CLEARING

(Dealcoholization)
OBJECTIVES:
1. Describe the purpose of clearing
2. Differentiate the use of clearing agents for the purpose of
embedding and for the purpose of mounting
3. Enumerate the advantages and disadvantages of the different
clearing agents
WE ARE NOW HERE
Purpose:
▪Clearing is the process whereby alcohol or 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
(paraffin)
▪Clearing agent is used after staining- it makes the tissue
transparent due to their high index of refraction
▪clearing agent is replaced by a medium for mounting (Canada Balsam)
▪Frozen sections do not use alcohol therefore there is no de-
alcoholization process involved. Clearing agents like GLYCERIN and
GUM SYRUP are the clearing agents used to provide a high index of
refractioning
▪Making tissues transparent makes internal structures visible hence
the tern ‘clearing’
▪Miscible alcohol to remove it from the tissue after
dehydration
▪Miscible with paraffin wax to facilitate its penetration
during embedding
▪High index of refraction to make tissue transparent when
used after tissue staining
Considerations
▪ Most clearing agents as flammable therefore safe methods of
use and storage should be adopted
▪ clearing agents with low boiling points are generally more
readily replaced by paraffin (except for chloroform vs xylene)
▪Viscosity affects speed of penetration of clearing agent
▪Prolonged exposure causes tissue to become brittle and more
difficult to cut
Common Clearing agents
1. Xylene- most common
2. Toluene
3. Benzene
4. Chloroform
5. Cedarwood oil
6. Aniline oil
7. Clove oil
8. Carbon tetrachloride
Xylene (Xylol)
▪ Most common and rapid (15-30 mins)
▪ Clearing for both embedding and mounting (does not dissolve
celloidin therefore compatible with celloidin sections)
▪Routine for processing tissues of less than 24 hours and when
tissue block is less than 5mm in thickness
▪Evaporates quickly in paraffin oven- readily replaced by wax
during impregnation and embedding
▪ Highly flammable

▪If used for more than 3 hours, it makes tissues excessively hard and brittle

▪Not for nervous tissues and lymph nodes- hardening and shrinkage

▪Becomes milky when used with incompletely dehydrated tissue

▪Carcinogenic
Toluene
▪ Substitute for xylene or benzene for clearing during both
embedding and mounting
▪Clearing time is 1-2 hours
▪Acts fairly rapidly hence may be used routinely
▪Does not make tissues excessively hard and brittle even when left in
toluene for 24 hours
▪Non carcinogenic
▪ it tends to acidify in a partially filled vessel
▪Highly concentrated solutions will emit fumes that are toxic on
prolonged exposure
▪It is more expensive
Benzene
▪ Penetrates and clears tissues rapidly, used for urgent biopsies (15-
60minutes)
▪Has sweet and gasoline like odor
▪Volatilizes rapidly in paraffin oven, therefore easily eliminated from
the tissue
▪Does not make tissue hard and brittle
▪Causes minimum shrinkage
▪Makes tissues transparent
▪Highly flammable
▪Causes tissue considerable tissue shrinkage when left in
benzene for a long time. Should be transferred promptly to
paraffin wax promptly
▪Toxicity:
▪Harmful to the bone marrow resulting in Aplastic Anemia (use only in
well ventilated laboratory)
▪Carcinogenic
Chloroform
▪ Slower than xylene but causes less brittleness (6-24 hours clearing)
▪ Thicker tissue blocks up to 1cm in thickness can be processed
▪ Tissues DO NOT become translucent (cannot be used for mounting)
▪ Only for clearing of tissues for embedding
▪ For tough tissue (Skin, fibroid and decalcified tissues)
▪ Minimal shrinkage and hardening, good for nervous tissue, lymph nodes
and embryo
▪ Suitable for large specimen
▪ NOT flammable
▪Toxic to the liver with prolonged inhalation (Prevented by adequate room
ventilation and proper caution in handling specimen)
▪Wax impregnation after chloroform clearing is relatively slow
▪Not volatile in paraffin oven therefore difficult to remove from paraffin sections,
may even cause deterioration
▪Its vapor may attack rubber seal used in vacuum impregnation bath
▪Complete clearing is difficult to evaluate
▪Tissues tend to float, avoided by wrapping tissue with absorbent cotton gauze
to sink the tissue in solution
▪Evaporates quickly from water bath
Cedarwood oil
▪ For clearing of both paraffin and Celloidin sections during
embedding
▪Especially recommended for:
▪ Central nervous system tissues
▪ Cytological studies of smooth muscles and skin

▪Requires 2 changes in clearing solution which is usually complete in

2-3 days
▪Excellent penetration
▪Clears Celloidin in 5-6 days
▪Minimal shrinkage and hardening of tissues
▪No considerable damage or distortion even after indefinite
immersion
▪ Makes tissue transparent
▪ Often improves cutting of sections
▪Extremely slow not for routine
▪Hard to eliminate from paraffin bath which makes impregnation very slow (hastened by
transferring specimen from oil to benzene for ½ hour before finally placing the tissue
in wax)
▪Inconsistent quality. Tissue ,may dry out before complete clearing (Tissues initially float
in the oil before gradually sinking to the bottom as clearing proceeds)- prevented by
superimposing absolute alcohol on the surface of the clearing agent. Once saturated,
the specimen should then be transferred to fresh solution of cedarwood oil
▪Oil used to clear acetic-alcohol fixed tissues produce crystals with a melting point of
35°C and interfere with clearing of tissues.- solution must be heated to 200°C to
dissolve the crystals and restore solution to normal state
▪It is very expensive
Aniline oil
▪Not routine
▪Recommended for clearing embryos, insects and very delicate
specimens, due to its ability to clear 70% alcohol without excessive
tissue shrinkage and hardening
Carbon Tetrachloride
▪May be used for clearing tissues for embedding
▪Properties and toxicities are the same as Chloroform but relatively
cheaper
▪Causes considerable tissue hardening
▪Highly toxic on prolonged exposure by inhalation
Tetrahydrofuran
▪Dehydrates and clears simultaneously
▪Non-toxic but foul in odor
Methyl benzoate and Methyl salicylate
▪Slow acting clearing agents
▪Used for double embedding techniques are required
Double embedding:any processing system which uses two support media at the
same time.
▪ Peterfyi's celloidin-paraffin wax technique.
▪ Gelatin-paraffin wax and agar-paraffin wax methods

▪Purpose: obtain increased support to the tissues, the first inclusion procedure
often being used to hold the tissues in place while the second infiltrates.