IMPREGNATION sectioned and break up when floated out in a water

bath.
What is Impregnation? • Tissues that are difficult to infiltrate, e.g. bones,
→ Also called “infiltration”. teeth, brains and eyes, need long immersion for
→ Process whereby the clearing agent is completely proper support; otherwise, they will crumble on
removed from the tissue and replaced by a medium sectioning. Prolonged immersion in paraffin, on the
that will completely fill all the tissue cavities and other hand, is not advisable.
give a firm consistency to the specimen.
→ Allows easier handling and cutting of suitably thin • Paraffin processing is not recommended
sections without any damage or distortion to the for fatty tissues. The dehydrating and
tissue and its cellular components. clearing agents used in the process dissolve
and remove fat from the tissues.
TYPES OF IMPREGNATION AND Points to Remember!
EMBEDDING MEDIUM • Common waxes have melting points of
45C, 52C, 56C, and 58C.
I. PARAFFIN WAX IMPREGNATION o 56C is the temperature for routine
→ The simplest, most common and best embedding work.
medium. o Laboratory temperature between
→ Paraffin wax is a polycrystalline mixture of solid 20−24C = melting point of wax to be
hydrocarbons produced during the refining of coal
used should be between 54−58C.
and mineral oils.
o Laboratory temperature between
Advantages 15−18C = melting point of wax to be
• Thin individual serial sections may be cut with ease used should be between 50−54C.
from the majority of tissues without distortion. • Hard tissues require wax with a higher
• The process is very rapid, allowing sections to be melting point than soft tissues.
prepared within 24 hours.
• Tissue blocks and unstained mounted sections may WAYS OF PROCESSING
be stored in paraffin for an indefinite period of time 1. Manual Processing
after impregnation without considerable tissue → At least four changes of wax are required
destruction. at 15 minutes intervals in order to insure
• Because formalin-fixed, paraffin-embedded tissues complete removal of the clearing agent
may be stored indefinitely at room temperature, from the tissue.
and nucleic acids (both DNA and RNA) may be
→ The specimen is then immersed in
recovered from them decades after fixation, they another fresh solution of melted paraffin
are an important resource for historical studies in
for approx. 3 hours to insure complete
medicine. embedding or casting of tissue.
• Many staining procedures are permitted with good
→ The following is an example of a time
results.
schedule for manual processing of tissues
Disadvantages about 3 mm. thick:
• Overheated paraffin makes the specimen brittle.
Fixation
• Prolonged impregnation will cause excessive tissue
10% Buffered Formalin 24 hours
shrinkage and hardening, making the cutting of
Dehydration
sections difficult.
70% Alcohol 6 hours
• Inadequate impregnation will promote retention of 95% Alcohol 12 hours
the clearing agent. Tissues become soft and 100% Alcohol 2 hours
shrunken, and tissue blocks crumble when 100% Alcohol 1 hour
 100% Alcohol 1 hour
Clearing Precautions with Automatic Processing
Xylene or Toluene 1 hour • Fluids are changed on the number and sizes of the
Xylene or Toluene 1 hour tissues processed.
Impregnation • Odor indicates the need for change of paraffin.
Paraffin wax 15 minutes • Dehydrating solution should be changed frequently
Paraffin wax 15 minutes since dehydration is the most critical stage of
Paraffin wax 15 minutes tissue processing.
Paraffin wax 15 minutes • The first 100% ethanol bath should always be
Embedding changed.
Paraffin wax 3 hours • Clearing agent and diluted ethanol should be
changed at least once a week.
• To avoid spillage, fluid and wax should be filled to
2. Automatic Processing the appropriate level and must correctly mounted.
→ Uses an automatic tissue processor. • Wax bath thermostats should be set at least 3
o Fixes, dehydrates, clears and infiltrates tissue. above the melting point of the wax.
o Decreases time and labor. • Due to the viscosity of molten paraffin wax, some
o Usually needs only 2 changes of paraffin. form of gentle agitation is highly desirable.
o Works through constant agitation.
→ Machine is mounted on rollers to permit the turning 3. Vacuum Processing
of platforms and easy access to beakers and wax → Recommended for urgent biopsies.
baths. → Involves wax impregnation under
o 12 individual processing steps, with ten 1-liter negative atmospheric pressure inside an
capacity glass beakers. embedding oven.
o Two thermostatically controlled wax baths with
→ The time required for complete
a safety device cut-out switch to protect the wax
impregnation is reduced by 25% − 75%
against over-heating.
of the normal time required for tissue
Reagent Processing Time processing.
Schedule Schedule Schedule
→ Vacuum embedding oven consist of flat-
1 2 3
bottomed heavy brass chamber covered
10% Buffered 2 hours 2 hours 2 hours
Formalin with a heavy glass lid resting on wide and
10% Buffered 2 hours 2 hours 2 hours thick rubber valve.
Formalin → The vacuum chamber is enclosed in a
70% Alcohol 2 hours 2 hours 2 hours thermostatically controlled water- jacket,
Absolute Alcohol (1) 1 hour 3 hours 2 hours usually maintained at 2−42C above the
Absolute Alcohol (2) 1 hour 3 hours 2 hours melting point of the wax.
Absolute Alcohol (3) 1 hour 3 hours 2 hours → The degree of the vacuum should not > 500
Xylene or Toluene (1) 1 hour mm. Hg.
Xylene or Toluene (2) 1 hour → A stopcock is provided to prevent water
Benzene (1) 30 from being sucked back into the trap
minutes bottle and vacuum chamber when the
Benzene (2) 1 hour water or suction pump is closed.
Chloroform (1) 2 hours ✓ After fixation and dehydration, proceed as
Chloroform (2) 2 hours follows:
Chloroform (3) 2 hours 1. Clear in two changes of xylene, for 1 hour
Wax (1) 2 hours 3 hours 2 hours each.
Wax (2) 3 hours 3 hours 2 hours
 2. Place the tissue in molten wax, in vacuum → It is soluble in common clearing agents and follows
chamber and make the oven airtight. the same time schedule for paraffin impregnation,
3. Exhaust the air slowly by means of a and does not tend to crack like other paraffin wax
vacuum pump or Venture suction pump substitutes.
until there is a negative pressure of 400 to → Paraplast with a melting point of 56−58C is
500 mm. mercury. recommended.
4. Leave for 15 minutes, then slowly readmit o During the winter, 54−56C Paraplast may be
air until normal atmospheric pressure is used if the tissue is cut in a cool room.
reached. o During the summer, it may be necessary to use
5. Place the tissue in fresh wax. 60−63C, although this is to be avoided in order
6. Repeat steps 3 and 4. to not to "cook" the tissue (does not section well
7. Place the tissue into fresh wax. or does not stain well).
8. Repeat step 3 and leave for 30 to 45 B. Embeddol
minutes. → A synthetic wax substitute similar to Paraplast with
9. Bring to normal atmospheric pressure and a melting point of 56−58°C.
embed the tissue.
→ It is less brittle and less compressible than
NOTE: The exhaustion and readmission of air
Paraplast.
must be gradual or the specimen may be ruined.
▪ Bio/aid − a semisynthetic wax recommended
Factors affecting Paraffin wax impregnation for embedding eyes.
• Size of the tissue ▪ Tissue Mat − a product of paraffin, containing
• Type of clearing agent rubber, with the same property as Paraplast.
• Numbers of changes of clearing agent C. Ester Wax
Precautions observed in Paraffin wax → Has a lower melting point (46−48°C), but it is
impregnation harder than paraffin.
• Prolonged treatment in melted paraffin → It is not soluble in water, but is soluble in 95%
causes tissue shrinkage. Ethyl Alcohol and other clearing agents; hence, it
• Infiltration above the melting point can be used for impregnation without prior clearing
produces shrinkage and hardening Paraffin of the tissue.
wax should be pure. → Cellosolve (ethylene glycol monoethyl ether) or
xylene may be used as clearing agents.
• Wax that has been trimmed can be recycled (filter → Three to four changes of wax are required to ensure
before use). complete tissue impregnation.
• Paraffin with water can be removed by heating the → Sectioning of ester wax-impregnated tissues should
wax (100−105C). be done on a heavy-duty microtome (e.g., sliding
• Paraffin wax can only be used twice. or sledge type microtome) due to the relative
hardness of the wax.
SUBSTITUTES FOR PARAFFIN WAX
I. Paraplast
→ A mixture of highly purified paraffin and synthetic D. Water Soluble Wax
plastic polymers, with a melting point of 56−57°C. → Plastic polymers, mostly polyethylene
→ It is more elastic and resilient than paraffin wax glycols with melting points of 38−42°C
thereby permitting large dense tissue blocks such or 45−56°C.
as bones and brain to be cut easily with the same → Polymer waxes are incorporated in the
result as in double embedding. majority of proprietary histological
→ Blocks obtained are more uniform than with any paraffin wax blends to improve adhesion,
other medium, with better ribboning of sections. hardness and plasticity.
 → The most commonly used is Carbowax, and speed of technique (w/c can take
a polyethylene glycol containing 18 or several weeks or months).
more carbon atoms, which appears solid Advantages
at room temperature. • It permits cutting of tissue sections which
→ It is soluble in and miscible with water; are thicker than in paraffin wax, and is
hence does not require dehydration and recommended for processing of
clearing of the tissue. neurological tissues.
→ Carbowax technique is suitable for • Its rubbery consistency allows tissue blocks that are
many enzyme histochemical studies. either very hard or of varying consistency, to be cut
→ For routine processing, 4 changes of without undue distortion.
Carbowax, one each in 70% and 90% • Dense tissues which are hard to infiltrate (e.g.,
and 2 times in 100% concentration, at a bones and brain) and specimens which tend to
temperature of 56°C are used, at 30 collapse easily due to air spaces (e.g., eyes) are
minutes, 45 minutes and 1 hour (with supported better, thereby avoiding the crumbling of
agitation). tissues during sectioning. When eye sections are
→ Specimens are then embedded in fresh embedded by the paraffin method, the retina may
Carbowax at 50°C and rapidly cooled in be detached from the harder tissues (e.g., sclera and
a refrigerator. choroid) that encircle it. The cedarwood oil used
→ Carbowax is very easily dissolved in in the dry celloidin technique helps to soften the
water due to its hygroscopic nature. brittle layers.
→ Tissue sections are very difficult to float • It does not require heat during processing; hence,
out and mount due to its extreme producing minimum shrinkage and tissue distortion
solubility in water, dehydrating and especially for cutting large bone sections. It is,
clearing agents. therefore, recommended in cases when minimum
→ Adding soap to water or using 10% shrinkage is required and when frozen section
Polyethylene Glycol 900 in water will technique cannot be done.
reduce tissue distortion.
Dis Advantages
II. CELLOIDIN IMPREGNATION
• Celloidin impregnation is very slow (lasting for
→ Also called as “Collodion”. several days or weeks).
→ A purified form of nitrocellulose soluble • Very thin sections (less than I 0 μ) are difficult to
in many solvents, suitable for specimens cut.
with… • Serial sections are difficult to prepare.
▪ large hollow cavities which tend to • Vapor of the ether solvent is very flammable;
collapse. hence, it should never be used near an open flame.
▪ for hard and dense tissues such as • Photomicrographs are difficult to obtain.
bones & teeth. • It is very volatile and therefore must be kept in
▪ for large tissue sections of the whole bottles with ground−glass stoppers to prevent
embryo. evaporation.
→ Used mainly for preparing soft tissue
sections of mixed consistency such as Methods Used for Celloidin Impregnation of
eyes and brain. Tissue
→ No heat is required, and the resultant ✓ Wet Celloidin Method
block has a rubbery consistency which o Recommended for bones, teeth, large brain
gives good support to the tissues. sections and whole organs.
→ Disadvantages include inability to cut ✓ Dry Celloidin Method
o Preferred for processing of whole eye sections.
thin sections, storage of blocks in alcohol
✓ Nitrocellulose Method EMBEDDING
o Low Viscosity Nitrocellulose (L.V.N.) is - After impregnation, the tissue is placed into a
another form of celloidin soluble in equal mold containing the embedding medium and
concentration of ether and alcohol, with a lower this medium is allowed to solidify
viscosity, allowing it to be used in higher - the embedding medium should match the
concentrations and still penetrate tissues rapidly. tissue type in strength and hardness.
o It forms a harder tissue block and makes cutting • too soft for the material: the tissue will
of thinner sections possible. not be supported and sections will be torn
o The tendency of tissues to crack may be or shredded.
prevented by adding plasticizers (e.g., oleum • too hard for the tissue: sections will be
ricini or castor oil) when embedding chrome- brittle and will shatter.
mordanted tissues. - To infiltrate the tissues with supporting
o Low viscosity nitrocellulose is more explosive embedding medium, tissues must be free of
than celloidin. all water (since usually embedding medium
is not miscible with water).
III. GELATIN IMPREGNATION - Paraffin embedded tissues are arranged at
→ Rarely used except when dehydration is the bottom of the mold together with their
to be avoided and when tissues are to be proper labels and immersed in melted
subjected to histochemical and enzyme paraffin at a temperature between 5-10°C
studies. above its melting point and then cooled
→ It is used as an embedding medium for rapidly in a refrigerator at -5°C or immersed
delicate specimens and frozen tissue in cold water to solidify.
sections because it prevents - This will allow hardening of tissues, giving
fragmentation of tough and friable tissues them a firmer consistency and better support,
when frozen sections are cut. thereby facilitating the cutting of sections.
→ It is water-soluble, and does not require - The process by which a tissue is arranged in
dehydration and clearing, although precise positions in the mold during
fixatives (such as 10% formalin) should embedding, on the microtome before cutting,
still be washed out by running water and on the slide before staining, is known as
whenever indicated. Orientation.
→ It has a low melting point and does not - the surface of the section to be cut should be
cause over-hardening of tissues by placed parallel to the bottom of the mold in
heating. which it is oriented.
→ Tissues should not be > 2-3 mm thick - Several types of Blocking-out Molds may be
since gelatin-embedded specimens are used:
harder to freeze than non-impregnated 1. Leuckhart’s Embedding Mold:
tissues. consists of two L-shaped strips of heavy
→ 1% phenol serves to prevent the growth brass or metal arranged on a flat metal
of molds. plate and which can be moved to adjust
→ The volume of the impregnating medium the size of the mold to the size of the
should be at least 25 times the volume of specimen. Blocks produced are even,
the tissue. with parallel sides, and with a fairly
shaped initial setting of the wax. The
mold is adjustable to give a wide variety
of sizes to fit the size of the tissue block
for casting. It is recommended for routine
use, although, too slow and cumbersome
for use in a busy laboratory.
 2. Compound Embedding Unit: made up 4. Disposable Embedding Molds
of a series of interlocking plates resting a. Peel-Away: disposable thin plastic
on a flat metal base, forming several embedding molds, available in 3
compartments. It has the advantage of different sizes, are simply peeled off
embedding more specimens at a time, one at a time, as soon as the wax has
thereby reducing the time needed for solidified, giving perfect even block
blocking. without trimming. It may be placed
3. Plastic Embedding Rings and Base directly in the chuck or block holder
Mold: consist of a special stainless steel of the microtome.
base mold fitted with a plastic embedding b. Plastic Ice Trays: such as those used
ring, which later serves as the block in ordinary refrigerators may be
holder during cutting. recommended for busy routine
laboratories. Each compartment may
be utilized for embedding one tissue
block, which may then be removed
• Tissue Tek by bending the plastic tray once the
- equipped with a warm plate to manage the wax has solidified or by smearing the
impregnated specimen, and a cold plate at - inner mold with glycerin or liquid
5°C for rapid solidification of the block. paraffin before embedding.
- It consists of a white plastic cassette mold c. Paper Boats: normally utilized for
with detachable, perforated stainless steel embedding celloidin blocks but are
hinge and Snap-On lid, used to hold the tissue equally useful for paraffin wax
specimen through-out fixation, dehydration, blocks. They have the advantage of
clearing and wax impregnation. being cheap and easy to make. They
- the specimen is placed on the base mold, the provide easy and accurate
plastic embedding ring is placed in position, identification of specimen, thereby
filled up with wax, and then placed on a small avoiding confusion and interchange
cool area to allow the wax in the base of the of tissue blocks. Rapid embedding of
mold to semi-harden. This will allow easy small or large volume of individual
orientation of the block. specimen is possible, since paper
- Once the tissue has been properly oriented, molds can be made to suit any size of
the base of the cassette is placed on top and tissue.
together, they are placed on the cold plate so - To mark the position of small tissues in the
that the paraffin wax can cool and harden paraffin block, a mark such as an "X" is
quickly. drawn with soft lead pencil on the inner
- After the paraffin wax has solidified (usually surface of the bottom of the boat. This will
5 minutes), the block is taken out together attach and be visible on the wax block when
with the embedding ring and is immediately solidified and removed from the paper boat.
ready for cutting without having to undergo - Embedding molds should bear the case
trimming or mounting, thereby saving time number, and other identification data of the
and effort. tissue block within. Once tissues have been
- Advantages: include ease of use, less embedded, they may be stored in a cool place
paraffin wax needed, faster embedding, indefinitely until they are cut. T
firmly attached tissue and holder, and - The choice of embedding mold will depend
permanent identification. on the type of chuck in the microtome you
- It produces easier orientation when will use to section the tissue. Stainless steel,
resectioning of tissue is required, and blocks ceramic, paper, plastic, and aluminum foil
can be filed immediately after molds can be used.
 1. Open cassette to view tissue sample and oven immediately and close oven door
choose a mold that best corresponds to between transfers.
the size of the tissue. A margin of at least
2 mm of paraffin surrounding all sides of
the tissue gives best cutting support.
Discard cassette lid.
2. Put small amount of molten paraffin in - Orientation of tissues in the Paraplast block is
mold, dispensing from paraffin reservoir. important for tissues such as an artery or
3. Using warm forceps, transfer tissue into fallopian tube so that they can be properly
mold, placing cut side down, as it was placed in a predetermined plane, such as
placed in the cassette. cross sections.
4. When the tissue is in the desired - Trimming is excessively difficult in a block
orientation, add the labeled tissue embedded with two or more tissues if they are
cassette on top of the mold as a backing. not carefully lined up before the Paraplast is
Press firmly. cooled.
5. Hot paraffin is added to the mold from - Celloidin or Nitrocellulose Embedding
the paraffin dispenser. Be sure there is Method used to be recommended for
enough paraffin to cover the face of the embedding hard tissues such as bones and
plastic cassette. teeth, and for large sections of whole organs
6. Cool the top surface of the Paraplast by like the eye, since the delicate layers of the
blowing gently on it. Tissues at this stage eyeball are difficult to keep intact when other
are very brittle and should be handled media are used.
with care. If necessary, fill cassette with - Tissues are embedded in shallow tins of
paraffin while cooling, keeping the mold enamel pans which are covered by sheets of
full until solid. weighted glass.
7. Cool thoroughly in cold running tap - Bell jars can be used to control the rate or
water. If you use ice water for the final evaporation of the solvent.
cooling, you may split the block owing to - The use of celloidin is discouraged now
too rapid shrinkage. Paraplast naturally because of the special requirements needed
splits in the line of least resistance-right for processing and the limited use of these
through the tissue types of sections in neuropathology
8. Paraffin should solidify in 30 minutes. - Double-Embedding: the process by which
When the wax is completely cooled and tissues are first embedded or fully infiltrated
hardened (30 minutes) the paraffin block with a supporting medium such as agar or
can be easily popped out of the mold; the nitrocellulose, then infiltrated a second time
wax blocks should not stick. If the wax with paraffin wax in which they are
cracks or the tissues are not aligned well, subsequently embedded.
simply melt them again and start over. - This is used to facilitate cutting of large
9. If you use plastic cups, the Paraplast blocks of dense firm tissues like the brain.
block can be removed as soon as it is - They are also recommended for making small
cooled. The stainless steel mold should sections of celloidin blocks.
slip off easily when cool and can be used - A shortcoming of using agar as the pre-
again. embedding media is that certain tissues
- It is important to work quickly while shrink during the embedding process, and the
transferring specimens or wax from oven agar-based pre-embedding media limits
because wax hardens quickly. Always tissue expansion during slide mounting,
remember to put wax container back into
 resulting in difficulties with the tissue sample carcinogenic. For protection, gloves should
adhering to the microscope slide. always be worn when handling these plastics,
PLASTIC (RESIN) EMBEDDING and adequate facilities including an
- The introduction of plastic resin embedding operational fume hood must be provided to
media has provided superior results for light remove the toxic vapors and properly dispose
microscopic studies, particularly in hard of toxic waste.
tissues such as undecalcified bone and for - Polyester plastics: originally introduced for
high resolution light microscopy of tissue electron microscopy in the mid- 1950s, but
sections thinner than the usual 4-6 µm, such have been superseded by more superior
as renal biopsies and bone marrow biopsies. epoxides, and are now seldom used.
- Plastics are classified into epoxy, polyester, - Acrylic plastics: made up of esters of acrylic
or acrylic, based on their chemical or methacrylic acid, and are used extensively
composition. for light microscopy.
- Epoxy embedding plastics are made up of a - Polyglycol methacrylate (GMA): has
carefully balanced mixture of epoxy plastic, proved to be a popular embedding medium
catalysts and accelerators. for light microscopy because it is extremely
- Three types of epoxy plastics are used in hydrophilic, allowing many staining methods
microscopy, i.e., those based on either to be applied, yet tough enough when
bisphenol A (Araldite), or glycerol (Epon), dehydrated to section well on most
or cyclohexene dioxide (Spurr). microtomes.
- Infiltration by Araldite is slow, partly - The polar water soluble, 2-hydroxyethyl
because the epoxy plastic itself is a large methacrylate, commonly known as "glycol
molecule. methacrylate", or GMA, has found an
- The glycerol-based epoxy plastics (Epon) increasing number of applications for the
have a lower viscosity but are often sold as embedding of biological tissue for
mixtures of isomers. transmission electron microscopy (TEM), for
- Cyclohexene dioxide-based plastics (Spurr) the preservation and observation of fine
can be obtained pure, have very low structure not previously subjected to solvent
viscosity, and infiltrate fastest. dehydration.
- Spurr’s Resin is a Low Viscosity mixture - GMA forms only non-crosslinked straight
which provides rapid infiltration of tissues. chains on polymerization and therefore
It's easy to prepare and mixes rapidly. requires no hardener.
- This resin is compatible with ethanol so no - GMA and specifically low acid GMA offers
change to propylene oxide is needed prior to a number of advantages over other systems:
infiltration. Polymerization at 60°C is • Dehydration of tissues can be made
recommended. directly in aqueous dilutions of GMA or
- Epoxy plastics disadvantages: hydrophobic optionally in organic solvents.
and subsequent oxidation by peroxide to • GMA does not need to be water-free and
correct this may produce tissue damage. indeed it works best with at least some
Epoxide groups may reduce antigenicity of water present.
embedded tissue, and may compromise the • Infiltration of tissue with monomer can
result of immunohistochemical staining. be performed at room temperature or
More importantly, epoxy resins may cause lower.
sensitization if absorbed by skin or • Polymerization of GMA can be
inhalation. performed at ambient temperature of 0°C
- The components of many epoxy plastics are with UV radiation to 60°C in an oven.
toxic and one of its components, vinyl • Thin sections of polymerized GMA can
cyclohexane dioxide (VCD) is known to be be cut with glass or diamond knives.
 • Sections from Low Acid GMA, unlike results in superior staining characteristics
ordinary technical grade GMA, resist the and excellent morphological detail.
uptake of stain, thereby reducing greatly - conventional MMA embedding causes
the occurrence of non-specific almost complete loss of enzyme activity and
background staining. protein antigenicity in the tissues, and
• Enzyme digestion, a variety of stains and therefore precludes the use of histochemical
immunological localizations may be and immunohistological methods
performed on thick sections of GMA - it is preferable to use acrylic plastic sections
without removal of the plastic. when high resolution light microscopy is
- Benzoyl peroxide: added to the plastic as a required, because of their ease of handling
catalyst that decomposes to form phenyl and the quality of staining achieved.
radicals acting as an active site for the - However, all acrylic hydrophilic media
polymerization of acrylics. (including glycol methacrylate) are insoluble
- Unlike epoxy plastics, the viscosity of acrylic so that all staining occurs with the plastic in
plastic is low so that short infiltration times situ. Because of this, the embedding medium
are possible, although the size and nature of itself may become stained, or the matrix may
tissue, along with processing and embedding act as a physical barrier to particular
temperature will affect the times required for molecules causing problems during
infiltration and embedding. immunohistochemical staining.
- Radicals can be produced spontaneously by - The alternative use of hydrophobic methyl
heat or light, so that acrylic plastics and their methacrylate permits the plastic to be
monomers should be stored in dark bottles in dissolved, and for certain techniques, this
a cool place to prevent premature may be a very useful property
polymerization. PRACTICAL CONSIDERATIONS:
- Acrylic plastics based on methyl • Specimen should only be processed
methacrylate (MMA) are also widely used under an operational fume hood.
because of its hardness as the ideal • Processing is best achieved if the
embedding medium for undecalcified bone specimen is agitated continuously on a
and is widely used for bone roller mixer.
histomorphometry and bone marrow • Small aliquots of benzoyl peroxide
hematopathology. should be dried carefully away from
- Compared to water-soluble methacrylates direct heat and sunlight as it is potentially
(e.g., glycol methacrylate, GMA), MMA explosive. It is important that no water is
offers a variety of advantages. present before dissolving the catalyst (2
• MMA penetrates tissues better than GMA minutes) in the infiltrating solution. It
and the histological quality of bone must be completely dissolved in the
sections is generally higher in MMA - infiltrating solution, and this may take up
embedded bone samples compared to to 30 minutes.
GMA – embedded specimens. • The acrylic plastic mixes are best
• GMA forms crosslinks during prepared only in the quantity required,
polymerization of the plastic so that preferably using a large glass vial. It is
removal of the resin from tissue sections advisable to measure the quantities
is not possible for GMA - embedded volume by weight.
material. • Any waste solutions containing plastic
• MMA, can easily and completely be components must be handled and
removed from tissue sections. This discarded in accordance with local and
legal requirements.