SEM100 - MTAP100 - MLSCI 100 | MEDICAL LABORATORY SCIENCE INTERNSHIP

CLEARING, IMPREGNATION, EMBEDDING

Lorraine Mission, RMT and Neil John Maravilla, RMT August 16 & 28, 2021

OUTLINE Xylene/Xylol
I. Clearing III. Embedding ● Colorless clearing agent that is most commonly used
II. Impregnation INDEX: APPENDIX ● Clearing time is ½ to 1 hour
RECAP: Processing of Tissues → can shorten depending on the procedures or specimen
● Used for clearing, both for embedding and mounting
● Fixation
procedures
● Decalcification
● Dehydration Advantages Disadvantages
● Clearing/Dealcoholization ● Most rapid, 15-30 mins, ● Highly inflammable
● Infiltration/Impregnation for urgent biopsies ● If used longer than 3
● Embedding → routine clearing time: hours, it makes tissues
● Trimming 30-60 mins excessively hard and
● Section Cutting ● Evaporates quickly in brittle
● Staining paraffin oven and can be → 45 minutes each
● Mounting readily replaced by wax station is ideal
during impregnation and ● Causes hardening and
I. CLEARING embedding shrinkage; not suitable
● Also known as dealcoholization ● It is cheap and readily for nervous tissue and
● Process of replacing the dehydrating fluid with a fluid that available lymph nodes
is miscible with BOTH the dehydrating fluid and the ● Becomes milky when an
impregnating/embedding medium incompletely dehydrated
● Serves as a bridge between dehydration and infiltration tissue is immersed in it
● When a dehydrating agent has been entirely replaced by → may serve as an
the solvent, the tissue has a translucent appearance indicator
● Clearing agents should be miscible with paraffin and
mounting medium Troubleshooting milky xylene
● Remove substantial amount of fat from tissue ● Cause: Incomplete dehydration.
→ fat presents a barrier to wax infiltration ● Action: Place the tissue sample to absolute alcohol, do
● In tissue processing, clearing is done to prepare the tissue not repeat the entire dehydration process
for infiltration → re-dehydration should be 30 minutes maximum to
✓ Note: prevent samples from becoming brittle
→ In H.E staining, the first 3 clearing agents are for final ● First station of milky xylene should be discarded
deparaffinization ● Move the second station xylene to the first, move the
→ in Pap smear, when used after the tissue has been third xylene station to second, the new xylene should be
stained, the clearing agent will make microscopic tissue placed on the third (last) station.
preparation transparent due to their high index of → Newest reagents should be put on the last station,
refraction similar to dehydration stations.
A. IDEAL CHARACTERISTICS OF A CLEARING AGENT Toluene
● Should be miscible with alcohol to promote rapid removal ● Clearing time is 1-2 hours
of the dehydrating agent ● Substitute for xylene and benzene
● Should be miscible with and easily removed by melted ● Should not be placed or stored in plastic
paraffin wax and/or by mounting medium to facilitate → All clearing agents should be stored in metal tin cans
impregnation and mounting sections Advantages Disadvantages
● Should not produce excessive shrinkage, hardening, or
● Tissue do not become ● Slower than xylene and
damage of tissue
excessively hard and benzene
● Should not dissolve aniline dyes
brittle even if left in ● More expensive
● Should not evaporate quickly
toluene for 24 hours
→ However, clearing agent should evaporate quickly in
● It is not carcinogenic
the oven
● Should make tissues transparent Chloroform
B. CLEARING AGENTS SUITABLE FOR ROUTINE USE ● Clearing agents can be placed in glass container
● Below are the advantages and disadvantages of each Advantages Disadvantages
clearing agent ● Recommended for tough ● Prolonged, continuous
→ Xylene tissues (e.g. skin, fibroid inhalation can be toxic to
→ Toluene and decalcified tissues), the liver
→ Chloroform nervous tissues, lymph ● Does not make tissues
→ Methyl benzoate and methyl salicylate nodes and embryos transparent
→ Cedarwood oil ● It is not flammable ● Difficult to remove from
→ Benzene paraffin sections
→ Aniline oil
Methyl benzoate and Methyl salicylate
● Slow-acting clearing agents that can be used when double
embedding techniques are required
● Placed in a glass container

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Cedarwood oil ● Wax that has been trimmed away may be melted and
filtered for future use with a coarse filter paper
● Used to clear both paraffin and celloidin sections during → Not recommended in certain laboratories
embedding process ● Reused wax
● Recommended for CNS tissues and cytological studies → may contain water
(smooth muscles and skin) ▪ removed by heating the wax to 100-105°C
● Expensive clearing agent ● Paraffin wax may be used only twice
● Clearing time is 2-3 days
A. TYPES OF IMPREGNATION
Benzene
AND EMBEDDING MEDIA
● Rapid acting; 15-60 mins
● Includes:
● Does not make tissues hard and brittle
→ Paraffin wax
● Causes minimum shrinkage
→ Celloidin
● Highly flammable
→ Gelatin
● Excessive exposure can cause aplastic anemia
→ Plastic Resin
→ However not proven, no studies that support such claim
Paraffin Wax
Aniline oil
● Simplest, most common, and the best embedding
● Recommended for embryos, insects, and very delicate
infiltration medium
specimens
● Introduced by Butschlii
● Used for research laboratories
● Is not recommended for fat specimens
Nota bene! ● Paraffin oven/Incubator Temp: 55-60°C
● Organic solvents include: → 56°C - usually used for routine
→ Xylene ● Paraffin oven must maintain a temperature 2-5°C ABOVE
→ Toluene the melting point of the paraffin wax
→ Methyl benzoate and methyl salicylate → to avoid overheating, hardening, and destruction of
→ Chloroform tissue
→ Benzene ● After clearing, the tissue is submerged in two or more
● Oils include: changes of melted paraffin wax - either in a paraffin oven
→ Cedarwood oil or an incubator (regulated at 55-60°C)
→ Aniline oil Advantages Disadvantages
● Suitable for CNS: ● Very rapid, allowing ● Overheated paraffin
→ Chloroform sections to be prepared makes the specimen
→ Cedarwood oil within 24 hours brittle
● Suitable for embryos: ● Tissue blocks and ● Prolonged: causes
→ Aniline oil unstained mounted excessive tissue
→ Chloroform sections may be stored shrinkage and hardening
● Clearing agents that can cause deleterious effects to in paraffin for an ● Inadequate: retention of
health: indefinite period of time the clearing agent
→ Liver disease: chloroform → tissue become soft
→ aplastic anemia: benzene and shrunken, and
C. CHOICE OF CLEARING AGENTS tissue blocks crumble
● Depends on: when sectioned and
→ the type of tissue break up when floated
→ type of processing and processor system out in a water bath
→ Intended processing conditions ● Tissues difficult to
▪ e.g. temperature, vacuum, and pressure infiltrate (bones, teeth,
→ safety factors, cost, and convenience brain, and eyes) need
long immersion for
II. IMPREGNATION proper support
● Also known as infiltration ● Not recommended for
● Process of replacing the clearing agent with the infiltrating fatty tissues
medium that will completely fill all the tissue cavities
● Giving firm consistency to the specimen Substitute for Paraffin Wax
● Allows easier handling and cutting of suitably thin sections ● Mixture of highly purified paraffin +
without any damage or distortion synthetic plastic polymers
● The medium used to infiltrate the tissue is usually the ● MP: 56-57oC
Paraplast
same medium used for embedding ● More elastic & resilient than paraffin
● Time for infiltration: 1 hour per station ● Used for dense tissue block like
● Double Embedding Technique bones and brains
→ Celloidin infiltrate, base for embedding is paraffin wax ● MP: 56-58oC
Embeddol ● Less brittle and less compressible
Factors Affecting Impregnation
than paraplast
● Larger and denser tissue blocks require longer periods Bioloid ● Recommended for embedding eyes
and more frequent changes of wax
● A product of paraffin, containing
● Benzene and xylene are easily removed
Tissue Mat rubber, with the same property as
● Chloroform and cedarwood oil
paraplast
→ more difficult to remove
● MP: 46-48oC
Precautions on Impregnation ● Harder than paraffin
● Paraffin wax must be pure; free from dust, water droplets, ● Insoluble in water, Soluble in 95%
Ester wax
and other foreign matter ETOH and other clearing agents
● Fresh wax should be filtered in a wax oven at a ● Can be used in impregnation
temperature 2-5°C higher than melting point without passing clearing
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 ● MP: 38-42oC or 45-56oC Celloidin
● mostly polyethylene glycols
● most commonly used: Carbowax ● Made up of nitrocellulose
(brand name) ● Suitable for specimens with large hollow cavities, hard and
Water-soluble ● Carbowax is soluble and miscible dense tissues (bone and teeth), large tissue sections of
waxes with water; does not require whole embryo
dehydration and clearing of the ● usually used in double embedding technique
tissue ● 2 Methods for celloidin impregnation
● Suitable for many enzyme → Wet Celloidin: for bones, teeth, large brain sections,
histochemical studies and whole organs
→ Dry Celloidin: preferred for processing of whole eye
Additional Notes on Paraffin Wax sections
● inadequate → promote retention of the clearing agent → ● Substitute:
tissue become soft and shrunken, and tissue blocks → Low Viscosity Nitrocellulose (LVN)
crumble when sectioned and break up when floated out ▪ another form of celloidin
in water bath ▪ more preferable
● Additional notes: ▪ soluble in equal concentration of ether and alcohol,
→ tissues difficult to infiltrate (bones, brain, teeth, eyes) with lower viscosity, allowing it to be used in higher
▪ need long immersion for proper support concentrations and still penetrate tissues rapidly
▪ have many cavities that need to be filled Gelatin
→ paraffin wax is not recommended for fatty tissues ● Not a wax
Ways to perform impregnation and embedding ● Rarely used except when dehydration is to be avoided
● Manual processing ● Used when tissues are for histochemistry and enzyme
→ old method studies
→ At least 4 changes of wax are required at 15 minutes ● Embedding medium of delicate specimens and frozen
interval to ensure complete removal of the clearing sections because it prevents fragmentation of tough and
agent friable tissues when frozen sections are cut
→ The specimen is then immersed in another fresh ● Water-soluble
solution approx 3 hours to ensure complete ● Does not require dehydration and clearing
embedding or casting of tissue Plastic/Resin
● Automatic processing ● Used for electron microscopy
→ Elliot Bench-Type Processor ● Classified into:
→ Accommodates approx. more than 200 tissue → epoxy
cassettes → polyester
→ 2-3 changes of wax are required to remove the → acrylic
clearing agent and properly impregnate the specimen III. EMBEDDING
→ Made possible due to constant tissue agitation which ● Also known as molding, casting, or blocking
accelerates and improves tissue penetration ● Process by which the impregnated tissue is placed into a
→ Precautions: precisely arranged position in a mold containing a medium
▪ The frequency with which fluids are changed (usually paraffin) which is then allowed to solidify
depends on the number and sizes of the tissues ● Orientation:
processed → a process by which a tissue is arranged in precisely
▪ Presence of any odor in the clearing agent during arranged positions in the mold during embedding, on
final paraffin wax bath the microtome before cutting, and on the slide before
− Paraffin wax should be changed staining
▪ Dehydrating agent - should be changed frequently - ● The process by which a tissue is arranged in a precise
− is the most critical stage of tissue processing positions:
▪ The first 100% ethanol should be discarded, and the → in the mold
others moved down, so that the final bath has fresh → on the microtome
100% ethanol → on the slide
▪ The clearing agent and the diluted ethanol should
be changed at least once a week Additional Notes
▪ Wax bath thermostat should be set 2-5°C above the ● The melted paraffin should have the temp 5-10°C above
melting point of wax its melting point
● Vacuum embedding → This is so more wax can be dispensed at a faster rate
→ Wax impregnation under negative atmospheric as there must be an overflowing amount of wax in the
pressure inside an embedding oven to hasten removal mold
of air bubbles and clearing agent from tissue block ● (-5°C) for 5-10 minutes is the temperature and time of
→ Promotes rapid wax penetration reference for rapid solidifying of embedded tissue
→ Recommended for urgent biopsies and delicate → Immersion on cold water can also be done
tissues (lungs, brain, connective tissues, decalcified ● The surface of the section should be placed parallel to
bones, eyes, spleen, and CNS) the bottom of the mold in which it is oriented
→ Required time for complete impregnation
▪ reduced from 25-75% of the normal time required
for tissue processing
→ Tissue is not over-exposed to heat
▪ due to negative pressure
→ The bigger and denser the tissue blocks are, the
longer it takes to impregnate

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 A. TYPES OF EMBEDDING MOLD
Leuckhart’s Embedding Mold
● Consists of 2 L-shaped strips of heavy brass of 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

Compound Embedding Unit

● Made up of a series of interlocking plates resting on a flat
metal base, forming several compartments
● Can embed more specimens at a time
● not recommended

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

Disposable Embedding Molds

● Peel away
● Plastic ice trays
● Paper boats
→ must be hollow

B. OTHER EMBEDDING METHODS

Celloidin/Nitrocellulose
● Used to be recommended for embedding hard tissues
such as bones, teeth and for large sections of whole
organs like eyes
Double-embedding Method
● Process in which tissues are first infiltrated with celloidin
and subsequently embedded in paraffin wax
● Used to facilitate cutting of large blocks of dense firm
tissues like the brain
Plastic/Resin Embedding
● Provide superior results for light microscopic studies,
particularly in hard tissues such as undecalcified bone and
for high resolution light microscopy of sections thinner
than 4-6um
● Epoxy
● Polyester
● Acrylic

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 INDEX: APPENDIX

Clearing Agent Advantages Disadvantages

● Most rapid, for urgent biopsies ● Highly inflammable

● Evaporates quickly in paraffin oven and ● If used longer than 3 hours, it makes
can be readily replaced by wax during tissues excessively hard and brittle
Xylene impregnation and embedding ● Causes hardening and shrinkage; not
(30 mins - 1 hour) ● It is cheap and readily available suitable for nervous tissue and lymph
nodes
● Becomes milky when an incompletely
dehydrated tissue is immersed in it

● Tissue do not become excessively hard ● Slower than xylene and benzene
Toluene and brittle even if left in toluene for 24 ● More expensive
(1 - 2 hours) hours
● it is not carcinogenic

● Recommended for tough tissues (e.g. skin, ● Prolonged inhalation can be toxic to the
fibroid and decalcified tissues), nervous liver
Chloroform
tissues, lymph nodes and embryos ● Does not make tissues transparent
● It is not flammable ● Difficult to remove from paraffin sections

Methyl benzoate ● Can be used when double embedding ● Slow clearing agent
and Methyl salicylate techniques are required

● Recommended for CNS tissues and

Cedarwood Oil
cytological studies (smooth muscles and
(2 - 3 days)
skin)

● Rapid clearing agent ● Highly flammable

Benzene ● Does not make tissue hard and brittle ● Excessive exposure can cause aplastic
(15 - 60 mins) ● Causes minimum shrinkage anemia

● Recommended for embryos, insects and

Aniline Oil very delicate specimens
● Used for research laboratories

Infiltrating & Embedding Agent Advantages Disadvantages

● Very rapid, allowing sections to be ● Overheated paraffin makes the specimen

prepared within 24 hours brittle
● Tissue blocks and unstained mounted ● Prolonged: causes excessive tissue
sections may be stored in paraffin for an shrinkage and hardening
indefinite period of time ● Inadequate: retention of the clearing agent
→ tissue become soft and shrunken, and
Paraffin Wax tissue blocks crumble when sectioned
and break up when floated out in a
water bath
● Tissues difficult to infiltrate (bones, teeth,
brain, and eyes) need long immersion for
proper support
● Not recommended for fatty tissues

● Suitable for specimens with large hollow

cavities, hard and dense tissues (bone and
Celloidin
teeth), large tissue sections of whole
embryo

● Embedding medium of delicate specimens

and frozen sections because it prevents
fragmentation of tough and friable tissues
Gelatin
when frozen sections are cut
● Water soluble; does not require
dehydration or clearing

Plastic/Resin ● Used for electron microscopy

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