Name: DAYAGAN, GWYNETH MARIE M.

Date: JULY 8, 2021

EXERCISE 5
IMPREGNATION and EMBEDDING

Impregnation or Infiltration involves removal of the clearing agent from the tissue
and saturating the tissue with a liquid that will fill natural cavities and tissue spaces. This
allows easier handling and cutting of suitably thin sections without any damage or distortion
to the tissue and it cellular components.

Embedding or Blocking or casting is the process by which the impregnated tissue

is placed precisely arranged position in a mold containing melted wax which is allowed to
solidify. The volume of the embedding medium should be at least 25 times the volume of the
tissue.

Impregnating and Embedding medium should be soluble in processing fluid. It

should be easy to section and make ribbons molten between 30 to 60° C. Common medium
used is the paraffin wax that can fill the tissue spaces so that airholes will not form upon
sectioning. After embedding, a paraffin block is produced which will be cut using the
microtome.

1. OBJECTIVES
At the end of the laboratory exercise, the students should be able to:

1.1 Demonstrate the proper infiltration of the tissue with paraffin wax.

1.2 Demonstrate the proper placing of tissue in a precisely arranged position in an

embedding mold using manual method.

2. MATERIALS

2.1 Cleared Kidney, Liver & Lung tissue 2.6 Embedding mold
2.2 Paraffin pitcher with thermal jacket 2.7 Ruler
2.3 Paraffin bath 2.8 Scissors
2.4 Stirring rod 2.9 Thermometer
2.5 Forceps

3. REAGENT
3.1 Paraffin wax pellets ……….….. 1 kg
4. PROCEDURE

4.1 IMPREGNATION / INFILTRATION:

4.1a Dissolve paraffin wax in five separate beakers. The maximum temperature should
be only 2 – 5°C higher than the melting point of the wax which is 60°C.
4.1b While waiting for the wax to melt, prepare the embedding mold.

4.1c Immerse previously cleared specimens in 4 changes of wax 1 hour each,

having 15-minute interval per change for a total of 4 hours impregnation.
4.1d Leave the specimen in the paraffin bath and prepare materials for embedding.

4.2. EMBEDDING:

4.2a Dissolve paraffin wax pellets in the pitcher with thermal jacket. The maximum
temperature should be only 2 – 5°C higher than the melting point of the wax
which is 60°C.
4.2b Prepare the label using a pre-measured strip of paper. Using a pencil, write the
family name, initials of the patient & type of specimen (represented by block
number).
4.2c Seal the label by immersing it in the paraffin pitcher with melted wax.
4.2d Prepare the hinge pop-out mold, making sure that it is clean and dry prior to
use. The size of the mold must fit the tissue with allowance for at least 2 mm
margin of wax at the four sides.
4.2e Fill the mold with melted wax up to the rim. Using a clean and preheated
forceps, transfer the impregnated tissue from the paraffin bath to the mold.
Orient the tissue in the perfect central position.
4.2f. Place the label in such a way that it is facing outside on one-side of the mold,
avoiding the slit part.
4.2g Allow the wax to cool using ice about 3-5 mins for complete cooling and
hardening. Do not over-expose the block in the ice for it may crack or break.
4.2h. Remove the block from the mold.
4.2i. Cross-check the block if the tissue was oriented correctly and transfer the label
at the side of the block using the pre-heated paraffin knife.
4.2j. You must redo the procedure if there is any error in the embedded tissue.
4.2k. If everything is done properly, the tissue block is now ready for cutting using
a rotary microtome.

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5. RESULT/OBSERVATION

5.1 Draw and label the set up for Infiltration / Impregnation:

5.2 Draw and label the set up for Embeddi

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6. STUDY QUESTIONS

6.1 What are the different types of molds used in tissue processing?

1. Leuckhort’s Embedding Mold

2. Compound Embedding Unit
3. Plastic Embedding Rings & Base Mold – Tissue Tek
4. Disposable Embedding Molds
a. Peel-Away
b. Plastic Ice
c. Paper Boats
5. Pop-out Hinge Type
6.2 Enumerate substitutes for paraffin wax used in infiltration or impregnation? Give its
advantages and disadvantages

1. PARAPLAST

Advantages – more elastic and resilient than paraffin wax thereby permitting large dense tissue
blocks such as bones and brain to be cut easily with the same result as in double embedding.
Blocks obtained are more uniform than with any other medium, with better ribboning of sections.
does not tend to crack like other paraffin wax substitutes

Disadvantages - During the winter, 54° to 56°C Paraplast may be used if the tissue is cut in a
cool room. During the summer it may be necessary to use 60 to 63°C, although this is to be
avoided if possible in order to not to "cook" the tissue. "Cooked" tissue does not section well or,
if it does, it does not stain well and most details are destroyed.

2. EMBEDDOL – it has similar properties with Paraplast, and it less brittle and less
compressible than Paraplast.

3. WATER SOLUBLE WAXES (CARBOWAX) - used when dehydration is to be

avoided and when tissues are to be subjected to histochemical and enzyme studies

Advantages - blends presently available to improve adhesion, hardness and plasticity, soluble in
and miscible with water; hence does not require dehydration and clearing of the tissue.
Processing time is reduced with the special advantage that harmful effects produced by ordinary
dehydrating agents are consequently avoided. Tissues are not exposed to too much heat so that
excessive hardening, shrinkage and brittleness of tissue is avoided

Disadvantages - Due to its hygroscopic nature, Carbowax is very easily dissolved in water.
Hence care must be taken to avoid contact of the block with water or ice. Tissue sections are
very difficult to float out and mount due to its extreme solubility in water, dehydrating and
clearing agents.
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 4. DIMETHYL SULPHOXIDE (DMSO)

Advantages - reduces infiltration times and facilitates thin sectioning

Disadvantages - Some individuals who handle DMSO-paraffin wax may experience an

unpleasant and annoying oyster or garlic taste probably caused by DMSO metabolites. Possible
health risks associated with the use of DMSO-paraffin wax are minimal

5. ESTER WAX

Advantages - lower melting point but it is harder than paraffin not soluble in water but in 95%
ethyl alcohol and other clearing agents; can be used for impregnation without the need for
clearing

Disadvantages - Sectioning of ester wax-impregnated tissues should be done on a heavy-duty

microtome (e.g. sliding or sledge type microtome) due to the relative hardness of the wax.

6.3 Aside from paraffin, what are the other media which can be used for impregnation and
embedding? When are these media used?

1. CELLOIDIN – used when specimens are dense tissues (hard to infiltrate) and collapse
easily due to air spaces, since it avoids crumbling of tissues during sectioning, thus
supported better; also allows very hard tissue blocks and those of varying consistency
to be cut without distortion.

2. GELATIN IMPREGNATION – used when dehydration is to be avoided and when

tissues are to be subjected to histochemical and enzyme studies

3. PLASTIC (RESIN) EMBEDDING – used when high resolution of light microscopy is

required in hard tissues and tissue sections thinner than the usual 4–6 um

6.4 Discuss the myriad factors affecting tissue processing.

Based on Lo, Raymundo W. et al (2015) Basic Histopathologic Techniques:

• SOLVENT EFFECTS
– Especially the dissolution of lipid-rich structures such as cell membranes and fat
droplets, increase tissue porosity that results in shrinkage.
• FIXATION
– May shrink the tissues to a certain degree, but completely fixed tissues are
protected against further significant contraction and hardening during processing.

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 • TISSUE MODIFIERS
– Such as phenol are added during fixation and processing to stabilize and soften
hard tissues. Phenol acts swelling collagen and elastic fibers, and enhance protein
polymer formation and tissue porosity.
• TISSUE POROSITY
– It is influenced by the nature and composition of the tissues and the effects of
fixatives, modifiers and processing reagents to which the tissues are subjected.
Tissue porosity involves natural and artifactual pores, and the swelling and
shrinkage of the biopolymer matrix.
Based on Gregorios, Jocelyn H. (2006) Histopathologic Techniques. 2nd ed.:
• TISSUE DENSITY AND THICKNESS
– Tissue size and tissue type will determine how quickly the reagents penetrate the
sample. Spongy tissues (like lungs) are more rapidly infiltrated than hard and
dense tissues. Thickness of the tissue also influences the rate of reagent diffusion
hence, the processing time which should be adjusted to accommodate thick, thin
or large tissue blocks.
• AGITATION
– Agitation using manual or automated processors increases the flow of fresh fluids
in and around the tissues. Most tissue processing protocols utilize automated
processors with vertical or rotary oscillation mechanisms to speed fluid exchange.
Without agitation, tissues tend to settle to the bottom of the processing device or
become too tightly packed, thus reducing surface area available for fluid
exchange.
• TEMPERATURE
– High temperature can cause the tissue to shrink and to become hard and brittle,
while low temperature increases the viscosity of reagent used in tissue processing,
thereby reducing the rate of diffusion and increasing processing time.
• VACUUM AND PRESSURE
– Reduced pressure can increase the infiltration rate and decrease the time needed to
complete steps in tissue processing protocols. High pressure facilitates infiltration
of dense specimens with the more viscous embedding media. Vacuum application
during tissue infiltration improves processing quality and can aid in removal of
trapped air from porous tissues.
6.5 Discuss the automated tissue processing.
Tissue processing can be done manually (hand processing), but when a significant
number of tissues needs to be processed, an automated tissue processing system is far more
convenient and efficient. This equipment allows for the infiltration of specimens with a series of
various solvents, finishing in molten paraffin wax. The specimens start off in an aqueous
environment (water-based) and must go through a series of dehydrating and clarifying solvents

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(usually ethanol and xylene) before being deposited in molten wax (which is hydrophobic and
immiscible with water). The duration and step details of the “processing schedule” chosen for a
particular batch of specimens will depend on the nature and size of the specimens. As a result,
the diagnosis can be made more quickly and with less complication. To remove the clearing
agent and properly impregnate the specimen, just two to three changes of wax are usually
required. This is made possible by the continual agitation of the tissue, which speeds up and
enhances tissue penetration, resulting in more consistent results.

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Student’s Name : DAYAGAN, GWYNETH MARIE M. Group& Sec: TABLE 2- 2C Date: JULY 8, 2021

Exercise No. 5: INFILTRATION and EMBEDDING

CRITERIA BEGINNING DEVELOPING ACCOMPLISHED EXEMPLARY SCORE

(5) (10) (15) (20)
Ability to Answer Able to answer Able to answer Able to answer Able to answer
Questions 25% of the 50% of the 75% of the item 100% of the
Able to answer all items required. items required. required. item required.
the required items
in the activity .
Content/Knowledge Presents 25% Presents 50% Presents 75% Presents 100%
Presented of the of the of the of the
Presents knowledge knowledge knowledge knowledge
scientific/practical required. required. required. required.
facts based on
knowledge of the
subject.
Finding Relevant 25% of the 50% of the 75% of the 100% of the
Information answers were answers were answers were answers were
Presents facts and supported by supported by supported by supported by
relevant facts & relevant facts & relevant facts & relevant facts & relevant
information to information. information. information. information.
support the
answers.
Sentence Fluency & 25% of the 50% of the 75% of the 100% of the
Organization of sentences were sentences were sentences were sentences were
Thoughts clear & clear & clear & clear &
Sentences are understandable. understandable. understandable. understandable.
very clear & Lacks Few ideas Most of the All ideas
understandable. spontaneity of showed ideas showed showed
ideas. spontaneity. spontaneity. spontaneity.
Ideas were
presented in order
and spontaneous.
Punctuality Did not able to Submitted on Submitted on Submitted on or
Submitted the submit on the the closing date the closing and before the due
output on the due date & but exceed 1 a day after the and closing
specified date & closing date. day on the due due date. dates.
time. date.
TOTAL SCORE

Preceptor’s Signature: __________________