IMPREGNATION AND

EMBEDDING
GLENMAYNE Y. SABALLA, RMT
IMPREGNATION AND EMBEDDING
Impregnation (Infiltration) is the process whereby
the clearing agent is completely removed from the
tissue and replaced by a medium that will completely
fill all the tissue cavities and give a firm consistency to
the specimen.
Embedding (Casting or Blocking) is the process by
which the impregnated tissue is placed into a
precisely arranged position in a mold containing a
medium which is then allowed to solidify.
 IMPREGNATION AND EMBEDDING
oEmbedding Medium oCharacteristics of good embedding
omedium used to infiltrate the tissue medium
is usually the same medium utilized oprocessing fluids
for impregnation osuitable for sectioning and ribboning
omolten between 30°C and 60°C
otranslucent or transparent; colorless
ostable
ohomogeneous
ocapable of flattening after ribboning
onon-toxic
oodorless
oeasy to handle
oinexpensive
 ADVANTAGES
1.Thin individual serial sections may be cut with ease from the
majority of tissues without distortion.
2. The process is very rapid, allowing sections to be prepared within
24 hours.
3. Tissue blocks and unstained mounted sections may be stored in
paraffin for an indefinite period of time after impregnation without
considerable tissue destruction.
4. Because formalin-fixed, paraffin-embedded tissues may be stored
indefinitely at room temperature, and nucleic acids (both DNA and
RNA) may be recovered from them decades after fixation, they are an
important resource for historical studies in medicine.
5. Many staining procedures are permitted with good results.
 Disadvantages
1. Overheated paraffin makes the specimen brittle.
2. Prolonged impregnation will cause excessive tissue shrinkage and
hardening, making the cutting of sections difficult.
3. Inadequate impregnation will promote retention of the clearing
agent. Tissues become soft and shrunken, and tissue blocks crumble
when sectioned and break up when floated out in a water bath.
4. Tissues that are difficult to infiltrate, e.g. bones, teeth, brains and
eyes, need long immersion for proper support; otherwise, they will
crumble on sectioning. Prolonged immersion in paraffin, on the other
hand, is not advisable.
5. Paraffin processing is not recommended for fatty tissues. The
dehydrating and clearing agents used in the process dissolve and remove
fat from the tissues.
FOUR TYPES OF IMPREGNATION AND
EMBEDDING MEDIUM
1. Paraffin wax
2. Celloidin (collodion)
3. Gelatin
4. Plastic
PARAFFIN WAX IMPREGNATION
ois the simplest, most common and best
embedding medium used for routine tissue
processing.
oPolycrystalline mixture of solid
hydrocarbons produced during the refining
of coal and mineral oils.
oSolid at room temperature but melts at
temperatures up to about 65°C or 70°C.
oMelting point: 56°C to 58°C.
 PARAFFIN WAX IMPREGNATION
oThere are three ways by which paraffin wax impregnation and
embedding of tissues may be performed:
1. By manual processing- At least four changes of wax are required
at 15 minutes intervals in order to insure complete removal of the
clearing agent from the tissue.
2. By automatic processing- use of an automatic tissue processing
machine (i.e., Autotechnicon)
3. By vacuum embedding- involves wax impregnation under
negative atmospheric pressure inside an embedding oven. It
reduces the time.
Vacuum embedding oven Tissue processor with vacuum and fume control
Embedding Procedure: After fixation and dehydration, proceed as follows:
1. Clear in two changes of xylene, for 1 hour each.
2. Place the tissue in molten wax, in vacuum chamber and make the oven
airtight. Exhaust the air slowly by means of a vacuum pump or
Venture suction pump until there is a negative pressure of 400 to 500
mm. mercury.
3. Leave for 15 minutes, then slowly readmit air until normal atmospheric
pressure is reached.
4. Place the tissue in fresh wax.
5. Repeat steps 3 and 4.
6. Place the tissue into fresh wax.
7. Repeat step 3 and leave for 30 to 45 minutes.
8. Bring to normal atmospheric pressure and embed the tissue.
NOTE: The exhaustion and readmission of air must be gradual or
the specimen may be ruined.
CELLOIDIN (COLLODION)
opurified form of nitrocellulose soluble in many solvents,
suitable for specimens with large hollow cavities which tend
to collapse, for hard and dense tissues such as bones and
teeth and for large tissue sections of the whole embryo.
oNo heat is required, and the resultant block has a rubbery
consistency which gives good support to the tissues.
Two methods are used for celloidin
impregnation
Wet Celloidin Method - is recommended for bones, teeth, large
brain sections and whole organs.
• After the usual fixation and dehydration of the tissue, it is placed in
equal parts of ether and alcohol for 12-24 hours.
Dry Celloidin Method is preferred for processing of whole eye
sections.
• The principle and procedure of this method is similar to wet
celloidin method, except that 70% alcohol is not used for storage
before cutting.
GELATIN IMPREGNATION
orarely used except when dehydration is to be
avoided and when tissues are to be subjected to
histochemical and enzyme studies.
oIt is used as an embedding medium for delicate
specimens and frozen tissue sections because it
prevents fragmentation of tough and friable tissues
when frozen sections are cut.
 PLASTIC (RESIN) EMBEDDING
oprovided superior results for light microscopic studies
oPlastics are classified into epoxy, polyester, or acrylic, based on
their chemical composition.
oEpoxy embedding plastics are made up of a carefully balanced mixture of
poxy plastic, catalysts and accelerators.
o those based on either bisphenol A (Araldite)
o glycerol (Epon)
o cyclohexene dioxide (Spurr).
oPolyester plastics were originally introduced for electron microscopy in the
mid- 1950s, seldom used
oAcrylic plastics are made up of esters of acrylic or methacrylic acid, and are
used extensively for light microscopy.
EMBEDDING
othe tissue is placed into a mold containing the embedding medium and
this medium is allowed to solidify.
oSeveral types of Blocking-out Molds may be used:
1. Leuckhart’s Embedding Mold - consists of two L-shaped strips of
heavy brass or metal arranged on a flat metal plate and which
can be moved to adjust the size of the mold to the size of the specimen .
2. Compound Embedding Unit - made up of a series of interlocking
plates resting on a flat metal base, forming several compartments.
3. Plastic Embedding Rings and Base Mold -consist of a special
stainless steel base mold fitted with a plastic embedding ring, which
later serves as the block holder during cutting.