Pre-Finals
CLEARING DISADVANTAGES:
Highly flammable,
longer than 3 hours make tissue excessively
Is the process whereby alcohol or dehydrating agent brittle
is removed from the tissue and replaced with a Not suitable for nervous tissues and lymph
substance that will dissolve the wax with which the nodes
tissue is impregnated or the medium on which the Becomes milky when incomplete dehydrated
tissue is mounted (canada balsam) tissue is immersed
Tissue has translucent appearance
Because of high refractive indices embryos and TOULENE
parasites become transparent, internal structures
become visible to the naked eye. TIME- 1-2 hours, for routine, NOT carcinogenic
Prolonged exposure causes the tissue to become Miscible with absolute alcohol and paraffin
brittle and difficult to cut Tissues do not become excessively hard and
Low boiling points are readily replaced by melted brittle even if left for 24 hours
paraffin
Viscosity affects the speed of penetration of clearing DISADVANTAGES:
agent Slower than xylene n benzene
More expensive
CHARACTERISTICS Acidify the partially filled vessels
Emit toxic fumes if high conc.
Miscible with alcohol, for rapid removal of
dehydrating agent BENZENE
Miscible and easily removed with melted paraffin
wax for impregnation and mounting Clears 15-60 minutes, routine, makes tissue
Should not produce excessive shrinkage, hardening transparent
or damage of tissue Rapid acting, urgent biopsies
Should not dissolve out aniline dyes Miscible with abs alcohol
Should not evaporate quickly in a water bath Volatile, Easily eliminated
Make tissue transparent Minimum shrinkage and does not tissues hard and
brittle
COMMON CLEARING AGENTS
DISADVANTAGES:
1. Xylene Highly flammable, well ventilated room needed
2. Toluene
Toxic, carcinogenic, may damage bone marrow
3. Benzene
(aplastic anemia)
4. Chloroform
5. Cedarwood oil
CHLOROFORM
6. Aniline oil
7. Clove oil
Routine (6-24 hours)
8. Carbon tetrachloride
Miscible with abs alcohol, large tissue specimens,
not flammable
XYLENE
Recommended for tough and dense tissues (skin,
uterus, fibroid and decalcified)
Colorless, most commonly used
Nervous, lymph nodes, embryos (due to minimum
Time ½ - 1 hour
shrinkage and hardening of tissues)
Most rapid, cheap
Slow penetrating transition solvent
Urgent biopsies 15-30 minutes
Tissue transparent
DISADVANTAGES:
Miscible with absolute alcohol and paraffin
TOXIC TO LIVER, expensive
Does not extract out aniline dyes
Does not dissolve celloidin WAX IMPREGNATION is slow
Evaporates quickly in paraffin oven, readily replaced Does not make tissue transparent
by wax during impregnation and embedding Difficult to remove from paraffin section, not
volatile
Vapor attacks the rubber seal used in vacuum ESTERS
impregnating bath
Evaporates quickly from water bath Colorless, flammable reagents
Tissue floats; to avoid-wrap it with absorbent Miscible with most organic solvents and
cotton gauze paraffin
CEDARWOOD OIL N-BUTYL ACETATE
Requires 2 changes Used as xylene substitute and nitrocellulose
Clears tissue from 95% alcohol solvent
Recommended for dense tissues (uterus) CNS,
cytologic studies, smooth muscles and skin LIMONENE
Tissues can remain indefinitely 2-3 days
Clears celloidin 5-6 days Derived from citrus fruit,
Does not dissolve out aniline dyes Similar to the esters in clearing action and
Makes tissue transparent eliminating from wax
DISADVANTAGES: TERPINEOL
SLOW, becomes milky upon prolonged storage,
very expensive, no uniform quality, turns milky Clear, almost colorless mixture of isomers
upon prolonged storage Very low evaporation rate
Expensive Substitute for cedarwood oil
Used oil can be restored by filtering then heating
to 60°C under vacuum for 30-60 minutes IMPREGNATION AND
ANILINE OIL EMBEDDING
For embryos, insects, very delicate specimens IMPREGNATION
CLOVE OIL Is the process whereby the clearing agent is
completely removed from the tissue and replaced
Wax impregnation is slow, expensive by a medium that will completely fill all the tissue
Causes minimum shrinkage cavities, thereby a firm consistency to the specimen
Tissues become brittle aniline dye is removed,
celloidin wax is dissolved EMBEDDING CASTING OR BLOCKING
CARBON TETRACHLORIDE Process whereby the impregnated tissue is placed
into a precisely arranged position in a mold
Properties similar to chloroform containing a medium
Cheaper
Highly toxic PARAFFIN WAX
Amyl acetate METHYL BENZOATE AND Is a polycrystalline mixture of solid
METHYL SALYCILATE hydrocarbons
Melting poin 39-68oC
Slow-acting
Simplest, most common and best embedding
Double embedding techniques
medium
Chiefly used as nitrocellulose solvents in
Individual serial sections may be cut with ease
double embedding techniques
Rapid process, prepared w/in 24 hours
TERPENES Tissue blocks and unstained mounted sections
maybe stored for indefinite period of time
Are isoprene polymers (natural plants & Many staining procedures are permitted w/ good
synthetic) staining results
Biodegradable, not water soluble
Disposed by recycling or incineration
DISADVANTAGES: Plastic embedding rings and base mold
Overheated makes tissue brittle - Consist of a special stainless steel base mold
Prolonged will cause excessive tissue shrinkage and fitted with a plastic embedding ring.
hardening
Inadequate impregnation promotes retention of Leuckhart’s embedding mold
clearing agents, tissues become soft and shrunken, - Consists of 2 L-shaped strips of heavy brass or
tissue blocks crumble when sectioned and break up metal arranged on a flat metal plate and which
when floated in water bath can be moved to adjust the size of the mold to
the size of the specimen.
PARAFFIN
Paper boat
Bones, teeth, brain eyes are difficult to - Cheap and easy to make
impregnate, need long immersion for proper support
otherwise they will crumble Peel-Away
Not recommended for fatty tissues - Peeled off one at a time, as soon as the wax has
After completely cleared, the tissue is submerged in solidified
2 or more changes of melted paraffin wax either in
oven or incubator at 55-60°C Common waxes have the melting points of 45°C,
52°C, 56°C and 58°C. The 56°C wax is usually used
MODIFIED PARAFFIN WAXES in routine work
Hard tissues require wax with higher melting
Increase hardness due to stearic acid point than soft tissues
Decrease melting point due to phenanthrene
Enhance tissue wax adhesion 3 WAYS OF PARAFFIN IMPREGNATION AND
Added with piccolyte 115, plastic polymers and EMBEDDING
dimethyl sulphoxide
By manual processing
Paraffin wax impregnation is the simplest, most By automatic processing
common and best embedding medium used for By vacuum embedding
routine processing.
MANUAL PROCESSING
FACTORS AFFECTING PARAFFIN WAX
IMPREGNATION At least 4 changes of waxes are required at 15
1. Nature of tissues minutes interval (for removal of clearing agent)
2. Size of tissues Specimen is immersed into another fresh
3. Type of clearing agent solution of melted paraffin for 3 hours for
completely embedding/casting
CELLOIDIN IMPREGNATION
AUTOMATIC PROCESSING
Recommended for neurological tissues
Does not require heating during processing; hence, Fixes, dehydrates, clears and infiltrates tissues
producing minimum shrinkage and tissue distortion Only 2 to 3 changes of waxes are required
especially for cutting large bone tissues There is constant agitation w/c accelerates and
Frozen section technique cannot be done improve tissue penetration
Example: Elliott Bench-Type Processor
Blocking-out Molds Leuckhart’s, Compound, Plastic
Embedding Rings and Base Mold, Peel Away, Plastic The machine is mounted on rollers to permit the
Ice Trays, Paper Boats turning of platforms and easy access to beakers and
wax baths
Compound embedding unit The presence of odor in the clearing agent during
- Made up of series of interlocking plates resting final paraffin wax bath indicates that the paraffin
on a flat metal base, forming several should be changed
compartments. Dehydrating agents should be changed regularly
since it is the most critical stage
Inadequate dehydration is difficult to correct since
tissue is in paraffin
Clearing agents and dilute ethanol should be Tubular and walled specimens such as cyst,
changed at least once a week fallopian tubes and GIT are embedded en face so as
Avoid spillage, fluid and wax containers should be to provide cross sections showing all tissue layers
filled to the appropriate level Tissues w/ epithelial surface such as skin are
Wax bath thermostats should be at least 3 degrees embedded to provide sections in a plane at a right
above melting point of the wax. angle to the surface
Multiple tissue pieces are aligned across the long
VACUUM EMBEDDING axis and the center of the mold and not placed
randomly.
Fastest result
Involves wax impregnation under negative
atmospheric pressure inside a embedding oven to
hasten removal of air bubbles and clearing agent
form the tissue block thereby promoting a more
rapid wax impregnation
Removes residual air bubbles in lungs
For urgent biopsies, brain connective tissue, decal
bone eyes spleen CNS
Time required is reduced by 25-75%
Controlled water jacket usually maintained at 2-4°C
above melting point of wax
SUBSTITUTES FOR PARAFFIN WAX
Paraplast
Ester wax
Water soluble media - such as polyethylene glycols
are used to investigate heat-and solvent-labile lipids
and proteins
AQUEOUS MEDIA
Includes agar, gelatin, sodium carboxymethyl
cellulose and polyvinyl alcohol
4 MAIN TYPES OF MOLD-EMBEDDING
Traditional
Leuckart or dimmock irons or metal containers
Peel-away system using disposable plastic molds
Embedding rings or cassette bases w/c become
integral part of the block and serve as the block
holder in the microtome
ORIENTING TISSUE IN THE BLOCK
Most important step in embedding
Improper placement may result in missed or
damaged tissue portions during microtomy
Tissues are blocked with the surface to be cut facing
down in a mold
Elongated tissues are placed diagonally across the
block
