IMPREGNATION (INFILTRATION)

IMPREGNATION 1. PARAFFIN WAX 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.
This 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.
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
• simplest, most common and best embedding medium used for routine
tissue processing
• Paraffin wax
o polycrystalline mixture of solid hydrocarbons produced during the
refining of coal and mineral oils
o solid at RT, melts at 65°C or 70°C.
o can be purchased with melting points at different temperatures
o most common for histological use: 56°C to 58°C
o The traditional advice with paraffin wax is to use this about 2°C above
its melting point
Advantages:
1. Thin individual serial sections may be cut with ease from the majority
of tissues without distortion.
2. 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 (permanent)
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:
The medium used to infiltrate the tissue is usually the same medium 1. NOT recommended for FATTY TISSUES.
utilized for impregnation, and for general purposes is known as an • The dehydrating and clearing agents used in the process dissolve
Embedding Medium. and remove fat from the tissues.
• Frozen sections can demonstrate fatty tissues (lipids and fats)
FOUR TYPES OF IMPREGNATION AND EMBEDDING MEDIUM 2. Overheated paraffin → brittle specimen
1. PARAFFIN WAX 3. Inadequate impregnation → retention of the clearing agent
• most commonly used; best medium 4. Tissues that are difficult to infiltrate, e.g. bones, teeth, brains and eyes,
• 3 types: need long immersion for proper support; otherwise, they will crumble
o Manual – 4 changes of wax at 15 minutes interval on sectioning.
o Automatic – Autotechnicon, Elliot Bench Type 5. Prolonged impregnation → excessive tissue shrinkage and
o Vacuum – fastest, negative atmospheric pressure hardening, making the cutting of sections difficult (should not be left
for more than 4 hours)
2. CELLOIDIN (COLLODION)
• suitable for specimens with large hollow cavities which tend to After clearing, the tissue is submerged in 2 or more changes of melted
collapse paraffin wax, either in a paraffin oven or in an incubator which has been
• for hard and dense tissues such as bones and teeth and for large regulated at 55-60°C.
tissue sections of the whole embryo
The duration and number of changes required for thorough impregnation
• two methods: of tissue depends on:
o wet • Size and type of tissues: longer time is required for thicker tissues.
o dry • Use of vacuum imbedding: Vacuum reduces the time required for
complete impregnation.
3. GELATIN • Clearing agent employed
• used when the dehydration process is avoided

• used in histochemical and enzyme studies METHODs OF PARAFFIN WAX

IMPREGNATION AND EMBEDDING
4. PLASTIC • By manual processing
• used for electron and light microscopy studies • By automatic processing
• By vacuum embedding
Infiltrating medium to tissue ratio: 25:1

1. MANUAL PROCESSING
At least 4 changes of wax are required at 15 minutes intervals to insure
complete removal of the clearing agent from the tissue

Time schedule for manual processing of tissues about 3 mm thick:

Xylene or Toluene 1 hour

Clearing
Xylene or Toluene 1 hour
Paraffin wax 15 minutes
Paraffin wax 15 minutes
Impregnation
Paraffin wax 15 minutes
Paraffin wax 15 minutes
Embedding Paraffin wax 3 hours
2. AUTOMATIC PROCESSING
• uses an automatic tissue processing machine which fixes, dehydrates,
clears and infiltrates tissues, thereby decreasing the time and labor
needed during the processing of tissues.
• results a more rapid diagnosis with less technicality
• 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
Example:
• Autotechnicon
• Elliot Bench-Type Processor
makes use of 12 individual processing steps, with 10 1-liter capacity
glass beakers and 2 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).
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 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.
3. VACUUM EMBEDDING
• involves wax impregnation under negative atmospheric pressure inside
an embedding oven.
• reduces the time when tissues are subjected to high temperatures →
minimizing heat-induced tissue hardening.
• 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 → more rapid wax penetration of the tissue.
• recommended for urgent biopsies, for delicate tissues such as lung,
brain, connective tissues, decalcified bones, eyes, spleen and central
nervous system.
• 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 processors are equipped to deliver vacuum, or vacuum
and pressure, to all or some reagent stations during the processing
cycle.
• 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 overexposed 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 mmHg. 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.
• 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 clearing 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.
PARAFFIN WAX CONSIDERATIONS
Paraffin wax must be
• Pure → free from dust, water droplets and other foreign matter.
• Fresh wax should be filtered before use
• Wax that has been trimmed away from the impregnated tissue may be
melted and filtered for future use
o with a coarse filter paper, e.g. Green's No. 904.
• Paraffin wax may be used only twice, after which, fresh wax must be
utilized
o When reused, some amount of water inevitably is mixed with it.
o removed by heating the wax to 100-105°C, thereby raising its melting
point.
 SUBSTITUTES FOR PARAFFIN WAX 2. CELLOIDIN (COLLODION) IMPREGNATION
Paraplast Water Soluble Wax • A purified form of nitrocellulose soluble in many solvents
Embeddol • suitable for:
Dimethyl sulphoxide
Ester wax 1. specimens with large hollow cavities which tend to collapse
2. hard and dense tissues such as bones and teeth
1. PARAPLAST 3. for large tissue sections of the whole embryo
• a mixture of highly purified paraffin and synthetic plastic polymers,
• a melting point of 56-57°C. • It is supplied in thin (2%), medium (4%) or thick (8%) solutions of
• more elastic and resilient than paraffin wax → permitting large dense cellulose dissolved in equal parts of ether and alcohol.
tissue blocks (bones and brain) to be cut easily with the same result
with double embedding. ADVANTAGES
1. It permits cutting of tissue sections which are thicker than in paraffin
wax and is recommended for processing of neurological tissues.
2. EMBEDDOL 2. Its rubbery consistency allows tissue blocks that are either very hard
• synthetic wax substitute like Paraplast or of varying consistency, to be cut without undue distortion.
• melting point of 56-58°C. 3. Does not require heat during processing → minimum shrinkage and
• less brittle and less compressible than Paraplast. tissue distortion

BIOLOID DISADVANTAGES
• semisynthetic wax recommended for embedding eyes. 1. Very thin sections (less than I0 µ) are difficult to cut.
2. It is very volatile and therefore must be kept in bottles with ground
TISSUE MAT glass stoppers to prevent evaporation.
• a product of paraffin, containing rubber, with the same property as
Paraplast 2 METHODS
WET CELLOIDIN DRY CELLOIDIN
recommended for bones, teeth, large preferred for processing
3. ESTER WAX brain sections and whole organs of whole eye sections
• lower melting point (46-48°C)
• harder than paraffin
• not soluble in water but is soluble in 95% Ethyl Alcohol and other
clearing agents
o it can be used for impregnation without prior clearing of the tissue.
• 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

4. WATER SOLUBLE WAXES

• plastic polymers, mostly polyethylene glycols
• melting points of 38-42°C or 45-56°C

• Carbowax
o most commonly used – solid at RT
o soluble in and miscible with water → does not require dehydration
and clearing
o Processing time is reduced
The specimen is removed from the celloidin, transferred to an embedding
o special advantages:
medium containing freshly poured thick celloidin and kept in a tightly
▪ harmful effects produced by ordinary dehydrating agents are covered jar or dessicator to evaporate the alcohol-ether solvent. The
consequently avoided. tissue block is then stored in 70-80% alcohol until ready for cutting.
▪ does not remove neutral fats and lipids
▪ Tissues are not exposed to too much heat → suitable for many
The principle and procedure of this method is similar to wet celloidin
enzyme histochemical studies (excessive hardening, shrinkage method, except that 70% alcohol is not used for storage before
and brittleness of tissue is avoided) cutting. Instead, Gilson's mixture, made up of equal parts of
chloroform and cedarwood oil, is added to the celloidin block before
o4 CHANGES OF CARBOWAX hardening, to make the tissue transparent. The dry method does not
70% carbowax 30 minutes make use of alcohol due to the presence of cedarwood oil in the block.
90% carbowax 45 minutes
100% carbowax 1 hour NITROCELLULOSE METHOD
100% carbowax 1 hour Low Viscosity Nitrocellulose (L.V.N.)
• another form of celloidin with a lower viscosity, allowing it to be used in
HYGROSCOPIC NATURE → Tissue sections are very difficult to
float out and mount due to its extreme solubility in water, dehydrating higher concentrations
• forms a harder tissue block and makes cutting of thinner sections
and clearing agents. Adding soap to water or using 10%
Polyethylene Glycol 900 in water will reduce tissue distortion and possible.
• DISADVANTAGE: more explosive than celloidin and should therefore
promote flattening and "floating out" of sections.
be handled with care. When dry, striking or dropping the container will
cause the substance to explode
• When no longer needed for future use, the nitrocellulose should be
5. DIMETHYL SULPHOXIDE (DMSO)
reduces infiltration times and facilitates thin sectioning carefully destroyed, since the material becomes increasingly
dangerous as the alcohol continues to evaporate.
 3.GELATIN IMPREGNATION a. TISSUE TEK
• rarely used except when dehydration is to be avoided • Cold and hot area
o no need for dehydration and clearing o Cold area → contains the cold

• for histochemical and enzyme studies plate set at -5 degrees Celsius

• embedding medium for delicate specimens and frozen tissue (rapid solidification)
sections o Warm area → warm plate

• water-soluble, and does not require dehydration and clearing, although o Paraffin dispenser cassette Base mold
fixatives (such as 10% formalin) should still be washed out by running
water • Cassette – can be used in fixation,
• has a low melting point and does not cause over-hardening of tissues dehydration, clearing and
by heating. impregnation

• Once the tissue has been properly oriented, the base of the cassette
Fixative/Washing Out 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
infiltration 10% gelatin + 1% phenol (24 hours) wax has solidified (usually 5 minutes), the block is taken out together
↓ with the embedding ring and is immediately ready for cutting without
20% gelatin + 1% phenol (24 hours) having to undergo trimming or mounting, thereby saving time and
↓ effort.
embedding 20% gelatin + 1% phenol
↓ ADVANTAGES
(Refrigerator – until impregnation and embedding) • ease of use

↓ • less paraffin wax needed

10% formalin for 12-24 hours • faster embedding

(to harden the tissue) • firmly attached tissue and holder

• permanent identification

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.
EMBEDDING
• aka casting or blocking
• Process by which tissue is placed into
a precisely arranged position in a mold
containing a medium which is then
allowed to solidify.
• Orientation – tissue is arrange in a
precise position in the mold during
embedding; most important step in
embedding
• The surface of the section to be cut should be placed parallel to the
bottom of the mold in which it is oriented.
• 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
TYPES OF BLOCKING OUT MOLDS
• Leuckhart’s Embedding Mold
• Compound Embedding Unit
• Plastic Embedding Rings and Base Mold
• Disposable Embedding Molds
1. LEUCKHART’S EMBEDDING MOLD
• consists of two L-shaped strips of heavy brass or metal arranged on
a flat metal plate
• can be moved to adjust the size of the
mold to the size of the specimen.
• recommended for routine use, although,
too slow and cumbersome for use in a
busy laboratory
4. DISPOSABLE EMBEDDING MOLDS
a. PEEL-AWAY
Thin plastic mold that are simple peeled off as soon
the wax has solidified
b. PLASTIC ICE TRAYS
place more tissue molds; more specimens
c. PAPER BOATS
for celloidin blocks but also useful for paraffin blocks;
cheap and easy to make
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.
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
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. 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.
7. If the wax cracks or the tissues are not aligned well, simply melt them
again and start over.

2. COMPOUND EMBEDDING UNIT

• Series of interlocking plates resting on a flat
metal base
• Embed more specimen at a time

3. PLASTIC EMBEDDING RINGS AND BASE MOLD

Stainless steel base mold fitted with a plastic embedding rings
 OTHER EMBEDDING METHODS
Celloidin or Nitrocellulose Embedding Method
Recommended for hard tissues (bones, teeth) and for large section and
whole organs (eye)

DOUBLE EMBEDDING METHOD

• process by which tissues are first embedded/infiltrated with agar or

nitrocellulose are then infiltrated/embedded with paraffin wax

• Facilitate cutting of large blocks of dense firm tissues (brain)

PLASTIC (RESIN) EMBEDDING

• Epoxy

• Polyester

• Acrylic

• Recommended for

o light microscopic studies

o hard tissues such as undecalcified bone
o for high resolution light microscopy of tissue sections thinner than
the usual 4-6 μm (renal biopsies and bone marrow biopsies)

a.EPOXY
• Three types of epoxy plastics are used in microscopy

• bisphenol A (Araldite) – component of baby bottle

• glycerol (Epon)

• cyclohexene dioxide (Spurr)

DISADVANTAGES:
• hydrophobic, compromise immunohistochemical staining, can

cause sensitization
• components are toxic and one of its components, vinyl

cyclohexane dioxide (VCD) is known to be carcinogenic.

• gloves should always be worn when handling these plastics

b.POLYESTER PLASTICS
originally introduced for electron microscopy but are seldom used now

c.ACRYLIC PLASTICS
•made of esters of acrylic or methacrylic
•Used extensively for light microscopy

c.1.POLYGLYCOL METHACRYLATE (PGMA)

• popular embedding medium for light microscopy

• extremely hydrophilic, allows many staining methods and tough

enough

C.2.GLYCOL METHACRYLATE (GMA)

• for transmission electron microscopy (TEM)

C.3.METHYL METHACRYLATE
• for undecalcified bone and for bone histomorphometry and bone

marrow hematopathology

AFTER ALL THESE, MICROTOMY!!!!