Clearing

Day 16
Learning Outcome

1. To describe the principle of clearing procedure of

histotechnique.
2. To identify the process of clearing.
3. To list down the different clearing agent used in the process.
4. To discuss the different advantages and disadvantages of
each learning agent.
Definition

Clearing (dealcoholization) is the

process whereby alcohol or a
dehydrating agent is removed from the
tissue and replaced with a substance
that will dissolve the wax with which
the tissue is to be impregnated (e.g
paraffin) or the medium on which the
tissue is to be mounted (e.g. Canada
balsam).
Definition

When the dehydrating agent has

been entirely replaced by the
solvent, the tissue has a translucent
appearance; hence, the use of the
term "clearing agent".
Definition

It must be both miscible with

dehydrating agent and infiltrating
and embedding agent.
Definition

When used after the tissue section

has been stained, the clearing agent
will make microscopic tissue
preparations transparent due to
their high index of refraction.
Definition

Aside from removing alcohol, a

clearing agent must also be miscible
with Canada balsam and other
resins that are used for mounting
sections.
Definition

The clearing agents merely improve

the refractive index of the tissue.
Tissues become transparent so that
the internal structures become
visible to the naked eye.
But not all clearing agents, however,
exhibit this property.
Characteristics of a Good Clearing Agent:

1. It should be miscible with alcohol to promote rapid removal of the dehydrating agent from
the tissue.
2. It should be miscible with, and easily removed by melted paraffin wax and or by mounting
medium to facilitate impregnation and mounting of sections
3. It should not produce excessive shrinkage, hardening, or damage of tissue.
4. It should not dissolve out aniline dyes.
5. It should not evaporate quickly in a water bath.
6. It should make tissues transparent.
Caution

Most clearing agents are flammable liquids that warrant

considerable caution in their use, and the histotechnologist
should be aware of the large quantities used in routine
processing so that the safest method of use and storage can be
adopted.
Caution

● Clearing fluids with a low boiling point are generally more

readily replaced by melted paraffin.
● Chloroform which has a lower boiling point than xylene in
fact takes longer than the latter to clear.
● Viscosity also affects the speed of penetration of the clearing
agent.
● Prolonged exposure to most clearing agents causes the
tissue to become brittle and therefore more difficult to cut.
Among the Common Clearing Agents Used Are:

1. Xylene (most common)

2 Toluene
3. Benzene
4. Chloroform
5. Cedarwood oil
6. Aniline oil
7 Clove oil
8. Carbon tetrachloride
A. Xylene (Xylol)

● Xylene is a colorless clearing agent that is most commonly

used in histology laboratories.
● Clearing time is usually 1/2 to 1 hour.
● It is used for clearing, both for embedding and mounting
procedures.
● It is generally suitable for most routine histologic processing
schedules of less than 24 hours, and when the tissue block
size is less than 5 mm. in thickness.
A. Xylene (Xylol)

ADVANTAGES:
1. It is the most rapid clearing agent, suitable for urgent biopsies which
2 it clears within 15-30 minutes.
3. It is miscible with absolute alcohol and paraffin.
4. It does not extract out aniline dyes.
5. For mounting procedures, it does not dissolve celloidin and can, therefore, be
used for celloidin sections.
6. It evaporates quickly in paraffin oven and can, therefore, be readily replaced by
wax during impregnation and embedding.
7. It is cheap.
8. It makes tissues transparent
A. Xylene (Xylol)

DISADVANTAGES:
1. It is highly inflammable and should be appropriately stored.
2. If used longer than 3 hours, it makes tissues excessively hard
and brittle.
3. It causes considerable hardening and shrinkage of tissues;
hence, is not suitable for nervous tissues and lymph nodes.
4. Xylene becomes milky when an incompletely dehydrated
tissue is immersed in it.
B. Toluene

Toluene may be used as a substitute for xylene or benzene for

clearing both during embedding and mounting processes. The
time recommended for clearing is 1-2 hours.
B. Toluene

ADVANTAGES:
1. It is miscible with both absolute alcohol and paraffin.
2. It acts fairly rapidly and is recommended for routine purposes.
3. Tissues do not become excessively hard and brittle even if left
in toluene for 24 hours.
4. It is not carcinogenic.
B. Toluene

DISADVANTAGES:
1. It is relatively slower than xylene and benzene.
2. It tends to acidify in a partially filled vessel.
3. Highly concentrated solutions will emit fumes that are toxíc
upon prolonged exposure.
4. It is more expensive.
C. Benzene

Benzene is preferred by some as clearing agent in the embedding

process of tissues because it penetrates and clears tissues
rapidly.
C. Benzene

ADVANTAGES:
1. It is rapid-acting, hence is recommended for, urgent biopsies (15-60
minutes) and routine purposes.
2. It volatilizes rapidly in a paraffin oven and is therefore easily
eliminated from the tissue.
3. It is miscible with absolute alcohol.
4. It does not make tissues hard and brittle.
5. It causes minimum shrinkage.
6. It makes tissues transparent.
C. Benzene

DISADVANTAGES:
1. It is highly inflammable.
2. If a section is left in benzene for a long time, considerable tissue
shrinkage may be observed; hence, tissues should be transferred to
paraffin wax as soon as possible.
3 Excessive exposure to benzene may be extremely toxic to man and
may become carcinogenic or it may damage the bone marrow
resulting in aplastic anemia. If ever benzene is to be used for clearing,
the laboratory should be well-ventilated.
D. Chloroform

Chloroform, when used for clearing of tissues during the

embedding process, is slower in action than xylene, but causes
less brittleness.
Thicker tissue blocks, even those up to 1 cm. in thickness, can be
processed. However, tissues placed in chloroform do not become
translucent.
D. Chloroform

ADVANTAGES:
1. It is recommended for routine work (6-24 hours).
2. It is miscible with absolute alcohol.
3. It is recommended for tough tissues (e.g. skin, fibroid and decalcified
tissues) for nervous tissues, lymph nodes and embryos because it
causes
4. minimum shrinkage and hardening of tissues. It is suitable for large
tissue specimens.
5. It is not inflammable.
D. Chloroform

DISADVANTAGES:
1. Itis relatively toxic to the liver after prolonged inhalation; this may be prevented by
adequate room ventilation and proper caution when handling the specimen.
2. Wax impregnation after chloroform clearing is relatively slow.
3. It does not make tissues transparent.
4. It is not very volatile in paraffin oven; hence, is difficult to remove from paraffin sections. It
may even produce considerable deterioration of the wax.
5. Its vapor may attack the rubber seal used in vacuum impregnating bath.
6. Complete clearing is difficult to evaluate.
7. Tissues tend to float in chloroform; this may be avoided by wrapping the tissues with
absorbent cotton gauze to facilitate sinking of the section in solution.
8. It evaporates quickly from a water bath.
E. Cedarwood oil

Cedarwood oil is used to clear both paraffin and celloidin

sections during the embedding process.
It is especially recommended for central nervous system tissues
and cytological studies, particularly of smooth muscles and skin.
It requires two changes in clearing solution.
Clearing is usually complete in 2-3 days.
E. Cedarwood oil

ADVANTAGES:
1. It is very penetrating.
2. It is miscible with 96% alcohol which it removes readily.
3. Lclears celloidin in 5-6 days.
4. It causes minimal shrinkage and hardening of tissues.
5. Tissues may be left in oil indefinitely without considerable damage and
distortion.
6. It does not dissolve out aniline dyes.
7. It makes tissues transparent.
8. Clearing with cedarwood oil often improves cutting of the sections.
E. Cedarwood oil

DISADVANTAGES:
1. It is an extremely slow clearing agent, hence, is not recommended for routine purposes.
2. It is hard to be eliminated from the tissues in paraffin bath, making the wax impregnation
process very slow. This may be improved or hastened by transferring the specimen from oil
to benzene for 1/2 hour before finally placing the tissue in wax.
3. Quality is not always uniform and good. Tissues cleared in cedarwood oil initially float
before gradually staying to the bottom as clearing proceeds. Hence, the tissue may dry out
before it is completely cleared. This can be prevented by superimposing absolute alcohol on
the surface
of the clearing agent. Once saturated, the specimen should then be transferred to a fresh
solution of cedarwood oil.
E. Cedarwood oil

DISADVANTAGES:
4. Cedarwood oil becomes milky upon prolonged storage and should
be filtered before use.
5. Cedarwood oil that has been previously used to clear acetic-alcohol
fixed tissues may produce crystals with a melting point of
approximately 35°C and therefore interfere with adequate clearing of
tissue. The solution must be heated to 200°C in order to dissolve the
crystals and restore the solution to its normal state.
6. It is very expensive.
F. Aniline oil

This is not normally utilized as a routine clearing agent but is

rather recommended for clearing embryos, insects and very
delicate specimens, due to its ability to clear 70% alcohol without
excessive tissue shrinkage and hardening.
G. Clove oil

This reagent causes minimum shrinkage of tissues. However, its

qualityis not guaranteed due to its tendency to become
adulterated. Wax impregnation after clearing with clove oil is
slow and difficult. Tissues become brittle, aniline dyes are
removed, and celloidin is dissolved. All of these, in addition to the
expensiveness of the solution, make it unsuitable for routine
clearing purposes.
H. Carbon tetrachloride

Carbon Tetrachloride may be used in clearing, tissues for

embedding. Its propeties are very similar to that of chloroform
although it is relatively cheaper. Its disadvantage is the same as
that of chloroform. It produces considerable tissue hardening,
and is dangerous to inhale on prolonged exposure due to its
highly toxic effects.
I. Methyl benzoate and methyl salicylate

These are slow-acting clearing agents that çan_be used when

double embedding techniques are required.