Histopathologic and Cytologic A.Y.

2023-
techniques
MT 2024
2nd
Impregnation and Embedding 113 Semester

IMPREGNATION 4 TYPES OF IMPREGNATION

• Also known as infiltration
• Process whereby the clearing
agent is completely removed
from the tissue and replaced by
a medium that will completely
fill the tissue cavities and give
firm consistency to the
specimen.
• This allows easier handling and
cutting of suitably thin sections
without any damage or
distortion to the tissue and its
cellular components.
• 25 times the tissue volume
• After clearing, tissue is
submerged in 2 or more
changes of melted paraffin
wax
• Process of replacing the
clearing agent with infiltrating
medium
• The medium used to infiltrate
the tissue is usually the same
medium used for embedding.
• Four types of tissue
impregnation and embedding
media
o a. Paraffin wax
o b. Celloidin (Cellodion)
o c. Gelatin
o d. Plastic
• Filling in tissue
cavities/holes/spave by using
melted wax
EMBEDDING
• (Casting or Blocking)
• It 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.
AND EMBEDDING MEDIUM
• Paraffin wax
• Celloidin (collodion)
• Gelatin
• Plastic
INFILTARING AND EMBEDDING
MEDIUM SHOULD BE:
• Soluble in processing fluids
• Suitable for sectioning and
ribboning
• Molten between 30°C and 60°C
translucent or transparent;
• Colorless stable
• Homogeneous
• Capable of flattening after
ribboning
• Non-toxic
• Odorless
• Easy to handle
• Inexpensive
example of a time schedule for
manual processing of tissues about 3
mm thick:
Fixation:
• 10% Buffered Formalin 24 hours
• Dehydration:
• 70% Alcohol 6 hours
• 95% Alcohol 12 hours
• 100% Alcohol 2 hours
• 100% Alcohol 1 hour
• 100% Alcohol 1 hour
Clearing:
• Xylene or Toluene 1 hour Xylene
or Toluene 1 hour
Impregnation:
• Paraffin wax 15 minutes
• Paraffin wax 15 minutes
• Paraffin wax 15 minutes
• Paraffin wax 15 minutes

BEVERLY MALANA 1
 Histopathologic and Cytologic
techniques
Impregnation and Embedding
Embedding:
• Paraffin wax 3 hours
II.
PARAFFIN
• Butschlii- first used the paraffin
wax
• Simples/most common and BEST
infiltrating/embedding medium
• Is NOT recommended for fatty
tissues- frozen section
• The 56°C wax is normally used
for routine work.
• Temperature of paraffin oven-
55 to 60C-paraffin oven must be
maintained at a temperature 2-
5C above the MP of the
paraffin wax.
• To remove excess water- heat
the wax to 100-105C
• Common waxes have melting
points of 45°C, 52°C, 56°C and
58°C.
• Hard tissues require wax with a
higher melting point than soft
tissues.
WAYS TO PERFORM PARAFFIN
WAX IMPREGNATION
1. By manual processing
2. By automatic processing
3. By vacuum embedding
I. MANUAL PROCESSING
• At least four changes of wax
are required at 15 minutes
III.
intervals in order to insure
complete removal of the
clearing agent from the tissue.
The specimen is then immersed
in another fresh solution of
melted paraffin for
approximately 3 hours to insure
complete embedding or
casting of tissue.
1. 15 MINS
2. 15 MINS
3. 15 MINS
BEVERLY MALANA
A.Y. 2023-
MT 2024
2nd
113 Semester
4. 15MINS
AUTOMATIC PROCESSING
• This method makes use of an
automatic tissue processing
machine (Ex. Autotechnicon,
Elliot Bench Type Processor)
which fixes, dehydrates, clears
and infiltrates tissues, thereby
decreasing the time and labor
needed during the processing
of tissues.
• Number of stations: 12
• 10 L container
• Wax Bath Thermostat: set at 3
°C higher than the melting point
of the wax Histopathologic
techniques performed: fixation,
dehydration, clearing,
impregnation
o Steps in autotechnicon
o Station1 and 2: 10%
formalin Station 3-6:
increasing grade of
alcohol
o Station 7-8: acetone for
further dehydration
o Station 9-10:
chloroform/xylene
o Station 11-12: Liquid
Paraffin
o Main advantage:
constant agitation
VACUUM EMBEDDING
• Principle: Wax impregnation
under negative atmospheric
pressure inside an embedding
oven
• Hastens removal of air bubbles
and clearing agent
• Promotes rapid penetration of
impregnating medium
• It is the fastest method and
used for urgent biopsies,
delicate tissues, CNS tissues.
2
 Histopathologic and Cytologic
techniques
Impregnation and Embedding
Substitute for paraffin wax- xylene
based
1. paraplast-MP=56-57 C
• Mixture of highly purified
paraffin and synthetic plastic
polymers
• More elastic and resilient than
paraffin
• For large dense tissue blocks
such as bones and brain
2. Embeddol- MP-56-58C
• Less brittle and less
compressible than paraplast
3. Bioloid
• recommended for embedding
eyes MP-56-58C
4. Tissue mat
• a product of paraffin,
containing rubber, with the
same property as paraplast
5. Ester wax- MP-46-48 C
• Harder than paraffin
• Not soluble in water
• Soluble in 95% EOH and other
clearing agents
• Can be used for impregnation
without prior clearing of tissue
(can omit clearing)
6. Water soluble waxes- MP 38-42 C or
45-56C
• Mostly polyethylene glycols
• Most commonly used:
Carbowax
• Soluble and micible with water
(hence does not require
dehydration and clearing of the
tissue)
• Suitable for many enzyme
histochemical studies
BEVERLY MALANA
A.Y. 2023-
MT 2024
2nd
113 Semester
CELLOIDIN
• Purified form of nitrocelluloase
• Suitable for specimens with
large hollow cavities and dense
tissues (bone and teeth), large
tissue section of the whole
embryo.
2 methods for celloidin impregnation
1. Wet
• recommended for bones,
teeth, large brains and whole
organ
2. Dry
• preferred for processing whole
eye sections.
• L.V.N. (low viscosity
Nitrocellulose) another form of
celloidin
• Soluble in equal concentration
of ether and alcohol with lower
viscosity, allowing it to be used
in higher conc. And still
penetrate tissues rapidly.
GELATIN
• Rarely used when dehydration
is to be avoided
• Used when tissues are for
histochem. and enzyme studies
• Embedding medium for
delicate specimens and frozen
sections because it prevents
fragmentation of tough and
friable tissues when frozen
sections are cut
• Water soluble
• Does not require dehydration
and clearing
PLASTIC/RESIN
Classified into epoxy, polyester, acrylic
• Epoxy brand
1. Bisphenol A (araldite)
3
 Histopathologic and Cytologic
techniques
Impregnation and Embedding
2. Glycerol (Epon)
3. Cyclohexene dioxide (spurr)
EMBEDDING
• Casting or Blocking
• 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.
• ORIENTATION- process by which
tissue is arranged in precise
positions in the mold during
embedding, on the microtome
before cutting, and on the slide
before staining.
• Temperature of melted paraffin
used for embedding = 5-10C
above its melting point.
• To solidify embedded tissue-
cooled rapidly in a ref (-5 C) or
immersed in cold water
• The surface of the section to be
cut should be placed parallel to
the bottom of the mold in which
it is oriented
• It is done after Wax
Impregnation
• Orientation: positioning the
impregnated tissue in the
embedding mold
MOLDS USED
✓ Leuckhart's Embedding Mold: 2
L shaped Strips of Heavy brass
or metal
• Advantage: the size of the mold
• Is adjustable
✓ Plastic embedding rings and
Base molds: special stainless
steel base mold fitted with
plastic embedding ring. It is
used is tissue tek
BEVERLY MALANA
A.Y. 2023-
MT 2024
2nd
113 Semester
✓ Disposable molds: Peel away (3
sizes: 22x22 mm, 22x30mm,
22x40mm), Ice tray, Paper boat
TRIMMING
• process of removing excess wax
after embedding
• Excess wax is cut off from the
block to expose the tissue
surface in preparation for
actual cutting
• Knife/blasé may be used
• Four sided prism/truncated
pyramid
• At least 2 mm of wax should
surround the tissue blocked.
SECTIONING
• CUTTING or MICROTOMY
• The process by which a
processed tissue is cut into
uniformly thin slices (sections) to
facilitate studies under the
microscope
• 4-5um- routine histologic
procedure (rotary microtome)
Binconcave knife
• 10-15 um- frozen sections
(cryostat)unfixed
• 0.5 um- electron microscopy
1. Rocking Microtome
(Cambridge Rocking
mucrotome)
o simplest among the
microtomes
o Disadvantage: difficult in
re orienting the block
o Inventor: Paldwell Trefall-
1881
o Can cut 10-12 um tissue
sections
o For large paraffin
embedded blocks
4
 Histopathologic and Cytologic A.Y. 2023-
techniques
MT 2024
2nd
Impregnation and Embedding 113 Semester

2. Rotary/Minot microtome- o Cutting sections as thin as 0.5

inventor um
o Minot 1885-1886
o Most common type used 7. Freezing Microtome
today especially for o invented by Queckett in 1848
paraffin-embedded o CO2 is used as
o tissue propellant/freezing agent
o Can cut 4 um or 10 um
3. Sliding microtome o Use alcohol to clean the knife in
o MOST DANGEROUS type due freezing microtome
to movable expose knife
MICROTOME KNIVES
o Inventor: Adams 1789
o Mostly made of stainless steel.
o For celloidin sections and
hard rough tissue blocks 3 basic types:
TYPES OF SLIDING MICROTOME 1. Plane concave (25 mm)
o Less concave side: celloidin
a. Base-sledge
o More concave side: for paraffin
o For all forms of media
o Use: base sledge, rotary or
o Block holder: moving
rocking microtome.
o Knife: stationary
2. Biconcave knife (120mm)
b. Standard Sliding microtome
o Both sides are concave
o Block: stationary
o Used for cutting paraffin
o Knife: moving
sections
o Used for rotary microtome
4. Rotary rocking microtome
o Routinely used
3. Plane concave knife (100mm)
o Invented by Minot
o For frozen sections or very hard
o Cutting of paraffin embedded
tissue.
sections
o Used for base sledge or sliding
o Excellent for serial sections
microtome
5. Vibrotome Other types of knives:
o used for unfixed, unfrozen
1. Disposable blades: more
specimen sectioning for
commonly used
enzyme demonstrations
2. Glass and diamond knives: for
o Disadvantage: sections are
Ultrathin microtome, glass
liable to disintegrate
knives: "ralph knives
o Clearance angle-0-15 degrees
6. Ultrathin Microtome
o Bevel angle-27-32 degrees
o for cutting sections for Elec.
o For routine work: 76x25mm slides
Microscopy
that are 1.0-1.2 mm thick are
o Uses DIAMOND knives or broken
usually preferred because they
plate glass Specimen is small,
do not break easily.
fixed in osmium tetroxide,
o Water bath 45-50C
embedded in plastic.

BEVERLY MALANA 5
 Histopathologic and Cytologic A.Y. 2023-
techniques
MT 2024
2nd
Impregnation and Embedding 113 Semester

o Approximately 6-10C lower than

the MP

HONING
• Knife hard sharpening
• Process of removing nicks/
irregularities of knife.
• Movement: from heel to toe
• Number of strokes per surface:
10-20 strokes

Hones

• Dimensions: 8inx3in

Types:

• Belgium yellow (provides best

result)
• Arkansas
• Fine carborandum (for badly
nicked)
• Plate glass (8x3x1inches)
excellent substitute for stone
• Lubricants: (soapy water,
mineral oil, clove oil, xylene, or
liquid paraffin)
• Clean knife with xylene

STROPPING
• Polishing
• Removal of burrs (irregularities if
knofe after honing)
• Movement: toe to heel
• Use paddle strope made of
horse leather
• Dimensions: 3-4x18in
• Strops should be oiled on the
back (DO NOT USE MINERAL
OIL)
• 40-120 DOUBLE STROKES

BEVERLY MALANA 6