CLEARING, INFILTRATION AND EMBEDDING

CLEARING (DEALCOHOLIZATION) UNIT

 Alcohol/dehydrating fluid is removed from the tissue and replaced with an intermediate PLASTIC
solvent that is fully miscible with both ethanol and paraffin wax EMBEDDING
RINGS AND
BASE MOLDS
APPLICATIONS DISPOSABLE
 To make tissue, embryos and parasite transparent EMBEDDING
 For dealcoholization of tissue preparatory to wax impregnation FOUR TYPES OF IMPREGNATION AND EMBEDDING MOLDS
 For dealcoholization of stained sections prior to mounting in PARAFFIN WAX
o Permount Three Ways of Paraffin Impregnation and Embedding
o Clarite  At least four changes of wax is required at 15 minute-interval in
o Canada Balsam order to ensure complete removal of the clearing agent from
COMMON CLEARING AGENTS the tissue
Manual or Hand  The specimen is immersed in another fresh solution of melted
 Most commonly used colorless clearning agent
Processing paraffin for approximately 3 hours to ensure complete
 Most rapid clearing agent
embedding or casting of tissue
 Used for clearing, embedding and mounting
XYLENE (XYLOL)  Requires paraffin oven set at approximately 2-5C above the
 De-waxing agent during staining
 Causes considerable hardening and shrinking tissues is immersed MP
in it Automatic  More rapid than manual
 For tough tissues, nervous tissues, lymph nodes and embryo Processing  Requires two to three changes of paraffin wax set at 3C above
o Skin (Autotechnicon) the MP
o Fibrinoid  Wax impregnation under negative atmospheric pressure inside
CHLOROFORM an oven
o Decalcified tissues Vacuum
 Fastest method which is recommended for urgent biopsies,
 Does not make tissue transparent Embedding
delicate tissues such as lungs, connective tissues, decalcified
 Toxic to the liver after prolonged inflammation
bones, eyes, spleen and central nervous system
 Rapid acting clearing agent
Substitutes for Paraffin Wax
 Highly inflammable
BENZENE  Mixture of highly purified paraffin and synthetic plastic polymers
 Excessive exposure to benzene may be extremely toxic Paraplast
 For large dense tissue blocks (bones and brain)
 May damage the bone marrow (Aplastic Anemia)
 Synthetic wax substitute similar to paraplast
 Clearing time 1 – 2 hours
 Less brittle and less compressible than paraplast
TOLUENE  May be used as a substitute for xylene or benzene for clearing both
during embedding and mounting process
 Mused to clear both paraffin and celloidin sections during Embeddol
embedding process - Semi-synthetic wax
Biloid
CEDARWOOD  Recommended for CNS tissues and cytological studies - Recommended for embedding eyes
OIL  Extremely low acting - Production of paraffin containing rubber with
Tissue Mat
 Becomes MILKY upon prolonged storage and should be filtered same property as paraplast
before use
Ester Wax
 Recommended for clearing embryos, insects and very delicate
specimens
ANILINE OIL Water Soluble Wax
 Due to its ability to clear 70% alcohol without excessive tissue
shrinkage and hardening Dimethyl
 Quality is not guaranteed due to its tendency to become sulphoxide (DMSO)
CLOVE OIL
adulterated
CELLOIDIN
CARBON  Same properties to chloroform but cheaper
TETRACHLORIDE
METHYL  Slow acting clearing agents that can be used when double
BENZOATE AND embedding techniques are required Three Methods of Embedding
METHYL Dry Celloidin
SALICYLATE
OTHER CLEARING AGENTS Wet Celloidin
M – Butyl-Acetate Carbon Disulfate
- Xylene substitute - Smooth muscle Nitrocellulose
- Nitrocellulose solvent - Foul odor
Terpenes (Plant Oil) GELATIN
- Earliest transition solvent used in histology Carbon Xylene
- Terpentene oils of Bergamot, clove, lemon, - Foul odor
oreganum, sandalwood PLASTIC (RESIN)
Orange Oil Terpineol
Chlorinated Hydrocarbons Phenol
Oil of Wintergreen
Coconut Oil
- Delicate tissues
Bleached Palm Oil Plastic Classification
EPOXY
IMPREGNATION (INFILTRATION)
POLYESTER
 Clearing agent is completely removed from the tissue and replaced by a medium that will
completely fill all the tissue cavities
ACRYLIC
EMBEDDING (CASTING, BLOCKING, MOLDING)
 Paraffin impregnated tissues are placed into a mold containing the embedding medium
together with their proper labels DOUBLE EMBEDDING METHOD
 Immersed in melted paraffin at a temperature between 5 – 10C above the melting point
 Cooled at -5C or immersed in cold water to solidify
MOLDS FOR EMBEDDING
LEUCKHART’S
ORIENTATION – tissue is arranged in a precise position in the mold during embedding, on the EMBEDDING
microtome before cutting and on the slide before staining COMPOUND
EMBEDDING