INFILTRATION/IMPREGNATION
Using AutoTechnicon
A. DEFINITION/PURPOSE
It is the process whereby clearing
agent is completely removed from
the tissue and replaced by a medium
that will completely fill all the tissue.
Purpose:
1. To give a firm consistency to the
specimen
2. To allow easy handling and cutting of
suitably thin sections w/out any damage
to the tissue and its cellular components
B. INFILTRATION METHODS
1. Paraffin Wax Infiltration
Advantages
Thin individual serial sections
may be cut w/ ease
Preparation of sections= w/in
24 hours
Tissue blocks & unstained
mounted sections= may be
stored for long time after
impregnation w/out
considerable tissue destruction
Disadvantages
Overheated paraffinbrittle
specimens
Prolonged impregnation
excessive tissue hardening &
shrinkage
Inadequate
impregnationretention of the
clearing agent
Prolonged immersion in
paraffinnot advisable
Paraffin processingNOT
recommended for fatty tissues
MELTING POINTS OF PARAFFIN
WAX
56OC For routine work
Lab Wax
Temperature temperature
20-24OC 54-58OC
15-18OC 50-54OC
METHODS OF PARAFFIN WAX
INFILTRATION
MANUAL METHOD
4 changes of wax at 15
minutes interval=for complete
removal of clearing agent
Immersed in another fresh
solution of melted paraffin=for
complete embedding/casting
of tissue
AUTOMATIC METHOD
Uses only 2-3 changes of wax
to remove the clearing agent
and properly impregnate the
specimen
Parts of Autotechnicon
o Glass beakers
o Transfer arm
o Cover plate
o Electric clock/Timing Disk
o Spring loaded plunger
Types of Automated Tissue
Processor
o Tissue transfer/ Dip and
dunk-specimen containing
cassettes to be processed
are moved from container
to container
o Fluid Transfer/ Enclosed
Type- cassettes stay in a
single chamber and fluids
are pumped in and out as
required
Vacuum Method- wax
impregnation under negative
atmospheric pressure inside an
embedding oven to hasten
removal of air bubbles and
clearing agent from tissue block.
This technique is
recommended for urgent
biopsies, for delicate
tissues (lung, brain,
connective tissues,
decalcified bones, eyes,
spleen and central nervous
tissue)
FACTORS AFFECTING PARAFFIN
WAX IMPREGNATION
Nature and size of the tissues
Clearing agents to be used
Notes:
1. Larger & Dense tissue blocks=
require longer periods and more
frequent changes of wax
2. Benzene & Xylene- easily removed
from the tissues
3. Chloroform & cedarwood oil-more
difficult to remove
PRECAUTIONS OBSERVED IN
PARAFFIN WAX IMPREGNATION
Prolonged treatment in melted
paraffin=causes shrinkage &
hardening of tissues
 Infiltration in overheated
paraffin=produce shrinkage &
hardening of tissues
Paraffin wax must be pure
Coarse filter paper=used to melt
and filter used paraffin wax
When using an automatic tissue
processing machine, water must
be discarded
For fixed knife microtome: hard
wax w/ a higher melting point is
recommended.
SUBSTITUTES FOR PARAFFIN WAX
Paraplast-mixture of highly
purified paraffin & synthetic plasma
polymers
o melting point: 56-57
o permits large dense tissue
blocks (bones & teeth) to be
cut easily w/ the same result
as double embedding
Embeddol- similar to paraplast w/
a melting point of 56-58OC
Bioloid-semisynthetic wax
recommended for embedding eyes
Tissue Mat-product of paraffin,
containing rubber
Ester wax
o Melting point: 46-48OC
o Not soluble in water
o Soluble in 95% Ethyl Alcohol
& other clearing agents
o Can be used for
impregnation w/out prior
clearing of tissue
o 3-4 changes of wax-for
complete tissue
impregnation
o Sectioning of ester wax
impregnated tissues- should
be done on a HEAVY DUTY
MICROTOME
Carbowax-a polyethylene glycol
o Soluble in water
o Does not remove neutral fats &
lipids
o Tissues are not exposed to too
much heat
o For ENZYME HISTOCHEMICAL
STUDIES
o Routine processing: 4 changes
of carbowax
1 each in: 70% & 90%
conc.
2 times: 100% conc.
@ 56OC
At 30, 45 & 60 minutes
respectively
o Adding soap to water/Using
10% PEG in water=reduce
tissue distortion & promote
flattening & floating out of
sections
2. CELLOIDIN IMPREGNATION
CELLOIDIN/COLLODION-Purified
form of nitrocellulose soluble in many
solvents
Suitable for specimens w/
hollow cavities which tend to
collapse
For hard & dense tissues such as
bones & teeth
Large tissue sections of the
whole embryo
Supplied in Thin (2%), medium
(4%), thick (8%)
ADVANTAGES & DISADVANTAGES
Permits cutting of tissue section
thicker than in paraffin wax
For processing of neurological
tissues
Its rubbery consistency allows
tissue blocks w/ varying
consistency to be cut w/out
undue distortion
Does not require heat during
processing
Celloidin impregnation is very
slow
Very thin sections=difficult to cut
Serial sections=difficult to
prepare
Very volatile
Photomicrographs-difficult to
obtain
2 METHODS
WET METHOD-recommended
for bones, teeth, large brain
sections & whole organs
70-80% alcohol- used to
store tissue until ready for
cutting
DRY METHOD- for processing of
whole eye sections
Gilsons Mixture
(chloroform + cedarwood
oil) added to the celloidin
block before hardening
 NITROCELLULOSE METHOD
LOW VISCOSITY NITROCELLULOSE-
another form of equal
concentration of ether & alcohol
Forms harder tissue blocks & makes
cutting of thinner sections possible
Adding tissue plasticizers (oleum
ricini or castor oil)-may prevent
tendency of tissues to crack
LVN- more explosive than celloidin
& should therefore be handled w/
care
3. GELATIN IMPREGNATION
Used when dehydration is to be
avoided and when tissues are to be
subjected to histochemical &
enzyme studies
Used as embedding medium for
delicate specimens & frozen tissue
sections
Does not require dehydration &
clearing, although fixatives should
still be washed by running water
After washing out of fixatives:
Tissue is placed in 10% gelatin
w/ 1% phenol for 24 hours
Transferred to 20% gelatin w/ 1
% phenol for the next 12 hours
Finally, to another fresh solution
of 20% gelatin w/ 1% phenol.
Gelatin embedded tissues=placed in 10%
formalin for 12-24 hours
Tissues should not be more than 2-3 mm
thick
1% Phenol- prevents growth of mold
Volume of impregnating medium=at least
25 times the volume of the tissue
4. PLASTIC RESINS
For light microscopic studies (hard tissues &
undecalcified bone & for high resolution
microscopy of tissue sections thinner than
usual (4-6 um): renal biopsies, bone marrow
biopsies
CLASSIFICATIONS
Epoxy
1. Bisphenol A (Araldite)- slowest
infiltration
2. Glycerol (Epon)
3. Cyclohexene dioxide (Spurr)-
fastest infiltration
Polyester-for electron microscopy,
seldom used
Acrylic
1. Polyglycol methacrylate(GMA)-
for light microscopy
2. Methyl Methacrylate (MMA)-for
undecalcified bone & other hard
tissues
EMBEDDING
a. Definition/ Purpose
Process by which impregnated tissue is
placed into a precisely arranged position in
a mold containing a medium which is then
allowed to solidify
b. Requirements for embedding
EMBEDDING MOLDS
i. Leuckharts Embedding Mold-
consists of 2 L-shaped heavy brass or
metal arranged on a flat metal plate
ii. Compound Embedding Unit-
made up of series of interlocking
plates resting on a flat metal base,
can embed more specimens at a
time
iii. Plastic Embedding Rings and
Base Mold- consist of a stainless
steel base mold fitted w/ a plastic
embedding ring
o TISSUE TEK- equipped w/
1. warm plate( to manage
impregnated specimen)
2. Cold plate (rapid
solidification of the block)
*Advantages:
ease of use
less paraffin wax
needed
faster embedding
firmly attached tissue
& holder
permanent
identification
iv. Disposable Embedding molds
1. PEEL-AWAY-give perfect even
block w/out trimming, may be
placed directly in the chuck of
the microtome
2. PLASTIC ICE TRAYS- those used
in refrigerators, tissue block is
removed by bending the plastic
tray once the wax has solidified
or by smearing the inner mold
w/ glycerin or liquid paraffin
before embedding
3. PAPER BOATS- for embedding
celloidin blocks but are equally
useful for paraffin wax blocks
c. POINTS IN ORIENTING TISSUE IN THE
BLOCK
1. ELONGATED tissues must be
placed diagonally
2. Tubular or walled organs such as
cysts, fallopian tubes &
gastrointestinal tissues are
embedded EN FACE so as to
provide cross-sections.
3. Tissue w/ an epithelial surface
such as skin are embedded to
provide sections in a plane at A
RIGHT ANGLE TO THE SURFACE.
4. Multiple tissue pieces are
aligned ACROSS THE LONG AXIS
and the center of the mold.
d. PROCEDURES TO ALLOW
SOLIDIFCATION OF BLOCKS
1. Immersed in cold water
2. Cooled rapidly in refrigerator

e. DOUBLE EMBEDDING
Tissues are first infiltrated w/
celloidin and subsequently
embedded in paraffin mass
Used to facilitate cutting of large
blocks of dense firm tissues like
brain
Recommended for making small
sections of celloidin blocks.

f. TRIMMING
Process of removing paraffin to form
a four-sided prism