CLEARING
● 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 used
as the medium on which the tissue
is to be mounted (e.g. Canada
balsam)
Characteristics of a Good Clearing Agent:
It should be miscible with alcohol to promote rapid removal of the
dehydrating agent from the tissue.
It should be miscible with, and easily removed by melted paraffin wax and/or
by mounting medium to facilitate impregnation and mounting of sections.
It should not produce excessive shrinkage, hardening or damage of tissue
It should not dissolve out aniline dyes.
It should not evaporate quickly in a water bath.
It make tissues transparent.
The choice of a clearing agent depends upon the ff:
The type of tissues to be processed, and the type of processing to be
undertaken
The processor system to be used
Intended processing conditions such as temperature, vacuum and pressure
Safety factors
Cost and convenience
Speedy removal of dehydrating agent
Ease of removal by molten paraffin wax
Minimal tissue damage
Clearing fluids with a low boiling point
are generally more readily replaced by
melted paraffin
A. Xylene (Xylol)
● is a colorless clearing agent that is most commonly used in
histology laboratories
● Clearing time is usually 1/2 to 1 hour
● Xylene is reasonably cost effective and works well for short-term
clearing of small tissue blocks.
● It is mostly used as a clearing agent during tissue processing and
as a dewaxing agent during staining
Methyl benzaldehyde
intermediate product of xylene metabolism believed to cause
central nervous system disorders, respiratory depression,
abdominal pain, dryness and redness of skin, dermatitis, liver
diseases, nephrotoxicity, conjunctivitis, and teratogenic and
fetotoxic effects.
B. Toluene
is better at preserving tissue structure and is more tolerant of
small amounts of water left behind in the tissues than xylene.
However, toluene is more expensive than xylene and more
toxic. Time recommended for clearing is 1 -2 hours.
C. Benzene
is preferred by some as clearing agent in the embedding process of
tissues because it penetrates and clears tissues rapidly. It used to be
a popular routine clearing agent until recently when its highly
carcinogenic properties were recognized. Its use for clearing
purposes is therefore strongly discouraged.
It clears overnight
D. 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 I cm. in thickness, can be
processed. However, tissues placed in chloroform do not become
translucent.
E. Cedarwood Oil
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.
F. Aniline oil
This is not normally utilized as a routine clearing agent but it
is 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 quality is 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
Its properties 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. Tetrahydrofuran
Tetrahydrofuran is superior to ordinary dehydrating and
clearing agents due to its ability to perform two processes at
the same time, thereby shortening the total processing time
and allowing more time for fixation. It is non-toxic but has
offensive odor and should be used in a well-ventilated room.
J. Dioxane
is miscible both with water and paraffin. It is used primarily when time is
important because the tissues may be embedded with paraffin within 4
hours after fixation. The tissues are transferred to dioxane straight from
Bouin's fluid or a formalin fixative. The dioxane is changed 3 times within
4 hours and the tissues are transferred directly to paraffin (3 changes
are made in a total of 90 minutes). Dioxane causes greater shrinkage
than xylene does. In addition, it is dangerous. Fumes of dioxane are toxic
to human especially to the liver.
Other Xylene Substitutes
The reported toxicity and environmental pollution from unsafe
disposal of xylene led to its substitution with other less-toxic
substitutes such as limonene reagents, mineral oil mixtures, 1.7%
dish washing solution, vegetable oils and coconut oil. Though these
substitutes exist, their availability in commercial quantities in
developing countries has hampered their use. All the xylenesubstitutes have to be analyzed thoroughly, before concluding which
alternative is better.
Terpenes
isoprene polymers found in essential oils originally derived from
plants, though some are now synthesized. They are the earliest
transition solvents to be used in histology and include
turpentine and oils of bergamot, cedarwood, clove, lemon,
oreganum and sandalwood. Terpenes are moderately effective
solvents, but they too are considered toxic. Solvents in this class
also dry slowly, leave an oily residue on slides and are relatively
expensive.
Limonene
One of the recommended xylene substitutes from the terpene family.
It is a volatile oil found in citrus peels which goes by several trade
names. It is a natural oil found in the skins of citrus fruits, such as
lemons or oranges, and in cooking is usually referred to as lemon or
orange zest. Limonene is obtained industrially by the steam
distillation of orange peel which is a byproduct of the orange juice
industry. It is a clear, colorless fluid with a distinctly citrus aroma, not
unpleasant to most people, although some do not like it.
Orange oil based clearing agents
offer the clearing action with the lowest hazard rating of all
xylene alternatives. It is excellent for preserving fine tissue
structure, and can often be used in place of xylene with no
alteration of protocol. In using a product containing orange
oils, it is important to use a product which has been rigorously
purified then stabilized
Chlorinated hydrocarbons
can be effective solvents, but they are considered toxic
chemicals, posing serious health risks. Government
regulations have restricted most of the effective
solvents in this class.
Coconut oil
an efficient substitute for xylene, as it is
non-hazardous, less expensive and causes less
shrinkage of the tissue. It can be used as a
dealcoholization agent in the histopathological
laboratory, without losing the quality of the
histological details. The only drawback associated
with coconut oil, is its tendency to get solidified at a
lower temperature.
Bleached palm oil
Substitution of the conventional xylene with bleached
palm oil as a clearing agent during tissue processing and
as a dewaxing agent during staining gives good tissues,
sections and histological slides. In addition, bleached
palm oil is nontoxic, nonhazardous, nonflammable,
bio-degradable, economic, easy to handle, and readily
available.