30 minutes. When the wax is
completely cooled and hardened (30 minutes) the paraffin block can be
easily popped out of the mold; the wax blocks should not stick. If the wax
cracks or the tissues are not aligned well, simply melt them again and
start over.
9. If you use plastic cups, the Paraplast block can be removed as soon as
it is cooled. The stainless steel mold should slip off easily when cool and
can be used again.

It is important to work quickly while transferring specimens or wax from

oven because wax hardens quickly. Always remember to put wax container back
into oven immediately and close oven door between transfers.

Figure 11-3. Orienting and embedding on the block

Orientation of tissues in the Paraplast block is important for tissues such as

an artery or fallopian tube so that they can be properly placed in a predetermined
plane, such as cross sections. Also, trimming is excessively difficult in a block
embedded with two or more tissues if they are not carefully lined up before the
Paraplast is cooled.
Celloidin or Nitrocellulose Embedding Method used to be recommended
for embedding hard tissues such as bones and teeth, and for large sections of
whole organs like the eye, since the delicate layers of the eyeball are difficult to
keep i ntact when other media are used. Tissues are em bedded in shallow tins
of enamel pans which are covered by sheets of weighted glass. Bell jars can be
used to control the rate or evaporation of the sol vent. The use of celloidin is

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discouraged now because of the special requirements needed for processing
and the limited use of these types of sections in neuropathology.

Double-Embedding is the 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. This is used to facilitate cutting of large blocks of dense firm tissues
like the brain. They are also recommended for making small sections of celloidin
blocks. A shortcoming of using agar as the pre-embedding media is that certain
tissues shrink during the embedding process, and the agar-based pre-embedding
media limits tissue expansion during slide mounting, resulting in difficulties with
the tissue sample adhering to the microscope slide. The availability of paraffin
waxes containing different types of resins has made this technique obsolete.

PLASTIC (RESIN) EMBEDDING

The introduction of plastic resin embedding media 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, such as renal biopsies a n d bone marrow
biopsies. Plastics are classified into epoxy, polyester, or acrylic, based on their
chemical composition.
Epoxy embedding plastics are made up of a carefully balanced mixture of
epoxy plastic, catalysts and accelerators. Three types of epoxy plastics are used
in microscopy, i.e., those based on either bisphenol A (Araldite), or glycerol
(Epon), or cyclohexene dioxide (Spurr). Infiltration by Araldite is slow, partly
because the epoxy plastic itself is a large molecule. The glycerol-based epoxy
plastics (Epon) have a lower viscosity but are often sold as mixtures of isomers.
Cyclohexene dioxide-based plastics (Spurr) can be obtained pure, have very
low viscosity, and infiltrate fastest.
Spurr’s Resin is a Low Viscosity mixture which provides rapid infiltration
of tissues. It's easy to prepare and mixes rapidly. This resin is compatible with
ethanol so no change to propylene oxide is needed prior to infiltration.
Polymerization at 60°C is recommended.
Epoxy plastics have several disadvantages. They are hydrophobic and
subsequent oxidation by peroxide to correct this may produce tissue damage.
Epoxide groups may reduce antigenicity of embedded tissue, and may
compromise the result of immunohistochemical staining. More importantly,
epoxy resins may cause sensitization if absorbed by skin or inhalation. The
components of many epoxy plastics are toxic and one of its components, vinyl

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cyclohexane dioxide (VCD) is known to be carcinogenic. For protection, gloves
should always be worn when handling these plastics, and adequate facilities
including an operational fume hood must be provided to remove the toxic
vapors and properly dispose of toxic waste.
Polyester plastics were originally introduced for electron microscopy in
the mid- 1950s, but have been superseded by more superior epoxides, and are
now seldom used.
Acrylic plastics are made up of esters of acrylic or methacrylic acid, and
are used extensively for light microscopy. Polyglycol methacrylate (GMA) has
proved to be a popular embedding medium for light microscopy because it is
extremely hydrophilic, allowing many staining methods to be applied, yet
tough enough when dehydrated to section well on most microtomes. The polar
water soluble, 2-hydroxyethyl methacrylate, commonly known as "glycol
methacrylate", or GMA, has found an increasing number of applications for
the embedding of biological tissue for transmission electron microscopy
(TEM), for the preservation and observation of fine structure not previously
subjected to solvent dehydration. GMA forms only non-crosslinked straight
chains on polymerization and therefore requires no hardener. GMA and
specifically low acid GMA offers a number of advantages over other systems: •
Dehydration of tissues can be made directly in aqueous dilutions of GMA or
optionally in organic solvents.
• GMA does not need to be water-free and indeed it works best with at
least some water present.
• Infiltration of tissue with monomer can be performed at room
temperature or lower.
• Polymerization of GMA can be performed at ambient temperature of
0°C with UV radiation to 60°C in an oven.
• Thin sections of polymerized GMA can be cut with glass or diamond
knives.
• Sections from Low Acid GMA, unlike ordinary technical grade
GMA, resist the uptake of stain, thereby reducing greatly the
occurrence of non-specific background staining.
• Enzyme digestion, a variety of stains and immunological localizations
may be performed on thick sections of GMA without removal of the
plastic.
Benzoyl peroxide is added to the plastic as a catalyst that decomposes to
form phenyl radicals acting as an active site for the polymerization of acrylics.
Unlike epoxy plastics, the viscosity of acrylic plastic is low so that short
infiltration times are possible, although the size and nature of tissue, along with

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processing and embedding temperature will affect the times required for
infiltration and embedding. Radicals can be produced spontaneously by heat or
light, so that acrylic plastics and their monomers should be stored in dark
bottles in a cool place to prevent premature polymerization.
Acrylic plastics based on methyl methacrylate (MMA) are also widely
used because of its hardness as the ideal embedding medium for undecalcified
bone and is widely used for bone histomorphometry and bone marrow
hematopathology. Compared to water-soluble methacrylates (e.g., glycol
methacrylate, GMA), MMA offers a variety of advantages.
MMA penetrates tissues better than GMA and the
histological quality of bone sections is generally
higher in MMA-embedded bone samples compared to
GMA-embedded specimens.
GMA forms crosslinks during polymerization of the
plastic so that removal of the resin from tissue
sections is not possible for GMA-embedded
material. MMA, on the other hand, can easily and
completely be removed from tissue sections. This
results in superior staining characteristics and
excellent morphological detail.
However, conventional MMA embedding causes almost complete loss of
enzyme activity and protein antigenicity in the tissues, and therefore precludes
the use of histochemical and immunohistological methods.
In general, it is preferable to use acrylic plastic sections when high
resolution light microscopy is required, because of their ease of handling and
the quality of staining achieved. However, all acrylic hydrophilic media
(including glycol methacrylate) are insoluble so that all staining occurs with
the plastic in situ. Because of this, the embedding medium itself may become
stained, or the matrix may act as a physical barrier to particular molecules
causing problems during imm unohistochemical staining. The alternative use of
h ydrophobic methyl methacrylate permits the plastic to be dissolved, and for
certain techniques, this may be a very useful property.

PRACTICAL CONSIDERATIONS:
Specimen should only be processed under an operational fume hood.
Processing is best achieved if the specimen is agitated continuously on
a roller mixer.
Small aliquots of benzoyl peroxide should be dried carefully away from
direct heat and sunlight as it is potentially explosive. It is important that

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 no water is present before dissolving the catalyst (2 minutes) in the
infiltrating solution. It m ust be completely dissolved in the infiltrating
solution, and this may take up to 30 minutes.
The acrylic plastic mixes are best prepared only in the quantity
required, preferably using a large glass vial. It is advisable to measure
the quantities volume by weight.
Any waste solutions containing plastic components must be handled
and discarded in accordance with local and legal requirements.

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Theory and Practice of Histological Techniques. 5'h Ed., Churchill Livingstone, London, 100.
Atwood K, Farmilo AI, Stead Rh, Boenisch T. (2003) Fixation & tissue processing. From: handbook
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Baker FJ. (1962) Progress in Medical Laboratory Technology, Vol. 1, Butterworths and Company,
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Bancroft J.D., Cook H.C. (1994) Manual of Histological Techniques and their Diagnostic Application.
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