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 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

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