HISTOPATHOLOGIC AND CYTOLOGIC TECHNIQUES - LECTURE

LESSON 6: IMPREGNATION & EMBEDDING

MIDTERMS | A.Y. 2022-2023 | PROF. DOREN VENUS OTOD

OUTLINE PARAFFIN WAX

I. Impregnation ADVANTAGES DISADVANTAGES
A. 3 Types of General Tissue Impregnation ● Rapid (24 hours) ● Prolonged
1. Paraffin Wax Impregnation ● Maybe cut with ease impregnation will
2. Celloidin Impregnation without undue cause excessive
3. Gelatin Impregnation distortion shrinkage
II. Embedding ● Many staining ● Not recommended to
A. Orientations procedures are fatty tissues
B. Types of Molds permitted (ex. breast)
C. Other Embedding Methods ● Overheated paraffin
makes the specimen
IMPREGNATION & EMBEDDING brittle

I. IMPREGNATION

IMPREGNATION (a.k.a. Infiltration)

● Process that removes the clearing agent
● Fills the tissue cavities
● Permeates tissue with a support medium
● Incomplete impregnation ➨ tissue airholes
● In this process, we are still using different changes
to remove the clearing agent then eventually fill the Figures 1 & 2. Paraffin Wax in Solid and Liquid form
tissue cavities.
● We need to fill the holes in order for us to create a
support and it could be easier during sectioning.

A. 3 TYPES OF GENERAL TISSUE IMPREGNATION

1. PARAFFIN WAX IMPREGNATION

● Paraffin wax is solid in form upon procurement thus

we need to melt it first in order for us to have it in
liquid form. Figure 3. Paraffin Oven
● Requires 2 or more changes of melted paraffin wax
● Melting point: 45°C, 52°C, 56°C, 58°C @RT (20- 3 Ways of Paraffin Wax Impregnation:
24°C) ➔ By manual processing
○ Upon performing, we need to use a hot ◆ This is what we are performing in the
plate in order to maintain the liquid state of laboratory. In this illustration, there are 3
the paraffin wax since it quickly solidifies at changes of paraffin; 1 hour per
room temperature. beaker/change.The changes will depend
● Never overheat; >60°C causes brittleness, on the protocol of the laboratory.
shrinkage, hardening; destruction of lymph tissue
● Paraffin oven must be maintained 2-5°C above
the melting point of wax
● Used pure
○ Wax must be filtered first using coarse filter
paper such as Green’s No. 904 in oven at
2°C higher than the melting point of wax.
○ Reusable only once, but heat it first @
100-105°C (remove water)
○ In the actual practice, it can be actually
used a multiple of times until a cloudiness
can be seen.

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 ➔ By automatic processing
◆ Our automatic processor is usually
designed with 12 stations. The infiltration
will take place up at stations 11 & 12.
◆ The infiltration time will be faster because
there is constant agitation in the machine.
Hence, a shorter processing time.
Figure 4 & 5. Autotechnicon & Elliott bench-type
➔ By vacuum embedding
◆ Among these 3 processes, this type is the
fastest. However, it is expensive because
we need to acquire first the machine itself,
◆ it can only perform the embedding/
infiltration process. Unlike with our
automatic processing, from filtration down
to infiltration.
◆ This is faster because it will remove the air
directly on our tissue thus allowing the
infiltration to happen.
◆ 25-75% reduced time than the usual
processing time.
○ Harder than paraffin thus used with
sliding/sledge-type microtome
○ Water insoluble but soluble in 95%
ethanol, thus prior clearing is not needed
○ But Cellosolve, and xylene may be used if
indicated
● Water Soluble Waxes (Polyethylene glycol)
○ Melting Point: 38-42°C or 45-56°C
○ Ex. Carbowax - most common
■ No need for dehydration and
clearing thus sections are difficult
to float out during the fishing
method and mount
■ Remedy: add soap to water or
10% PEG 900 in water
■ Neutral fats and lipids can be
demonstrated
■ For enzyme histochemistry
■ Used for URGENT
2. CELLOIDIN IMPREGNATION
● A.k.a Colloidin
● Purified form of nitrocellulose/gun cotton
● Specimen with large and hollow cavities which tend
to collapse; hard and dense tissues; neurologic
tissues
● Concentration: in 2%, 4%, 8% dissolved in equal
parts of ether and alcohol - increasing
CELLOIDIN
ADVANTAGES DISADVANTAGES
● Does not require heat; ● Very slow (days or
less shrinkage week)
● Cutting of thicker ● Thin sections are
tissues difficult to cut
● Recommended for ● Very volatile
neurological tissues

METHODS OF CELLOIDIN INFILTRATION

Fixation & Dehydration
⬇
Figure 6. VEU (Vacuum Embedding Unit) Place tissue in ether-alcohol
⬇
Thin celloidin (2%)
SUBSTITUTE FOR PARAFFIN WAX ⬇
● Paraplast
Medium celloidin (4%)
○ Melting Point: 56-57°C
⬇
○ Mixture of pure paraffin and synthetic
Thick celloidin (8%)
plastic polymer (Dimethyl sulfoxide); more
⬇
elastic and resilient
Remove specimen and put in a fresh thick celloidin
● Embeddol
⬇
○ Melting Point: 56-57°C
Keep in jar or desiccator until ether-alcohol evaporates
○ Less brittle, and less compressible
⬇
● Bioloid
WET: Store tissue block in
○ semisynthetic; for embedding of dyes
70%-80% alcohol - Wet
● Ester Wax
Gilson’s Mixture (chloroform and cedarwood oil) - Dry
○ Melting Point: 46-48°C

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➔ Wet - for bones, brain, teeth II. EMBEDDING
◆ Store tissue block in 70%-80% alcohol
◆ The purpose of storing it in this ● a.k.a. Casting, Blocking, Molding
concentration is to avoid dehydration and ● Placing the impregnated tissue into a mold with
shrinkage of the tissue. embedding media, and then allowing the media to
solidify
➔ Dry - for whole eye sections ● Orientation: Arrangement of the tissue in a precise
◆ Store tissue block in Gilson’s Mixture position in the mold during embedding
(chloroform and cedarwood oil) ● Surface to be cut should be parallel to bottom of the
mold
● Molds should bear the accession number
➔ Nitrocellulose/Low Viscosity Nitrocellulose
◆ Has lower viscosity, thus can be used in
higher concentration, and rapid tissue
penetration
◆ Advantages: Harder tissues blocks, thus
thinner sections are possible
◆ Disadvantages: Explosive when dry due
to nitrates
● That is why this is usually
suspended with alcohol to
maintain its liquid state Figure 7. Embedded Tissues
◆ It consists Plasticizer
● E.g. oleum ricini or castor oil A. ORIENTATIONS
● Needed to prevent tissue cracking
● Arrangement of the tissue in a precise position in
in chrome mordanted tissues
the mold during embedding
● This promotes plasticity as well as
● We need to follow a specific type of orientation for a
flexibility in order to reduce
specific type of tissue sample:
brittleness.

3. GELATIN IMPREGNATION ➔ Tubular tissue:

◆ All layers in transverse sections
● Rarely used ◆ If not: we will only see the sides of the
● For histochemical, enzyme studies, and frozen sec. sample, not the layers
● Advantage: Water soluble (no dehydration and ◆ Ex. Fallopian tube, Appendix
clearing needed)
● Disadvantage: may decay
● Tissue must be <2-3mm thick
● 1% Phenol must be added to prevent molds and
prevent further damage such as tissue decay

METHODS OF CELLOIDIN INFILTRATION

Flash out fixative
➔ Skin:
⬇
◆ All layers should come (pahigda)
Put tissue in 10% gelatin with 1% phenol
◆ We need to follow the proper orientation to
⬇
appreciate the layers of the skin
20% gelatin with 1% phenol
⬇
Fresh 20% gelatin with 1% phenol
⬇
Cool in refrigerator
⬇
10% formalin

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➔ Endometrial curetting: 2. Compound Embedding Unit
◆ Keep in center ● This used for batch embedding thus one of
◆ Ex. scrapings in endometrial lining its disadvantages is being prone to the
interchanging of samples.
● This is made of interlocking plates and
there is a heavy base at the bottom.

➔ Long tissue:
◆ Keep diagonally
◆ It should be kept in a diagonal position for
it to stay continuous for the ease of the Figure 10. Compound Embedding Unit
pathologist’s readings.
3. Plastic Embedding Rings and Base Mold
● (11) This is made of silicon.
● (12) This is made of metal.

➔ Intestine:
◆ All layers should come Figure 11 & 12. Plastic Embedding ring; Base Mold

4. Disposable Embeding Molds

● In plastic ice trays, we need to grease it
first with glycerin/liquid paraffin wax
before the process of embedding.
● This is for the ease of separation of the
➔ Membrane: tissue block from the mold after it solidifies.
◆ Swiss roll

Figure 13-15. Peel away; Plastic Ice Tray; Paper boat

B. TYPE OF MOLDS
C. OTHER EMBEDDING METHODS
1. Leuckhart’s Embedding Mold
● It has two (2) L-shaped metal plates, strips DOUBLE-EMBEDDING METHOD
of heavy brass metal. ● Tissues are first infiltrated with Celloidin and
● This is adjustable depending on the size of subsequently embedded in a paraffin mass
the tissue. ● For large blocks of dense tissues; obsolete

PLASTIC RESIN EMBEDDING

● For high resolution light microscopy of thinner than
usual sections, renal biopsies, BM biopsies
○ (1) Epoxy, (2) Polyester, (3) Acrylic Plastic

➔ EPOXY
◆ For electron microscopy
Figure 8 & 9. Leuckhart’s Embedding Mold ◆ Most widely applied, but carcinogenic due
to vinylcyclohexane dioxide (VCD)
component

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 ◆
Types:
● Bisphenol A (Araldite) - slow
● Glycerol (Epon) - low viscosity
● Cyclohexene dioxide (Spurr) -
very low viscosity; fastest
➔ POLYSTER
◆ For electron microscopy; seldom used

➔ ACRYLIC PLASTICS
◆ For high resolution light microscopy
◆ Examples:
● Plyglycol methacrylate (GMA)
● Methyl methacrylate (MMA)
◆ Benzoyl peroxide: catalyst; forms radicals
which are site for polymerization
◆ Acrylic plastics must be stored in dark
bottles to prevent radical formation and
premature polymerization
◆ Embedding media may be stained, thus
use hydrophobic MMA
◆ The tissue block cannot be decayed
because it already underwent fixation
down to infiltration thus it can outstand up
until 10 years time.

Reference: Bruce-Gregorios, J.H.(2016)/ Histopathologic

techniques. (2nd Revised Edition). Makati, PH: Katha
/N83)
Publishing (611.0185/b83)

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