Clearing Agent
XYLENE (xylol)
-Colorless
- Most commonly used
Used for clearing, both mounting
and embedding procedures
- Generally suitable for routine
histologic processing schedules of
<24 hours and when tissue block is
<5mm in thickness
TOLUENE
- may be used as substitute for
xylene or benzene for clearing
both during embedding and
mounting processes
BENZENE
- Preferred by some in the
embedding process because it
penetrates and clears tissues
rapidly
CHLOROFORM
- when used for clearing in
embeds process, slower than
xylene
- Causes less brittleness
- Can be used for thicker tissue
blocks (up to 1 cm)
- Tissues don't be on translucent
Advantages
- most rapid clearing - for urgent biopsies
(15-30 mins)
- Makes tissue transparent
- Miscible w abs alcohol and paraffin
- Doesn't extract out aniline dyes
- For mounting procedures - doesn't
dissolve celloidin
- During embedding and impregnation -
evaporates quickly in paraffin oven - can
be readily replaced by wax
- Cheap
1. Miscible w both absolute alcohol
and paraffin
2. Acts fairly rapidly - recommended
for routine purposes
3. Doesn't make tissues excessively
hard and brittle if left for 24 hours
1. Rapid acting - for urgent biopsies
(15-60 mins) and routine purposes
2. Volatilizza rapidly in paraffin oven -
easily eliminated from the tissue
3. Doesn't make tissues hard and
brittle
4. Causes minimum shrinkage
5. Makes tissues transparent
1. For routine processing (6-24 hrs)
2. Miscible w abs alcohol
3. Causes min shrinkage - for tough
tissues (e.g skin, fibroid, decalcified
tissues) for nervous tissues, lymph
nodes and embryos
4. For large tissue specimens
5. Not flammable
Disadvantages Clearing Time
- highly flammable 1/2-1 hour
- Makes tissue excessively hard of
used over 3 hrs
- Not suitable for nervous tissues and
lymph nodes - causes considerable
hardening and shrinkage
1. Slower than xylene and 1-2 hours
benzene
2. Acidified in a partially filled
vessel
3. Highly conc solns will emit
fumes that are toxic upon
prolonged exposure
4. More expensive
1. Highly flammable
2. Causes considerable tissue
shrinkage if left in benzene for a
long time
3. Excessive exposure may
extremely be toxic to man - may
damage bone marrow (aplastic
anemia)
1. Toxic to the liver after
prolonged inhalation
2. Wax impregnation after
chloroform clearing relatively
slow
3. Doesn't make tissues
transparent
4. Not very volatile in paraffin
oven
5. Vapor may attack rubber seal
used in vacuum impregnating
bath
6. Complete clearing is difficult to
evaluate
7. Tissues tend to float in
chloroform
8. Evaporates quickly from a
water bath
 Clearing Agent Advantages Disadvantages Clearing Time
CEDARWOOD OIL

- used to clear both paraffin and
cellodin sections during the
embedding process
- Especially recommended for
CNS tissues and cytological
studies (smooth muscles and
skin)
- Requires 2 changes in clearing
solution
ANILINE OIL
1. Very penetrating
2. Miscible w 96% alcohol w/c it
removes readily
3. Clears celloidin in 5-6 days
4. Causes minimal shrinkage and
hardening
5. Tissues may be left indefinitely w/o
causing considerable damage and
distortion
6. Makes tissues transparent
7. Improves cutting of sections
1. Extremely slow - not for 2-3 days
routine purchases
2. Hard to be eliminated from
tissues in paraffin bath quality
not always uniform and good
3. Cedarwood oil becomes milky
upon prolonged storage, should
be filtered before use
4. Previously used to clear acetic
acid and alcohol fixed tissues may
be produce crystals
5. Very expensive

- Not normally utilized as a

routine clearing agent
- Recommended for clearing
embryos, insects, and very
delicate specimens - ability to
clear 70% alcohol w/o excessive
shrinkage and hardening
CLOVE OIL

- Causes min shrinkage - Expensive

- Quality not guaranteed- - Unsuitable for routine clearing
tendency to become purposes
adulterated
- Was impregnation slow and
difficult tissues become brittle,
aniline dyes removed, celloidin
is dissolved

CARBON TETRACHLORIDE

- used in clearing tissues for - Highly toxic - dangerous to

embedding inhale on prolonged exposure
- Properties similar to
chloroform
- Produces considerable tissue
hardening

METHYL BENZOATE AND

METHYL SALICYLATE

- slow-acting clearing agents

- Can be used when double
embedding techniques are
required