Process where the 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 or the
medium on which the tissue is to be
mounted
When the dehydrating agent has been
entirely replaced by the solvent, tissue
has a translucent appearance
Characteristics of a Good Clearing Agent:
1. Should be miscible with alcohol
2. Should be miscible with and easily
removed by melted paraffin wax and/or
mounting medium
3. Should not produce excessive shrinkage,
hardening or damage of tissue
4. Should not dissolve out aniline dyes
5. Should not evaporate quickly in a water
bath
6. Should make tissues transparent
Most clearing agents are flammable
liquids
Common Clearing Agents Used:
1. Xylene
2. Toluene
3. Benzene
4. Chloroform
5. Cedarwood oil
6. Aniline oil
7. Clove oil
8. Carbon tetrachloride
A . Xylene (xylol)
Colorless agent – most commonly used in
histo lab
Clearing time is ½ to 1 hour
Used for clearing, both for embedding and
mounting procedures
Suitable for most routine histologic
processing schedules of <24 hours
- tissue block is <5mm in thickness
Advantages:
- Most rapid clearing agent
- Makes tissues transparent
- Miscible with absolute alcohol and
paraffin
- Does not extract out aniline dyes
- For mounting procedures, does not
dissolve celloidin
- Evaporates quickly in paraffin oven and
can be replaced by wax during
impregnation and embedding
- Cheap
Disadvantages:
- Highly inflammable and should be
appropriately stored
- Makes tissues excessively hard and brittle
if used > 3 hours
- Causes considerable hardening and
shrinkage of tissues; not suitable for
nervous tissues and lymph nodes
- Becomes milky when incompletely
dehydrated tissue is immersed in it
B. TOLUENE
May be used as substitute for xylene or
benzene for clearing both during embedding
and mounting process
Clearing time : 1 – 2 hours
ADVANTAGES:
- Miscible with both absolute alcohol and
paraffin
- Acts fairly rapidly; recommended for routine
purposes
- Tissues do not become excessively hard and
brittle even if left for 24 hours
- Not carcinogenic
DISADVANTAGES:
- Relatively slower than benzene and
xylene
- Tends to acidify in a partially filled vessel
- Highly concentrated solutions emit fumes
that are toxic upon prolonged exposure
- More expensive
C. BENZENE
Penetrates and clear tissues rapidly
ADVANTAGES:
- Rapid acting; recommended for urgent
biopsies (15-60 minutes) and routine
purposes
- Volatilizes rapidly in paraffin oven (easily
sliminated from the tissues)
- Miscible with absolute alcohol
- Does not make tissues hard and brittle
- Causes minimum shrinkage
- Makes tissues transparent
DISADVANTAGES:
- Highly inflammable
- Cause tissue shrinkage when left in a long
period of time
- Excessive exposure will be toxic to man
and become carcinogenic or may damage
the bone marrow causing aplastic anemia.
Room should be well ventilated if used.
D. CHLOROFORM
Slower in action than xylene, but cause less
brittleness
Thicker tissue blocks (up to 1 cm thickness) can
be processed
Tissues do not become translucent
ADVANTAGES:
- Recommended for routine work (6 – 24 hours)
- Miscible with absolute alcohol
- Recommended for tough tissues( ex., skin,
fibroid and decalcified tissues) for nervous
tissues, lymph nodes and embryos – causes
minimum shrinkage and hardening of tissues
- Suitable for large tissue specimens
- Not inflammable
DISADVANTAGES:
- Toxic to the liver after prolonged
inhalation
- Wax impregnation is slow
- Does not make tissues transparent
- Not very volatile in paraffin; difficult to
remove from paraffin sections
- Complete clearing is difficult to evaluate
- Evaporates quickly from the water bath
- Tissues tend to float in chloroform
E. CEDARWOOD OIL
Used to clear both paraffin and celloidin sections during
embedding process
Recommended for central nervous system tissues and
cytological studies, particularly smooth muscles and skin
Clearing time : 2 – 3 days
ADVANTAGES:
- Very penetrating
- Miscible with 96% alcohol
- Clears celloidin in 5 – 6 days
- Causes minimal shrinkage and hardening of tissues
- Tissues may be left in oil indefinitely without considerable
damage and distortion
- Does not dissolve out aniline dyes
- Makes tissues transparent
- Improves cutting of the sections
DISADVANTAGES:
- Slow clearing agent, not recommended
for routine purposes
- Hard to be eliminated from tissues in
paraffin bath, making impregnation slow
- Quality is not always uniform and good
- Oil becomes milky upon prolonged storage
(filtered before use)
- Very expensive
F. ANILINE OIL
Not normally utilized as routine clearing
agent
Recommended for clearing embryos, insects
and very delicate specimens due to inability
to clear 70% alcohol without excessive tissue
shrinkage and hardening
G. CLOVE OIL
Causes minimum shrinkage of tissues
Wax impregnation is slow and difficult
Tissues become brittle, aniline dyes are
removed and celloidin is dissolved
Expensive, unsuitable for routine clearing
purposes
H. CARBON TETRACHLORIDE
Properties is similar to chloroform;
cheaper
Disadvantages is similar to chloroform
I. METHYL BENZOATE AND METHYL
SALICYLATE
Slow acting clearing agents; used when
double embedding techniques are
required
